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Influence of the fat content of Cheddar cheese on retention and localization of starters
ELSEVIER
Int. Dairy Journal 6 (1996) 729-740 Copyright 0 1996 Published by Elsevier Science Limited Printed in Ireland. All rights reserved 0958-6946/96/U 5.00 + 0.00 0958-6946(95)00068-2
Influence of the Fat Content of Cheddar Cheese on Retention and Localization of Starters
Edith Laloy,” Jean-Christophe Vuillemard,” Morsi El Sodab & Ronald E. Simard” Yentre de Recherche STELA, Universitk Lava1 QuCbec, Canada GlK 7P4 ‘Department of Agricultural Industries, University of Alexandria, Alexandria, Egypt (Received 20 July 1995; accepted 29 October 1995)
ABSTRACT Microbiological counts of fat-free, 50% reduced-fat and full-fat Cheddar cheeses were obtained to determine the population of starter bacteria in the different cheeses. Microbiological counts and observations on the cheese curd by transmission electron microscopy indicated that bacterial populations in the curd were directly related to the fat content of cheese. Electron microscopy examinations of cheeses ripenedfor less than one month showed that cells were either directly in contact with the fat globule membrane or located at the casein-fat interface, However, a higher number of bacterial cells appeared to be in close contact with the fat globule membrane. After one and two months of ripening, the number of ghost cells increased and bacteria seemed to be embedded in the milk fat globule membrane or directly in contact with the inside of fat globules. Copyright 0 1996 Elsevier Science Limited
INTRODUCTION Cheese ripening is largely determined by enzymatic reactions which can be divided into three main groups: glycolysis, lipolysis and proteolysis. The last, usually the most complex, also appears to be the major event in cheese maturation (Manning & Nursten, 1985; Visser, 1993). While texture is correlated to the breakdown of the protein network (Lawrence et al., 1987), peptides, amino acids and products of amino acids catabolism have been shown to contribute directly to Cheddar cheese flavour (Manning & Nursten, 1985). The contribution of starter bacterial proteinases and peptidases to this process is important (Law, 1984). Law et al. (1993) showed that starter proteinases were required for the accumulation of small peptides and free amino acids, involved in flavour development of Cheddar cheese. 729
E. Laloy et al.
The role of lipolysis in Cheddar cheese flavour formation is more equivocal (Law, 1984). There is no clear evidence that volatile fatty acids are directly involved in Cheddar flavour. However, it is known that low-fat and fat-free Cheddar cheeses are characterized by poor and atypical flavour (Banks et al., 1989). If lipolysis is not a noteworthy event of cheese flavour formation, then fat content of cheese may have other indirect effects on this process. Considering that starter bacteria play an important role in Cheddar flavour formation and that fat is necessary for flavour expression, a relationship between the fat content of cheeses and starter bacteria should exist. The retention and localization of starter bacteria in the cheese curd during ripening must be studied to explain this potential relationship. Few data dealing with this subject are available in the literature. Dean et al. (1959) were the first to note the tendency of cheese bacteria to congregate at the fat droplet surfaces in Cheddar cheese. The same phenomenon was observed by Umemoto et al. (1978) in Cheddar and Tunick et al. (1993) in Mozzarella cheeses. The present study aimed at determining the role of fat content in cheese milk on starter bacteria retention, localization and evolution during ripening.
MATERIALS
AND METHODS
Bacterial strains Lactococcus lactis subsp. cremoris KB was isolated in our laboratories from the commercial starter MA016 (RhGne Poulenc, Brampton, Ontario). The mixed starter, containing L. lactis subsp. cremoris and L. lactis subsp. lactis strains, was the Redi-Set DVS culture (Chr. Hansen’s Laboratory, Milwaukee, Wisconsin, USA). The strain KB was prepared by subculturing twice (12 h at 3O”C), from a frozen stock, in autoclaved (1 15”C, 10 min) 10% (w/w) reconstituted skim milk. The Redi-Set DVS culture was thawed and diluted (l/10) in milk immediately prior to its incorporation to the cheese milk.
