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Evolution of the Microbiological and Biochemical Characteristics of Afuega’l Pitu Cheese During Ripening
DAIRY FOODS Evolution of the Microbiological and Biochemical Characteristics of Afuega’l Pitu Cheese During Ripening PALOMA CUESTA, ESTRELLA FERNANDEZ-GARCiA, DOLORES GONZALEZ DE LLANO, ANTONIA MONTILLA, and ANA RODRfGUEZ’ lnstituto de Productos Lacteos de Asturias (IPLA), Consejo Superior de lnvestigaciones Cientificas (CSIC), Apdo. de Correos 85, 33300 Villaviciosa, Asturias, Spain
ABSTRACT
INTRODUCTION
Changes in the microflora and biochemical characteristics of Afuega’l Pitu cheese were studied throughout a 60-d ripening period. Total viable counts were high in milk (7.62 f 0.95 loglo cfdml) and in cheese throughout ripening (9.26 f 0.56 loglo cfidmg in 3-d-old cheese; 8.06 .t 0.47 loglo cfidg in 60-d-old cheese). La.ctic acid bacteria were the main microbial group responsible for the substantial pH decrease during coagdation and for the further maintenance of low pH. Lactococcus lactis ssp. lactis, citrate-utilizing Lactococcus lactis ssp. lactis, Leuconostoc mesenteroides ssp . mesenteroides, Leuconostoc mesenteroides ssp. dextranicum, Leuconostoc citreum, Leuconostoc paramesenteroides, Lactobacillus plantarum, and Lactobacillus brevis were the species identified. Mean TS and the peircentage of salt in the moisture phase increased significantly during ripening to reach 72.32 and 7.51%, respectively, in 60-d-old cheeses. The mean fat and protein contents were 46.67% and 41.77% of TS, respectively. Proteolysis was weak, water-soluble N attained 16% of total N at the end of ripening, and phosphotungstic acid-soluble N increased froin 0.36 to 2.12% of total N during 60 d of ripening. The P-CN remained relatively intact, and, consequentlly, the y-CN fraction did not significantly increase. Residual (r,l-CN also remained quite high throughout ripening compared with other varieties of traditional raw milk cheeses. ( Key words: cheese, ripening, proteolysis, microflora)
Afiiega’l Pitu cheese is an artisanal, acidcoagulated cheese; body is characteristically pasty, and flavor is sharp and aromatic. Afuega’l Pitu is manufactured by traditional methods in Asturias, northern Spain; annual production is about 150,000 kg. Two variants, atroncao (troncoconic shape) and de trupo (pear shape), are distinguished. The latter variant is red because paprika is added t o the curd. Afuega’l Pitu is made throughout the year from raw bovine milk and without starter. Mixed evening and morning milk is preripened to 0.2% acidity before addition of 2 t o 5 g of calf renneU100 kg of milk. The coagulation process takes about 16 h at ca. 22°C. At the end of this process, acidity reaches 0.8 to 0.9%, and the whey begins t o separate from the curd, being removed carefully with a saucepan. In the troncoconic variant, the curd is filled into troncoconic molds, and whey is allowed t o drain for over 20 h. The cheese is then rubbed on the surface with dry salt and placed again in smaller molds. In the pear-shaped variant, drained curd is mixed thoroughly with salt and paprika and poured into a muslin cloth, which is tied over the curd and hung until drainage is complete. This procedure provides the typical flattened pear shape. The cheese is then ripened in a well-ventilated room at 15 to 16°C and 85 t o 90% relative humidity. A wrinkled and creamish layer of yeasts grows on the surface during ripening. Troncoconic pieces weigh 200 to 300 g, and pear-shaped pieces weigh 500 to 600 g. The optimal consumption period of this variety of cheese ranges from 15 t o 30 d, but much is consumed Abbreviation key: Aw = water activity, PTASN = earlier. The present study reports the changes observed in 5% phosphotungstic acid-soluble N, WSN = waterthe microbiological and biochemical characteristics of soluble N. Afuega’l Pitu cheese throughout ripening in order to enable the design of a specific starter from the lactic microflora isolated from the traditional cheese and to Received May 4, 1995. establish quality standards for the manufacture of Accepted June 10, 1996. this cheese from pasteurized milk. ‘Corresponding author. 1996 J Dairy E M 79:1&3-1698
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CUESTA ET AL.
MATERIALS AND METHODS Cheese Sampling
Twelve batches of Afuega’l Pitu cheese ( 6 batches of troncoconic white cheese and 6 batches of red cheese) were manufactured by traditional methods at four different farms. Three batches of white and 3 batches of red cheese were collected from one farm. One batch of white and 1 batch of red cheese were collected from each of the others. Each batch consisted of milk, curd, and five regular size cheese pieces (ripened for 3, 8, 15, 30, and 60 d ) that were sent to our laboratory under refrigeration (4°C)for microbiological and biochemical analyses. Chemical Analysis
The death rate of microbial groups was calculated from the linear regression of loglo colony-forming units per gram on cheese age and expressed as the mean decrease in loglo cfdg per week of ripening, as indicated by Medina et al. (17). Identification of Isolates
Identification tests were mainly undertaken on lactic acid bacteria (genera Lactococcus, Leuconostoc, and LactobaczZZus) because those microorganisms were to be used as the source of starter strains. Colonies were taken at random from plates that were specific for lactic acid bacteria, inoculated in MRS or M17 broth, and examined microscopically after growth at 30°C. The isolated strains were subcultured twice in the corresponding broth for activation prior to the identification tests ( 12). Identification of surface yeasts and Micrococcaceae was carried out according to Barnett et al. (1) and Holt et al. (12), respectively.
