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mean proportion of a-lactalbumin (LA) from caprine wheys from the manufacture of Chevre and Cheddar- type cheeses was higher than values previously.

Seasonal Changes in Protein Composition of Whey from Commercial Manufacture of Caprine and Ovine Specialty Cheeses J. L. CASPER,1 W. L. WENDORFF,1,2 and D. L. THOMAS3 University of Wisconsin-Madison, Madison 53706

ABSTRACT Pooled whey from the production of one variety of ovine cheese and two varieties of caprine cheeses was studied for gross composition and individual whey protein composition over one production season. Individual proteins were quantified by sodium dodecyl sulfate-PAGE and digital imaging technology. The mean proportion of a-lactalbumin ( L A ) from caprine wheys from the manufacture of Chevre and Cheddartype cheeses was higher than values previously reported for bovine whey from Cheddar cheese; proportions of serum albumin, immunoglobulin (Ig)G, and b-lactoglobulin ( L G ) were lower. Ovine whey from Manchego-type cheese showed a higher proportion of b-LG, about the same proportion of aLA, and lower proportions of serum albumin and IgG than did the bovine whey. Relative amounts of a-LA decreased throughout the season, but b-LG rose in midlactation and then gradually decreased toward the end of lactation. Relative proportions of serum albumin remained fairly stable throughout the year, and IgG decreased. ( Key words: specialty cheese, caprine, ovine, whey protein) Abbreviation key: SA = serum albumin. INTRODUCTION The demand in the US for specialty cheeses made from caprine and ovine milks recently has increased over 20%/yr (4, 28) as consumers have become more adventurous and as various ethnic populations have grown (18). This increased demand for specialty cheeses has been particularly beneficial to small producers of cheese, many of whom have begun producing cheeses from caprine and ovine milks rather than competing with larger plants in the

Received November 5, 1997. Accepted August 28, 1998. 1Department of Food Science. 2Corresponding author. 3Department of Animal Sciences. 1998 J Dairy Sci 81:3117–3122

production of less expensive commodity cheeses made from bovine milk. There is great potential for growth in both the caprine and ovine cheese markets; however, the processing of caprine and ovine milks brings with it the problem of whey disposal. Currently, whey processors do not accept caprine or ovine whey for processing, including caprine or ovine whey that has been commingled with bovine whey, because of concerns related to flavor, consistent volume, and lack of knowledge about the compositional and functional properties of caprine or ovine whey. Because of these reasons, specialty cheese manufacturers have turned to land spreading as the primary means of disposal, but this disposal method results in both environmental and economic costs that lower the economic return to the specialty cheese maker. The production of foodgrade products from caprine and ovine wheys would provide a market for these by-products and eliminate the environmental problems of whey disposal for the small producers of specialty cheeses. Information on the gross composition, relative proportions of individual whey proportions, and lactational variation of whey is important in predicting the functional attributes of whey products (12). Considerable information exists on caprine and ovine milks from areas traditionally known for high production, such as India, the Middle East, southern Europe, and North Africa (3, 16, 21, 22, 23). In comparison, little information is available regarding whey composition from these species (10). The chemical composition of whey is dependent upon chemical composition of the milk, which varies with stage of lactation, feeding, breeding, individual animal differences, and climate (20). The lactational variance of individual whey proteins from the caprine and ovine milks has not been widely reported (10, 20). Limited studies have shown that the whey protein composition of these milks follows the general lactational pattern of the cow (17, 20). In addition, whey composition varies according to slight changes in milk processing parameters ( 7 ) . For these reasons, a study that examines the composition of caprine and ovine wheys after the manufacture of specialty cheeses is neces-

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sary for evaluation of the potential for food-grade products from these sources of whey. The objectives of this study were to determine the seasonal variance in composition of both gross and individual whey proteins from commercial caprine and ovine specialty cheeses. Results from this study help to identify trends and relative proportions of individual whey proteins that may be unique and conducive to the production of specialty food ingredients. MATERIALS AND METHODS

for electrophoretic analysis were stored at –80°C until used. Compositional Analysis Ash, total nitrogen (Kjeldahl), and fat (Mojonnier) were determined by standard methods (14). Nonprotein nitrogen was determined by Kjeldahl analysis after selective precipitation in TCA (15). Moisture was determined using a microwave equipped with an analytical balance (AVC 80 automatic volatility computer; CEM, Indian Trail, NC) (14). Lactose was determined by difference. Samples were analyzed in duplicate.

