Farkye, N. Y.

Harwalkar, V. R.

Imafidon, G. I.

Tong, Phillip S.

English

DigitalCommons@CalPoly

321 ·~:~~~;~y?:·:, . . ~· Cheddar cheese .. -~r:.:... : !liflu.ence of gene~ic variants of K-casein and ~-lactoglobulin in IJlilk on proteolysis in Cheddar cheese . · .j·" ··· . ~-~.J.IMAFIDON1 •3,N.Y. FARKYE1.4, P.S. TONG1 and V.R. HARWALKAR2 ... ~ ..· ;!)airy Products Technology Center, California Polytechnic State University, San Luis Obispo, CA 93407 lGeOtre for Food and Animal Research, Agriculture Canada, Ottawa, Ontario, Canada spresent address: USDA, ARS, SRRC, 1100 Robert E. Lee Blvd, New Orleans, LA 70124 ·~nding author .·:~/· ·.. .. .. ,... 1: Jntroduction Genetic variants of all·the major milk proteins in­fujence the composition (3, 13) and processing char­acteristics of milk (8, 11, 19), and the composition and .yield of cheese therefrom (11, 13, 23). Differ­ences in milk protein variants are reported (14, 18) to affectthe sensory qualities ofcheese also. SCHAAR eta/. (1B) repqrted that soluble-N levels in cheese wadefrom ~-LG AA milkwere higher than in cheese IJtm ~LG AB or BB milks. However, van den BERG . et aJ. (19) found that genetic variants of ~-LG orK-CN . in milk did not significantly affect the levels of solu­·ble-N or amino-N in Gouda cheese during ripening. Because flavor development of rennet-coagulated cneeses, like Cheddar, is greatly influenced by the rate and extent ofproteolysis that occurduring ripen­Big (5), it is necessary to ascertain the effects of ge­netic variants of.milk proteins on cheese proteolysis. Therefore, the purpose of this study was to evaluate . proteolysis in Cheddar cheese manufactured from milk containing different geneticvariants of ~~LG and .:-CN. 2. Materials and methods · 2.1 Cheddar cheese samples .Three replicates of Cheddar cheese manufac­tured for another study (21) were obtained and used for this investigation. The cheese was manufactured by the traditional 4.25 h method {20) using single­strength calf renne~ as coagulant. The milk used for d!eese manufacture contained the following milk proteinvariants: K-CN A +~~LG A, K-CN A + ~LG B, (-eN A+ P-LG AB, K-CN AB +~-LGA K-CN AB + ~­lG AB, or i~CN AB + ~-LG B. Cheese made from bulk mUk obtained from a mixed herd served as con­trol. The cheeses were ripened at soc, and sampled a!ter1 d, and 1, 3 or-6 mo of ripening for assessment ofproteolysis. · 2.2 Assessment ofproteolysis Proteolysis in cheese was followed by polyacry­lamide gel electrophoresis {PAGE), and by analysis O{water-soluble e"tract (WSN) from the cheese. The WSN was prepared as described by KUCHROO and FoX_(9). The % N.in the extract was quantified by lhtrsemi-macro-Kjeldahl method (1 ). Freeze-dried llOrtions ot-theWSN_were analy_zed by PAGE, high­llertoirnarice liquid -chromatography {HPLC), or fast P_roteinliquid chromatography (FPLC). . -. -: .. PAGE of the cheese was performed as described by FARKYE eta/. (4) using the Mini-PROTEAN II™ (Bio-Rad, Hercules, CA) system. The gels were run for 1 h 10 min at 200 V. The gels were fixed and stained in acetic acid/methanoV water, 7/40/53, con­taining 1.4 mM of Coomassie brilliant blue R250. Gels were destained in acetic acid/methanol/water, 7/40/53. Reversed-phase (RP) HPLC was run on the Sys­tem Gal™ (Beckman