Microscopic crystalline inclusions in Mahón cheese

Food Science and Technology International (1997) 3, 43-47
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Microscopic crystalline inclusions in Mahón cheese
Inclusiones cristalinas microscópicas en el queso Mahón
M. Frau1, A. Mulet2, S. Simal1, J. Massanet1 and C. Rosselló*1
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Department of Chemistry, University of Illes Balears, Ctra de Valldemosa km 7,5, 07071 Palma de Mallorca, Spain
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Food Technology Department, Universidade Politécnica, Cno. Vera s/n, 46071, Valencia, Spain
The incidence, structure and composition of microscopic crystalline inclusions have been studied
in 32 samples of Mahón cheese (non-cooked pressed cheese manufactured from cow’s milk) at
different ripening times. Samples were observed by light microscopy (LM), transmission electron
microscopy (TEM) and scanning electron microscopy (SEM). Circular, oval or kidney-shaped structures approximately 20 mm in diameter (range 4–65 mm), were observed in all Mahón cheese samples.
The quantity of crystalline inclusions per unit surface observed in Mahón cheese was heterogeneous, with an average of 30 crystals/m2. It was possible to differentiate two different structures
in the crystals: the nucleus with disorganized morphology and the cortex with radial laminar
morphology. The ratio between nucleus and inclusion diameters was 0.2–0.5. Crystalline inclusions observed in Mahón cheese were studied by X-ray microanalysis in SEM and TEM. Calcium
and phosphorous were found to be the most common elements present in these inclusions.
Keywords: cheese, crystaline inclusions, electron microscopy
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Se ha estudiado la incidencia, estructura y composición de inclusiones cristalinas detectadas en
queso Mahón mediante técnicas de microscopía óptica, microscopía electrónica de transmisión y
de barrido. Se analizaron 32 muestras de queso Mahón (queso de leche de vaca de pasta prensada no cocida) en diferentes estadios de su maduración. Se observaron estructuras circulares,
ovales o arriñonadas de aproximadamente 20 mm de diámetro (encontrados entre 4 y 65 mm), con
una distribución heterogénea en todas las muestras (con una media de 30 cristales/m2). En las
inclusiones se pudo diferenciar dos estructuras: el núcleo, de morfología desorganizada, y la
corteza, de morfología laminar radial. La relación entre el tamaño del núcleo y de la inclusión fue
de 0,2–0,5. La composición de las inclusiones cristalinas observadas se determinó mediante
microanálisis de rayos X. El calcio y el fósforo fueron los elementos más detectados.
Palabras clave: queso, inclusiones cristalinas, microscopía electrónica
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INTRODUCTION
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Microscopic crystalline inclusions in different cheese
types have often been reported. The presence of calcium phosphate crystalline inclusions was described
in Cheddar cheese (Brooker et al., 1975; Kalab, 1980),
Edam and Gouda cheeses (Kalab, 1977), processed
cheese (Kalab et al., 1987), mould-ripened cheese
(Brooker, 1987) and semi-hard sheep cheese (Fontecha,
1991). Calcium lactate crystals were observed in
* To whom correspondence should be sent.
Received 25 May 1996; accepted 23 September 1996.
1082–0132 © 1997 Chapman & Hall
Cheddar cheese (Brooker et al., 1975) while trisodium
phosphate (Kalab et al., 1987), sodium citrate (Rayan
et al., 1980, Kalab et al., 1987), tetrasodium pyrophosphate and aluminium and sodium phosphate crystals
(Rayan et al., 1980) have been reported in cheese
processed with emulsifying salts.
The origin of calcium phosphate crystals in cheese
has not been clearly defined in the literature although
there is some evidence of different effects of its formation. Brooker (1987) concluded that in mould-ripened
cheese, the escape of cell contents from moribund
hyphae contributed not only to a rise in the pH, but
also that the released cell organelles may act as nucleating centers. Kalab (1980) demonstrated that bacteria
M. Frau et al.
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have similar effects in Cheddar cheese. Brooker et al.
(1975) observed the presence of calcium phosphate
crystalline inclusions in cheeses made without salting,
pressing or starter bacteria. These authors considered
the development of acid in the curd as a determinant
of the Ca and P content.
Calcium phosphate may be present in different
compositions and forms, either amorphous or
crystalline (Walstra and Jenness, 1987). Therefore,
observed inclusions in milk and cheese could contain
amorphous calcium phosphate (Brule and Lenoir,
1990). Microscopic crystalline inclusions in noncooked, pressed cow’s milk cheese have not been
described previously. The present study was carried
out in order to examine their incidence, structure and
composition by light microscopy (LM), transmission
electron microscopy (TEM) and scanning electron
microscopy (SEM).