Moisture, pH, TS, ash, ratio of salt t o moisture, fat, protein, total N, and water-soluble N ( WSN) were determined as described by GonzAlez de Llano et al. ( 11). Nitrogen soluble in 5% phosphotungstic acid ( PTASN) was determined according t o methods of Doie et al. ( 3 ) . Water activity (A,) was calculated Statistical Analyses from the aqueous phase NaCl ( 1 5 ) . Analyses were Data were subjected to regression analysis and performed in duplicate. Triplicate native PAGE of the one-way analysis of variance by using SPSS software WSN and urea-PAGE of the casein fractions were ( 2 2 1. Regression analysis was conducted t o deterperformed in milk and in cheeses at 3, 8, 15, and 30 d mine the linear relationship between different variaof ripening as described previously ( 6 ) . Measure- bles (microbial group counts and some gross composiments were based on the peak heights of densito- tion parameters; casein fractions and some gross grams obtained using a Ultrascan XL densitometer composition parameters) at each stage of ripening. (LKB-Pharmacia, Bromma, Sweden), and peaks One-way analysis of variance was conducted to deterwere quantified as relative percentages of the total mine separately the effect of the ripening time and sum. the cheese variant (white or red) on the dependent variables (microbial group counts, gross composition, Microbiological Analysis and casein fractions). Duncan’s multiple range test Sampling was conducted according to the IDF stan- has been applied for comparison of means. dards (13). Cheese rind was discarded, and samples RESULTS AND DISCUSSION were blended in 2% sodium citrate solution. Decimal dilutions were made in quarter-strength Ringer’s solution and pour-plated or streak-plated onto specific Composition media used to isolate microorganisms from milk and Mean values for composition of cheeses throughout ripening cheese. Total viable counts were determined on standa.rd methods agar, enterococci on KF agar, ripening are shown in Table 1. During the coagulacoliforms on violet red bile agar, staphylococci and tion process, milk pH dropped from 6.71 to 4.42 and micrococci on Baird-Parker agar supplemented with continued to decrease during whey drainage, reaching egg yolk-tellurite, and yeasts and molds on acidified the lowest values in 8-d-old cheeses, as a result of the potato-dextrose agar. In addition, several media were lactic acid produced by the metabolic activity of the used for counting lactic acid bacteria: lactobacilli on indigenous lactic acid microflora. The pH increased acidified MRS agar (Biokar, Beauvais, France), leu- slightly at the end of ripening as a consequence of the conostocs on MSE agar (Biokar), and lactococci on growth of microorganisms that utilize lactic acid. This M17 agar (Biokar), Elliker agar (Biokar) sup- effect was mainly attributed t o yeasts ( P < 0.01; r = plemented with 0.1% tallium acetate, and Chalmers -0.995)at 60-d of ripening. Total solids increased ( P < 0.01) during the riperiing period; mean TS was medium ( 2 1. Plates were prepared in duplicate. Journal of Dairy Science Vol. 79, No. 10,1996
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CHARACTERISTICS OF AFUEGA'L PlTU CHEESE
TABLE 1. Mean' composition of milk and of Afuega'l Pitu cheese throughout ripening. Cheese ripening time Curd
Milk
PH TS, % Protein, % of' TS Fat, % of TS NaCl,* % Ash, % of T9 Aw3
X SD 6.71 0.09 12.01 0.60 25.48 0.86 30.03 2.34
3 d
-
X SD 4.42 0.28 18.85 1.16 31.83 8.17 40.80 7.87
15 d
8 d
-
X 4.34 33.34 41.24 44.34 1.21 4.02 0.993
-
SD
X
SD
0.21 4.25 3.70 38.60 8.57 39.46 8.03 45.67 0.58 1.61 0.51 4.52 0.004 0.991
0.18 3.79 9.12 8.42 0.80 1.09 0.005
30 d
X
4.27 49.50 41.61 48.20 2.53 4.87 0.986
SD 0.26 6.14 9.04 9.96 1.14 0.76 0.007
X
60 d
X 4.73 0.18 6.28 72.32 9.36 44.07 9.74 44.87 7.51 1.74 0.72 4.77 0.012 0.956 SD
4.27 67.65 42.48 50.28 5.41 4.57 0.969
-
SD
0.42 4.79 8.08 8.13 3.21 0.73 0.019
'Twelve batches of cheese. 2The NaCl in the moisture phase. W a t e r activity.
72.32%at (30 d of maturation, which was higher than 2. The high total viable counts (loglo) detected in the TS values reported for other cheeses at the same raw milk (7.62 f 0.95) and the high numbers of stage of ripening (16, 19), because of the small size of coliforms (5.51 f 0.71) and enterococci (4.90 1.49) the pieces. The mean fat and protein contents were are indicative of the poor hygienic quality of the raw 46.67 and 41.77% of TS, respectively. The percentage material used in the manufacture of this variety of of salt in the moisture phase increased, and, conse- cheese. Nevertheless, the low pH throughout ripening quently, AIHdecreased ( P < 0.011, throughout ripen- could be responsible for the continuous decrease of ing. The percentage of salt t o moisture was higher ( P coliforms. This microbial group showed the highest < 0.01) for red cheeses, the curd of which is mixed death rate (0.565/wk, loglo), disappearing by d 30 of with salt, than for the surface-salted white cheeses, ripening. Enterococci initially showed a decrease, as for which salt diffuses slowly from the surface into the indicated by the death rate of 0.047/wk (loglo) during cheese core ( 9 ) . The ash content was also higher in the 1st wk of ripening, followed by an increase ( < 1 log unit) in 8-d-old and 15-d-old cheeses. Afterward, an red than in white cheeses. additional decrease was observed until the end of the ripening period. These results differed from those Microbiolo!gicalCharacteristics reported by other researchers for cheese from raw The evolution of the main microbial groups in- ovine milk, such as Manchego ( 18) or La Serena ( 4 1, volved in the ripening of this cheese is shown in Table or from raw bovine milk, such as Mah6n cheese ( 2 1) .
*
TABLE 2. The main microbial groups1 during ripening of Afuega'l Pitu cheese. Cheese ripening time Milk
TVC2 Lactococci3 Leuconostocs Lactobacilli Enterococci Micrococcaceae Coliforms Yeasts Molds
Curd
X
SD
7.62 7.49 6.99 6.08 4.90 4.31 5.51 3.77
0.95 1.19 1.31 1.71 1.49 0.45 0.71 0.32
05
-
X 9.48 9.34 8.89 7.75 4.56 3.79 5.09 4.31 05
3 d
SD 0.27 0.42 0.80 0.47 1.79 0.70 2.02 0.18
15 d
8 d
X
SD
-X
SD
9.26 8.98 8.84 8.19 4.46 3.93 4.06 6.56 5.69
0.56 0.43 0.58 0.64 0.62 1.13 0.52 0.12 0.46
9.06 8.35 8.67 8.66 5.39 3.69 3.28 6.59 5.84
0.44 0.40 0.38 0.50 1.68 0.86 1.17 0.70 0.97
-
-
30 d
X
SD
8.74 8.18 8.13 8.42 5.43 4.25 3.24 6.68 5.94
0.56 0.36 0.15 0.31 1.25 0.36 1.23 0.71 0.84
-
X 8.29 7.19 7.32 8.20 4.49 4.81 04 6.70 6.63
60 d
SD 0.16 0.24 0.22 0.29 1.62 1.16 0.97 0.63
-
X 8.06 6.77 6.76 8.01 4.07 4.69 04 6.22 6.11
SD 0.47 0.48 0.89 0.18
0.63 0.44 0.99 0.65
'Mean of 112 batches of cheese expressed as loglo colony-forming units per gram. 2Total viatile counts. 3Mean logl,~counts for lactococci obtained in three different media (M17 agar, Elliker agar, and Chalmers medium). 4Coliform were only detected in one batch a t this stage of ripening. SMolds were only detected in milk and curd from one batch. Journal of Dairy Science Vol. 79, No. 10, 1996
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CUESTA ET AL.
