Milk Fermentation by Lacticaseibacillus rhamnosus GG and Streptococcus thermophilus SY-102: Proteolytic Proﬁle and ACE-Inhibitory Activity

: Health beneﬁts of probiotics and production of inhibitors of angiotensin converting enzyme (ACE) released during milk fermentation are well known. That is why in this investigation the proteolytic proﬁle and ACE inhibitory capacity of peptide fractions from protein hydrolysis of milk during fermentation processes was analyzed. Milk fermentation was carried out inoculating 10 6 CFU of L. rhamnosus GG, S. thermophilus SY-102 and with both bacteria. The proteolytic proﬁle was determined using: TNBS, SDS-PAGE and SEC-HPLC techniques. In vitro ACE inhibition capacity was measured. The pH of 4.5 was reached at 56 h when the milk was fermented with L. rhamnosus , at 12 h with S. thermophillus and at 41 h in the co-culture. Production of free amino groups corresponded with the proﬁle of low molecular weight peptides observed by SDS-PAGE and SEC-HPLC. Co-culture fermentation showed both the highest concentration of low molecular weight peptides and the ACE inhibitory activity (>80%). Results indicated that the combination of lactic cultures could be useful in manufacture of fermented milk with an added value that goes beyond basic nutrition, such as the production of ACE-inhibitory peptides.


Introduction
Fermented milks are products that have been generated since millennia and in the beginning, fermentation was the simplest, cheapest and safest technique for preserving milk [1]. The term "fermented milk" includes dairy products obtained from a technology equivalent to that of the manufacture of yogurt, but that uses different microorganisms than Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus [2].
In addition to the research carried out on the technology of fermented milk, there are studies conducted to the diversification of starter cultures [3][4][5]. These starter cultures are chosen for their potential in transforming lactose into lactic acid, generating flavors, The bacteria Streptococcus thermophilus SY-102 and Lacticaseibacillus rhamnosus GG used were obtained from the Food Biotechnology Laboratory of the Universidad Autónoma Metropolitana campus Iztapalapa. Bacteria were conditioned in MRS broth incubating for 24 h at 42 • C. Then, one milliliter of the inoculated broth was added to 9 mL of skimmed milk powder solution (Dairy Gold at 10% (w/v)) pasteurized at 90 • C for 10 min. It was incubated for 24 h at 42 • C. From this solution, 1 mL was added to an Erlenmeyer flask with 100 mL of a 10% (w/v) pasteurized skim milk powder solution (90 • C for 10 min). Each microorganism was conditioned in the same way independently. After incubating for 24 h at 42 • C, the solution was refrigerated. This solution was the starter culture and before each inoculation a viable count was performed to determine the initial inoculum concentration for each fermentation.

Fermentation
For the fermentation process, three systems were prepared: one inoculated with Lacticaseibacillus rhamnosus GG (LLR), another inoculated with Streptococcus thermophillus SY-102 (LST) and a third inoculated with both microorganisms (LLS). The initial concentration of bacteria was approximately 1 × 10 6 CFU for each of the systems. The solution for the fermentation was prepared with skimmed milk powder at 10% (w/v) added with 2% lactose (w/v). This solution was pasteurized at 90 • C for 10 min before inoculation and incubation at 42 • C. Pre-fermentations were performed to explore sampling points, fermentation time, and pH changes. In this way, the fermentation process was standardized. After the exploratory fermentations, it was sampled each two hours stopping when it reached a pH of 4.5. Samples were centrifuged at 24,600× g for 10 min at 4 • C (Eppendorf centrifuge) to eliminate biomass and high molecular weight proteins. The supernatants of the centrifuged samples were stored at −4 • C for later analysis. All measurements were made in triplicate.

Proteolytic Profile Analysis
Proteolytic profile characterization was carried out through three different studies. Free amino groups determination was performed with the TNBS technique to know the concentration of peptides produced. Tris-Tricine polyacrylamide gel electrophoresis (SDS-Tris-Tricine-PAGE) was used to separate peptides released during fermentation. Finally, with the objective to observe the presence of peptides with aromatic amino acids, especially those of low molecular weight produced during milk fermentation, an HPLC analysis by size exclusion column was carried out.

