A buffalo yogurt fermented with commercial starter and Lactobacillus plantarum orignied from breast milk and its lowering blood pressure to Pregnant hypertensive rats

Nutritional therapy, which may have advantages over medication, is being investigated as a novel treatment for pregnancy-induced hypertension. Several studies have shown that probiotic yogurt supplementation during pregnancy has beneficial effects on maternal and fetal health. In this study, fermented buffalo milk was produced with yogurt culture and Lactobacillus plantarum B, a probiotic isolated from healthy breast milk with high angiotensin-converting enzyme inhibitory activity. The fermentation conditions under which the ACE inhibitory activity reached 84.51%, were optimized by the response surface method as follows: 2 × 10 6 cfu/mL of L. plantarum B, yogurt culture 2.5 × 10 5 cfu/mL, and 8 h at 37°C. The distribution of ACE inhibitory peptides from fermented buffalo milk and fermented cow milk were further analyzed by liquid chromatography-mass spectrometry. By searching according to the structural features of ACE inhibitory peptides, 29 and 11 peptides containing ACE inhibitory peptide features were found in fermented buffalo milk and fermented cow milk, respectively. To investigate the in vivo antihypertensive activity of fermented buffalo milk, 18 pregnant rats were divided into 3 groups (n = 6 in each group) and administered 10 mL of normal saline, yogurt (20 mg/kg) or labetalol hydrochloride (4 mg/kg) daily from the beginning of pregnancy to parturition. To induce hypertension, methyl nitrosoarginine (125 mg/kg) was injected subcutaneously every day from d 15 of pregnancy to the day of delivery. Blood pressure was not significantly changed in the yogurt and labetalol groups after induction of hypertension and was lower compared with the normal saline group ( P < 0.05), but there was no difference between the yo-gurt and labetalol groups. This implied that the buffalo yogurt had a preventive and antihypertensive effect in the pregnancy-induced hypertensive rat model. Further studies to determine the mechanism of action, as well as a randomized control trial, are


INTRODUCTION
Pregnancy-induced hypertension (PIH) is a common disease during pregnancy that increases the risk of placental abruption, cerebral hemorrhage, and other diseases, and is one of the leading causes of maternal, fetal, and neonatal mortality and morbidity (Webster, 2019).The pathogenesis of PIH involves multiple factors and the placenta plays a central role, with abnormalities in the placenta leading to the production of anti-angiogenic factors and cytokines that promote PIH pathogenesis (Saleh, 2016).The renal angiotensin system (RAS) plays an important role in regulating placental development and uteroplacental circulation, monitoring blood pressure and bodily fluids by affecting placental intervillous blood flow and assisting local helical artery remodeling and enzymatic reactions controlled by various enzymes and receptors (Shaheen, 2019).The RAS is maintained by renin and angiotensin-converting enzyme (ACE).Renin converts serotoninogen to angiotensin I, while ACE, a key rate-limiting enzyme in the regulation of human blood pressure, hydrolyzes angiotensin I to angiotensin II, which has vasoconstrictive effects (Wei, 2022).Chemical drugs such as methyldopa, labetalol, and nifedipine may cause some maternal or fetal side effects and are usually not recommended for pregnancies with mild to moderate PIH (American College of Obstetricians and Gynecologists, 2013).Clinical studies have shown that nutritional interventions can also play an important role in the treatment of hypertension in pregnancy, with a higher safety profile compared with pharmacological treatment (Poon et al., 2019).The MoBa study showed that high intake of fermented dairy products during pregnancy reduced the incidence of severe preeclampsia by up to 40% (Griffin et al., 2015).
Probiotics can be added to foods as nutritional supplements to treat a variety of diseases such as hypertension, cardiovascular disease, and inflammation (Federation of Gynecology and Obstetrics, 2019).Fermented dairy products such as yogurt, which are considered good carriers of probiotics, have been increasingly used as functional foods to promote human health and prevent high levels of stress (Prasanna et al., 2014;Castellano et al., 2013).Lactic acid bacteria release a variety of bioactive peptides including hypotensive peptides, antioxidant peptides, and antibacterial peptides during milk fermentation, remain active during digestion, and are absorbed into specific organs and tissues through blood circulation (Beltrán-Barrientos et al., 2016).Among the different bioactive peptides produced by fermented milk, ACE inhibitory activity peptides (ACEIPs) have received a lot of attention for their application in the treatment of hypertension and the hypotensive properties of some products have been demonstrated in clinical medicine and animal studies (Rai et al., 2017).Lactobacillus plantarum is a common type of probiotic ferment in the food industry.Because of its probiotic properties, it is also used to improve food function and prolong the safety and shelf-life of fermented foods (Zdolec et al., 2018).To date, research on the production of ACEIPs by probiotic fermentation has been reported for different milk sources such as cow milk (Rendón-Rosales et al., 2022), camel milk (Ayyash et al., 2018), and goat milk (Sathya et al., 2019).However, buffalo milk is richer in nutrients, such as protein and fats, and is therefore theoretically a better raw material for fermented dairy products than cow milk (Vargas-Ramella et al., 2021).
In this study, we used Lactobacillus plantarum B (LPB) isolated from breast milk to co-ferment buffalo milk with yogurt culture, and the fermentation conditions were optimized to obtain fermented buffalo milk (FBM) with high ACE inhibitory activity.We also used a liquid phase-mass spectrometry system to analyze the composition of ACEIPs, which laid the foundation for the development of hypotensive buffalo milk products.Finally, in vivo experiments verified that FBM has therapeutic benefits for hypertension during pregnancy.