Cheesemaking Three independent Cheddar cheese manufacturing trials were carried out following the procedure described by Kosikowski (1977). Small Cheddar cheeses of 200 g or 1 kg were manufactured either in beakers placed in a controlled water bath (trials 1 and 2) or using a laboratory computer controlled cheese plant (INRA, Poligny, France) for trial 3. In the latter case, temperature was monitored by an automated control station (AGIL, Gleize, France). Before each trial, fresh milk was pasteurized at 72°C for 15 s and skimmed by centrifugation at 32°C. Skimmed milk was divided into three parts. Part 1 was used for the fat-free cheese production. Cream was added to Parts 2 and 3 of skimmed milk to produce 50% reduced-fat cheese and full-fat cheese, respectively. The final volume of each part of milk was the same, and the casein/fat ratios of Part 1, Part 2 and Part 3 were adjusted to 25f2, 1.6f0.2 and 0.7f0.1, respectively. The same preparation of L. lactis subsp. cremoris KB was used as starter (2% v/v) for cheeses in trial 1. Trials 2 and 3 were carried out with the Red&Set DVS, taken from the same can, diluted as described above and added to a 2% (v/v)
Fat content of Cheddar cheese
731
final ratio. The pH was monitored during cheese production; the Cheddaring was stopped when pH reached 5.2fO.l; the cheeses were vacuum packaged and ripened for 60 days at 12°C. Bacterial enumeration Using a Lab-Blender 400 (Stomacher, London, England), fresh cheese samples (10 g) were homogenized in a sterile bag with a 100 mL final volume of 0.10% peptonized water. Total lactic acid bacteria population was determined by counting diluted samples on Ml7 agar (Difco Laboratories, Surrey, UK) after 48 h at 30°C. Total bacterial enumerations were also carried out on the fresh milk used for each cheese production. Transmission electron microscopy Cheese samples were taken for electron microscopy after 24 h, one week, one month and two months ripening. They were cut into small cubes of about 1 mm sides and fixed in 3% (v/v) glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2, Caco buffer) for 15 h at 4°C washed with Caco buffer and then in 1% (v/v) osmium tetroxide (in Caco buffer) for 10 h at 4°C. Samples were then dehydrated in a graded ethanol series (30, 50, 70, 80,95 and 100%) and embedded in Epon 812 (Benhamou, 1991). Ultrathin sections (0.1 pm) were cut with an ultramicrotome, collected on formvarcoated nickel grids, stained with uranyl acetate during 5 min, intensively washed with water, lightly dried, stained with lead citrate during 5 min, washed again and dried before examination with a JEOL 1 200 EX (Tokyo, Japan) electron microscope at 80 kV. At least three cheese cubes per treatment and sample time were cut, and five to 10 ultrathin sections per cube were observed. Photomicrographs show the most representative fields of all the sections examined for one sample.
RESULTS AND DISCUSSION Bacterial population in fresh cheese curds Table 1 shows the effect of fat content on the population of starters in Cheddar cheese, just before pressing. It appears that bacterial counts were related to the fat content of cheeses. Compared to the fat-free cheese, starter populations were 30100% higher and 4-lo-fold higher in 50% reduced-fat cheese and in full-fat cheese, respectively. Bacterial counts of fresh milk were always less than lo2 cfu mL_‘. Considering that the same amount of starter bacteria was added to each of the three parts of milk and that the starter bacteria were the major bacterial population, it is possible to conclude that the amount of milk fat directly influenced the retention and/or the growth of starter bacteria in the fresh curd. These results contradict the work of Tunick et al. (1993), who reported that low-fat Mozzarella cheese contained about 50% more starter than high-fat cheeses. However, in their work, they prepared low-fat cheese at a temperature more conducive to bacterial survival and proliferation than was the temperature of high-fat cheese. Moreover, bacterial strains (Luctobacillus bulgaricus and Streptococcus thermophilus) were different than ours. Similarly to our results,
732
E. Laloy
et al.
TABLE 1
Starter Bacteria Population in Cheddar Cheese Curds Cheese
Fat-free 50% reduced-fat Full-fat
Trial I Strain KB
log 2*10g 4*10g
Trial 2 Mixed starter (cfu g-’ of cheese)
lo6 1,3*106 10*106
Trial 3 Mixed starter
1,1*106 1,5*106 8*106
Quinones et al. (1994) reported that counts of Salmonella and Listeria, initially present in milk in the same quantity, were lower in low-fat compared to full-fat Cheddar cheeses. The higher bacterial counts found in high-fat cheese might be due to a higher retention rate of starter bacteria in the curd or an optimal growth of starter in the high-fat curd during the 6-8 h of production. The first hypothesis is more likely, since temperature and pH, the two major growth-limiting factors for lactic acid bacteria in Cheddar cheese, were controlled and maintained identically for the different cheese production trials. Moreover, there was no difference in the moisture in non-fat-substance between the different types of cheeses (54.2f1.5). The higher retention rate of starter bacteria in high-fat cheese curd could be explained by an active relationship between fat globules and cells, which are retained during the syneresis in high-fat cheeses more than in low-fat ones. Another hypothesis concerns the role of milk fat globules as ‘stoppers’, which could interfere with the flow of whey containing starter cells. In the latter situation, bacteria would be stopped by the fat globules and then mechanically aggregated around them. However, the microscopic observations show that the active relationship should not be excluded. Detection of starter bacteria in the cheese microstructure Figures 1 and 2 show electron micrographs, with about the same magnification, of fat-free and full-fat cheeses, respectively, produced with the commercial mixed starter. The cheese microstructure is consistent with previous observations of lowfat and full-fat Cheddar cheese (Mistry & Anderson, 1993; Kalab, 1993) consisting mainly of a protein matrix in which fat globules are dispersed. Fat globule numbers and area were found to decrease as the cheese fat content decreased. In fat globules, the fat globule membrane was identified as the grey structure, either as a thin circle at the periphery of the globule or a whole web covering the globule. This could be explained by differences in the orientation of the section plane toward the membrane. Figures 1 and 2 show the presence of starter bacteria in the curd after 1 day of ripening. They constituted the major bacterial population observed, and their morphology was similar to the morphology of L. Zactis starter, detected in SaintPaulin cheese by Chapot-Chartier et al. (1994). These microscopic observations also provide an illustration of the differences in starter quantity between fat-free (Fig. 1) and full-fat (Fig. 2) Cheddar cheeses.
