Approaches to Aging Control - Sociedad Española de Medicina

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APPROACHES TO AGING CONTROL. VOL 17. SEPTEMBER 2013
Approaches to
Aging Control
Journal of Spanish Society of Anti-Aging Medicine and Longevity
Nº 17
September 2013
SEMAL
www.semal.org
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. 3%04%-"%2 - Editorial ..................................................................................................................................................
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- Articles....................................................................................................................................................
7
- Replicative Senescence and the Telomere Connection ........................................................
7
Jerry W. Shay
- The Future Role of Testosterone in Treating Prostate Cancer.................................................... 13
Edwin Lee, M.D., F.A.C.E.
- Aging and sleep, and vice versa......................................................................................................... 17
María Amparo Lluch, MD, PhD; Tomás Lloret, MD, PhD. Vicenta Llorca, MD.
- Calcium and Vitamin D. Health implications ................................................................................ 22
Pascual Garcia Alfaro, Máximo Izquierdo Sanz, Montserrat Manubens Grau
- Lifestyle and breast cancer. A review............... ............................................................................ 27
Màxim Izquierdo, Pascual García, Montserrat Manubens.
- Melatonin and Cancer ......................................................................................................................... 33
Gilberto E. Toniolo Chechile
- Silent Players of Health in a Broader Age-management Medicine Understanding:
A Dissertation on Bacteriocin of Lactic Acid Bacteria. ................................................................. 48
R. Catanzaro, M. Milazzo, C. Tomella, U. Solimene, A. Polimeni, F. Marotta
- Dermotological Diseases and Human Placental Extracts Psoriasis Case
Study in Europe............... ................................................................................................................ 90
Vicenta Llorca, MD; Tomás Lloret, MD, PhD; Jesús Ballesteros, FD.
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WWWAPPROACHESTOAGINGCONTROLORG
%DITORIAL
For a healthy aging, it is important to know the relative influence of genes versus environment in
determining life expectancies. Over the last two decades, scientists have found that certain genes can play
important parts in determining the rate of aging in model organisms. However, isolating a genetic link to
active life is complicated by human genetic heterogeneity, and a wide variety of patterns of aging.
Genetics, in contrast to life style and environmental agents, generally has minor influence on age-related
disorders and health, although there are exceptions: certain deleterious gene loci have been associated
with accelerated changes characteristic of aging (presenilin, APOE e4, etc). From identical twins studies,
it is generally accepted that aging is determined 70 % by life style and 30 % by genetic. Thus, about onethird of life span can be associated with particular gene differences between individuals.
Identical twins have exactly the same genetic but many times they show great differences in life span and
they do not have the same diseases, even if those diseases have a genetic component like diabetes, heart
diseases. How these twins, under the same environment are so different? This is because life style and
environment affect our genes through epigenetic, which is a simple process by which we have chemical
switches that can turn our genes “on and off”. For instance, epigenetic turns “on and off” our defense
mechanisms. Two features of these chemical switches are: they can last during several generations
and they are not permanent. This means that sometimes the changes in our genes occur without “our
permission”. Also, our bad habits may affect our grandchildren.
Folic acid is the most important “epigenetic vitamin”. The right intake has many health advantages but
if we take too much of it we can get increased risk of cancer. In addition to these popular protective
compounds having effect on our epigenetic, there are many toxins and chemicals (coming from plastics,
paint, pollution, etc) with powerful negative epigenetic actions, for instance, on the hormone and
neurotransmitters levels and this can be passed on to offspring. As a conclusion, we know that many
environmental factors affect our genes and longevity but in many cases keeping our health is not under
our control.
6
Replicative Senescence and the Telomere Connection
Jerry W. Shay, The University of Texas Southwestern Medical Center at Dallas.
Department of Cell Biology, 5323 Harry Hines Boulevard, Dallas, Texas, USA 75390-9039
Abstract:
Telomeres are repetitive DNA sequences at the
ends of linear chromosomes that serve as essential
protective structures that maintain the integrity of
chromosomal DNA. Each time a normal human
cell divides some telomeric DNA sequences are
lost. When telomeres are short, cells enter an
irreversible growth arrest state called replicative
senescence (or aging).
There is mounting evidence that short telomeres
correlate with age-associated diseases by limiting
the ability of tissues to regenerate. This has led to
the idea that telomere length could be a good and
highly reliable indicator (a biomarker) of biological
(not necessarily chronological) aging. Telomere
length measurements, especially measurements
of the shortest telomeres, provide a molecular
determinant about overall health. It has been shown
that environmental stressors can lead to increases
in oxidative damage and premature telomere
shortening. Smoking, inflammatory disease,
lack of modest levels of exercise, and excessive
drinking can all contribute to increases in the rate
of telomere shortening but in some instances these
may be reversed by behavior modification. Just
as cholesterol and blood pressure measurements
provide an indication of overall health, newly
introduced highly quantitative telomere length
assays also provide a window into one’s overall
health.
While no one can predict how long any individual
person will live, quantitative telomere tests are
scientifically proven biological assays that correlate
with the ability of human cells to proliferate and
replenish tissues.
Background:
Aging is associated with the gradual decline in
the performance of organ systems, resulting in the
loss of reserve capacity, leading to an increased
chance of death (1). In some organ systems, this
loss of reserve capacity with increasing age can be
attributed to the loss of functional cells (2).
Chronic localized stress to specific cell types results
in increased cell turnover, focal areas of replicative
(aging) senescence (3) followed by predictable
alterations in patterns of gene expression. This
results in reduced tissue regeneration, culminating
in many of the clinical pathologies that are largely
associated with increased age.
Evidence that telomere shortening leads to
replicative senescence:
Telomere length is a record of the history of and
the potential for replication of human cells (4).
Telomere dynamics in proliferating tissues might
provide insight into not only the biology of aging
but also the pathology of age-related diseases. A
body of epidemiological and clinical data suggests
that relatively short telomeres and accelerated
telomere attrition are linked to factors that define
aging and diseases of aging in humans (5-12).
There is also experimental support that most
human proliferative tissues and organs including
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most somatic cells (even stem cells of renewal
tissues) undergo progressive telomere shortening
throughout life (13). While there have been
many studies demonstrating correlations between
telomere shortening and proliferative failure of
human cells, the evidence that it is causal has
now been directly demonstrated (14). Telomerase
is a ribonucleoprotein enzyme that functions
to lengthen telomere length during early fetal
development and in some proliferative adult stem
cells.
However, almost all adult tissues do not have
detectable telomerase activity and telomere lengths
decrease throughout life. Introduction of the
telomerase catalytic protein component into normal
human cells results in detection of telomerase
activity (14). Normal human cells stably expressing
transfected telomerase demonstrate telomere
maintenance and extension of life span, providing
direct evidence that telomere shortening controls
cellular aging. The cells with introduced telomerase
maintain a normal chromosome complement and
continue to grow in a normal manner (15). These
observations provide the first convincing evidence
for the hypothesis that telomere length determines
the proliferative capacity of human cells.
Evidence that telomere shortening is
important in organ or tissue ageing:
There is a relationship between replicative aging
and skin pressure ulcers (16) that are believed to be
caused initially by decreased circulation, leading
to localized areas of necrosis. This is followed by
attempts of the skin cells to regenerate. In the areas
of pressure ulcers there is dramatic shortening of
telomeres compared to adjacent normal areas.
Other examples include patients with advanced
and progressive human immunodeficiency virus
infections who have specific T-cell deficiencies,
patients with liver cirrhosis, patients with muscular
dystrophy, and patients with bone-marrow
exhaustion in myeloproliferative diseases. In spite
of a diverse etiology, a common pathological
mechanism is an increased turnover of stem-like
cells leading to cellular senescence and then to a
disease state. Thus, in patients with progressive
8
AIDs there is increased turnover of certain types
of mature T-cells and at least initially the patient
regenerates more T-cells.
Proliferative failure due to telomere erosion
may ultimately result in the patient having low
T-cell counts, leading to opportunistic infections.
In Duchenne muscular dystrophy, children are
capable of walking for a few years but, because they
have inherited a mutated dystrophin gene, their
muscle fibers degenerate. To compensate, their
muscle stem-like cells (satellite cells) regenerate
new muscle fibers but these also degenerate and
eventually the stem cells cannot keep dividing
and the muscle is replaced with fat (adipocytes).
More recently alterations in key genes involved
in maintaining telomeres have been demonstrated
to be involved in human genetic diseases such
as dyskeratosis congenita, sporadic bone marrow
failure and idiopathic pulmonary fibrosis (17-21).
In summary, there is mounting evidence that in
some aged-related disorders telomere decline in
specific tissues and organs may contribute to aging
and cancer vulnerability (22).
Gradual shortening of telomeres coincide
with the long term aging process:
Under normal conditions most tissues can last a
typical life span. However, with the improvement
in sanitation, the development of antibiotics,
vaccines, and modern pharmaceutical drugs,
humans are living longer. A proliferative capacity
for good maintenance and repair for 80-100 years
would not have been selected for in evolutionary
terms, when the average human lived at most
30-40 years. Today there is an increase in agedrelated cellular decline in normal people who
live to an exceptionally old age, while in the
past problems from a limited cellular proliferative
capacity was only observed in disease states (23).
However in older individuals without diseases,
there is an increased incidence of immunological
deficiencies, chronic ulcers, wearing down of the
vascular endothelium leading to arteriosclerosis,
proliferative decline of retinal pigmented epithelial
cells leading to age-related blindness and cancer.
In order to track telomere lengths and potentially
slow down or reverse the increased probability
of telomere associated diseases, it is fair to ask
if telomere tests would have utility as a clinical
diagnostic assay.
Are telomere tests ready for prime time?
We still know little about the dynamics of telomere
length changes over multiple years in large human
populations. It would be difficult for government
funding agencies in today’s financially challenged
environment to support large scale longitudinal
telomere research studies. Thus, the private sector
has taken the lead and developed a series of telomere
tests to determine if the multiple associations of
diseases with telomere length measurements hold
up in placebo controlled studies. Since there are
emerging classes of natural products (telomerase
activators) (24) and genetic manipulations that
may influence telomere biology and aging (2526), we need rigorous scientific telomere tests to
prove the mechanism of actions. The ability of
the private sector to start large scale longitudinal
telomere measurements, including patients
completing detailed questionnaires, will permit
the scientific community to assemble databases
that will allow for determining statistically relevant
sub-populations of patients that may benefit from
telomere length modifications.
Telomere length measurement tests:
The basic laboratory research telomere test is
known as TRF (terminal restriction fraction).
This test is at present not suited for high throughput
scale up, but provides a visual representation of the
distribution of populations of cells using a Southern
blot electrophoresis approach. Other laboratory
assays being developed include a modification of
the TRF assay using a dot blot approach which is
similar but does not provide visualization of the
range of telomere lengths
(Figure 1). Neither the TRF nor dot blot method
provides information about the shortest telomeres
in individual cells. However, as long as one can
get sufficient DNA, data can be obtained. Another
newly developed method is called STELA (single
telomere length analysis).
This is very low throughput but the advantage
is that it can provide information on the shortest
telomeres in a population of cells. It cannot easily
be adapted to a commercial test at the present time
due to turnaround is more than a week per assay,
and this method does not provide information
about the shortest telomeres in individual cells.
Figure 1. Summary of current telomere tests (modified
from Life Length, Inc.)
The qPCR method is not only used in some
research laboratories but is also being used in
some commercial situations (Telomehealth.com;
Spectracell.com). The advantage of the qPCR
method is that it is relatively fast and capable of
high throughput. The disadvantage is that this test
does not give information about individual cells
so the results are generally averages of telomere
lengths in populations of cells. The flow FISH
method almost exclusively uses lymphocytes and is
beginning to have some commercial development
(Repeatdiagnostics.com; Figure 1). This method
uses a FACS (fluorescence activated cell sorter)
to analyze cells with different signals after
hybridization with a fluorescence telomere probe.
This method provides the distribution of telomere
lengths one cell at time but only on the average
of telomere lengths (not telomere lengths within
individual cells). This method is CLIA certified for
measuring telomeres as part of genetic counseling.
The highthroughput microscopic Q-FISH
(Quantitative Fluorescence In Situ Hybridization)
9
method is a highly reliable approach to quantitating
telomere lengths and has the advantage over other
methods of providing not only average telomere
length per cell but also the number and distribution
of the shortest telomeres in individual cells (27,
28; Figure 2). This method permits visualization
and quantitation in a microscope of individual
cells so one can distinguish between a subset of
cells containing very short telomeres and those
that have very long telomere lengths (Figure 2).
A commercial test (HT Q-FISH) has now been
developed (Lifelength.com). With the exception
of HT Q-FISH, no other commercial laboratory
method is available which can distinguish a single
critically short telomere within one cell that
may be triggering senescence. While initially a
laboratory test with relatively low throughput, this
approach has recently been commercialized into a
high throughput method with an accuracy of 5%
between tests (Lifelength.com).
Figure 2. In situ FISH of interphase cells (3 at the top)
and one metaphase spread (lower part of figure). Fixed
cells were hybridized with a fluorescence labeled telomere
probe.
The last method is called Telomapping (Lifelength.
com) (US Patent Nº 8,084,203 B2). This is similar
to Q-FISH but determines the telomere lengths
on chromosomes from tissues (thus maintaining
the topology of the samples). The advantage of this
method is that archival formalin embedded paraffin
sections can be used to determine if specific cells
within a tissue have short telomeres. This is likely
to have important implication in the precancerous
detection field. This method is slightly more time
intensive and is not scaled up to high throughput
analysis at the present time.
Bottom-line, telomere tests are ready for prime
time and while there several options, one needs
to ask if the method delivers results that provide
insights in critically short telomeres in individual
cells which are universally regarded as the principal
cause of replicative cell aging and age-related
diseases (Dr. Carol Greider, Nobel Prize winner,
2009; 29).
Summary and future challenges:
Telomere biology is important in human aging and
cancer. Cancer cells need a mechanism to maintain
telomeres if they are going to divide indefinitely,
and telomerase solves this problem.
Thus, inhibition of telomerase may have utility in
cancer therapeutics (30). Since almost all tissues
show progressive shortening of telomeres with
increased age, in some instances, organ failure
may occur in chronic diseases of high cellular
turnover.
Therefore, telomere manipulations in cells of
regenerative tissues may have utility in treating
certain disorders in the aging population (24, 28).
While the aging process is complex and certainly
cannot be explained solely on the basis of telomere
biology, there is a growing consensus that in some
situations telomere biology and telomere tests may
have important utility similar to cholesterol assays
or blood pressure monitoring measurements.
This is still a young field and improvements will
continue to occur.
10
With longitudinal studies in individuals over many
years trends will emerge that are likely to provide
important insights into human disease. The
challenge is to understand how telomere biology
leads to increased aging vulnerability and to learn
how to intervene in these processes.
8. Benetos A, Okuda K, Lajemi M, Kimura M,
Thomas F, Skurnick J, Labat C, Bean K, Aviv A.
2001
Conflict of Interest:
and pulse wave velocity. Hypertension. 37(2 Part
2):381-5.
Dr. Shay is a Scientific Consultant to Life Length,
Inc. (www.lifelength.com), Madrid Spain
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JR, Goodall AH. 2001 Telomere shortening in
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17. Armanios, M, and Alder, JK. 2009. Short
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Coffman JR, Platz EA, March GE, De Marzo
AM. 2002 Telomere length assessment in human
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12
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The Future Role of Testosterone in Treating Prostate
Cancer
Edwin Lee, M.D., F.A.C.E.
Institute for Hormonal Balance. Orlando, FL 32819-5150
Keywords. testosterone, prostate cancer, androgen deprivation, BPH benign prostate hypertrophy, alkaline phosphatase, hypogonadism, LHRH luteinizing hormone releasing hormone, PSA prostate-specific
antigen, dihydrotestosterone, vitamin D, IGF-BP3 insulin growth factor binding protein 3.
Abstract
For more than 70 years, the use of testosterone
replacement in men has been thought to cause
prostate cancer. In 1941, Dr. Charles B. Huggins
became the founder of the theory that testosterone
replacement can cause prostate cancer. The theory
was based on one patient - who, after having
undergone castration for the treatment of prostate
cancer, received testosterone for only 18 days. The
patient was then found to have an elevation of acid
phosphatase, and so began the theory. Current
medical studies have shown that men with low
testosterone levels have a higher risk for prostate
cancer, and that higher levels of testosterone do
not increase prostate cancer growth. In addition,
there is absolutely no current data supporting
the Huggins theory. This article will review the
saturation model of testosterone on the prostate
gland, as theorized by Dr. Abraham Morgentaler
from Harvard University. Early testosterone
replacement in treating early hypogondal men will
most likely show reduced rates of prostate cancer;
but, future studies will be needed to confirm this
new theory.
Main Text
After more than 70 years, the use of testosterone
replacement in men is still overshadowed by the
fear that testosterone may cause prostate cancer.
On the basis of one article in 1941 by Dr. Charles
B. Huggins - in which one of three men with
prostate cancer was treated with testosterone and
it made the prostate cancer worse - many doctors
have been trained to believe that giving testosterone
can increase the risk of prostate cancer.
An interesting medical finding regarding eunuchs
has contributed to the myth that testosterone can
cause prostate cancer. It was found that eunuchs
(boys who undergo castration before puberty) do
not develop prostate cancer because they have
no source of testosterone production. Even the
US Food and Drug Administration stated that,
“Known or suspected carcinoma of the prostate is
a contraindication for testosterone products.”
Even as late as 2001, the then director of the
National Cancer Institute in the United States
refused to fund a large testosterone replacement
trial by stating that he was, “Concerned that
testosterone could spur the growth of prostate
cancer among some men in the study.” [1]
The origin of Huggins’ original article - and the
first association of testosterone with prostate cancer
- began in 1940 when Huggins, a urologist from
Chicago, was working toward understanding what
causes the prostate to grow. He was interested in
shrinking the prostate, or treating benign prostate
hypertrophy. He worked on dogs since dogs have
a prostate gland. Dogs also develop benign prostate
hypertrophy and, unfortunately, also prostate
cancer.
Because his work had shown that castration in
dogs with an enlarged prostate had caused a
reduction in prostate size, he made the connection
that testosterone had a role in prostate growth. By
luck, one of his dogs had prostate cancer, and he
13
noted that after castration the prostate cancer was
cured. Huggins published his findings in the 1940
issue of the Journal of Experimental Medicine.
Then, in 1941, Huggins used androgen deprivation
therapy (either with castration or estrogen therapy)
in men with metastatic prostate cancer and showed
a rapid reduction of markers of prostate cancer in
acid and alkaline phosphatase (markers of prostate
cancer in the 1940s).
At the time, men with metastatic prostate cancer
had a very high mortality rate, and it was apparently
associated with significant pain of the bones. By
having his patients consent to castration for the
treatment of metastatic prostate cancer, Huggins
showed that it significantly helped reduce mortality,
and that it helped with the pain. The tradeoff of
having low testosterone was worth the sacrifice,
at that time, to live longer and to reduce the pain.
This connection - that castration and lowering
the testosterone level to zero - was the beginning
of androgen deprivation therapy. Fortunately
today, there are better ways (LHRH, Luteinizing
Hormone Releasing Hormone) to achieve this
without undergoing surgical castration.
During his clinical studies, Huggins had three
men with metastatic prostate cancer undergo
surgical castration, and receive testosterone
replacement therapy. Of those three, the data
on patient one was lost, patient two had an
equivocal result, and patient three (with only 18
days of testosterone replacement) had worsening
of his condition. Huggins reported that one
man’s testosterone treatment caused an elevation
of a blood test called acid phosphatase, and then
he concluded that testosterone causes prostate
cancer. That was the beginning of the theory that
testosterone replacement causes prostate caner.
[2] Unfortunately, mainstream medicine has since
embraced this as gospel.
It is interesting to note that when Huggins received
the Noble Prize in 1966 for his discovery that there
is a chemical modulator for cancer, his Noble Prize
was based on androgen deprivation therapy helping
patients with prostate cancer. He also made the
claim that testosterone aggravates prostate cancer
14
based on only one patient after receiving 18 days of
testosterone replacement. Then, in 1967, only one
year later, there was a study published in which 26
patients with advanced prostate cancer were given
testosterone replacement after receiving androgen
deprivation therapy. The patients did not show
any progression of the cancer (several patients
even reported an improved sense of well-being,
increased appetite and decreased bone pain) with
testosterone replacement. [3]
Millions of men suffer from prostate cancer, and
have been told that testosterone replacement will
make the prostate cancer worse. However, if that
is true, and higher testosterone levels are indeed
linked to prostate cancer, then why is that we
see prostate cancer mostly in the elderly (when
testosterone levels are low), and prostate cancer
rarely occurs in the youth (when testosterone
levels are high)?
In regard to the myth that testosterone causes
prostate cancer, there have been many studies in
different urology journals unable to support this
theory. In a study of 75 men with a high risk of
prostate cancer (prostatic intraepithelial neoplasia)
and with low testosterone, the men were treated
with testosterone for one year. None of them
experienced an increased incidence of prostate
cancer. [4] Also, to date, there is absolutely no data
supporting the theory that restoring testosterone
increases prostate cancer. [5]
In addition, a large meta analysis of 19 prospective
double-blind randomized placebo controlled
studies showed that testosterone replacement
showed there was no significant difference for men
receiving testosterone, versus the placebo group,
in developing prostate cancer. [6]
For over 20 years now, Dr. Abraham Morgentaler
from Harvard University has been questioning the
validity of the testosterone/prostate cancer myth which has undergone many mutations since 1941
(when Huggins concluded from one case that
testosterone activates prostate cancer). Morgentaler
best summarizes the prevailing mutations of the
myth in this way: “In the 1980s, it was believed
high testosterone caused prostate cancer. In the
1990s, the argument became that high testosterone
stimulated growth only of existing prostate cancer.
In the early 2000s, high testosterone was proposed
to affect risk only over a period of years.” All this
misinformation, despite the fact that all of the
above beliefs have been disproved with medical
studies published in peer reviewed journals. [7]
The Journal of the National Cancer Institute
looked at 3,886 men with prostate cancer and
found no association with testosterone, or free
testosterone, and prostate cancer. [8] In a study
that looked at men being treated with LHRH
(medical castration) for prostate cancer, the LHRH
will eventually cause the testosterone level drop to
a castration level; however, during the first seven
days of being treated with LHRH, there is a flare
(or rise) of testosterone level before the level drops.
It has been shown in this study that even with that
rise in the testosterone level, the prostate-specific
antigen does not change or increase with the rise
of the testosterone level [9], as illustrated in Table
A.
In addition it has even been shown that low
testosterone levels are associated with an increased
risk of prostate cancer, and have a higher Gleason
scores. [10] As for the rate of prostate cancer being
published in testosterone replacement trials at
approximately one percent [11], the rate is similar
to the cancer detection rate in prostate cancer
screening trials. While this is the current data we
have about prostate cancer rates, a large and longterm study of testosterone replacement therapy
will be needed to confirm this rate.
In addition, Morgentaler has also noted a paradox
of testosterone and prostate cancer: While men
treated with castration (medical or surgical)
do receive a benefit from castration, higher
testosterone levels do not increase prostate cancer
growth. So, if higher levels of testosterone do not
cause prostate cancer to grow, then what is the
saturation or plateau level?
Such a saturation model of testosterone on the
prostate gland has been theorized, and Morgentaler
says, “Testosterone receptors in the prostate are
completely bound at low levels of testosterone
by dihydrotestosterone. By increasing the serum
testosterone level, no changes occur due to the
fact the receptors are saturated.” An analogy of the
saturation model is that of giving water to a dying
plant. Once the plant’s “thirst” has been quenched,
the additional water has no further effect.
Recently, Dr. Roberto L. Muller and his
colleagues proved this saturation model with their
study published in the 2012 Journal of European
Urology. In the study, there were 3,255 men
undergoing regular biopsy of the prostate. The men
were followed over time, and it was found that
there was no association between prostate cancer
with testosterone and dihydrotestosterone. It was
also shown that low testosterone levels increased
the risk, and that prostate cancer declines with the
upper testosterone levels. [12] See Table B for the
saturation model described by Morgentaler. The
saturation model does support the original finding
by Huggins that prostate cancer is dependent of
testosterone at low levels; however, his claim that
higher testosterone levels activate prostate cancer is
no longer supported by any medical studies. Having
higher levels of testosterone does not increase the
risk or stimulate the growth of prostate cancer.
It is important to mention that prostate tissue whether it is benign or malignant – is exquisitely
sensitive to changes in low levels of testosterone.
According to Morgentaler, “The androgen
receptor in the prostate is maximally bound with
testosterone at 60-90 ng/dl. Higher levels of
testosterone have no effect on the prostate gland
whether it is benign or malignant.” [13]
Conclusion
In conclusion, the development of prostate cancer
is multifactorial. It may be linked to a low vitamin
D level, low insulin growth factor binding 3 (IGF
BP-3), damage to any cancer suppressor genes,
genetic defects in the prostate, high insulin levels,
environmental toxins, or nutritional deficiencies.
In regard to Huggins’ case, I suspect that all
his patients had low testosterone before being
diagnosed with prostate cancer – and they may
also have had low Vitamin D, low IGF BP-3, high
15
insulin levels, environmental toxins and nutritional
deficiencies.
I believe that future long-term studies on using
testosterone replacement in treating early
hypogonadal men will most likely show reduced
rates of prostate cancer. Furthermore, I predict that
if you are able to maintain optimal testosterone
levels with testosterone replacement - while
maintaining low insulin levels, optimizing vitamin
D, optimizing IGF BP-3, optimizing the liver to
detox well (or improving phase 1 and phase 2 of
the liver detoxification system) - then you will
have a low chance in developing prostate cancer.
I look forward to future studies showing that
early replacement therapy with testosterone will
decrease the risk of prostate cancer.
Acknowledgements
I would like to acknowledge the extensive and
tireless work of Dr. Abraham Morgentaler of
Harvard Medical School. For more than 20 years
now, Dr. Morgentaler has broken significant
ground in his studies of male testosterone therapy.
I believe in his work, and wish him success as he
continues to advance our understanding of prostate
cancer.
References
[1] Kolata, G. (2002). Male hormone therapy
popular but untested. NY Times Aug 19: A10.
[2] Spark, R. (2000). Sexual Health for Men: 338339.
[3] Prout, G.R.; Brewer, W.R. (1967). Response
of men with advanced prostatic carcinoma to
exogenous administration of testosterone. Cancer
(20): 1871-1878.
[6] Calof, O.M.; Singh, A.B.; Lee, M.L.; Kenny,
A.M.; Urban, R.J.; Tenover, J.L.; Bhasin, S.
(2005). Adverse events associated with testosterone
replacement in middleaged and older men: a metaanalysis of randomized, placebo-controlled trials. J
Gerontol (60): 1451-1457.
[7] Morgentaler, A. (2012). Goodbye Androgen
Hypothesis, Hello Saturation Model. Eur
Urol, Nov; 62(5): 765-767, doi: 10.1016/j.
eururo.2012.06.027. Epub 2012 Jun 16.
[8] Endogenous Hormones and Prostate Cancer
Collaborative Group (2008). Endogenous sex
hormones and prostate cancer: a collaborative
analysis of 18 prospective studies. J Natl Cancer
Inst (100): 170-183.
[9] Tomera, K.; Gleason, D.; Gittelman, M.;
Moseley, W.; Zinner, N.; Murdoch, M.; Menon,
M.; Campion, M.; Garnick, M.B. (2001). The
gonadotropin-releasing hormone antagonist
abarelix depot versus luteinizing hormone
releasing hormone agonists leuprolide or goserelin:
initial results of endocrinological and biochemical
efficacies in patients with prostate cancer. J Urol;
165(5): 1585-1589.
[10] García-Cruz, E.; Piqueras, M.; Huguet, J.;
Peri, L.; Izquierdo, L.; Musquera, M.; Franco,
A.; Alvarez-Vijande, R.; Ribal, M.J.; Alcaraz,
A. (2012). Low testosterone levels are related
to poor prognosis factors in men with prostate
cancer prior to treatment. BJU Int May 15, doi:
10.1111/j.1464-410X.2012.11232.x. Epub ahead
of print.
[11] Rhoden, E.L.; Morgentaler, A. (2004).
Risks of testosterone-replacement therapy and
recommendations for monitoring. N Engl J
Med;350:482-492.
[4] Rhoden, E.; Morgentaler A. (2003).
Testosterone replacement therapy in hypogonadal
men at high risk for prostate cancer: results of 1 year
of treatment in men with prostatic intraepithelial
neoplasia. J Urol, Dec 170; (6 Pt 1): 2348-2351.
[12] Muller, R.L.; Gerber, L.; Moreira,
D.M.; Andriole, G.; Castro-Santamaria, R.;
Freedland, S.J. (2012). Serum Testosterone and
dihydrotestosterone and prostate cancer risk in
the placebo arm of the reduction by dutasteride
of prostate cancer events trial. Eur Urol May 18.
Epub ahead of print.
[5] Morgentaler, A. (2008). Guilt by association:
a historical perspective on Huggins, testosterone
therapy, and prostate cancer. J Sex Med, Aug;
5(8): 1834-1840.
[13] Morgentaler, A.; Traish, A.M. (2009). Shifting
the paradigm of testosterone and prostate cancer:
the saturation model and the limits of androgendependent growth. Eur Urol (55): 310-320.
16
Aging and sleep, and vice versa
María Amparo Lluch 1, MD, PhD; Tomás Lloret 2, MD, PhD. Vicenta Llorca 2, MD.
Institute for Hormonal Balance. Orlando, FL 32819-5150
1Division of Pulmonology. Consorcio Hospital General Universitario, Valencia
2Hospital de Levante, Benidorm. Antiaging Unit, IMED Hospitals.