Free Amino Groups Analysis
Free amino groups derived from milk fermentation were measured with 2,4,6trinitrobenzenesulphonic acid (TNBS). A volume of 125 µL of sample was mixed with 1 mL of phosphate buffer solution of 0.21 M, pH 8.2 in test tubes wrapped with aluminum foil. After that, 1 mL of 0.10% TNBS (Sigma Aldrich, San Louis, MO, USA) was added to phosphate buffer 0.21 M, pH 8.2, and each tube was agitated in vortex. Tubes were incubated for 1 h at 50 • C in the dark. The reaction was interrupted after 60 min, adding 2 mL of chloride acid 0.1 N. It was read in a spectrophotometer at 340 nm wavelength against control. Control was prepared with deionized water and a glycine concentration curve (0.05 to 0.25 mg/mL) was used.

Peptides Separation by SEC-HPLC
The González et al. [26] technique was used with some modifications. Samples were deep frozen (−40 • C) and they were lyophilized (Labconco DrySystem/Freezone 4.5). The lyophilizates were re-suspended in 100 µL of Milli-Q deionized water (18.2 MΩ·cm) and they were filtered using a syringe filter of 0.22 µm (Whatman ® UNIFLO ® ) before HPLC analysis. A 20 µL of sample was injected into a modular HPLC equipment (Perkin Elmer, Series 200), with a size exclusion column (SEPAX Technologies, Inc., Newark, NJ, USA, SRT SEC-150, particle size 5µm, pore size 150 Å, 300 × 7.8 mm). The mobile phase used was a phosphate buffer pH 6.8. The run was carried out with an isocratic flow of 0.25 mL/min for 70 min at room temperature. The detection of residues with phenylalanine was carried out at 257 nm and at 280 nm for the detection of tryptophan and tyrosine residues. A diode array detector (Applied Biosystems 1000S) was used.

ACE-Inhibitory Activity
The inhibitory effect of ACE-I (EC 3.4.15.1; Sigma-Aldrich, San Louis, USA) was evaluated spectrophotometrically according to Cushman et al. (1977), with some modifications. The substratum, hippuril-histidyl-leucine (HHL; Sigma-Aldrich, San Louis, MO, USA), was dissolved in sodium borate buffer (0.1M, pH 8.3, with 0.3M sodium chloride) at a concentration of 5 mM substrate. Then 10 µL of ACE (EC 3.4.15.1, 5.1 U/mg; Sigma-Aldrich) was added to 100 µL substrate and 40 µL sample (AbsM). The reaction was carried out for 75 min at 37 • C. The formed hipuric acid was extracted with ethyl acetate, re-suspended in deionized water, and measured at 220 nm in a Power Wave XS UV-Biotek (software KC Junior, Kansas, MO, USA) spectrometer. The same treatment was performed for a sample of the positive control (AbsC) prepared instead with 40 µL of borate buffer instead, and for the negative control (AbsB) prepared with 50 µL of borate buffer and 100 µL of substratum (HHL). The inhibitory activity of ACE was calculated using the following formula according to absorbance of each measurement:

pH Changes during Fermentation
The pH decreased steadily in the three fermented milks of the study until reaching a pH between 4.5 and 5. In the LLR milk the pH was reached at 56 h, while for the LST and LLS milks the time was 12 and 41 h (Table 1). The decreased time to reach the ending pH could be due to the type of fermentation process and the stress of the microorganism to environmental conditions. It is known that the decrease in pH is mainly due to two factors: lactic acid production from lactose conversion and biomass generation [27]. In the case of the rapidity of biomass generation, this is due to the metabolic capacity not only of the use of carbon but also of the hydrolysis of proteins. That is, the differences observed between fermentation times are dependent on the ability of each bacterium to use carbon and nitrogen sources [28].
On the other hand, some research results showed that the pH decrease in a milk fermentation system is similar to that observed in this study [28][29][30][31]. Additionally, S. thermophilus SY-102 in LST showed a decrease at pH 4.51 in a period of 12 h. This decrease is due to the adaptation of the microorganism to the fermentation medium and to the source of free amino acids available for its growth [20,32]. Due to the fact in LLR milk the pH did not decrease beyond 5, it was decided to stop the fermentation at this value since in preliminary tests the same microorganism did not manage to lower the pH to 4.5. In the case of co-culture fermentation, the value of pH obtained after 41 h of fermentation was of 4.56. This value was obtained despite presence of L. rhamnosus in the medium. It is known that in symbiotic systems in which S. thermophilus participates, both the growth and the adaptation are dependent on the metabolism of the other microorganism (carbon source competition) since the release of some essential amino acids is necessary for their growth with the consequent delay in the formation of biomass and mainly of the lactose conversion [30,33,34].
The decrease in pH in dairy systems fermented with S. thermophilus is retarded using two different carbon sources. First, lactose hydrolysis is carried out and it is known that this microorganism has a diauxic growth curve. This is since S. thermophilus can use the liberated galactose as a carbon source, fermenting it via the pentose phosphate pathway. However, in co-culture fermentation the acidification of the medium is limited by the fermentation of only the glucose released from the hydrolyzed disaccharide [33]. Additionally, the proteolytic capacity has a direct impact on the metabolic behavior of each microorganism due to its auxotrophies.

