The objective of this investigation was to verify whether biologically active peptides (BAPs) could be obtained from water solutions of
Not only are milk proteins appreciated for their good nutritional quality, but they are perceived also as a reach precursor for a wide range of
It is worth to mention that the cow milk proteins are also one of the major food allergens; however, the activity of effective proteolytic system of lactic acid bacteria (involved in milk fermentation) allows to reduce the antigenicity of the proteins [
Currently, a number of desired biological effects have been attributed to various sequences of BAPs (also depending on the type of the food matrix constituting a source of proteins that are precursors for biopeptides) [
Active amino acid sequences of biopeptides can be released from the native protein structure by enzymatic hydrolysis (
It is worth to emphasize that among all the lactic acid bacteria,
Although it has been proven that certain biopeptides have significant nutritional and functional importance for the regulation of certain physiological functions in the organism, they have not been fully described and widely discussed in terms of their production, diverse mechanisms of action, and the wide range of multibiological activities [
The beneficial properties of BAPs derived from milk products are perceived as significant factors influencing a decrease in the risk of development of some civilization diseases [
Three strains of
The bacterial inoculums used in the study were prepared according to Beganović et al. [
Water solutions of
The analysis was performed using an Agilent HPLC chromatograph series 1290 coupled to Agilent 6550 iFunnel Q-TOF equipped with a Jest Stream Technology electrospray ion source. An Agilent HPLC
From the wide range of amino acid sequences obtained after hydrolysis of the analyzed protein preparations, products in the mass range from 400 to 8000 Da were selected for further
Samples of 13% regenerated skim milk (OSM Krasnystaw, Poland) enriched by the addition of 1% (
The texture profile analysis (TPA) was performed in order to compare the textural properties of final products (fermented beverages containing the addition of CGMP or
The protein content in the fermented products obtained was determined with the Kjeldahl method according to EN ISO 8968-1:2014 [
Statistical analyses were performed using the Statistica 13.1 software package (StatSoft, Krakow, Poland). The hierarchical cluster analysis (HCA) was used to explore similarity between the examined samples. Clustering was performed using the Ward minimum variance method with Euclidean distance as a similarity measure. The principal component analysis (PCA) was applied to show the relationships between the variables. PCA was conducted using the correlation matrix. The results of PCA and the calculated Pearson correlation coefficients allowed choosing variables for HCA. Also, the post hoc test was applied in order to explore differences between the mean values in the analysis of texture parameters, determination of the protein, and nitrogen content and in the analysis of the proteolysis index (at 0.05 level).
Nowadays, an increasing interest of new food-derived sources of biopeptides precursors has been observed. Moreover, the development of interdisciplinary investigations focused on isolation and identification short amino acid sequences (containing from 2 to 20 residues) from various food matrixes in order to determine the peptide bioactivities is clearly discernible [
Sequences of biopeptides detected in the hydrolysates.
