Significance of inoculation density and carbon source on the

Process Biochemistry 43 (2008) 576–586
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Significance of inoculation density and carbon source on the mycelial
growth and Tuber polysaccharides production by submerged
fermentation of Chinese truffle Tuber sinense
Ya-Jie Tang *, Ling-Li Zhu, Dong-Sheng Li, Zhi-Yuan Mi, Hong-Mei Li
Hubei Provincial Key Laboratory of Industrial Microbiology, College of Bioengineering, Hubei University of Technology, Wuhan 430068, China
Received 23 November 2007; received in revised form 14 January 2008; accepted 28 January 2008
Abstract
Truffles are among the most valuable gourmet mushrooms on the market. By taking Chinese truffle Tuber sinense as a typical example, a
submerged fermentation process for the production of mycelia and Tuber polysaccharides was developed for the first time. Significances of
inoculation density, carbon source and its initial concentration were studied in details. For inoculation density within the range of 160–653 mg dry
weight (DW)/L, a maximal biomass of 15.59 0.59 g DW/L was obtained at its lowest level of 160 mg DW/L, while the maximal extracellular
polysaccharides (EPSs) production of 1.97 0.08 g/L was attained at its highest level of 653 mg DW/L. The maximal intracellular polysaccharides (IPSs) production of 1.40 0.10 g/L was obtained at its level of 487 mg DW/L. The carbon sources examined were glucose, maltose,
sucrose and lactose, and sucrose was suitable for the cell growth and IPS production. Lactose was beneficial for EPS production although the cell
could not grow well. There was no b-galactosidase activity when T. sinense grew in lactose, which was the reason why lactose was not favorable for
the cell growth. Initial sucrose concentration within the range of 20–125 g/L significantly affected the process. At sucrose 125 g/L, both biomass
(i.e., 24.07 1.94 g/L) and EPS production (i.e., 2.85 0.04 g/L) reached their peak values. The maximal IPS production of 2.92 0.20 g/L was
obtained at sucrose 80 g/L. This work demonstrated the submerged fermentation of Chinese truffle T. sinense is a potential alternative for the
efficient production of mycelia and Tuber polysaccharides.
# 2008 Elsevier Ltd. All rights reserved.
Keywords: Chinese truffle; Tuber sinense; Tuber polysaccharides; Submerged fermentation; Process optimization
1. Introduction
Truffles, also known as ‘‘black diamonds’’, are hypogeous
gourmet mushroom, belonging to the family Tuberaceae, the
order Tuberales, and the phylum Ascomycotina, which grow in
symbiosis with certain trees [1–3]. There are more than 60
different kinds of truffles around the world, most of which grow
in various parts of Europe, particularly in France [1–3]. They
are thought to be a ‘‘miracle of nature’’ and have been since
ancient times the ultimate in gastronomy because of their
highly nutritional attributes [2]. Through chemical and
pharmacological studies, many ingredients including androstenol, ceramides and Tuber polysaccharides have been isolated
from the fruiting-bodies of truffles and their biological
activities have been identified. The androstenol could modulate
* Corresponding author. Tel.: +86 27 88015108; fax: +86 27 88015108.
E-mail address: [email protected] (Y.-J. Tang).
1359-5113/$ – see front matter # 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.procbio.2008.01.021
the female catamenia and heighten female sexual arousal [4,5].
Recently, new ceramides were isolated from the fruiting-body
and their bioactivities such as moisture-retaining, apoptosisinducing, antitumor and immunostimulatory have been
reported [6–8]. Tuber polysaccharides isolated from the
fruiting-body also have immunomodulating and antitumor
activity [9]. More interestingly, the ability of pigs to detect
truffles underground has been associated with the existence of
trace amounts of aromatic steroidal pheromone in both black
and white truffles [10].
There is a growing market for the products derived from
truffles, while annual world production of natural truffle over
the past century has dropped dramatically from ca. 1000 to
200 tons [11,12]. This drop has led to the establishment of
truffle orchards, while truffle production begins 5–10 years
following orchard establishment [13]. Currently, commercial
truffle products are mostly derived from the fruiting-bodies of
field-cultivated truffles. Truffle filed-cultivation involves raising infected plants in glasshouses and then planting these into
Y.-J. Tang et al. / Process Biochemistry 43 (2008) 576–586
suitable sites. Many ectomycorrhizal plants can be produced
commercially using pure isolates in axenic mass culture
under controlled conditions. However, because it is very
difficult to obtain pure mycelial cultures of Tuber spp., spore
inoculum is usually used to produce truffle-infected plants.
Unfortunately, such techniques often result in plants being
contaminated with the wrong Tuber spp. or other ectomycorrhizal fungi such as Pulvinula constellatio and Sphaerosporella brunnea [13,14].
Submerged fermentation of mushrooms is a promising
alternative for the efficient production of mycelia and
metabolites and has received increasing attention around the
world [15–20]. By taking Chinese truffle Tuber sinense as a
typical example, a bioprocess of simultaneous production of
mycelia and Tuber polysaccharides was developed for the first
time by submerged fermentation of T. sinense. In this work, the
significances of inoculation density and carbon sources on the
submerged fermentation of T. sinense were investigated in order
to improve the biomass accumulation and Tuber polysaccharides production. The kinetics of cell growth, substrates
consumption, and metabolites biosynthesis were monitored
during the submerged fermentation process. The information
obtained is considered fundamental and useful for the efficient
production of truffle mycelia and Tuber polysaccharides by the
submerged fermentation of T. sinense and other truffles on a
bioreactor scale.