Fat content of Cheddar cheese
733
Fig. 1. Transmission electron micrograph of fat-free Cheddar cheese produced with a commercial mixed starter, after 1 day of ripening. Large and thin arrows indicate, respectively, starter bacteria and fat globules. Bar represents 1 pm.
Localization of starter bacteria in the cheese microstructure Electron microscopic examination of about 400 bacteria on several sections of full- and low-fat fresh cheeses containing both types of starter showed that about 85% of starter bacteria (L. Zactis subsp. cremoris KB or mixed starters) were localized in the peripheric area of fat globules. Figure 2 illustrates this observation and confirms earlier studies on starter localization in Cheddar cheese curd (Dean et al., 1959; Umemoto et al., 1978). When the cheese sample preparation used by Dean et al. (1959) was followed, it was difficult to observe the bacteria in the cheese curd. However, the tendency of bacteria to congregate at the fat globule surface was noticeable. On the other hand, the method used by Umemoto et al. (1978) was similar to ours and the contrast obtained for the cheese microstructure elements made it very easy to observe the bacteria, clearly shown to be localized in limited areas of the cheese curd close to fat globules. It is possible to contrast these results with the work of Brooker et al. (1975), who identified crystalline inclusions in spaces between the fat and casein phases of l-month-ripened cheeses and hypothesized that the inclusions corresponded to former pockets of residual whey. Therefore, the area between fat globules and casein matrix would correspond to a hydrophilic medium suitable for bacteria to be dispersed, at least during the early stage of the ripening. Moreover, the phenomenon of milk coagulation has been shown to induce the formation of large interconnected clusters of caseins (Kalab, 1993), which become very dense
734
E. Ldoy et al.
Fig. 2. Transmission electron micrograph of full-fat Cheddar cheese produced with a commercial mixed starter, after 1 day of ripening. Large and thin arrows indicate, respectively, starter bacteria and fat globules. Bar represents 1 pm.
after pressing. As well, Kimber et al. (1974) suggested that the reduction of spaces between casein clumps would bring fat globules and bacteria, which initially occupy these spaces, closer together. Detailed microscopic observations of the periphery of fat globules also gave more information about the relationship between starter bacteria and the fat globule membrane (Figs 3 and 4). During the early stage of ripening (less than 14 days), bacteria appeared either directly in contact with the external face of fat globule membranes (Fig. 3) or were found in a limited area around the fat globule (Fig. 4), that was probably filled with residual whey (Brooker et al., 1975). Consequently, it seems possible that an active interaction between starter bacterial cells and the fat globule membrane is occurring.
Evolution of starter bacteria in the cheese microstructure during ripening The morphologies of intact (Fig. 5a, b), lysed (Fig. 6) and ghost cells (Figs 7 and 8) are easily differentiated. After one month ripening, some cells appeared lysed or were already transformed into ghost cells (Figs 6, 7 and 8). After two months ripening, the proportion of ghost cells was higher. No differences can be seen between the two types of starters used. These results agree with Chapot-Chartier et al. (1994), who noticed that less than 10% of Lactococcus luctis subsp. cremoris starter cells were still viable after two months of ripening. showed that ghost cells lost their cell walls and sometimes to yield larger units.