Correspondence: María Amparo Lluch. Division of Pulmonology .Consorcio Hospital General Universitario,
Valencia . Av. Tres Cruces,2 . 46014 Valencia (España). [email protected]
Abstract
50% of the elderly complain of sleep problems.
As a person ages the circadian rhythms
desynchronize and lose amplitude, the melatonin
secretion lessens, phase changes occur and
the prevalence of sleep disordered breathing
increases. The apnoea-hypopnoea syndrome
is the primary sleep disorder most commonly
found among the geriatric population. The
possible influence it has on the progression of
aging could be due to not only to the metabolic
and cellular effects of intermittent hypoxia and
the oxidative stress, but also due to the added
alteration produced by sleep fragmentation. This
alteration affects the circadian rhythms of several
hormones that regulate the metabolism and
cellular functions. Conclusion: The cutoff point
in the apnoea-hypopnoea index (AHI) at which
it should be considered pathological for the
geriatric population is currently undetermined.
However, in view of the possible effects of
untreated Sleep Apnoea Syndrome (SAS) and
given the increasing longevity of the population,
the current recommendation is that age should
not be an obstacle when diagnosing and treating
SAS in the elderly.
Introduction
Sleep is natural, periodic, transient and reversible
behaviour which is virtually universal in the
animal kingdom but, as yet, its full biological
meaning is not still known. Both homeostatic
and circadian mechanisms influence our sleep
regulation. Homeostatic mechanisms maintain
internal balance, so that the more hours spent
awake, the greater the need for sleep is. This need
to sleep appears to be mediated by substances
such as adenosine or interleukin-1 (IL-1),
among others. The suprachiasmatic nucleus of
the hypothalamus is the anatomical substrate
of the circadian clock regulating sleep; the
activity of this nucleus is modulated by various
external stimuli, the most important of which
is the natural light. Apart from the homeostatic
and circadian factors, age is another important
element in sleep organization. The effects of sleep
on human physiology include: the reduction of
the sympathetic tone (which increases during
arousals or micro-arousals), reduced cardiac
frequency and output, lower blood pressure,
lower glomerular filtration rate with increased
water reabsorption and a decrease in the body
temperature. Also the cortical process of memory
consolidation, the inhibition of TSH production,
and the production of growth hormone (GH)
which increases especially during the first few
hours of sleep and increases the secretion of IL-1
occur during sleep.
Lack of sleep not only translates into fatigue,
tiredness and excessive daytime sleepiness but it
also exerts deleterious effects on several systems
causing metabolic, endocrine, and immune
17
changes. Evidence has accumulated over recent
decades to suggest that both a lack and an excess
of sleep are associated with several adverse effects,
including cardiovascular disease, type 2 diabetes,
high blood pressure, respiratory disorders,
.
obesity, and a poorer quality of life1
Sleep changes associated with aging.
Approximately 50% of the elderly complain of
problems related to sleep.
Although sleep requirements do not alter with
age the ability to fulfill these requirements
decreases, but this is due mainly to age associated
co morbidities rather than aging itself.
The poor sleep quality has significant consequences
in the elderly population. It increases the risk of agerelated problems, psychophysical deterioration and
mortality.
With age the percentage of REM sleep is reduced
and the light sleep stages increase; these changes are
not considered pathological and may reflect neural
degeneration associated with age. Phase advance is
also common, and this results in the elderly waking
up earlier in the morning and being drowsier in the
evening2.
Another age-related phenomenon is the
desynchronisation and amplitude loss of the circadian
rhythms1 such as melatonin and other hormones and
mediators. This not only affects the regulation of the
sleep-wake rhythms but, as we shall see later, it also
has an impact on the metabolism regulation, thereby
promoting aging.
Primary sleep disorders must also be considered.
The most common amongst the elderly population
include Sleep Apnoea Syndrome (SAS) on which
this article is based, REM sleep behaviour disorder
(RBD) and Restless Leg Syndrome and Periodic
Limb Movement of Sleep (RLS/PLMS).2.
SAS in the elderly
18
The number of respiratory sleep disorders increase
linearly with age, whether due to aging itself (increased
airways collapsibility) or due to a true pathological
condition (SAS). The cutoff point in the AHI
(Apnoea-Hypopnoea Index) at which SAS should be
considered pathological is currently undetermined. It
is an issue of particular epidemiological importance
given the increasing longevity of the population.
The symptoms of SAS sometimes differ from the
norm because the impact of SAS in the elderly can
be more centered in the neurocognitive sphere. The
Epworth questionnaire 3 has not been validated in
the elderly, and this should be taken into account
when evaluating the results. This questionnaire is
used in clinical practice to assess subjective daytime
sleepiness. The presence of a score equal to or
higher than 12 points (out of 24 points) indicates
pathological hypersomnia. There are no studies
with sufficient levels of evidence which analyze the
effect of CPAP treatment (Continuous Positive Airway
Pressure) or other treatments in the elderly4.
This article briefly reviews the two sides to the sleep
respiratory disorder, as it becomes more prevalent
with increasing age and may promote aging itself,
thereby causing a vicious circle.
Pathogenesis of SAS and its relationship to
aging.
Subjects with SAS experience repeated episodes
of reduced ventilation (hypopnoea) or cessation
(apnoeas) due to obstruction of the upper airway.
These obstructions result in: sleep disruption with
frequent arousals (sleep fragmentation); loss of REM
sleep and slow wave sleep (already reduced due to
age); repetitive oxygen desaturations with rapid
reoxygenation causing cyclical deoxygenation/
reoxygenation; repeated intrathoracic pressure
changes; and hypercapnic episodes.
1. Intermittent Hypoxia
Much of the attention placed on the effects caused by
SAS has focused on the role of chronic intermittent
hypoxia: it is generally associated with increased
sympathetic activity and hypertension, increased
catecholamine levels, liver dysfunction, learning
deficits with associated hippocampal and cortical
neuronal damage, insulin resistance, atherosclerosis
and vascular remodeling.
More specifically, hypoxia/reoxygenation has been
compared to ischemia-reperfusion injury with an
increase of Reactive Oxygen Species (ROS). This
has lead SAS and its effects to be considered as an
oxidative stress disorder. It is known that one of the
most prominent theories on aging attributes it to the
accumulation of free radicals. According to this model
an increase of free radicals activates the pathways of
the proinflammatory transcription factor: nuclear
factor kappaB (NF-kB), and the hypoxia-induced
factor-1 (HIF-1_), which eventually leads to an
increase of proinflammatory cytokines and adhesion
molecules, thereby linking the inflammatory and
hypoxic response pathogenic pathways.
All of the above leads to higher blood pressure, an
increase in triglyceride levels, endothelial dysfunction
and consequent cardiovascular disease5.
In turn the end product of phospholipids oxidation,
namely (carboxyalkyl) pyrrole (CAP), promotes the
angiogenesis process. On the one hand it contributes
to oedemas and vascular fragility and on the other to
a possible, controversial implication in carcinogenesis
and other age-related processes such as macular
degeneration6.
In addition age is another important consideration
when considering the relevance of oxidative
damage caused by intermittent hypoxia. Alterations
in dopaminergic activity of the prefrontal-striatal
circuit are important in intermittent hypoxia induced
memory deficits and subjects with SAS have decreased
volume of hippocampal grey matter. So younger
subjects with SAS may have cognitive deficits similar
to those that are produced by aging. The combined
presence of SAS and old age may lead to greater
disability than either factor on its own7, 8.
Finally, intermittent hypoxia through oxidative
stress, lipid peroxidation and the induction of stress
responsive proteins induces cellular apoptosis at
a neuronal, myocardial and vascular level, which
has an impact on functional decline and the aging
progression.7.
2. Sleep fragmentation
It is not only intermittent hypoxia that occurs during
apnoeic events; sleep fragmentation with multiple
microarousals is also produced. Each arousal is
followed by a burst of sympathetic activity, with
changes in the blood pressure, elevated cortisol and
lipid levels, and increased neurocognitive deficits as
a consequence of sleep fragmentation. The role of
sleep fragmentation has not, however, received as
much attention as oxidative stress, but it is known
that short sleep duration and sustained partial sleep
deprivation are associated with an increased risk of
hypertension, weight gain, insulin resistance and
type 2 diabetes, neurocognitive deficits and increased
inflammatory markers such as Tumour Necrosis
Factor alpha (TNF-_.)5.
The Tumour Necrosis Factor alpha (TNF-_) and
Interleukin-1 (IL1) are cytokines that are released in
response to many stimuli, including sleep loss, tissue
injury and infection. They are pleiotropic and their
function includes modulation of memory and mood.
Altered cytokine levels are associated with enhanced
sensitivity to pain, fatigue, sleepiness, metabolic
syndrome and impaired cognition. Altered cytokine
levels have been observed in situations of sleep loss,
including SAS9.
With aging, the circadian rhythms become weaker,
desynchronised and lose amplitude. The nocturnal
secretion of melatonin, a hormone secreted by the
pineal gland with a regulatory function of the sleepwake cycle, gradually decreases with age, possibly
reducing sleep efficiency2. Melatonin levels are
higher at night and lower during the day. Exposure
to light at night quickly suppresses its secretion. This
hormone has sleep promoting properties (a natural
hypnotic), it is thermoregulatory and antioxidant,
as well as being several times more potent than
vitamin E in neutralising peroxyl radicals10,11. Its
immunomodulatory action and its oncostatic action
in experimental models12 have also been observed.
The cause for the reduction of melatonin observed
with aging is not known, and the question whether the
alteration to its rhythm is the cause or the consequence
of the aging process is unanswered. However it is
hypothesized that the reduction of melatonin levels
with age contributes to the aging process, and to an
19
increased risk of age-related diseases11: the lack of its
natural hypnotic effect may be related to geriatric
insomnia. Strong reductions of circulating melatonin
are also observed in Alzheimer’s disease, Parkinson’s
disease, other types of senile dementia, stressful or
painful conditions, cancer, cardiovascular diseases
and type 2 diabetes13. The melatonin secretion
pattern in patients with SAS shows values in a plateau
without the nocturnal peak found in health patients,
accompanied by decreased levels in the morning10.
Thus, SAS could again contribute to promoting the
aging process through this pathway.
Furthermore, as the melatonin rhythm deteriorates,
other circadian rhythms desynchronize, which in turn
can contribute to aging and increase the susceptibility
to age-related diseases. Evidence shows that SAS also
induces additional changes in the secretory patterns
of several hormones. These changes are not only
related to obesity (a risk factor for SAS) but may also
be due: to sleep fragmentation induced by apnoea
and hypopnoea; to frequent arousals that break the
circadian rhythms and increase stress hormone levels;
to direct effects of hypoxia on hypothalamus-pituitary
axes and in the secretion of the peripheral endocrine
glands; and to the effects of episodic hypercapnia
which may increase the ACTH levels and adrenal
hormones.14.
The temporal organization of the release of the
counterregulatory hormones growth hormone (GH)
and cortisol as well as the release of appetite regulatory
hormones, such as leptin and ghrelin, is partially
dependent on sleep duration. Glucose tolerance and
insulin secretion are also markedly modulated by
the sleep-wake cycle. The main GH pulse occurs
during slow wave sleep, while the cortisol profile is
characterised by a morning maximum, with levels
declining throughout the day. Awakenings induce a
pulse of cortisol. Leptin, the satiety hormone secreted
by adipocytes, and ghrelin, a potent orexigenic
hormone secreted in the digestive tract, both increase
during sleep15.
The sleep deficient subjects have lower levels of leptin,
which signals the need for unnecessary extra caloric
intake and increases the risk of obesity. Subjects with
SAHS show reduced GH secretion coupled with a
20
reduction of the insulin-like growth factor (IGF-I),
while GH concentrations rise upon awakening. Sleep
loss also involves insulin-resistance, which leads to an
increased risk of obesity and type 2 diabetes.
As we have seen, nocturnal awakenings are associated
with pulsatile cortisol release and autonomic
activation. This hypothalamus-pituitary-adrenal
axis hyperactivity may be a risk factor for metabolic
syndrome, insomnia and depression.
Changes of gonadal axis are common in patients
with SAS, with hypogonadotropic hypogonadism,
showing in particular decreased morning and
nocturnal testosterone concentrations. Hypoxemia
and sleep fragmentation may also affect the rhythm
of prolactin, which under normal conditions shows
a sleep-dependent pattern, with higher levels during
sleep and lower levels when awake.14
Therapies
Different types of insomnia especially sleep/wake
cycle alterations of chronobiological origin, i.e. those
related to primary rhythm alterations or secondary
to conditions that induce phase changes (e.g. jet lag
or shift work) respond well to the administration
of melatonin. Apart from its sleep-inducing effect
(administered 30-60 minutes before bedtime) it
prolongs the period of sleep, decreases night time
awakenings and improves the subjective sleep
quality (in slow-release formulations). It readjusts the
biological clock (chronobiotic effect) so that it induces
phase advance if it is administered at midday or in the
afternoon, and induces phase delay if administered in
the morning. This is obviously very useful in older
subjects. Also thanks to the antidepressant properties
of melatonin it has also been useful in depressive
episodes associated with sleep disturbances.16
On the other hand, once the beneficial properties
of melatonin are known, it should be mentioned
that SAHS treatment with CPAP has been shown to
normalize the serum levels, so this information could
be of importance when deciding whether to start
CPAP treatment in the elderly patient with SAS.10
Conclusions
Although the cutoff point in the apnoea-hypopnoea
index (AHI), at which it should be considered
pathological for the geriatric population, is currently
undetermined. However, in view of the possible effects
of untreated SAS and given the increasing longevity
of the population, the current recommendation is
that age should not be an obstacle when diagnosing
and treating SAS in the elderly (except under extreme
circumstances). CPAP testing for a few months
followed by a subsequent evaluation of the clinical
response could be a good alternative in case of doubt.
It is unknown whether there is an age after which the
CPAP may be withdrawn, so therefore, this practice
is not advised at this moment4.
(11) Melatonin and Ageing: prospects for human
treatment. Journal of Physiology and Pharmacology
2011, 62, 1, 13-19.
References
(14) Neuroendocrine alterations in obese patients
with sleep apnea síndrome. Internatinal Journal of
endocrinology. Volume 2010, Article ID 474518.
Alvarez-Sala Walther JL, González Mangado N.
Trastornos Respiratorios del Sueño. Monografías
Neumomadrid. Volumen VI. 2004.
Sleep disorders in the Older Adult – a Mini-Review.
Gerontology 2010;56:181-189.
Johns MW. Daytime sleepiness, snoring, and
obstructive sleep apnea. The Epworth Sleepiness
Scale. Chest. 1993;103:30–6.
(12)Neurobiology, pathophysiology, and treatment of
melatonin deficiency and disfunction. The scientific
World Journal Volume 2012, Article ID 640389.
(13) Melatonin in Aging and disease-multiple
consequences of reduced secretion, options and limits
of treatment. Aging and disease. Volume 3, Number
2, April 2012.
(15) Role of Sleep Loss in Hormonal Release and
Metabolism. Endocr. Dev. 2010; 17: 11-21.
(16) Melatonina, análogos sintéticos y el ritmo sueño/
vigilia. REV NEUROL 2009; 48(5): 245-254.
(4) Normativa SEPAR Diagnóstico y
tratamiento del síndrome de apneashipopneas del sueño P. Lloberesetal/
ArchBronconeumol.2011;47(3):143–156.
(5) Molecular Signatures of Obstrutive Sleep Apnea
in Adults: A Review and Perspective. SLEEP 2009;
32(4):447-449.
(6) Oxidation as ”The stress of life” Aging, September
2011, Vol 3 No 9.
(7) Can O2 Dysregulation induce premature aging?
Physiology 23: 333-349, 2008.
(8) Obstrutive Sleep Apnea and Age. A double insult
to brain function?American Journal of Respiratory
and Critical Care Medicine Vol 182 2010.
(9) Biochemical Regulation of Sleep and Sleep
Biomarkers. Journal of Clinical Sleep Medicine
Supplement to Vol. 7, No 5, 2011.
(10) Nocturnal melatonin plasma levels in patients
with OSAS: the effect of CPAP. Eur Respir J 2007;
30: 496-500.
21
Calcium and Vitamin D. Health implications
Pascual Garcia Alfaro*, Máximo Izquierdo Sanz, Montserrat Manubens Grau
Department of Obstetrics, Gynaecology and Human Reproduction. Institut Universitari Dexeus. Gran Vía Carlos
III, 71-75 08028 Barcelona, España
*Corresponding author: [email protected]
Abstract
Calcium and Vitamin D are essential for bones
health maintenance. They are also involved
in muscular function, nerves conduction,
coagulation, cardiovascular physiology, immunity
mechanisms and in different enzymatic processes.
Understanding the calcium absorption mechanisms
and the vitamin D synthesis is very important
in order to understand the reasons of their lack.
It is widely known that this lack is involved in
osteoporosis, increased incidence of breast cancer,
colon cancer, hypertension and other diseases. It is
quite important to identify the population at risk
in order to assess supplementation and decrease the
risk of these pathologies, because in some countries
it has become a real public health problem.
Introduction
Calcium and vitamin D have a very important
role in the health of women, especially in the
maintenance of good bone mass. Calcium is the
main component of bone and vitamin D which
is involved in regulating bone metabolism and
homeostasis. Without this vitamin is practically
impossible intestinal calcium absorption.
It’s also involved in maintaining multiple
biological functions such as muscle function,
nerve conduction, coagulation, cardiovascular
physiology, immune mechanisms and many other
enzymatic processes1.
20-35% in duodenum and jejunum. Calcium
absorption across intestinal epithelia has a dual
transport mechanism, one paracellular and other
transcellular. The paracellular transport by passive
diffusion is performed through the intercellular
spaces driven by the electrochemical gradient
of calcium. It’s a non-saturable mechanism and
a less controlled physiological regulation than
transcellular pathway. The transcellular transport
by active diffusion is performed through the
enterocyte joined to a carrier protein called
calbindin and subsequently a step is performed
towards the extracellular fluid by an ATP dependent
pump. Vitamin D is involved in this process by
stimulating the synthesis of calbindin and activating
the calcium pump. As we can see this transport
mechanism is controlled metabolically and every
step in the transcellular movement of calcium
has an energy expenditure and has a dependent
component of vitamin D2,3.
Calcium needs and types of calcium salts
Calcium absorption mechanism
We have daily calcium losses through the skin,
intestine and renal system ranging from 200300 mg. In the process of intestinal calcium
absorption factors intervene such as gastric acidity,
the presence of food, the type of calcium salt and
age4. This absorption decreases as we get older,
especially after age 50. Given the daily loss and
with age increase the requirements and reduces
the absorption, it’s necessary to adapt the daily
calcium intake.
Calcium ingested through diet or supplements,
once in a soluble and ionized form, clearly absorbed
It’s advisable to maintain a daily dose of 1000 mg
calcium / day in adults up to age 65. From this
22
Calcium carbonate 40% of calcium element
-Calcium oxalate renal lithiasis by joining calcium
with oxalate diet, reducing intestinal absorption.
Regarding calcium supplementation, recent
studies Bolland MJ et al. have found an association
between increased risk of myocardial infarction
and calcium supplementation without the
complement of vitamin D10,11, whereas in the
randomized controlled trial of Rejnmark L et al.
calcium supplementation with vitamin D shows a
null effect on health cardiovascular12.
Calcium citrate ...............30% of calcium element
Synthesis of vitamin D
Calcium lactate ...............13% of calcium element
During exposure to the sun, on our skin the
photons from sunlight produce the opening of the
B ring of 7 dehydrocholesterol, between carbons
9 and 10, transforming in previtamin D. This
previtamin is very unstable and quickly it turns in
cholecalciferol (vitamin D3).
age the dose should increased to 1500 mg / day.
Calcium supplements were co administered with
the diet to achieve the necessary levels. When
choosing a calcium supplement it should be taken
into account the mineral content of calcium
contained in each type of salt and tolerability.
Among the most commonly used calcium salts
are:
Calcium pidolate.............13% of calcium element
Calcium gluconate ...........9% of calcium element
The citrate salt absorption is less dependent on
the acidity gastric and can be administered at any
time of day. The carbonate salt should be taken
with meals to ensure higher absorption of HCl
as required to become ionized calcium soluble.
Small amounts of calcium (500 mg per serving) are
better absorbed The reaction of carbonate salt with
HCl, produces CO2 and consequently abdominal
distension and flatulence. Most common side
effects are produced early in the treatment 5,6,7.
Effects of dietary calcium supplementation
Calcium is associated with following different
benefits or health risks.
A diet low in calcium is associated with:
* Calcium metabolism disturbance, resulting in a
loss of bone mass, predisposition to osteoporosis
and increased fracture risk.
* Obesity increased lipid synthesis in adipocytes.
* High blood pressure by an action of muscle
cells8,9..
Instead, a diet rich in calcium may reduce the risk
of:
- Colon cancer, since calcium is not absorbed in
the intestine may exert a protective effect against
colonocyte cell proliferation induced by bile salts.
This has not been demonstrated in human.
Cholecalciferol circulates in blood bound to
carrier proteins (DBuildingProtein) deposited in
adipose tissue part and another is released in liver,
where the first hydroxylation occurs becoming
25 (OH) vitamin D3. Has a half life of 2 weeks
and a second level after renal hydroxylation
becomes 1.25 (OH)2 vitamin D3, which is the
active form and acts on specific receptors and
vitamin D. (Fig.1) Vitamin D is inactivated in the
liver by glucoconjugation and sulfoconjugation.
After completion of the synthesis of vitamin D,
we can talk about complex hormonal D, as it is
synthesized by the body itself, different metabolites
were detected and effect specific receptors in
different organs.
It is estimated that a sun exposure up to 10-15 minutes
daily on face and arms during the spring, summer
and fall, is enough to maintain vitamin D deposits
in adequate levels13. An excess of sun exposure
does not cause vitamin D intoxication due to that
solar energy itself destroys any excess of production.
Also a number of factors influence the synthesis
of vitamin D: latitude, the use of sunscreens and
skin color. In latitudes above the parallel 35 ° N
and S of Ecuador, especially in winter and in the
early morning and late afternoon the sun’s rays fall
on the earth in an oblique angle, the ozone layer
absorbs more photon and reducing skin synthesis of
23
vitamin D. With respect to the use of sun creams
with protective factor 8 lower than about 95%
the capacity of the skin to produce vitamin D. In
people tanned and in black race is also decreased
synthesis and increased sun exposure needed to
obtain good levels of vitamin D14,15,16.
To obtain normal levels of vitamin D, the
National Osteoporosis Foundation recommends
the administration of 800 IU vitamin D daily to
people at risk or deficit without exceeding 2000 IU
vitamin D daily. Studies have shown that vitamin
D deficiency is associated with increased multiple
pathologies in different organs and systems of the
organism18,19,20,21,22,23,24.
Below are the problems associated with
vitamin D:
Bone.- Decreases bone mineralization, producing
osteoporosis and increased fracture risk.
Breast.- Observed increase breast density on
mammograms and increased incidence of breast
cancer.
Blood vessels.- Dysfunction in the vasodilation
that results an increase of the blood pressure levels
Skin.- Decreased skin thickness epidermal growth
and nails.
Intestine.- Decreases the calcium transport and
increase the risk of colon cancer.
Kidney.- Decreases kidney phosphate reabsorption,
increases the production of rennin, ontributing to
the implementation of HTA.
Figure 1. Synthesis of vitamin D
Requirements and effects of vitamin D
deficiency
In 2010 the International Osteoporosis
Foundation considered that the best indicative
parameter of the conditions of vitamin D deposits
is the determination of 25 (OH) vitamin D317.
Analyzing blood value we can know whether the
deposits are normal or insufficient.
The current criteria for defining normal
values are:
Optimal levels of vitamin D: > 30 ng/ml
Insufficient vitamin D: <30 ng/ml
Vitamin D deficiency: <20 ng/ml
Vitamin D intoxication: > 150 ng/ml
24
Muscles.- Decreases muscle contractile force
resulting in weakness and risk of falls.
Neurons.-Decreases
neurotransmitters.
the
production
of
Pancreas.- Pancreatic insulin production decreases
by increasing the risk of diabetes mellitus.
Parathyroid.- Increases Parathyroid PTH
production. Inmune System.- Enhance IL-1, IL-6,
TNF, inflammation, and also increases the risk of
leukemia.
Pregnancy.- Increases the risk of preeclampsia,
gestational diabetes and children with low
birthweight.
Biological Basis of vitamin D involvement
with breast cancer
In 1979 was identified the vitamin D receptor
(VDR). It’s a nuclear receptor that after activation
maintains extracellular calcium levels and also
influences in 200 genes involved in cell growth,
differentiation, apoptosis and other mechanisms
clearly implicated in oncogenesis. 1.25 (OH)2 D3
stimulates the expression of cell cycle inhibitors
=> inhibits the transcriptional activity of the
beta-catenin. 1.25 (OH)2 D3 activates cellular
calcium signals dependent of proteases µ-calpain
and caspase-12 inducing apoptosis. It regulates
the expression of oncogenes (c-myc and c-fos) and
various growth factors. It may inhibit the synthesis
and biological actions of estrogens by decreased
expression of the gene encoding aromatase. As
we see vitamin D has a protective effect on the
development of cancer by multiple mechanisms
with inhibitory effect on cell cycle25,26,27,28.
Status of vitamin D insufficiency in Spain
It’s affects more than 50% of the all age population.
In elderly affected with steoporosis fractures, the
prevalence of hypovitaminosis D reaches 100%.
The information to be considered before this
situation would be:
1. - The diet is not sufficient to achieve adequate
levels of vitamin D. 2. - It is widely believed that
in our country, is apparently easy get vitamin D
with taking unscheduled sun.
3. - The great “paradox” is that in patients being
treated for osteoporosis, it is evident the high
prevalence of insufficient levels of calcifediol
in> 63%29,30. Indications of vitamin D
supplementation. It’s recommended supplement
to the most at-risk population: Elderly, Low
exposure to sunlight, Vegetarians, Pregnancy and
lactation, Intestinal malabsorption, Liver diseases,
Kidney diseases, Obesity, Osteoporosis.
Conclusions
Calcium and vitamin D are essential for multiple
body functions. Calcium supplementation without
the complement of vitamin D is associated with
an increased risk of myocardial infarction. The
main source of vitamin D is the skin synthesis
under the stimulus of solar ultraviolet. Vitamin
D deficiency is implicated in the increased risk of
various cancers. It’s recommended supplement the
population with highest risk of default.
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1. Holick M.F. Vitamin D deficiency. N Engl J
Med 2007;357:266-81
2. Wasserman RH. Vitamin D and the dual
proesses of intestinal calcium absorption. J Nutr.
2004;134:3137-3139
3. Hoenderop JG, Nilius B, Bindels RJ. Calcium
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4. Gueguen L, Pointillart A. The bioavailabily of
dietary calcium. J Amm Coll Nutr. 2000;19(suppl
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5. Harvey JA, Zobitz MM, Pack CY. Dose
dependence of calcium absortion: a comparison
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Miner Res. 1988;3:253-8
6. Kenny AM, Prestwood KM, Biskup B, et al.
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calcium citrate or calcium carbonate on bone
turnover in postmenopausal women. Ost Int
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7. Chustecka Z. Calcium supplements: citrate
better than carbonate? In: www.jointandbone. org
2004;Juny 8
8. Barger Lux MJ, Heaney RP. The role of calcium
intake in preventing bone fragility, hypertension
and certain cancers. J Nutr. 1994;124:1406S-11S
9. Shea B, Wells G, Cranney A, Zytaruk N, Robinson
V, Griffith L, el al. Calcium supplementation on
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10. Bolland MJ, Avenell A, Baron JA, Grey A,
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supplements and cardiovascular risk. J Bone Miner
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Calcium supplements with vitamin D do not
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13. Holick MF. Vitamin D. Shils ME, editor.
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14. Gilchrest BA. Sun exposure and vitamin D
sufficiency. Am J Clin Nutr. 2008; 88:570S-7S
15. Hagenau T, Vest R, Gissel TN, Poulsen CS,
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levels in relation to age, gender, skin pigmentation
and latitude: an ecologic meta-regression analysis.
Osteoporosis Int. 2009;20:133-40
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Dermatol Ther. 2010;23:48-60
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Boonen S, Burckhardt P, Fuleihan GE, et al. IOF
position statement: vitamin D recommendations
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18. Glerup H, Mikkelsen K, Poulsen L, Hass E,
Overbeck S, Thomsen J ET, et al. Commonly
recommended daily intake of vitamin D is not
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20. Pérez-López FR. Sunlight, the vitamin D
endocrine system, and their relationships with
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Tremollieres F, Gambacciani M, et al. Vitamin D
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1,25-dihydroxyvitamin D3 and retonic acid
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cells with function and cell-specific additive effects.
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Tissueselective regulation of aromatase expression
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Lifestyle and breast cancer. A review
Màxim Izquierdo, Pascual García, Montserrat Manubens.
Department of Obstetrics, Gynecology and Reproduction.
Institut Universitari Dexeus. Barcelona. Spain.
Breast cancer, like all neoplasia is caused by
genetic mutations in oncogenes and suppressor
genes. In clinical practice, the fact that two
patients with the same genetic mutation do not
develop the neoplasia at the same time even they
do not develop the process at all, is explained by
Epigenetics. Epigenetic alterations are changes
in the DNA without altering its sequence. The
genes are activated or deactivated through the
methylation of DNA or acetylation of histones.
There are genes that do not act in inhibiting
cancer because they are methylated. This is the
reason why more than 90% of tumors are legacy.
(1) Epigenetics explains why gene expression can
be affected by lifestyle and explains how lifestyle
can act on genes.
The body mass index, exercise and diet affect the
incidence and prevalence of breast cancer.
BMI and breast cancer. The Body Mass Index or
Quetelet index, devised by the Belgian Adolphe
Quetelet between 1830 and 1850 (2), is a measure
of association between weight and height, is
calculated BMI = kg/m2. The World Health
Organization (WHO) (3) established in 1995 the
following values: Underweight <18.5, Normal
18.5 - 24.9, Overweight 25 - 29.9, Obesity > 30.