577
2.3. Carbon source experiments
Effect of carbon source on Chinese truffle T. sinense culture was studied by
using various carbon sources, i.e., glucose, maltose, sucrose, and lactose. The
fermentation medium was composed of 5 g/L of peptone, 5 g/L of yeast extract,
1 g/L of KH2PO4H2O, 0.5 g/L of MgSO47H2O, 0.05 g/L of vitamin B1, and
35 g/L of a certain kind of sugar as investigated. The 45-mL medium in a 250mL shake flask was inoculated with 5-mL second seed preculture broth (with ca.
350–450 mg DW of cells per liter). The fermentation was conducted at 25 8C on
a rotary shaker at 120 rpm. For the investigation on the initial sucrose concentration, its levels of 20, 35, 50, 65, 80, 95, 110, and 125 g/L were tested to
obtain an optimal cell growth and metabolites’ accumulation. The other culture
conditions were the same as above.
2.4. Sampling, determination of dry weight and residual sugar
For sampling, three flasks were taken each time. Dry cell weight (DW) was
obtained by filtering through a mesh with 30 mm pore size, washing the cells for
three times with distilled water, and drying at 60 8C for sufficient time to a
constant weight. After sampling, the fermentation supernatant was stored at
20 8C, and then thawed for analyses of residual sugar. Residual sugar level
was assayed by phenol–sulfuric acid method [21,22].
2.5. Measurements of extracellular and intracellular polysaccharides
2. Materials and methods
For the determination of extracellular polysaccharides (EPSs), after removal
of mycelia by filtration, the crude EPS was precipitated with the addition of 95%
(v/v) ethanol by four times of volume, and then separated by centrifugation at
13,000 rpm. The insoluble components were suspended in 1 M NaOH at 60 8C
for 1 h, and the supernatant was measured by phenol–sulfuric acid method
[21,22]. For the analysis of intracellular polysaccharides (IPSs), the dried
mycelia (ca. 100 mg) were extracted by using 1 M NaOH at 60 8C (1 h),
and then the supernatant was assayed by phenol–sulfuric acid method [21,22].
2.1. Maintenance and preculture of T. sinense
2.6. Computational methods [22,23]
The strain of T. sinense was kindly provided by Mianyan Institute of Edible
Fungi (Sichuan Province, China). It was maintained on potato-agar-dextrose
slants. The slant was inoculated with mycelia and incubated at 25 8C for 5 days,
then stored at 4 8C for about 2 weeks. Preculture medium consisted of the
following components (g/L): glucose, 35; peptone, 5; yeast extract, 2.5;
KH2PO4H2O, 1; MgSO47H2O, 0.5; vitamin B1, 0.05. For the first-stage seed
preculture, 40-mL medium with initial pH of 5.0 was prepared in a 250-mL
flask, and then 10-mL mycelium suspension from a slant culture was inoculated,
and followed by 5-day incubation at 25 8C on a rotary shaker (120 rpm). For the
second-stage seed preculture, 180-mL medium was prepared in a 500-mL flask,
and inoculated with 20-mL first preculture broth (with ca. 400–500 mg dry cell
weight (DW)/L), then followed by 2-day incubation at 25 8C on a rotary shaker
(120 rpm).
Average growth rate was calculated as: (maximum DW initial DW)/
(maximum DW + initial DW)/2/culture time.
Average sugar consumption rate was calculated as: (initial sugar concentration residual sugar concentration when the maximal DW obtained)/culture
time.
Metabolite productivity was calculated as: (maximum metabolite production initial metabolite production)/culture time.
Cell yield on sugar was calculated as: (maximum DW initial DW)/(initial
sugar concentration residual sugar concentration when the maximal DW
obtained).
Metabolite yield on sugar was calculated as: (maximum metabolite production initial metabolite production)/(initial sugar concentration residual
sugar concentration when maximum metabolite production obtained).
2.2. Inoculation density experiments
The seed preculture process were strictly controlled to make sure that cell
density in the second-stage seed preculture process was kept almost constant.
Then, the amount of inoculation density for the fermentation process could be
controlled by the control of the second-stage seed volume. The effects of
inoculation density on the cell growth and the production of Tuber polysaccharides including extracellular polysaccharides (EPSs) and intracellular polysaccharides (IPSs) by T. sinense cells were studied by controlling inoculation
density at 160, 320, 487, and 653 mg DW/L, respectively. The fermentation
medium was composed of 35 g/L of glucose, 5 g/L of peptone, 5 g/L of yeast
extract, 1 g/L of KH2PO4H2O, 0.5 g/L of MgSO47H2O, and 0.05 g/L of
vitamin B1. The dynamic profiles of the cell growth, substrate consumption,
and Tuber polysaccharides biosynthesis were monitored during the submerged
fermentation process. Multiple flasks were run at the same time, and three flasks
were taken at each sampling point. Each data point was expressed by an average
with an error bar (i.e., standard deviation from three independent samples).