Umemoto et al. (1978) fused with one another
Fat content of Cheddar cheese
Fig. 3. Starter bacteria (strain KB) in close contact with the fat globule membrane
(arrow), in full-fat Cheddar cheese ripened for 7 days. Bar represents 200 nm. Electron micrographs, taken after one month (Figs 5a, b and 6) and two months (Figs 7 and 8) of ripening, show that the interaction, noted above, between starter cells and the fat globule membrane became more and more intimate with time. In Fig. 5a, the fat globule membrane appears altered in the contact area with the starter cell. This deformation could be explained by proteolysis resulting from the starter proteinases, leading to a weakening of the casein matrix in the proximity of fat globules. The pressure on the fat globule exerted by the integrity of the casein network would then decrease and fat globule membranes would tend to stretch out. This hypothesis has already been proposed by Kiely et al. (1993) in an explanation of the coalescence of fat globule membranes between different fat globules in Mozzarella cheese. However, Fig. 5b suggests yet another type of relationship between starter cells and the fat globule membrane. In this electron micrograph, starter bacteria seem to directly alter the organization of the membrane, which becomes thinner and curves in, towards the inside of the fat globule. This phenomenon is difficult to explain and the hypotheses are numerous: mechanical pressure of bacteria on the fat globule membrane, enzymatic activity, etc. Even though viable cells still appeared at the external face of the fat globule membrane after one month of ripening, the localization of ghost cells was very different. Figure 6 shows that ghost cells could be included into the membrane. Observations of more than 100 ghost cells, on several sections, led to the conclusion that all ghost cells, after one or two months of ripening, were either included into the fat globule membrane (Figs 6 and 7) or appeared directly in contact with the inside of fat globules (Fig. 8).
136
E. Laloy et al.
Fig. 4. Starter bacteria (strain KB) in a limited region near the fat globule membrane (arrow), in full-fat Cheddar cheese ripened for 14 days. The effect of bacterial proteolysis of casein is also visible (PC). Bar represents 400 nm.
CONCLUSIONS This study showed that the fat content of cheese milk directly influenced the number of starter cells retained in the curd and subsequently the potential extent of proteolysis during Cheddar cheese ripening. Consequently, the lower number of starter cells in low-fat cheese could explain the lack of flavour of low-fat Cheddar cheeses. Moreover, the observation of a dynamic evolution of structure and localization of fat globules and starter cells during ripening suggested an active interaction between them. This observation raised numerous questions, such as the role of the fat globule membrane on cell activity and autolysis and the nature of the ghost cell compounds retained in the fat globules. A better understanding of this overall interaction could provide the basis for the optimization of low-fat Cheddar cheese flavour and texture. ACKNOWLEDGEMENTS This work would have never been realized without the advice and the expertise of Dr Nicole Benhamou (Departement de Phytologie, Universite Laval, Quebec, Canada), who also allowed us to use her electron microscopy equipment. Special thanks to her. The authors also thank Dr M. El Abboudi for his help in the cheese production and L. Lamboley for providing the Lactococcus lactis ssp. cremoris KB strain.
Fat content of Cheddar cheese
Fig. 6. Lysed (thin arrow) and ghost (large arrow) cells of strain KB, in fuI1-fat Cheddar cheese ripened for 1 month. Bar represents lPm*
Fig. 7. Ghost cells (arrows) included in the fat globule membrane, of full-fat Cheddar cheese produced with a commercial mixed starter and ripened for 2 months. Bar represents 1 m.
Fat content of Cheddar cheese
739
Fig. 8. A ghost cell in direct contact with the inside of a fat globule, in full-fat Cheddar cheese produced with a commercial mixed starter and ripened for 2 months. Bar represents 200 nm.
REFERENCES Banks, J.M., Brechany, E.Y. & Christie, W.W. (1989). The production of low fat Cheddartype cheese. J. Sot. Dairy Technol., 42, 69. Benhamou, N. (1991). Cell surface interactions between tomato and Clavibacter michiganense subsp. michiganense: Localization of some polysaccharides and hydroxyproline-rich glycoproteins in infected host leaf tissues. Physiol. Molec. Plant Pathol., 38, 15-38.
Brooker, B.E., Hobbs, D.G. & Turvey, A. (1975). Observations on the microscopic crystalline inclusions in Cheddar cheese. J. Dairy Res., 42, 341-348. Chapot-Chartier, M.-P., Deniel, C., Rousseau, M., Vassal, L. & Gripon, J.-C. (1994). Autolysis of two strains of Lactococcus lactis during cheese ripening. Int. Dairy J., 4, 251-269. Dean, M.R., Berridge, N.J. & Mabbitt, L.A. (1959). Microscopical observations on Cheddar cheese and curd. J. Dairy Res., 26, 77-82. Kalab, M. (1993). Practical aspects of electron microscopy in dairy research. Food Struct., 12, 95-114. Kiely, L.J., Kindstedt, P.S., Hendricks, G.M., Levis, J.E., Yun, J.J. t Barbano, D.M. (1993). Age related changes in the microstructure of Mozzarella cheese. Food Strut., 12, 13-20.
Kimber, A.M., Brooker, B.E., Hobbs, D.G. & Prentice, J.H. (1974). Electron microscope studies of the development of structure in Cheddar cheese. J. Dairy Res., 41, 389-396.