Collection of Whey Electrophoresis Samples of caprine and ovine wheys from specialty cheeses that were prepared by commercial manufacturers were supplied at monthly intervals over a 1-yr period. Caprine whey was obtained from an acidcoagulated cheese (Chevre) produced from pasteurized whole milk and from a rennet-coagulated cheese (Cheddar-style) produced from pasteurized whole milk. Ovine whey from Manchego-type cheese manufactured (rennet coagulated) from raw milk was supplied from 3 separate d of production over the 1-yr period. This production schedule allowed for whey samples from cheese produced from milks of early lactation (March), midlactation (June), and late lactation (August). The procedures for manufacturing cheese were consistent throughout the study period. All samples were immediately frozen upon collection and shipped in coolers to Madison, Wisconsin. Samples were stored at –22°C until analysis for gross composition, and samples that were prepared

The SDS-PAGE (SE 600 vertical slab gel unit; Hoefer Scientific, San Francisco, CA) of whey proteins was carried out on untreated liquid whey using the discontinuous method of Laemmli ( 9 ) . The stacking and separating gels were prepared with 4.2 and 12.5% acrylamide, respectively, and the concentration of crosslinks relative to the total concentration was 5 and 4%, respectively. Bovine serum albumin, caprine IgG, b-LA, and a-LA (Sigma Chemical Co., St. Louis, MO) were used as standards. Results from the previous research (11, 22) were used to estimate the appropriate amount of each protein needed in order to obtain a representative range of proteins in the samples for the standard curve. The exponential curves [2, 4, 8, and 16 g/ml each for serum albumin ( SA) , IgG, and a-LA and 4, 8, 16, and 32 g/ml for b-LG] covered the range in which all proteins in the samples occurred. Samples and appropriate standards were denatured at 100°C for 5 min in Tris·HCl buffer at pH

TABLE 1. Gross composition of caprine and ovine wheys from the manufacture of specialty cheeses and bovine whey from the manufacture of Cheddar cheese. Component pH

Cheddartype1 6.2

Total solids Water Fat Ash Lactose (by difference) Total protein (TN – NPN × 6.38) NPN 1Means

6.61 93.39 0.51 0.61 4.71 0.77 0.05

Chevre2 4.6

Manchegotype3

6.3 (%, wt/wt) 6.40 7.46 93.60 92.54 0.03 0.82 0.76 0.43 5.07 5.16 0.53 1.05 0.06 0.08

Cheddar4 ND5 6.7 93.30 0.36 0.52 4.50 0.60 ND

from February, March, April, May, July, September, October, and December. from January, March, April, May, July, and September. 3Means from March, June, and August. 4Source: Webb (26). 5Not determined. 2Means

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6.8 containing 1% SDS (wt/vol) and 4% 2mercaptoethanol. Immediately upon loading samples and standards, gels were run at 30 mA per gel until the leading bands were approximately 1 cm from the bottom of the gel. Gels were immediately removed from the plates and were placed in a fixative solution of 40% methanol (vol/vol) and 10% TCA (wt/vol). Gels were then stained as described by the procedure of Basch et al. ( 1 ) . Gels were destained in a solution of 40% methanol (vol/vol) and 10% acetic acid (vol/ vol). Serum albumin, IgG, b-LG, and a-LA in whey samples were quantified using the Foto/Analyst computer imaging system (Fotodyne Inc., New Berlin, WI) as described by Bogenrief et al. ( 2 ) and Bogenrief and Olson ( 3 ) . The integrated intensities of the sample bands were then compared with known standard bands and converted to percentage of total whey protein using total protein from gross compositional analysis as the basis. Values that are presented for individual whey proteins are means of four gels, each with its own internal standard curve to account for gel variation. Dated whey samples were assigned to gels using a randomized block format, and each gel was relegated to its own plant to prevent confounding variation among gels with variation in processing between plants.