However, our figures were higher than those reported for Gredos checse from caprine milk ( 17 and Casar de Caceres cheese from ovine milk ( 2 0 ) . Similar proportions of Enterococcus durans and Enterococcus faecalis were identified. Micrococcaceae (loglo) decreased during the first week of ripening from 4.31 f 0.45 to 3.69 f 0.86 as a consequence of the low pH during this period. The increase observed afterward might have been due to the growth of some population that was more tolerant of acidic and very salty conditions. Micrococcus species were cllearly predominant, and no pathogenic species of Staphylococcus were detected. Total viable counts (loglo) increased by 1.66 during milk coagulation and then decreased; death rate was 0.157Iwk (loglo). Counts were affected ( P c 0.01) by the ratio of salt to moisture. Lactic acid bacteria behaved similarly. This microbial group constituted the main microflora in milk, curd, and cheese throughout, ripening. Counts of lactococci (r2 = 0.792; P c O.OOl:i, lactobacilli (r2 = 0.520; P c 0.01),and leuconostocs ( r 2 = 0.554; P c 0.01) were related t o the pH decline during milk coagulation. Worth noting is the change of dominance of the different lactic genera: lactococci were the major group in milk, curd, and 3-d-old cheese but decreased progressively afterward. Leuconostac was the major group in 8-d-old cheese, suggesting a significant role for this genus in the final flavor of Afuega'l Pitu cheese. The importance of leuconostocs in ripening of Spanish artisanal cheeses was also reported by Fontecha et a1 ( 7 ) . As with lactococci, leuconostocs showed a slight but continuous decrease in the following stages of ripening. From d 15 until the end of ripening, lactobacilli were the main lactic acid microflora and contributed to the maintenance of low pH (r2 = 0.443; P e O.OOl), as observed for other cheeses (4, 17). Among lactic acid bacteria, the most abundant species identified were
Lactococcus lactis ssp. lactis, citrate-utilizing Lactococcus lactis asp. lactis, Leuconostoc mesenteroides ssp. mesenteroides, Leuconostoc mesenteroides ssp. clextranicum, Leuconostoc citreum, Leuconostoc paramesenteroides, Lactobacillus plantarum, and Lactobacillus brevis. Yeasts increased -2 log units from curd t o 3-d-old cheese and continued to rise until 30 d of ripening. Initially, a lactose-fermenting species, Khyueromyces marxianus biovar lactis, was predominant, contributing to the low pH. From 15 d of ripening onward, species utilizing lactate, such as Debaryomyces hansenii, Candida lipolytica, and Pichia ohmeri, grew and utilized the available lactic acid. Those species contributed to the formation of the typical creamish rind of this cheese variety and probably produced volatile substances, which were desirable for the development of a complete flavor. Mold counts followed a similar evolution, although they were usually not detected in milk or curd. When white and red cheeses were compared, significant differences ( P e 0.05) were detected for mold counts in 8-d- and 30-d-old cheeses. In 60-d-old cheeses, red and white cheeses differed in enterococci counts. In all of these cases, white cheeses showed higher counts, indicating a possible antimicrobial action of paprika. N Fractions Table 3 shows the mean values for WSN, PTASN, and residual casein fractions, obtained by urea-PAGE analysis, in milk and in cheese during ripening. The low WSN was very likely due to the low pH of this cheese variety ( ~ 4 . 6 1which , would have caused the precipitation of caseins and also peptides. The WSN values increased during ripening and attained a mean value of 16% of TN in 60-d-old cheese. These
TABLE 3.Water-soluble N (WSN), phosphotungstic acid-soluble N (PTASN), and relative percentage of ol,l-CN, P-CN, and degradation products detectable by PAGE in Afuega'l Pitu cheese during ripening.' Cheese ripening time Milk
WSN2 PTASN P-CN' (~al-CN3 Degradation products3
X
SD
43.198 45.228 2.05C
1.73 3.06 0.74
-3 d X
SD 6.57c 1.83 0.356C 0.180 41.47ab 2.86 41.37ab 5.20 6.25b 4.43
-8 d
X 7.28a 0.424C 41.628b 40.74b 7.26b
15 d
SD 1.79 0.20 3.15 5.41 5.30
aJ',cMeans within the same row not followed by the same letter differ ( P c 0.05). lMeane for 12 batches of cheese. 2Expreseed as percentage of total N. 3Expreseed as percentage of total caseins. Journal of Driiry Science Vol. 79, No. 10, 1996
-
X 8.24a 0.785c 39.29c 38.98b 10.68
30 d
SD 2.45 0.73 3.75 9.04 7.97
-
X 10.30b 1.213b 40.73a 39.32b 9.408
SD 3.07 0.71 3.58 8.20 6.70
CHARACTERISTICS OF AFUEGA’L PlTU CHEESE
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results were similar to those reported for Roncal the sometimes undesirable, sometimes appreciated cheese frorn ewe milk ( 1 9 ) and lower than those for characteristic of artisanal products: their heterogeneMajorero cheese from goat milk ( 1 6 ) and Gamonedo ous nature. The final aim of this study would be to blue cheese (11); these results are indicative of a develop a unique methodology for the elaboration of a moderate degree of proteolysis. Also, F’TASN in- homogeneous product, which would improve both creased very slowly throughout ripening, from 0.36 to microbiological and sensory characteristics, opening 2.12% of TN. Recently, Gonzalez de Llano et al. ( 1 0 ) new markets for traditional products. studied the peptide patterns of the WSN from f f i e ga’l Pitu cheeses and reported similar low concentraACKNOWLEDGMENTS tions for white and red variants. This work was supported by grants ALI900233-C02-02 and AL193-0873-C02-02 from the ComiPAGE Analysis si6n Interministerial de Ciencia y Tecnologia of Native PAGE of WSN fractions did not show an Spain. P. Cuesta was the recipient of a scholarship appreciable difference among cheeses during ripen- from the Fundacion para el Foment0 en Asturias de la ing. Investigacih Cientifica Aplicada y Tecnologica, Urea-PAGE showed that, in general, casein frac- Spain. We also thank Helena Pazo for her laboratory tions undexwent a weak primary proteolysis in this assistance. type of cheese (Table 3 ) . The 0-CN remained relatively intact, and, consequently, the y-CN fraction did REFERENCES not increase ( P > 0.05), a n indication that the in1 Barnett, J. A., W. Payne, and D. Yarrow. 