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Kosikowski, F. V. (1982). Cheese and Fermented Milk Food, 2nd edn. F. V. Kosikowski and Assoc., Brooktondale, NY. Law, B. (1984). Flavour development in cheese. In Advances in Microbiology and Biochemistry of Cheese and Fermented Milk, eds L. Davies & B. A. Law. Elsevier Applied Science Publishers, London, pp. 187-208. Law, J., Fitzgerald, G.F., Uniacke-Lowe, T., Daly, C. & Fox, P.F. (1993). The contribution of lactococcal starter proteinases to proteolysis in Cheddar cheese. J. Dairy Sci., 76, 2455-2467.
Lawrence, R.C., Creamer, L.K. & Gilles, J. (1987). Texture development during cheese ripening. J. Dairy Sci., 70, 1748-1760. Manning, D. J. & Nursten, H. E. (1985). Flavour of milk and milk products. In Developments in Dairy Chemistry-3, ed. P. F. Fox. Elsevier Applied Science Publishers, London, pp. 217-238. Mistry, V.V. & Anderson, D.L. (1993). Composition and microstructure of commercial full-fat and low-fat cheeses. Food Struct., 12, 259-266. Quinones, A,, Degnan, A.J., Luchansky, J.B. & Johnson, E.A. (1994). Pathogen survival in reduced-fat and full-fat Cheddar cheese containing antimicrobials. J. Dairy Sci., 77, (Suppl. 1) 48. Tunick, M. H., Mackey, K. L., Shieh, J. J., Smith, P. W., Cooke, P & Malin, E. L. (1993). Rheology and microstructure of low-fat Mozarella cheese. Znt. Dairy J., 3, 649-662. Umemoto, Y., Sato, Y. & Kito, J. (1978). Direct observation of fine structures of bacteria in ripened Cheddar cheese by electron microscopy. Agr. Biol. Chem., 42, 227-232. Visser, S. (1993). Proteolytic enzymes and their relation to cheese ripening and flavor: An overview. J. Dairy Sci., 76, 329-350.
Int. Dairy Journal 6 (1996) 729-740 Copyright 0 1996 Published by Elsevier Science Limited Printed in Ireland. All rights reserved 0958-6946/96/U 5.00 + 0.00 0958-6946(95)00068-2
Influence of the Fat Content of Cheddar Cheese on Retention and Localization of Starters
Edith Laloy,” Jean-Christophe Vuillemard,” Morsi El Sodab & Ronald E. Simard” Yentre de Recherche STELA, Universitk Lava1 QuCbec, Canada GlK 7P4 ‘Department of Agricultural Industries, University of Alexandria, Alexandria, Egypt (Received 20 July 1995; accepted 29 October 1995)
ABSTRACT Microbiological counts of fat-free, 50% reduced-fat and full-fat Cheddar cheeses were obtained to determine the population of starter bacteria in the different cheeses. Microbiological counts and observations on the cheese curd by transmission electron microscopy indicated that bacterial populations in the curd were directly related to the fat content of cheese. Electron microscopy examinations of cheeses ripenedfor less than one month showed that cells were either directly in contact with the fat globule membrane or located at the casein-fat interface, However, a higher number of bacterial cells appeared to be in close contact with the fat globule membrane. After one and two months of ripening, the number of ghost cells increased and bacteria seemed to be embedded in the milk fat globule membrane or directly in contact with the inside of fat globules. Copyright 0 1996 Elsevier Science Limited
INTRODUCTION Cheese ripening is largely determined by enzymatic reactions which can be divided into three main groups: glycolysis, lipolysis and proteolysis. The last, usually the most complex, also appears to be the major event in cheese maturation (Manning & Nursten, 1985; Visser, 1993). While texture is correlated to the breakdown of the protein network (Lawrence et al., 1987), peptides, amino acids and products of amino acids catabolism have been shown to contribute directly to Cheddar cheese flavour (Manning & Nursten, 1985). The contribution of starter bacterial proteinases and peptidases to this process is important (Law, 1984). Law et al. (1993) showed that starter proteinases were required for the accumulation of small peptides and free amino acids, involved in flavour development of Cheddar cheese. 729
E. Laloy et al.
The role of lipolysis in Cheddar cheese flavour formation is more equivocal (Law, 1984). There is no clear evidence that volatile fatty acids are directly involved in Cheddar flavour. However, it is known that low-fat and fat-free Cheddar cheeses are characterized by poor and atypical flavour (Banks et al., 1989). If lipolysis is not a noteworthy event of cheese flavour formation, then fat content of cheese may have other indirect effects on this process. Considering that starter bacteria play an important role in Cheddar flavour formation and that fat is necessary for flavour expression, a relationship between the fat content of cheeses and starter bacteria should exist. The retention and localization of starter bacteria in the cheese curd during ripening must be studied to explain this potential relationship. Few data dealing with this subject are available in the literature. Dean et al. (1959) were the first to note the tendency of cheese bacteria to congregate at the fat droplet surfaces in Cheddar cheese. The same phenomenon was observed by Umemoto et al. (1978) in Cheddar and Tunick et al. (1993) in Mozzarella cheeses. The present study aimed at determining the role of fat content in cheese milk on starter bacteria retention, localization and evolution during ripening.