MATERIAL AND METHODS
Samples
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Mahón cheese, a non-cooked pressed cheese manufactured in Menorca (Spain) from cow’s milk, was
used. Cheeses are parallelepiped with round edges,
of approximately 0.2 m 3 0.2 m 3 0.08 m and 2.5 kg
weight. It is manufactured according to the methodology described by the Mahón cheese Appellation of
Origin, where regulation differentiates four different
kinds of Mahón cheese according to their ripening
time: fresh (less than 10 d of ripening), half-ripened
(from 2 to 5 months of ripening), ripened (from 5 to
10 months of ripening) and old-ripened (more than 10
months of ripening).
Thirty two different samples of Mahón cheese
purchased from local markets have been used in this
study: eight fresh, eight half-ripened, eight ripened
and eight old-ripened samples. Slices of cheese
0.01 m thick and perpendicular to the larger surface
were taken from Mahón cheese. Rectangular blocks,
2 3 5 3 5 mm3 were cut from the centre of the cheese
slices, using a razor blade. Samples were immediately
immersed in 2.5 ml/L glutaraldehyde solution in
0.1 M cacodylate buffer, pH 7.3 for 24 h.
series of ethanol/water mixtures, 100% ethanol and
100% xylol before embedding in paraffin; sections of
1–4 mm thick were cut on a Leitz-1512 microtome and
stained with haematoxylin and eosin to visualize the
general structure, and by the von Kóssa technique to
visualize phosphate and carbonate groups; (ii)
sections (1–2 mm thick) of samples embedded in
Epon-Araldite (for observation by a transmission electron microscope) were stained with hot Richardson
colouring to visualize the general structure, and by
the von Kóssa technique to visualize phosphate and
carbonate groups.
Scanning electron microscopy and X-ray analysis (SEM)
All specimens were examined by SEM following the
modified method proposed by Frau et al. (1993) which
involves cryofracturing the samples before mounting
and coating them with gold. All X-ray microanalysis
was carried out using a Kevex 7500 microanalyser,
using Quantex EV software. A semi-quantitative
determination of the composition of the crystalline
inclusions and the spatial distribution of a number of
elements including calcium (Ca), phosphorus (P),
magnesium (Mg), sodium (Na) and chlorine (Cl) were
determined using digital X-ray mapping procedure.
Transmission electron microscopy and X-ray analysis
(TEM)
The cheese samples fixed in the glutaraldehye were
trimmed to particles slightly over 0.6 mm in diameter
and placed into fresh glutaraldehyde fixative for 24 h.
The particles were then washed with distilled water
and transferred to a solution of osmium tetroxide
(20 g/L) in 0.1 M cacodylate-HCl buffer, pH 7.2, for 2
h post-fixation. The post-fixed particles were washed
with water, dehydrated in a graded series of acetone/
water mixtures and 100% acetone, transferred to
propylene oxide (100%) for 1 h, and embedded
in Epon-Araldite. Sections (approximately 90 nm
thick) were cut on an Ultracut-E (Reichert-Jung) ultramicrotome, stained with lead citrate and examined
in a Hitachi 600 transmission electron microscope
operated at an accelerating voltage of 50–70 kV. All
X-ray microanalysis was done using a Link Pentafet
microanalyser with DEMON Plus software.
Methods
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Light microscopy (LM)
Samples were observed with a Zeiss D-7082 photo
microscope. Two different methodologies were used:
(i) after fixing, samples were dehydrated in a graded
RESULTS AND DISCUSSION
Circular, oval or kidney-shaped structures approximately 20 mm in diameter (range 4–65 mm), were
observed in all Mahón cheese samples using LM
FOOD SCIENCE AND TECHNOLOGY INTERNATIONAL (1997) 3(1)
Crystalline inclusions in Mahón cheese
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Figure 1. Light microscopy image. Bar represents
200 mm. Large arrows, bacterial colonies; small arrows,
crystalline inclusion.
Figura 1. Imagen obtenida con microscopio óptico.
La barra representa 200 mm. Flecha grande, colonias
bacterianas; flecha pequeña, inclusión cristalina.
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Figure 2. SEM image of integer calcium phosphate
crystal. Bar represents 20 mm.
Figura 2. Imagen obtenida mediante SEM de una
cristal de fosfato cálcico. La barra representa 20 mm.
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(Figure 1), SEM (Figure 2), and TEM (Figure 3). Their
morphology and size are similar to those of the
crystalline inclusions observed in Cheddar (Brooker
et al., 1975, Kalab, 1980), Edam and Gouda cheeses
(Kalab,1977), processed cheese (Kalab et al., 1987),
Figure 3. TEM image. Microstructure of calcium phosphate crystal. Bar represents 5 mm.
Figura 3. Imagen obtenida mediante TEM. Microestructura de un cristal de fosfato cálcico. La barra
representa 5 mm.
mould-ripened cheese (Brooker, 1987) and semi-hard
sheep cheese (Fontecha, 1991).
The quantity of crystalline inclusions per unit
surface observed in Mahón cheese was heterogeneous, with an average of 30 crystals/m2. In the
literature, it was found that the figures reported by
different authors for Roquefort, Tilsit and Emmental
cheeses were 50, 115 and 200 crystals/m2 respectively
(Brooker et al., 1975).