Probiotics and screening
Four strains, Lactobacillus plantarum B (LPB), Lactobacillus plantarum R5 (LPR5), Lactobacillus plantarum R7 (LPR7), and Lactobacillus gasseri A, with high ACE inhibitory activity (>40%) were isolated from the breast milk of healthy women in China by the National Engineering Center of Dairy for Maternal and Child Health, and a commercial strain Lactobacillus plantarum 299253 (LP299253) was obtained from this same center.The different strains were activated by inoculation (4% inoculum, vol/vol) into de Man, Rogosa and Sharpe (MRS) broth medium 3 times (Xiaolong, 2023).The strains were then inoculated (5% inoculum, vol/vol) into 12% skimmed cow milk with 0.5% glucose and yeast extract, and were incubated at 37°C until curdled.ACE inhibitory activity and pH were measured to screen the strains.

Determination of ACE inhibitory activity
ACE derived from rabbit lungs and hippuryl-histidyl-leucine (Hip-His-Leu) hydrate obtained from Sigma were dissolved in borate buffer (pH = 8.3) to 0.1 U/ mL and 5.0 mM/L, respectively.The ACE inhibitory activity of probiotics and yogurt were determined according to a previously established method (Cushman et al., 1971) with some modifications.The sample (100 µL) was dissolved in 200 µL Hip-His-Leu (5.0 mM, pH = 8.3).After incubation at 37°C for 5 min, the ACE solution (0.1 U/mL, 20 µL) was added to the sample and incubated for 30 min at the above temperature.Then, 250 µL of HCl (1 M) was added to terminate the reaction.Next, 1.7 mL of ethyl acetate was added, the solution was centrifuged at 4000 × g for 15 min, 1 mL of the ethyl acetate layer was absorbed, then dried at 120°C for 30 min.After cooling, 2.0 mL of ultrapure water was added and mixed thoroughly.The absorbance of the mixture was measured at 228 nm using a spectrophotometer (SP-756P Shanghai Spectrum Instrument Co., Ltd., China).The enzyme solution for control group B (in which the sample was replaced with borate buffer) and the blank control for group C (in which 1 M of HCl was added before the reaction to inactivate the enzyme) were treated in the same manner.The ACE inhibitory activity was determined according to the following equation (Eq 1): ACE inhibitory activity (%) = (OD B − OD A ) / (OD B − OD C ) × 100% (1) where OD A , OD B and OD C represent the absorbance of group A, group B and group C, respectively.ACE inhibitory activity was indicated as the peptide concentration required to inhibit 50% (IC 50 ) of the ACE enzyme activity under certain conditions.The peptide content of yogurt was determined using a modification of the method by Cotton et al. (1997).The 5% trichloroacetic acid (m/v) solution was added to the yogurt and then left for 20 min to precipitate the protein.The supernatant was collected by centrifugation at 4000 × g for 10 min and the peptide content was determined by the Biuret method (Dan et al., 2012).The supernatant solution was diluted 0, 2, 4, 6 and 8 times to obtain a standard curve with the peptide content as the horizontal coordinate and the ACE inhibition rate as the vertical coordinate.