MATERIALS
AND METHODS
Bacterial strains Lactococcus lactis subsp. cremoris KB was isolated in our laboratories from the commercial starter MA016 (RhGne Poulenc, Brampton, Ontario). The mixed starter, containing L. lactis subsp. cremoris and L. lactis subsp. lactis strains, was the Redi-Set DVS culture (Chr. Hansen’s Laboratory, Milwaukee, Wisconsin, USA). The strain KB was prepared by subculturing twice (12 h at 3O”C), from a frozen stock, in autoclaved (1 15”C, 10 min) 10% (w/w) reconstituted skim milk. The Redi-Set DVS culture was thawed and diluted (l/10) in milk immediately prior to its incorporation to the cheese milk.
Cheesemaking Three independent Cheddar cheese manufacturing trials were carried out following the procedure described by Kosikowski (1977). Small Cheddar cheeses of 200 g or 1 kg were manufactured either in beakers placed in a controlled water bath (trials 1 and 2) or using a laboratory computer controlled cheese plant (INRA, Poligny, France) for trial 3. In the latter case, temperature was monitored by an automated control station (AGIL, Gleize, France). Before each trial, fresh milk was pasteurized at 72°C for 15 s and skimmed by centrifugation at 32°C. Skimmed milk was divided into three parts. Part 1 was used for the fat-free cheese production. Cream was added to Parts 2 and 3 of skimmed milk to produce 50% reduced-fat cheese and full-fat cheese, respectively. The final volume of each part of milk was the same, and the casein/fat ratios of Part 1, Part 2 and Part 3 were adjusted to 25f2, 1.6f0.2 and 0.7f0.1, respectively. The same preparation of L. lactis subsp. cremoris KB was used as starter (2% v/v) for cheeses in trial 1. Trials 2 and 3 were carried out with the Red&Set DVS, taken from the same can, diluted as described above and added to a 2% (v/v)
Fat content of Cheddar cheese
731
final ratio. The pH was monitored during cheese production; the Cheddaring was stopped when pH reached 5.2fO.l; the cheeses were vacuum packaged and ripened for 60 days at 12°C. Bacterial enumeration Using a Lab-Blender 400 (Stomacher, London, England), fresh cheese samples (10 g) were homogenized in a sterile bag with a 100 mL final volume of 0.10% peptonized water. Total lactic acid bacteria population was determined by counting diluted samples on Ml7 agar (Difco Laboratories, Surrey, UK) after 48 h at 30°C. Total bacterial enumerations were also carried out on the fresh milk used for each cheese production. Transmission electron microscopy Cheese samples were taken for electron microscopy after 24 h, one week, one month and two months ripening. They were cut into small cubes of about 1 mm sides and fixed in 3% (v/v) glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2, Caco buffer) for 15 h at 4°C washed with Caco buffer and then in 1% (v/v) osmium tetroxide (in Caco buffer) for 10 h at 4°C. Samples were then dehydrated in a graded ethanol series (30, 50, 70, 80,95 and 100%) and embedded in Epon 812 (Benhamou, 1991). Ultrathin sections (0.1 pm) were cut with an ultramicrotome, collected on formvarcoated nickel grids, stained with uranyl acetate during 5 min, intensively washed with water, lightly dried, stained with lead citrate during 5 min, washed again and dried before examination with a JEOL 1 200 EX (Tokyo, Japan) electron microscope at 80 kV. At least three cheese cubes per treatment and sample time were cut, and five to 10 ultrathin sections per cube were observed. Photomicrographs show the most representative fields of all the sections examined for one sample.
RESULTS AND DISCUSSION Bacterial population in fresh cheese curds Table 1 shows the effect of fat content on the population of starters in Cheddar cheese, just before pressing. It appears that bacterial counts were related to the fat content of cheeses. Compared to the fat-free cheese, starter populations were 30100% higher and 4-lo-fold higher in 50% reduced-fat cheese and in full-fat cheese, respectively. Bacterial counts of fresh milk were always less than lo2 cfu mL_‘. Considering that the same amount of starter bacteria was added to each of the three parts of milk and that the starter bacteria were the major bacterial population, it is possible to conclude that the amount of milk fat directly influenced the retention and/or the growth of starter bacteria in the fresh curd. These results contradict the work of Tunick et al. (1993), who reported that low-fat Mozzarella cheese contained about 50% more starter than high-fat cheeses. However, in their work, they prepared low-fat cheese at a temperature more conducive to bacterial survival and proliferation than was the temperature of high-fat cheese. Moreover, bacterial strains (Luctobacillus bulgaricus and Streptococcus thermophilus) were different than ours. Similarly to our results,
732
E. Laloy
et al.