1983. Yeasts, Characdigenous alkaline milk proteinase was not active in teristics and Identification. Cambridge Univ. Press, London, this type of cheese. The low pH over the ripening England. period is not suitable for casein hydrolysis by plasmin 2 Chalmers, C. H.1962. Bacteria in h l a t i o n to the Milk Supply 4th ed. Edward Arnold, London, England. ( 8 ) . Residual asl-CN also remained much higher 3 Doie, E., S. Shibata, and T. Matoba. 1981. Modified colorimetric than in other varieties of traditional raw milk cheeses ninhydrin methods for peptidase assay. Anal. Biochem. 118: ( 5 , 7, 17, 21). The degradation products of higher 173. 4 Fernandez del Pozo, B., P. Gaya, M. Medina, M. A. Rodriguez electrophoretic mobility, asl-I-CN included, increased Marin, and M. Nufiez. 1988. Changes in the microflora of the La as ripening time increased ( P < 0.05). Most of the Serena ewe’s milk cheese during ripening. J. Dairy Res. 55:449. traditional cheeses have a rennet-based coagulation, 5 Fernandez del Pozo, B., P. Gaya, M. Medina, M. A. Rodriguez Marin, and M. Nufiez. 1988. Changes in chemical and rheologibut Afuega’l Pitu cheese milk is coagulated very cal characteristics of the La Serena ewe’s milk cheese during slowly at relatively low temperatures, mainly as a ripening. J. Dairy Res. 55457. result of the acidification by lactic acid bacteria. The 6 Fernandez-Garcia, E., R. Lopez-Fandifio, D. Cabezudo, P. Martin-Alvarez, and M. Ramos. 1993. Accelerated ripening of decrease in aS1-CN was accompanied by a n increase Manchego type cheese by added commercial enzyme preparain WSN, but a low inverse correlation ( r = -.6221; P < tion from Aspergillus orytcre. Enzyme Microb. Technol. 15:519. 0.01) was observed between asl-CN and WSN con7 Fontecha, J., C. Pelaez, M. Juarez, T. Requena, C. G m e z , and tents. No correlation was detected between asl-CN M. Ramos. 1990. Biochemical and microbiological characteristics of artisanal hard goat cheese. J. Dairy Sci. 73:1150. and PTASN, indicating that PTASN was more related FOX, P. F. 1981. Proteinases in dairy technology. Neth. Milk to the exopeptidase activity of the primary and seconDairy J. 35:233. dary microflora ( 1 41 than to rennet activity. In the 9 Guinee, T. P.,and P. F. Fox. 1987. Page 251 in Salt in cheese: physical, chemical and biological aspects. Cheese, Chemistry, 3-d-old cheese, a n inverse correlation ( r = -0.9299; P Physics and Microbiology. Vol. 1. General Aspects. P. F. Fox, ed. < 0.001) between residual asl-CN and pH was deterElsevier Appl. Sci. Publ., London, England. mined, indicating that a higher pH could favor a,l10 Gonzalez de Llano, D., M. C. Polo, and M. Ramos. 1995. Study of proteolysis in artisanal cheeses: high performance liquid CN hydrolysis by proteinases. The correlation coeffiof peptides. J. Dairy Sci. 78:1018. cient decreased to nonsignificance as ripening time 11chromatography Gonzalez de Llano, D., M. Ramos, A. Rodriguez, A. Montilla, increased. Significant correlation ( r = -0.9493; P < and M. J u k e z . 1992. Microbiological and physicochemical characteristics of Gamonedo blue cheese during ripening. Int. 0.001) was also established between casein degradaDairy J. 2:121. tion products and asl-CN. 12 Holt, J. G., N. R. Krieg, P.H.A. Sneath, J. T. Staley, and S. T. In Asturias (northern Spain), Afuega’l Pitu is a Williams. 1994. Page 527 in Bergey’s Manual of Determinative Bacteriology. 9th ed. Williams and Wilkins, Baltimore, MD. popular cheese that possesses very special sensory properties. Through this study we have found some 13 International Dairy Federation. 1985. Milk and milk products. Methods of Sampling. Int. Dairy Fed., Brussels, Belgium. differences among cheeses from different manufac- 14 Law, B.A. 1987. Proteolysis in relation to normal and accelerturers and manufacturing processes, which confirm ated cheese ripening. Page 365 in Cheese, Physics and MicrobiJournal of Dairy Science Vol. 79, No. 10, 1996
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ology. Vol. 1. General Aspects. P. F. Fox, ed. Elsevier Appl. Sci. Publ., London, England. 15 Marcos, A., M. Alcali, F. Le6n, J. Fernlndez-Salguero, and M. A.Esteban. 1981. Water activity and chemical composition of cheese. J. Dairy Sci. 64622. 16Martin-Hernlndez, M. C., M. Juarez, and M. Ramos. 1992. Biochemical characteristics of three types of goat cheese. J. Dairy Ski. 75:1747. 17 Medina, M., P. Gaya, and M. Ndiiez. 1992. Gredos goat’s milk cheese: microbiological and chemical changes throughout ripening. J. Dairy Res. 59:563. 18 Ndfiez, M., and J. L. Martinez-Moreno. 1976. Flora microbiana del queso Manchego. I. Evolucion de la flora microbiana de
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quesos Manchegos artesanales. Ann. Inst. Nac. Invest. Agar./ Ser Gen. 4:ll. 19 Ordbfiez, J. A., J. A. Masso, M. P. Mbrmol, and M. Ramos. 1980. Contribution a E t u d e du fromage Roncal. Le Lait 60:283. 20 Poullet, B., M. Huertas, A. S h c h e z , P. Caceres, and G. Larriba. 1991. Microbial study of Casar de Caceres cheese throughout ripening. J. Dairy Res. 58231. 21 Ramos, M., R. Barneto, J. A. Suarez, and B. Ifiigo. 1982. Contribution to the Study of Mah6n Cheese. I. Microbiological and Biochemical Aspects. Chem. Mikrobiol. Techno]. Lebensm. 7: 167. 22 SSPS-PCt Version 4.0 Software. 1984-1990. Software, Inc., Chicago, IL.