3. Results and discussion
3.1. Effect of inoculation density
Inoculation density (or inoculum size) is an important
culture factor for the submerged fermentation of many
mushrooms (e.g., Refs. [23–25]). The effect of inoculation
density on the cell growth in the submerged fermentation of
Chinese truffle T. sinense in shake flasks is shown in Fig. 1A. In
all cases, no lag phase was observed within the range of
inoculation density as investigated (e.g., 160–653 mg DW/L).
For the inoculation density within the range of 70–670 mg DW/
L, a lag phase of 1 day was observed for all cases during the
submerged fermentation of medicinal mushroom Ganoderma
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Y.-J. Tang et al. / Process Biochemistry 43 (2008) 576–586
Fig. 1. Effect of inoculation density on the cell growth (by dry cell weight,
DW) (A), culture pH (B) and glucose consumption (C) in the submerged
fermentation of Chinese truffle T. sinense in shake flasks. Symbols for
inoculation density (mg DW/L): 160 (*), 320 (*), 487 (~), and 653 (~).
The error bars in the figure indicate the standard deviations from three
independent samples.
lucidum in shake flasks [23]. This suggested that T. sinense cell
grows faster than G. lucidum cell. At the beginning of the
culture process, higher specific cell growth rate of T. sinense
was observed at larger inoculation density. The cell growth at
an inoculation density of 160 mg DW/L appeared to be slow as
the culture period continued for up to 5 days, where is was 4
days for the other cultures. And, the average growth rate kept
almost constant (e.g., 0.46–0.48 day 1) at relatively larger
inoculation density (e.g., 320–653 mg DW/L), which was
higher than that (0.39 day 1) at the lowest inoculation
density of 160 mg DW/L. At an inoculation density of 160,
320, 487, and 653 mg DW/L, a maximum cell concentration
was 15.59 0.59, 14.85 0.12, 14.10 0.51, and 14.10 0.13 g DW/L, respectively. It appeared that the maximal
biomass of 15.59 0.59 g DW/L obtained at the lowest
inoculation density of 160 mg DW/L was enhanced by 11%
compared with the minimal biomass of 14.10 0.51 g DW/L
obtained at the largest inoculation density of 653 mg DW/L.
This indicated that a low inoculation density resulted in a high
final cell concentration, and an inoculation density of 160 mg
DW/L was favorable for the cell accumulation of T. sinense in
shake flasks fermentation. In contrast to the above results, Fang
et al. [23] reported that a small inoculum size led to a low final
cell density during the submerged fermentation of G. lucidum
in shake flasks, an inoculation size of 170 mg DW/L was
necessary for the fermentation production, and a maximal cell
concentration of 15.7 g DW/L was obtained at an inoculation
density of 330 mg DW/L.
Fig. 1B clearly shows the time profiles of culture pH during
the submerged fermentation of T. sinense in shake flasks were
quite similar despite various inoculation densities. Culture pH
remained almost constant during the first day cultivation. On
the second day culture, a sharp decrease in culture pH was
observed. After that, culture pH remained relatively constant
for about 2–3 days. When the residual glucose was almost
exhausted on day 4 or 5 (Fig. 1C), culture pH quickly increased
to about 8.0 on day 6. At the later stage of fermentation, culture
pH kept constant. This suggested that there is no significant
effect of inoculation density on the variation of culture pH
during the submerged fermentation of T. sinense. Similar
phenomenon has also been claimed in the case of G. lucidum
cultivation [26]. During the culture process, the culture pH
showed a sharp decrease to 3.2 during the first 4 days of
cultivation. After that, the pH remained relatively constant
for about 1 week. At the end of fermentation (days 10–14),
when the residual glucose was almost exhausted, the pH
values quickly increased to 7.0. The pH increase towards the
end of the culture was considered to be related with glucose
consumption [26].
Fig. 1C shows the dynamic profiles of glucose consumption
under various inoculation densities. The glucose consumption
corresponded well to the cell growth of T. sinense. While
medium glucose was almost consumed on day 4 or 5, the cell
density reached the peak. Both the cell growth rate and glucose
consumption rate were relatively lower at an inoculation
density of 160 mg DW/L, while they kept almost constant at
higher inoculation density. The average glucose consumption
Y.-J. Tang et al. / Process Biochemistry 43 (2008) 576–586
rate was 7.07, 8.21, 8.23, and 8.35 g glucose/L per day, and the
corresponding cell yield on glucose was 0.45, 0.44, 0.41, 0.40 g
DW/g glucose at an inoculation density of 160, 320, 487,
653 mg/L, respectively. It seemed that there is no significant
effect of inoculation density on the cell yield against glucose
during the submerged fermentation of T. sinense in shake flasks.
Fig. 2A clearly shows the control of inoculation density
significantly affected EPS production. At an inoculation density
of 160, 320, 487, and 653 mg DW/L, the total EPS
accumulation was 0.97 0.04, 1.15 0.06, 1.62 0.07, and
1.97 0.08 g/L, the EPS productivity was 0.13, 0.16, 0.21, and
0.26 g/L per day, the corresponding EPS specific productivity
was 8.97, 12.11, 18.00, and 23.14 mg/g DW per day, and the
EPS yield on glucose was 21.59, 27.56, 41.49, and 51.63 mg/g
glucose, respectively. This indicated that not only EPS
production, productivity, and specific productivity, but also
EPS yield against glucose was increased with the inoculation
density within the range as investigated. It was also shown that a
relatively high EPS production was obtained at a large inoculum
size in the G. lucidum fermentation [23], and the maximal
Ganoderma EPS production titer of 0.88 g/L was obtained at the
highest inoculation density of 670 mg/L on day 8.