TABLE 1
Starter Bacteria Population in Cheddar Cheese Curds Cheese
Fat-free 50% reduced-fat Full-fat
Trial I Strain KB
log 2*10g 4*10g
Trial 2 Mixed starter (cfu g-’ of cheese)
lo6 1,3*106 10*106
Trial 3 Mixed starter
1,1*106 1,5*106 8*106
Quinones et al. (1994) reported that counts of Salmonella and Listeria, initially present in milk in the same quantity, were lower in low-fat compared to full-fat Cheddar cheeses. The higher bacterial counts found in high-fat cheese might be due to a higher retention rate of starter bacteria in the curd or an optimal growth of starter in the high-fat curd during the 6-8 h of production. The first hypothesis is more likely, since temperature and pH, the two major growth-limiting factors for lactic acid bacteria in Cheddar cheese, were controlled and maintained identically for the different cheese production trials. Moreover, there was no difference in the moisture in non-fat-substance between the different types of cheeses (54.2f1.5). The higher retention rate of starter bacteria in high-fat cheese curd could be explained by an active relationship between fat globules and cells, which are retained during the syneresis in high-fat cheeses more than in low-fat ones. Another hypothesis concerns the role of milk fat globules as ‘stoppers’, which could interfere with the flow of whey containing starter cells. In the latter situation, bacteria would be stopped by the fat globules and then mechanically aggregated around them. However, the microscopic observations show that the active relationship should not be excluded. Detection of starter bacteria in the cheese microstructure Figures 1 and 2 show electron micrographs, with about the same magnification, of fat-free and full-fat cheeses, respectively, produced with the commercial mixed starter. The cheese microstructure is consistent with previous observations of lowfat and full-fat Cheddar cheese (Mistry & Anderson, 1993; Kalab, 1993) consisting mainly of a protein matrix in which fat globules are dispersed. Fat globule numbers and area were found to decrease as the cheese fat content decreased. In fat globules, the fat globule membrane was identified as the grey structure, either as a thin circle at the periphery of the globule or a whole web covering the globule. This could be explained by differences in the orientation of the section plane toward the membrane. Figures 1 and 2 show the presence of starter bacteria in the curd after 1 day of ripening. They constituted the major bacterial population observed, and their morphology was similar to the morphology of L. Zactis starter, detected in SaintPaulin cheese by Chapot-Chartier et al. (1994). These microscopic observations also provide an illustration of the differences in starter quantity between fat-free (Fig. 1) and full-fat (Fig. 2) Cheddar cheeses.
Fat content of Cheddar cheese
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Fig. 1. Transmission electron micrograph of fat-free Cheddar cheese produced with a commercial mixed starter, after 1 day of ripening. Large and thin arrows indicate, respectively, starter bacteria and fat globules. Bar represents 1 pm.
Localization of starter bacteria in the cheese microstructure Electron microscopic examination of about 400 bacteria on several sections of full- and low-fat fresh cheeses containing both types of starter showed that about 85% of starter bacteria (L. Zactis subsp. cremoris KB or mixed starters) were localized in the peripheric area of fat globules. Figure 2 illustrates this observation and confirms earlier studies on starter localization in Cheddar cheese curd (Dean et al., 1959; Umemoto et al., 1978). When the cheese sample preparation used by Dean et al. (1959) was followed, it was difficult to observe the bacteria in the cheese curd. However, the tendency of bacteria to congregate at the fat globule surface was noticeable. On the other hand, the method used by Umemoto et al. (1978) was similar to ours and the contrast obtained for the cheese microstructure elements made it very easy to observe the bacteria, clearly shown to be localized in limited areas of the cheese curd close to fat globules. It is possible to contrast these results with the work of Brooker et al. (1975), who identified crystalline inclusions in spaces between the fat and casein phases of l-month-ripened cheeses and hypothesized that the inclusions corresponded to former pockets of residual whey. Therefore, the area between fat globules and casein matrix would correspond to a hydrophilic medium suitable for bacteria to be dispersed, at least during the early stage of the ripening. Moreover, the phenomenon of milk coagulation has been shown to induce the formation of large interconnected clusters of caseins (Kalab, 1993), which become very dense
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Fig. 2. Transmission electron micrograph of full-fat Cheddar cheese produced with a commercial mixed starter, after 1 day of ripening. Large and thin arrows indicate, respectively, starter bacteria and fat globules. Bar represents 1 pm.
after pressing. As well, Kimber et al. (1974) suggested that the reduction of spaces between casein clumps would bring fat globules and bacteria, which initially occupy these spaces, closer together. Detailed microscopic observations of the periphery of fat globules also gave more information about the relationship between starter bacteria and the fat globule membrane (Figs 3 and 4). During the early stage of ripening (less than 14 days), bacteria appeared either directly in contact with the external face of fat globule membranes (Fig. 3) or were found in a limited area around the fat globule (Fig. 4), that was probably filled with residual whey (Brooker et al., 1975). Consequently, it seems possible that an active interaction between starter bacterial cells and the fat globule membrane is occurring.