whey from Cheddar cheese manufacture (26). Chevre whey, although produced from whole milk, had a lower concentration of fat than did Cheddar-type whey, possibly because of lower fat recovery in the Cheddar-type cheese from cooking and stirring and a higher pH at whey drainage (5, 6). Ovine whey from Manchego-type cheese manufacture had the highest fat, protein, and lactose contents of the three wheys. This result would be expected because ovine milk contained higher TS, fat, protein, and lactose than did bovine or caprine milks (8, 24, 27). The ash content of the ovine whey was lower than that of the whey from other cheeses, which was unexpected because the ash content of ovine milk has been reported to be higher than both bovine and caprine milks (13, 27). The higher pH at the time of whey drainage would result in a higher recovery of colloidal calcium phosphate in the curd. Curd grain size and rate of acid development in the cheese manufacturing procedure have been reported as being of great importance for the retention of minerals in cheese (18). Whey Protein Analysis Figure 1 shows the typical electrophoretic pattern of whey proteins from the specialty cheeses manufac-

Statistical Analysis Results were analyzed using one-way ANOVA and Fischer’s difference test on Minitab statistical software (release 11; Minitab, Inc. State College, PA). The level of significance was determined at P < 0.05. RESULTS AND DISCUSSION Compositional Analysis Mean values for pH and gross composition of ovine and caprine wheys and reported values for bovine whey are shown in Table 1. The whey from the two caprine cheeses varied from each other mainly in fat and ash contents. Ash content of goat cheeses has been reported to be higher in soft varieties, such as Chevre, than in harder varieties, such as Cheddar (18). Park ( 1 8 ) reported that process differences such as the degree and speed of acidification, temperature, and curd grain size were the important determinants of ash levels in caprine whey. As shown in Table 1, the lower pH at time of whey drainage for Chevre resulted in increased ash content. The composition of caprine milk is comparable with that of bovine milk (24). Results of compositional analysis of caprine whey from Cheddar-type cheese manufacture are comparable with those for bovine

Figure 1. Typical SDS-PAGE patterns of individual whey proteins on a 12.5% gradient gel. Samples contained 2-mercaptoethanol; gels were stained with Coomasie blue R250. Lanes 2, 7, and 8 are reference lanes containing 4, 16, and 2 mg ( ×2 for b-LG) of reference protein, respectively. Remaining lanes are caprine whey from Cheddar-type cheese. Journal of Dairy Science Vol. 81, No. 12, 1998

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TABLE 2. Distribution1 of whey proteins in specialty cheese whey from caprine and ovine milks compared with that in Cheddar whey from bovine milk. Cheddar-type2

Chevre3

Manchego-type4

Whey protein fraction

X

SD

Range

X

SD

Range

X

SD

Range

Cheddar5

Serum albumin IgG b-LG a-LA

4.0a 9.7a 58.6b 27.0b

0.2 1.0 3.4 3.9

3.7–4.2 8.8–11.5 55.4–63.9 22.0–31.7

3.8a 6.4b 59.2b 31.7a

1.3 1.9 5.6 2.1

1.8–5.5 4.4–9.0 50.9–63.8 29.8–34.7

4.1a 7.3b 74.0a 14.8c

0.8 1.8 3.2 1.9

3.6–5.1 5.5–9.0 71.9–77.7 13.5–17.0

6.5 13.0 64.9 15.6

a,b,cMeans

within the same row without a common superscript differ ( P < 0.05). reported as mean percentages of total whey protein. 2Means from February, March, April, May, July, September, October, and December, replicated on four individual gels. 3Means from March, April, May, July, and September, replicated on four individual gels. 4Means from March, June, and August, replicated on four individual gels. 5Reported values in bovine Cheddar whey. Source: Marshall (13). 1Values