Time profile of IPS content under different inoculation
densities is compared in Fig. 2B. On the first day of culture, IPS
content rapidly increased from 3.0 to about 7.0 mg/100 mg
DW. And then IPS content was increased slowly. After medium
glucose was exhausted and the cell density reached the peak
values, IPS content still increased. The maximal IPS content of
11.39 0.49 mg/100 mg DW was obtained at an inoculation
density of 487 mg/L on day 7, and the other three kinetic
profiles of IPS content was quite similar. Kinetic profile of IPS
accumulation under various inoculation densities is compared
in Fig. 2C. IPS production reached its peak on day 6, 6, 6, and 4,
their maximum level at an inoculation density of 160, 320, 487,
and 653 mg DW/L was 1.21 0.08, 1.24 0.03, 1.40 0.10,
and 1.17 0.03 g/L, and the IPS yield on glucose was 34.83,
36.18, 39.98, and 35.04 mg/g glucose, respectively. These
results indicated that higher IPS production could be achieved
at a relatively large inoculums size (e.g., 487 mg/L) in the T.
sinense cultures. IPS productivity was 0.20, 0.21, 0.23, and
0.29 g/L per day at an inoculation density of 160, 320, 487, and
653 mg DW/L, and the corresponding IPS specific productivity
was 14.47, 15.64, 18.49, and 24.54 mg/g DW per day,
respectively. It appeared that not only IPS productivity, but
also the IPS specific productivity increased with inoculation
density. While, in the submerged fermentation of G. lucidum in
shake flasks, both the maximal IPS content of 10.9 mg/100 mg
DW and the maximal IPS production of 1.22 g/L were obtained
at the largest inoculation density of 670 mg/L within the range
as investigated [23].
The effect of inoculation density on the cell growth and
metabolites biosynthesis in the culture of T. sinense is
summarized in Fig. 3. A maximal biomass of 15.59 0.59 g
DW/L was obtained at the lowest inoculation density of 160 mg
DW/L, while the maximal EPS production of 1.97 0.08 g/L
was attained at the largest inoculation density of 653 mg DW/L.
Not only the maximal IPS content of 11.39 0.49 mg/100 mg
579
Fig. 2. Effect of inoculation density on the production of EPS (A), the content
(B) and production (C) of IPS in submerged fermentation of T. sinense. Symbols
for inoculation density are the same as those in Fig. 1.
DW, but also the maximal IPS production of 1.40 0.10 g/L was
obtained at an inoculation density of 487 mg DW/L. There are
several other reports investigating the effect of inoculation
density measuring by seed volume on the submerged fermentation of mushroom. During the submerged fermentation of
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Y.-J. Tang et al. / Process Biochemistry 43 (2008) 576–586
Fig. 3. Effect of inoculation density on the cell growth (hatched bar), the
production of EPS (blank bar) and IPS (dark bar). The error bars in the figure
indicate the standard deviations from three independent samples.
medicinal mushroom Cordyceps jiangxiensis, both mycelial
biomass and intracellular polysaccharides (IPSs) production
were both near optimal values at inoculum sizes of 4–6% and
declined rapidly outside this range [24]. During the submerged
fermentation of mushroom Agaricus blazei, the optimum
inoculum size for both mycelial growth and EPS production
was identified to be 10% (v/v) in shake flask cultures [25]. These
works indicate that the control of inoculation density was very
important to both the cell growth and polysaccharides production
during the submerged fermentation of mushroom. In conclusion,
control of inoculation density was significant for cell growth, the
production of EPS and IPS, and an inoculation density of
487 mg/L was favorable for the submerged fermentation of
Chinese truffle T. sinense.
3.2. Effect of carbon source
Carbohydrates are a major component of the cell
cytoskeleton and a key nutritional requirement for mushroom
fermentation. In order to develop the novel submerged
fermentation of Chinese truffle T. sinense, it is essential to
optimize carbon source for the cell growth and Tuber
polysaccharides production. Effects of different carbon
sources, i.e., glucose, maltose, sucrose, and lactose, on the
submerged fermentation of T. sinense were investigated in
shake flasks. Fig. 4A clearly shows the significance of carbon
source on the cell growth during the submerged fermentation of
T. sinense. The cell growth patterns were quite similar when
glucose, maltose and sucrose were used as carbon source in the
culture medium. Compared with glucose, maltose and sucrose,
when lactose was used, a much lower final cell density (by
DW), i.e., 4.65 0.08 g DW/L, was obtained on the first
culture day. After then, there was no increase in the cell density
until the end of the fermentation. The maximum cell density of
13.92 0.15, 14.00 0.23, and 14.97 0.08 g DW/L was
obtained on day 6, 5, and 5 when glucose, maltose and sucrose
were used, respectively, and the corresponding average growth
rate was 0.31, 0.38, and 0.38 day 1. From the viewpoint of the
Fig. 4. Effect of carbon source on the cell growth (A), culture pH (B) and sugar
consumption (C) in the submerged fermentation of T. sinense. Symbols for
carbon source: glucose (*), maltose (*), sucrose (~), and lactose (~).