Evolution of starter bacteria in the cheese microstructure during ripening The morphologies of intact (Fig. 5a, b), lysed (Fig. 6) and ghost cells (Figs 7 and 8) are easily differentiated. After one month ripening, some cells appeared lysed or were already transformed into ghost cells (Figs 6, 7 and 8). After two months ripening, the proportion of ghost cells was higher. No differences can be seen between the two types of starters used. These results agree with Chapot-Chartier et al. (1994), who noticed that less than 10% of Lactococcus luctis subsp. cremoris starter cells were still viable after two months of ripening. showed that ghost cells lost their cell walls and sometimes to yield larger units.
Umemoto et al. (1978) fused with one another
Fat content of Cheddar cheese
Fig. 3. Starter bacteria (strain KB) in close contact with the fat globule membrane
(arrow), in full-fat Cheddar cheese ripened for 7 days. Bar represents 200 nm. Electron micrographs, taken after one month (Figs 5a, b and 6) and two months (Figs 7 and 8) of ripening, show that the interaction, noted above, between starter cells and the fat globule membrane became more and more intimate with time. In Fig. 5a, the fat globule membrane appears altered in the contact area with the starter cell. This deformation could be explained by proteolysis resulting from the starter proteinases, leading to a weakening of the casein matrix in the proximity of fat globules. The pressure on the fat globule exerted by the integrity of the casein network would then decrease and fat globule membranes would tend to stretch out. This hypothesis has already been proposed by Kiely et al. (1993) in an explanation of the coalescence of fat globule membranes between different fat globules in Mozzarella cheese. However, Fig. 5b suggests yet another type of relationship between starter cells and the fat globule membrane. In this electron micrograph, starter bacteria seem to directly alter the organization of the membrane, which becomes thinner and curves in, towards the inside of the fat globule. This phenomenon is difficult to explain and the hypotheses are numerous: mechanical pressure of bacteria on the fat globule membrane, enzymatic activity, etc. Even though viable cells still appeared at the external face of the fat globule membrane after one month of ripening, the localization of ghost cells was very different. Figure 6 shows that ghost cells could be included into the membrane. Observations of more than 100 ghost cells, on several sections, led to the conclusion that all ghost cells, after one or two months of ripening, were either included into the fat globule membrane (Figs 6 and 7) or appeared directly in contact with the inside of fat globules (Fig. 8).
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Fig. 4. Starter bacteria (strain KB) in a limited region near the fat globule membrane (arrow), in full-fat Cheddar cheese ripened for 14 days. The effect of bacterial proteolysis of casein is also visible (PC). Bar represents 400 nm.
CONCLUSIONS This study showed that the fat content of cheese milk directly influenced the number of starter cells retained in the curd and subsequently the potential extent of proteolysis during Cheddar cheese ripening. Consequently, the lower number of starter cells in low-fat cheese could explain the lack of flavour of low-fat Cheddar cheeses. Moreover, the observation of a dynamic evolution of structure and localization of fat globules and starter cells during ripening suggested an active interaction between them. This observation raised numerous questions, such as the role of the fat globule membrane on cell activity and autolysis and the nature of the ghost cell compounds retained in the fat globules. A better understanding of this overall interaction could provide the basis for the optimization of low-fat Cheddar cheese flavour and texture. ACKNOWLEDGEMENTS This work would have never been realized without the advice and the expertise of Dr Nicole Benhamou (Departement de Phytologie, Universite Laval, Quebec, Canada), who also allowed us to use her electron microscopy equipment. Special thanks to her. The authors also thank Dr M. El Abboudi for his help in the cheese production and L. Lamboley for providing the Lactococcus lactis ssp. cremoris KB strain.
Fat content of Cheddar cheese
Fig. 6. Lysed (thin arrow) and ghost (large arrow) cells of strain KB, in fuI1-fat Cheddar cheese ripened for 1 month. Bar represents lPm*
Fig. 7. Ghost cells (arrows) included in the fat globule membrane, of full-fat Cheddar cheese produced with a commercial mixed starter and ripened for 2 months. Bar represents 1 m.
Fat content of Cheddar cheese
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Fig. 8. A ghost cell in direct contact with the inside of a fat globule, in full-fat Cheddar cheese produced with a commercial mixed starter and ripened for 2 months. Bar represents 200 nm.
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