Free Amino Groups Determination by TNBS
During fermentation with Lacticaseibacillus rhamnosus GG, LLR fermentation showed a higher initial proteolysis compared to that observed in LST (Table 2). However, during fermentation, despite the concentration of free amino groups remaining apparently constant (from 0.544 ± 0.011 to 0.639 ± 0.006 mg/L), a significant difference (p = 0.05) was presented. These statistical results showed that there was an increase in the concentration of free amino groups at the end of the fermentation. Table 2. Free amino groups production during fermentation of milk with Lactticaseibacillus rhamnosus GG (LLR); Streptococcus thermophilus SY-102 (LST) and co-culture with both microorganisms (LLS).  In general, the differences in the concentration of free amino groups are due to the specificity of the proteolytic system of each microorganism. Some investigations similar to this work carried out with the genus Lactobacillus casei, showed that there is not variation in the concentration of free amino groups until 21 h of fermentation [30,32]. It has also been determined that the differences may be due to the method, since in the case of fermentations with casein hydrolysis, the decrease in free amino groups may be largely due to the precipitation of high molecular weight proteins. These proteins, despite being fractionated, undergo a flocculation process because they are in a medium close to their isoelectric point. This phenomenon is caused by the generation of lactic acid, which destabilizes the caseins [25,32].

Stage of Fermentation
At the beginning of the LST milk fermentation, a high proteolytic activity of the microorganism was found, which decreased after 8 and at the end of the process a concentration of free amino groups of 0.24 ± 0.012 mg/L was obtained. This behavior may be since some species of S. thermophilus have low proteolytic activity associated with cell wall proteases. Furthermore, it has been shown that increased proteolytic activity is due to intracellular peptidases that are released by cell lysis [20,21,35,36].
It was observed that the differences between the proteolytic systems of lactic acid bacteria are mainly due to the initial proteolysis [21]. Streptococcal species, having higher nutritional amino acid requirements compared to lactobacillus species, must have a greater capacity for initial protein hydrolysis and this is associated with the activity of the PrtS protease linked to the cell wall [20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36]. Furthermore, unlike lactobacilli, most streptococcal peptidases and aminopeptidases are intracellular and this causes a lower hydrolysis rate. For this reason, the S. thermophilus peptidase system is more complex compared to that of lactobacilli, and all peptide cleavage is carried out within the cell with greatly decreased excretion of short-chain peptides [20].
Despite the low proteolytic capacity of S. thermophilus SY-102, in LLS milk an increase in the concentration of free amino groups was observed throughout the fermentation process. The final concentration could be due to two important factors: (1) groups exposed because the partial denaturation of caseins for the formation of lactic acid, which cause a decrease in pH until the isoelectric point of caseins exposing their amino groups [33] and (2) action of the proteolytic system of L. rhamnosus GG, which generates the initial release of peptides and free amino acids for the development and growth of S. thermophilus SY-102. Various investigations [31,32] showed that the growth and adaptation of streptococcal species are dependent on the metabolism of the other microorganism [37]. After having the amino acids necessary for its growth, the proteolytic action of S. thermophillus continues until the end of fermentation [27].