Production of buffalo milk yogurt
In this experiment, a 4-factor, 3-level response surface optimization of buffalo milk yogurt fermentation conditions was performed using ACE inhibitory activity as an indicator.The buffalo milk yogurt obtained under optimal conditions was then analyzed for its hypotensive effect in vitro using ACE inhibitory peptide analysis and in vivo with an animal experimental model for the treatment of PIH.The flowchart of yogurt fermentation and ACE inhibitory peptide identification is shown in Figure 1.

Fermented milk preparation
Two types of commercially available milk, cow milk and Mora buffalo milk, produced by Sanyuan and Huangshi, respectively, were purchased from the Jingdong platform.LPB that was stored at −80°C was inoculated at 1% (vol/vol) in sterilized MRS at 120°C for 20 min and then incubated for 24h in a 37°C incubator and repeated twice.The buffalo and cow milks were sterilized at 105°C for 15 min, and then fermented with a yogurt ferment (consisting of S. thermophilus and Lactobacillus delbrueckii) (2.92 × 10 9 cfu/mL) and LBP (2.39 × 10 10 cfu/mL) at a ratio of 8:1 with a 3% inoculum (vol/vol) for 8 h at 37°C.The FBM and fermented cow milk (FCM) were placed at 4°C overnight.

Peptide analysis by liquid chromatography-mass spectrometry (LC-MS/MS)
Separation and extraction of peptides.The buffalo and cow yogurts were adjusted to pH 3.4-3.6,centrifuged at 5000 × g for 15 min, and then the supernatant was collected and adjusted to pH 8.3.The fermented milk supernatant, prepared according to the procedure for probiotics, was separated by ultrafiltration membranes with molecular weight cutoffs of 10 K and 3 K, in turn, to obtain 3 components: FBM I or FCM I (>10 kDa), FBM or FCM II (3-10 kDa), and FBM or FCM III (<3 kDa).Their ACE inhibitory activities were determined, and then components II and III were further extracted with a mixture of methanol: chloroform: water (3:1:4,V:V:V), concentrated under a vacuum, and dried for inspection.
Identification of the ACEIPs.LC-MS/MS analysis of the peptides was performed by Wuhan Anlong Kexun Technology Co (http: / / www .anachro.com.cn).A high-performance liquid chromatography system (RIGOL L-3000, Beijing Puyuan Jingdian Technology Co., Ltd., Beijing, China) was used for gradient separation of peptide components.The mobile phases used were liquid A (100% mass spectrometry water, 0.1% formic acid) and liquid B (80% acetonitrile, 0.1% formic acid) with a separation flow rate of 600 nL/min.Then, an Orbitrap Exploris 480 mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) was used with Nanospray Flex (NSI) as the ion source to perform a full scan of m/z 350-1200, and the primary mass spectrometer resolution was set to 60, 000 (200 m/z), the automatic gain control was set to custom, and the maximum injection time was 50 ms.The precursor ions were fragmented by the high-energy collisional fragmentation method, and secondary mass spectrometry detection was performed.For this, the automatic gain control was set to custom, the maximum injection time was 22 ms, and the peptide fragmentation collision energy was set to 30% to generate the original data of mass spectrometry detection.The liquid phase-mass spectrometry analysis of peptides was completed by Wuhan Anlong Kexun Technology Co., Ltd.Identified peptide sequences ranged from 7 amino acids upwards.
Proteome Discoverer 2.4 software (https: / / www .thermofisher.com/proteomediscoverer) was used to search for information related to peptide sequence and source, and the search parameters were set as follows: static modification was set to carbamidomethyl (C), dynamic modification to M Oxidation (15.995Da), acetyl (protein N-terminal), precursor ion mass tolerance of ± 15 ppm, fragment ion mass tolerance of ± 0.5 Da.BIOPEP-UWM (https: / / biochemia .uwm.edu.pl/biopep -uwm) was used to screen out identified ACEIPs while combining features of ACEIP sequences to find potential ACEIPs.