Instruments, Fullerton, CA) setup, which consisted of 2 modei110B solvent de­livery pumps, sample injector, 20 f.l.l loop, and a model116 UVNIS detector. An Ultraspher™-ODS guard column, 4.6 mm x 4.5 em, attached to an Ul­traspher™-ODS (5 J.LITl, 80 A) analytical column, 4.6 mm x 25 em (Beckman}, were used for the analysis. A binary solvent system: A, 0.1 % (v/v) trifluoroacetic acid (TFA) in water, and 8 , 0.1 % TFA (v/v} in 75 % acetonitrile was used. Samples were eluted by a stepwise gradientfrom 0 ~ 18-+ 35-+ 45-+ 80-+ 0% B for 5, 5, 15, 30, 5, and 40 min, respectively, at a flow rate of 0.75 mVmin. Total run time was 100 min. The eluatewas monitored at230 nm. For HPLC, 5.0 mg of the freeze-dried WSN were dissolved in 1 ml of solvent A. The resultant solution was filtered successively through 0.2 J.lm membrane filter (Gelman Sciences, Ann Arbor, Ml), and a Sep­pak™ C18 plus cartridge (Waters Chromatography Division, Milford, MA), and 20 J.LI of the filtrate was loaded onto the column. FPLC of the WSN extracts was done using a Mono-Q HR5/5 anion exchange column (Pharmacia Ltd, Montreal, Canada). The freeze-dried WSN ex­tracts were re-hydrated to 6.3 % solids, and 200 J.d of the solution was loaded onto the column. Chroma­tography was run using a nonlinear gradient of buff­ers A (0.01 M lmidazole/HCI (pH 7) containing 8 M urea) and B {buffer A plus 1 M NaCI). The gradient was varied as follows: A = 0. 4.0, 7.5, 10.0, 24.0, 28.0, 32.0, 32.0, and 35.0 ml which corresponded to % B = 0, o, 12.5, 14.8, 35.0, 100.0, 100.0, o, and 0, respectively. Samples were eluted at a flow rate of 1 mllmin and the eluate monitored at 280 nm. 2.3 Statistical analysis The Kjeldahl WSN data were analyzed by least­squares analysis of variance using the SAS com­puter software package (SAS Institute Inc., CaryNq. ' 322 lmafidon, Cheddarcheese 3. Results and discussion 3.1 Water-soluble nitrogen Table 1 shows the mean ± S.E. of the WSN con­tent of the cheeses. Average WSN levels increased at similar rates in all cheeses. At each ripening pe­riod, the differences in % WSN were not statistically significant (P >0 .05), suggesting that the levels of WSN in the cheeses were not affected by the genetic types of 1e-CN (A or AB} or ~-LG (A, AB or 8). Similar results were reported by van den BERG eta/. (19) who found that genetic variants of 1e-CN or ~-LG did not influence the levels of soluble-N or amino-N in Goudacheese. Table 1:	Water-soluble N1 levels duringripening ofChed­dar cheese containing different variants of "' casein and (l-lactoglobulin [Type of protein Water-soluble Nltotal N (%) variants in cheese 1d 1 mo 3mo 6mo Control (bulk milk) 4.56±0.45 13.69±0.25 20.45±0.33 29.34±0.74 x:-CN A+/3-LG A 6.15±0.59 13.18±0.35 19.83±0.67 27.74±0.85 lC-CN A+/3-LG AB 5.19±0.61 11 .90±0.63 18.37±0.47 25.85±0.43 x:·CN A+I}-LG B 5.50±0.76 12.00±0.62 19.36±0.65 27.17±0.49 JC-CN AB+/3-LG A 4.92±0.20 11.39±0.09 19.51±0.65 29 .32±2.15 IC-CN AB+Il·LG AB 5.43±0.21 12.52±0.30 21.49±0.78 27.06±2.1 0 IC-CN AB+I}-LG B 5.67±0.28 12.05±0.09 21.43±0.31 28.08±1.07 1Means±SE of duplicate d et erminations on 3 cheeses Because of scarcity, enough milk containing 1e-CN B could not be obtained for inclusion in this study. From a limited number of cheese making trials using milk containing 1e-CN 8, we found that