Peptide Separation byTris-Tricine SDS-PAGE
The electrophoresis gel to the samples obtained from LLR milk and showed bands corresponding to peptides less than 1.4 kDa ( Figure 1). Many of the peptides with bioactive functions that are reported in the bibliography are less than 10 kDa [32]. An increase in the concentration of low molecular weight peptides and a decrease in both the concentration of caseins (c) and serum proteins (a and b) were observed.
teases. Furthermore, it has been shown that increased proteolytic activity is due to intracellular peptidases that are released by cell lysis [20,21,35,36].
It was observed that the differences between the proteolytic systems of lactic acid bacteria are mainly due to the initial proteolysis [21]. Streptococcal species, having higher nutritional amino acid requirements compared to lactobacillus species, must have a greater capacity for initial protein hydrolysis and this is associated with the activity of the PrtS protease linked to the cell wall [20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36]. Furthermore, unlike lactobacilli, most streptococcal peptidases and aminopeptidases are intracellular and this causes a lower hydrolysis rate. For this reason, the S. thermophilus peptidase system is more complex compared to that of lactobacilli, and all peptide cleavage is carried out within the cell with greatly decreased excretion of short-chain peptides [20].
Despite the low proteolytic capacity of S. thermophilus SY-102, in LLS milk an increase in the concentration of free amino groups was observed throughout the fermentation process. The final concentration could be due to two important factors: (1) groups exposed because the partial denaturation of caseins for the formation of lactic acid, which cause a decrease in pH until the isoelectric point of caseins exposing their amino groups [33] and (2) action of the proteolytic system of L. rhamnosus GG, which generates the initial release of peptides and free amino acids for the development and growth of S. thermophilus SY-102. Various investigations [31,32] showed that the growth and adaptation of streptococcal species are dependent on the metabolism of the other microorganism [37]. After having the amino acids necessary for its growth, the proteolytic action of S. thermophillus continues until the end of fermentation [27].

Peptide Separation byTris-Tricine SDS-PAGE
The electrophoresis gel to the samples obtained from LLR milk and showed bands corresponding to peptides less than 1.4 kDa ( Figure 1). Many of the peptides with bioactive functions that are reported in the bibliography are less than 10 kDa [32]. An increase in the concentration of low molecular weight peptides and a decrease in both the concentration of caseins (c) and serum proteins (a and b) were observed. In the analysis, a decrease in the concentration of proteins with a molecular weight greater than 14.4 kDa was shown. Likewise, from the first time of monitoring, up to 5 peptides less than 14.4 kDa were identified. Several authors have highlighted that the origin of peptides from serum proteins is in general of greater importance because the fractionation of β-lactoglobulin is higher due to its concentration of proline, since the proteinases and peptidases of the proteolytic system of lactic bacteria are proline-specific [20,38]. In the analysis, a decrease in the concentration of proteins with a molecular weight greater than 14.4 kDa was shown. Likewise, from the first time of monitoring, up to 5 peptides less than 14.4 kDa were identified. Several authors have highlighted that the origin of peptides from serum proteins is in general of greater importance because the fractionation of β-lactoglobulin is higher due to its concentration of proline, since the proteinases and peptidases of the proteolytic system of lactic bacteria are prolinespecific [20,38].
In the case of LST (Figure 2), a high concentration of peptides less than 1.4 kDa was not observed despite having found a decrease in the concentration of proteins with a molecular weight greater than 14.4 kDa. These data coincide with the results obtained during the analysis of free amino groups by the TNBS method. Very high proteolysis was observed at the beginning of the fermentation, related to the decrease in the casein concentration.