Anti-PIH effect of FBM
Animal experiments were performed in part at Zhonghong Boyuan Biotechnology Co. (http: / / www .zvast-bio .cn).All rat experiments were conducted in accordance with internationally accepted animal welfare and ethical guidelines.Specific pathogen free grade Sprague-Dawley (SD) female rats and male rats aged 10 weeks, were purchased from Spelford (Beijing) Biotechnology Co. Female rats were caged with males at 1:1 (female: male) after a one-week acclimatization period and the day of observation of the vaginal plug was recorded as d 0 of pregnancy.To verify the anti-PIH effect of FBM, 18 pregnant SD rats at the first day of pregnancy were randomly divided into the yogurt group, labetalol group, and control group (n = 6), and gavaged with 10 mL of yogurt (20 mg/kg), labetalol hydrochloride (4 mg/kg), and normal saline, respectively.Gavage was administered once a day until the day of delivery.To maintain the PIH model, methyl nitrosoarginine (125 mg/kg) was injected subcutaneously every day from d 15 of pregnancy to the day of delivery.Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were recorded on d 0, 14, and 21 of pregnancy in female SD rats.

Data analysis
RSM analysis was performed using Design-Expert 11.1.2software and Origin 2018 software was used for mapping.Data for ACE inhibitory activity and blood pressure were independently repeated 3 times and expressed as the mean values ± standard deviation.SPSS (version 25, SPSS/IBM, Chicago, IL) was used for Oneway ANOVA (ANOVA) and Tukey tests to compare ACE inhibitory activity and blood pressure differences (P < 0.05).

Probiotic screening
Dairy products are considered good carriers of probiotics to reduce the prevalence of hypertension-related diseases in pregnancy (Brantsaeter et al., 2011).Since researchers first isolated lactic acid bacteria from breast milk in the early 21st century and confirmed their ability as potential probiotics, human milk-derived probiotics have received increasing attention (Hu et al., 2021).L. plantarum, an extensively studied probiotic, has been shown to be effective in lowering blood pressure in animal studies and clinically.Sharafedtinov et al. (2013) showed a significant reduction in body weight and blood pressure levels in obese hypertensive patients after supplementation with cheese containing the probiotic L. plantarum TENSIA.Marek Naruszewicz et al. (2002) studied the effect of L. plantarum 299v supplementation on cardiovascular disease risk factors in smokers and found that 6 weeks of L. plantarum supplementation resulted in a significant reduction in SBP.L. plantarum can be found in a mother's mammary gland or pancreas after intake from fermented foods (Maldonado et al., 2021).In this study, a L. plantarum strain, LPB, with high ACE inhibitory activity was screened from human milk.As shown in Figure 2, LPB had a higher ACE inhibitory activity compared with strain LPR5, LPR7, and L. gasseri A. There was no significant difference in ACE inhibitory activity between LPB (76.84 ± 0.95%) and the commercial strain LP299253 (77.78 ± 1.59%).Furthermore, fermented milk inoculated with LPB showed a significantly lower pH (Figure 2).This suggested that LPB had a higher acid-producing ability that promoted protein coagulation.Therefore, LPB was selected as the target strain for subsequent studies.

