Peptides in fermented Finnish milk products Minna Kamala, Eero Pahkala and Anne Pihlanto-Leppälä Kamala, M., Pahkala, E. & Pihlanto-Leppälä, A. 1993. Peptides in fermented Finnish milk products. Agric. Sei. Eini. 2: 379-386. (Agric. Res. Centre of Finland, Food Res. Inst., FIN-31600 Jokioinen, Finland.) This study was conducted to investigate the rate of proteolysis and peptide profiles of different Finnish fermented milk products. The highest rate of proteolysis was observed in Biokefir, while the greatest change in the rate of proteolysis was observed in Gefilus®. Differences in starters and manufacturing processes reflected on the peptide profiles of the products. Most of the identified peptides originated from either the N- or C-terminal region of (5-casein or from the N-terminal region of as i-casein. Key words: fermented milk products, proteolysis, peptides Introduction The enzymatic degradation of milk proteins yields amino acids and peptides of varying size. The qual- ity and quantity of the products of degradation de- pend on the components of the milk protein fraction and the enzymes involved , i.e. bacterial proteases and plasmin. The temperature and pH of the pro- cesses and products also have their effect on the liberation of amino acids and peptides. The starters of fermented milk products contain proteolytic enzymes, which cause proteolysis dur- ing manufacture and storage. The products formed by proteolysis have a great effect on the final prod- uct. They can contribute to its texture, nutritional properties and flavour of the product. Peptides and amino acids are not solely responsible for the fla- vour, but they can act as precursors of the flavour- producing enzymatic reactions (Tamine and Ro- binson 1989). Certain peptides contribute to a bit- ter taste in the product. The formation of bitter peptides in the manufacture of yoghurts is favoured by a temperature below 38°C as well as by the activity of the enzymes during cold storage (Robin- son and Tamine 1981). Only a few reports have been published on the peptide content of fermented milk products. Tanev and Zivkova (1977) investigated the peptide con- tent of milk and Bulgarian yoghurt during storage using an electrophoretic method. Free amino acids have been the subject of several studies (Miller et al. 1964, Alm 1982). Also, the degradation of ca- sein components with different lactic acid bacteria has been studied by Pahkala et al. (1989a, 1989b) The aim of this study was to investigate the peptide profiles of various fermented milk products with high performance liquid chromatography (HPLC) methods. Material and methods The investigated fermented milk products were Gefilus®, plain yoghurt, Bulgarian yoghuit, viili, soured skimmed milk, acidophilus milk and Bioke- fir. The samples were obtained from two dairies in 379 Agric. Sei. Fin!. 2 (1993) https://www.c-info.fi/en/info/?token=6euAlW-6PHdbGLYp.VadauwFDLxxy6h1r-hA1AQ.2Mcbmn_LLpCquYHMQ24r1YMXP6X2nUJzhMqGpgHTWvJ5Bz2VDcGb1vaJ1ZsdGfMnb1sFBNtX1f93ZooxcKm2-a7t4wLNjbnXBD808GhaKf0oSXljk_dYW7ZyGbLo86yoVFFswaufzg2nBgHbfkcno3o1AcuAciOVxgTeuwidlmSPUUCP8fA3AnuVDcJhkUi0NfVmfRsGCF62FNwhByY0Xd2XkILzQKNDgrhjdipo9JgzDLZcf_jTAwicyrcFMt2hVL5Ctk8EUw6DwMIvMWicJ7AXw5T7KBTBk7szjPV7hj5nfuAmi5uO0Y29KF4kvhlYJ92FG1w Table 1. Micro-organisms used in the fermented milk products. Product Starter type Micro-organisms 1. Bulgarian yoghurt thermophilic Streptocoocus salivarius ssp. thermophilus, Lactobacillus