Brooker et al. (1975) observed an increase in the
quantity of crystalline inclusions during the first
month of ripening in Cheddar cheese. In this work,
it was not possible to establish any relationship
between the number of crystalline inclusions or their
size, and the ripening time in Mahón cheese.
When samples treated with paraffin wax were
examined by LM, specific localization of the crystals
was not observed, their distribution inside the curd
particles and on the lines of curd particle fusion was
similar. On the other hand, Brooker et al. (1975)
observed structures with analogous morphology in
Cheddar cheese that were noticeably concentrated in
areas probably corresponding to the lines of curd
particle fusion.
From the observation of the crystalline inclusions
using SEM (Figure 4) and TEM (Figure 5) it was
possible to differentiate two different structures:
a central zone of disorganized morphology, the
nucleus, and a peripheral zone, the cortex that
had radial laminar morphology. The ratio between
nucleus and inclusion diameters was 0.2–0.5.
FOOD SCIENCE AND TECHNOLOGY INTERNATIONAL (1997) 3(1)
M. Frau et al.
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Figure 4. SEM Image. Microstructure of two calcium
phosphate crystals. Two different structures are
apparent: a nucleus and a cortex. Bar represents 20 mm.
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Figura 4. Imagen obtenida mediante SEM. Microestructura de dos cristales de fosfato cálcico o fraccionados. Se diferencian el núcleo y la corteza. La barra
representa 20 mm.
mainly by protein. Crystals were approximately
30 nm in width (range 20–50 nm); length was not
measured. This description agrees with that proposed
by Brooker et al. (1975) for calcium phosphate crystals observed by TEM in Cheddar cheese. According
to these authors crystal size is very variable, up to
4–5 mm long and 25 nm diameter, and each crystal is
surrounded by casein submicelles of approximately
25 nm diameter. Posner et al. (1984) experimentally
obtained crystals formed by hydroxyapatite of 20 3 5
3 5 nm with similar morphology to those described
before.
In sections of material 1–2 mm thick embedded in
Epon-Araldite for electron microscopy, crystalline
inclusions were not stained by the von Kóssa technique; nevertheless, samples treated with paraffin
partially coloured using this technique. This singular
behaviour could be a consequence of the solubilization of the crystals during sample processing (this
hypothesis was advocated by Brooker et al., 1975), or
due to their release from the sample surface if they
were not embedded in the resin.
Crystalline inclusions observed in Mahón cheese
were studied using microanalysis of X-rays in SEM
and TEM and Ca and P were found to be the most
common elements. Through SEM, by digital X-ray
mapping, higher calcium and phosphorous concentrations were shown to correspond to the location of
the inclusions (Figures 6 and 7). In the microanalysis
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Figure 5. TEM image of microstructure of a calcium
phosphate crystal. Bar represents 1 mm.
Figura 5. Imagen obtenida mediante TEM. Microstructura de un cristal de fosfato cálcico. La barra representa 1 mm.
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The cortex structure, according to the TEM observations (Figure 5), could be formed by units with prismatic morphology, radially distributed around the
nucleus. The amorphous material which surrounded
the crystals and kept them grouped could be formed
Figure 6. Diagram of energy spectrometric analysis of
calcium phosphate crystals in Mahón cheese. Peaks
of phosphorus (P), chlorine (Cl) and calcium (Ca) are
identified.
Figura 6. Análisis espectrométrico. Diagrama energético de un cristal de fosfato cálcico del queso Mahón.
Se han identificado los picos de fósforo (P), cloro (Cl)
y calcio (Ca).
FOOD SCIENCE AND TECHNOLOGY INTERNATIONAL (1997) 3(1)
Crystalline inclusions in Mahón cheese
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REFERENCES
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Figure 7. Digital X-ray mapping. Ca location in the
calcium phosphate crystal.
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Figura 7. Registro digital de rayos X. Localización del
Ca en el cristal de fosfato cálcico.
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carried out on samples prepared for TEM examination, higher Ca and P concentrations at these points
were not observed. These results are totally opposed
to those presented by Pommert et al. (1988) who
obtained a P/Ca ratio of 0.7, which corresponded to
calcium phosphate. Nevertheless, the methodology
for sample preparation used by these authors for crystals microanalysis observation by TEM of processed
cheese samples was different to that described in this
work. The solubilization or release of calcium phosphate crystals during sample preparation for TEM
could account for negative microanalysis results.
Brooker B.E. (1987). The crystallization of calcium phosphate
at the surface of mould-ripened cheeses. Food Microstructure 6: 25–33.
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(1988). Observation and analysis of crystalline phases in
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and structure of precipitated hydroxyapatites. In: Nriagu
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ACKNOWLEDGEMENTS
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The authors wish to thank Dr F. Hierro and M. Pocovi
for their technical assistance and the Mahón cheese
Appellation of Origin Council for their collaboration.
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