tured in this study. This pattern is similar to those presented in other studies of bovine and caprine wheys (1, 11, 25). Identification of individual whey proteins occurred using standards as markers. Because of the dissociating conditions of SDS-PAGE, the order of the individual whey protein bands are on a molecular weight basis only and are as follows: lactoferrin (86,000), SA (67,000), IgG, heavy chain ( ∼55,000), b-LG (18,400), and a-LA (14,300). bLactoglobulin appeared as a doublet in each gel, even though only one genetic variant was present. This phenomenon was reported by Basch et al. ( 1 ) for all genetic variants of bovine b-LG and was thought to be caused by buffer fronts. Both bands in the doublet were used in the quantification of b-LG. In a similar system, Law and Brown ( 1 1 ) reported that caprine and ovine b-LG comprised only genetic variant A, unlike bovine b-LG, which comprised both A and B variants. The relative proportions of individual whey proteins from the whey of each specialty cheese that was analyzed are shown in Table 2. Caprine whey protein had less SA, IgG, and b-LG than bovine Cheddar whey, although caprine whey protein was higher in aLA. Using gel scanning, Storry et al. ( 2 4 ) found that caprine whey protein contained less b-LG than bovine whey protein but contained a similar amount of a-LA.

Law and Brown ( 1 1 ) reported that caprine whey protein contained less b-LG, more a-LA, and similar amounts of the minor proteins compared with bovine whey protein. Their study employed gel permeation FPLC (Pharmacia Biotech, Uppsala, Sweden). Pintado and Malcata ( 1 9 ) reported that a-LA constitutes 42% of caprine whey proteins, which is markedly higher than the 21.4% reported by Law and Brown ( 1 1 ) or those percentages observed in other studies (20, 24). The concentrations (grams per liter) of individual whey proteins in caprine whey from specialty cheese manufacture were lower than those reported ( 1 1 ) in studies in which unheated, bulk caprine skim milk was used. Specifically, there was a large reduction in SA and IgG, the two most heat-labile whey proteins. Concentrations of b-LG in caprine whey from Cheddar-type and Chevre cheeses in our study showed a moderate reduction from values reported from unheated, bulk skim milk. The concentrations of a-LA in Cheddar-type and Chevre wheys were consistent with values reported from unheated bulk skim milk (11). Ovine whey contained more b-LG and less SA and IgG as a percentage of total whey protein than did bovine whey. a-Lactalbumin in ovine whey was found in about the same proportion as in bovine whey but

TABLE 3. Seasonal variation in whey protein composition1 of caprine whey from Cheddar-type cheese. Whey protein fraction

April

May

June

July

September

October

December

Serum albumin IgG b-LG a-LA

4.2a 9.1a 55.4a 31.7a

3.9a 8.8a 56.3a 31.0a

4.0a 8.8a 57.1a 30.1a

3.7a 9.3a 55.7a 25.6ab

3.9a 11.5a 59.4a 25.2ab

4.0a 10.5a 62.2a 23.5ab

4.1a 10.1a 63.9a 22.0b

a,bMeans 1Values

( n = 4 ) within the same row without a common superscript differ ( P < 0.05). reported as mean percentages of total whey protein.

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WHEY PROTEIN COMPOSITION TABLE 4. Seasonal variation in whey protein composition1 of caprine whey from Chevre cheese. Whey protein fraction

March

April

May

July

September

Serum albumin IgG b-LG a-LA

3.2ab 6.7b 61.2a 31.4a

5.5a 9.0a 50.9a 34.7a

4.1ab 7.0ab 56.3a 32.9a

1.8b 4.6c 63.8a 29.8a

4.1ab 4.4c 63.8a 30.0a

a,b,cMeans 1Values

( n = 4 ) within the same row without a common superscript differ ( P < 0.05). reported as mean percentages of total whey protein.

was significantly lower than that found in both types of caprine whey. Ovine whey contained significantly more b-LG than did caprine whey. These relationships were consistent with previous reports (10, 19). The concentrations (grams per liter) of the individual proteins in ovine whey from specialty cheese manufacture differed from those reported in studies ( 1 0 ) in which unheated bulk ovine skim milk was used. As with caprine whey, there was a large reduction in the concentration of SA and IgG. The concentrations of a-LA and b-LG were greater than concentrations reported from unheated bulk skim milk (10). Seasonal Variance The seasonal variance in the composition of the whey protein between species is shown in Tables 3, 4, and 5 from the manufacture of Cheddar-type (caprine), Chevre (caprine), and Manchego-type (ovine) cheeses, respectively. Values for the four individual proteins were converted from concentration to percentage of whey protein so that relative changes could be observed. The changes in proportions of individual whey proteins from the whey of caprine cheeses analyzed in this study were somewhat variable throughout the season. b-Lactoglobulin showed no significant changes; however, proportions increased slightly after the season, a-Lactalbumin decreased significantly after September in Cheddar-type whey (Table 3 ) but showed no significant change in Chevre whey (Table 4). Pintado and Malcata ( 1 9 ) reported similar trends in caprine whey proteins and identified a decrease in total whey protein as the animals made a transition from a stored feed diet to pasture in the middle of lactation. Relative proportions of SA and IgG remained constant in caprine whey from Cheddar-type cheese manufacture. In Chevre whey, IgG decreased significantly after May. The SA remained stable in Chevre whey except in July, when SA decreased. This result is in contrast to the findings of Law and Brown (11), who reported gradual increases in the propor-