cell growth, our results indicated that lactose was the worst and
sucrose was the best in the submerged fermentation of T.
sinense. During the submerged cultivation of Cordyceps
militaris, a low cell density and growth rate was also observed
Y.-J. Tang et al. / Process Biochemistry 43 (2008) 576–586
when lactose was used as carbon source, whereas the cell
growth in galactose medium was preferred [27]. In the previous
reports on the submerged fermentation of G. lucidum, Tang and
Zhong [22] found that lactose was the best carbon source while
sucrose was the worst from the viewpoint of cell growth of G.
lucidum. This indicated that the utilization of carbon sources
varies among the mushrooms.
Time profiles of culture pH during the submerged
fermentation of T. sinense in shake flasks under different
carbon source are compared in Fig. 4B. The culture pH
variation pattern in the case of glucose and sucrose was similar.
After inoculation, culture pH value decreased quickly from 6.0
to about 2.5 in the first three days culture, and then kept almost
constant at around 2.5 until the end of fermentation. However,
the pH variation pattern in the case of lactose and maltose were
quite different from those in the case of glucose and sucrose.
When lactose was used as carbon source, the culture pH
increased to 8.56 in the first 2 days culture, then kept constant
around a very high level (about 8.8). In the case of maltose,
culture pH increased to 7.56 in the first day culture, which was
the same with the lactose. After then, culture pH descended a
little bit in the second day culture, and decreased quickly to
2.52 in the third day culture. After then, there was almost no
change in culture pH until the end of fermentation. At the later
stage of fermentation (i.e., after day 3), the pH variation patter
in the case of maltose was quite similar with the cases of
glucose and sucrose.
Dynamic profiles of sugar consumption under different
carbon source are described in Fig. 4C. General speaking, the
sugar consumption pattern corresponded well to the cell growth
pattern (Fig. 4A). In the case of lactose, the maximal cell
density was obtained on the first day culture, and simultaneously the lactose concentration in the culture broth was
decreased only in the first day culture, after then the medium
lactose level kept almost constant, and at the end of
fermentation (day 9) there still remained a high level of
medium lactose (i.e., 30.3 g/L), which well corresponded to the
kinetics of cell growth (Fig. 4A). For the other three carbon
sources, the sugar consumption patter in the case of glucose and
sucrose was quite similar, which was a little bit faster than the
case of maltose. In the case of glucose and sucrose, almost all
the sugar amount was utilized at the end of fermentation (day
9), while there was still about 5.4 g/L residual maltose in the
fermentation broth on day 9. The average sugar consumption
rate was 5.50, 4.75, 5.46, and 1.97 g/L per day for the cases of
glucose, maltose, sucrose and lactose, respectively, and the
corresponding cell yield against sugar was 0.36, 0.43, 0.48, and
1.28 g DW/g sugar.
Based on the above results, it was concluded that lactose was
not beneficial for the cell growth of T. sinense. Lactose can be
hydrolyzed into glucose and b-galactose if there is bgalactosidase activity in the fermentation system, so two
possible reasons could be related to the inhibition effect of
lactose on the T. sinense cell growth, one may be the inhibition
effect of b-galactose on the cell growth, and the other could be
no b-galactosidase activity in the fermentation broth and T.
sinense cells. In order to explain the reason why lactose was not
581
favorable for the cell growth of T. sinense, an experiment was
conducted by using glucose, b-galactose, and lactose as carbon
source. Fig. 5A clearly showed that T. sinense cells grew well in
the existence of b-galactose as sole carbon source, while they
Fig. 5. Effect of carbon source on the cell growth (A), culture pH (B) and sugar
consumption (C) in the submerged fermentation of T. sinense. Symbols for
carbon source: glucose (*), b-galactose (*), and lactose (~).
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Y.-J. Tang et al. / Process Biochemistry 43 (2008) 576–586
could not grow in lactose. This indicated that there is no
inhibition effect of b-galactose on the T. sinense cell growth. In
this respect, the inhibition effect of lactose on the T. sinense cell
growth should not result from b-galactose. When lactose was
used as carbon source, the activity of b-galactosidase in the
fermentation broth and T. sinense cells was determined
according to the method described by Tang and Zhong [28].
There was no activity of b-galactosidase determined in the
fermentation broth and T. sinense cells in the existence of
lactose as carbon source (data not shown). Tang and Zhong [28]
demonstrated the existence of a lactose permease system in the
cells of G. lucidum and the induction of b-galactosidase activity
by lactose feeding when G. lucidum was cultured in the lactose
as a suitable carbon source. In our case, the inhibition effect of
lactose on the cell growth of T. sinense was considered to be
related with no b-galactosidase in the fermentation broth and T.
sinense cells.