WHO changed the classification in 2000, with
different subgroups in obesity (4): Underweight
<18.5, Normal weight 18.5 - 24.9, Pre obesity
25’0 – 29.9, Obesity class I 30’0 - 34.9, Obesity
class II 35’0 - 39’9, Obesity class III> 40.
Premenopausal obese women have a lower risk
of breast cancer than women of healthy weight,
but obese postmenopausal women have a 1.5
times greater risk than women of healthy weight.
This is because the levels of sex hormone binding
globulin in premenopausal women is high and the
free estradiol is lower in postmenopausal women
and the level of binding globulin is decreased and
high free estrogen. (5,6)
Estrogen levels in postmenopausal women are 50%
- 100% higher in obese women. (7)
BMI is a prognostic factor in patients with breast
cancer. A higher BMI is associated with diseasefree survival (DFS) and overall survival (OS) lower.
(8) There are studies in which the relationship of
BMI and postmenopausal breast cancer is only
present in women who have not used hormone
replacement therapy. (5)
Diet and breast cancer. The overall intake of
fruits and vegetables is not associated with a lower
risk of breast cancer. (9) There is no relationship
between high intakes of beta carotene and Vitamin
C, to breast cancer in postmenopausal women
taking exogenous hormones. (9,10) A metaanalysis of studies published from 1982 to 1997
(11) suggests a moderate protective effect for high
consumption of vegetables and micronutrients.
The Mediterranean diet protects against breast
cancer. (12,13,14) This is characterized by
a prominent role for fruit, vegetables and
olive oil, with a diet rich in monounsaturated
fats and low in saturated fat, making tissues
less susceptible to oxidative damage. (15)
Alcohol consumption is associated with an increased
risk of breast cancer. Additional consumption of 10
27
grams a day was associated with an increase of 9%
in the risk of breast cancer. (16) No relationship
was found between green tea consumption and
the incidence of breast cancer. (17,18)
Most of the vitamin D is produced when the
7-dehydrocholesterol in the skin is exposed to
ultraviolet B. By the action of hydroxylase in
liver and kidney, it is produced 1.25 (OH) 2 D
(Calcitriol), which is the active form of vitamin D.
(19) The active form of vit D binds to the vitamin
D receptor, a nuclear receptor that can regulate
the expression of several genes. (20)
Vitamin D has a protective effect in vitro and
in animal models of breast cancer development.
(21-30) Intake of 200UI/day of vitamin D and
moderate exposure to sunlight raises the 25 (OH)
D and is associated with a 50% reduction in the
incidence of breast cancer (31,32)
Antiinflammatory effects. 1,25 (OH) 2D decreases
COX2, which is an important factor in the synthesis
of prostaglandins in breast cancer cells and enhances
the expression of 15-hydroxyprostaglandin
dehydrogenase, which catalyzes the conversion
of prostaglandin ketoderivatives into biologically
inactive (40) . Prostaglandins are involved in the
development and progression of breast cancer. (41)
The prostaglandins released by breast cancer cells
stimulate cell proliferation and inhibit apoptosis
(41). A high expression of COX 2 tumors correlates
with high grade and poor prognosis (42)
Inhibition of estrogen pathway. 1,25 (OH)
2D may inhibit the synthesis and the biological
actions of estrogen, decreased expression of the
gene encoding aromatase. (43-45) and inhibits
the estrogen receptor _, the nuclear receptor that
mediates the actions of estrogen. (44-45)
Inhibition of Growth and Apoptosis. The
mechanism involves cyclin dependent inhibitors
of kinase, p21 and p27. (33,34), the expression of
oncogenes (c-myc and fos) and various growth
factors (35) and morphological changes associated
with apoptosis in breast cancer cells. (36)
In epidemiological studies there are an association
between low sun exposure and the incidence
and mortality from breast cancer. (46-48). In 87
counties of the United States there was a correlation
between low exposure to sunlight and mortality
from breast cancer adjusted for age, with higher
rates in the Northeast, compared to the southwest.
(46)
Studies in vivo and in vitro have shown
that oleic acid (the major monounsaturated fatty
acid of extra virgin olive oil) prevented expression
of HER2. Studies in vivo and in vitro have shown
that oleic acid (the major monounsaturated fatty
acid of extra virgin olive oil) prevented expression
of HER2.(49-55)
Inhibition of invasion and metastasis. Vitamin
D deficiency promotes cell growth of human
breast cancer in mouse bone, suggesting that
vitamin D can stimulate the growth of cancer by
altering the bone microenvironment. (37) 1 , 25
(OH) 2D increases the expression of E-cadherin
(transmembrane glycoprotein involved in cell
adhesion), preventing the invasion and metastasis
(38). It has potent anti-angiogenic activity by
inhibiting tumor invasion (35). Also, it decreases
the activity of the plasminogen activator and
increase the expression of plasminogen activator
inhibitor (39)
Alcohol consumption is associated with a risk
of breast cancer(56), a 3% increase in the risk of
breast cancer for each increment of 10 g / day
in drinking (57). Two meta-analyzes estimate a
12% increase in risk with a daily consumption of
alcohol (58,59). The red wine is associated with a
higher level of free Testosterone, lower levels of
SHBG and higher levels of LH vs white wine in
healthy premenopausal women (60). Red wine is a
nutritional aromatase Inhibitor (AI) does not seem
to increase the risk of breast cancer (60). AI activity
in red wine has been attributed to phytochemicals
(61,62). The consumption of red wine, but not
Several mechanisms have been proposed for the
inhibitory effects of vitamin D 1,25 (OH) vit
D on the growth of breast cancer: inhibition of
Growth and Apoptosis, Inhibition of invasion
and metastasis, Anti-inflammatory effects and
Inhibition of estrogen pathway.
28
white wine, was inversely related to breast density
in postmenopausal women (63)
cancer risk. Journal of the American Medical
Association 1997; 278(17):1407–1411
Sport and breast cancer. There are several studies in
which women who exercise have a lower prevalence
of breast cancer (64,65) In a meta-analysis, 76% of
the studies were a protective association between
high physical activity and breast cancer incidence.
(66). A high physical activity shortens the luteal
phase, progesterone levels and may increase the
number of an ovulatory cycle, reducing your risk
of breast cancer. (67) Also in postmenopausal
women high physical activity is associated with a
lower risk of breast cancer. (68, 69) The exercise
increases sex hormone binding globulin (SHBG)
decreasing estrogen levels and free androgen(70)
8.- G. Berclaz, S. Li, KN Price, et al. Body mass
index as a prognostic feature in operable breast
cancer: the International Breast Cancer Study
Group experience. Ann Oncol 2004;15 (6):875884
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Melatonin and Cancer
Gilberto E. Toniolo Chechile
Instituto de Enfermedades Prostáticas. Hospital Universitario Quirón-Dexeus. Instituto Médico Tecnológico. Barcelona
Introduction
Melatonin is a hormone produced mainly by
the pineal gland, but it is also produced in small
amounts in other places in the body: the retina,
bone marrow, gastrointestinal tract, respiratory
epithelium, lymphocytes, etc. It also is widely
distributed in the plant kingdom (banana,
cucumber, tomato, beetroot)1. In chemical terms,
melatonin is N-acetyl-5-methoxytryptamine, a
derivative of serotonin, which in turn is a derivative
of the amino acid tryptophan.
The secretion of melatonin by the pineal gland
follows a daily cycle (the circadian rhythm), with
very low levels during the day (10-12pg/ml), while
at night synthesis increases, leading to higher blood
plasma levels (80-150pg/ml), and the highest levels
being reached between midnight and 3am. The
process of melatonin secretion is controlled by the
light-dark cycle.
There are two visual systems in the retina: in
one, light entering the eye stimulates the visual
receptors - rods and cones - (sight), and in the other
specialised photoreceptors act on the biological
clock (circadian vision). The photoreceptor
axons of circadian vision exit the eye through
retinohypothalamic tract in the optic nerve and
reach the suprachiasmatic nucleus (SCN) in the
anterior hypothalamus and from there, they reach
the pineal gland through the neurons of the
sympathetic nervous system2.
At night, in darkness, the SCN sends an electrical
signal to the pineal gland which stimulates the
release of norepinephrine in the pinealocytes
(pineal gland cells), which start synthesising
melatonin. Light exerts an inhibitory effect,
whereas darkness stimulates the production and
release of melatonin. Blind people have lost visual
sight but still retain circadian vision, thus the SCN
maintains the stimulus for the pineal to secrete
melatonin. People who have lost both eyeballs,
however, lack both visual systems.
Melatonin levels in the blood are higher in
young people (55-75pg/ml) and start to decline
after the age of 40, although the fastest decrease
is found from 60 years of age onwards, reaching
very low levels in the elderly (18-40pg/ml)3.
Melatonin production also varies according to
season and gender – it is higher in winter than in
summer, and in elderly women than elderly men.
In many diseases, such as coronary artery disease,
schizophrenia and Alzheimer’s, there are found to
be low levels of melatonin. Exposure to artificial
light or electromagnetic fields at night blocks the
secretion of melatonin. As about a quarter of the
world’s population works at night, and in Western
countries there is also excessive night lighting (light
pollution), melatonin secretion is dangerously
reduced in humans, leading to serious alterations in
people’s biological clocks, which are almost solely
regulated by the natural light cycles determined by
the rising and setting of the sun5.
The melatonin released into the blood has a very
short half-life (less than 30 minutes); it enters
the cells and diffuses into all body fluids, such as
saliva, semen, follicular fluid, cerebrospinal fluid
33
and bile. Melatonin is metabolised into6-hydroxymelatonin-sulphate in the liver, and is subsequently
excreted in urine. Smokers have lower levels
of melatonin than non-smokers since cigarette
smoke contains certain elements (polycyclic
hydrocarbons) that boost one of the enzymes that
metabolise melatonin.
The effects of melatonin.
Melatonin keeps the rhythm of the internal clock
that regulates the biological cycles involved in basic
physiological and physiopathological processes
in the body. Examples of such processes are: the
control of the sleep-wake cycle; the production
and secretion of other hormones (hypophysary
hormones, testosterone by the testes, cortisol by
the adrenal glands) and neurotransmitters; seasonal
reproductive cycles; immune system modulation;
bone metabolism; functions of the cardiovascular
system; gastrointestinal physiology; protection
against oxidative damage, and the inhibition of
certain tumours4.
Melatonin acts through receptors located in the
cell membrane, although its anti-oxidising action
is direct, neutralising free radicals and thereby
protecting DNA from oxidative damage without
the involvement of receptors. Two melatonin
receptors (MT1 and MT2) have been identified.
Depending on cell type, these receptors activate a
series of second messengers which act on particular
genes in the nucleus that are involved in the
inhibition of inflammatory processes and in the
stimulation of antioxidant enzymes.
Melatonin and the immune system.
Aging is associated with a decline in immune
function, leading to greater susceptibility to
infection, degenerative diseases and cancer6.
With aging there is also a decrease in a number
of hormones, such as the growth hormone,
oestrogen, the androgen dehydroepiandrosterone,
and melatonin. It has been shown that melatonin
stimulates the immune system in humans and is
a natural antioxidant with significant anti-aging
properties6.
34
Natural killer cells (NK) play an important role
in inhibiting cancer and metastases. People living
to advanced ages (over 90) have been observed to
have a high number of NK cells.
B lymphocytes (which play an important role in the
production of antibodies -humoral immunity), and
T lymphocytes (which are responsible for cellular
immunity) are greatly reduced in the elderly.
Furthermore, the decrease in T lymphocytes is one
of the factors leading to a decrease in interleukin-2,
which also leads to a decrease in the production of
antibodies. People aged over 85 show an increase
in levels of interleukin 6 and 10, which is associated
with a higher incidence of disease (cancer) and
mortality.
The progressive deterioration of the immune
system caused by the decrease of B and T
lymphocytes and by the decrease of interleukin
2 occurs around the age of 60; these changes are
consistent with the decrease in melatonin levels in
the blood. Melatonin, therefore, is considered to
play an important modulatory role in the immune
system7. There are changes in the immune system
according to time of day and season that have been
found to correlate with the synthesis and secretion
of melatonin. Moreover, human lymphocytes
produce melatonin, which also gives support to
the role melatonin plays in the immune system.
Experimental studies have shown that inhibition
of melatonin in mice causes the inhibition of a
cellular and humoral immune response, and
the effects are reversed by the administration of
melatonin at nightfall6. Other studies have shown
that administration of melatonin increases nonspecific immunity against tumours (NK cells and
monocytes)8. Melatonin has also proven useful for
correcting the deficiencies in the immune system
caused by stress, viral infections and treatments
with drugs.
Melatonin and sleep
Sleep is not necessary for the production of
melatonin; however, what is essential for
maintaining the circadian rhythm of melatonin
secretion is darkness. Higher levels of melatonin
in the dark contribute significantly to the decrease
in body temperature, the decrease in systolic and
diastolic blood pressure and the propensity to
sleep9. The drop in blood pressure produced by
melatonin at night is of great importance - it has
been shown that individuals whose blood pressure
falls at night show higher survival rates than
individuals whose blood pressure does not fall in
this way10.
Melatonin production precedes the onset of
sleepiness by about two hours. It is believed
that more than inducing sleep, melatonin blocks
the mechanisms generating wakefulness, thus
the secretion of melatonin at night ensures a
slow transition between wakefulness and sleep.
Individuals who sleep for more than 8-9 hours a
night show elevated levels of nocturnal melatonin
for longer (about one hour) than healthy subjects
who sleep less than 6 hours a night11. Several
studies have shown that people who sleep less are
more likely to develop breast or prostate tumours
than those who sleep for longer12-14. Lack or
disturbance of sleep can lead to immune system
suppression with a reduction in anti-tumoural
cytokines, such as interleukin-2 and interferon
gamma, and an increase in tumour-inducing
cytokines, such as interleukin-6 and -1015. One of
the first applications of melatonin was to minimise
the effects of jet lag. The American Academy
of Sleep Medicine currently recommends using
melatonin for jet lag and other situations that
disturb sleep by disrupting the circadian rhythm16.
Melatonin is also used successfully with children
with neurodevelopmental disabilities17.
nitric oxide and hypohydrochloric acid18; the
metabolites of melatonin also have antioxidant
effects. One of the intracellular sites where the
greatest quantity of free radicals is produced, and
therefore where there is greater oxidative damage,
is the mitochondria. If free radical generation
within the mitochondria reaches a critical level,
cellular apoptosis (programmed cell death) results.
Melatonin needs to penetrate the mitochondria
to exert its antioxidant effect and prevent
apoptosis19.
The anti-apoptotic activity of melatonin is of
great importance in the central nervous system
since in neurodegenerative diseases neuron
death occurs mainly by apoptosis. As well as its
direct antioxidant activity, melatonin stimulates a
number of antioxidant enzymes and inhibits prooxidant enzymes, such as nitric oxide synthase,
catalase, superoxide dismutases, glutathione
peroxidase and reductase, etc. The beginning of,
and continued neuronal loss as a result of normal
aging, as also occurs in some forms of dementia,
is usually a consequence of apoptotic neuronal
death. Administration of melatonin to patients
with neurodegenerative disease or brain lesions
caused by cerebral aging decelerates the process20.
Melatonin’s antioxidant effects are superior to those
produced by other molecules, such as vitamins E
and C, glutathione, mannitol, etc21.
Melatonin and artificial light
The antioxidant effects of melatonin
Exposure of any animal species and humans to
artificial light after nightfall, or a considerable
reduction in the period of darkness, produce
disturbance in sleep, in the circadian rhythm and
in melatonin production22.
The antioxidant effects of melatonin are produced
without involving the melatonin receptors:
melatonin only needs be in proximity to where
free radicals are being formed. The fact that
melatonin protects lipids, proteins and DNA from
oxidative damage indicates that its intracellular
distribution is ample4. Its antioxidant effect is
produced by supplying electrons. The most toxic
compounds neutralised by melatonin are: hydroxyl
radicals, peroxynitrite anion, hydrogen peroxide,
The World Health Organization’s International
Agency for Research on Cancer recently
concluded that shift workers who break the light/
dark circadian cycle through exposure to artificial
light at night, and individuals subjected to chronic
jet lag, such as air crews, are more vulnerable to
developing certain types of cancer23. In one study,
the incidence of breast cancer was related to light at
night (detected with satellite images of 146 Israeli
towns and villages). It was seen that where there
35
was more intense light at night, the incidence of
breast cancer was 73% higher than in communities
with less intense night time light24.
The light intensity necessary for inhibiting
melatonin production varies among different
species. In rats, less than 1 lux (the light given by
a full moon) for 30 minutes is enough to block
melatonin synthesis, while in humans it takes at
least 15 lux (equivalent to street lighting) to block
melatonin production25. This means that the
normal lighting in a home (100-200 lux) or intense
office or factory lighting (> 700 lux) produces a
marked suppression of melatonin production by
the pineal gland.
In several studies in which tumours were
implanted into rats, the animals exposed to
24-hour a day electric lighting showed increased
growth of the implanted tumours or spontaneous
tumours compared to animals kept in complete
darkness26-27. If tumours were put in contact with
blood obtained from individuals exposed to intense
electric light at night (2800 lux for 90 minutes),
this blood with low melatonin levels stimulated
tumour growth compared with tumours treated
with blood obtained from subjects who had been
in total darkness during the night 26. These effects
were seen only in well differentiated tumours,
whereas the growth of poorly differentiated
tumours did not vary.
It has recently been suggested that the use of
glasses that filter light of the specific wavelengths
that suppress melatonin production, or the use of
electric light bulbs which do not generate these
wavelengths (460-480nm), can avoid light pollution
and reduce the incidence of breast and prostate
cancer28. Although these recommendations may
be useful, it is likely that the final solution to the
problem is far more complex. The anti-tumoural
effects of melatonin. Although the relationship
between the pineal gland and the growth and
spread of cancer has been known for more than 80
years, scientific bases for the relationship were not
established until 197729. In 1978 the theory was
put forward that the hyperoestrogenism produced
by the reduction of pineal gland function with
36
a resulting reduction of melatonin could play a
role in carcinogenesis of the mammary gland30.
Since then, numerous studies have suggested
the association of low levels of melatonin in the
progression of several cancers29, 31, 32. The effects
of melatonin have been studied in the following
tumours: breast, prostate, colon and rectum, ovary,
endometrium, lymphomas and leukaemia, lung,
melanoma, sarcomas, hepatocellular carcinomas,
skin, neural tumours, cervix, and laryngeal
carcinomas. In general, melatonin inhibits cell
proliferation, induces apoptosis (programmed cell
death), reduces carcinogenesis and slows tumour
growth.
The anti-tumoural
melatonin
mechanisms
of
1 - Experimental data Melatonin exerts its antitumoural effects through a series of cellular
mechanisms such as:
a) cell-cycle modulation, b) inhibition of
proliferation, c) induction of apoptosis, d)
telomerase inhibition, e) aromatase inhibition, f)
anti-angiogenesis, g) interference with oestrogen
receptor, h) inhibition of metastases.
a) Cell-cycle modulation
In several studies with different tumour
cells, it has been shown that melatonin
increases the duration of the cell cycle by
extending the G1 phase, which reduces cell
proliferation, allows the repair of damaged
DNA and delays the cell entering S phase,
thereby decreasing DNA synthesis33, 34.
b) Inhibition of cell proliferation
This is one of melatonin’s most important
anti-tumoural effects, and has been
observed in the tumours of various organs
(breast, prostate, ovary, endometrium,
liver). In in vitro studies with breast cancer
cells, melatonin has been shown to suppress
the proliferation of all oestrogen receptorpositive cell lines and some oestrogen
receptor-negative cell lines35, 36.
c) Induction of apoptosis
If there is a significant amount of damaged
DNA within a cell, apoptosis is induced
(induced cell death). For a cell to enter
into apoptosis, a series of genes must be
activated, one of which is the p53 tumour
suppressor gene, which in turn activates
other pro-apoptotic genes, such as Bax and
Bcl-237. Other genes that are activated are
the caspases, whose function is to digest
numerous proteins in the cell cytoplasm,
bringing about cell death. Some studies
show that melatonin prevents normal cells
entering apoptosis while inducing apoptosis
in the cells of several cancers (breast,
prostate, colon, liver)38-39. However, other
studies have not confirmed these results40.
Melatonin may occasionally enhance the
apoptotic effects of some chemotherapy
drugs, such as ifosfamide or vincristine41.
d) Telomerase inhibition
Telomerase is an enzyme that synthesises
telomere extensions in normal cells and
is essential in maintaining chromosome
structure. In most cells, telomerase activity
is very low, and so telomere length
decreases with successive cell divisions,
which causes the chromosome to become
increasingly unstable and susceptible to
damage, and there is a higher likelihood
of apoptosis occurring. Telomerase is
activated during carcinogenesis, and while
the capacity of unlimited division of tumour
cells is maintained, telomerase generates
new chromosome extensions, making the
chromosomes more stable thus preventing
apoptosis4. Telomerase is activated in 90%
of cancers42, thus inhibition of telomerase
in tumour cells may have a potential
therapeutic anti-tumoural use. In mice
implanted with MCF-7 breast cancer
cells, the animals receiving melatonin for
five weeks showed a significant decrease
in telomerase activity and tumour size was
significantly reduced, as was the number of
metastases43.
e) Aromatase inhibition Aromatase is an
enzyme involved in the conversion of weak
androgens, such as dehydroepiandrosterone
(DHEA) and dehydroepiandrosterone
sulphate (DHEA-S), to oestrogens. It
has recently been shown that melatonin
inhibits the expression and activity of
P450 aromatase and other enzymes,
such as oestrogen sulphatase and the 17`
hydroxysteroid dehydrogenase, involved
in the synthesis and transformation of
oestrogens from androgens in MCF-7
breast cancer cells44, 45. The anti-aromatase
effects of melatonin are mediated via the
MT1 melatonin receptor46.
f) Angiogenesis inhibition
Hypoxia is the principal mechanism
by which more than 70% of tumours
progress, through angiogenesis activation,
which is essential for a tumour to grow
over 200 micras47. However, unlike what
occurs with normal tissue vascularisation,
the tumoural microvessels formed through
angiogenesis are highly disorganised
and so more hypoxia occurs with the
subsequent activation of transcription
factors associated with cell hypoxia, such
as the hypoxia-inducible factor 1-_ and
1-` (HIF-1_ and HIF-1`). These in turn
activate the expression of different genes
related to angiogenesis which lead to
greater progression and aggressiveness of
tumours48.
Tumour-induced angiogenesis is regulated
by factors produced by macrophages,
neutrophils and by tumour cells themselves,
such as the vascular endothelial growth
factor (VEGF), the platelet-derived growth
factor (PDGF) and the transforming
growth factor alpha (TGF- _)47. VEGF
induces angiogenesis by acting directly
on the endothelium. Tumours showing
high VEGF levels are more aggressive and
the onset of metastasis is earlier. Several
studies have shown that melatonin inhibits
angiogenesis by inhibiting VEGF and the
37
transcription factor HIF-1_49-51. Melatonin
has recently been shown to inhibit the
proliferation and migration of pancreatic
cancer cells through the suppression
of VEGF expression52. In hormonerefractory prostate cancer cells (PC-3),
it has been shown that, under hypoxic
conditions, melatonin inhibits transcription
factor HIF-1_ through the inhibition of
sphingosine kinase 1 (SPHK1)53. This is
a recently described HIF-1_ modulator
whose expression is increased in many
malignant cells and which has various
biological actions on cell growth, invasion,
angiogenesis and carcinogenesis. Inhibition
of the activity of these enzymes means
blocking the proliferation and induction
of apoptosis in malignant cells; therefore,
SPHK1 inhibitors could be of therapeutic
potential54.
g) Interference with the oestrogen
receptor
Several studies have shown that melatonin
inhibits the oestrogen receptor, which is
one of the mechanisms through which
the proliferation of breast cancer cells is
blocked33,55,56. The anti-oestrogenic effect
of melatonin is produced by its action of
decreasing the expression of the oestrogen
receptor and not by either the binding
of melatonin to the receptor nor by
interfering with the binding of oestrogen
to its receptor57,58. The anti-oestrogen
receptor action of melatonin is realised
through binding to its ML1 receptor59.
h) Inhibition of tumour invasion and
metastases
For some years experimental research with
breast cancer cells has shown melatonin’s
ability to inhibit tumour invasion and
metastases60. More recently it has been
shown that with the administration of
melatonin, tumour cell invasion reduced
between 65 and 85% and these effects
were mediated by over-expression of the
38
MT1 melatonin receptor33. For tumours
to infiltrate surrounding tissue, tumour
cells need to secrete proteolytic enzymes,
such as metalloproteinases, to digest the
extracellular matrix. Recent research
suggests that one of the mechanisms by
which melatonin acts on tumour invasion
is modulation of metalloproteinase61, 62.
Microfilaments are important structures
in the cytoskeleton of epithelial cells and
in cell adhesion. These microfilaments
are responsible for the cell taking on a
polyhedral shape, which is important
in its function as a water-impermeable
barrier. Melatonin acts as a modulator of
the microfilaments of cells’ cytoskeleton
in both normal and malignant cells.
Melatonin has been shown to be able to
change the phenotype of microfilaments of
invasive breast cancer cells to a phenotype
of microfilaments typical of cells that
cannot migrate and produce metastasis63.
2 - Clinical evidence
Several clinical studies have shown that levels of
melatonin in plasma are lower in patients with
certain types of tumours (such as breast, prostate,
uterus, colon and rectum, lung, etc)64-65. Although
melatonin was first used as an anti-tumoural
agent in 1963, it was not until 1999 when the
results of a study of 250 patients with metastatic
tumours (lung, breast, gastrointestinal tract, head
and neck) were presented. These patients had
been treated either with chemotherapy alone
(126 cases) or chemotherapy plus melatonin
(124 cases), with a 20mg oral dose at dusk started
seven days before the start of chemotherapy and
continued until the progression of the disease66.
After one year of treatment, the patients treated
with chemotherapy plus melatonin showed longer
survival rates than those receiving chemotherapy
alone66. Furthermore, the patients receiving the
combination therapy showed a lower incidence
of cardiac, neurological or haematological side
effects.
In another study of patients with lung cancer,
tumour regression and five-year survival rates
were significantly higher in patients treated with
chemotherapy and melatonin 67. None of the
patients treated with chemotherapy alone was
alive after two years, while after five years, 6% of
patients treated with chemotherapy and melatonin
were alive67.
Barceló Sánchez et al collected 20 published clinical
studies that analysed melatonin treatment in various
tumours (lung, colorectal, prostate, breast, kidney,
leukaemia, thyroid)68. In metastatic colorectal
carcinoma which had progressed following initial
chemotherapy treatment, the combination of
melatonin and irinotecan produced 36% partial
relapse and 50% stable disease, compared to 12%
partial response and 12% stable disease in patients
treated with irinotecan alone69. In another study
the combination of interleukin-2 (IL-2) and
melatonin produced a partial response in 12% of
patients with metastatic colorectal cancer whose
initial chemotherapy treatment had failed 70.
In three studies with patients with advanced or
metastatic lung cancer receiving chemotherapy
(cisplatin and etoposide) alone or combined
with melatonin (20mg/day at dusk), a greater
response was observed in patients treated with the
combined chemotherapy and melatonin71-73. The
percentage of tumour regression and survival at
two years was higher in the combined treatment.
One of the studies showed complete regression
and partial regression in 3% and 30% respectively
of the patients treated with chemotherapy and
melatonin, whereas in those patients treated
with chemotherapy alone complete regression
was not observed, and a partial regression of the
disease was observed in only 17% of cases 71.In
addition, patients treated with melatonin had a
lower incidence of side effects from chemotherapy
(neurotoxicity and thrombopenia). In a study of
30 patients with metastatic renal cell cancer, the
association of interleukin 2 (IL-2), oral morphine
and melatonin (20mg/day at dusk) was superior
to the combination of IL-2 and morphine74.
The partial response rate in patients treated with
IL-2 and morphine was significantly lower than
in those concomitantly treated with melatonin.
The three-year survival rate was also higher for
patients receiving melatonin. In a study in patients
with metastatic lung cancer, 50 cases were treated
with chemotherapy (cisplatin and gemcitabine)
combined with melatonin (20mg/day at dusk),
while 100 cases were treated with cisplatin and
gemcitabine alone75.The response was significantly
higher in patients receiving chemotherapy plus
melatonin (42%) compared to those who received
chemotherapy alone (24%). Objective tumour
regression was significantly higher in patients who
as well as receiving chemotherapy plus melatonin
also held religious beliefs75.
Seely et al searched seven databases up to
February 2010 and found 21 randomised clinical
trials in which melatonin was combined with
chemotherapy or radiotherapy for solid tumours
with or without metastases76.The results of 3697
patients with breast, colorectal, lung, liver, kidney
cancer and glioblastomas were analysed. It was
observed that patients who received melatonin had
a lower risk of mortality at one year and greater
likelihood of complete response, partial response
or stable disease than patients who had not received
melatonin. Furthermore, patients who had received
melatonin showed a lower incidence of side effects
caused by chemotherapy or radiotherapy (nausea
and vomiting, fatigue, leucopenia, thrombopenia
and hypotension) than patients who had not been
treated with melatonin.
Wang et al reviewed databases up to November
2011 and found eight randomised controlled
clinical trials in patients with solid tumours in which
melatonin was combined with chemotherapy or
radiotherapy77.The research included 760 patients.
The combination of melatonin showed objective
response (complete and partial) in 33% of cases,
compared to 17% in those who had not received
melatonin. The incidence of adverse effects due
to radiotherapy or chemotherapy was lower
with melatonin: thrombopenia (2.2% vs 19.7%),
neurotoxicity (2.5% vs 15.2%) and fatigue (17.2%
vs 49, 1%).