ABSTRACT
INTRODUCTION
Changes in the microflora and biochemical characteristics of Afuega’l Pitu cheese were studied throughout a 60-d ripening period. Total viable counts were high in milk (7.62 f 0.95 loglo cfdml) and in cheese throughout ripening (9.26 f 0.56 loglo cfidmg in 3-d-old cheese; 8.06 .t 0.47 loglo cfidg in 60-d-old cheese). La.ctic acid bacteria were the main microbial group responsible for the substantial pH decrease during coagdation and for the further maintenance of low pH. Lactococcus lactis ssp. lactis, citrate-utilizing Lactococcus lactis ssp. lactis, Leuconostoc mesenteroides ssp . mesenteroides, Leuconostoc mesenteroides ssp. dextranicum, Leuconostoc citreum, Leuconostoc paramesenteroides, Lactobacillus plantarum, and Lactobacillus brevis were the species identified. Mean TS and the peircentage of salt in the moisture phase increased significantly during ripening to reach 72.32 and 7.51%, respectively, in 60-d-old cheeses. The mean fat and protein contents were 46.67% and 41.77% of TS, respectively. Proteolysis was weak, water-soluble N attained 16% of total N at the end of ripening, and phosphotungstic acid-soluble N increased froin 0.36 to 2.12% of total N during 60 d of ripening. The P-CN remained relatively intact, and, consequentlly, the y-CN fraction did not significantly increase. Residual (r,l-CN also remained quite high throughout ripening compared with other varieties of traditional raw milk cheeses. ( Key words: cheese, ripening, proteolysis, microflora)
Afiiega’l Pitu cheese is an artisanal, acidcoagulated cheese; body is characteristically pasty, and flavor is sharp and aromatic. Afuega’l Pitu is manufactured by traditional methods in Asturias, northern Spain; annual production is about 150,000 kg. Two variants, atroncao (troncoconic shape) and de trupo (pear shape), are distinguished. The latter variant is red because paprika is added t o the curd. Afuega’l Pitu is made throughout the year from raw bovine milk and without starter. Mixed evening and morning milk is preripened to 0.2% acidity before addition of 2 t o 5 g of calf renneU100 kg of milk. The coagulation process takes about 16 h at ca. 22°C. At the end of this process, acidity reaches 0.8 to 0.9%, and the whey begins t o separate from the curd, being removed carefully with a saucepan. In the troncoconic variant, the curd is filled into troncoconic molds, and whey is allowed t o drain for over 20 h. The cheese is then rubbed on the surface with dry salt and placed again in smaller molds. In the pear-shaped variant, drained curd is mixed thoroughly with salt and paprika and poured into a muslin cloth, which is tied over the curd and hung until drainage is complete. This procedure provides the typical flattened pear shape. The cheese is then ripened in a well-ventilated room at 15 to 16°C and 85 t o 90% relative humidity. A wrinkled and creamish layer of yeasts grows on the surface during ripening. Troncoconic pieces weigh 200 to 300 g, and pear-shaped pieces weigh 500 to 600 g. The optimal consumption period of this variety of cheese ranges from 15 t o 30 d, but much is consumed Abbreviation key: Aw = water activity, PTASN = earlier. The present study reports the changes observed in 5% phosphotungstic acid-soluble N, WSN = waterthe microbiological and biochemical characteristics of soluble N. Afuega’l Pitu cheese throughout ripening in order to enable the design of a specific starter from the lactic microflora isolated from the traditional cheese and to Received May 4, 1995. establish quality standards for the manufacture of Accepted June 10, 1996. this cheese from pasteurized milk. ‘Corresponding author. 1996 J Dairy E M 79:1&3-1698
1693
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CUESTA ET AL.
MATERIALS AND METHODS Cheese Sampling
Twelve batches of Afuega’l Pitu cheese ( 6 batches of troncoconic white cheese and 6 batches of red cheese) were manufactured by traditional methods at four different farms. Three batches of white and 3 batches of red cheese were collected from one farm. One batch of white and 1 batch of red cheese were collected from each of the others. Each batch consisted of milk, curd, and five regular size cheese pieces (ripened for 3, 8, 15, 30, and 60 d ) that were sent to our laboratory under refrigeration (4°C)for microbiological and biochemical analyses. Chemical Analysis
The death rate of microbial groups was calculated from the linear regression of loglo colony-forming units per gram on cheese age and expressed as the mean decrease in loglo cfdg per week of ripening, as indicated by Medina et al. (17). Identification of Isolates
Identification tests were mainly undertaken on lactic acid bacteria (genera Lactococcus, Leuconostoc, and LactobaczZZus) because those microorganisms were to be used as the source of starter strains. Colonies were taken at random from plates that were specific for lactic acid bacteria, inoculated in MRS or M17 broth, and examined microscopically after growth at 30°C. The isolated strains were subcultured twice in the corresponding broth for activation prior to the identification tests ( 12). Identification of surface yeasts and Micrococcaceae was carried out according to Barnett et al. (1) and Holt et al. (12), respectively.