the % WSN in 1e-CN 88 cheeses w ere slightly lower(15.44 to 16.29 % ) than those in ~e-CN AA (18.83 to 19.83 % ) or JC­CN AB (19.51 to 21.49 % ) cheeses after 3 mo of ripening. MORINI eta/. (13} and SCHAAR eta/. (18) also reported that cheese made from JC-CN 88 milk had a slower rate of proteolysis than that made from 1e-CN AA milk. 3.2 Electrophoresis Figs. 1 A, 8 and C show typical electrophoretic patterns of the cheeses after 1 d, 3 mo or 6 mo of ripening, respectively. The mostdistinct difference is the band, x , which was present between the bands corresponding to y,- and y3-CN in the gels of ~e-CN AB cheeses (lanes 5, 6 and 7), but absent from the gels of 1e-CN AA cheeses (lanes 1 to .4). Similarly, electophoretograms of the WSN fraction from JC-CN AA or AB cheeses showed differences in the intensi­ties of some bands (Fi g. 2). For example, after 3 mo of ripening, bands a to e (Fig. 2) were of higher or lower intensities in the gels of the WSN from K-CN AA cheeses than in 1e-CN AB cheeses. This sug­gests that the specificities of the major proteolytic enzymes (i.e., residual rennet or plasmin} toward caseins differ in cheese containing different milk protein variants. 3.3 FPLC of water-soluble fraction Results of FPLC analysis of the WSN in the cheeses <;ire shown in Table. 2. The peptid.e peaks ..~. ~ . : ....., .. , . ........:<". ..  . ..4. • • •• ,.•• • • , . ·'. , . .... . : Rg. 1: Urea:PAGE (12% T, 4 % C; pH 8.3) of Cheddar cheese made from control milk (1), or.milk contain· ing K·CN A + j3-LG A (2), JC-CN A + IRG AB(2) , IC· CN A+ J3·LG 8 (4), K-CN AB + J3-LG A (5), K-CN AB '+ j3-LG AB (6), K-CN AB + J3·LG 8 (7) after rip~ning . :1 d (A), 3 mo (B), or 6 mo (C) of iipening. · Milchwissenschaft 50 (6) 1995 323 /fr¢idon, Cheddar cheese fig. 2: 	 Urea-PAGE (12 o/o T, 4 o/o C, pH 8.3} of water­soluble N extracts from Cheddar cheese made from control milk (1), or milk containing K-CN A+ 1}-L~ A (2), K-CN A+ ~-LG A8 (2) , IC-CN A+ IJ-LG 8 (4), K· CN A8 + 1}-LG A (5), IC-CN AB + IJ-LG A8 (6), K-CN AB + P-LG B (7) after 3 mo of ripening. were subdivided into 3 groups, on the basis of their retention times (At) ofOto7.5 min, 7.5to 16 min, and >16 min, which corresponds to peptides characteri­zed as hydrophobic, intermediate, or very ionic, re­spectively. Most of the peaks had Rt of< 7.5 min, suggesting a high . concentration of hydrophobic peptides in the WSN fraction of the cheeses. The differences between FPLC profiles of the WSN from cheeses containing the different milk protein vari­ants were minimal. However, after 1 mo of ripening, peak areas for the intermediate peptides were lower in the WSN from cheese containing K-CN A than in those containing K-CN AB. 3.4 RP-HPLC of water-soluble fraction Table 3 summarizes quantitative RP-HPLC data for the WSN from the cheeses. The number of pep­tides peaks and their corresponding areas were different among the different cheeses. In general, the average number of peptide peaks in the WSN from K-CN AA cheeses were more than those from 1e-CN AB cheeses, suggesting that proteolysis·in JC­CN AA cheeses was more extensive than in K-CN AB cheeses. Fig. 3 shows typical RP-HPLC chroma­tograms of the WSN from the cheeses. The most distinct difference in the chromatograms is the peak, c, which was