ABSTRACT
Camembert cheese was manufactured from pasteurized cow's milk by the traditional method in order to determine changes in the microflora, physicochemical, and biochemical characteristics over a 30-day ripening period. The total bacteria counts were high in cheese throughout ripening, with lactic acid bacteria being the main microbial group both on the surface and the center of the curd. However, the microbial activity was more important on the surface than in the center. Each group of microorganisms showed a typical variation during ripening on the surface and in the center. External heterogeneous microflora containing yeasts, molds and halophilic bacteria induced a higher rate of proteolysis and lipolysis on the surface than in the curd of the cheese at the end of ripening (30 days). Migration of salt from the curd reached equilibration after 23 days of ripening. A fast increase in the pH of the surface was observed and the pH differential was maintained between the surface and the center during the ripening period. Rheological analysis demonstrated the softening of Camembert cheese during ripening due to extensive proteolysis.
INTRODUCTION
The purpose of aging in cheese is to develop specific flavor, body, and texture qualities. These characteristics result from the activity of microorganisms and enzymes. For such development to take place, the cheese must be maintained under the conditions favorable to the desired growth and activity. The aging conditions can also result in objectionable changes if the original milk is contaminated with undesirable microorganisms or if improper manufacturing procedures are used. Thus knowledge of the main physicochemical, biochemical and microbiological characteristics at various stages of ripening is required for the development of an acceptable product.
The effects of ripening on the chemical and physical characteristics of cheese have been studied by numerous scientists.Citation1-4 However, most of this research was focused on hard cheeses with soft cheeses being rather neglected in this respect. Soft cheeses, such as Camembert, ripen very quickly because of the high moisture content and the rapid growth of surface mold.Citation[5] The action of the mold protease, in addition to the proteolytic action of the coagulant and the protease from the starter culture, transforms the insoluble casein into acid-soluble nitrogen compounds.
Studies related to the ripening of traditional Camembert obtained from raw cow's milk have been the subject of numerous publications.Citation1-3, Citation6-9 These researchers showed that the development of microbial flora and the main physicochemical and biochemical characteristics during ripening of Camembert were not comparable on the surface and the center of the cheese.
There is scanty information in the literature on Camembert made from pasteurized cow's milk. The present study deals with the quantitative changes of the microbial flora and the main physicochemical and biochemical properties of Camembert made from pasteurized cow's milk during ripening.
MATERIALS AND METHODS
Cheese Manufacture and Sampling
Camembert cheese was manufactured following the method of Kosikowski.Citation[10] Whole milk from the Agricultural Experimental Station of Sultan Qaboos University was pasteurized (85°C/15 s) and cooled to 32°C. The milk was inoculated at 2.5% with a mixed lactic starter culture (a combination of Lactococcus lactis ssp. lactis and Lactococcus lactis ssp. cremoris) and 0.01% Penicillium caseicolum spore powder (Chr. Hansen's Lab, Denmark). Extracts of single strength rennet from calf were added at a ratio of 4.5–6 mL per 45 kg of milk. Following formation of the curd, it was held 3 times the length of coagulation period and then cut. The curd was maintained at 32°C for 1 h and then loaded into plastic cheese molds. The molds were maintained for about 3 h to allow whey separation and build strength and pliability to the curd. A fine mist of P. camemberti was then sprayed over the full curd. After 30 min, the curd was dry-salted and left to air-dry for 1–2 days at 14°C.
The cheese was then held in a curing chamber (Fisons Environmental Equipment, Loughborough, UK) set at 14°C and about 85% RH. Blocks of cheese were turned every 2 to 3 days and wrapped at 14 days. The size of each block was approximately 130 mm in diameter and 60 mm high. Three samples were taken randomly from the surface and the center (inner part) of the curd for analysis at regular intervals during the ripening process which lasted 30 days. Samples in the center were taken by slicing the cheese block. Analyses were run in duplicates.