delbrueckii ssp. bulgaricus 2. Plain yoghurt thermophilic Streptocoocus salivarius ssp. thermophilus, Lactobacillus delbrueckii ssp. bulgaricus 3. Gefilus mixed mesophilic + special Laclococcus lactis ssp. lactis, L. lactis ssp. cremoris, L. lactis ssp. lactis biovar. diacetylactis, Leuconostoc mesenteroides ssp. cremoris, Lactobacillus GG 4. Viili mixed mesophilic + special Laclococcus lactis ssp. lactis, L. lactis ssp. cremoris, L. lactis ssp. lactis biovar. diacetylactis, Leuconostoc mesenteroides ssp. cremoris, Geotrichum candidum 5. Biokefir mixed mesophilic + special Laclococcus lactis ssp. lactis, L. lactis ssp. cremoris, L. lactis ssp. lactis biovar. diacetylactis, Leuconostoc mesenteroides ssp. cremoris, Kefir grains 6. Acidophilus milk mixed mesophilic + special Laclococcus lactis ssp. lactis, L. lactis ssp. cremoris, L. lactis ssp. lactis biovar. diacetylactis, Leuconostoc mesenteroides ssp. cremoris, Lactobacillus acidophilus 7. Soured skimmed milk mixed mesophilic Laclococcus lactis ssp. lactis, L. lactis ssp. cremoris, L. lactis ssp. lactis biovar. diacetylactis, Leuconostoc mesenteroides ssp. cremoris southern Finland and stored at +4°C during the study period. The micro-organisms used in the manufacture of these fermented milk products are listed in Table 1. Sample preparation The proteins and larger peptides were precipitated with 6% trichloroacetic acid (TCA). The supernat- ant was centrifuged at +7°C for 15 minutes and 6(X)0 Gand filtered through a 0.45 pm filter. The samples were precipitated on the second and eighth day after manufacture as well as on the sell-by date. The proteolysis was monitored by de- termining the total nitrogen in the product and in the filtrate with the Kjeldahl method. Separation of peptides with HPLC The HPLC equipment consisted of two pumps (Waters 510), an automatic sample processor (Wa- ters Wisp 710), a UV detector (Pharmacia LKB VWM 2141) and a personal computer (NEC APV IV) with an analysis programme (Waters Baseline 810). The column was SuperPac Pep-S (4.0 x 250 mm, 5 pm; Pharmacia LKB) equipped with precol- 380 Agric. Sei. Fint. 2 (1993) umn Pep-S (4.0 x 10 mm, 5 pm; Pharmacia LKB). The column temperature was 30°C and flow rate 1.0 ml/min. The eluants were: A 0.05% trifluoro- acetic acid (TFA) in waterand B 0.05% TFA, 90% acetonitrile (CH3CN) in water. The gradient grew linearly from 2% to 60% B in 45 minutes. Before FIPLC analysis the samples were filtered once again through a 0.45 pm filter. The injection volume was 100 pi and the peptides were detected at wavelengths of 214 and 280 nm. The peptides were collected manually after the detector. Peptide identification and amino acid analysis After collection the peptides were dried under va- cuum. The total amino acid content and the N-ter- minal sequence of three amino acid residues were determined for each peptide. Free amino acids were analyzed in the TCA soluble fraction. The peptides were hydrolyzed for the total amino acid analysis in gas phase (6 N HCI + 1% phenol) at 110°C for 24 hours. The amino acids were analyzed as phenyl- tiocarbamate (PTC) derivatives using the Waters Pico Tag method. The preparation of the deriva- tives and the HPLC analysis were performed ac- cording to the instructions of Millipore Corporation (1987). The equipment was the same as in the pep- tide analysis, except that the column was Waters Pico Tag (3.9 x 150mm) and the temperature 40°C. The manual N-terminal sequence analysis