prominent in 3- or 6-mo-old cheeses containing K-CN A, but was almost lacking in cheeses containing K-CN AB. The percentage of peptide peaks that were eluted in0.1% TFAwere36to42% after1 d, and 10to 11 %after6 mo of ripening in cheese containing JC-CN A + {1-LG variants. The corresponding values in cheeses containing IC-CN AB + ~-LG variants were 29 to 38 % and 13 to 17% , respectively, suggesting that the levels of hydrophilic peptides decreased during ripening, and the greatest decrease occurred in cheese containing 1e-CN A. Table2: Summary of FPLC data on water-soluble N in cheeses made from milk containing different variants of ~lactoglobulin and JC-casein Type of proteins Cheese in cheese age lC<:N A+~-LG A 1 d lC<:N A+~·LG A8 1d lC<:N A+fJ-LG B . 1 d lC.CN AB+IJ-LG A 1 d ~e.CN AB+j)-LG AB 1 d lC.CN AB+~·LG 8 ·1 d JC.CNA+lHGA 1 mo JC.CN A+IJ-LG AB 1 mo lC.CN A+~·LG 8 .  .1 ina ~.CN AB+IJ-LG A ·  1 mo · ,;.CN AB+!J-LG AB 1 mo JC·CN AB+~·LG 8  1 mo 3mo· t·CN A+~LG A IC.CNA+ LG AB 3mo t.CN A+~-LG 8 · 3mo • I(.CN AB+~·LG A 3mo IC·CN AB+IJ-LG AB 3rno IC.CN AB+j)~LG B · 3mo 6mo · .IC.CN A+~-L~ A · IC.CN A+j)·LG AB 6mo .lC·CN A+jHG 8 . 6mo IC.CN AB+~:LG .A 6mo 6mo·JC-tNA.B+~·LG AB · · . a:.CN AB+ -LG 8 . 6mo . . . - . . . 1·Ave~age of 3 replieates .. Mi!chwisse~schaft 50 (6) 1995 . · . Peak areas1 at retention times of 	 Total area <7.5 min 7.5 to 16.0 min > 16.0 min (arbitrary (hydrophobic) (intermediate) (ionic) units) 65.5 12.3 - 77.9 53.9 15.6 1.1 77.9 71.3 10.9 0.2 82.4 54.9 17.9 0.5 73.3 58.9 15.4 0.5 74.8 55.1 13.3 0.7 69.1 56.7 7.7 14.8 79.2 55.1 7.9 17.4 80.4 52.7 8.8 15.0 76.5 53.7 11.5 14.8 80.0 55.7 10.0 11.3 77.0 51.9 10.2 17.4 79.5 51.1 4.4 28.3 83.8 48.5 6.4 22.6 77.5 58.5 7.0 19.2 ·84.7 54.1 8.7 19.2 82.0 59.2 10.2 17.2 86.6 52.5 7.4 18.5 78.4 65.8 6.8 13.6 86.2 64.3 · 7.3 15.0 86.6 59.8 6.8 . 18.3 84.9 61.6 6.2 15.3 83.1 62.5 5.9 16.6 85.5 62.9 7.3 17.1 87.3 324 	 tmafidon, Chectdar cheese c "-CN A+ ~-LG B K-CN AB + ~-LG 8 6mo 6mo ~e-CN 	A8 + jJ-LG 8 3mo IC-CN A +~LGB 3mo IC•CN A + ~-LG B · .C-CN AB + ji-LG 8 1d 0 5 10 15 20 25 30 35 40 0 5 10 15 20 25 30 . 35 40 Time (min) 	 Time (min) Rg. 3: 	 Reversed-phase HPLC peptide profiles in WSN extracts from Cheddar cheese made from milk containing K-CN A + jJ-LG A, K-CN A + 13-LG AB, K-CN A + 13-LG 8, l\-CN AB + 1}-LG A, K-CN AB + 13-LG AB, or K-CN A8 + ,13-LG 8 after 1 d, 3 mo, or 6 mo of ripening. Peak C distinguishes K-CN AA cheeses from K-CN AB cheeses. 4. Conclusions The % WSN in Cheddar cheese during ripening was not affected by the genetic variants of K-CN plus ~-LG in the milk used for manufacture. However, t~e peptide patterns in the cheeses made from m1~k containing different genetic types of K-CN were dif­ferent. Specific peptide bands with slower electro­phoretic mobilities than ~-CN were present in the electrophoretograms of cheeses made from milk containing K·CN AB but were absent in cheese made from K-CN AA milk. Similar1y, HPLC and FPLC peptide profiles of the.WSN from K-CN AA cheese were different from those of K-CN AB cheeses. The results suggest that the specificities of proteolytic eniymes towards milkproteins in cheese depend on the genetic type present. · . The significance of the observed differences in proteolytic patterns in Cheddar cheese made. from