Kinetics of EPS accumulation under different carbon source
is shown in Fig. 6A. To surprise, when T. sinense cells grew the
worst in the existence of lactose as carbon source, the highest
EPS production of 1.61 0.40 g/L was obtained. The EPS
production titer was 1.57 0.16, 1.18 0.19, 1.21 0.13, and
1.61 0.40 g/L when glucose, maltose, sucrose and lactose
were used as carbon source, respectively, and the corresponding
EPS productivity of 0.20, 0.13, 0.13, and 0.80 g/L per day. The
results indicated that lactose was favorable for EPS production
and productivity. Similar phenomenon was also claimed by
Tang and Zhong [22] in the submerged fermentation of G.
lucidum, the highest EPS of 0.63 0.02 g/L was obtained
when G. lucidum cells grew the worst in the existence of
sucrose as carbon source. During the submerged fermentation
of mushroom Agaricus brasiliensis, Zou [29] demonstrated that
sucrose was suitable for EPS production although the cells
could not grow well. Tang and Zhong [22] reported that many
short mycelia were observed by microscope when sucrose was
used as carbon source. The poor cell growth and more release of
polysaccharide to the medium may be related with the
morphological change.
Time profiles of IPS content under different carbon source are
compared in Fig. 6B. The specific IPS (i.e., content) accumulation pattern was quite similar when glucose, maltose and lactose
were used as carbon source. While, IPS content in the case of
sucrose was higher than the other three cases after the third day
culture. The maximum IPS content was 11.12 0.24,
10.59 0.53, 12.73 0.30, and 10.60 0.79 mg/100 mg
DW when glucose, maltose, sucrose, and lactose was used as
carbon source, respectively, and the corresponding specific IPS
productivity was 22.80, 20.87, 25.99, and 33.70 mg/g DW per
day. Kinetics of IPS production under different carbon source is
indicated in Fig. 6C. Because of the lowest cell density in the
existence of lactose as carbon source, IPS production was much
lower than the other three cases; despite the IPS content in the
case of lactose was comparable with the case of glucose and
maltose. The IPS accumulation pattern in the case of glucose and
maltose was quite similar, and the IPS production in the case of
sucrose was higher than the other three cases. The total
production of IPS reached 1.55 0.03, 1.43 0.03,
Fig. 6. Effect of carbon source on the production of EPS (A), the content (B),
and production (C) of IPS in the submerged fermentation of T. sinense. Symbols
for carbon source are the same as those in Fig. 4.
1.91 0.05, and 0.38 0.01 g/L for the cases of glucose,
maltose, sucrose, and lactose, respectively, and their corresponding productivity was 0.31, 0.29, 0.38, and 0.13 g/L per day. Not
only IPS content and production, but also IPS productivity
reached the peak values when sucrose was used as carbon source
during the submerged fermentation of T. sinense. Tang and
Y.-J. Tang et al. / Process Biochemistry 43 (2008) 576–586
Fig. 7. Effects of carbon source on the cell growth (hatched bar), the production
of EPS (blank bar) and IPS (dark bar). The error bars in the figure indicate the
standard deviations from three independent samples.
Zhong [22] demonstrated that the maximal IPS production of
1.43 g/L was obtained in the existence of lactose as carbon source
in the submerged fermentation of G. lucidum.
Fig. 7 summarized the significance of carbon source on the
submerged fermentation of Chinese truffle T. sinense. It is
concluded that lactose was favorable for EPS production but
seriously inhibited the cell growth of T. sinense, and sucrose
was the best for both the cell growth and IPS production during
the submerged fermentation of T. sinense. In the submerged
fermentation of G. lucidum, Tang and Zhong [22] demonstrated
that sucrose was favorable for EPS production but seriously
inhibited the cell growth of G. lucidum, while lactose was
beneficial for the cell growth and IPS production. During the
submerged fermentation of a culinary-medicinal mushroom
Lyophyllum decastes in shake-flasks, lactose, glucose and
fructose were identified to be the top three best carbon sources
for mycelial growth with corresponding yields of 6.73, 6.36,
and 6.10 g/L, respectively. Glucose was the best for production
of EPS and IPS with 1.65 g/L and 317 mg/g dry mycelia,
respectively [30]. In the submerged culture of Phellinus gilvus,
Hwang et al. [31] reported that glucose was the most suitable
carbon source for both mycelial biomass and EPS production.
Thus, the response of different culture system to carbon source
was different and a detailed research should be done for a
specific case.
3.3. Effect of initial sucrose concentration
Based on the above results, sucrose was selected as the
suitable carbon source for further studies. The effects of initial
sucrose concentration on the kinetics of the cell growth and
metabolites’ production were investigated in shake flasks.