In the case of LST (Figure 2), a high concentration of peptides less than 1.4 kDa was not observed despite having found a decrease in the concentration of proteins with a molecular weight greater than 14.4 kDa. These data coincide with the results obtained during the analysis of free amino groups by the TNBS method. Very high proteolysis was observed at the beginning of the fermentation, related to the decrease in the casein concentration. It has been observed that due to its nutritional requirements, S. thermophilus is a species that has a high activity of PrtS proteinase at the beginning of fermentations to obtain the necessary amino acids for its survival [20,21]. This activity decreases through the fermentation time since the peptide cut is carried inside the cell. In contrast, in other lactic acid bacteria the peptide fractionation occurs by hydrolases linked to the cell wall and peptidases inside the cell [21]. Figure 3 shows that in LLS milk the pattern of peptides released was different from that found in mono-culture systems, generating a higher concentration of low molecular weight peptides. At the same time, a decrease in the concentration of proteins with a molecular weight greater than 14.4 kDa was observed. The analysis demonstrated the release of peptides with molecular weight less than 1.4 kDa. It is well known that in some cocultures fermentation of milk there is a protocooperation mechanism, which is regulated by the release of essential amino acids for one of the species present in the environment [39]. It has been observed that due to its nutritional requirements, S. thermophilus is a species that has a high activity of PrtS proteinase at the beginning of fermentations to obtain the necessary amino acids for its survival [20,21]. This activity decreases through the fermentation time since the peptide cut is carried inside the cell. In contrast, in other lactic acid bacteria the peptide fractionation occurs by hydrolases linked to the cell wall and peptidases inside the cell [21]. Figure 3 shows that in LLS milk the pattern of peptides released was different from that found in mono-culture systems, generating a higher concentration of low molecular weight peptides. At the same time, a decrease in the concentration of proteins with a molecular weight greater than 14.4 kDa was observed. The analysis demonstrated the release of peptides with molecular weight less than 1.4 kDa. It is well known that in some co-cultures fermentation of milk there is a protocooperation mechanism, which is regulated by the release of essential amino acids for one of the species present in the environment [39].
In the case of LST (Figure 2), a high concentration of peptides less than 1.4 kDa was not observed despite having found a decrease in the concentration of proteins with a molecular weight greater than 14.4 kDa. These data coincide with the results obtained during the analysis of free amino groups by the TNBS method. Very high proteolysis was observed at the beginning of the fermentation, related to the decrease in the casein concentration. It has been observed that due to its nutritional requirements, S. thermophilus is a species that has a high activity of PrtS proteinase at the beginning of fermentations to obtain the necessary amino acids for its survival [20,21]. This activity decreases through the fermentation time since the peptide cut is carried inside the cell. In contrast, in other lactic acid bacteria the peptide fractionation occurs by hydrolases linked to the cell wall and peptidases inside the cell [21]. Figure 3 shows that in LLS milk the pattern of peptides released was different from that found in mono-culture systems, generating a higher concentration of low molecular weight peptides. At the same time, a decrease in the concentration of proteins with a molecular weight greater than 14.4 kDa was observed. The analysis demonstrated the release of peptides with molecular weight less than 1.4 kDa. It is well known that in some cocultures fermentation of milk there is a protocooperation mechanism, which is regulated by the release of essential amino acids for one of the species present in the environment [39]. The concentration of peptides obtained in the combined system increased throughout the study. These results are consistent with those observed in the analysis of free The concentration of peptides obtained in the combined system increased throughout the study. These results are consistent with those observed in the analysis of free amino groups. It was also found that the release of peptides during fermentation followed the same pattern as the study carried out by Rojas-Ronquillo et al. [32], where they also found that antihypertensive activity was related to the production of low molecular weight peptides.