Source of peptides (type of analyzed hydrolysate) | Identified peptide sequence | Mass (Da) | ID of the bioactive peptide in the database | |
---|---|---|---|---|
CGMP | T105 | LPYPYY | 814.30 | biopep00859/BioPepDBb |
CGMP | T80 | LRFa | 434.26 | biopep00880/BioPepDB |
CGMP | T105 | DKIHPF | 755.39 | biopep00136/BioPepDB |
CGMP | T105 | AVPYPQR | 829.44 | biopep00091/BioPepDB |
CGMP | DSMZ, T80, T105 | DKIHP | 608.33 | biopep00135/BioPepDB |
CGMP | T80 | EMPFPKYPVEP | 1332.64 | biopep00178/BioPepDB |
CGMP | B734 | RPKHPI | 746.45 | biopep01209/BioPepDB |
CGMP | B734 | PYVRYL | 809.44 | biopep01134/BioPepDB |
CGMP | B734 | LTLTDVE | 789.41 | biopep00907/BioPepDB |
CGMP | T80 | KYPVEPFTESQSLTL | 1737.88 | biopep00721/BioPepDB |
DSMZ | ||||
CGMP | B734 | GPFPIIV | 741.44 | 8159/BIOPEP-UWM |
CGMP | T105 | FVAPFPEVFGK | 1236.65 | biopep00304/BioPepDB |
CGMP | B734, T80 | IPNPIGSE | 825.42 | biopep00561/BioPepDB |
CGMP | B734, DSMZ, T105 | ARHPHPHLSF | 1197.61 | biopep00073/BioPepDB |
CGMP | B734, DSMZ, T80, T105 | SPPEIN | 655.30 | biopep01254/BioPepDB |
T105 | YQKFPQY | 972.47 | biopep01624/BioPepDB | |
CGMP; | B734, DSMZ, T80, T105 | ERFa | 450.22 | biopep00189/BioPepDB |
CGMP | B734, DSMZ, T80, T105 | VRSP | 457.26 | biopep01460/BioPepDB |
T80 | ||||
CGMP | T80, T105, B734, | SRYa | 424.21 | biopep01260/BioPepDB |
T80, T105, B734, DSMZ | ||||
CGMP; | B734, DSMZ, T80, T105 | RPKHPIKHQGLPQEVLNEN | 2233.22 | biopep01215/BioPepDB |
B734 | RPKHPIKH | 1011.60 | biopep01211/BioPepDB | |
CGMP | T105 | RPKHPIKHQGLPQEV | 1762.99 | biopep01214/BioPepDB |
CGMP | B734, DSMZ, T105 | RPKHPIKHQGLPQ | 1534.88 | biopep01213/BioPepDB |
CGMP | B734, DSMZ, T105 | RPKHPIKHQ | 1139.6 | 7483/BIOPEP-UWM |
B734, T80, T105 | ||||
CGMP | B734, T80, DSMZ, T105 | HKEMPFPKYPVEPF | 1744.86 | biopep00457/BioPepDB |
CGMP | B734, T105 | MAIPPK | 655.37 | biopep00945/BioPepDB |
CGMP | B734, T80, DSMZ, T105 | MAIPPKK | 783.46 | biopep00946/BioPepDB |
CGMP | B734, T80 | IPNPIGSE | 825.42 | biopep00561/BioPepDB |
CGMP | B734, T105 | RELEE | 674.32 | biopep01181/BioPepDB |
CGMP | T80, T105 | QKAVPYPQRDMPI | 1541.80 | biopep01145/BioPepDB |
CGMP; | B734, T80, DSMZ, T105 | AYPS | 436.20 | 8472/BIOPEP-UWM |
CGMP | B734, DSMZ | LLRa | 400.28 | 8484/BIOPEP-UWM |
CGMP | T80, DSMZ | RELEELNVPGEIVESLSSSEESITR | 2801.39 | biopep04772/BioPepDB |
CGMP; | B734, T80, DSMZ, T105 | YQEPVLGPVRGPFPIIV | 1880.06 | biopep04801/BioPepDB |
CGMP | B734, DSMZ T105 | NLHLPLP | 802.47 | 2669/BIOPEP-UWM |
CGMP; | B734, T80, DSMZ, T105 | VTSTAV | 576.30 | 7481/BIOPEP-UWM |
CGMP; | B734, T80, DSMZ, T105 | LLYQEPVLGPVRGPFPIIV | 2106.22 | 8174/BIOPEP-UWM |
T105, DSMZ, | LLYQEP | 761.40 | biopep00829/BioPepDB | |
CGMP | DSMZ | YPFPGPI | 789.41 | biopep04798/BioPepDB |
CGMP; | B734, T80, DSMZ, T105 | MAIPPKKNQDKTEIPTINTIASGEPTSTPTTEAVESTVATLEDSPEVIESPPEINTVQVTSTAV | 6703.37 | biopep03480/BioPepDB |
CGMP; | DSMZ | YYQQKP | 825.40 | 8383/BIOPEP-UWM |
CGMP | DSMZ | YIPIQY | 795.42 | biopep01570/BioPepDB |
CGMP | B734, T80, DSMZ, T105 | MAIPPKKNQDK | 1268.69 | biopep04784/BioPepDB |
CGMP | T105 | VLPVPQK | 779.49 | biopep01419/BioPepDB |
CGMP | B734, T80, DSMZ, T105 | VPSERYL | 862.45 | 9250/BIOPEP-UWM |
CGMP | T80 | VVPP | 410.25 | biopep01483/BioPepDB |
T105 | TKKTKLTEEEKNRL | 1716.97 | biopep01292/BioPepDB | |
CGMP | B734, T80, | |||
CGMP | B734, T80, DSMZ, T105 | VQVTSTAV | 803.40 | biopep01445/BioPepDB |
CGMP | B734, T80 | RPKHPIKHQGLPQEVLNENLLRF | 2762.54 | biopep03712/BioPepDB |
B734 |
aBased on accurate mass matching, <5 ppm; bdatabase:
It is worth to mention that
The analysis of obtained peptide sequences (generated by
