Microbial transglutaminase enhances antioxidant activity of yogurt ...

International Journal of Food Science and Technology 2017

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Original article Microbial transglutaminase enhances antioxidant activity of yogurt through altering pattern of water-soluble peptides and increasing release of amino acids HaiNa Yuan,1 JianMin Lv,2 JinYan Gong,1* HaiLong Xiao,3 GuangSheng Zhao,4 GongNian Xiao,1 Hui Xu1 & 1 WenChao Wang 1 School of Biological and Chemical Engineering/School of Light Industry, Zhejiang University of Science and Technology, Zhejiang Provincial Collaborative Innovation Center of Agricultural Biological Resources Biochemical Manufacturing, Zhejiang Provincial Key Lab for Chem & Bio Processing Technology of Farm Produces, Hangzhou, Zhejiang 310023, China 2 Laboratory Animal Research Center, Zhejiang Chinese Medical University, Hangzhou, Zhejiang 310053, China 3 Hangzhou Institute for Food and Drug Control, Hangzhou, Zhejiang 310022, China 4 Hangzhou New Hope Bimodal Dairy Co. Ltd, Hangzhou, Zhejiang 310000, China (Received 4 August 2017; Accepted in revised form 25 October 2017)

Summary

This study compared the antioxidant capacity of yogurt with and without microbial transglutaminase treatment after milk fermentation, and investigated the correlation between antioxidant property and the water-soluble peptides and amino acids composition. Results showed that small molecular peptide fraction exhibited stronger antioxidant activity than large peptides. Microbial transglutaminase yogurt isolated fraction had stronger antioxidant capacity than that from the control. Microbial transglutaminase altered the peptides composition, and resulted in higher amount of small molecular peptides (30 kDa, second fraction: 10–30 kDa, third fraction: 3–10 kDa, and fourth fraction: 30 kDa, 10–30 kDa, 3–10 kDa and 30 kDa, 10–30 kDa, 3–10 kDa, and 30 kDa) showed the highest IC50 value, followed by the second peptide fraction (10–30 kDa) and then the third peptide fraction (3– 10 kDa). However, the fourth peptide fraction (30 kDa) possessed the least DPPH antioxidant activity, whereas the highest DPPH antioxidant capacity was found in the fourth peptide fraction (30 kDa) isolated from the yogurt with the microbial transglutaminase treatment showed higher DPPH antioxidant property than that isolated from the control yogurt. The similar observation was also found in the third (3–10 kDa) and fourth peptide fractions (30 kDa) showed the least reducing power value, whereas the fourth peptide fraction ( 30 kDa –4

–3

–2

–1

0 1 PC 1 (70.2%)

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peptide fraction (>30 kDa) (Fig. 2d). More importantly, each peptide fraction isolated from the yogurt treated with the microbial transglutaminase showed higher lipid peroxidation inhibitory activity than that isolated from the control yogurt except for the 3– 10 kDa fraction (Fig. 2d). These indicated that the microbial transglutaminase might alter the cross-linking location of amino acids in the sequence of peptides, which resulted in the formation of more hydrophobic sites in the peptides (O’Sullivan et al., 2013). Besides, the microbial transglutaminase has also been reported to play a positive role in enhancing the emulsifying property of peptides through inter- and/or intramolecular polymerisation of peptides (Yang et al., 2016). Such interactions could form a barrier in the oil–water interface to block interactions between free radicals and lipid, which could prevent lipid peroxidation (Donnelly et al., 1998). Free amino acids in isolated peptide fractions

Table 1 shows the composition and concentration of free amino acids in each isolated peptide fraction from the yogurt treated with and without the microbial transglutaminase. All the peptide fractions isolated from the yogurt with the microbial transglutaminase treatment exhibited higher concentration on the free amino acids compared to those from the control yogurt. It was observed that the predominant free amino acids in the peptide fractions isolated from the microbial transglutaminase-treated yogurt included glutamic acid, leucine and arginine. These three amino acids represented 30.9%, 22.6% and 18.9% of the total free amino acid content, and existed mainly in the third (3–10 kDa) and fourth (30 kDa, 10–30 kDa, 3–10 kDa, and 30 kDa, 10–30 kDa, 3–10 kDa, and 30 kDa) peptide fraction isolated from the microbial transglutaminase-treated yogurt. Meanwhile, this peptide fraction exhibited a peptide band with the molecular weight between 25 kDa and 50 kDa compared to that isolated from the control yogurt. The alteration on the peptide molecule patterns indicated that the microbial transglutaminase might specifically cause the transamination and deamination of amino acids in proteins, resulting in the different peptide molecules in this fraction. In addition, the microbial transglutaminase did not significantly affect the pattern of the second isolated peptide fraction (10–30 kDa) from the yogurt as this peptide fraction between the control and the treated yogurt had the similar peptide pattern. Regarding the third isolated peptide fraction, only a peptide band (around 10 kDa) was observed in this fraction. However, the intensity of this band was greater in the peptide fraction isolated from the microbial transglutaminasetreated yogurt, indicating that such a treatment enhanced the concentration of the peptides in yogurt. No visible bands were observed in the fourth peptide fraction isolated from the yogurt. This might be because that these smaller molecular peptides could be easily eluted from the gel during the electrophoresis or the staining process.