was performed according to the method of Tarr (1986). Phenyltiohydantoin (PTH) amino acids were analyzed with the above-mentioned HPLC equipment on a Waters Pico Tag column. The run- ning conditions were according to Lemieux and AMIOT (1990). Results and discussion Table 2 shows the change in the rate of proteolysis of the different fermented milk products between the first day and the sell-by date. In each product the rate of proteolysis increased during the storage pe- riod. The greatest change after one week of storage was observed in Gefilus (+2.3%), while there was Table 2. Rate of proteolysis (%) in fermented milk products during storage. (6% TCA soluble N/total N x 100). Product age 1 day 7 days sell-by (days) date 1. Bulgarian yoghurt 6.37.8 8.5 (15) 2. Plain yoghurt 6.8 7.7 8.1 (14) 3. Gefilus 6.2 8.3 8.5 (18) 4. Viili 8.2 9.4 9.6 (13) 5. Biokefir 9.1 9.5 10.1 (13) 6. Acidophilus milk 8.0 - 9.6 (11) 7. Soured skimmed milk 7.8 - 8.1 (10) only a slight change in soured skimmed milk after 10 days of storage. The highest rate of proteolysis was found in Biokefir both at the age ofone day and on the sell-by date. Figures 1-7 show the peptide profiles of the dif- ferent fermented milk products at the age of one day and on the sell-by date. The suggested sequence of the identified peptides is also indicated. Figure 8 shows the peptide profile of a pasteurized milk sample on the day of manufacture and after four days’ cold storage. Fig. 1. Peptide profile of Bulgarian yoghurt at the age of I and 15 days. Identified fractions: 1. Leu, 2. Tyr, 3. Phe, 4. otsi-CN 1-14, 5. P-CN 47-57, 6. 0-CN 166-175, 7. 0-CN 176-188. 381 Agric. Sei. Finl. 2 (1993) 1 Day 14 Days Fig. 2. Peptide profile of plain yoghurt at the age of I and 14 days. Identified fractions: 1. Leu, 2. Tyr, 3. Phe, 4. aS i-CN 1-14, 5. P-CN 47-57, 6. P-CN 166-175, 7. P-CN 176-188. Fig. 3. Peptide profile of Gefilus at the age of I and 18 days. Identified fractions: I. lie, 2. Leu, 3. Tyr, 4. Phe, 5. P-CN 176-182, 6. p-CN 7-15. Fig. 4. Peptide profile of viili at the age of 1 and 13 days. Identified fractions: 1.Tyr, 2. Phe, 3. P-CN 176-182,4. P-CN 169-175, 5. P-CN 7-15, 6. P-CN 1-9, 7. P-CN 44-52. Fig. 5. Peptide profile of biokefir at the age of 1 and 13 days. Identified fractions: 1. Tyr, 2. Phe, 3. P-CN 22-28, 4. P-CN 176-182,5. P-CN 47-57, 6. P-CN 169-175,7. P-CN 7-15,8. P-CN 1-9,9. P-CN 44-52. 382 Agric. Sei. Fint. 2 (1993) A wide peak caused by TCA can be seen at the beginning of each chromatogram. This is followed by the peaks of free amino acids and peptides. Only a few peaks are found on the chromatogram of pasteurized milk (Figure 8). A comparison between fermented milk products and pasteurized milk shows the great effect of the fermentation process on the peptide content of the product. Also, the increase in both the height and area of the peptide peaks after storage gives information on the intens- ity of proteolysis. In the free amino acid determinations, a high content of proline compared to other amino acids was observed, showing the highest content of all amino acids in all the fermented milks except in Gefdus (Figures 9 and 10). Glutamine content was also high in the products. During the manufacture and storage the content of all amino acids and pep- tides increased. Alm (1982) also found great varia- tions in the amino acid content of different prod- ucts. In yoghurt the proline content was found to be higher than average. Kefir was