The findings demonstrated the presence of a wide range of BAPs with antihypertensive, ACE inhibitory, and antithrombotic activities in the fermented protein preparations. This is particularly relevant especially in the context of prevention and treatment of atherothrombotic disease, which affects a substantial part of society nowadays. It is considered that coronary artery disease and stroke are one of the world’s leading causes of death. In our study, the antihypertensive effect was the most common bioactivity in all the detected sequences of BAPs (Table
Number of detected biopeptide sequences (with identified bioactivity) generated from the tested protein preparations by the analyzed
Bioactivity | Source of peptides (type of analyzed hydrolysate) | Number of bioactive peptide sequences generated by the tested | |||
---|---|---|---|---|---|
B734 | DSMZ | T80 | T105 | ||
Antihypertensive | CGMP | 24 | 17 | 18 | 24 |
7 | 7 | 7 | 9 | ||
ACE inhibitory | CGMP | 8 | 9 | 5 | 8 |
3 | 3 | 4 | 4 | ||
Antithrombotic | CGMP | 3 | 2 | 2 | 3 |
— | — | — | — | ||
Antioxidative | CGMP | 3 | 2 | 3 | 4 |
1 | 1 | 1 | 1 | ||
Antimicrobial/antibacterial | CGMP | 4 | 3 | 4 | 3 |
3 | 2 | 2 | 2 | ||
Immunomodulating/immuno- and cytomodulatory peptides | CGMP | 2 | 3 | 2 | 3 |
2 | 2 | 2 | 2 | ||
Mineral binding | CGMP | — | 1 | 1 | — |
— | — | — | — | ||
Opioid agonist | CGMP | — | 1 | — | — |
— | — | — | — | ||
Total number of identified different biopeptide sequences | CGMP | 29 | 24 | 26 | 29 |
The desirable cardiovascular effects of BAPs derived from milk are associated with the presence of opioid peptides (generated from whey proteins
ACE (peptidyldipeptide hydrolase, EC 3.4.15.1) is an exopeptidase that performs an important role in the blood pressure regulation through the renin-angiotensin and bradykinin pathways [
It was revealed that AVPYPQR (casokinin) obtained through enzymatic hydrolysis (by trypsin) of
The YQKFPQY sequence is an example of another unique biopeptide that was obtained only through application of the particular
Unlike the above-mentioned BAPs (generated from the specific protein preparation only by some strains of the tested bacteria), the sequence MAIPPKK was released by all of the analyzed strains from CGMP (Table
It was noted that the CGMP fermentation by
The YQEPVLGPVRGPFPIIV is an example of a biopeptide that was generated by all the
Another biopeptide (that is a precursor of YQEPVLGPVRGPFPIIV) with immunomodulatory properties was LLYQEPVLGPVRGPFPIIV detected in all samples of the tested preparation fermented by the analyzed
The result revealed that only strains T80 and DSMZ were able to generate the biopeptide with mineral-building activity (RELEELNVPGEIVESLSSSEESITR), which was released from CGMP (Tables
The peptide MAIPPKKNQDK was the only sequence with antithrombotic activity detected in the present study. It was generated from CGMP by all the tested strains (Table
The present results revealed the ability to release individual peptide sequences by
In our study, CGMP proved to be a better source of precursors for these biologically active peptides than
The results indicate that certain sequences were released from the specified protein preparations only by one (determined, single) strain of the tested
The obtained findings correspond to other study indicating that in
It was revealed that addition of selected milk or whey protein preparations (in a proper concentration) to milk may improve the texture parameters of fermented milk beverages and stimulate the growth of probiotic microorganisms [
In terms of textural properties, there were differences among the fermented milk beverages (Table
Texture parameters for analyzed fermented products.