39
Melatonin and prostate cancer
The inhibitory effects of melatonin on tumours
produced in mice with hormone-sensitive prostate
cancer cells (Dunning R 3327) was shown
in 198878. It was later observed that the antitumoural effects of melatonin was accentuated if
associated with castration in mice implanted with
hormone-sensitive LNCaP prostate cancer cells,
but was not effective in mice implanted with
hormone-resistant prostate cancer cells (PC3 and
DU-145 )79. The mechanism of melatonin’s antiproliferative effect is mediated by the ML1 receptor
present in LNCaP cells but defective in PC3 and
DU-145 cells. Other recent studies have shown
that melatonin has anti-proliferative effects on
hormone-resistant prostate cancer cells (PC3 and
22Rv1) 80.81.The anti-proliferative effects were
blocked by luzindole, a non-selective MT1 and
MT2 melatonin receptor antagonist, but were not
blocked by a selective MT2 antagonist (4-phenyl2-propionamidotetralin). These results suggest that
melatonin’s anti-proliferative effects in prostate
cancer are mediated exclusively by the MT1
receptor. Furthermore, melatonin has been shown
to reduce the number of prostate cancer cells
and halt the progression of the cell cycle in both
hormone-sensitive cells and hormone-independent
cells, and these effects are not mediated by MT1
and MT2 receptors80.
In pharmacological doses, melatonin has been
shown to possess anti-angiogenenic effects in
prostate cancer cells in that it inhibits the expression
of the hypoxia-inducible factor 1-_ (HIF-1 _)
both in the presence of normal concentrations
of oxygen (normoxia ) as well as in hypoxic
conditions. These effects were observed in both
hormone-sensitive cells (LNCaP) and hormoneindependent cells (PC3 and DU-145)82.
Another of melatonin’s anti-tumoural effects on
prostate cancer cells is through the activation of
apoptosis. It has been shown that cell death by
apoptosis occurs through the activation of several
genes (p38, p53, p21) that in turn transactivate
pro-apoptotic proteins39,83. Moreover, melatonin
reduces the response of anti-apoptotic proteins.
40
It has recently been shown that melatonin exerts
anti-proliferative effects by reducing androgen
receptor expression in LNCaP, VCap and 22Rv1
prostate cancer cells, which could potentially be
used as a strategy for chemoprevention of prostate
cancer84.
In various prostatic pathologies, such as prostate
cancer, intraepithelial neoplasia (PIN), and even
benign hyperplasia (BPH), there has shown to be
an alteration of some genes that encode proteins
involved in the regulation of circadian rhythms85.
Thus, Per2 and Clock gene expression is significantly
decreased, while the expression of the BMAL1
gene is significantly increased85. When prostate
cancer cells were treated with melatonin, there
was observed to be an increase in the expression of
Per2 and Clock genes, and a reduction in BMAL1
gene expression. Overexpression of Per2 caused
the arrest of malignant cell growth and decreased
the viability of these malignant cells85. The ability
of melatonin was also observed in re-synchronising
the circadian rhythms of malignant cells.
Recently, it has been shown that one of the members
of the sirtuin family (SIRT1) regulates Clock and
BMAL1 genes86. This has led to the idea that
SIRT1 may be of great importance in age-related
tumours and its modulation can re-synchronise the
deregulated biological clock at cell level, which
would have implications in the treatment of various
cancers. SIRT1 is overexpressed in human prostate
cancer cells, while expression in non-malignant
prostatic tissue is normal87. SIRT1 inhibition
causes growth arrest in prostate cancer cells while
not affecting normal prostate cells88. Inhibition of
SIRT1 also induces apoptosis in prostate cancer
cells89. Treatment of prostate cancer cell lines with
melatonin inhibited the activity of SIRT1 protein,
leading to a significant decrease in the proliferation
of malignant cells but not normal cells90. When
transgenic mice implanted with prostate cancers
were treated with oral melatonin, a decrease in
SIRT1 and tumorigenesis was observed90.
Several epidemiological studies have shown that
individuals living in the Arctic Circle and subjected
to long periods of darkness, and likewise blind men,
show a lower incidence of prostate cancer91,92.
It has also been shown that melatonin levels in
plasma are lower in elderly men, coinciding with
the increase in the incidence of prostate cancer93.
Furthermore, patients with prostate cancer have
lower levels of melatonin in blood than those found
in patients with benign prostatic hyperplasia94.
Based on these experimental data, the use of
melatonin has been proposed for the prevention
and treatment of prostate cancer95.
In one patient with hormone-resistant prostate
cancer, daily treatment with 5mg of melatonin
at 8pm stabilised the tumour and slowed the
biochemical progression of the disease96.
In patients with metastatic prostate cancer whose
initial treatment with LH-RH analogues had
failed, the combination of melatonin (20mg every
day at dusk) and a LH-RH analogue significantly
decreased PSA levels (more than 50%) in 57% of
patients, normalised platelet numbers in three out
of five patients with thrombopenia, and achieved
survival for longer than one year in 64% of
cases97.
Melatonin as a chemo- and radioprotector
Several studies have shown that melatonin reduces
the toxicity of various chemotherapeutic agents,
such as cisplatin, etoposide, anthracyclines and
5-fluorouracil. A statistically significant reduction
in neurological, renal, cardiac, and bone marrow
toxicity has been observed 98-103.
Similarly, cells exposed to ionizing radiation and
melatonin show fewer genetic alterations compared
to cells exposed only to radiation104. Radiation
disrupts the chemical structure of molecules
producing free radicals that immediately react with
nearby molecules so producing oxidative damage.
It is considered that 60-70% of tissue damage
produced by radiation is due to free radicals and
oxidative damage. A major part of that damage
is done to DNA structure105. Melatonin is a
powerful antioxidant that has a triple action: a) by
directly scavenging free radicals, b) by increasing
the activity of antioxidative enzymes (superoxide
dismutase, glutathione peroxidase, etc.), and c)
by decreasing the activity of the pro-oxidative
enzymes106. To be effective against the effects of
radiation, it has been shown that melatonin should
be within the cell at the time of irradiation, and so
needs to be administered prior to the radiotherapy
session.
Clinical studies with melatonin
In the U.S. National Institutes of Health, 142
clinical trials have been registered that study
the effects of melatonin in different clinical
situations107. Ten of the 142 trials analysed patients
with cancer. In four studies with breast cancer
patients, melatonin is assessed for the treatment
of sleep disturbance. One study on patients with
lung cancer analyses the effects of melatonin on
the prevention of recurrence and mortality. Other
studies analyse patients with brain metastases,
and with primary brain cancer. Two studies treat
patients with advanced cancer cachexia, and in the
remaining study, the patients in treatment suffer
from advanced cancer fatigue.
Conclusions
The increased incidence of different cancers, as well
as decreased immunity due to aging, has led to the
search for substances that stimulate the immune
system. Melatonin has proven useful for effectively
increasing immune function in both animals and
humans. Altered circadian rhythms are also a risk
factor for developing tumours. Treatment with
melatonin, which acts at cell level on the genes
involved in circadian rhythms, has re-synchronised
the altered rhythms, and this may be of potential
therapeutic value for treating several tumours.
Melatonin’s ability to neutralise free radicals may
partly explain how it reduces carcinogenesis and
inhibits the growth of various types of tumour.
Other anti-tumoural effects of melatonin,
demonstrated in vivo and in vitro are: cell cycle
modulation, cell differentiation, induction of
apoptosis, inhibition of telomerase activity,
blocking angiogenesis and tumour invasion, and
regulation of the organism’s circadian rhythm.
Furthermore, melatonin is the only molecule that
41
acts on the oestrogen receptor and on the enzymes
involved in oestrogen synthesis, which gives
potential for the treatment of hormone-dependent
tumours. The low toxicity of melatonin, together
with its proven anti-tumoural effects, makes it
potentially valuable in treating tumours; further
clinical studies are needed to confirm the results
obtained from experimental research. Reducing
the toxicity of cancer treatment (chemotherapy
and radiotherapy) is another of the applications
of melatonin that requires further multi-centre
randomised double-blind studies.
Although many clinical trials with melatonin are
underway, its potential therapeutic effects in cancer
have not yet been extensively studied.
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47
Silent Players of Health in a Broader Age-management
Medicine Understanding: a Dissertation on Bacteriocin
of Lactic Acid Bacteria.
1R. Catanzaro, 1M. Milazzo, 2C. Tomella, 3U. Solimene, 2A. Polimeni, 2F. Marotta
1Gastroenterology
Unit, Dept. of Internal Medicine, University of Catania, Italy; 2ReGenera Research Group for Aging
Intervention, Milan, Italy; 3WHO-cntr for Biotechnology and Traditional Medicine, University of Milano, Milan, Italy
The preservation of foods in healthy and safe
condition has long been used and still it remains
an on-going challenge for food microbiologists.
Drying, salting and fermentations were the
traditional methods of preservation. Canning and
freezing were relatively recent developments.
The role of fermented milk in human diet in well
known since Vedic times but the scientific interest
arose only after the publication of a book“Prolongation
of Life” (Metchnikoff, 1908). In developed societies,
food preservation is viewed as a ‘convenience’ of an
efficient food system, and food preservation is the
key to ensure the availability of food as vital benefit.
Food fermentations, developed by default rather
than by design. Lactic acid bacteria (LAB) play
an important role in food fermentations, causing
the characteristic flavor, changes and exercising
a preservation effect on the fermented product
(Caplice and Fitzgerald, 1999). It is estimated that
25% of the European diet and 60% of the diet in
many developing countries consists of fermented
foods (Holzapfel et al., 1995). The spice trade was
the start of addition of the chemicals adjunct to
foods.With the industrial revolution and subsequent
development of food industries, food processing
moved from kitchen or cottage industries to largescale technological operations with increased need
for food preservation. This stimulated the use of
food additives, especially those that preserve the
foods and enhance food quality.This has resulted in
the emergence of a new generation of chill stored,
minimally processed foods (de Souza et al., 2005).
Hurst, (1973) reviewed the preservation of foods
by the antagonistic growth of microorganisms. He
48
showed the growth of lactic acid bacteria (LAB)
in milk, saurkaut and vacuum packaged meats as
examples of protective and antagonistic growth. In
recent times this has been termed as ‘biopreservation’
to differentiate it from the chemical (artificial)
preservation of foods.
Biopreservatives such as lactic acid bacteria (LAB)
and their metabolites have been investigated by
several authors (Buncic et al., 1997; Pirttijarvi et
al., 2001; Sakhare and Narasimha Rao, 2003).
Considerable research has been done on the
ability of LAB to inhibit growth of pathogenic
microorganisms (Winkowski et al., 1993; MinorPerez et al., 2004). The capability of these bacteria
to control growth of spoilage microorganisms has
not been investigated to the same extent. To be
successful in biopreservation, a bacteriocinogenic
LAB culture must compete with the relatively high
indigenous microbial load of raw meat, to actively
inhibit pathogenic and spoilage bacteria (Sakhare
and Narasimha Rao, 2003; Minor-Perez et al.,
2004).
Bacteria preserve foods as a result of competitive
growth, products of their metabolism and
bacteriocin production. Biopreservation refers to
extended storage life and enhanced safety of foods
using their natural or controlled microflora and
(or) their antibacterial products. It may consist
of (i) adding bacterial strains that grow rapidly
and (or) produce their antibacterial products; (ii)
adding purified antagonistic substance(s); (iii)
adding the fermentation liquor or concentrate
from an antagonist microorganism; or (iv) adding
mesophilic LAB and other related bacteria as a
‘fail-safe’ protection against temperature abuse.
LAB and other related bacteria produce lactic acid
or lactic and acetic acid, and they may produce other
inhibitory substances such as diacetyl, hydrogen
peroxide, reuterin ( -hydroxypropionaldehyde) and
bacteriocins (de Vuyst and Degeest, 1999).
Bacteriocins:Bacteriocins have been described as ribosomally
synthesized extracellular macromolecular precursor
polypeptides or proteins produced by one bacterium
that are active against other bacteria, either in the
same species (narrow spectrum), or across genera (
broad spectrum) and, as with host defence peptides
(Jack et al., 1995; Russell and Mantovani, 2002;
Bowdish et al., 2005).Bacteriocins have bacteriocidal
activity (Tagg et al., 1976) due to the combined
action of the bacteriocin and the host autolysis
(Martinez-Cuesta et al., 2000), or bacteriostatic
against other species, usually closely related to the
producer strain (Russell and Mantovani, 2002). In
some cases, they are also active against other species
(Klaenhammer, 1993; Jack et al., 1995).
Bacteriocins are heterogeneous group of bacterial
antagonists that vary considerably in molecular
weight, biochemical properties, range of sensitive
hosts and mode of action. Klaenhammer (1988)
defined them as, proteins or protein complexes with
bactericidal activity directed against species that are
usually closely related to the producer bacterium.
As peptides, bacteriocins are of low molecular
weight, but larger than antibiotics. This makes
them susceptible to biochemical reactions, which
may limit their antimicrobial activity (Muriana,
1996). CAST (Council of Agricultural Science and
Technology) (1998) reported that concentration,
microorganisms, pH, temperature and interactions
affect the activity of bacteriocins.
Bacteriocins are produced by both Gram-positive
and Gram-negative bacteria including LAB, which
are used in food fermentations (Klaenhammer,
1988; Daeschel, 1989; Ray and Daeschel, 1992;
Nettles and Barefoot, 1993; Klaenhammer, 1993;
Sahl et al., 1995; Muriana, 1996) Two well-known
representatives of bacteriocins produced by Gramnegative bacteria are colicins and microcins.The first
desription of bacteriocin-mediated inhibition was
reported 80 years ago, when antagonism between
strains of Escherichia coli was first discovered (Gratia
1925), they were named as colicins (Fredericq,
1948), bacteriocins produced by Escherichia coli
and usually showing activity against other strains
of E. coli and very closely related members of the
Enterobacteriaceae. Induction usually accurs under
stressful conditions such as nutrients depletion or
over crowding (Riley and Gordon 1999). Colicins
have been studied for over six decades and are
well characterized (Akutsu et al., 1989). Colicins
differ from bacteriocins that are preduced from
Gram-positive bacteria in the sense that they
have 3 general mechanisms of action: channel
formation in the cytoplasmic membrane, (The
common mechanism found with Gram-positive
bacteriocins), degradation of cellular DNA, and
inhibition of protein synthesis. It is estimated that
about 30% of natural population of E.coli produce
bacteriocins (Riley, 1998). Colicins are plasmid
encoded bacteriocins and classified into groups on
the basis of the receptor to which they bind. Over
25 colicin types have been identified (Pugsley,
1984). It is also estimated that about 65% of the
cells in a population of E. coli are resistant to any
one colicin, and 30% are resistant to all colicins
produced in a population with the remaining
cells colicin-sensitive (Smarda, 1992). The relative
numbers of colicin-producing cells have been
found dependent on the energy costs associated
with colicin synthesis (Riley and Gordon, 1999).
The Gram-negative bacteriocins are colicin,
which are produced by strain of E. coli (Braun
et al., 1994; Riley and Gordon, 1999). These are
large, complex proteins, that inhibit bacterial
growth through the inhibition of cell synthesis,
permeabilizing the cell membrane or inhibiting
Rnase or Dnase activity (Cleveland et al., 2001)
20-90 Kda, with characteristic structural domains
involved in cell attachment, translocation and
bactericidal activity.They bind to specific receptors
on the outer membrane of the target cell. The
bacteriocins produced by Gram-positive bacteria
are small peptides 3-6 Kda, in size (Nes et al.,
1996), although there are exceptions (Joerger and
Klaenhammer, 1990). They fall with in two broad
classes, viz (namely) the lantibiotics (Jack et al.,
49
1995) and the non-lantibiotic bacteriocins (Nes et
al., 1996). Most of the Gram-positive bacteriocins
are membrane active compounds that increase the
permeability of the cytoplasmic membrane (Jack
et al., 1995). They often show a much broarder
spectrum of bactericidal activity than the colicins.
There is currently much interest in the application
of bacteriocins in both food preservation and the
inhibition of pathogenic bacteria (Liao et al., 1994;
Delves-Broughton, 1996; Cleveland et al., 2001;
de Souza et al., 2005). Most of the bacteriocins
have been isolated from organisms involved in
food fermentation. Bacteriocin production and
resistance is considered as an important property in
strains used as commercial inoculants to eliminate
or reduce growth of undesirable or pathogenic
organisms.
Microcins, produced by the Gram-negative
bacteria of family Enterobacterioaceae, are posttranslationally modified. They are active against
other Gram-negative bacteria and act through
inhibition of DNA replication or protein synthesis
(Bacquero and Moreno, 1984).
Lactic acid bacteria (LAB):- Lactic acid
bacteria (LAB) have played a long and important
role in food technology. These microorganisms are
industrially important and have been used as starter
cultures in various foods–fermentation processes.
Global production of cheese starter cultures, for
example, already 1.5 × 106 tons per year (Fox,
2000).The LAB include a wide variety of cell types
and physiological and biochemical characteristics
(Yanagida et al., 2005). Lactic acid bacteria (LAB)
are a group of bacteria united by a constellation
of morphological, metabolic and physiological
characteristics. The currently recognized genera
of LAB are Aerococcus, Alloicoccus, Carnobacterium,
Dolosigranulum, Enterococcus, Globicalella, Lactococcus,
Lactosphaera, Leuconostoc, Melissioccus, Oenococcus,
Pediococcus, Strepcoccus, Tetragenococcus, Vagococcus and
Weissella (Axelsson, 1998). This classification is
largely based on phenotypic characteristics such as
morphology, mode of glucose fermentation, growth
at different temperature, configuration of lactic acid
produced, ability to grow at high salt concentration,
and acid or alkaline tolerance (Axelsson, 1998).
Generally, LAB are described as Gram-positive, non-
50
motile, non-spore forming and microaerophilic rods
(singly or in chains) or cocci (diplococci, tetracocci,
streptococci). These bacteria usually belong to the
family Lactobacteriacae and are characterized by the
production of lactic acid as a major metabolic end
product of carbohydrate fermentation (Axelsson,
1998), hydrogen peroxide, diacetyl secondary
reaction products and bacteriocins, which may be
important for starter culture functions of the bacteria
(Daeschel, 1989). The characteristics of LAB used
as a starter culture are well documented (Tramer
and fowler, 1964). Lactic acid bacteria (LAB) are
low-GC-content, Gram-positive bacteria, which
are found in nutrient-rich environments such as
milk, meat, decomposing plant material and the
mammalian gastrointestinal tract, (Carr et al., 2002).
Surface growth on most media is very poor. The
nutritional requirement of this group is complex;
they need amino acids and vitamins.
Lactic acid bacteria may be homofermentative
or heterofermentative. Those bacteria which
ferment only lactic acid from lactose are known
as homofermentative and those that ferment other
than lactic acid, e.g., acetic acid alcohol and produce
CO2 are heterofermentative. They are catalase
negative, acid tolerant, and lack cytochromes and
porphyrins (Adams et al., 1995).
Lactic acid bacteria (LAB) in the form of
fermentative organisms are traditionally used to
preserve food and feed. It is well known that many
species of Lactobacillus and Lactococcus used in the
manufacture of fermented dairy products inhibit
the growth of other bacteria including intestinal
pathogens like Escherichia coli, Enterobacter faecalis,
etc. Bacteriocins produced by LAB are of great
interest to the food industry because of their
antagonistic effect against food borne pathogenic
and spoilage microorganisms (Matchikoff, 1908;
Ray et al., 2001). The inhibitor part is a protein
that could not be destroyed in milk even on
heating at 100°C for 30 min and was inhibitory
to several strains of Streptococcus lactis. Among the
microorganisms inhibited by certain bacteriocins,
numerous reports have included the fatal pathogen
Listeria monocytogenes (Spelhang and Harlander,
1989; Muriana, 1996; Klaenhammer, 1993; Ennahar
et al., 2000; Hechard et al., 2002).
Over the years, several publications have reviewed
colicins, bacteriocins, bacteriocins from LAB and
applications of specific bacteriocins. Examples
include, Reeves (1972), Franklin and snow (1975),
Hardy (1975), Tagg et al., (1976), Konisky (1982),
Klaenhammer (1988, 1993), Jack et al., (1995),
de Vos et al. (1995b), Sahl et al. (1995), Venema
et al. (1995), Abee et al. (1995), Nes et al. (1996),
Cleveland et al. (2001).
Taxonomy of lactic acid bacteria:- The
classification of LAB was initiated in 1919 by Orla
Jensen and was until recently primarily based on
morphological, metabolic and physiological criteria.
Lactic acid bacteria comprise a diverse group of
Gram positive, non-spore forming, non-motile
rod and coccus shaped, catalase-lacking organisms.
They are chemoorganotrophic and only grow in
complex media. Fermentable carbohydrates and
higher alcohols are used as the energy source to
form chiefly lactic acid. LAB degrades hexoses
to lactate (homofermentatives) or lactate and
additional products such as acetate, ethanol, CO2,
formate or succinate (heterofermentatives). They
are widely distributed in different ecosystems and
are commonly found in foods (dairy products,
fermented meats and vegetables, sourdough, silage,
beverages), sewage, on plants but also in the genital,
intestinal and respiratory tracts of man and animals
whose they play important roles as symbionts.
Current methodolgies used for classification of
LAB mainly rely on 16S ribosomal ribonucleic
acid (rRNA) analysis and sequencing (Olsen et al.
1994). Based on these techniques, Gram-positive
bacteria are divided into two groups depending on
their G + C content. The Actinomycetes have a G
+ C content above 50 mol% and contain genera
such as Atopobium, Bifidobacterium, Corynobacterium
and Propionibacterium. In contrast, the Clostridium
branch has a G + C content below 50 mol% and
include the typical LAB genera Carnobacterium,
Lactobacillus, Lactococcus, Leuconostoc, Pediococcus and
Streptococcus.
Table 1. Orla-Jensen (1919) key to
differentiation of the lactic acid bacteria and
current taxonomic classification.
Genusa
Shape
Catalase
Betabacterium
Thermobacterium
Streptobacterium
Rod
Rod
Rod
-
Nitrite
reduction
-
Streptococcus
Coccus
-
-
Homo
Betacoccus
Microbacterium
Tetracoccus
Coccus
Rod
Coccus
+
+b
+
+
Heter
Homo
Homo
Fermentation
Current genera
Hetero
Homo
Homo
Lactobacillus Weissella
Lactobacillus
Lactobacillus Carnobacterim
Streptococcus Enterococcus
Lactococcus Vagococcus
Leuconostoc Oenococcus Weissella
Brochothrix
Pediococcus Tetragenococus
aAccording to Orla-Jensen (1919).
bIn genera Pediococci are catalase negative but some strains produce a pseudocatalase that results in false positive reactions.
+ =Positive result, - = Negative result
Bcteriocins from lactic acid bacteria:The bacteriocins from LAB are mostly small, heatstable, hydrophobic and cationic peptides (Jack et
al., 1995). Several bacteriocins of LAB have been
characterized biochemically and genetically and in
a number of cases their mode of action has been
studied (Hoover and steenson, 1993; Klaenhammer,
1993; Chen and Yanagida, 2006; Kabuki et al.
2007).
Ever since, the publication of the first review
on the bacteriocins of Gram-positive bacteria
by Tagg et al. (1976), there has been a renewed
interest in the field of bacteiocins of Grampositive bacteria. The researches on bacteriocins
produced by a heterogeneous group of Gram
positive bacteria comprising genera, Lactobacillus,
Lactococcus, Leuconostoc, Pediococcus, Streptococcus
and Carnobacterium, collectively known as lactic
acid bacteria (LAB) has witnessed a tremendous
growth in the past one and half decade as evident
from the publications of several review articles
(Klaenhammer, 1988; Piard and Desmazeaud, 1992;
Klaenhammer, 1993; Nettles and Barefoot, 1993)
and books (Ray and Daeschel, 1992; Hoover and
Steenson, 1993) dealing with various aspects of
bacteriocins produced by lactic acid bacteria. The
interest in microorganisms occurring in foods is
primarily due to the biotechnological potential
of new bacterial species and strains (Leisner et al.,
1999).
In the dairy products, the species composition of
lactic acid bacteria is more varying and inconsistent
when compared with those of the trade products.
In biotechnological aspects, the “wild” strains of the
LABs are prospective bacteriocin producers (Nikupaavola et al., 1999) and probiotics (Rinkinen et al.,
2003).
Bacteriocin producing LAB in food preservation
51
has led to the isolation and characterization of
several bacteriocins. The bacteriocin producing
LAB have been isolated from various sources
such as vegetables, meat and meat products, milk
and milk products etc. In some cases, an identical
bacteriocin may be produced by different subspecies
of the same species as observed for lactococcin A
(Neve et al., 1984; Holo et al., 1991). There are
also incidents where a single strain produces more
than one bacteriocin as recorded for L. lactis subsp.
cremoris 9B4 (van Belkum et al., 1991a, 1992) and
Lactobacillus plantarum LPC010 (Jimenez-Diaz et
al., 1993).
Numerous bacteriocins produced by species and
strains of LAB were identified in 1980s and 1990s.
These include lactocin and helveticin (Lactobacillus
helveticus), lactocin B and F (Lactobacillus acidophilus),
curvacins (Lactobacillus curvatus), propionicin
(Propinibacterium spp.), plataricin A (Lactobacillus
plantarum), Las 5 and diplococcin (Streptococcus
cremoris), mesenterosins and leuconosins (Leuconostoc
spp.) and pediocins (Pediococcus acidilactici and
Pediococcus pentasaceous) (Klaenhammer, 1988;
Daeschel, 1989; CAST, 1998).
Classification of bacteriocin from LAB:During recent years, a large number of novel
bacteriocins have been identified from several
different LAB. Based on their amino acid sequences,
stability to heat, size, mode of action, biological
activities, secretion mechanism and the presence of
modified amino acids, LAB bacteriocins have been
classified into three classes of which the first two
classes have further been subtyped (Klaenhammer,
1993; Nes et al., 1996).
Figure 1 Lanthionine synthesis. As shown in a, lanthionine
residues are formed when an enzymatically dehydrated serine
52
(dehydroalanine, Dha) condenses with the sulphydryl group
of a neighbouring cysteine (Cys). This forms a bridge between
the two residues, thereby creating a ring within the modified
peptide or lantibiotic. When the partners are threonine (Thr)
and cysteine, the novel residue is a `-methyllanthionine. The
resulting lanthionine and `-methyllanthionine bridges are
indicated in pink as Ala–S–Ala (alanine–S–alanine) and
Abu–S–Ala (aminobutyrate–S–alanine), respectively. Many
lantibiotics also contain dehydrated serines (Ser) and threonines
(dehydrobutyrine, Dhb). Source:- Cotter et al. (2005).
Class I- Lantibiotices (from lanthionine-containing
antibiotic) are small (< 5kDa) peptides containing
the unusual amino acids lanthionine (Lan),
_-methyllanthionine (melan), dehydroalanine, and
dehydrobutyrine.These bacteriocins are grouped in
class I. Class I is further subdivided into type A and
type B lantibiotics according to chemical structures
and antimicrobial activities (Moll et al., 1999).Type
A lantibiotics are elongated peptides with a net
positive charge that exert their activity through the
formation of pores in bacterial membranes. Type B
lantibiotics are smaller globular peptides and have
a negative or no net charge; antimicrobial activity
is related to the inhibition of specific enzymes.
They are heat stable protein. e.g. nisin, lacticin 481,
lactocin 5, Carnocin U 149 etc.
Class II- Small (< 10kDa), heatstable, nonlanthionine containing peptides are contained in
class II. The largest group of bacteriocins has been
included in this classification system.These peptides
are divided into 3 subgroups.
Class IIa includes pediocin-like peptides having
N-terminal consensus sequence –Tyr-Gly-AsnGly-Val-Xaa-Cys. This subgroup has attracted
much of the attention due to their anti-listeria
activity (Ennahar et al., 2000b).
Class IIb contains bacteriocins requiring two
different peptides for activity, e.g. lactococcin G and
lactocin F.
Class IIc contains the remaining peptides of this
class, including sec-dependent secreted bacteriocins
(Worobo et.al., 1995). e.g. divergicin A.
Class III- These bacteriocins are not well
characterized. This group contains large (> 30kDa)
heat-labile proteins that are of lesser interest to
food scientists. (Joerger and Klaenhammer, 1986;
Vaughan et al., 1992) e.g., helveticin I, caseicin 80,
lacticins A and B.
A class IVth class consisting of complex bacteriocins
that require carbohydrate or lipid moieties for
activities has also been suggested by Klaenhammer
(1993); however, bacteriocins in this class have not
been characterized adequately at the biochemical
level to the extent that the definition of this
class requires additional descriptive information
(Jimenez-Diaz et al., 1995).
Cotter et al. (2006) have proposed a new scheme of
classification for bacteriocins, which is reproduced
below in Figure 2.
Figure-2: Proposed
classification
scheme
bacteriocins. Source:- Cotter et al. (2006) .
for
Chen and Hoover (2003) have summarized
different classes of bacteriocins and their producer
strain, which are reproduced below in Table 2.
53
Table 2:Examples of bacteriocin producing
by lactic acid bacteria: BACTERIOCINS
PRODUCER
REFERENCES
CLASS I-typeA lantibiotics
Nisin
Lactococcus lactis
Hurst 1981
lactocin S
Lactobacillus sake
Mortvedt et al., 1991
Epidermin
Staphylococcus epidermidis
Allgaier et al., 1986
Gallidermin
Staphylococcus gallinarum
Kellner et al., 1988
lacticin 481
Lactococcus lactis
Piard et al., 1992
CLASS I-typeB lantibiotics
mersacidinj
Bacillus subtilis
Altena et al., 2000
cinnamycin
Streptomyces cinnamoneus
Sahl and Bierbaum 1998
ancovenin
Streptomyces spp.
duramycin
Streptomyces cinnamoneus
actagardin
Actinoplanes spp.