Moisture, pH, TS, ash, ratio of salt t o moisture, fat, protein, total N, and water-soluble N ( WSN) were determined as described by GonzAlez de Llano et al. ( 11). Nitrogen soluble in 5% phosphotungstic acid ( PTASN) was determined according t o methods of Doie et al. ( 3 ) . Water activity (A,) was calculated Statistical Analyses from the aqueous phase NaCl ( 1 5 ) . Analyses were Data were subjected to regression analysis and performed in duplicate. Triplicate native PAGE of the one-way analysis of variance by using SPSS software WSN and urea-PAGE of the casein fractions were ( 2 2 1. Regression analysis was conducted t o deterperformed in milk and in cheeses at 3, 8, 15, and 30 d mine the linear relationship between different variaof ripening as described previously ( 6 ) . Measure- bles (microbial group counts and some gross composiments were based on the peak heights of densito- tion parameters; casein fractions and some gross grams obtained using a Ultrascan XL densitometer composition parameters) at each stage of ripening. (LKB-Pharmacia, Bromma, Sweden), and peaks One-way analysis of variance was conducted to deterwere quantified as relative percentages of the total mine separately the effect of the ripening time and sum. the cheese variant (white or red) on the dependent variables (microbial group counts, gross composition, Microbiological Analysis and casein fractions). Duncan’s multiple range test Sampling was conducted according to the IDF stan- has been applied for comparison of means. dards (13). Cheese rind was discarded, and samples RESULTS AND DISCUSSION were blended in 2% sodium citrate solution. Decimal dilutions were made in quarter-strength Ringer’s solution and pour-plated or streak-plated onto specific Composition media used to isolate microorganisms from milk and Mean values for composition of cheeses throughout ripening cheese. Total viable counts were determined on standa.rd methods agar, enterococci on KF agar, ripening are shown in Table 1. During the coagulacoliforms on violet red bile agar, staphylococci and tion process, milk pH dropped from 6.71 to 4.42 and micrococci on Baird-Parker agar supplemented with continued to decrease during whey drainage, reaching egg yolk-tellurite, and yeasts and molds on acidified the lowest values in 8-d-old cheeses, as a result of the potato-dextrose agar. In addition, several media were lactic acid produced by the metabolic activity of the used for counting lactic acid bacteria: lactobacilli on indigenous lactic acid microflora. The pH increased acidified MRS agar (Biokar, Beauvais, France), leu- slightly at the end of ripening as a consequence of the conostocs on MSE agar (Biokar), and lactococci on growth of microorganisms that utilize lactic acid. This M17 agar (Biokar), Elliker agar (Biokar) sup- effect was mainly attributed t o yeasts ( P < 0.01; r = plemented with 0.1% tallium acetate, and Chalmers -0.995)at 60-d of ripening. Total solids increased ( P < 0.01) during the riperiing period; mean TS was medium ( 2 1. Plates were prepared in duplicate. Journal of Dairy Science Vol. 79, No. 10,1996
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CHARACTERISTICS OF AFUEGA'L PlTU CHEESE
TABLE 1. Mean' composition of milk and of Afuega'l Pitu cheese throughout ripening. Cheese ripening time Curd
Milk
PH TS, % Protein, % of' TS Fat, % of TS NaCl,* % Ash, % of T9 Aw3
X SD 6.71 0.09 12.01 0.60 25.48 0.86 30.03 2.34
3 d
-
X SD 4.42 0.28 18.85 1.16 31.83 8.17 40.80 7.87
15 d
8 d
-
X 4.34 33.34 41.24 44.34 1.21 4.02 0.993
-
SD
X
SD
0.21 4.25 3.70 38.60 8.57 39.46 8.03 45.67 0.58 1.61 0.51 4.52 0.004 0.991
0.18 3.79 9.12 8.42 0.80 1.09 0.005
30 d
X
4.27 49.50 41.61 48.20 2.53 4.87 0.986
SD 0.26 6.14 9.04 9.96 1.14 0.76 0.007
X
60 d
X 4.73 0.18 6.28 72.32 9.36 44.07 9.74 44.87 7.51 1.74 0.72 4.77 0.012 0.956 SD
4.27 67.65 42.48 50.28 5.41 4.57 0.969
-
SD
0.42 4.79 8.08 8.13 3.21 0.73 0.019
'Twelve batches of cheese. 2The NaCl in the moisture phase. W a t e r activity.
72.32%at (30 d of maturation, which was higher than 2. The high total viable counts (loglo) detected in the TS values reported for other cheeses at the same raw milk (7.62 f 0.95) and the high numbers of stage of ripening (16, 19), because of the small size of coliforms (5.51 f 0.71) and enterococci (4.90 1.49) the pieces. The mean fat and protein contents were are indicative of the poor hygienic quality of the raw 46.67 and 41.77% of TS, respectively. The percentage material used in the manufacture of this variety of of salt in the moisture phase increased, and, conse- cheese. Nevertheless, the low pH throughout ripening quently, AIHdecreased ( P < 0.011, throughout ripen- could be responsible for the continuous decrease of ing. The percentage of salt t o moisture was higher ( P coliforms. This microbial group showed the highest < 0.01) for red cheeses, the curd of which is mixed death rate (0.565/wk, loglo), disappearing by d 30 of with salt, than for the surface-salted white cheeses, ripening. Enterococci initially showed a decrease, as for which salt diffuses slowly from the surface into the indicated by the death rate of 0.047/wk (loglo) during cheese core ( 9 ) . The ash content was also higher in the 1st wk of ripening, followed by an increase ( < 1 log unit) in 8-d-old and 15-d-old cheeses. Afterward, an red than in white cheeses. additional decrease was observed until the end of the ripening period. These results differed from those Microbiolo!gicalCharacteristics reported by other researchers for cheese from raw The evolution of the main microbial groups in- ovine milk, such as Manchego ( 18) or La Serena ( 4 1, volved in the ripening of this cheese is shown in Table or from raw bovine milk, such as Mah6n cheese ( 2 1) .
*
TABLE 2. The main microbial groups1 during ripening of Afuega'l Pitu cheese. Cheese ripening time Milk
TVC2 Lactococci3 Leuconostocs Lactobacilli Enterococci Micrococcaceae Coliforms Yeasts Molds
Curd
X
SD
7.62 7.49 6.99 6.08 4.90 4.31 5.51 3.77
0.95 1.19 1.31 1.71 1.49 0.45 0.71 0.32
05
-
X 9.48 9.34 8.89 7.75 4.56 3.79 5.09 4.31 05
3 d
SD 0.27 0.42 0.80 0.47 1.79 0.70 2.02 0.18
15 d
8 d
X
SD
-X
SD
9.26 8.98 8.84 8.19 4.46 3.93 4.06 6.56 5.69
0.56 0.43 0.58 0.64 0.62 1.13 0.52 0.12 0.46
9.06 8.35 8.67 8.66 5.39 3.69 3.28 6.59 5.84
0.44 0.40 0.38 0.50 1.68 0.86 1.17 0.70 0.97
-
-
30 d
X
SD
8.74 8.18 8.13 8.42 5.43 4.25 3.24 6.68 5.94
0.56 0.36 0.15 0.31 1.25 0.36 1.23 0.71 0.84
-
X 8.29 7.19 7.32 8.20 4.49 4.81 04 6.70 6.63
60 d
SD 0.16 0.24 0.22 0.29 1.62 1.16 0.97 0.63
-
X 8.06 6.77 6.76 8.01 4.07 4.69 04 6.22 6.11
SD 0.47 0.48 0.89 0.18
0.63 0.44 0.99 0.65
'Mean of 112 batches of cheese expressed as loglo colony-forming units per gram. 2Total viatile counts. 3Mean logl,~counts for lactococci obtained in three different media (M17 agar, Elliker agar, and Chalmers medium). 4Coliform were only detected in one batch a t this stage of ripening. SMolds were only detected in milk and curd from one batch. Journal of Dairy Science Vol. 79, No. 10, 1996
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CUESTA ET AL.