milkcontaining different genetic variants of ~-LG and K-CN to the quality of the cheeses is not known and needs further investigation. The relationship of K..:CN . or 1}-LG variants to proteolysis .in Cheddar cheese is difficult to explain because under normal conditions, very little of either protein is presen! in t~e cheese. Milk containing K-CN A has more subm1celles and larger micelles (12), that are less cluster~~ or fused during coagulation (16), than that contaJf!Ing K·ct'l AB or B. Therefore, differences in the rate of prate. clysis in cheese made from JC-CN AA milk comp?red to K-AB milk may be due to differences in the d1st~. bution of casein particles in cheese. An open prote,n structure in curd from K-CN AA milk may result in more accessible peptide bonds for taster proteoly. sis, than in curd containing a compact structure. . Acknowledgments . . · . This work was supported by Califomia Dauy Ae- ·. search Foundation. The ·authors also thank Dr. Juan Medrano, University of California, Davis. for gene~ typing of animals; Dr. Stan Henderson, ~al Poly State University, for animal care; Mr. Sean V1nk. Cal Poly · State University, for cheese making; and M~- Dorot~y E.· Raymond, Agriculture . Canada, fo-r assrst_ance It) · FPLC analysis. · Mllchwissenschaft 50 (6) 199s · 325 ~~os, Soft cheese s. References 6. Summary · N~iation of Official Analytical Chemists (AOAC): IMAFIDON, G.l., FARKYE, N.Y., TONG, P.S., HAR­· · Official Methods of Analysis 15th ed Arlington WALKAR, V.R.: Influence of genetic variants of K­· 	 VA (1990). . · ., ' casein and ~-lactoglobulin in milk on proteolysis(2} AALT0NEM, M.L, ANITA, V.:Milchwissenschaft in Cheddar cheese. Milchwissenschaft 50 (6) 321­.·· 42490-492(1987) 325 (1995).{3) ALEANDRI, R., BUTTAZZONI, L.G., SCHNEI­ 54 Cheddar cheese (proteolysis, genetic protein • DER, J.C., CAROl!, A., DAVOLI, A.: J. Dairy Sci. variants · · ·.· 73241-255 (1990) (4) FARKYE, N.Y., KIELY, L.J., ALLHOUSE, A.D., Effects of genetic variants of 11:-casein (A or AB) and . KINDSTEDT, P.S.: J Dairy Sci. 74 1433-1438 !}-lactoglobulin (A, .AB or B) in milk on proteolysis in : (1991) . Cheddar cheese were evaluated. Average water-sol­(5) 	FOX, P.F.: J. Dairy Sci. 721379-1400 (1989). uble nitrogen levels (expressed at % total nitrogen) in the cheeses did not depend, significantly (p >0.05), on (6) GRAHAM, E.R.B., MCLEAN, D.M., ZVIEDRANS, P.: Int. Dairy Congress, The Hague, Posters. D. 	 genetic types of 11:-casein or J3-lactoglobulin. However, qualitative differences were noted in the electrophoretic· · Reidel Publ. Co., Dordrecht, Netherlands, p. 68. . · (1986) . . 	 patterns of the cheese made from 11:-CN AA or A8 milks . (7) 	 IMAFIDON, G.l., NG-KWAI-HANG, K.F.: Int. Da1ry Similarty, electrophoretic patterns or HPLC profiles of peptides in the water-soluble extracts from K-casein AAJ.2275-285 (1992) cheeses were different from those from 11:-CN AB(8) JAKOB, E., PUHAN, Z.: lnt Dairy J. 2 157-178 cheeses. 	 · . (1992) . . . . The results suggest that proteolytic enzymes in(9) KUCHROO, C.N., P.F. FOX, P.F.: M llchWtssen­cheese may have different specificities towards milk . 	 