tions of SA and IgG throughout lactation. Differences in seasonal trends of whey protein composition in pooled and commercial whey samples may be compounded by variation in breeds, husbandry, and processing methods. Although the frequency of commercial production of Manchego-type cheese did not allow for monthly samples throughout the season, it did provide a general view of individual whey protein concentrations through early, middle, and late lactation. Proportions of b-LG grew significantly during midlactation and then fell to concentrations that were similar to those in early lactation; a-LA proportions gradually decreased throughout lactation. Both SA and IgG proportions decreased significantly in midlactation; however, IgG proportions rose again in late lactation. These trends were similar to those previously reported ( 1 1 ) in caprine whey proteins from unheated skim milk. CONCLUSIONS Results of this study show that caprine and ovine wheys from specialty cheese manufacture have a unique whey protein composition that might be useful to provide unique food-grade products for the food industry. For example, the consistently high proportions of a-LA in caprine whey have the potential for use for unique whey products with enhanced emulsifi-

TABLE 5. Seasonal variation in whey protein composition1 of ovine whey from Manchego-type cheese. Whey protein fraction

March

June

August

Serum albumin IgG b-LG a-LA

3.8b 7.4a 71.9b 17.0a

3.6b 5.5b 77.7a 13.9b

5.1a 9.0a 72.4b 13.5b

a,bMeans ( n = 4 ) within the same row without a common superscript differ ( P < 0.05). 1Values reported as mean percentages of total whey protein.

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cation. The consistently high proportions of b-LG in ovine whey may offer the potential for whey products with enhanced foaming, gelation, and emulsification. Specialty whey products should be fairly consistent in whey protein composition throughout the production season. Further studies are being performed on caprine and ovine wheys to determine the commercial feasibility for production of these specialty whey products. ACKNOWLEDGMENTS We thank Bresse Bleu, Inc., Watertown, Wisconsin; Southwestern Wisconsin Dairy Goat Product Cooperative, Mt. Sterling, Wisconsin; Bass Lake Cheese Factory, Somerset, Wisconsin; and the Babcock Hall Dairy Plant, Madison, Wisconsin for their assistance with providing whey samples. This research was supported in part by the College of Agricultural and Life Sciences, University of Wisconsin, Madison and the US Department of Agriculture, Washington, DC. REFERENCES 1 Basch, J. J., F. W. Douglas, L. G. Procino, V. H. Holsinger, and H. M. Farrell. 1985. Quantification of caseins and whey proteins of processed milks and whey protein concentrates, application of gel electrophoresis, and comparison with HarlandAshworth procedure. J. Dairy Sci. 68:23–31. 2 Bogenrief, D. D., S. Gunasekaran, and N. F. Olson. 1993. Rheological evaluation of mozzarella cheese by uniaxial horizontal extension. J. Texture Stud. 24:437–453. 3 Bogenrief, D. D., and N. F. Olson. 1995. Hydrolysis of betacasein increases Cheddar cheese meltability. Milchwissenschaft 50:678–682. 4 Carter, D. 1992. Market size and potential. Pages 4–7 in Proc. 1992 Wisconsin Specialty Cheese Seminar, Madison. Wisconsin Ctr. Dairy Res., Madison. 5 Chen, C. M., J. J. Jaeggi, and M. E. Johnson. 1994. Effect of coagulum firmness at cutting on quality and yield for 50% reduced fat Cheddar cheese. J. Dairy Sci. 72(Suppl. 1): 13.(Abstr.) 6 Ernstrom, C. A. 1989. Effect of manufacturing practices on cheese yield. Pages 31–39 in Proc. 1989 Ctr. Dairy Res. Cheese Res. Technol. Conf., Madison, WI. Wisconsin Ctr. Dairy Res., Madison. 7 Hawks, S. E., L. G. Phillips, R. R. Rasmussen, D. M. Barbano, and J. E. Kinsella. 1993. Effect of processing treatment and cheese-making parameters on foaming properties of whey protein isolates. J. Dairy Sci. 76:2468–2477.