rich in lysine and proline at that study. Differences caused by starters during the fer- mentation process become obvious when the pep- tide profile and content of the products are com- pared. Aclear difference was between yoghurts and other fermented milk products. Yoghurts contained peptides which could not be found in the other products. Most of the identifiedpeptides in all prod- ucts originated from (Tcasein. However, opioid Fig. 6. Peptide profile of acidophilus milk at the age of I and 11 days. Identified fractions: 1. Tyr, 2. Phe, 3. (3-CN 176- 182, 4. (3-CN 169-175, 5. (3-CN 7-16, 6. (3-CN 164-175, 7. (3-CN 44- 52. Fig. 7. Peptide profile of soured skimmed milk at the age of 1 and 10 days. Identified fractions: I. Tyr, 2. Phe, 3. (3-CN 22-28,4. (3-CN 176-182,5. k-CN 161-169,6.(3-CN 169-175, 7. (3-CN 7- 15. Fig. 8. Peptide profile of pasteurized milk at the age of 1 day and after 4 days’ storage. 383 Agric. Sei. Finl. 2 (1993) p-casomorphines (P-CN f 60-70) were not ob- served. Table 3 summarizes the peptides which were identified during the study. Peptides typical for yo- ghurts include fragments 1-14from a si-casein and 47-57,166-175 and 176-188 from P-casein. Peptide P-CN 176-188 contains a fragment which, accord- ing to Maruyama et al. (1985), possesses antihy- pertensive activity (p-CN f 177-183). Despite the apparent similarity of the peptide profiles of Bul- Fig. 9. Free amino acid content (pmol/g product) of Bulgarian yog- hurt, Plain yoghurt, Gefdus and Biokefir. The three letters amino acid codes used. Fig. 10. Free amino acid content (pmol/g product) of acidophilus milk, soured skimmed milk and viili. The three letters amino acid codes used. 384 Agric. Sei. Fint. 2(1993) Table 3. Identified peptide sequences in fermented milk prod- ucts. 1. Bulgarian yoghurt, 2. Plain yoghurt, 3. Gefilus, 4. Viili, 5. Biokefir, 6. Acidophilus milk, 7. Soured skimmed milk. Sequence 1. 2. 3. 4. 5. 6. 7. 0,1-CN x x 1-14 P-CN x x 1-9 7-16 x x x x x 22-28 x x 44-52 xxx 47-57 xx x 164-175 x x 166-175 x x 169-175 xxxx 176-182 x x x x x 176-188 x x K-CN x 161-169 garian and plain yoghurt, slight differences exist which are presumably caused by differences in the combination of starter and the manufacturing proc- esses. The peptide profile of Gefilus differs very much from that of both yoghurts. The identified peptides are different and the free amino acid con- tent is also higher than in yoghurts. There is a certain similarity between the peptide profiles of viili, soured skimmed milk and Gefilus (Figures 3-7). Identical fragments of (3-casein were found in these products. The most common pep- tides were fragments 7-15, 44-52, 169-175 and 176-182 of (3-casein. Fragment 161-169 from the C-terminal region of K-casein appears in soured skimmed milk. This peptide was not identified in any other product. Fragment 22-28 from (3-casein appears only in kefir and soured skimmed milk. One of the peptides identified in viili and acidophi- lus milk was (3-CN f 164-175. The effect of plasmin on the peptide profile of the studied fermented milk products seems to be negli- gible. Its high specificity towards ots 2-casein should be easily recognizable (VISSER et al. 1989, Pah- kala et al. 1989a). The activity is obviously weak- ened by the high heat treatment ofmilk and low pH. An additional cause might be the relatively short storage period which may have led to an overlap- ping of the proteolytic activity of starters with the