However, our figures were higher than those reported for Gredos checse from caprine milk ( 17 and Casar de Caceres cheese from ovine milk ( 2 0 ) . Similar proportions of Enterococcus durans and Enterococcus faecalis were identified. Micrococcaceae (loglo) decreased during the first week of ripening from 4.31 f 0.45 to 3.69 f 0.86 as a consequence of the low pH during this period. The increase observed afterward might have been due to the growth of some population that was more tolerant of acidic and very salty conditions. Micrococcus species were cllearly predominant, and no pathogenic species of Staphylococcus were detected. Total viable counts (loglo) increased by 1.66 during milk coagulation and then decreased; death rate was 0.157Iwk (loglo). Counts were affected ( P c 0.01) by the ratio of salt to moisture. Lactic acid bacteria behaved similarly. This microbial group constituted the main microflora in milk, curd, and cheese throughout, ripening. Counts of lactococci (r2 = 0.792; P c O.OOl:i, lactobacilli (r2 = 0.520; P c 0.01),and leuconostocs ( r 2 = 0.554; P c 0.01) were related t o the pH decline during milk coagulation. Worth noting is the change of dominance of the different lactic genera: lactococci were the major group in milk, curd, and 3-d-old cheese but decreased progressively afterward. Leuconostac was the major group in 8-d-old cheese, suggesting a significant role for this genus in the final flavor of Afuega'l Pitu cheese. The importance of leuconostocs in ripening of Spanish artisanal cheeses was also reported by Fontecha et a1 ( 7 ) . As with lactococci, leuconostocs showed a slight but continuous decrease in the following stages of ripening. From d 15 until the end of ripening, lactobacilli were the main lactic acid microflora and contributed to the maintenance of low pH (r2 = 0.443; P e O.OOl), as observed for other cheeses (4, 17). Among lactic acid bacteria, the most abundant species identified were
Lactococcus lactis ssp. lactis, citrate-utilizing Lactococcus lactis asp. lactis, Leuconostoc mesenteroides ssp. mesenteroides, Leuconostoc mesenteroides ssp. clextranicum, Leuconostoc citreum, Leuconostoc paramesenteroides, Lactobacillus plantarum, and Lactobacillus brevis. Yeasts increased -2 log units from curd t o 3-d-old cheese and continued to rise until 30 d of ripening. Initially, a lactose-fermenting species, Khyueromyces marxianus biovar lactis, was predominant, contributing to the low pH. From 15 d of ripening onward, species utilizing lactate, such as Debaryomyces hansenii, Candida lipolytica, and Pichia ohmeri, grew and utilized the available lactic acid. Those species contributed to the formation of the typical creamish rind of this cheese variety and probably produced volatile substances, which were desirable for the development of a complete flavor. Mold counts followed a similar evolution, although they were usually not detected in milk or curd. When white and red cheeses were compared, significant differences ( P e 0.05) were detected for mold counts in 8-d- and 30-d-old cheeses. In 60-d-old cheeses, red and white cheeses differed in enterococci counts. In all of these cases, white cheeses showed higher counts, indicating a possible antimicrobial action of paprika. N Fractions Table 3 shows the mean values for WSN, PTASN, and residual casein fractions, obtained by urea-PAGE analysis, in milk and in cheese during ripening. The low WSN was very likely due to the low pH of this cheese variety ( ~ 4 . 6 1which , would have caused the precipitation of caseins and also peptides. The WSN values increased during ripening and attained a mean value of 16% of TN in 60-d-old cheese. These
TABLE 3.Water-soluble N (WSN), phosphotungstic acid-soluble N (PTASN), and relative percentage of ol,l-CN, P-CN, and degradation products detectable by PAGE in Afuega'l Pitu cheese during ripening.' Cheese ripening time Milk
WSN2 PTASN P-CN' (~al-CN3 Degradation products3
X
SD
43.198 45.228 2.05C
1.73 3.06 0.74
-3 d X
SD 6.57c 1.83 0.356C 0.180 41.47ab 2.86 41.37ab 5.20 6.25b 4.43
-8 d
X 7.28a 0.424C 41.628b 40.74b 7.26b
15 d
SD 1.79 0.20 3.15 5.41 5.30
aJ',cMeans within the same row not followed by the same letter differ ( P c 0.05). lMeane for 12 batches of cheese. 2Expreseed as percentage of total N. 3Expreseed as percentage of total caseins. Journal of Driiry Science Vol. 79, No. 10, 1996
-
X 8.24a 0.785c 39.29c 38.98b 10.68
30 d
SD 2.45 0.73 3.75 9.04 7.97
-
X 10.30b 1.213b 40.73a 39.32b 9.408
SD 3.07 0.71 3.58 8.20 6.70
CHARACTERISTICS OF AFUEGA’L PlTU CHEESE
1
1697
results were similar to those reported for Roncal the sometimes undesirable, sometimes appreciated cheese frorn ewe milk ( 1 9 ) and lower than those for characteristic of artisanal products: their heterogeneMajorero cheese from goat milk ( 1 6 ) and Gamonedo ous nature. The final aim of this study would be to blue cheese (11); these results are indicative of a develop a unique methodology for the elaboration of a moderate degree of proteolysis. Also, F’TASN in- homogeneous product, which would improve both creased very slowly throughout ripening, from 0.36 to microbiological and sensory characteristics, opening 2.12% of TN. Recently, Gonzalez de Llano et al. ( 1 0 ) new markets for traditional products. studied the peptide patterns of the WSN from f f i e ga’l Pitu cheeses and reported similar low concentraACKNOWLEDGMENTS tions for white and red variants. This work was supported by grants ALI900233-C02-02 and AL193-0873-C02-02 from the ComiPAGE Analysis si6n Interministerial de Ciencia y Tecnologia of Native PAGE of WSN fractions did not show an Spain. P. Cuesta was the recipient of a scholarship appreciable difference among cheeses during ripen- from the Fundacion para el Foment0 en Asturias de la ing. Investigacih Cientifica Aplicada y Tecnologica, Urea-PAGE showed that, in general, casein frac- Spain. We also thank Helena Pazo for her laboratory tions undexwent a weak primary proteolysis in this assistance. type of cheese (Table 3 ) . The 0-CN remained relatively intact, and, consequently, the y-CN fraction did REFERENCES not increase ( P > 0.05), a n indication that the in1 Barnett, J. A., W. Payne, and D. Yarrow. 