Pediococcus acidilactici
Henderson et al., 1992
sakacin A
Lactobacillus sake
Holck et al., 1992
sakacin P
Lactobacillus sake
Tichaczek et al., 1992
leucocin A-UAL187
Leuconostoc gelidum
Hastings et al., 1991
Mesentericin Y105
Leuconostoc mesenteroides
Hechard et al., 1992
enterocin A
Enterococcus faecium
Aymerich et al., 1996
divercin V41
Carnobacterium divergens
Metivier et al., 1998
lactococcin MMFII
Lactococcus lactis
Ferchichi et al., 2001
CLASS IIb
lactococcin G
Lactococcus lactis
Nissen-Meyer et al., 1992
lactococcin M
Lactococcus lactis
van Belkum et al., 1991
lactacin F
Lactobacillus johnsonii
Allison et al., 1994
plantaricin A
Lactobacillus plantarum
Nissen-Meyer et al., 1993a
plantaricin S
L. plantarum
Jimenez-Diaz et al., 1995
plantaricin EF
L. plantarum
Anderssen et al., 1998
plantaricin JK
L. plantarum
Anderssen et al., 1998
CLASS IIc
acidocin B
Lactobacillus acidophilus
Leer et al., 1995
carnobacteriocin A
Carnobacterium piscicola
Worobo et al., 1994
divergicin A
C. devergens
Worobo et al., 1995
enterocin P
E. faecium
Cintas et al., 1997
enterocin B
E. faecium
Nes and Holo 2000
CLASS III
helveticin J
Lactobacillus heleveticus
Joerger and Klaenhammer 1986
helveticin V-1829
L. heleveticus
Vaughan et al., 1992
CLASS IIa
pediocin PA-1/AcH
54
Bacteriocin produced by different group of
lactic acid bacteria:1 Lactococcus: - Lactococci are coccibacteria, which
form chains of variable length, They have a homofermentative metabolism and produce exclusively L
(+) lactic acid (Roissart, 1994), although Akerberg et
al (1998) reported that, D (−) lactic acid can also be
produced specially at low pH values. Furthermore,
Lactococcus lactis is sub-divided into other subspecies: lactis, cremoris and diacetylactis.(Schleifer and
Kilpper-Balz, 1987; Kim et al., 1999). Their most
important habitat is untreated milk, fermented milk
and cheeses. Lactococcus lactis subsp. lactis, either in
pure form or associated with other microorganisms,
is mesophilic strain most commonly used as a
starter culture for lactic products, thus, they fulfill
an irreplaceable role in ensuring the structure, taste,
conservation and healthfulness of these products.
(Jensen and Hammer, 1993; Salminen and Von
wright, 1993; Roissart, 1994; Boonmee et al., 2003;
Ziadi et al., 2005; Do-won et al., 2006).
They also play an important role in aroma
enhancement, the production of flavoured milk,
and in milk and cheese flavouring, and recently a
great deal of attention has been focused on their
probiotic properties (Salminen and Von wright,
1993; Boonmee et al., 2003). For these reason this
microorganism, has great commercial potential,
and that is why Lactococcus, and more especially
Lactococcus lactis, isolated in the lactic industry, is still
being studied exhaustively. The major product of
fermentation is lactic acid, a compound with a high
commercial value, with applications in the food,
cosmetic, medical and pharmaceutical industries
(Boonmee et al., 2003.
Several scientists (Mattick and Hirsch, 1947; Neve
et al., 1984; Holo et al., 1991 and Kojik et al.,
1991) have reported bacteriocinogenicity among
the different strains of the three most economically
important lactococcal species: Lactococcus lactis subsp.
lactis, Lactococcus lactis subsp. cremoris and Lactococcus
lactis subsp. lactis biovar.diacetylactis.
1.1Lactococcus lactis subsp. lactisNisin: The most extensively characterized
bacteriocin from lactic acid bacteria is produced by
several strains of Lactococcus lactis subsp. lactis. Mattick
and Hirsch coined the word ‘nisin’ to designate the
group ‘N’ inhibitory substance in 1947. Nisin, is
widely used bacteriocin, is normally ineffective
against Gram negative bacteria, yeast and moulds,
but effective against a wide range of Gram positive
bacteria including other lactic acid bacteria,
Staphylococcus aureus and Listeria monocytogenes.
Gram-positive spore formers i.e. Bacillus spp. and
Clostridium spp. are particularly sensitive to nisin
with spores being more sensitive than vegetative
cells (Ray, 1992). Hirsch et al (1951) first examined
the potential of nisin as a food preservative. In 1957,
nisin was reported to be commonly occurring in
farmhouse cheese (Chevalier et al., 1957). In that
same year, Aplin and Barrett developed commercial
preparations for use in foods (Delves-Broughton
et al., 1996). Nisin-like substances were found to
be commonplace among cheese cultures (Hurst
1967), and now it is understood (that lactococci
can produce other bacteriocins and inhibitory
substances in addition to nisin.
First elucidated by Gross and Morell in 1971, nisin
is a 34 amino, acid peptide. At least 6 different
forms have been discovered and characterized
(designated as A through E and Z), with nisin A,
the most active type. Nisin Z is a natural variant
of nisin differing from nisin A with substitution of
a histidine residue for an aspartic acid. The most
established commercially available form of nisin for
use as, a food preservative is NisaplinTM, with the
active ingredient 2.5% nisin A and the predominate
ingredients NaCI (77.5%) and nonfat dry milk (12%,
protein and 6% carbohydrate). Several companies
market antimicrobial products containing nisin. It
belongs to inhibitory substances called ‘lantibiotics’
a family of peptides containing unsaturated amino
acids, dehydroalanine and dehydrobutyrine and
thio-ether amino acids, lanthionine and -methyl
lanthionine (Gross and Morell, 1967, 1971). It is
a protein having 34 amino acids and molecular
weight of 3.5 KDa (Klaenhammer, 1988). Nisin has
five polypeptide variants, which are designated as A,
B, C, D, and E. International acceptance of nisin was
given in 1969 by the Joint Food and Agriculture
Organization/World Heatth Organizaton (FAD/
WHO) Expert Committee on Food Additives
(WHO 1969). The only other antibiotic-like
55
compound with similar approval as a preservative is
the surface-active antimycotic compound,pimaricin
(Henning et al., 1986). FAO/WHO Committee
recommended a maximum daily intake of nisin
for a 70-kg person to be 60 mg of pure nisin or
33000 Units (Hurst and Hoover 1993); however,
nisin is permitted in processed cheeses in Australia,
France, and Great Britain with no maximum limit.
In the U.S., the maximum limit is 10000 IU/g; in
Russia, the maximum limit is 8000 IU/g, while in
Argentina, Italy, and Mexico, the limit is 500 IU/g
for processed cheeses and other products (Chikinda
and Montville 2002).
LMG2081 (Nissen-Meyer et al., 1992); lactococcin
972 by Lactococcus lactis subsp. lactis (Martinez et
al., 1996); lactococcin 484 by Lactococcus lactis
subsp. lactis 484 has been reported to be effective
against members of the Lactococcus group, B. cereus,
Staphylococcus aureus and Salmonella typhi (Gupta
and Batish, 1992).
Structure of bacteriocin: - Nisin is well known
member of bacteriocin and is produced by strains
of Lactococcus lactis subsp. lactis. Its structure is
illustrated in Fig-4. (Kaletta and Entian, 1989).
The name “nisin” for this bacteriocin is derived
from the term “group N inhibitory substance”
(where group N refers to sero group N of bacteria
classified as member of the genus Lactococcus). Nisin
consists of 34 amino acids, however, it is initially
synthesized as prenisin, consisting of 23 amino acid
leader peptide and the 34- amino acid pronisin
peptide. (Nes, et al.1996).
Figure 3: Milestones in the commercial delopment of
nisin Source:- Cotter, et al. (2005).
In the USA, the FDA has affirmed a nisin preparation
as a GRAS (Generally recognized as safe) substance
for use in pasteurized cheese spreads for inhibition
of outgrowth of Clostridium botulinum. Shtenberg
and Ignatev already reported the toxicity of nisin in
1970.They suggested that the toxicity of nisin is low
and it is not used in animal or human medicine.
A bacteriocin, lactacin 481, produced by Lactococcus
lactis subsp. lactis CNRZ481 was found to be
effective against Lactococcus spp., some Lactobacillus
spp., Leuconostoc spp., and Closrtridium spp.
reported by Piard et al (1990) The production of
several lactococcins has been described in several
other Lactococcus lactis subsp. lactis which include:
Lactococcin by Lactococcus lactis subsp. lactis ADRI
85L030 (Dufour et al., 1991) has been found to
inhibit vegetative cells of Clostridium tyrobutyricum,
strains of Streptococcus thermophilus and Lactobacillus
helveticus but is rather inactive against other Gram
positive and Gram negative genara (Thuault et al.,
1991); lactococcin G by Lactococcus lactis subsp. lactis
56
Figure 4 : Nisin structure. Source:- Cotter et al. (2005).
Variants of nisin differing in 1 amino acid are
known. Certain serine and threonine residues in
the pronisin are converted to dehydroalanine and
dehydrobutyrine through dehydration. Thioether
bonds are then formed by reaction with the
sulfhydryl groups of cysteine residues in the pronisin
(lanthionine, Ala-S-Ala; `-methyllanthionine;
Ala-S –Aba; aminobutyric acid). Following these
chemical modifications in the pronisin segment of
prenisin, export and concomitant removal of the
leader sequence yield active nisin.
Normally, two nisin molecules form a dimer. The
early reports on the molecular weight of nisin as
7 kDa (approx) where, due to the formation of
dimers rather than the approximately 3.5 kDa
measured only (Joerger and Hoover, 2000), Sharma
(2002) also reported 3.5 kDa molecular weight
of nisin isolated from Lactococcus lactis subsp. lactis
CCSU1101.
Figure 5: Lacticin A1 and A2 structure.
1.2 Lactococcus lactis subsp. cremoris- The
first description of a proteinaceous inhibitor in
lactococci was from Lactococcus lactis subsp. cremoris.
The antimicrobial agent described by Whitehead
in 1933 was later on partly purified and shown
to be proteinaceous in nature. It was termed as
‘Diplococcin’to signify the diplococcal arrangement
of the producer cells. A number of lactococcins
have been described for Lactococcus lactis subsp.
cremoris. These include: lactococcin A from strain
LMG2130 (Holo et al., 1991) and strain 9B4 (Neve
et al., 1984). Later it was found that Lactococcus
lactis subsp. cremoris strain 9B4 produced two more
bacteriocins termed as lactococcin M (van Belkum
et al. 1991) and lactococcin B (van Belkum et al.
1992). Huot et al (1996) described the production
of a bacteriocin designated as Bacteriocin J46 by
Lactococcus lactis subsp. cremoris J46.
Many substances have been reported from lactococci,
which have been designated as lactococcin. These
include lactococcin I, A, B and M, lactococcin
I has been isolated from Lactococcus lactis subsp.
cremoris strain A and C. Purified lactococcin has
been reported to be heat stable (99°C, 33 min)
peptide with a molecular weight of 6,000 Da. It
is encoded by a 18.4 Kb fragment of DNA of a
60 Kb conjugative plasmid. It has been reported to
inhibit other Lactococci and some Clostridia. (Geise
et al. 1983).
Lactococcin A has been reported to be produced
by three different Lactococci, which include 1.8 Kb
region of plasmid p9 B4-6, from Lactococcus lactis
subsp. cremoris 9B4, associated with production of
another bacteriocin. It has also been cloned and
analyzed (Van Belkum, et al., 1991). Nucleotide
sequence analysis of the resulting plasmid (pMB225)
showed a ribosomal binding site followed by three
ORFs designated ORFA-1 ORFA-2 and ORFA3. The third ORF, ORFA-3 is suspected to encode
an immunity protein for lactococcin M (Van
Belkum et al. 1991).
Lactococcin B is a bacteriocin associated with a
1.2 Kb fragment present in plasmid p9B4--6 of
Lactococcus lactis subsp. cremoris 9B4 (Van Belkum et
al. 1991). The genes encoding this bacteriocin are
present on the same fragment on which the genes
for lactococcin M and lactococcin A are present.
Active bacteriocin is a 5300 Da protein.
Diplococcin is one of the earliest bacteriocin
isolated from LAB. It is protein with molecular
weight of 5.3 kDa and produced by Lactococcus lactis
subsp. cremoris in milk and M17 broth during early
stationary phase. It was partially purified by Oxford
(1944) and was found to be water-soluble and heat
stable under acidic conditions. It differs from nisin
in many of its characteristics. It does not conatain
sulphur containing amino acids, lanthionine and
-methyl lanthionine, which are the characteristics
of lantibiotics (Davey and Richardson, 1981). The
inhibitory spectrum of diplococcin from Lactococcus
lactis subsp. cremoris was restricted to lactococci only
(Davey and Pearce, 1980). Lactococcus lactis subsp.
cremoris starin 9B4-secreting lactococcins A, B and
M prevented the growth not only of other lactococci
but also of some Clostridia (Geise et al. 1983). Holo
et al. (1991) purified lactococcin A and found that
it inhibited the growth of only lactococci. Out of
over 120 strains of different Lactococci tested only
one was insensitive to lactococcin A as was the case
with all other Gram-positive bacteria. Bacteriocin
J46 has a wide spectrum of antibacterial activity
including anticlostridial activity (Gonzalaz et al.
1996).
Purified diplococcin is unstable at room temperature,
rapidly inactivated by heat and degraded by
proteolytic enzymes like chymotrypsin, trypsin
and pronase (Klaenhammer, 1988). It has a narrow
spectrum of activity against closely related Lactococcus
lactis and other strains of Lactococcus cremoris (Davey,
1981). It is encoded by 54 Mda plasmid (Davey,
1984). In addition to nisin and diplococcin,
57
lactococci have been reported to produce some
other bacteriocins. Hirsch and Grinsted (1951)
have reported that streptococci produced a variety
of bacteriocins but in comparision to nisin activity
of other bacteriocins was much lesser.
1.3 Lactococcus lactis subsp. lactis biovar.
diacetylactis-The bacteriocin described in Lactococcus
lactis subsp. lactis biovar. diacetylactis WM4 (Scherwitz
et al., 1983) has been found to be identical to the
lactococcin A produced by Lactococcus lactis subsp.
cremoris strains 9B4 and LMG2030 (Stoddard et al.,
1992). Kojic et al (1991) reported the production
of bacteriocin S50 by Lactococcus lactis subsp. lactis
biovar. diacetylactis S50. A strain of Lactococcus lactis
subsp. lactis biovar. diacetylactis UL720 isolated from
raw milk has been found to produce a bacteriocin
termed as diacetin B (Ali et al., 1995). Morgan et
al (1995) isolated Lactococcus lactis subsp. lactis biovar.
diacetylactis DPC398 from an Irish cheese factory
and observed the effect of all the three lactococcins
viz. A, B, and M in the strain DPC398.
Lactostrepsins are another group of inhibitory
substances, which have been reported to be
produced by non-nisin producing strains
of Lactococcus lactis subsp diacetylactis, Lactococcus
lactis subsp. cremoris and Streptococcus lactis subsp.
lactis (Kozak, et. al., 1978). These substances
show maximum activity in the pH range of
4.6 to 5.0, whereas at pH 7.0 the activity is
lost (Dobrzanski, 1982). Lactostrepsins are
stable at 121°C for 10 minutes and produced
in non-agitated broth cultures during early
logarithmic phase. These are inactivated by
proteolytic enzymes. Their molecular weight
exceeds 10,000 Da. These show inhibitory
action against other lactococci, group A, C
and G streptococci, Bacillus cereus, Lactobacillus
helveticus, Leuconostoc mesenteroides subsp. cremoris,
and Leuconostoc paracitrovorum.. Lactostrepsin
5 produced by Lactococcus lactis subsp. cremoris
202 disrupts the cell membrane, interferes
with uridine transport and inhibits DNA,
RNA or protein synthesis (Dabrzanski,
1982). Information on genetic determinants
responsible for production and immunity is
inconclusive.
2.2 Lactobacilli:- The bacteriocinogenicity
58
has been described for several of the obligate
homofermenters (Lactobacillus acidophilus, and
Lactobacillus helveticus), facultative heterofermenters
(Lactobacillus plantarum and Lactobacillus sake) and
for the heterofermentative Lactobacillus brevis.
2.2.1 Dairy lactobacilli:2.1.1 Lactobacillus helveticus-The bacteriocins
described from the species includes helveticin
J by the strain Lactobacillus helveticus 481 (Joerger
and Klaenhammer, 1986). Helveticin J is sensitive
to several proteolytic enzymes and heat and
shows its action against limited related Lactobacilli.
It is encoded by chromosomal determinants.
Crude protein has molecular weight of 30,000
Da but purified protein has a molecular weight
of 37,000 Da and helviticin V-1829 by the strain
Lactobacillus helveticus 1829 (Vaughan et al., 1992).
This bacteriocin has been found to be heat labile
(50°C for 30 min.), which is bactericidal against
other Lactobacilli. The partially purified preparation
is inactivated by proteinase K, trypsin, pronase, heat
and pH above 7.0. It is chromosomally encoded and
has no plasmids. (Vaughan, et al., 1992). Thompson
et al., (1996) identified a bacteriocin in the culture
supernatant of Lactobacillus helveticus CNRZ450.
2.1.2 Lactobacillus acidophilus:- Early investigations
into the antimicrobial activities of Lactobacillus
acidophilus suffered due to insufficient characterization
of the antagonistic agents, to determine whether or
not bacteriocins are responsible for the observed
inhibition (Klaenhammer, 1988). Barefoot and
Klaenhammer (1983) provided a more definitive
characterization of bacteriocins from the species
with the description of lactacin B, a bacteriocin
produced by Lactobacillus acidophilus N2. ten-Brink
et al. (1994) reported the production of acidocin
B, an atypical bacteriocin by Lactobacillus acidophilus
strain M46 isolated from human dental plaque
and Lactobacillus acidophilus TK9201 was found
to produce a bacteriocin termed as acidocin A
(Kanatani et al., 1995).
Two bacteriocins, which have been named as
lactacin F and B, are produced by Lactobacillus
acidophilus 11088 and Lactobacillus acidophilus
N2 respectively (Muriana and Klaenhammer,
1987; Barefoot and Klaenhammer, 1983). HPLC
purification of lactacin F preparations followed
by SDS-PAGE showed that activity is retained in
a 2500 Da band, however, this finding does not
correlate with amino acid composition analysis,
which indicates that the protein possesses 56
amino acids. (Muriana and Klaenhammer, 1991).
Lactacin F is sensitive to protinase K, trypsin, ficin
and subtilisin and is heat stable at 121°C for 15
minutes. It is active against Lactobacillus delbrueckii
subsp. bulgaricus, Lactobacillus helveticus Lactobacillus
acidophilus, Lactobacillus fermetum and one strain
of Lactobacillus faecalis. Genetic determinants for
lactacin F production and immunity are present on
an episome (Muriana and Klaenhammer, 1991).
Lactacin B is a 6,500 Da protein which is sensitive
to proteinase K and heat stable at 100°C for 3
minutes at pH 5, (Barefoot and Klaenhammer,
1983). It is inhibitory to Lactobacillus delbrueckii
subsp. lactis, Lactobacillus delbrueckii subsp. bulgaricus
and Lactobacillus helveticus. Lactacin B appears to be
encoded by chromosomal genes, as the producing
bacteria possess no plasmids. (Barefoot and
Klaenhammer, 1983).
2.2.2. Non-dairy lactobacilli:2.2.1 Lactobacillus plantarum:- Daeschel et al
(1990) reported the production of plantaricin
A by Lactobacillus plantarum C-11 isolated from
cucumber fermentations. Plantaricin A is produced
by Lactobacillus plantarum C-11 and is inhibitory to
other lactic acid bacteria. Its molecular weight has
been estimated to be more than 6000 Da. It is heat
stable at 100°C for 30 minutes and shows activity in
the pH range of 4 to 6.5. Investigations indicate that
it may not be encoded by plasmid. (Daeschel et al.,
1990). A protein with narrow spectrum of activity,
called plantaricin B is produced by Lactobacillus
plantarum NCDO 1193, which shows inhibitory
action against other strains of Lactobacillus plantarum,
Lactobacillus mesenteroides and Pediococcusҏ damnosus
(West and Waner, 1988). Plantaricin B is a protein
complexed with carbohydrate and lipid moieties,
as inhibitory activity is reduced by lipase and _
amylases. It is interesting to note that Lactobacillus
plantarum LPC010 isolated from a green olive
fermentation elaborated into the growth medium
by two bacteriocins designated as plantaricins S
and T (Jimenez-Diaz et al., 1993). A bacteriocin,
plantaricin KW30, producing strain of Lactobacillus
plantarum has recently been isolated from fermented
maize (Kelly et al., 1996).
Bacteriocinogenic Lactobacillus plantarum strains
from dairy and meat products have also been
reported. A Lactobacillus plantarum strain LTF154
isolated from a fermented sausage produced a
bacteriocin designated as plantacin 154 (Kanatani
and Oshimura, 1994). Rekhif et al. (1994) isolated
a bacteriocin producing Lactobacillus plantarum
strain LC74 from goat raw milk and named the
bacteriocin as plantaricin LC74. Recently Ennahar
et al. (1996) reported the production of a bacteriocin
identical to pediocin AcH by a strain of Lactobacillus
plantarum WHE92 isolated from a soft cheese.
2.2.2 Lactobacillus curvatus:Curvacin A
produced by Lactobacillus curvatus LTH 1174, an
isolate from meat, produces this bacteriocin which
shows inhibitory action against other Lactobacilli,
Leuconostoc, Cornybacteria, Listeria monocytogenes,
as well as a weak action against Micrococci and
Staphylococci. (Tichaczek et al. 1992). Like sakacin P,
curvacin A is destroyed by proteinase K and trypsin
but stable when treated with pepsin or heat (100°C,
3 min). Its molecular weight has been estimated to
be 3,000 to 5,000 Da.
2.2.3 Lactobacillus sake:– Bacteriocins produced
by the strains of Lactobacillus sake isolated from
meat and fermented sausages include: sakacin A by
Lactobacillus sake 706 (Schillinger and Lucke, 1989),
lactocin S by Lactobacillus sake L45 (Mortvedt and
Nes, 1990, 1991; Skaugen et al., 1994).
Sakacins are the bacteriocins produced by
Lactobacillus sake and include sakacin, A, M, S and P.
Lactobacillus sake is responsible for fermentation in
sausages. Sakacin A and M inhibit other Lactobacilli
as well as Listeria monocytogenes (Schillinger and
Lucke, 1989; Schillinger, et.al., 1991). Lactocin S
inhibits strains of lactic acid bacteria (Mortvedt and
Nes, 1990). Sakacin A is produced by Lactobacillus
sake 706 and is heat stable (100°C for 20 min.)
produced during the mid and late logarithmic
growth phase in liquid medium and is associated
with a 27.7 Kb plasmid. (Schillinger and Lucke,
1989).
Sakacin M was produced from Lactobacillus sakeҏ
̓isolated from Spanish dry fermented sausages.
59
(Sobrino et al. 1991). It is produced maximally
in a synthetic medium supplemented with 1.5%
tryptone during growth at 32°C. Molecular weight
of the bacteriocin has been estimated to be 4640
Da. Inhibitory activity of a partially purified
compound is diminised by trypsin, pepsin, papain
and protease XIV and II. Crude and partially
purified compounds are heat stable at 80°C for
60 minutes and 150°C for 9 minutes. Bacteriocin
shows inhibitory action against Lactobacilli,
Leuconostoc, Carbonobacteria, Listeria monocytogenes
and Staphylococcus aureus (Sobrino et al. 1991).
Another bacteriocin, sakacin P, was produced by
Lactobacillus sake LTH 673, isolated from meat and
it was found to inhibit Lactobacilli and spoilage
organisms like Leuconostoc, Cornybacteria, Enterococci,
Brochothrix thermosphacta and Listeria sp. (Tichaczek
et al. 1992). Bacteriocin is sensitive to proteinase
K and trypsin but insensitive to pepsin and heat at
100°C for 7 minutes. It is a protein of molecular
weight 3,000 to 5,000 Da with 36 to 41 amino
acid residues.
Lactocin S is a heat stable protein active against
Pediococcus, Leuconostoc, and Lactobacilli. It has been
isolated from Lactobacillus sake 245. (Mortvedt and
Nes, 1990). Molecular weight of crude Lactocin S
has been reported to be 30,000 Da, however, partially
purified active proteins have molecular weight less
than. 13,700 Da. Production is associated with an
unstable 50 Kb plasmid. (Mortvedt et al., 1991).
Mode of action studies indicated that lactosin S acts
bactericidally in a pH dependent fashion (Twomey
et al., 2002).
2.2.4 Lactobacillus brevis:– Production of brevicin
286 has been recently reported in Lactobacillus brevis
VB286 that was originally isolated from vaccum
packaged meat (Conventry et al., 1996).
Lactobacillus brevis produced an antibacterial
substance named brevicin 37, which was inhibitory
to Pediococcus sp., Leuconostoc sp., Lactobacillus sp. and
Nocardia carolina. The protein is stable at pH range
of 1 to 11. It is also stable to heat at 121ºC for 1 h
and is retained on a 10,000 molecular weight cut
off membrane. (Rammelsberg and Radler, 1990).
2.2.5 Lactobacillus casei- Rammelsberg and
Radler (1990) isolated Lactobacillus caseiҏҏ B 80 from
60
plants and fermenting materials. Lactobacillus casei
B 80 produced a heat sensitive protein with a
narrow spectrum of activity against other strains
of Lactobacillus casei. (Rammelsberg et al., 1990). Its
molecular weight has been reported to be 40,000
to 42,000 Da.
Other lactobacilli:- Lactacin F producing
Lactobacillus acidophilus 11088 (Muriana and
Klaenhammer, 1987) has been renamed as
Lactobacillus johnsonii as cited by Klaenhammer
(1993).
2.3 Leuconostoc:- The first evidence for bacteriocin
production in Leuconostoc spp. was provided by
Harding and Shaw in 1990. They reported the
production of a heat stable protein by a strain of
Leuconostoc gelidum that was active against other
lactic acid bacteria and three strains of Listeria
monocytogenes. In recent years, a number of
bacteriocin producing strains of Leuconostoc species
have been isolated from various sources such as
milk and meat products.
Hastings and Stiles (1991) reported the production
of a bacteriocin-designated leucocin A-UAL187
by Leuconostoc gelidum UAL187 isolated from
meat packed under elevated (30%) carbondioxide.
Leuconostoc paramesenteroides OX isolated by Lewus
et al (1991) from retail lamb was found to produce
a bacteriocin named as leuconocin S (Lewus et al.,
1992).
Bacteriocins, carnosin 44A, carnocin LA54A and
leucocin B-Talla, produced by Leuconostoc carnosum
LA44A from vaccum packagedVienna-type-sausage
(van Laack et al. 1992), Leuconostoc carnosum LA54A
from meat (Keppler et al. 1994) and Leuconostoc
carnosum Talla isolated from vacuum packaged
processed meat (Felix et al. 1994), repectively, have
been described in the strains of Leuconostoc carnosum.
Yang and Ray (1994a) observed the predominance
of Leuconostoc carnosum and Leuconostoc mesenteroides
in the spoiled low heat processed vacuum packaged
meat products. The notable feature of many of
these Leuconostoc isolates is their ability to produce
bacteriocins.
Bacteriocinogenic strains of Leuconostoc spp.have also
been isolated from milk and milk products. Strains
of Leuconostoc mesenteroides subsp. mesenteroides,
Y105 from goat milk and FR52 from raw milk
were found to produce bacteriocins, mesentericin
Y105 (Hechard et al., 1992) and mesentericin 52
(Mathiew et al. 1993), respectively. Dextranicin
J24 was a bacteriocin produced by an isolate of
Leuconostoc mesenteroides subsp. dextranicum J24 from
French soft cheese (Sudirman et al., 1994). Malik
et al (1994a) reported the detection and activity of
a novel bacteriocin, leucocidin R1, produced by
Leuconostoc paramesenteroides NM14 isolated from
an aged cream sample.
2.4 Pediococci:2.4.1 Pediococcus pentosaceus:- The bacteriocin
produced by Pediococcus pentosaceus FBB61 from
cucumber fermentations was designated as
pediocin A (Daeschel and Klaenhammer, 1985).
Hoover et al, (1988) observed bacteriocinogenic
activity in Pediococcus pentosaceus MC03 isolated
from pepperoni, a fermented sausage. Bacteriocin
production in Pediococcus pentosaceus strain N5p
from wine has been reported and the bacteriocin
was named as pediocin N5p (Strasser-de-Saad and
Manca-de-Nadra, 1993).
2.4.2 Pediococcus acidilactici:-The most extensively
characterized bacteriocins, pediocin AcH and
pediocin PA-1, after nisin have been produced
by strains of Pediococcus acidilactici. Gonzalez and
Kunka, (1987) reported pediocin PA-1 production
by Pediococcus acidilactici PAC1.0. Pediocin AcH
producing Pediococcus acidilactici H was isolated
by Bhunia et al, (1987) from fermented sausage.
Hoover et al, (1988) observed the production of
unnamed bacteriocins by Pediococcus acidilactici PO2
as pediocin PO2. Schved et al (1993) reported
the isolation of Pediococcus acidilactici SJ1 from a
naturally fermented meat product and designated
its bacteriocin as pediocin SJ1 while pediocin L50
producing Pediococcus acidilactici L50 was obtained
from Spanish dry fermented sausage (Cintas et al.,
1995).