© 2017 Institute of Food Science and Technology

Peptide characterisation by LC-ESI-QTOF-MS and relation with antioxidant activity

To investigate the antioxidant mechanism, these peptide fractions were further analysed using LC-MS and their chromatograms are displayed in Fig. 5. Table 2 lists the sequences of the dominant peptides in the yogurt without the microbial transglutaminase treatment, whereas the dominant peptide sequences found in the microbial transglutaminase-treated yogurt are shown in Table 3. The sequence of these main peptides was identified by comparing their mass spectrum with the milk proteins in the PLGS databank search system. It was observed that the peptides with the molecular weight below 1.5 kDa accounted for about 63.1% of the total peptides in the microbial transglutaminasetreated yogurt, whereas the control yogurt only contained about 55.2% of the peptides with the molecular weight below 1.5 kDa. It has been reported that small molecular peptides, compared to big molecular peptides, possessed stronger radical scavenging capacity (Irshad et al., 2013). This could explain why the microbial transglutaminase-treated yogurt displayed higher antioxidant activity than the control yogurt. In addition, the control yogurt had b-casein as the major peptides (Table 2), whereas the primary peptide fragments in the microbial transglutaminase-treated yogurt was from j-casein. In detail, these j-casein-derived peptides played important parts in the domains of the amino acid sequence number 13–20, 30–75 and 100– 160. The major peptide sequences in the control yogurt were derived from b-casein, and our results were consistent with the previous study (SabeenaFarvin et al., 2010b). We speculated that the proteinases in lactic acid bacteria resulted in the cleavages of b-casein (Hernandez-Ledesma et al., 2005). On the contrary, the microbial transglutaminase-treated yogurt only showed 10 b-casein-derived peptides. Meanwhile, numerous jcasein-derived peptides were found in the treated yogurt. These results indicated that the proteolytic mechanism by lactobacillus might be modified with the presence of the microbial transglutaminase (Wr oblewska et al., 2011; Li et al., 2012). The significant differences were also observed on the composition and distribution of the peptides from the yogurt with and without the microbial transglutaminase treatment. For instance, few peptides with molecular weight below 3 kDa were identified in both


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mTG enhances antioxidation of yogurt H. N. Yuan et al.

Table 3 Identification of peptides in yogurt with microbial transglutaminase treatment using LC-ESI-QTOF MS/MS Peak No

Retention time (min)

1

1.39

2 3 4 5 6

2.36 4.01 5.58 6.19 6.6

7 8

7.42 8.07

9

8.45

10

8.5

11 12 13 14

8.94 10.8 11.73 11.85

15

12.48

16 17 18 19 20 21 22 23

13.31 13.69 14.28 14.85 16.18 2.96 4.23 4.89

24

5.89

25 26

6.17 6.87

27

7.42

28

8.73

29

9.14

30 31

9.58 10.2

32

12.19

Peak mW (Da)

Sequence

Suggested fragment

390.2 486.26 1991.09 2607.55 1937.04 2308.08 1158.6 681.36 1052.45 1098.57 1881.97 436.23 2210.02 2009 1325.69 589.36 1126.77 945.57 799.51 616.34 2070.73 2278.27 1250.72 1492.58 1883.97 503.27 1403.69 641.34 1457.74 1630.82 1564.86 1743.9 2395.26 2144.04 1864.04 1031.49 905.49 950.47 1482.6 1892.66