Microbial Study
Changes in microflora during ripening were studied on the surface and the center of the cheese. Following separation of the surface from the center of the curd, each part was homogenized in a blender and 10 g were dissolved in 90 mL of sterile 2% sodium citrate solution heated at 45°C.Citation[3] Appropriate dilutions were prepared and incubated in duplicates. Plate counts of total aerobic organisms were carried out using plate count agar (30°C for 48 h);Citation[11] mesophilic lactic acid bacteria using Elliker medium (30°C for 48 h);Citation[12] halophilic flora using MSA agar (30°C for 48 h); enterobacteria using violet red bile agar (37°C for 24–48 h);Citation[13] coliform bacteria using brilliant green bile agar (37°C for 24–48); and molds and yeasts using potato dextrose agar acidified with tartaric acid to pH 3.5 (30°C for 3 days).Citation[11]
Physical and Chemical Study
Development of physicochemical and biochemical characteristics during ripening was measured on the surface and the center of the curd. Dry matter was determined by oven drying at 105°C.Citation[14] Total and soluble nitrogen at pH 4.6 in 0.5 M trisodium citrate with a pH of 7.0 were measured by the method described by Gripon et al.Citation[15] The kinetics of global proteolysis was followed by the ratio of soluble nitrogen (SN) to total nitrogen (TN). Fat was determined by the Gerber-Van Gulik method.Citation16-17 Free fatty acids were determined using the rapid method of Gallois and Langlois.Citation[18] Sodium chloride analysis was carried out using the method of the International Dairy Federation.Citation[14] The pH was measured by immersing the electrode of a Beckman meter into a blend of 10 g of grated cheese with 50 mL of distilled water.
Rheological Investigation
This was performed using the ARES, the Advanced Rheometric Expansion System (Rheometric Scientific, Piscataway, NJ, USA), which is a controlled strain rheometer. ARES has two Force Rebalance Transducers (FRT) covering a torque range of 0.02 to 2000 g cm. FRT is air-lubricated and essentially non-compliant thus ensuring that any inherent machine compliance was insufficient to significantly offset measured values from the cheese networks.
For precise control of the sample temperature, an air convection oven was used. The oven has a dual element heater with counter-rotating air flow covering a wide temperature range of −60 to 160°C. Samples were loaded onto the plate of the rheometer and analysed at ambient temperature (23°C). Parallel plate geometry of 40 mm diameter and 5 mm gap was used. Frequency sweeps of 0.1 to 100 rad s−1 were obtained at regular time intervals throughout the 30 days of sample aging. In a second set of experiments, strain sweeps were carried out to identify the area of linear viscoelastic response of the sample and potential changes in their elasticity with aging. Thus readings of the rigidity/storage modulus (G′), viscous/loss modulus (G′′) and complex dynamic viscosity (η*) variation with frequency and applied deformation were obtained.
RESULTS AND DISCUSSION
Development of Microbial Population During Ripening
The development of the main microbial groups involved in the ripening of cheese is shown in Table . It is apparent from the results that each group of microorganisms underwent a characteristic development on the surface and center of the sample. At the beginning of ripening, the total microflora count was comparable on the surface and in the center of the curd. After eleven days, however, it became more important on the surface (7.3×107 cfu/g) compared to that of the center (4.6×107 cfu/g). A similar trend persists to the end of ripening and it can be explained on the basis of progressive neutralization of the cheese surface by mold (P. caseicolum). This allows rapid implantation of the halotolerant and acid-sensitive flora, which is maintained at high levels during ripening.Citation[4]
Table 1. Main Microbial Groups During Ripening of Camembert Cheese
Mesophilic lactic flora is responsible for the degradation of residual lactose into lactic acid. It constitutes the dominant microflora of the surface and the center during the first 23 days of ripening where it represents about 50% of the total population. However, its relative importance decreases as ripening progresses, accounting for only 33% of the total flora at the end of ripening. The decrease may be the result of competition with halophilic flora comprising mainly micrococci, coryneform bacteria and fecal streptococci.Citation[1], Citation[6], Citation[8] Volumes of the halophilic flora reached 1.5×107 cfu/g of cheese at the end of the ripening period representing 19% of the total population on the surface.