Table 1 summarizes the effect of initial sucrose concentration
ranging from 20 to 125 g/L on the cell growth (by the maximum
DW) and Tuber polysaccharides production. General speaking,
T. sinense’s cell growth increased in parallel with an increase of
initial sucrose concentrations, while the increase became
slowly when the initial sucrose concentration exceeded 80 g/L
(Fig. 8A). The maximal cell density of 24.07 1.94 g/L
583
obtained at the highest initial sucrose concentration of 125 g/L
is increased by 93% compared to that (i.e., 12.50 0.17 g/L) at
the lowest initial sucrose concentration of 20 g/L. With the
increase of initial sucrose concentration, the cultivation period
appears to be extended and more culture time was needed to
obtain the maximal cell density. The average growth rate
decreased at an initial sucrose concentration of 20–80 g/L, then
kept almost constant with an increase of initial sucrose
concentration (95–125 g/L). The average growth rate (i.e., 0.12
day 1) at the highest initial sucrose concentration of 125 g/L
was only one quarter of that (i.e., 0.47 day 1) at the lowest
sucrose concentration of 20 g/L. The medium lactose was
exhausted at an initial sucrose concentration of 20–80 g/L,
while there was 15.78, 33.42, and 41.11 g/L of residual sucrose
at the end of fermentation (day 16) under initial sucrose level of
95, 110, and 125 g/L, respectively (data not shown). Both the
average sucrose consumption rate and the cell yield against
sucrose decreased at an initial sucrose concentration of 20–
80 g/L, then kept constant with an increase of initial sucrose
concentration (95–125 g/L). This suggested that more biomass
could be obtained at a relatively higher initial sucrose
concentration in the submerged fermentation of T. sinense.
Such a similar phenomenon was also claimed in submerged
cultivation of medicinal mushroom G. lucidum and C. militaris
[22,27]. Cell growth increased in parallel with an increase of
initial sugar concentrations, and the cell yield on sugar
decreased with an increase of initial sugar concentration
[22,27]. While, Zhong with his colleagues [22,27] reported that
the average growth rate kept almost constant when the initial
sugar concentration exceeded 25 g/L in submerged cultivation
of G. lucidum and C. militaris.
Fig. 8B clearly indicated that EPS production was
significantly affected by initial sucrose levels during the
submerged fermentation of T. sinense. General speaking, higher
initial sucrose concentration, more EPS production, and longer
culture time needed to obtain the maximal EPS production. The
maximal EPS production of 2.85 0.04 g/L obtained at the
highest initial sucrose concentration of 125 g/L is increased by
261% compared to that (i.e., 0.79 0.07 g/L) at the lowest
initial sucrose concentration of 20 g/L. Not only EPS
productivity and specific productivity, but also the EPS yield
on the sucrose reached the peak values at an initial sucrose
concentration of 65 g/L. The results suggest that a relatively
higher initial glucose concentration led to higher EPS
production. Such a similar phenomenon was also claimed in
submerged cultivation of medicinal mushroom G. lucidum by
Tang and Zhong [22], whose results indicated that a relatively
higher lactose concentration led to not only higher EPS
production but also higher EPS productivity and yield of EPS
when the initial lactose ranged from 20 to 65 g/L. Actually, in
our work, not only the productivity and specific productivity of
EPS, but also EPS yield on sugar was increased with the
increase of initial sucrose concentration when initial sucrose
levels was not more than 65 g/L. These suggest that our result is
consistent with the results about the fermentation of G. lucidum
[22]. During the submerged fermentation of mushroom A.
brasiliensis, Zou [29] also reported that a relativity higher
584
Table 1
Effect of initial sucrose concentration on the cell growth and production of EPS, IPS during submerged fermentation of T. sinense in shake flasks
Initial sucrose concentration
20 g/L
a
35 g/L
50 g/L
65 g/L
80 g/L
95 g/L
110 g/L
125 g/L
12.50 0.17 (day 4)a 15.97 0.37 (day 5) 18.75 0.46 (day 8) 19.40 0.52 (day 9) 22.81 1.03 (day 11) 22.40 1.04 (day 16) 23.29 1.87 (day 14) 24.07 1.94 (day 16)
0.47
0.38
0.24
0.21
0.18
0.12
0.14
0.12
6.47
6.16
6.11
5.47
5.70
5.21
5.10
5.34
0.60
0.51
0.38
0.42
0.32
0.27
0.28
0.28
0.79 0.07
1.17 0.06
1.99 0.15
2.25 0.17
2.33 0.22
2.19 0.05
2.65 0.07
2.85 0.04
0.07
0.12
0.22
0.23
0.13
0.12
0.15
0.17
29.53
28.67
36.20
45.48
25.63
24.39
29.25
31.13
7.43
8.44
12.04
12.12
7.98
6.35
6.79
7.00
10.02 0.40
12.02 0.33
12.72 0.57
12.78 0.53
12.99 0.83
12.48 0.16
11.66 0.80
12.19 1.11
1.21 0.07
1.92 0.08
2.26 0.10
2.31 0.07
2.92 0.20
2.67 0.08
2.69 0.01
2.69 0.31
0.30
0.38
0.28
0.29
0.26
0.19
0.19
0.17
59.46
61.97
46.01
52.58
41.83
36.44
36.00
31.36
31.15
26.40
15.54
15.15
11.75
9.09
8.32
7.05
Culture time when the maximum cell mass was reached.