1983. Yeasts, Characdigenous alkaline milk proteinase was not active in teristics and Identification. Cambridge Univ. Press, London, this type of cheese. The low pH over the ripening England. period is not suitable for casein hydrolysis by plasmin 2 Chalmers, C. H.1962. Bacteria in h l a t i o n to the Milk Supply 4th ed. Edward Arnold, London, England. ( 8 ) . Residual asl-CN also remained much higher 3 Doie, E., S. Shibata, and T. Matoba. 1981. Modified colorimetric than in other varieties of traditional raw milk cheeses ninhydrin methods for peptidase assay. Anal. Biochem. 118: ( 5 , 7, 17, 21). The degradation products of higher 173. 4 Fernandez del Pozo, B., P. Gaya, M. Medina, M. A. Rodriguez electrophoretic mobility, asl-I-CN included, increased Marin, and M. Nufiez. 1988. Changes in the microflora of the La as ripening time increased ( P < 0.05). Most of the Serena ewe’s milk cheese during ripening. J. Dairy Res. 55:449. traditional cheeses have a rennet-based coagulation, 5 Fernandez del Pozo, B., P. Gaya, M. Medina, M. A. Rodriguez Marin, and M. Nufiez. 1988. Changes in chemical and rheologibut Afuega’l Pitu cheese milk is coagulated very cal characteristics of the La Serena ewe’s milk cheese during slowly at relatively low temperatures, mainly as a ripening. J. Dairy Res. 55457. result of the acidification by lactic acid bacteria. The 6 Fernandez-Garcia, E., R. Lopez-Fandifio, D. Cabezudo, P. Martin-Alvarez, and M. Ramos. 1993. Accelerated ripening of decrease in aS1-CN was accompanied by a n increase Manchego type cheese by added commercial enzyme preparain WSN, but a low inverse correlation ( r = -.6221; P < tion from Aspergillus orytcre. Enzyme Microb. Technol. 15:519. 0.01) was observed between asl-CN and WSN con7 Fontecha, J., C. Pelaez, M. Juarez, T. Requena, C. G m e z , and tents. No correlation was detected between asl-CN M. Ramos. 1990. Biochemical and microbiological characteristics of artisanal hard goat cheese. J. Dairy Sci. 73:1150. and PTASN, indicating that PTASN was more related FOX, P. F. 1981. Proteinases in dairy technology. Neth. Milk to the exopeptidase activity of the primary and seconDairy J. 35:233. dary microflora ( 1 41 than to rennet activity. In the 9 Guinee, T. P.,and P. F. Fox. 1987. Page 251 in Salt in cheese: physical, chemical and biological aspects. Cheese, Chemistry, 3-d-old cheese, a n inverse correlation ( r = -0.9299; P Physics and Microbiology. Vol. 1. General Aspects. P. F. Fox, ed. < 0.001) between residual asl-CN and pH was deterElsevier Appl. Sci. Publ., London, England. mined, indicating that a higher pH could favor a,l10 Gonzalez de Llano, D., M. C. Polo, and M. Ramos. 1995. Study of proteolysis in artisanal cheeses: high performance liquid CN hydrolysis by proteinases. The correlation coeffiof peptides. J. Dairy Sci. 78:1018. cient decreased to nonsignificance as ripening time 11chromatography Gonzalez de Llano, D., M. Ramos, A. Rodriguez, A. Montilla, increased. Significant correlation ( r = -0.9493; P < and M. J u k e z . 1992. Microbiological and physicochemical characteristics of Gamonedo blue cheese during ripening. Int. 0.001) was also established between casein degradaDairy J. 2:121. tion products and asl-CN. 12 Holt, J. G., N. R. Krieg, P.H.A. Sneath, J. T. Staley, and S. T. In Asturias (northern Spain), Afuega’l Pitu is a Williams. 1994. Page 527 in Bergey’s Manual of Determinative Bacteriology. 9th ed. Williams and Wilkins, Baltimore, MD. popular cheese that possesses very special sensory properties. Through this study we have found some 13 International Dairy Federation. 1985. Milk and milk products. Methods of Sampling. Int. Dairy Fed., Brussels, Belgium. differences among cheeses from different manufac- 14 Law, B.A. 1987. Proteolysis in relation to normal and accelerturers and manufacturing processes, which confirm ated cheese ripening. Page 365 in Cheese, Physics and MicrobiJournal of Dairy Science Vol. 79, No. 10, 1996
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ology. Vol. 1. General Aspects. P. F. Fox, ed. Elsevier Appl. Sci. Publ., London, England. 15 Marcos, A., M. Alcali, F. Le6n, J. Fernlndez-Salguero, and M. A.Esteban. 1981. Water activity and chemical composition of cheese. J. Dairy Sci. 64622. 16Martin-Hernlndez, M. C., M. Juarez, and M. Ramos. 1992. Biochemical characteristics of three types of goat cheese. J. Dairy Ski. 75:1747. 17 Medina, M., P. Gaya, and M. Ndiiez. 1992. Gredos goat’s milk cheese: microbiological and chemical changes throughout ripening. J. Dairy Res. 59:563. 18 Ndfiez, M., and J. L. Martinez-Moreno. 1976. Flora microbiana del queso Manchego. I. Evolucion de la flora microbiana de
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quesos Manchegos artesanales. Ann. Inst. Nac. Invest. Agar./ Ser Gen. 4:ll. 19 Ordbfiez, J. A., J. A. Masso, M. P. Mbrmol, and M. Ramos. 1980. Contribution a E t u d e du fromage Roncal. Le Lait 60:283. 20 Poullet, B., M. Huertas, A. S h c h e z , P. Caceres, and G. Larriba. 1991. Microbial study of Casar de Caceres cheese throughout ripening. J. Dairy Res. 58231. 21 Ramos, M., R. Barneto, J. A. Suarez, and B. Ifiigo. 1982. Contribution to the Study of Mah6n Cheese. I. Microbiological and Biochemical Aspects. Chem. Mikrobiol. Techno]. Lebensm. 7: 167. 22 SSPS-PCt Version 4.0 Software. 1984-1990. Software, Inc., Chicago, IL.