Typical development of halotolerant flora was observed during ripening of Camembert cheese. Being aerobic and acid-sensitive it grows actively on the surface, as shown by the increase in the initial surface population from 6.3×104 to 1.5×107 cfu/g at the end of ripening. In the center of the curd, the proliferation of halotolerant bacteria is relatively low since the maximum measured population on day 23 of ripening amounted to 3.7×104 cfu/g. This is due to the dominance of lactic acid bacteria imparting considerable acidity to the center of the curd during ripening hence making it difficult for halotolerant and acid-sensitive bacteria to compete.
Besides their well-known role in the development of aroma and the modification of texture during ripening of Camembert, yeasts and molds (mainly P. camemberti) act as the agents responsible for progressive surface-neutralization of cheese. They metabolize residual lactic acid thus allowing the surface proliferation of micrococci. In the present study, the number of the latter at the end of ripening was a hundred times higher than for yeasts and molds. With respect to the contaminating flora (coliforms and enterobacteria in general), numbers were higher on the surface than in the center at each stage of ripening. Lenoir reported that the number of coliforms varied significantly from one Camembert to another and even from one sample to another for the same lot of cheese.Citation[1]
Physicochemical and Biochemical Properties During Ripening
Table shows data on the development of the main physicochemical and biochemical properties on the surface and the center of Camembert during ripening. Total solids increased continuously during the ripening period. The increase was due to surface evaporation and the exchange of volatile products (water, ammonia, fatty acids, etc.) between the cheese and its environment.Citation[4] At the end of ripening, total solids reached the mean value of 52.86 g/100 g of cheese, considered as normal for this type of cheese. The level of total solids is, of course, one of the determinants of texture in finished products.Citation[9], Citation[19]
Table 2. Development of Main Physico-chemical Characteristics in the Surface (S) and Center (C) During Ripening of Camembert Cheese
Table also depicts data on the concentration of sodium chloride on the surface and center of the curd during ripening. Immediately after salting, its concentration on the surface and center was 4.22% and 0.71%, respectively. A progressive diffusion of salt from the surface to the center took place thereafter due to the existence of a concentration gradient of NaCl between the two zones. This behaviour persisted until day 23 of ripening and should be accompanied by migration of water from the center to the surface of the sample.Citation[20] From day 23 to day 30, the salt/water ratio showed a stable value of approximately 49 g/kg for both zones of cheese.
The amount of fat, expressed on a dry basis, varied relatively little during the ripening process in both the surface and center of the curd. The relative constancy of the fat content may be attributed to the weak lipolysis reaction that takes place in the surface of mold-ripened cheeses. Thus it has been argued that in surface ripened cheeses, such as Camembert, lipolysis touches only 3 to 5% of the total fat.Citation[21] In this study, the total reduction of fat during the entire period of ripening was 0.99 and 1.05 g/100 g of total solids for the surface and the center of the curd, respectively.
The pH of cheese increased progressively on the surface and center of the curd during the ripening process. This could be attributed to the assimilation of lactic acid and the deamination of amino acids by mold (P. camemberti). It has been reported that as the mold neutralizes the acidity of the cheese, the pH increases.Citation[22] It is worth noticing that deacidification was more pronounced on the surface than in the center of the cheese at each stage of ripening. Thus the pH increased 1.57 units on the surface whereas the overall pH increase was 0.84 units in the center.
Table includes data on the variation of proteolysis during ripening of the surface and center of Camembert cheese. At the beginning of ripening, the soluble nitrogen was 6.14 and 7.16% of the total nitrogen on the surface and center, respectively. It has been reported that the soluble nitrogen fraction is mainly the result of the proteolytic action of rennet at pH 4.6.Citation[1], Citation[15] During maturation, a net difference in proteolytic activity appeared between the surface and center of the sample. On the surface, the soluble nitrogen increased within 6 days from 6.14% to 9.63% whereas the degree of proteolysis remained constant in the center. From day 6 to day 23, nitrogen solubilization occurred rapidly both on the surface (from 9.63 to 24.74%) and the center (from 7.12 to 13.72%). In the last week of ripening, surface proteolysis slowed down but it continued in the center at a slightly higher rate.