2.4.3 Characteristics of bacteriocins:Bacteriocins of LAB have been characterized with
respect to their (i) sensitivity to various proteolytic
and non-proteolytic enzymes (ii) stability to various
heat treatments (iii) pH stability (iv) mode of action
and (v) molecular weight etc. In most of these
characterization studies, either crude or partially
purified bacteriocin preparations have been used.
The fact that bacteriocins are proteins renders them
sensitive to at least one of the proteolytic enzymes.
Apart from protein moiety, some bacteriocins have
been found to contain an active lipid or carbohydrate
moiety which is also required for antibacterial
activity as revealed by loss of bacteriocin activity
upon treatment with lipases or amylases (Lewus et
al., 1992; van Laack et al., 1992; Jimenez-Diaz et
al., 1993; Schved et al., 1993; Keppler et al., 1994)
The term bacteriocin has been restricted to those
antibacterial proteins that exhibit a bactericidal
mode of action (Tagg et al. 1976). Although, a vast
majority of bacteriocins of LAB exert a bactericidal
mode of action, but a few have been found to
be bacteriostatic rather than bactericidal to the
sensitive cells (Lewus et al., 1992; Thompson et
al., 1996). Characteristic of bacteriocin and other
conventional antibiotics shown In Table 3.
Table 3:Characteristic aspects of bacteriocins
and other conventional antibiotics.
S.No.
1
2
3
4
5
6
Characteristics
Bacteriocins
Other antibiotics
Foods
Application
Ribosomally
Clinical
Synthesis
Limited spectrum
Secondary metabolism
Activity
Present
Wide spectrum
Presence of immune cells in the host The most through the channel
Absent
Mode of action
formation in the cell
Specific target
Toxicity/other effects in eukaryotic cells cytoplasmic membrane
Present
Absent
Most of the bacteriocins of LAB characterized
to date are small (< 10kDa) heat stable peptides,
however, the occurrence of large (> 30kDa) heat
labile proteins has also been reported (Joerger
and Klaenhammer, 1986; Vaughan et al., 1992).
Bacteriocins are extremely heat stable at low pH
(Hurst, 1981; Hastings et al., 1991; Felix et al., 1994)
becoming more sensitive to heat upon purification
(Davey, 1981; Hastings et al., 1991). Bacteriocins of
LAB, in general, are active over a wide pH range
with optimum being on acidic side.
Currently, over 20 bacteriocins of lactic acid
bacteria have been sequenced. Most contain less
than 60 amino acids, and all are devoid of lipid and
carbohydrate moieties. The molecules are cationic,
61
hydrophobic and have isoelectric points ranging
from 8.6 to 10.4. Their net positive charge varies
with pH, and this is important for both bactericidal
efficiency and purification (Ray et al., 2001). They
also reported that due to the hydrophobic nature of
these molecules they have a tendency to aggregate,
especially when stored at high concentration or
for a long time. Antibacterial potency is greater at
lower pH, destroyed at a pH above 10, relatively
heat stable but partially destroyed by heating above
100ƕC, and not affected after treatment with many
organic and inorganic chemicals. However, some
anions interfere with activity in a concentration
dependent manner, and many proteolytic enzymes
can hydrolyze the molecules resulting in loss of
antibacterial properties.
Bactericidal potency remains stable during storage
in dried or liquid forms at frozen and refrigeration
temperatures. However, it slowly decreases during
storage at room temperature in the presence of
air that can oxidize methionine residues to the
inactive methionine sulfoxide form (Ray 1992;
Klaenhammer 1993; Jack et al., 1995; Yang et al.,
1992; Ennahar et al., 2000b). Characteristics of
some common bacteriocins produced from LAB
are summarized in Table 4.
Table 4: Characteristics of some bacteriocins
produced by LAB.
Bacteriocin
Produced by
Number of
amino acids
Moecularlar Weight
(kDa)
Isoelectric
points
References
Klaenhammer et al.,
(1988)
Van Belkum et al.,
(1991a)
Van Belkum et al.,
(1991a)
Schillinger and Luke
(1989)
Nisin A
Lactococcus lactis subsp. lactis
34
3.5
10.1
Lactococcin A
Lactococcus lactis subsp. cremoris
55
5.8
8.6
Lactococcin B
Lactococcus lactis subsp. cremoris
47
5.3
9.1
Sakacin A
Lactobacillus sake 706
41
4.3
10.0
Sakacin P
Lactobacillus sake LTH 673
43
4.4
8.8
Tichaczek et al., (1992)
Lactocin S
Lactobacillus sake 245
37
3.9
N.A
Mortvedt and Nes,
(1990)
Pediocin ACH
Pediococcus acidilacticin H
44
4.6
9.6
Bhunia et al., (1987)
Leucocin A
Leuconostoc gelidium
37
3.9
9.5
Ahn and Stiles, (1990)
Enterocin A
Enterococcus faecium
47
4.8
9.6
Dutta et al., (1999)
The bacteriocins of lactic acid bacteria following
translation usually undergo very little structural
alteration, and thus, are regarded as ribosomally
translated peptides.At the translation level,a molecule
designated as prebacteriocin (such as prenisin and
prepediocin) contains a leader peptide segment at
62
the N-terminus and a propeptide segment at the
C-terminus. The molecules are transported out of
the cytoplasm through the membrane by specific
ABC (ATP binding cassette) transporters with the
expense of energy. (Figure 6).
Figure 6: Schematic depiction of transport of bacteriocins
and signaling pathway leading to bacteriocin expression. IF,
induction factor; HK; histidine kinase;P, phosphate; RR,
response regulator; ABC, Trans, ABC transporter system; (Nes
et al. 1996).
During transport endopeptidase activity of the
ABC transporter removes the leader peptide before
the propeptide is released into the environment.
(Jack et al., 1995; Liu and Hansen, 1990; Ennahar et
al., 2000). Ennahar et al., (2000) reported that the
fate of excised leader peptide is not known but it
has been speculated that they might act as signaling
molecules for the production of bacteriocins. It was
assumed earlier that leader peptides, besides helping
prebacteriocin molecules to interact with ABC
transporters, also inhibit activity of the attached
probacteriocin. Recent studies have shown that this
may be true for nisin, but is not the case for pediocin
PA-1/AcH, as prepediocin as well as pediocin PA1/
AcH with a fused protein at the N-terminus are
biologically active. (De-Vos et al., 1995). Following
release of the matured bacteriocin, it either remains
free or may bind via electrostatic attraction to the
surface of the producer cells.At around pH 6, a large
fraction of the molecules remain in the absorbed
state while at pH 2 or below most are released into
the environment (Yang et al., 1992).
The probacteriocins part of some bacteriocins
may undergo nonenzymatic as well as enzymatic
chemical modifications. Pre-nisin molecules, while
in the cytoplasm, undergo enzymatic dehydration
of serine and threonine residues converting-
Figure 7: The conversion of serine residues to threonine
residues in cytoplasm
They then form thioether linkages to cysteine
creating lanthionine (Lan; -Ala-S Ala) or `-methyl
lanthionine (MeLan; - Abu-S-Ala). Bacteriocins
with thioether rings, i.e., containing lanthionine
and/or methyl lanthionine, are grouped as
lantibiotics. In lantibiotics, these changes occur in the
probacteriocin sequence while the prebacteriocin
still resides in the cytoplasm. Lantibiotics can have
different numbers of thioether rings e.g., nisin has
five rings while lacticin 481 has three rings. The
thioether rings play a crucial role in the antibacterial
properties of a lantibiotic; however, the presence of
lanthionine does not necessarily make a bacteriocin
more potent than bacteriocin that lack lanthionine.
(Ray et al., 2001).
2.4.4 Mode of action of bacteriocin: - Due
to the great variety of their chemical structures,
bacteriocins affect different essential functions of
the living cell (transcription, translation, replication,
and cell wall biosynthesis), but most of them act by
forming membrane channels or pores that destroy
the energy potential of sensitive cells. The different
modes of action of various types of bacteriocin
produced by gram-positive bacteria have been
reviewed by several authors (Sahl and Brandis, 1982;
Abee, 1995; Ennahar et al., 2000).
“Nisin” (a compound belonging to group Ia,
according to Klaenhammer’s classification) is
the bacteriocin whose mode of action has been
studied the best. This cationic lantibiotic associates
electrostatically with the negatively charged
membrane phospholipids (Abee et al., 1995;
Driessen et al., 1995), which favors subsequent
interaction of bacteriocin’s hydrophobic residues
with the target cytoplasmic membrane. The
interaction between the hydrophobic part of nisin
and the bacterial target membrane generates unspecific ionic channels whose formation is aided by
the presence of high transmembrane potentials, and
by the presence of anionic and absence of cationic
lipids (Hancock, 1997). Pore formation, on the
other hand, decreases in the presence of divalent
cations (Mg2+ or Ca2+) because they neutralize
the negative charges of the phospholipids, reducing
the fluidity of the membrane. Nisin generated
membrane pores allow the passive efflux of ions (K
+ and Mg2+), amino acids (glutamic acid, lysin),
and A TP, but not of larger cytoplasmic proteins,
yielding membrane potential and proton-motivforce dissipation and subsequent cell death (Boman
et al., 1994).
Figure 8: Mode of action of lactic acid bacteria
bacteriocins. Lactic acid bacteria (LAB) bacteriocins can
be grouped on the basis of structure, but also on the basis of
mode of action. Some members of the class I (or lantibiotic)
bacteriocins, such as nisin, have been shown to have a dual
mode of action. They can bind to lipid II, the main transporter
of peptidoglycan subunits from the cytoplasm to the cell wall,
and therefore prevent correct cell wall synthesis, leading to cell
death. Furthermore, they can use lipid II as a docking molecule
to initiate a process of membrane insertion and pore formation
that leads to rapid cell death. A two-peptide lantibiotic, such
as lacticin 3147, can have these dual activities distributed
across two peptides, whereas mersacidin has only the lipid-IIbinding activity, but does not form pores. In general, the class
II peptides have an amphiphilic helical structure, which allows
them to insert into the membrane of the target cell, leading
to depolarisation and death. Large bacteriolytic proteins (here
called bacteriolysins, formerly class III bacteriocins), such as
lysostaphin, can functiondirectly on the cell wall of Gram-
63
positive targets, leading to death and lysis of the target cell.
Source: - Cotter et al. (2005).
2.4.5 Antibacterial properties: - Many
bacteriocin producing strains belonging to several
genera and species of lactic acid bacteria have
been isolated. Their bacteriocins are bactericidal to
sensitive cells and death occurs very rapidly at a low
concentration. Ray (1992) reported a range of Gram
positive bacteria sensitive to a bacteriocin, while the
producer strain is immune to its own bacteriocin
and often is sensitive to other bacteriocins.
Most of the class I bacteriocins have a fairly broad
inhibitory spectrum. They not only inhibit closely
related bacteria, such as species from the genera
Enterococcus, Lactobacillus, Lactococcus, Leuconostoc,
Pediococcus, and Streptococcus, but also inhibit many
less closely related Gram-positive bacteria, such as
Listeria monocytogenes, Staphylococcus aureus, Bacillus
cereus, and Clostridium botulinurn. Several bacteriocins
in this class, such as nisin and thermophilin 13,
prevent outgrowth of spores of Bacillus cereus
and Clostridium botulinum. Interestingly, acidocin
J1132 has a very narrow inhibitory spectrum and
sensitive strains are limited to members of the genus
Lactobacillus (Table 5), while at the other extreme,
plantaricin LP84 (produced by Lactobacillus plantarum
NCIM 2084) has demonstrated antagonism against
E. coli (Suma et al., 1998).
Compared to class I bacteriocins, most class IIa
bacteriocins have comparatively narrow activity
spectra and only inhibit closely related Grampositive bacteria. In general, members of the genera
Enterococcus, Lactobacillus, Pediococcus are sensitive to
class lIa bacteriocins, and members of the genus
Lactococcus are resistant (Table 5). For example,
Eijsink and others (1998) found that pediocin
PA-I was active against different species of Enterococcus, Lactobacillus, and Pediococcus; however, only
1 out of 11 Lactococcus strains tested (Lactococcus
lactis LMG 2070) was sensitive to the bacteriocin.
Some class lIa bacteriocins, such as pediocin PA-l,
have fairly broad inhibitory spectra and can inhibit some less closely related Gram-positive bacteria,
such as Staphylococcus aureus and vegetative cells
of Closlridium spp. and Bacillus spp. Some class lIa
bacteriocins, such as mundticin from Enterococcus
mundtii, even prevent the outgrowth of spores of
64
Clostridium botulinum (Table 5).
As evident in Table 5, class IIa bacteriocins are
generally active against Listeria. Eijsink et al (1998)
found that 9 strains of Listeria tested, including
Listeria monoeytogenes, Listeria innocua and Listeria
ivanovii were very sensitive to 4 class lIa bacteriocins
(pediocin PA-1, enterocin A, sakacin P, and
curvacin A). Moreover, the extent of sensitivity
varied from strain to strain.The minimal inhibitory
concentrations against Listeria monocytogenes for
the above 4 bacteriocins varied from 0.1 to 8 mg/
ml, however, some Listeria strains, such as Listeria
monocytogenes V7 and Listeria innocua LB 1, have
been found to be resistant to class lIa bacteriocins
(enterocin A, mesentericin Y I05, divercin V41, and
pediocin AcH) (Ennahar et al., 2000).
It might seem that bacteriocins with broader
activity spectra would always be preferable for use in
food preservation, but under certain circumstances
bacteriocins with narrower inhibitory spectra
may prove more desirable. For example, sakacin P,
which has limited activity against LAB but nearly
as effective as pediocin PA-1 against Listeria, might
find application in LAB fermentation products that
are prone to contamination by Listeria monocytogenes
(Eijsink et al., 1998).
Table 5: Activity spectra of some Class I and
Class IIa bacteriocins
(Chen, H. and Hoover, D.G. (2003).
Bacteriocins
Strain
Class lIa
Lactobacillus acidophilus TK9201
Acidocin A
Lactobacillus sake MI401
Activity spectra
References
Active against different species of Enterococcus (1/5), Lactobacillus Kanatani et al 1995
(13/32), Pediococcus (2/7), Streptococcus (8/13), and L. monocytogenes
(5/5). Not active against Bacillus subtilis (0/6) and S. aureus (0/2).
Active against different species of Enterococcus (2/2), Lactobacillus
Nissen-Meyer et al 1993
(11/25), Lactococcus (5/15), Leuconostoc
(4n), Pediococcus (215), and L. monocytogenes (9/10).
Not active against Carnobacterium (0/1), Streptococcus
Bavaricin A
Lactobacillus curvatus LTH1174
Curvacin A
Carnobacterium divergens V41
Divercin V41
Enterococcus faecium CTC492
Lactococcus lactis MMFII
Lactococcin MMFII
Leuconostoc mesenteroides Y105
Mesentericin Y105
Enterococcus mundtii ATO6
Mundticin
Pediococcus acidilactici PAC 1.0
Pediocin PA-1
Piscicocin V1 b
Eijsink et al 1998
Active against different species of Carnobacterium (3/3), Enterococcus
(1/2), Lactobacillus (10/23), Lactococcus (1/12), Pediococcus (5/8), L. Guyonnet et al 2000
monocytogenes (7n ), L. innocua (1/1), and L. ivanovii (1/1). Not active against Leuconostoc (0/3) and Clostridium spp. (0/12).
(2/5), Pediococcus (2/2), L.monocytogenes (1/1). L. innocua (1/1), Aymerich et al 1996
and L. ivanovi (1/1). Not active against Lactococcus (0/1) and Leuconostoc (0/3).
(2/2), Pediococcus (2/2), L.monocytogenes (4/4), and L. innocua (2/2). Ferchichi et al 2001
Enterocin A
Piscicocin V1a
(0/2), Brochothrix thermosphacta (0/1), Bacillus spp. (On), and Staphylococcus spp. (0/5).
Carnobacterium piscicolaV1
Carnobacterium piscicola V1
(2/2) , Lactococcus (2/6), and L. lanovi (1/1),
Active against different species of Enterococcus (3/4), L.actobacillus Guyonnet et al 2000
(1/5), Leuconostoc 2/3, ediococcus (2/2).L. monocytogenes (1 ),L. innocua (1/1), and L. ivanovi(1/1). Not active against Lactococcus (0/1)
Active against different species of Carnobacterium (1/1), Enterococcus (2/2), Lactobacillus (2/2), Leuconostoc (2/2), Pediococcus (2/2), L. Bennik et al 1998
monocytogenes (1/1), and L. innocua (1/1). Prevents the outgrowth of
spores and vegetative cells of C. botulinum.
Active against different species of Carnobacterium (3/3), Enterococcus (2/3), Lactobacillus (23/31), Lactococcus (1/14), Leuconostoc (3/4), Eijsink et al 1998
Pediococcus (8/11), L. monocytogenes (12/12), L. innocua (2/2). L. ivanovii (1/1), Staphylococcus spp. (2/6), B. cereus (1/1), and Clostridium
spp. (4/17).
Active against different species of Carnobacterium (2/2), Enterococ- Bhugaloo-Vial et al 1996
cus (1/1), Lactobacillus (3/3), Leuconostoc (1/1), Pediococcus (1/1), L.
monocytogenes (1/1), and L. innocua (1/1). Not active against Lactococcus (0/1), B. cereus (0/1), Clostridium spp. (0/3), and S. aureus
(0/1).
Bhugaloo-Vial et al 1996
Active against different species of Carnobacterium (2/2), Enterococcus (1/1), Lactobacillus (3/3), Leuconostoc (1/1), Pediococcus (1/1), L.
monocytogenes (1/1), and L. innocua (1/1). Not active against Lactococcus (0/1), B. cereus (0/1), Clostridium spp. (0/3), and S. aureus (0/1).
Jack et al 1995
Active against different species of Carnobacterium (1/1), Enterococcus (2/2), Lactobacillus (2/3), Leuconostoc (2/3), Pediococcus
(1/2), Streptococcus (2/2), L. monocytogenes (2/2), L. grayi (1/1),
L. ivanovii (1/1), L. seeligeri (1/1), and B. thermosphacta (1/1).
Aymerich et al 1996; Guyonnet et al 2000
Not active against Bacillus spp. (0/5), Clostridium spp. (0/2), Lactococcus (0/3), Listeria denitrificans (0/1), and Staphylococcus spp.
(0/3).
Piscicolin 126
Carnobacterium piscicola JG126
Sakacin A
Lactobacillus sake LB706
Active against different species of Enterococcus (7/8), Lactobacillus Aymerich et al 1996;
(317), Pediococcus (1/4), L. monocytogenes (5/5), L. innocua (3/3), and
L. ivanovi (1/1). Not active against Lactococcus (0/1) and Leuconostoc
(0/3).
Lactobacillus sake LB674
Active against different species of Enterococcus (7/8), Lactobacillus Guyonnet et al 2000
(317), Pediococcus (2/4), L. monocytogenes (5/5), L innocua (3/3), and
L. ivanovi (1/1). Not active against Lactococcus (0/1) and Leuconostoc
(0/3).
Sakacin P
65
Several other characteristics of bacteriocins and
bacteriocin producing lactic acid bacteria have
been noted (Ray 1992; Ennahar 2000; Ray and
Miller, 2001). A strain can sometimes produce
more than one type of bacteriocin (e.g. lactoccin A,
B and M by Lactococcus lactis subsp. cremoris). Strains
of the same species generally produce the same
bacteriocin (e.g. pediocin PA-1/AcH by different
Pediococcus acidilactici strains), however, strains of the
same species can also produce different bacteriocins
(e.g. Sakacin A and Sakacin P produced by two
strains of Lactobacillus sake), and the strains from
different species and different genera can produce
the same bacteriocin (e.g., pediocin PA-1/AcH
produced by Pediococcus acidilactici, Pediococcus
pentosaceus, Pediococcus parvulus, and Lactobacillus
plantarum strains). Strains from different subspecies
of the same species can produce different
bacteriocins (e.g. nisin A and lactocin 481 produced
by different strains of Lactococcus lactis subsp lactis)
different species in a genus can produce different
bacteriocins (e.g. Enterococcin EFS2 and enterocin
900 produced by strains of Enterococcus faecalis and
Enterococcus faecium, respectively). Natural variants of
the same bacteriocin can be produced by different
strains of the same species (e.g. nisin A and nisin
Z by Lactococcus lactis subsp lactis, strains ATCC
11454 and ATCC 7962, respectively) and also by
different species (e.g. leucocin A and mesenterocin
by Leuconostoc gelidum and Leuconostoc mesenteraides,
respectively). These generalizations are drawn
based on analysis of the amino acid sequences of
numerous bacteriocins.
2.4.6 Production of bacteriocins:- One
of the most important steps in the study of
bacteriocins is their production. The composition
of culture medium and cultural conditions such
as temperature, pH and time of incubation have
profound effect on the production of bacteriocins.
In general, conditions that provide high cell density
favour high bacteriocin concentration.
The culture media generally employed for the
growth of lactic acid bacteria such as MRS, APT,
TGE, M17G, ELB etc. have also been found to
support good bacteriocin production. Although,
bacteriocin production occurs over a wide
temperature range, it is greater at the optimum
66
temperature for the growth of the producer.
The production of bacteriocins by lactic acid
bacteria is strongly influenced by the pH of the
culture medium. The regulation of pH at a certain
value during the course of fermentation has been
found to have favourable (Hurst, 1981; Piard et
al., 1990) and detrimental (Biswas et al., 1991;
Coventry et al., 1996) effects on the final yield of
bacteriocins of lactic acid bacteria.
The maximum production of bacteriocins occurs
at different phases in the cell growth cycle. Most of
the bacteriocins of lactic acid bacteria are secreted
during the logarithmic growth phase with a slight
decline in the activity of some of them during the
stationary phase of the producer culture. However,
some bacteriocins for e.g. nisin (Hurst, 1981),
pediocin SJ-1 (Schved et al., 1993) are secreted
as secondary metabolites. The termination of
the incubation at appropriate time is essential to
prevent the loss of bacteriocin activity.
2.4.6.1 Growth medium: - Commonly used
media for the production of bacteriocins by lactic
acid bacteria include MRS (Ten Brink et al., 1994;
Coventry et al., 1996; Holo et al., 2001;Vaughan et
al., 2001;Yanagida et al., 2005), TGE (Biswas et al.,
1991;Yang and Ray, 1994),APT (Lewus et al., 1992),
GM17 (Parente et al. 1997), M17(Achemchem
et al., 2005), ELB (Geis et al., 1983; Piard et al.,
1990), BHI (Achemchem et al., 2005) etc. with or
without modifications. Although, a large number of
bacteriocins have been found to be identified and
several media have been used for the production of
bacteriocins, very few studies are available on the
comparision of bacteriocin production in different
media.
Geis et al. (1983) compared various media including
ELB, GM17, BHI, a synthetic medium and milk for
their ability to support bacteriocin production by
various lactococcal strains. All the strains produced
antibiotic activities in milk. Highest bacteriocin
activities were found in unbuffered ELB followed
by BHI, buffered M17 and synthetic medium (Geis
et al., 1983).
Lactococcus lactis subsp. lactis CNRZ481 produced
maximum bacteriocin (12800 AU/ml) in ELB
buffered with sodium `–glycerophosphate. The
observed titre was double than the value recorded
when the culture was grown in M17 or unbuffered
ELB (Piard et al., 1990).
Parente et al. (1997) formulated three media
(Tryptone-Yeast Extract-Tween) TYT10, TYT11
and TYT30 and compared with seven different
media [ELB, M17, M17 dialysate, Tryptose
phosphate (TP), tryptone yeast extract broth (TYB),
yeast glucose lemco (YGL) broth and MRS] for the
growth of and bacteriocin production by Lactococcus
lactis subsp. lactis DPC3286 and Lactococcus lactis subsp.
cremoris LMG2130. Good growth and bacteriocin
production were obtained for both in the TYT, M17
and MRS media. Bacteriocin production was very
poor in YGL. It was also observed that Lactococcus
lactis subsp. cremotis LMG2130 could not grow
or produce bacteriocins in M17 dialysate and TP
media (Parente et al, 1997). Although the cell mass
was greater in MRS broth, 15% less pediocin AcH
production by P. acidilactici LB42-923 produced
higher pediocin AcH titres in TGE broth than in
buffered TGE broth (Yang and Ray, 1994).
In contrast to pediocin AcH, higher levels of nisin,
sakacin A and leuconocin Lm1 were observed in
TGE buffer broth than inTGE (Yang and Ray, 1994).
Earlier Hechard et al (1992) observed consistently
higher levels of (x16) mesentericin Y105 in MRS
broth than in a semi-defined medium.
2.4.6.2 Effect of pH on bacteriocins
production: 2.4.6.2.1 Lactobacilli bacteriocins:- Barefoot
and Klaenhammer (1984) reported maximum
lacticin B production when Lactobacillus acidophilus
N2 was grown in MRS broth regulated at pH 6.0.
In contrast, lacticin F was produced maximally in
MRS broth held at a constant pH of 7.0 rather than
7.5, contrast, lacticin F was produced maximally in
MRS broth held at a constant pH of 7.0 rather than
7.5, 6.0 or 5.0 (Muriana and Klaenhammer, 1987).
Production of heveticin J and helviticin V-1829
was observed to be greatest in anaerobic MRS
cultures maintained at a pH 5.5 than at other pH
values tested in the range of 5.0 to 7.0 (Joerger and
Klaenhammer, 1986;Vaughan et al., 1992).Vaughan
et al. (1992) also reported a two-fold increase in
helveticin V-1829 when MRS broth was held at a
pH 5.0 than in pH-unregulated cultures.
Ten-Brink et al. (1994) observed that growth
of Lactobacillus acidophilus M46 in five fold
concentrated MRS broth held at a constant pH
5.5 resulted in eight fold increase in acidocin B
activity than that obtained after growth in normal
MRS broth without pH control. Regulation of
MRS broth at pH 5.0 resulted in maximum yield
of acidocin A produced by Lactobacillus acidophilus
TK9201 (Kanatani et al., 1995).
Maximum production of plantaricin S was obtained
in a fermenter system in unregulated pH in MRS
broth containing 4% NaCl. It was also reported that
regulation of pH at 4.0-7.0 during fermentation
had a detrimental effect on the production of
plantaricin S by Lactobacillus plantarum LPC010
(Jimenez-Diaz et al., 1993).
Coventry et al. (1996) studied the effect of pH
on the production of brevicin 286 by Lactobacillus
brevis VB286. No substantial cell growth or
brevicin 286 activity was detected in MRS broth
with an initial pH 4.5. In spite of substantial cell
growth, brevicin 286 production was minimal at
pH 5.0. Optimum production of brevicin 286 was
observed in MRS broth at an initial pH of 6.0-6.5.
It was also observed that regulation of pH at either
6.0 or 6.5 had no advantage over stirred culture
without pH control with respect to brevicin 286
(Coventry et al., 1996).
2.4.6.2.2 Lactococcal bacteriocins:- Nisin
production was maximum when medium was
maintained at pH 6.0 alongwith a large cell mass
(Hurst, 1981). Piard et al (1990) observed maximum
lacticin 481 production when the producer strain
Lactococcus lactis subsp. lactis CNRZ481 was grown
in buffered ELB 6.5 or growing the producer in
pH non-regulated medium resulted in decreased
bacteriocin yields (Piard et al., 1990). Bacteriocin
production by Lactococcus lactis subsp. lactis ADRI
85L030 was reported to be independent of the
initial pH of the medium in the range 5.0 to 7.0
(Thuault et al., 1991). Cock and Stouvenel in 2006
reported MRS liquid culture medium, after 48 h at
36C and 45C in anaerobic condition for lactic acid
production by a strain of Lactocoocus lactis subsp.
lactis isolated from sugarcane plants.
2.4.6.2.3 Leuconostocs bacteriocins:- Leuconostocs
gelidum UAL187 produced leucocin A-UAL187
67
maximally in APT broth at pH 6.0 and 6.5. At a
lower initial pH, growth of the producer organism
was slower and a concentration maximum was
lower (Hastings and Stiles, 1991). Lewus et al.
(1992) studied the effect of initial pH of APT broth
on the growth of and bacteriocin production by
Leuconostoc paramesenteroides OX. Leuconocin Swas
produced in detectable amounts at pH 6.0 and
appeared to be optimal (400 AU/ml) at pH 6.5
and 7.0. They observed slight depression in growth
and leuconocin S production at pH 7.5. Recently
Baker et al (1996) reported that the production
of leuconocin S was maximum (2000 AU/ml) in
fermenters maintained at pH 7.0 than at 6.0, 6.5
and 7.5. Van Laack et al. (1992) observed a 50%
decrease in the production of carnosin 44A when
the initial pH of MRS broth was lowered from 6.0
to 5.1.
Although, Leuconostoc carnosum Talla produced
leucicin B-Talla in MRS broth with an initial pH in
the range 4.5 to 7.5, the bacteriocin concentration
was found to be optimal at pH 6.0 to 6.5 (Felix et
al., 1994).
2.4.6.2.4 Pediococcal bacteriocins:- Pediococcus
acidilactici H produced maximum pediocin AcH
when grown in TGE broth with an initial pH of 6.5.
Pediocin AcH was produced in negligible amounts
when the pH of TGE broth was maintained at pH
5.0 or above. It was concluded that a terminal pH
below 4.0 alongwith a large cell mass was essential
for the production of pediocin AcH (Biswas et al.,
1991). High titres of pediocin N5P were observed
when P. pentosaceus N5P was grown in TGE
broth with an initial pH of 6.5 (Strasser-de-saad
and Manca-de-Nadra, 1993). It was also reported
that pediocin N5P could not be detected in TGE
broth at an initial pH below 5.0. Liao et al (1993)
reported optimum production of pediocin PO2 in
whey permeate medium with an initial pH of 6.5
without pH regulation during incubation.