activity of plasmin. The high heat treatment of milk during the manufacture of all the fermented milk products denaturates the whey proteins and causes the association of K-casein and (3-lactoglobulin (Dalgleish 1990). The effect of these phenomena on the proteolytic end products was, however, not distinguishable in this study. References Alm, L. 1982. The effect of fermentation on nutrients in milk and some properties of fermented liquid milk products. Stockholm, Sweden. (Diss.). Dalgleish, D.G. 1990. Denaturation and aggregation of serum proteins and caseins in heated milk. J. Agric. Food Chem. 38: 1995-1999. Lemieux, L. & Amiot, J. 1990. High-performance liquid chromatography of casein hydrolysates phosphorylated and dephosphorylated. 1. Peptide mapping. J. Chroma- togr.soo: 299-321. Maruyama, S., Naoami, K., Tomizuka, H. & Suzuki, H.K. 1985.Angiotensin I converting inhibitor derived froman enzymatic hydrolysate of casein. 11. Isolation and brady kin-potentiating activity on the uterus and the ileum of rats. Agric.Biol.Chem. 49: 1405-1409. Miller, I„ Martin, H. & Kandler, O. 1964. Das Ami- nosäurespektrum von Joghurt. Milchwissenschaft 19: 18- 25. Millipore Corporation 1987. Liquid chromatographic ana- lysis of amino acids in feeds and foods using a modifica- tion of the Pico-Tag method. Revision. Pahkala, E., Pihlanto-Leppälä, A., Laukkanen, M. & An tila, V. 1989a. Decomposition of milk proteins during the ripening of cheese. 1, Enzymatic hydrolysis of as-ca- sein. Meijeritieteellinen Aikakauskirja 47, 1: 39-47. , Pihlanto-Leppälä, A., Laukkanen, M. & Antila, V. 1989b. Decomposition of milk proteins during the ripen- ing of cheese. 2. Enzymatic hydrolysis of(3-casein. Mei- jeritieteellinen Aikakauskirja 47. 1: 63-70. Robinson, R.K. & Tamime, A.Y. 1981. Microbiology of fermented milks. In: Robinson, R.K. (ed.). Dairy Micro- biology, vol. 2, The Microbiology of Milk Products. Applied Science Publishers, Essex, England, p. 245-277. Tamime, A.Y. & Robinson, R.K. 1989. Yoghurt: Science and Technology. 2nd ed., Pergamon Press, Oxford. Tanev, G. & Zivkova, A. 1977. Study of short-chain pep- tides in Bulgarian yoghurt 1. Preparation of peptide maps, Milchwissenschaft 32; 280-282. 385 Agric. Sei. Finl. 2 (1993) Tarr, G.E. 1986. Manual Edman sequencing system. In: Shively, J.E, (ed.). Methods of Protein Microcharacter- ization, A Practical Handbook. Humana Press, Clifton, New Jersey, p. 155-193. Manuscript received July 1993 Minna Kahala Eero Pahkala Visser, S., Slangen, K.J., Alting, A.C. & Vreeman, H.J. 1989. Specificity of bovine plasmin in its action on bo- vine aS2-casein. Milchwissenschaft 44: 335-339. Anne Pihlanto-Leppälä Agricultural Research Centre of Finland Food Research Institute FIN-31600 Jokioinen, Finland SELOSTUS Hapanmaitotuotteiden peptideistä Minna Kahala, Eero Pahkala jaAnne Pihlanto-Leppälä Maatalouden tutkimuskeskus Suomalaisten hapanmaitotuotteiden valkuaisaineiden pilk- koutumista tutkittiin varastoinnin aikana. Tuotteista määritet- tiin proteolyysiaste sekä eristettiin ja identifioitiin peptidejä peptidikartalta. Suurin proteolyysiaste todettiin biokefiirissä, kun taas suurin proteolyysiasteen muutos todettiin gefilukses- sa. Erot käytetyissä hapatteissa ja valmistusmenetelmissä ku- vastuivat tuotteiden peptidikartoissa. Useimmat tunnistetuista peptideistä olivat peräisin (J-kaseiinin N- tai C-termmaalisesta päästä tai aS i-kaseiinin N-terminaalisesta päästä. 386 Agric. Sei. Finl. 2(1993)