Y.-J. Tang et al. / Process Biochemistry 43 (2008) 576–586
Maximum DW (g/L)
Average growth rate
(per day)
Average sugar
consumption
rate (g/L per day)
Cell yield on sugar
(g DW/g sugar)
Maximum EPS
production (g/L)
EPS productivity
(g/L per day)
EPS yield on sugar
(mg/g sugar)
EPS specific
productivity
(mg/g DW per day)
Maximum IPS content
(mg/100 mg DW)
Maximum IPS
production (g/L)
IPS productivity
(g/L per day)
IPS yield on sugar
(mg/g sugar)
IPS specific
productivity
(mg/g DW per day)
Y.-J. Tang et al. / Process Biochemistry 43 (2008) 576–586
585
sucrose concentration increased from 20 to 80 g/L. When the
initial sucrose concentration continued to increase up to 125 g/
L, there was a little bit decrease of the IPS production, which
almost kept constant at the level of 2.67–2.69 g/L. The results
suggest that a higher initial sucrose concentration (20–80 g/L)
led to higher IPS production, while relatively higher sucrose
concentration (>80 g/L) inhibited the IPS biosynthesis. In the
fermentation of G. lucidum, Tang and Zhong [22] also observed
the inhibitory effect of higher initial lactose concentration on
the IPS biosynthesis and indicated that an initial lactose
concentration of 50 g/L was favorable to IPS production. Mao
et al. [27] reported that the highest production and productivity
titers of cordycepin were obtained at 40 g/L initial glucose, and
indicated that a high initial glucose concentration (55 or 70 g/L)
was unfavorable to cordycepin biosynthesis. Here, the osmotic
pressure caused by a high sugar concentration may be
detrimental to the metabolite biosynthesis although the cell
growth was not inhibited. In ganoderic acid biosynthesis by G.
lucidum [22], the inhibitory effect of osmotic pressure caused
by a relatively high initial sugar concentration on the metabolite
biosynthesis was claimed.
In a conclusion, initial sucrose concentration (i.e., 20–125 g/
L) significantly affected the cell growth and metabolites’
production during the submerged fermentation of Chinese
truffle T. sinense. At the highest initial sucrose concentration of
125 g/L, both the maximal cell density (i.e., 24.07 1.94 g/L)
and EPS production (i.e., 2.85 0.04 g/L) reached their peak
values, which was enhance by 93% and 261% compared to
those (i.e., 12.50 0.17 g DW/L and 0.79 0.07 g EPS/L) at
the lowest initial sucrose concentration of 20 g/L. The maximal
IPS production of 2.92 0.20 g/L was obtained at an initial
sucrose concentration of 80 g/L, which was enhanced by 141%
compared to that (i.e., 1.21 0.07 g/L) at the lowest initial
sucrose concentration of 20 g/L. General speaking, an initial
sucrose concentration of 80 g/L was favorable for the
submerged fermentation of Chinese truffle T. sinense.
4. Conclusion
Fig. 8. Effects of initial sucrose concentration on the cell growth (A), the
production of EPS (B), and IPS (C) during the submerged fermentation of T.
sinense. The error bars in the figure indicate the standard deviations from three
independent samples.
initial glucose concentration with the range of 20–50 g/L as
investigated led to higher production, productivity and yield of
EPS.
Fig. 8C shows the significance of initial sucrose concentration on the total accumulation of IPS. The IPS production was
increased from 1.21 0.07 to 2.92 0.20 g/L when the initial
In this work, a novel submerged fermentation process of
Chinese truffle T. sinense for the efficient production of truffle
mycelia and bioactive metabolites—Tuber polysaccharides was
developed. The effects of inoculation density, carbon source
and initial sucrose concentration were studied in shake flasks in
order to enhance the cell density and Tuber polysaccharides
production and productivity. Control of inoculation density was
significant for the submerged fermentation of T. sinense.
Relatively higher inoculation density was favorable for Tuber
polysaccharides production and productivity, while the maximal cell density reached the peak value at the lower inoculation
density. Lactose was beneficial for EPS production but
seriously inhibited the cell growth of T. sinense, and sucrose
was the best for both the cell growth and IPS production.
General speaking, not only the maximal cell density but also the
EPS production increased in parallel with an increase of initial
sucrose concentrations with the range of 20–125 g/L as
investigated. While, relatively higher sucrose concentration
586
Y.-J. Tang et al. / Process Biochemistry 43 (2008) 576–586
(>80 g/L) inhibited the IPS biosynthesis and the maximal IPS
production was obtained at an initial sucrose concentration of
80 g/L. The fundamental information obtained in this work is
beneficial for further development of Chinese truffle T. sinense
submerged cultivation process for hyperproduction of Tuber
polysaccharides on a bioreactor scale. Such work may also be
helpful to other mushroom fermentations for bioactive
metabolites production.
Acknowledgements
The financial supports from the National Natural Science
Foundation of China (NSFC, project No. 20706012), National
High Technology Research and Development Key Program of
China (Project No. 2007AA021506), the Scientific Research
Foundation for the Returned Overseas Chinese Scholars
(Ministry of Personnel and State Education Ministry), Hubei
Provincial Innovative Research Team in University (Project
No. T200608), Hubei Provincial Science Foundation for
Distinguished Young Scholars (Project No. 2006ABB034),
Hubei Provincial International Cooperation Foundation for
Scientific Research (project No. 2007CA012), the Science and
Technology Commission of Wuhan Municipality ‘‘Chenguang
Jihua’’ (Project No. 20065004116-31), and the Scientific
Research Key Foundation from Hubei University of Technology (Project No. 306.18002) are gratefully acknowledged. YaJie Tang also thanks the Chutian Scholar Program from Hubei
Provincial Department of Education, China (2006).
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