Analyzed fermented product | Texture parameter | |||||
---|---|---|---|---|---|---|
Hardness (N) | Cohesiveness | Springiness | Gumminess (g) | Chewiness (g) | Resilience | |
CGMP_B734 | 0.318 | 0.038 | 0.050 | 0.012 | 0.001 | 0.019 |
±0.033 | ±0.005 | ±0.012 | ±0.002 | ±0.000 | ±0.003 | |
CGMP_DSMZ | 0.416 | 0.028 | 0.037 | 0.012 | 0.001 | 0.014 |
±0.054 | ±0.006 | ±0.009 | ±0.001 | ±0.000 | ±0.003 | |
CGMP_T80 | 0.390 | 0.03 | 0.042 | 0.010 | 0.001 | 0.015 |
±0.193 | ±0.020 | ±0.037 | ±0.002 | ±0.000 | ±0.011 | |
CGMP_T105 | 0.282 | 0.050 | 0.067 | 0.014 | 0.001 | 0.026 |
±0.040 | ±0.014 | ±0.031 | ±0.004 | ±0.001 | ±0.007 | |
0.186 | 0.223 | 0.758 | 0.035 | 0.034 | 0.131 | |
±0.063 | ±0.152 | ±0.602 | ±0.020 | ±0.029 | ±0.093 | |
0.356 | 0.051 | 0.072 | 0.018 | 0.001 | 0.026 | |
±0.01 | ±0.002 | ±0.007 | ±0.001 | ±0.000 | ±0.001 | |
0.236 | 0.179 | 0.544 | 0.042 | 0.029 | 0.100 | |
±0.006 | ±0.098 | ±0.346 | ±0.023 | ±0.024 | ±0.058 | |
0.273 | 0.145 | 0.477 | 0.038 | 0.023 | 0.079 | |
±0.051 | ±0.078 | ±0.340 | ±0.020 | ±0.020 | ±0.044 |
Explanation notes: the analyzed fermented products are described by the designations: the name of the additive introduced in the milk (
Considering that comparison of tested products in terms of all tested texture parameters simultaneously was also the interest of investigation, the HCA was applied to verify whether the groups of similar objects could be distinguished among all tested fermented products based on the analyzed texture features. Grouping the objects (fermented products) in terms of similarity of texture characteristics (Figure
Projection of variables on the principal component plane ((a) H: hardness; Ch: chewiness; G: gumminess; S: springiness; C: cohesiveness; R: resilience) and dendrogram showing the results of hierarchical cluster analysis comparing the texture profiles of fermented milk beverages (b).
The results revealed that, apart from hardness, other texture parameters constituted one group of strongly positively correlated variables (the analysis performed in 3 repetitions for tested combinations). Hardness and springiness (which represented the remaining variables and most strongly correlated with them) were selected for cluster analysis.
The analysis indicated that, in terms of textural similarity, the fermented milk beverages could be assigned into two separate groups (bonding distance equal to 2.25). The first group includes milk beverages containing the addition of
All products classified in the first group were characterized by the highest values of the springiness parameter (in a range from
In general terms, the profile of mean values of the tested texture parameters exhibited by products fermented by T105 containing
The differences observed in the texture characteristics between the fermented milk beverages containing
The highest values of the proteolytic index were noted in samples of CGMP-containing milk beverages fermented by T105 and an analogous variant of milk products fermented by strain B734 (Table
Proteolysis index, protein, non-protein nitrogen, and total nitrogen contents in the obtained milk products.
Analyzed product | TN (g/100 g) | P (g/100 g) | NPN (g/100 g) | PI (%) |
---|---|---|---|---|
RSM | ||||
CGMP_nf | ||||
CGMP_B734 | ||||
CGMP_DSMZ | ||||
CGMP_T80 | ||||
CGMP_T105 | ||||
Explanation notes: P: protein; TN: total nitrogen; NPN: non-protein nitrogen; PI: proteolysis index; the analyzed fermented products are described by the designations: the name of the additive introduced in the milk (
In our study, this was noticed that fermented products with the addition of CGMP exhibited generally higher value of PI than the analogous variants containing
The HCA was used to separate samples exhibiting similar properties in terms of P, TN, NPN, and PI. The principal component analysis was used to illustrate the structure of the variables (Figure
Projection of variables on the principal component plane (a) and dendrogram expressing the results of hierarchical cluster analyses of similarities of products in terms of the proteolysis index and contents of protein, total nitrogen, and non-protein nitrogen (b).
A very strong correlation (determined by the analysis of the values of Pearson correlation coefficients) was noted between TN and P and between NPN and PI (the analysis was performed in 2 repetitions for the tested combinations). Therefore, the TN and PI (which was weaker correlated with TN than NPN) were selected for the cluster analysis.
Clustering was carried out with the Ward method using a Euclidean distance matrix and the analysis of the bond distances (2 bond level) revealed that 4 groups of similar products can be distinguished (Figure
However, the greatest similarity was observed between the
The second group is represented only by CGMP-containing milk beverages fermented by
The other fermented milk beverages constituted the third cluster (group III) and exhibited high values of PI as well (Table
The differences between the fermented products observed in our study might be related to the different metabolic activities of the strains, their enzymatic preference for selected protein preparations influencing texture profiles, proteolysis index, and contents of protein, total nitrogen, and non-protein nitrogen. This is in agreement with the results reported by Ewe and Loo [
The tested strains of
The present obtained results suggest that the novel strains of
All the experimental data used to support the findings of this research are available from the corresponding author upon request.
The authors declare no conflict of interest.
The research was funded by the National Science Centre, Poland (Research Grant No. 2014/15/N/NZ9/04042). The authors would like to express their gratitude to Prof. Łucja Łaniewska-Trokenheim (University of Warmia and Mazury in Olsztyn, Poland) for providing the strains of