(V)SSKS(A) (G)LPQE(V) (A)RPKHPIKHQGLPQEVLN(E) (R)YLGYLEQLLRLKKYKVPQLEI(V) (I)PPKKNQDKTEIPTINTI(A) (E)DIKQMEAESISSSEEIVPNSV(E) (N)QDKTEIPTIN(T) (L)PQEVLN(E) (P)LGTQYTDAPS(F) (E)VIESPPEINT(V) (S)TVATLEASPEVIESPPEI(N) (K)HQGL(P) (P)SGAWYYVPLGTQYTDAPSFS(D) (P)NSVEQKHIQKDDVPSER(Y) (E)VIESPPEINTVQ(V) (K)IAKYI (P) (K)IAKYIPIQY(V) (K)IAKYIPIQ(Y) (K)IAKYIPI(Q) (K)YKVPQ(L) (D)CSGTMKCCNNGCIMSCM(D) (Y)AKPAAVRSPAQILQWQVLSNT(V) (K)PAAVRSPAQILQ(W) (A)SGEPTSTPTTEAVES(T) (E)VIESPPEINTVQVTSTAV(-) (N)NQFL(P) (N)NQFLPYPYYAK(P) (Y)PYYAK(P) (N)QFLPYPYYAKPA(A) (L)INNQFLPYPYYAK(P) (K)PAAVRSPAQILQWQ(V) (A)LINNQFLPYPYYAK(P) (Y)QQKPVALINNQFLPYPYYAK(P) (F)SDIPNPIGSENSGKTTMPLW(-) (K)PAAVRSPAQILQWQVLS(N) (S)GEPTSTPTIE(A) (N)TVQVTSTAV(-) (G)IHAQQKEP(M) (T)EDQAMEDIKQME(A) (I)AAGPCPKGNPCSIDSDCS(G)

j-CN 154-157 a-CN 26-29 a-CN 16-32 a-CN 106-126 j-CN 100-116 a-CN 71-91 j-CN 105-114 a-CN 27-32 a-CN 184-193 j-CN 143-152 j-CN 133-150 a-CN 23-26 a-CN 176-195 a-CN 89-105 j-CN 143-154 j-CN 13-17 j-CN 13-21 j-CN 13-20 j-CN 13-19 a-CN 119-123 a-lactalbumin 108-124 j-CN 53-73 j-CN 55-66 j-CN 118-132 j-CN 143-160 j-CN 44-47 j-CN 44-54 j-CN 50-54 j-CN 45-56 j-CN 42-54 j-CN 55-68 j-CN 41-54 j-CN 35-54 a-CN 195-214 j-CN 55-71 j-CN 119-128 j-CN 152-160 a-CN 142-149 a-CN 65-76 a-lactalbumin 92-109 a-CN 106-136

3740.31 1894.63 1403.68 2122.73 542.28 876.48 1098.57 1601.77 1325.69 2367 2890.23 1843.66 2390.26 898.45 755.43 2413.1

(R)YLGYLEQLLRLKKYKVPQLEIVPNSAEERLH(S) (D)SDCSGTMKCCNNGCIM(S) (N)TIASGEPTSTPTIE(A) (C)SGTMKCCNNGCIMSCMDP(K) (K)TEIPT(I) (K)YKVPQLE(I) (E)VIESPPEINT(V) (Y)PSGAWYYVPLGTQY(T) (E)VIESPPEINTVQ(V) (N)DMCCPSSCGRPCKTPVNIEV(Q) (L)SKDIGSESTEDQAMEDIKQMEAESIS(S) (K)CCNNGCIMSCMDPKP(D) (F)VAPFPEVFGKEKVNELSKDIGS(E) (V)IESPPEIN(T) (Q)GLPQEVL(N) (P)WNPIQMIAAGPCPKGNPCSIDS(D)


a-lactalbumin j-CN 115-128 a-lactalbumin j-CN 107-111 a-CN 119-125 j-CN 143-152 a-CN 175-188 j-CN 143-154 a-lactalbumin a-CN 56-81 a-lactalbumin a-CN 40-61 j-CN 144-151 a-CN 25-31 a-lactalbumin

106-121 109-126

58-77 114-128

85-106



Table 3 (Continued) Peak No

Retention time (min)

33 34 35

1.49 2.97 4.12

36 37

4.51 6.21

38

6.43

39 40

7.44 8.11

41 42

8.5 8.94

43

9.17

44 45 46 47 48 49 50 51

9.51 9.81 10.2 10.52 10.91 2.23 3.6 4.45

Peak mW (Da)

Sequence

Suggested fragment

705.38 1118.53 1750.69 770.41 1418.67 812.45 680.4 567.32 915.51 1158.6 1098.57 478.24 892.46 2278.27 1250.72 1628.63 1267.49 1094.52 1669.82 1843.66 1435.83 1241.6 898.45 1422.76 1067.66 560.29 790.47 529.3