schaft.37 331 -335 (1982) protein variants during cheese ripening. (10) MAAZIALI, A .S., NG-KWAI-HANG, K.F.: J. Dairy . Sci.691193-1201 (1986) (11) MARZIALI, A.S., NG-KWAI-HANG, K.F.: J. Dairy IMAFIDON, G.l., FARKYE, N.Y., TONG, P.S., HAA­Sci.692533-2542(1986) · WALKAR, V.A.: EinfluB genetischer Varianten von (12} MORINI, D., LOSI, G., CASTAGNETTI, G.B., . . K-Casein und ~-Laktoglobulin in Milch auf die Pro­. 	BENEVELLI, M., RESMINI, P., VOLONTERIO, G.: . ·. teolyse in Cheddarkise. Milchwissenschaft 50 (6) Sci. e Teen. Latt.-cas. 26437-444(1975) 321-325 (1995).(13) MORINI, D., LOSI, G., CASTAGNETTI, G.B., 	 (MARIANI, P. : Sci. e Teen. Latt.-cas. 30 243-262 54 Cheddarkise Proteo1yse, genetische Protein­. (1979) . varianten {14) NG-KWAI-HANG, K.F.: Mod. Dairy 69 (2) 14-15 Es wurde der EinfluB genetischer Varianten von K­(1990) . 	 Casein (A oder AB) und ~-Laktoglobulin (A, AB oder (15) NG-KWAI-HANG, K.F., IMAFIDON, G.l.: Int. Da1ry B) in Milch auf die Proteolyse in Cheddarkase be-Congr., Posters and Brief Commun. Vol. 2. p. 359. . wertet. Der durchschnittliche Gehalt an wasserlosli­. Int. Dairy Fed., Brussels, Belgium (1990) chem N (ausgedriickt in % Gesamt-N) in den Kasen (16) NIKI, R., ARIMA, S.: Jap. J. Zootech. Sci. ~ 409- war nicht signifikant (p >0,05) abha.ngig von den ge­415(1984) · netischen Typen des K-Casein oder ~-Laktoglobulin. (17) REHALI,V.,MENARD,J.L.:lait71275-279(1991} Jedoch wurden qualitative Unterschiede in den elek­(18) SCHAAR, J ., HANSSON, 8., PETTERSSON, H.- trophoretischen Mustem der Kase festgestellt, die aus E.:J. Dairy Res. 52429-437 (1985) ~-Casein AA- oder AS-Milch hergestellt worden waren. (19) VAN DEN BERG, . G., ESCHER, J.T.~.. £?E Ahnlich unterschieden sich die elektrophoretischen . KONING, P.J .• BOVENHUIS, H .: Neth. M1lk Da~ry Muster oder HPLC-Profile der Peptide in den wasser-J. 46145-1681992 	 loslichen Extrakten aus K-Casein-AA-Kasen von de­(20) VANSLYKE, l.L., PRICE, W .V.: In Cheese. Or- . nen aus lC-Casein-AB-Kasen. . ange Judd. Pub I. Co., NewYork, NY. (1952) Die Ergebnissen lassen vermuten, daB proteolytische (21) VINK S.• TONG, P.S., FARKYE, N.Y., IMAFIDON, Enzyme in Kase unterschiedliche Spezifitaten gegen-G.I., MEDRANO, J.F.: J. Dairy Sci. 75 (Suppl. 1) Gber Milchproteinvarianten wahrend der Kasereifung · • 123 (Abstr.) (1992) haben konnten. Production and evaluation of a fresh soft cheese (quark type) from·ewe and cow milk with selective cultures By I. VLACHOS Laboratory of Dairy Technology, Faculty of Agriculture, Aristotle University of Thessaloniki, 54006 Thessaloniki, G~e . · . · . . . . · t inu'oduction · The fresh: cheeses are products which are con­ of fresh cheese (quark type) obtained from ewe milk sumed immediatE!Iy. Differences between the vari­ with selective lacticcultures. Otis.types of cheeses. include fat content, milk ho­ 2. Materials and methods mogenization during cheese manufacturing, and the. .Presenc·e :of other.added ingredients. The objective Cheese milk Cow and ewe milk from evening and Of ~is·sfudy was theproduction and Characterization morning milkings was used for cheese production. ·Milchwissen~chaft· so (6) 1995 

Influence of genetic variants of κ-casein and β-lactoglobulin in milk on proteolysis in Cheddar cheese

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Influence of genetic variants of κ-casein and β-lactoglobulin in milk on proteolysis in Cheddar cheese

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