2.4.6.3 Effect of temperature on bateriocins
production:2.4.6.3.1 Lactococcal bacteriocins:- Nisin
production was maximum when the culture was
incubated between 25 and 30°C as opposed to 37°C.
Incubation of nisin producer at 37°C resulted in
386 AU/ml of nisin as compared to 542 AU/ml at
68
26°C (Hurst, 1981). Thuault et al. (1991) reported
that the bacteriocin production by Lactococcus lactis
subsp. lactis ADRI 85L030 was not significantly
dependent on the incubation temperature in the
range of 30 to 42°C.
2.4.6.3.2 Leuconostocs bacteriocins:- Leuconostoc
carnosum Talla produced bacteriocin, leucocin
B-Talla over a wide range of temperature i.e. 0°C
to 30°C, but the optimal production was observed
at 25°C (Felix et al., 1994).Van Laack et al. (1992)
reported that Leuconostoc carnosum LA44A could
grow and produce bacteriocins in the temperature
range of 4-10°C. Although bacteriocins by various
Leuconostoc spp. was observed both at 4°C and 25°C,
the bacteriocin titres, in general, were 2-3 times
higher at 25°C than at 4°C (Yang and Ray, 1994a).
Leucocin A-UAL187 production by Leuconostoc
gelidum UAL187 was observed over a wide range
of incubation temperatures (1-25°C) with more
time taken at low temperatures (Hastings and Stiles,
1991).
2.4.6.3.3 Pediococcal bacteriocins:- Pediococcus
acidilactici H produced same amounts of pediocin
AcH after 16 h of growth in TGE broth both at
30°C and 37°C. The cell mass and bacteriocin
production were slightly reduced at 40°C (Biswas
et al., 1991). Schved et al. (1993) observed the
production of pediocin SJ-1 at 20°C, 30°C, 40°C
and 45°C with optimal production in the range
of 35 to 40°C. It was reported that the maunt of
pediocin L50 fromed at 16°C was comparable
to that formed at 32°C, while considerably less
amount was produced at 8°C. The organism failed
to produce detectable amounts of bacteriocin at
45°C (Cintas et al., 1995).
2.4.6.4 Growth phase:2.4.6.4.1 Lactobacilli bacteriocins:- Joerger
and Klaenhammer (1986) observed accumulation
of helveticin J between late log phase and stationary
phase of growth of Lactobacillus helveticus 481.
Helveticin V-1829 was produced from the middle
log phase into the stationary phase of growth of
Lactobacillus helveticus V-1829 (Vaughan et al.,
1992).
Barefoot and Klaenhammer (1984) observed the
production of lacticin B during the logarithmic
phase of growth of Lactobacillus acidophilus N2.
Lactobacillus acidophilus M46 produced acidocin B
continuously during the logarithmic growth phase.
The level of inhibition reached maximum at the
beginning of the stationary phase and maintained
constant for at least 24 h (Ten Brink et al., 1994).
Lactobacillus plantarum C-11 was found to accumulate
maximum amount of plantaricin A during the mid
log phase of growth with a decrease in activity
thereafter (Daeschel et al., 1990). Maximum
production of plantaricin S was obtained in log
phase cultures of Lactobacillus plantarum LPC010.
It was also observed that Lactobacillus plantarum
PLC010 secreted another bacteriocin-designated
plantaricin T in the late-stationary phase (JimenezDiaz et al., 1993). Rekhif et al. (1994) reported
plantaricin LC74 production in exponential phase
of growth of Lactobacillus plantarum, however, the
bacteriocin, plantaricin KW30, was maximally
produced at the beginning of stationary phase
culture of Lactobacillus plantarum KW30 (Kelly et
al., 1996).
It was reported that the concentration of brevicin
286 was highest at the late exponential growth
phase (Coventry et al., 1996).
2.4.6.4.2 Lactococcal bacteriocins:- Davey and
Pearce (1980) observed diplococcin production by
Lactococcus lactis subsp. cremoris 346 throughout the
exponential growth phase. Nisin is synthesized as
a secondary metabolite at a high rate when the
cells have reached mid-exponential phase, and
continues to be synthesized during a greater part
of the stationary phase when the cells are grown at
a constant pH of 6.8 at 30°C for 20 to 24 h (Ray,
1992a).
Bacteriocin S50 by Lactococcus lactis subsp. lactis
biovar. diacetylactis S50 was produced continuously
during the growth, but the highest production was
observed after 8 h of incubation (Kojic et al., 1991).
Lacticin 481 production occurred in late-log phase
of growth of Lactococcus lactis subsp. lactis 481 (Piard
et al., 1990).
2.4.6.4.3 Leuconostocs bacteriocins:- Mathieu
et al (1993) reported that the biosynthesis of
mesenterocin 52 and its secretion into the medium
started early in the growth phase, continued over
the whole of that phase before reaching a maximum
at the end. A decrease upto one to two orders of
magnitude in the activity of carnocin LA54A was
recorded during the stationary phase (Keppler et
al., 1994). Yang and Ray (1994a) observed the
termination of bacteriocins production by various
Leuconostoc spp. in the stationary phase of their
growth. Leucocin B-Talla production occurred
during the exponential phase of growth of the
producer Leuconostoc carnosum Talla (Felix et al.,
1994). Production of leucocin A-UAL187 occurred
early in the growth cycle of the producer organism,
rather than as secondary metabolites of growth
(Hastings and Stiles, 1991).
2.4.6.4.4 Pedicoccal bacteriocins:- Biswas et al.
(1991) repoted that about 60% of the pediocin AcH
was produced by 8 h and the rest 40% was produced
during the next 8 h (stationary phase). The authors
have suggested that pediocin AcH appeared to be
a secondary metabolites. Later studies have shown
that post translational processing of prepediocin
to active pediocin AcH occurred efficiently at a
pH below 5.0 (Johnson et al., 1992). After 24 h of
growth, the pediocin AcH was slightly reduced at
all the temperatures studied (Biswas et al., 1991).
Production of pediocin during the logarithmic
and early stationary phases of growth suggested
that pediocin SJ-1 was a secondry metabolite and
after reaching maximum levels, in contrast to many
bacteriocins, the antibacterial activity of pediocin
SJ-1 remained stable in broth cultures over a period
of upto 48 h (Schved et al., 1993). Pediococcus
acidilactici L50 produced highest bacteriocin from
the onset of stationary phase and it remained
stable at 8°C and 16°C while at 32°C, a decrease
in antibacterial activity was seen throughout the
stationary phase (Cintas et al., 1995). Daba et al.
(1991) observed the secretion of pediocin 5 from
P. acidilactici UL5 during the late exponential phase
of growth and the activity dropped sharply (>90%
in 24 h) during the early stationary phase, however,
experiments with pH controlled at 5.0 did not
show this large decrease in activity during the
stationary phase.
2.4.7 Purification of bacteriocins: - An
extensive characterization with respect to physical
and chemical properties of bacteriocins is necessary
69
before considering them for application in foods.
The availability of bacteriocins in a pure form is
essential for characterization studies.
Purification of bacteriocins is a difficult task for
several reasons. Firstly, protein concentration in
the supernatant is very high while bacteriocin
concentration is low, meaning a very low specific
activity.Secondly,bacteriocins form a heterogeneous
group of substances, and the specific purification
protocol has to be developed by trial and error
for each bacteriocin. An additional problem
encountered with the purification of bacteriocins
of lactic acid bacteria is the use of media containing
tween 80, a surfactant that has been shown to
interfere with the precipitation procedures (Muriana
and Klaenhammer, 1991a; van Laack et al., 1992).
Vaughan et al. (2001) purified the bacteriocin to
homogeneity by ammonium sulphate precipitation,
cation exchange, hydrophobic interaction and
reverse-phase liquid chromatography.
During the recent years, the above mentioned
problems have been overcome and several
bacteriocins of lactic acid bacteria have been
purified to homogenecity by growing the producers
in semi-defiend media by minimizing the level of
contaminating proteins and peptides (Joerger and
Klaenhammer, 1986; Hastings et al., 1991; Hechard
et al., 1992). Also MRS broth has been generally
modified by omission of tween 80 (van Laack et al.,
1992; Mortvedt et al., 1991).
2.4.7.1 Lactobacilli bacteriocins:- Barefoot and
Klaenhammer, (1984) purified lacticin B by ionexchange chromatography, ultrafiltration and gel
filtration chromatography. Later, as mentioned by
Nettles and Barefoot, (1993) a simpler purification
protocol was devised for lacticin B. The protocol
involved lyophilisation of culture supernatants
followed by ultrafiltration and preparative
electrofocussing. Muriana and Klaenhammer,
(1991a) achieved a 474-fold increase in specific
activity of lactacin F by ammonium sulfate
precipitation, gel filtration and HPLC.
Lactocin S produced by Lactobacillus sake L45 was
purified to a 4000-fold increase in specific activity
with a recovery of just 3.0% by ammonium sulfate
precipitation, and sequential anion and cation
exchange, hydrophobic interaction, gel filtration,
70
phenyl superose and reverse-phase chromatographies
(Mortvedt et al., 1991). Holck et al. (1992) purified
sakacin A to a 9000-fold increase in specific
activity and a very good recovery of about 80% was
achieved by ammonium sulfate precipitation, ion
exchange, hydrophobic interaction and reversephase chromatography.
Plantaricin S from plantarum LPC010 was
purified to homogeneity by ammonium sulfate
precipitation, binding to SP-sepharose fast flow,
phenyl sepharose CL-4B and C2/C-18 reversephase chromatographies. The purification protocol
resulted in a final yield of 91.6% and 352, 617fold increase in specific activity (Jimenez-Diaz et
al., 1995).
A purification protocol comprising ammonium
sulfate precipitation and sequential cation exchange
and reverse-phase chromatographies has been used
for the purification of acidocin A with a recovery
of about 10% (Kanatani et al., 1995). The protocol
resulted in a more than 3000-fold increase in the
specific activity of acidocin A.
2.4.7.2 Lactococcal bacteriocins:- Diplococcin
was purified from the supernatant of Lactococcus
lactis subsp. cremoris 346. The procedure employed
included ammonium sulfate precipitation (60%
saturation) and cation exchange chromatography
on carboxy methyl cellulose (CMC) resulting
approximately 1000-fold purification (Davey and
Richardson, 1981). Dufour et al. (1991) purified
lactococcin from culture supernatant of Lactococcus
lactis subsp. lactis as a single band by dialysis, cation
exchange and gel filtration chromatographies.
The procedure employed resulted in a 14.5-fold
purification with about 3000-fold increase in
specific activity.
Ammonium sulfate precipitation of culture
supernatant obtained from Lactococcus lactis subsp.
lactis CNRZ481 resulted in a 455-fold increase in the
total lacticin 481 activity. Subsequent purification
by gel filtration chromatography and C18 reversephase high performance liquid chromatography
(HPLC) lead to a 107, 506-fold increase in the
specific activity of lacticin 481 (Piard et al., 1992).
Holo et al. (1991) purified lactococcin A with
about 2300-fold purification and yield of 16% by
a sequential protocol including ammonium sulfate
precipitation, cation exchange chromatography and
reverse-phase HPLC. Lactococcin G was similarly
purified to homogeneity by a four step protocol
which included ammonium sulfate precipitation,
binding to a cation exchanger and octyl-sepharose
CL-4B and reversed-phase chromatography
leading to a recovery of about 20% of the original
activity and a 7000-fold increase in specific activity
(Nissen-Meyer et al., 1992). The bacteriocin
diacetin B produced by Lactococcus lactis subsp.
lactis biovar.diacetylactis UL720 was purified by a
pH dependent adsorption-desorption procedure
followed by a reverse-phase HPLC with a yield
of just 1.25% of the original activity (Ali et al.,
1995).
2.4.7.3 Leuconostocs bacteriocins- Leucocin
A-UAL187 from Leuconostoc gelidium UAL-187
was purified by ammonium sulfate precipitation
followed by a sequential hydrophobic interaction,
gel filtration and reverse-phase HPLC with a yield
of 58% of the original activity and a purification
fold of 4500 (Hastings et al., 1991). Hechard et
al. (1992) employed a three-step protocol for the
purification of mesentericin Y105. The protocol
included affinity chromatography on a blue agarose
column, untrafiltration through a 5-kDa cut off
membrane and finally reverse-phase HPLC on a
C4 column. The purification procedure resulted
in a very low yield of 0.7% with a purification
fold of about 420. The purification procedure
consisting of ammonium sulfate precipitation, and
a sequential gel filtration, cation exchange and
hydrophobic interaction chromatography resulted
in a satisfactory increase of specific activity (1,135fold) but a very low recovery of 8% of mesenterocin
52 produced by Leuconostoc mesenteroides FR52.
Keppler et al. (1994) reported the purification to
homogeneity of carnocin LA54A by single step
hydrophobic interaction chromatography using
amberlite XAD-2. Revol-Juneless and Lefebvre,
(1996) reported the purification of dextrancin J24
to homogeneity by desorbing the bacteriocin from
the producer cells at pH 2.0 followed by a reversephase HPLC.
2.4.7.4 Pediococcal bacteriocins: - Pediocin
AcH from the culture supernatant of P.
acidilactici H was purified by ammonium sulfate
precipitation (70% saturation), fast protein liquid
chromatography (FPLC), gel filtration and anion
exchange chromatography leading to a 98.8-fold
purification with a single band on SDS-PAGE gel
(Bhunia et al., 1988). Yang et al. (1992) reported
the purification of pediocin AcH to homogeneity
as revealed by a single sharp band on SDS-PAGE
gel by a pH dependent adsorption/desorption
procedure. Pediocin AcH was adsorbed to the
producer cells at a pH of 6.0- 6.5, centrifuged; the
bacteriocin adsorbed onto the cells was extracted
at a low pH of 1.5-2.0. The purification protocol
resulted in the recovery of almost all the bacteriocin
produced. Henderson et al. (1992) reported a 470fold purification of pediocin PA-1 by gel filtration,
ion-exchange chromatography, dialysis and HPLC,
whereas Lozano et al. (1992) achieved a 80,000fold increase in specific activity of pediocin PA-1
by employing ammonium sulfate precipitation,
chromatography with a cation exchanger and octyl
sepharose and reverse-phase HPLC.
Daba et al. (1994) employed the pH dependent
adsorption/desorption procedure developed by
Yang et al. (1992) for the recovery of pediocin 5
produced by P. acidilactici UL5. The procedure
resulted in a partial recovery of the cell associated
bacteriocin fraction and even longer desorption
times exceeding 24 h could not result in the
recovery of more than 10% of the original activity.
Further purification to homogeneity was, however,
achieved by reverse-phase HPLC (Daba et al.,
1994).
Schved et al. (1993) reported a 262-fold purification
with a recovery of 50% of pediocin SJ-1 by the direct
application of cell free supernatant containing crude
bacteriocin to a cation exchange chromatography
column. The homogeneity of pediocin SJ-1 thus
purified was confirmed by SDS-PAGE. Cintas et
al. (1995) purified pediocin L50 to homogeneity
by ammonium sulfate precipitation, and sequential
cation exchange, hydrophobic interaction and
reverse-phase chromatographies resulting in the
recovery of more than 80% of the starting material
with a 114, 112-fold increase in specific activity.
2.4.8 Genetic determinants of bacteriocin
production and immunity:- An understanding
of the genetic control for bacteriocin production
71
and host immunity might be beneficial for their
effective use. This is also necessary for cloning
and sequencing of genes involved, and application
of genetic methods for the construction and
improvement of bacteriocin producing strains of
LAB.
The original criteria laid down for bacteriocins
specify the plasmid borne genetic determinants
of bacteriocin production and host cell immunity
(Tagg et al., 1976). Although, most of the
bacteriocins of lactic acid bacteria analysed to
date adhere to this criterion, a very few, especially
those produced by lactobacilli, have been found
to have chromosomal borne genetic determinants
(Barefoot and Klaenhammer, 1983; Joerger and
Klaeanhammer, 1986). The bacteriocin immunity
genes are generally borne on the plasmids
that encode bacteriocin production, however,
bacteriocin plasmids that do not carry immunity
genes have also been found in lactic acid bacteria
(Gonzalez and Kunka, 1987; Schved et al., 1993;
Kanatani and Oshimura, 1994).
2.4.9 Chemical nature of bacteriocins: - All
bacteriocins, which have been studied in sufficient
detail, are found to be macromolecular particulate
in nature and include, if not consist of polypetides
or protein, Currently, over 20 bacteriocins of lactic
acid bacteria have been sequenced. Most contain less
than 60 amino acids and all are devoid of lipid and
carbohydrate materials. The molecules are cationic
and hydrophobic. Due to the hydrophobic nature of
these molecules they have a tendency to aggregate,
especially when stored at high concentration or for
a long time (Ray and Miller, 2001).
Their high isoelectric point (Jack et al., 1995)
allows thcm to interact at physiological pH values
with the anionic surface of bacterial membranes.
This interaction can suffice, in the case of broadspectrum bacteriocins, or facilitate, in the case
of receptor-requiring compounds, insertion
of the hydrophobic moiety into the bacterial
membrane. Later, the cooperation between a
number of bacteriocin molecules will build up
the transmembrane pore responsible for gradient
dissipation and cellular death, These features have
favored the development of general purification
protocols for bacteriocins that include hydrophobic
72
interaction, cationic exchange and reverse-phase
chromatographic steps. The complex pattern of
monosulfide and disulfide intramolecular bonds
helps in the stabilization of secondary structures
by reducing the number of possible unfolded
structures (entropic effect). From a structural point
of view, the effect of the intramolecular bonds is
additive, and the higher their number, the higher the
global stability of the peptide. These facts and the
sensitivity of nisin to digestive enzymes discouraged
the clinical application of this compound but made
it a product of choice as food preservative (Barnbysmith, 1992).
The studies on the kinetics of killing by bacteriocins
show that killing begins as soon as the bacteriocin is
added to a culture and suggest that all bacteriocins
are bacteriocidal, as opposed to bacteriostatic
(Reeves, 1965). Bacteriocidal effect remains stable
during storage in dried or liquid forms at frozen
and refrigeration temperatures. It slowly decreases
during storage at room temperature in the presence
of air that can oxide methionine residues to the
inactive methionine sulfoxide form Molecules
with disulphide bond can undergo bond exchange
in the presence limited amounts of reducing agent
and form dimer and trimers that retain bactericidal
activity. (Klaenhammer, 1993). The first stage is
specific irreversible adsorption of the bacteriocion
and that in some instances at least; only one molecule
may be required to kill a sensitive cellBactericidal
nature of bacteriocins has also been observed by
Toora et al. (1994), as inhibition zone remained
clear of indicator colonies for two weeks in case of
Yersinia enterocolitica.
2.4.10 Bacteriocin biosynthesis: - Bacteriocins
are synthesized as pre-propeptide which are
processed and externalised by dedicated transport
machinery (Nes et aI.,1996). Bacteriocin
production in LAB is growth associated: it usually
occurs throughout the growth phase and ceases
at the end of the exponential phase or sometimes
before the end of growth (Parente et aI., 1997).
Bacteriocin production is affected by type and level
of the carbon, nitrogen and phosphate sources,
cations surfactants and inhibitors. Bacteriocins
can be produced from media containing different
carbohydrate sources. Nisin Z can be produced
from glucose, sucrose and xylose by Lactococcus
lactis IO-1 (Matsuaki et aI., 1996) but better results
were obtained with glucose compared to xylose.
Glucose followed by sucrose, xylose and galactose
were the best carbon sources for the production of
Pediocin AcH in an unbuffered medium Biswas et
aI., 1991).
All bacteriocins are synthesized with an N
terminal leader sequence and until recently only
the double glycine type of leader was found in
class II bacteriocins (Holo et at., 1991; Muriana
and Klaenhammer, 1991; Klaenhammer, 1993).
However, it has now been disclosed that some
small, heat stable and non modified bacteriocins
are translated with sec dependent leaders (Leer
et al., 1995). The structural bacteriocin gene
encodes a preform of the bacteriocin containing
an N-terminal leader sequence (termed double
glycine leader) whose function seems to prevent
the bacteriocin from being biologicalhy active
while still inside the producer and provide the
recognition signal for the transporter system.
A number of genes, often found in close proximity
to each other are required for production of
lantibiotics. These genes include:
(a) The structural gene, Ian A,
(b) Immunity genes (Lan I and in some cases Lan
E, Lan F and Lan G) encoding proteins that protect
the producer from the producer lantibiotic,
(c) A gene Lan T encoding what appears to be
a membrane associated ABC transporter that
transfers, the lantibiotic across the membrane,
(d) A gene, Ian P, encoding a serine proteinase,
which removes the leader sequence of the lantibiotic
prepeptide,
(e) Two genes, Ian B and Lan C (or in some cases
only one gene, Lan M), with no sequence similarity
to other known gens thought to encode enzymes
involved in the formation of lanthionine and
methyllanthionine, and
(f) Two genes Ian k and Ian R encoding two
component regulatory proteins that transmit an
extracellular signal and therby inducing lantibiotic
production.
Each gene cluster appears to contain all the genes
necessary for translation and posttranslational
modifications, when necessary, of a prebacteriocin,
secretion and removal of the leader peptide, and
self-immunity. (Ray et al., 2001).
The promoter upstream of the nis A gene is activated
by the nis R and nis K proteins which induce
transcription of the gene cluster. (de vos et al.,
1995). Following translation of prenisin, the leader
peptide directs the precursor to the membrane
located nis B protein that dehydrates serine to
dhA and threonine to dhB. Nis C, which forms a
complex with nis B, then forms thioether linkages
between dehydration residues and cysteines (Ray
et al., 2001).
The modifying enzymes, nis B and nis C are
encoded directly downstream of nis A. These
enzymes act on the prepeptide, modifying only the
mature protein, which is then transported (Allison
and Klaenhammer, 1999). Nis T encodes a protein
that shares significant homology with ATPdependent translator proteins, and is involved in the
translocation of fully modified precursor nisin across
the cytoplasmic membrane (Qiao and Saris, 1996).
Once outside the membrane, the leader peptide is
removed from the biologically active precursor by
the nis P which is an extracellular serine protease.
With the removal of leader peptide, the matured
pronisin (or nisin) molecule becomes biologically
active and is released into the environment. Nis I
encode a lipoprotein that is involved in immunity.
Proteins F, E, and G also provide cells with additional
protection against nisin. (Bukhtiyarova and Yand,
1994).
2.4.11 Storage studies:- Gandhi and Nambudripad
(1981) reported that crude and partially purified
antibiotic from Lactobacillus acidophilus was stored
at –25°C for 6 months without any loss of activity.
While lactacin B was stable during storage at
room temperature or at -20°C for several months
(Barefoot and Klaenhammer, 1984) without any
loss of activity. ten-Brink et al. (1994) found that
filter sterilized culture supernatant fluids containing
acidocin B could stored at –20°C or 4°C for at least
90 days without loss of acidocin B activity while
during storage at 37°C some inactivation occurred,
possibly caused by the action of proteolytic
enzymes present in culture supernatant. Dave and
Shah (1997) repoted that acidophilicin LA-1 was
73
stable for >15 days at 37°C, >3 months at 4°C and
>8 months at -18°C.
2.4.12 Applications: - The single most important
reason behind the recent interest in isolating
bacteriocin producing lactic acid bacteria and in
studying bactericidal effectiveness of bacteriocin is
their potential applications as food biopreservations
(Ray et al., 2001).
2.4.12.1 Food applications: - Food processors
face a major challenge in an environment in which
consumers demand safe foods with a long shelf
life, but also express a preference for minimally
processed products that do not contain chemical
preservatives. Bacteriocins are an attractive option
that could provide at least part of the solution.
They are produced by food-grade organisms, they
are usually heat stable and they can inhibit many of
the primary pathogenic and spoilage organisms that
cause problems in minimally processed foodstuffs,
however, at present, only nisin and pediocin PAl/
AcH have found widespread use in food. The
form of nisin used most widely in food is Nisaplin
(Danis co), which is a preparation that contains
2.5% nisin with NaCl (77.5%) and non-fat dried
milk (12% protein and 6% carbohydrate). The use
of pediocin PAl for food biopreservation has also
been commercially exploited in the form of ALTA
2431 (Quest), which is based on LAB fermentates
generated from a pediocin PAl-producing strain of
Pediococcus acidilactici (Rodriguez et al., 2002). Its
use is covered by several US and European patents
(Ennahar et al., 2000; Rodriguez et al., 2002) when
screening for a bacteriocin With a food application
in mind, there are several important criteria: first,
the producing strain should preferably have ‘generally recognized as safe’ (GRAS) status; and second,
the bacteriocin should have a broad spectrum of
inhibition that includes pathogens, or have activity
against a particular pathogen.Third, the bacteriocin
should be heat stable; fourth, have no associated
health risks; fifth, its inclusion in products should
lead to beneficial effects such as improved safety,
quality and flavour; and sixth, it should have
high specific activity (Holzapfel et al., 1995).
Bacteriocins have been shown to have potential in
the biopreservation of meat, dairy products, canned
food, fish, alcoholic beverages, salads, egg products,
74
high-moisture bakery products, and fermented
vegetables, either alone, in combination with
other methods of preservation, or through their
incorporation into packaging film/food surfaces
(Chen and Hoover, 2003).
Although, bacteriocins with a wide spectrum of
activity are usually the most sought after, other
factors including pH optima, solubility and stability
are as important and are major considerations in
choosing a particular inhibitor for a particular food
or target bacterium. Furthermore, the antimicrobial
spectra of a variety of LAB bacteriocins can be
extended to encompass Gram-negative bacteria
through their use in combination with measures
that affect the integrity of the outer membrane, such
as temperature shock, high pressure, chelators and
eukaryotic antimicrobial peptides (Stevens et al.,
1991; Delves-Broughton et al., 1996; Suma et al.,
1998).There are also rare natural and bioengineered
bacteriocins (Yuan et al. 2004) that possess inherent
activity against Gram-negative microorganisms.
Bacteriocins can also be used to promote quality,
rather than simply to prevent spoilage or safety
problems. For example, bacteriocins can be used
to control adventitious non-starter flora such as
non-starter lactic acid bacteria (NSLAB) in cheese
and wine.The uncontrolled growth of NSLAB can
cause major economic losses owing to calciumD-lactate formation and slit defects in cheeses,
and the production of detrimental compounds in
wine. Bacteriocins producing starters and adjuncts
(one- or two-strain strategies) have been found to
significantly reduce these problems (Daeschel et al.,
1991; Ryan et al., 1996). However, as some NSLAB
such, as lactobacilli and other starter adjuncts
in cheese, and Leuconostoc oenos and Pediococcus
damnosus in some red wines can improve flavour,
the complete elimination of NSLAB is not always
desirable.This problem has been overcome through
the use of a three-strain system in which an adjunct
strain with reduced bacteriocin sensitivity (obtained
on repeated exposure to increasing concentrations
of the bacteriocin) is used with a bacteriocinproducing starter (Ryan et al., 2001) Figure 9.
Bacteriocins can also be applied in other ways to
enhance food fermentation. This has been shown
dur ing semi-hard and hard cheese manufacture
in which bacteriocin production brings about the
controlled lysis of starter LAB, which results in
the release of intracellular enzymes and ultimately
accelerated ripening and even improved flavour
(Martinez-Cuesta et al., 2000).
Although traditionally, the use of bacteriocins is
associated with the preservation of food, in the
near future food might merely act as a vehicle for
the delivery of bacteriocin-producing probiotic
bacteria. The production of antimicrobials by
a probiotic culture is a desirable trait as they
are thought to contribute to the inhibition of
pathogenic bacteria in the gut (Dunne et al., 1999),
whereas bacteriocins in food are degraded by the
proteolytic enzymes of the stomach, probiotic
bacteria might be ingested in a form that facilitates
gastric transit, allowing the in vivo production
of the bacteriocin in the small or large intestine.
It has also been speculated that recombinant
probiotic strains that can be induced to produce
bacteriolysin could be developed to facilitate the
in vivo delivery of bioactive compounds that are
produced intracellularly (Hickey et al., 2004).
Three approaches are commonly used in the
application of bacteriocins for biopreservation of
foods (Schillinger et al., 1989):
1) Inoculation of food with LAB that produces
bacteriocin in the products. The ability of the LAB
to grow and produce bacteriocin in the products is
crucial for its successful use.
2) Addition of purified or semi-purified bacteriocins
as food preservatives.
3) Use of a product previously fermented with a
bacteriocin-producing strain as an ingredient in
food processing.
Unlike most other preservation methods,
such as heat or low pH, which are essentially
indiscriminate in their antimicrobial effect, it is
this ability to precisely influence the developing
flora in an otherwise perishable food that led us
to describe the use of bacteriocins as a form of
‘innate immunity’ for food. As already described,
the inclusion of Listeria-active class IIa bacteriocins
can specifically prevent the growth of this pathogen,
without affecting harmless LAB, or bacteriocin-tolerant strains can be introduced into an
otherwise hostile food environment. It is unlikely
that the use of bacteriocins in food will negatively
impact on the natural flora of either the human (or
animal) host, or on the environment. The low level
of bacteriocins required eliminating or reducing
small numbers of pathogenic or spoilage organisms
in food are unlikely to have an impact on more
microorganism-rich environments. In any event,
bacteriocins are unlikely to survive gastric transit,
as they are sensitive to proteolytic degradation.
2.4.12.2 Clinical applications:- In particular, the
elucidation of the precise mechanism of action of
some lantibiotics and their activity against multidrug
resistant pathogens by a novel mechanism makes
them an attractive option as possible therapeutic
agents.