Besides the obvious action of chymosin on the degradation of casein during ripening, the kinetics and the nature of proteolysis in Camembert depend mainly on the action of proteases secreted by Penicillium Citation[21], Citation[23] and mesophilic streptococci.Citation24-26 Many micrococci also possess important proteolytic power.Citation[2], Citation[6], Citation[21] In the present investigation, total proteolysis appears to be 1.2 to 1.5 times higher on the surface than in the center. The protein content at the end of ripening averaged 500 g kg−1 of dry solids which is considered to be normal for this type of cheese.Citation[19] It is worth noting that at the end of ripening the average soluble protein content (23%) was relatively similar to that reported by Lenoir for pasteurized Camembert aged 24 days but less than that reported for raw Camembert aged 34 days.Citation[2] Heat-treatment of milk could slow down the proteolysis of Camembert by modifying the total flora and the proteolytic system of raw milk. Such an effect was observed for pressed cooked cheeses obtained from mildly heated milk.Citation27-28 Finally, a parallel is recorded for the variation of soluble nitrogen and that of pH during ripening. This should be due to the double activity, i.e. proteolytic and deacidifying, of P. camemberti during its growth.Citation[9]
The increase in degree of proteolysis during ripening is reflected in the mechanical spectra of Camembert cheese in Fig. . These reveal solid structures with the storage modulus dominating over the loss modulus and complex viscosity descending rapidly with increasing frequency of oscillation. Furthermore, the traces of shear modulus show modest frequency dependence. Structures are stronger at the beginning of ripening and lose about 50% of their rigidity at the end of the thirty-day period of observation. Despite the loss of strength with ripening, samples retain unaltered their large deformation properties. As shown in Fig. , catastrophic fracture commences at about 1% applied deformation both at the beginning and end of the ripening process, a value which characterizes brittle networks. Frequency sweeps were carried out at 0.1% strain which, according to Fig. , is well within the linear viscoelastic region of the samples. Recent work on the rheological properties of Greek feta cheese showed a transformation from a soft and elastic consistency (yield stress: 2.3 kPa; yield strain: 18% deformation) to a hard and brittle body (yield stress: 6.9 kPa; yield strain: 7% deformation) as a function of aging from day 110 to day 300 of production.Citation[29]
Figure 1. Mechanical spectra of Camembert cheese at day 1 [G′ (▪); G′′ (•); η* (▴)] and day 30 [G′ (□); G″ (○); η* (▵)] of the ripening period obtained at 23°C (strain: 0.1%).
![Figure 1. Mechanical spectra of Camembert cheese at day 1 [G′ (▪); G′′ (•); η* (▴)] and day 30 [G′ (□); G″ (○); η* (▵)] of the ripening period obtained at 23°C (strain: 0.1%).](/cms/asset/51a6a0ed-97aa-44ae-8e2c-8356b7fbf6a7/ljfp_a_10345049_o_f0001.gif)
Figure 2. Strain sweeps of Camembert cheese at day 1 [G′ (▪); G′′ (•)] and day 30 [G′ (□); G′′ (○)] of the ripening period obtained at 23°C (frequency: 1 rad s−1).
![Figure 2. Strain sweeps of Camembert cheese at day 1 [G′ (▪); G′′ (•)] and day 30 [G′ (□); G′′ (○)] of the ripening period obtained at 23°C (frequency: 1 rad s−1).](/cms/asset/1da93bcc-06ef-4f2f-be62-c7f11f9b3c12/ljfp_a_10345049_o_f0002.gif)
CONCLUSIONS
Quantitative microbiology demonstrated that microorganisms undergo a characteristic growth on the surface as well as the center of Camembert cheese made from pasteurized cow's milk. Proteolysis and lipolysis were greater on the surface of the cheese than in the center. Due to these processes, cheese becomes softer at the end of the ripening period of observation but maintains its brittleness. Processes were mainly due to the presence of a surface flora rich in yeasts and molds (P. camemberti), and in halotolerant bacteria equipped with an important proteolytic and lipolytic power. Neutralization of the surface of the cheese by yeasts led to the formation of a pH gradient between the surface and the center, which was maintained during the ripening period. In return, the gradient of salt concentration disappeared after about 3 weeks.
Acknowledgments
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