Separation of Peptides with Aromatic Amino Acid Residues
The peptide fractions corresponding to the beginning, the middle and the end of each fermentation were analyzed. Figure 4-I shows that at the beginning of the fermentation with L. rhamnosus GG (Figure 4-IA) at 257 nm, 8.5 kDa molecular weight fractions were found with the presence of phenylalanine, with a retention time (Rt) of 35 min. However, Figure 4-IB shows how the concentration of this peptide fraction decreased and completely disappeared at the end of the study (Figure 4-IC). weight peptides.

Separation of Peptides with Aromatic Amino Acid Residues
The peptide fractions corresponding to the beginning, the middle and the end of each fermentation were analyzed. Figure 4-I shows that at the beginning of the fermentation with L. rhamnosus GG (Figure 4-IA) at 257 nm, 8.5 kDa molecular weight fractions were found with the presence of phenylalanine, with a retention time (Rt) of 35 min. However, Figure 4-IB shows how the concentration of this peptide fraction decreased and completely disappeared at the end of the study (Figure 4-IC).
In the case of the analysis at 280 nm (Figure 4-II), at the beginning of the fermentation two peptide fractions with molecular weights of 5.  The presence of aromatic amino acids in the peptide fractions is due to the proteolytic system of each lactic acid bacteria. At the end of the proteolytic process, intracellular peptidases cut the peptide fractions inside the cell and excrete the fractions they do not use. Petidases such as PepX, PepO, PepF, and PepT can cut peptides in the hydrophobic part of the peptide sequence and sometimes the accumulated excreted peptides have aromatic amino acids in their structure [20,[35][36][37]. Additionally, it is known that this type of structure is better coupled to the active site of ACE, causing its inhibition [40].
In the analysis of the peptide fractions obtained during the fermentation with S. thermophilus, a protein of 46.9 kDa was observed with the presence of Phe (Figure 5-IA). This fraction could come from bovine serum albumin that was fractionated during fermentation or from some associated proteins in polymers, which is very common under certain temperature and pH conditions for some caseins, mainly β-casein [20,41].  The presence of aromatic amino acids in the peptide fractions is due to the proteolytic system of each lactic acid bacteria. At the end of the proteolytic process, intracellular peptidases cut the peptide fractions inside the cell and excrete the fractions they do not use. Petidases such as PepX, PepO, PepF, and PepT can cut peptides in the hydrophobic part of the peptide sequence and sometimes the accumulated excreted peptides have aromatic amino acids in their structure [20,[35][36][37]. Additionally, it is known that this type of structure is better coupled to the active site of ACE, causing its inhibition [40].
In the analysis of the peptide fractions obtained during the fermentation with S. thermophilus, a protein of 46.9 kDa was observed with the presence of Phe (Figure 5-IA). This fraction could come from bovine serum albumin that was fractionated during fermentation or from some associated proteins in polymers, which is very common under certain temperature and pH conditions for some caseins, mainly β-casein [20,41]. In the same way, at the beginning of the fermentation, a peptide fraction of 8.8 kDa was observed, which remained in an apparently constant concentration ( Figure 5-IB,C). Many of these fractions could be produced from the first protein breakdown by the S. thermophilus SY-102 PrtS proteinase. From the start of the fermentation, two peptides with Tyr and Trp residues ( At the beginning of the fermentation, peptides with molecular weight less than 10 kDa were also found, with Tyr and Trp residues (Figure 6-IIA). At the end of the process there was an apparent increase in the concentration of the 5.0 kDa fraction (Figure 6-IIB). As in the analysis at 257 kDa, some fractions disappeared in the intermediate stage of fermentation ( Figure 6-IIB) while others appeared at the end, mainly a 9.2 kDa fraction ( Figure 6-IIC).
During the fermentation process in a co-culture system, the proteolytic system of L. rhamnosus GG broke casein to obtain essential amino acids for its metabolism, releasing low molecular weight peptide fractions that S. thermophillus SY-102 could metabolize. This behavior was described mainly for L. delbrueckii sub. bulgaricus and S. salivarius sub. thermophilus during the yogurt process [23,30,34]. PrtS proteinase of S. thermophilus disintegrates the peptide fraction, using only some essential amino acids for its growth and survival in the medium. On the other hand, this microorganism contains special peptidases such as PepS, which breaks oligopeptides and releases residues with aromatic and hydrophobic amino acids, mainly Phe [20,21,[35][36][37]. At the beginning of the fermentation, peptides with molecular weight less than 10 kDa were also found, with Tyr and Trp residues ( During the fermentation process in a co-culture system, the proteolytic system of L. rhamnosus GG broke casein to obtain essential amino acids for its metabolism, releasing low molecular weight peptide fractions that S. thermophillus SY-102 could metabolize. This behavior was described mainly for L. delbrueckii sub. bulgaricus and S. salivarius sub. thermophilus during the yogurt process [23,30,34]. PrtS proteinase of S. thermophilus disintegrates the peptide fraction, using only some essential amino acids for its growth and survival in the medium. On the other hand, this microorganism contains special peptidases such as PepS, which breaks oligopeptides and releases residues with aromatic and hydrophobic amino acids, mainly Phe [20,21,[35][36][37].

Determination of ACE Inhibition
At the beginning of fermentation in LLR milk (Figure 7) an ACE inhibition of 46.9% was determined. Inhibition increased in the middle of the process, since at 21 h of fermentation an inhibition of 74.6% was obtained. However, despite the accumulation of low molecular weight peptides, the percentage of inhibition decreased at the end (59.9%).
It is known that at the beginning of fermentation L. rhamnosus requires essential aromatic amino acids (Phe, Trp) for its growth [42]. However, these needs apparently prevent the accumulation of peptides with aromatic amino acid residues, which can lead to decreased ACE inhibition. On the contrary, the peptide fractions from fermentation LST showed an increase in ACE inhibition from 53.2% to 80.4% at 21 h and at the end of fermentation the inhibition decreased until 73.3%. These results could be due to the fact that S. thermophilus has different nutritional requirements than other lactic acid bacteria [20,43] and also has a very advanced system as an operon for the production of amino acids (leucine, isoleucine and valine) . Low molecular weight peptides are also generated, although during the

Determination of ACE Inhibition
At the beginning of fermentation in LLR milk (Figure 7) an ACE inhibition of 46.9% was determined. Inhibition increased in the middle of the process, since at 21 h of fermentation an inhibition of 74.6% was obtained. However, despite the accumulation of low molecular weight peptides, the percentage of inhibition decreased at the end (59.9%).