The broad-spectrum lantibiotics could theoretically
be of use against any clinical Gram-positive human
or animal pathogen. For example, the two-peptide
lantibiotic lacticin 3147 has in vitro activity against
Staphylococcus aureus [including methicillin-resistant
S. aureus (MRSA)], enterococci (including VRE),
streptococci (S. pneumoniae, Streptococcus pyogenes,
Streptococcus agalactiae, Streptococcus dysgalactiae,
Streptococcus uberis, Streptococcus mutans), Clostridium
botulinum, and Propionibacterium acnes (Galvin et al.,
1999). Initial in vivo trials with animal models have
demonstrated the success of lantibiotics in treating
infections caused by S. pneumoniae (Goldstein et al.,
1998), and MRSA (Niu and Neu, 1991; Kruzewska
et al., 2004), and in preventing tooth decay and
gingivitislss (Howell et al 1993; Blackburn and
Goldstein, 1995, Ryan et al., 1999).
Figure 9. Selected applications of bacteriocins. a | Food
quality. A cheese made with a commercial starter culture (Bac
75
– ) will develop an undefined flora called non-starter lactic
acid bacteria (typified by different fingerprints generated by
random amplified polymorphic DNA patterns). However, a
cheese inoculated with the same commercial strain that can
produce a bacteriocin (Bac + ) (lacticin 3147, in this example)
and a resistant adjunct strain of Lactobacillus, chosen for a
flavour attribute, will develop a single defined culture once the
starter culture has died off, offering the cheese manufacturer
control over previously adventitious flora development. b |
Food safety. A simple example of the role of bacteriocins in
food safety is the production of cottage cheese with a starter
culture that produces a bacteriocin with activity against Listeria
monocytogenes, which results in a cheese that is inherently antiListeria. c | Veterinary medicine. A teat seal is a physical barrier
against infection. Here, a bacteriocin was incorporated into
the teat seal and the teat was challenged with Staphylococcus
aureus. The number of staphylococci recovered from 14 teats
with or without bacteriocin is shown. d | Human medicine.
A Streptococcus mutans strain that cannot produce acid, but that
produces the lantibiotic mutacin (shown), can competitively
exclude acidogenic S. mutans, thereby offering protection
against tooth decay ( Hilman, 2002). Source:- Cotter et al.
(2005).
The use of nisin for human clinical applications
has been licensed to Biosynexus Incorporated by
Nutrition 21 and Immu Cell Corporation has
licensed the use of the anti-mastitic nisin-containing
product Mast Out to Pfizer Animal Health. Bovine
mastitis is defined as an inflammation of the udder
and is the most persistent disease in dairy cows.
Nisin is also used as an active agent in WipeOut (a teat wipe), and lacticin-3147-containing
Teat Seals (Cross Vetpharm Group Ltd) have been
shown to prevent deliberate infection by mastitic
staphylococci and streptococci in animal challenge
trials (Ryan et al., 1999) Figure 8. A strain that
produces the lantibiotic mutacin 1140 is entering
Phase I clinical trials in the US with a view to
replacement therapy, and the dietary supplement
BUS K12 throat guard, which contains a Streptococcus
salivarius that produces two lantibiotics salivaricin
A2 and B, is sold in New Zealand as an inhibitor of
the bacteria responsible for bad breath (Tagg, 2004).
From a nonantimicrobial medical perspective, the
cinnamycin-like lantibiotics have attracted interest
76
owing to their novel activities against the functions
of medically important specific human enzymes,
such as phospholipase A2 and angiotensinconverting enzyme, and nisin has also been found
to have contraceptive efficacy (Aranha et al., 2004;).
The effectiveness of probiotics as agents in the
treatment of various gastrointestinal disorders has
also been shown in several recent studies (Fedorak
and Madsen, 2004).
For commercial and industrial reasons, the selected
probiotics and starter cultures must be produced under the most stringent fermentation and
manufacturing conditions. In general, the selection
criteria for starter cultures are at their acidification
rate and flavour-producing characteristics. For
probiotics, selection is based on a detectable health
effect on the host. In addition, the use of low-cost
industrial growth substrates and microbial strains
with low phage sensitivity are regularly considered
as important objectives for both strain and process
improvement. Most of these strain and processimprovement strategies are based on screening and
trial-and-error approaches.
Genome-sequencing and functional-genomics
studies that focus on LAB which are used as part of
industrial starter cultures (Kleerebezem et al., 2003)
or exploited for their probiotic properties are rapidly
revealing the molecular basis of relevant traits,
including stress-response-adaptation mechanisms
and amino-acid and vitamin auxotrophies (Siezen
et al., 2004).
Some LAB secretes vitamins, including
riboflavin (vitamin B2), folate (vitamin B11) and
cyanocobalamine (vitamin BI2). This unique
characteristic offers the food industry the possibility
to fortify raw food materials such as soy, milk, meat
and vegetables with B vitamins without adding food
supplements (Kleerebezem et al., 2003). Currently,
in the fermentation industry, starter cultures are
selected on the basis that they can produce and
secrete high levels of vitamins B. In addition to
natural strain selection, overproduction of vitamin
B2 and vitamin B11 has been achieved by genetic
engineering of the corresponding biosynthesis
pathways of L. lactis (Sybesma et al., 2003).
However, metabolic engineering of such complex
biosynthesis pathways can often lead to unexpected
phenotypes because the products, being co-factors
in various biochemical reactions, impact directly on
many other pathways. LAB are also good candidates
for the production and delivery of heterologous
proteins and peptides that have potential therapeutic
activity (Miyoshi et al., 2002). Many LABs are acid
and bile resistant, and are therefore, well adapted to
function as vehicles for the oral delivery of vaccine
antigens (Mercenier et al., 2000). The best-studied
LAB used, as vaccine vectors are Lactococcus lactis
and Lactobacillus plantarum. Robinson et al, (1997)
showed that intragastric or intranasal administration
of recombinant lactococci expressing tetanus toxin
fragment C resulted in the induction of systemic
antibody responses in mice at levels sufficient to be
protective against a lethal challenge with tetanus
toxin. Lactococcal immunization was also found
to induce mixed immunoglobulin-G and T-helper
responses, allowing the induction of protection
against various infectious agents and potentially at
several mucosal surfaces (Robinson et al., 2004),The
impact of overproduction of heterologous proteins
on the lactococcal host cells can be significant, as
evident from the general stress response usually
associated with protein overproduction (Schweder
et al., 2002). In addition, the drain on aminoacids
used in heterologous protein biosynthesis can cause
global effects on the metabolism of the host cell.
Thus, keeping in view the wide application
of bacteriocins in food industry and clinical
microbiology, it was felt desirable to undertake
the present studies with an aim to isolate and
characterize high bacteriocin producing strains
of LAB from milk and milk products. It was also
aimed to standerize various parameters for cheaper
and optimum production of bacteriocin from the
selected strain. The effects have also been made
to purify and identify the bacteriocin during the
present investigation.
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90
Dermatological Diseases and Human Placental Extracts.
Psoriasis Case Study in Europe
Vicenta Llorca 1, MD; Tomás Lloret 2, MD, PhD; Jesús Ballesteros3, FD
1 Hospital de Levante Benidorm. Unidad Antiaging. IMED Hospitales. 2 Servicio de Neumología. Consorcio
Hospital General Universitario de Valencia. 3 Fundación Instituto Europeo para el Estudio de la Longevidad
Correspondence:Vicenta LLorca.Unidad Antiaging Hospital de Levante Benidorm. Imed Hospitales
c/ Santiago Ramón y Cajal Nº 7. 03503 Benidorm. [email protected]
Keywords: “placental extracts”,
“rejuvenation”, “psoriasis”
Abbreviations: BSA Body Surface Affectance,
PASI Index of Severity and Psoriasis Area and
PGA Global clinical assessment of Psoriasis
Abstract
Introduction: Placenta is the source of a large
number of biologically active molecules, Benefits:
Wound healing and tissue repair, antioxidant,
inducing melanogenesis1,2, immunoregulatory
effect3, and rejuvenation procedures.4
Methods: Patients treated for 12 weeks, with the
following selection criteria
Main variable to value: Percentage of patients to
response to the treatment with two criteria:a)
Global clinic evaluation by the doctor: “practically
clean” or “clean”.
b) Number of patients whose the Psoriasis Area
Severity Index (PASI) improve in more than 75%
over the basal status (PASI>75%).
Results: In the preliminary results look great
hydration of psoriasis plaques, leaving the clean
area by 50% in the first two weeks of treatment
and gradually improved.
In photoaging, it was found that with mesotherapy
sessions each week during the first month and
continue with a monthly session, the wrinkles are
smoothed, appearance of the skin is hydrated, soft
and bleached.
Discussion: We have tested placental extracts in
psoriatic patients considering placental extract could
improve the patient status due to the improvement
of water retention capacity of the skin cells. It
remains to establish the appropriate maintenance
dose to prevent or ameliorate exacerbations in
patients with psoriasis.
In patients with photo aged skin pose cycles of 4
weekly treatments 2 times a year and a monthly
maintenance. Side effects have not appeared in any
case.
Introduction
Placenta is the source of a large number of biologically
active molecules, hepatocyte growth factor (HGF),
epidermal growth factor (EGF), transforming
growth factor _ (TGF_) and transforming growth
factor ` (TGF-`), and others such as vitamins,
minerals, aminoacids, glucopolysacharides, …)5
Hormone age is a key evidence for aging and
IGF1 (Insuline-like Growth Factor 1) is the most
important marker of hormone age5. Benefits:
Wound healing and tissue repair, antioxidant,
inducing melanogenesis, immunoregulatory effect,
and rejuvenation procedures.6,7,8
We have made this pilot study “12 weeks study to
evaluate the evolution of adult patients diagnosed
with psoriasis in moderate to serious plaques after
the Laennec® use” (24 cases)
Objectives
Primary objectives: a) To establish the percentage
of responders under this treatment , after using it for
12 weeks. b) To establish time for the treatment’s
reaction.
91
Secondary objectives: a) A record of the
adverse effects. b) The number of the medication’s
administration until a therapeutic response is
reached
Furthermore, we note for the first time a response
to Laennec ® with intramuscular administration in
Psoriasis cases
Justification
We know that psoriasis is a chronic inflammatory
illness9 characterized by erythematosus plaques,
with clear borders. It is more frequent on limbs
and scalp10,11.It has two components: an epidermal
hyperproliferation and a swelling on the dermis12.
Diagnosis is clinical. It does not need any histological
checking. We do not have any specific analysis 13
We know that psoriasis is a genetical disorder
(there is a mutation in the PSORS-1’s genome,
in the chromosome ·6), with a strong influence of
environmental factors.
It is an autoimmune illness, with immune cellular
system activation. It is an immunepathogenetic
process mediated by T lymphocytes14
This affects a 1-3% of the European population and
has a strong impact on the life’s quality: pain, itching,
psychological problems.15The most frequent way
is a psoriasis with plaques. Today’s treatments
have a potentially serious toxicity, as hepatoxicity,
nephrotoxicity, bone marrow depression, neoplasias.
16,17. Furthermore, these are doses without a
security in the treatment’s duration18. We need a
treatment for psoriasis that has guarantees to be
safe and efficient in the long-term. Now we have
some alternatives as the biological agents, that are
efficient, but they have some risks of malignancy
and infection 19. Our main working hypotheses is
evaluating the human placenta extracts as a safe and
efficient treatment on psoriasis.
We have got many clinical and experimental
researches with several treatments for psoriasis.
BSA and PASI are the most usual scale to measure
the results. But if we search on PubMed for some
researches on the psoriasis treatments with placenta
extracts we only find four studies that link psoriasis
to topical treatment and none of these extracts
92
of human placentas are considered as a systemic
treatment. 20,21,21,23
Maybe the most researched medication is
STELARA. A monoclonal antibody therapy, with
the Phoneix-1, Phoenix-2 and Accept trials in the
long term.24 25
Pilot study design
This Pilot Study Design is a multicentre study
with a 12 weeks duration of active treatment. This
study’s population have been 24 patients, but it is
still open.The form of administration and posology:
2 intramuscular injections, twice a week for 12
weeks.
In the visit “zero” a first evaluation is done and
the patient is properly informed. A study and
classification is done. Likewise, the patients sign
to consent informed that they agree to participate
and they do not have any problems that pictures
of them are taken and that they will follow the
treatment. After this every visit is evaluated for
the next 12 weeks. At the end of the treatment,
we wait for 2 weeks and a final evaluation is done
at the 13rd visit. The trademark is Laennec inj,®
injection. The composition is 112 mgs of a watersoluble substance of an enzymatic human placenta
product.
Design - Development and evaluation
Evaluation criteria.
We have used a PASI, PDI and PGA scale and the
adverse events. The main evaluation variables are
these two: firstly, a global clinical evaluation by
the researcher where patients are considered as
“virtually clean” and “clean”. Secondly, the patients
whose PASI improves over a 75% on the baseline.
This lets us to establish the percentage’s responder
according to the EMA (European Medicines
Agency). Furthermore, we have introduced some
more variables (PDI, etc.)
Design- development
Zero Visit (selection): Explanation/CI, criteria for
inclusion/exclusion. Features: sex, race, weight (kg),
size (cm), smokers, Date of the Psoriasis Diagnose,
health record, previous medication. Visits 1-12:
Researcher’s evaluation. Adverse events with no
evaluation. Pictures of visits 1 and 4. Visit 13 (2
weeks after the end). Researcher’s evaluation and
pictures.
Researcher’s evaluation
Visual evaluation of representative injuries. Index
(>). Body surface affected area (PASI scoring).
Clinical global evaluation. PASI scoring. The
researcher will evaluate according to the PASI scale
the two most important injuries
Design- selection
In order to include a patient in this research
we need that they fully meet the requirements,
otherwise they are not included. Patients can be
of both genders, between 18 to 65 years, with a
psoriasis diagnose with moderate to severe plaques,
a negative pregnancy test and they should not
follow any hormonal treatment.
Our researches has been done with 24 patients and
it is still open. There are 8 men and 16 women.
The ages are: 1 patient: 20-30 years old, 5 patients:
31-40 years, 10 patients: 41-50 years and 8 patients:
51-60 years old.
Psoriasis Seriousness Evaluation
Psoriasis: slight to moderate: Good control of
damages only with external treatments. BSA>10%
or PASI 10 or higher.
Moderate Psoriasis: It is still possible to control the
illness with external treatments. BSA>10% or PASI
10 or higher.
Moderate to serious psoriasis: External treatments
cannot be control the illness. BSA>10% or PASI
10-20
Serious psoriasis: It needs a systemic treatment to
control the illness. BSA>20% or PASI>20
Estimates of the global clinical evaluation of
psoriasis for doctors (PGA) for the whole
body
For the global clinical psoriasis evaluation on the
body, the PGA scale is used, and the “virtually
clean” or “clean” criteria are included.
Serious: rise of the skin plaque, desquamation and
erythema
Moderate to serious: rise of the skin plaque,
desquamation and erythema
Moderate: moderate rise of the skin plaque,
desquamation and erythema
Slight: slight rise of the skin plaque, desquamation
and erythema
Virtually clean: from slight to clean
Clean: without rests of psoriasis (sometimes,
it is possible to find a little post-inflammatory
hyperpigmentation)
Clinical psoriasis
There are several forms of psoriasis and these can
coexist.
With erythema-scaly plaques, drops,
psoriasic erythrodermia,
inverted,
Localized pustular form, a spread pustular form,
an active psoriasis induced by medication, and the
medication’s name is recorded.
Estimates of the index surface and severity
of psoriasis
PASI is a surface index and the psoriasis seriousness.
It has a scoring from 1 to 4 according to the
seriousness, and from 1 to 6, depending on the
affected skin surface.
It’s an evaluation system of the psoriasic damages
and the patient’s answer to treatments. It represents
a rate from 0 to 72. This organism can be divided
in 4 regions: head (c=), upper limbs (s), axioappendicular (t) and legs (i), which represent a
10%, 20%, 30% and 40% of the body surface.
Neck is here considered as a part of head. Axillas
and groin are here considered as part of the axioappendicular surface and nates are considered as a
part of the legs. All of these parts of the body are
evaluated separately according to the seriousness of
the injuries depending on the erythemas (E), the
infiltration (I) and desquamation (D), in a scale
from 0 to 4. 26
93
PSORIASIS DISABILITY INDEX (PDI)
extracts (laennec inj)
PDI is a disability index of psoriasis. It is a
questionnaire with 15 items that the patient
must answer . It consists of 15 items divided in
five dimensions: activities for everyday (5 items),
work/researches (3 items), personal relations (2
items), leisure (4 items) and treatment (1 item).The
reaction goes from 0 (psoriasis interference) to 3
(maximal psoriasis interference) and a total rate
from 0 to 45 (the higher the rate, the bigger the
impact on the life’s quality)
HPE (human placental extracts) is the only
component of Laennec inj. Several are the properties
associated to the mechanism of action of this active
ingredient in relation to the skin functionality. This
is a summary of scientific publications:
Psoriasis treatment
Topical therapy
Fototherapy
Topical corticoids
Fotochemical therapy PUVA
Combined or isolated Fototherapy UVB
Inner injuries
Fototherapy UVA1
Calcipotriol
Tazarothene
tar
dithranol
Combined therapy
Systematic therapy
Metotrexate
Ciclosporine
Acitretin
Fumarate*
*It is only available in Europe
Biological agents
Alefacept Efalizumab
Etanercept Infliximab
Adalimumab
Modificado de Yamauchi PS, Rizk D, Kormeili T,
Patnaik R, Lowe NJ.
Current systemic. Therapies for psoriasis when are
we know.
J Am Acad Dermatol. 2003: 49:S66-6727
Working hypotheses: to analyze the human
placenta’s extracts on psoriasis, as a safe,
effective and convenient treatment.
Our working hypotheses is to analyze the human
placenta’s extracts on psoriasis, as a safe, effective
and convenient treatment. Medical effectiveness:
Curative. Specific action on the psoriasis
pathogenesis. Quick clinical reactions. Constant
control of the illness with long-term drugs. Effective
as monotherapy. Effective as associated illness
Security. Safe as a chronic treatment and limited
use . Maximal control. Suitable for all ages and
population groups. Minimal medical interaction.
Minimal contra-indication
Hypersensitivity
-Reduce the number of CD4(+) T cells in
peripheral blood. Decrease peripheral production
of Th2-Type cytokines in Ag-challenged site by
the cyclo-trans-4-L-hydroxyproplyl-L-serine28
-Reduce lymphocyte infiltration.Decrease of tissue
infiltrating lymphocytes29
-Protective effect associated with down-regulation
of serum IgE level and inflammatory cytokine
production (IL-1`, IFN-a TNF_, IL-4 and IL-17)
in total lymph node cells and CD4(+) cells.30
Immunomodulating Activity
-Decrease of initially high levels of neutrophils31,32
-Decrease the level of eosinophils, serum IgE,
serum cryoglobulines, number of monocytes, titre
of R-protein and the number of mediomolecular
peptides33,34
Wound Healing and Tissue Repair35,36
- Increase of fibroblast proliferation37
- Increase of the content of fibronectine type III
line peptide (responsible of curing process)38
- Contents glutamate which induces chemotasis
of neutrophils accompanied by polarization of
the actin cytoskeleton and by polymerization of
F-actin
- Promotes cell adhesion39
- Increase of TGF-` in the early stage of wound
healing and VEGF in the late phase40
Convenience. Convenient and well accepted by
patients. Easy application
- Free and bound NADPH in HPE is a potent
wound healer and it’s the substitute of the
glutathione reductase41
Mechanism of action of human placental
Moisturizing
94
Increase water retention of the epidermal cells42
Antioxidant
Glycine (g)-XY aminoacid (derived from collagen)
is an antioxidant component of HPE43.
L-Tryptophan, an aminoacid on HPE, suppress the
lipid peroxidation in the oxidative stress44
For a clearer exhibition of the results, we have chosen
the “typical” patient. She is a selected patient with a
the most spread psoriasis and, furthermore, several
types of psoriasis.We will need know to work with
the patient on the process to the healing
CONCLUSIONS
In an 80% of patients the primary objective of this
research has been reached. Signs and symptoms of
the evaluated psoriasis cases have been reduced.
We have followed the PASI scale (PASI-75) after
twelve treatment weeks.
Reached results with two blisters of LAENNEC ®
(112 mgs/blister), twice a week/12 weeks.
We have noticed a 75% healing from third to
sixth week. This kept like this until the end of our
treatment
The reached results were after a medical evaluation
two weeks after the end of the treatment
We have noticed that the plaques can come back
on the elbows in a patient after a stress time, but
not on the rest of her body surfaces
There is only a patient who did not improve
with regard to the beginning of the treatment.
However he has some differences with the rest
of the patients: he is a social drinker and takes
sintrom (acenocumarol)
We have not noticed any adverse symptoms
A weekly INR was asked to the patient with
sintrom. We have not noticed any disturbances
associated with Laennec®
As a collateral effect we have noticed an
hypopigmentation in the clean plaque
As a positive collateral effect we have noticed an
improvement in the quality of the skin on the
body and especially on the face and even a kind of
antiaging effect
We have noticed a 100% healing from thorax,
abdomen, back and scalp in all patients
The human placentas extracts of Laennec® of JBP
can be a good help on psoriasis patients in plaques
in a systematic way; it improves the damages
and prevents negative side effects or also more
treatments
We need to establish the way we track the results
after a short or a long time after the treatment
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97
INFORMACIÓN
DE PRENSA
Las claves del éxito de mesoestetic Pharma Group
mesoestetic Pharma Group se ha convertido en uno de los laboratorios farmacéuticos líderes en el
mundo gracias a su incansable labor de investigación, desarrollo tecnológico y presencia internacional
mesoestetic Pharma Group es una multinacional española especializada en el desarrollo, la fabricación
y comercialización de productos farmacéuticos y cosmecéuticos, medicamentos tópicos e equipos
médicos-estético con marcado CE. Durante casi treinta años, la compañía ha desarrollado productos
que proporcionan soluciones estéticas integrales que ofrecen un alto nivel de respuesta terapéutica.
En 1984, en la rebotica de una pequeña farmacia de Barcelona, Joan Carlos Font, fundador del mesoestetic Pharma Group y actual Presidente, comenzó a desarrollar sus propios cosméticos. Actualmente, la
empresa vende el 85% de sus productos en más de 60 países, con una cifra de ventas de 40 millones
de euros.
Desde sus inicios, la compañía se ha comprometido a cumplir con estrictos estándares farmacéuticos
y las normativas europeas más rigurosas. La empresa posee las certificaciones ISO 9001:2008 e ISO
13485:2003 y cumple las Buenas Prácticas de Fabricación de productos médicos. La innovación se
considera un punto de referencia en el mesoestetic Pharma Group. Como prueba de su compromiso
estratégico, la compañía invierte anualmente el 40 % de sus beneficios en Investigación y Desarrollo
(I+D), y gastó 23 millones de euros el año pasado en la construcción de su nueva sede.
Autorizadas por la Agencia Española del Medicamento y Productos Sanitarios (AEMPS), las instalaciones cuentan con tres líneas de fabricación de productos farmacéuticos (para medicamentos tópicos,
productos médicos estériles y cosmecéuticos), un avanzado laboratorio de control analítico, departamentos de I+D y control de calidad, y un gran almacén logístico.
Recientemente, la compañía ha inaugurado su nueva Unidad de Biotecnología para realizar ensayos de
cultivos celulares para buscar nuevas moléculas y combinaciones de principios activos más efectivos.
Las principales líneas de investigación de la Unidad de Biotecnología son las dos líneas prioritarias en
la empresa: antienvejecimiento y despigmentación. Además, la unidad servirá para establecer contacto
con compañías y grupos científicos.
Con su Unidad de Biotecnología y su Unidad Médica de seguimiento y control observacional, la compañía abarca el ciclo de producción completo y puede llevar a cabo estudios in vitro e in vivo para garantizar la seguridad y eficacia de sus productos.
La compañía es actualmente el único laboratorio farmacéutico español que fabrica dispositivos médicos de administración intradermal de clase III con el sello de conformidad de la CE, lo que indica que
se ajustan a los estándares de calidad, seguridad y eficacia de la Unión Europea. Su línea mesohyal™
ofrece una completa gama de dispositivos médicos intradermales diseñados para tratamientos mesoterapéuticos faciales y corporales. Además hay que subrayar que sus tratamientos cosmelan® y dermamelan® son los productos despigmentantes más vendido en todo el mundo.
Joan Carlos Font, Fundador y Presidente
de mesoestetic Pharma Group
La sede de mesoestetic Pharma Group
mesohyalTM, la última generación de productos inyectables para tratamientos de mesoterapia
Con 10 productos, mesohyal™ es actualmente la gama de productos sanitarios inyectables para la realización de tratamientos de mesoterapia y con marcado CE más completa del mercado.
mesohyal™: la gama más amplia del mercado
1. mesohyal™ HYALURONIC: rehidratación y rejuvenecimiento dérmico.
2. mesohyal™ NCTC 109 : biorrevitalización celular intensiva.
3. mesohyal™ VITAMIN C: con acción antioxidante e iluminadora.
4. mesohyal™ DMAE: aporta elasticidad y firmeza.
5. mesohyal™ ORGANIC SILICON: regeneración y reestructuración del tejido cutáneo.
6. mesohyal™ BIOTIN: reactiva el metabolismo celular cutáneo y del cuero cabelludo
7. mesohyal™ CARNITINE: Indicado para todos los tipos de celulitis, especialmente la compacta o
fibrosa. Favorece el metabolismo de los ácidos grasos.
8. mesohyal™ ARTICHOKE: Indicado para todos los tipos de celulitis, especialmente la blanda. Acción
drenante y detoxíficante.
9. mesohyal™ MELILOT: Indicado para todos los tipos de celulitis, especialmente la edematosa con
componente vascular. Activa la micro-circulación
10. NOVEDAD: mesohyal™ OLIGOELEMENTS: estimulación de la matriz extracelular dérmica
Además de poder utilizarse de forma individual, las diferentes soluciones de la gama mesohyal™, pueden combinarse entre si para realizar cócteles de mesoterapia a medida y tratar así de manera personalizada el caso de cada paciente.
Todas las soluciones de la gama tienen el mercado CE que avala su calidad como seguridad, y ofrece a
los médicos como pacientes la garantía de productos supervisados y de eficacia contrastada.
mesoestetic Pharma Group entra en el mercado chino
mesoestetic Pharma Group ha llegado a un acuerdo para entrar en el mercado chino y empezar a comercializar sus
productos y tratamientos a principios de 2014. Actualmente los productos de la firma están en proceso de registro
y se espera que estén listos para finales del último trimestre del año. El acuerdo con un socio local incluye la apertura durante 2014 de unos 300 puntos de venta en el país asiático incluyendo centros de estética, clínicas hoteles y
spas. Para el presidente de mesoestetic Pharma Group, Joan Carles Font, se trata de un “acuerdo estratégico para
la compañía porque China es un mercado con un enorme potencial de crecimiento en el sector de la estética”.
En pasado mes de julio la empresa consiguió reunir en Barcelona a los más influyentes periodistas y blogueros
chinos. Entre ellos, redactores de revistas como Harper’s Bazaar o el primer periódico de Shanghai, The Shanghai
Times, o el blog QQ.com, o key opinion leaders como Roger Zhou Xin.
La nueva Unidad de Biotecnología de la firma
La línea mesohyal
3OCIEDAD %SPA×OLA DE -EDICINA !NTIENVEJECIMIENTO Y ,ONGEVIDAD
El objetivo y finalidad de la Sociedad es fomentar y llevar a cabo, en interés público y sin ánimo de lucro, la
Medicina Antienvejecimiento (Anti-Aging) como procedimiento terapéutico, procurando la cooperación
y la unión de especialidades médicas y de todos los profesionales de la salud, farmacéuticos, psicólogos,
biológos, odontólogos, etc..., que por su actividades y dedicaciones, manifiestan expresamente su interés en
la Medicina Antienvejecimiento, que básicamente es un sistema integral preventivo y curativo, que a partir
del estudio del envejecimiento natural, descarta los factores perjudiciales que producen un envejecimiento
prematuro, proponiéndose un sistema de vida de promoción de la salud, aplicando técnicas correctoras de
los signos estéticos y orgánicos de decaimiento corporal.
Para cumplir estos objetivos, vamos a proporcionar a los miembros, la información necesaria sobre la
práctica y avances de las técnicas que nos ocupan, mediante congresos, symposium, cursos y actos (siempre
en unas condiciones especiales para sus miembros), además recibiras la Approaches to Aging Control,
órgano oficial de la S.E.M.A.L.
Para mayor información puedes conectarte a nuestra página web: www.semal.org
BOLETÍN DE INSCRIPCIÓN A LA SEMAL
Nombre ..................................Apellidos ..............................................................................Edad..........
Dirección ...............................................................................................................................................
Localidad ...............................................................................................................................................
C.P. ........................ Provincia......................................................................... País ..............................
Teléfono ...........................Fax ............................. E-mail.....................................................................
Especialidad.............................................................................................................................................
Nº Colegiado ........................Colegio de: .............................................................................................
Hospital/Clínica ....................................................................................................................................
Consulta privada ....................................................................................................................................
Miembros Fundadores, de la Junta Directiva y Numerarios: 200 euros/año
Miembros españoles y miembros correspondientes
extranjeros: 100 euros/año
La inscripción a la sociedad incluye la suscripción a la
revista de la misma.
Entidad Bancaria:
La Caixa. Nº de cta: 2100 2112 11 0200500924
Titular: Sociedad Española de Medicina Antienvejecimiento y Longevidad (S.E.M.A.L.)
Para formar parte de la sociedad, envíe este boletín de
inscripción y el justificante de ingreso o transferencia,
junto con una copia del título de Licenciado o Doctor
en Medicina y Cirugía o de Especialista, a la siguiente
dirección:
Sociedad Española de Medicina Antienvejecimiento y
Longevidad
Real e Ilustre Colegio de Médicos de Sevilla
Avda. de la Borbolla, 47 - 41013 Sevilla
Tlf.: 954 084 700 - Fax: 954 084 700
e-mail: [email protected]
web: www.semal.org
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Approaches to Aging Control - Sociedad Española de Medicina

Transcription

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