(V)QVTSTAV(-) (A)SGEPTSTPTIE(A) (G)PCPKGNPCSIDSDCSG(T) (H)QGLPQEV(L) (A)PKHEEMPFPKY(P) (G)LPQEVLN(E) (G)LPQEVL(N) (G)LPQEV(L) (D)KTEIPTIN(T) (N)QDKTEIPTIN(T) (E)VIESPPEINT(V) (G)KTTM(P) (G)KTTMPLW(-) (Y)AKPAAVRSPAQILQWQVLSNT(V) (K)PAAVRSPAQILQ(W) (P)KGNPCSIDSDCSGTM(K) (S)ESTEDQAMEDI(K) (F)TESQSLTLTD(V) (-)DELQDKIHPFAQTQ(S) (K)CCNNGCIMSCMDPKP(D) (K)VLPVPQKAVPYPQ(R) (-)DELQDKIHPF(A) (V)IESPPEIN(T) (P)NSLPQNIPPLTQT(P) (S)LSQSKVLPVP(Q) (S)TPTIE(A) (T)TIQTTNI(P) (N)TVQVT(S)

j-CN 154-160 j-CN 118-128 b-CN 95-110 a-CN 24-30 b-CN 62-72 a-CN 26-32 a-CN 26-31 a-CN 26-30 j-CN 106-113 j-CN 104-113 j-CN 41-50 a-CN 208-211 a-CN 208-214 j-CN 53-73 j-CN 55-66 b-CN 98-112 b-CN 62-72 b-CN 78-87 b-CN 1-14 a-lactalbumin 114-128 b-CN 128-140 b-CN 1-10 j-CN 144-151 b-CN 26-38 b-CN 123-132 j-CN 124-128 j-CN 133-139 j-CN 152-156

control and treated yogurt (Fig. 5). This result was in an agreement with our SDS-PAGE result (Fig. 4). Tyrosine, cysteine, histidine and methionine have been confirmed to possess antioxidant property (SabeenaFarvin et al., 2010b). These amino acids existed in different levels in the fourth peptide fraction isolated from the yogurt treated with and without the microbial transglutaminase (Table 1), which resulted in the different antioxidant properties to these fractions. In the other three isolated peptide fractions (3–10 kDa, 10–30 kDa, and >30 kDa) from the treated yogurt, some peptides have been reported to display the antioxidant activity. For example, the j-casein-derived peptide IAKYIPIQY (Peak No.9) shared a sequence IPIQY with a known antioxidant peptide IPIQYVL (Table 3) (Hernandez-Ledesma et al., 2005). This peptide sequence exhibited an intense mass spectrum signal in these fractions from the treated yogurt, indicating that these isolated fractions were rich in this peptide. It should be noted that the first (>30 kDa) and second (10–30 kDa) isolated peptide fractions from the control yogurt also contained this sequence (Table 2) (Peak No.4). Besides, it has been reported that the peptide YY, YYL, YYI, TTYY, LGFEYY, SGYYMH, WVYY, YYDPL, YYLVS, YIPIQY and


YLGAK also showed the antioxidant properties, and these peptides contained the sequence of YY, YL, or YI (Beermann et al.,2009; Guo et al.,2009; Shen et al., 2010; Chen et al., 2012; Amadou et al.,2013; Girgih et al.,2014). The j-casein-derived peptides, such as PYYAK, NQFLPYPYYAK, INNQFLPYPYYAK, QFLPYPYYAKPA, IAKYI and IAKYIPI, in these peptide fractions from the treated yogurt also contained these sequences, indicating that these peptides might play important roles in contributing to the overall antioxidant properties (Table 3). It should be noted that many peptides in the treated yogurt contained hydrophobic amino acid residues, such as valine, leucine, proline, cysteine, histidine and methionine (Table 3). It has been confirmed that tyrosine and cysteine residues can contribute the radical scavenging property to peptides, whereas proline, valine, leucine, phenylalanine and histidine in peptides play roles in inhibiting lipid peroxidation (Tagliazucchi et al., 2016). Different from the microbial transglutaminasetreated yogurt, the b-casein-derived peptides, including PYPQ, VLPVPQKAVPYPQ and PVPQKAVPYPQ, were considered the antioxidative peptides in the control yogurt (Table 2), as their domain PYPQ has been confirmed to participate in antioxidant activity (Rival


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et al.,2001). Additionally, the j-casein-derived peptide VLSRYPSYGLN and PIQYVLSRYPSY were also considered to have antioxidant properties as the sequence YPS was reported to provide antioxidative feature to the peptide AYPS, SRYPS, and RYPS (De Gobba et al., 2014; Tagliazucchi et al., 2016). Moreover, a long peptide QPTTMARHPHPHLSFMAI (j-casein 82-99) found in the control yogurt might be the precursor of an antioxidant peptide HPHPHLSF (G omez-Ruiz et al., 2008). It was speculated that microbial transglutaminase might cross-link different proteins within casein and/or whey proteins, which resulted in the alteration of their solubility or altered their reaction with the proteinases during yogurt production. As a result, the composition and concentration of peptides and amino acids in yogurt were significantly altered (Perez-Mateos & Carmen G omez-Guiuen, 2002). Conclusions

In conclusion, the antioxidant activity of yogurt was enhanced by microbial transglutaminase treatment during fermentation. Microbial transglutaminase application resulted in yogurt with higher amounts of small molecular peptides (