Determination of ACE Inhibition
At the beginning of fermentation in LLR milk ( Figure 7) an ACE inhibition of 46.9% was determined. Inhibition increased in the middle of the process, since at 21 h of fermentation an inhibition of 74.6% was obtained. However, despite the accumulation of low molecular weight peptides, the percentage of inhibition decreased at the end (59.9%).
It is known that at the beginning of fermentation L. rhamnosus requires essential aromatic amino acids (Phe, Trp) for its growth [42]. However, these needs apparently prevent the accumulation of peptides with aromatic amino acid residues, which can lead to decreased ACE inhibition. On the contrary, the peptide fractions from fermentation LST showed an increase in ACE inhibition from 53.2% to 80.4% at 21 h and at the end of fermentation the inhibition decreased until 73.3%. These results could be due to the fact that S. thermophilus has different nutritional requirements than other lactic acid bacteria [20,43] and also has a very advanced system as an operon for the production of amino acids (leucine, isoleucine and valine) . Low molecular weight peptides are also generated, although during the It is known that at the beginning of fermentation L. rhamnosus requires essential aromatic amino acids (Phe, Trp) for its growth [42]. However, these needs apparently prevent the accumulation of peptides with aromatic amino acid residues, which can lead to decreased ACE inhibition.
On the contrary, the peptide fractions from fermentation LST showed an increase in ACE inhibition from 53.2% to 80.4% at 21 h and at the end of fermentation the inhibition decreased until 73.3%. These results could be due to the fact that S. thermophilus has different nutritional requirements than other lactic acid bacteria [20,43] and also has a very advanced system as an operon for the production of amino acids (leucine, isoleucine and valine) . Low molecular weight peptides are also generated, although during the experiment it was observed that the low molecular weight peptides were presented in a lower proportion than in the fermentation with L. rhamnosus. Furthermore, the null need for aromatic amino acid peptides by S. thermophilus allows their accumulation in the fermentation medium [20,43,44].
In LLS milk, a 38% inhibition was observed; this percentage increased gradually, reaching 80.6% inhibition at 21 h and remained without significant change until the end of the process (41 h). This increase could be due to the presence of a specific intracellular peptidase of S. thermophilus that constantly generates peptides containing Phe [45], since it has been reported that this microorganism excretes two and three amino acid peptides generated by the action of intracellular dipeptidases. [20,21,45]. This increase in ACE inhibition is consistent with the results obtained during the electrophoresis analysis, in which accumulation of low molecular weight peptides was found. In the same way, this activity of inhibition could be related with the presence of peptides with aromatic amino acid residues observed in the HPLC analysis.
An increase in ACE inhibition has been observed during milk fermentation with lactic acid bacteria [46]. However, it is known that the antihypertensive capacity of fermented milks in which co-cultures were used is greater [31] than in those produced with monocultures. Additionally, since S. thermophilus has the ability to release peptides with antihypertensive capacity from the beginning of protein hydrolysis [21], it is likely that this activity will not be lost as long as this bacterium is found in combination with other microorganisms during lactic fermentation.
In the case of the co-culture fermentation studied in the present investigation, the microorganisms used showed a symbiotic growth as observed in proteolysis. The release of peptides by these microorganisms exhibited a behavior similar to that observed during fermentation with S. thermophilus, however, according to electrophoresis analysis, there is a greater diversity and concentration of peptides in the co-culture system and these residues could present other biological activities of importance, which were not studied in this investigation.

Conclusions
The combination of L. rhamnosus GG and S. thermophilus SY-102 in milk fermentation favors the acidification of the medium in shorter times, compared to fermented systems inoculated only with L. rhamnosus GG. Likewise, the use of a mixed system promotes greater proteolytic activity, favoring the increase in the concentration of low molecular weight peptides released by the hydrolysis of proteins. Furthermore, the co-culture fermentations do not produce negative effects on the release of peptides with aromatic amino acids, which creates an advantage in the bioactivity of these peptide fractions.
The antihypertensive activity of the peptide fractions obtained in this work is related to the presence of low molecular weight peptides as well as to the presence of aromatic amino acids in these fractions. In addition, using a mixed system for milk fermentation increases the antihypertensive capacity of the peptides derived from proteolysis. Co-culture fermentations are a field of study with challenges and opportunities yet to be discovered, especially in those investigations directed towards the elaboration of nutraceutical and functional food products.