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8 Jenness, R. 1980. Composition and characteristics of goat milk: review, 1968–1979. J. Dairy Sci. 63:1605–1630. 9 Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (Lond.) 227: 680–685. 10 Law, A.J.R. 1995. Heat denaturation of bovine, caprine, and ovine whey proteins. Milchwissenschaft 50:384–388. 11 Law, A.J.R., and J. R. Brown. 1994. Compositional changes in caprine whey proteins. Milchwissenschaft 49:674–678. 12 Mangino, M. E. 1992. Gelation of whey protein concentrates. Food Technol. 46:114–117. 13 Marshall, K. R. 1982. Industrial isolation of milk proteins: whey proteins. Page 341–373 in Developments in Dairy Chemistry 1. P. F. Fox, ed. Appl. Sci. Publ., London, United Kingdom. 14 Marshall, R. T. 1993. Standard Methods for the Evaluation of Dairy Products. 16th ed. Am. Publ. Health Assoc., Inc., Washington, DC. 15 McKellar, R. C. 1981. Flavors in ultra-high temperature and pasteurized milk as a function of proteolysis. J. Dairy Sci. 64: 2138–2145. 16 Muir, D. D., D. S. Horne, A.J.R. Law, and W. Steele. 1993. Ovine milk: 1. Seasonal changes in composition of milk from a commercial Scottish flock. Milchwissenschaft 48:363–366. 17 Nunez, M., M. Medina, and P. Gaya. 1989. Ewe’s milk cheese: technology, microbiology, and chemistry. J. Dairy Res. 56: 303–321. 18 Park, Y. W. 1990. Nutrient profiles of commercial goat milk cheeses manufactured in the United States. J. Dairy Sci. 73: 3059–3067. 19 Pintado, M. E., and F. X. Malcata. 1996. Effect of thermal treatment on the protein profile of whey from ovine and caprine milk throughout lactation. Int. Dairy J. 6:497–518. 20 Quiles, A., C. Gonzalo, Y. Barcina, F. Fuentes, and M. Hevia. 1994. Protein quality of Spanish Murciano-Granadina goat milk during lactation. Small Ruminant Res. 14:67–72. 21 Sakul, H., and W. J. Boylan. 1992. Evaluation of U.S. sheep breeds for milk production and milk composition. Small Ruminant Res. 7:195–201. 22 Singh, S., K. H. Rao, S. K. Kanawjia, and L. Sabikhi. 1992. Goat milk products technology—a review. Indian J. Dairy Sci. 45: 572–587. 23 Singh, V. B., and S. N. Singh. 1980. Total protein, whey protein, and casein content of milk of four Indian goat breeds during lactation. Int. Goat Sheep Res. 1:118–124. 24 Storry, J. E., A. S. Grandison, D. Millard, A. J. Owen, and G. D. Ford. 1983. Chemical composition and coagulating properties of renneted milks from different breeds and species of ruminant. J. Dairy Res. 50:215–229. 25 Strange, E. D., E. L. Malin, D. L. Van Hekken, and J. J. Basch. 1992. Chromatographic and electrophoretic methods used for analysis of milk proteins. J. Chromatogr. 624:81–102. 26 Webb, B. H. 1972. Recycling whey for profitable uses. Am. Dairy Rev. 34:32A–32D. 27 Williams, A. P., D. R. Bishop, J. E. Cockburn, and K. J. Scott. 1976. Composition of ewe’s milk. J. Dairy Res. 43:325–329. 28 Wisconsin Agriculture Statistics Service. 1997. Specialty cheese production, Wisconsin, 1993–1996. Wisconsin Farm Rep. 29:2.

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