Effect of optimization of fermentation on the ACE inhibitory activity of RSM
The data in Table 1 were fitted by regression to obtain quadratic multinomial regression models for the ratio of LPB to the yogurt culture number (6, 8, 10) (X 1 ), LPB inoculum concentration (2%, 3%, 4%) (X 2 ), fermentation time (6 h, 8 h, 10 h) (X 3 ), and fermentation temperature (34.5°C, 37°C, 39.5°C) as: Y = −1339.8571+ 13.4548x 1 + 54.8598x 2 + 30.0331x 3 + 63.4507x 4 − 0.3750x 1 x 2 − 0.2338x 1 x 3 + 0.0465x 1 x 4 − 0.1138x 2 x 3 − 0.0240x 2 x 4 − 0.2835x 3 x 4 − 0.7092x 1 2 − 8.7000x 2 2 − 0.9658x 3 2 − 0.8409x 4 2 (2) The absolute value of each coefficient in the equation directly reflects the degree of influence of each factor on the index value, and the positive and negative coefficients reflect the direction of influence.From Eq 2, it is clear that fermentation temperature and LPB inoculum concentration have the greatest effect on ACE inhibitory activity.The response surface design results of the buffalo milk fermentation process showed that the term R 2 of the polynomial model was 0.9795, indicating that more than 97.95% of the actual values could be explained (Table 2).The model accurately predicted the effect of these 4 factors on ACE inhibitory activity (P < 0.01).The fermentation time, fermentation temperature, and inoculum concentration of LPB significantly (P < 0.05) correlated with the ACE inhibitory activity of FBM.The optimal conditions were an LPB inoculum of 3% (8:1 ratio with yogurt culture agent bacteria), a fermentation time of 8 h, and a temperature of 37°C to obtain a maximum predicted Y-value of 84.10%.For the experiments conducted under optimal conditions, the Y-value of 84.51% was obtained.

ACE inhibitory activity of yogurt
A variety of different milk sources have been developed into blood pressure-lowering fermented milk products.ferment camel milk and the ACE inhibitory activity of its water-soluble extract was > 80%, which was significantly higher than that of fermented cow milk (<60%).In our study, the FBM obtained under optimal fermentation conditions reached 84.51 ± 1.11% ACE inhibitory activity, which was significantly higher than that of FCM (79.31 ± 0.70%) under the same conditions.This indicates that of the animal milks studied to date, goat milk and buffalo milk are good milk sources to make a blood pressure-lowering fermented milk product.To determine the source of ACE inhibitory activity in FBM, we detected the ACE inhibitory activity of the LPB MRS centrifugation supernatant (LPB supernatant), unfermented buffalo milk, and FBM without LPB.As shown in Figure 3, the ACE inhibitory activities in buffalo milk and LPB supernatant were significantly lower than those in buffalo yogurt fermented with common ferment only or mixed with LPB (P < 0.05).This suggested that the ACE inhibitory activity in buffalo yogurt may be related to peptides produced by fermentation (Begunova et al., 2020).During the fermentation process, lactic acid bacteria break down milk proteins through their unique cell wall proteases and intracellular peptidases to produce ACEIPs.In addition, the ACE inhibitory activity of fermented buffalo milk supplemented with LPB was significantly higher than that of buffalo yogurt fermented with yogurt culture only (P < 0.05), confirming the ability of LPB to produce ACEIPs.

ACEIPs of yogurt
The peptides produced in FBM and FCM were sequentially separated by ultrafiltration membranes of 10 kDa and 3 kDa, respectively.The 3 components showed significant differences in ACE inhibitory activity (P < 0.05, Figure 4).The III fraction exhibited significantly higher ACE inhibitory activity, indicating that the smaller molecular weight peptide fragments are the main ACEIPs, which was consistent with a previous report (Rubak et al., 2020).Meanwhile, the ACE inhibitory activity of FBM was higher than that of FCM for the same molecular mass fraction, indicating that FBM contains more ACEIPs.There were significant differences in the composition in casein (CN) among the different fractions (<10 kDa).The α s1 -CN content was significantly reduced in the fractions, and the β-CN content was significantly increased.Comparing the same fractions between the fermented milks, it was clear that the amount of casein was higher in FBM than in FCM, especially for β-CN and α s1 -CN (Figure 5).This may account for the higher ACE inhibitory activity of FBM than FCM.To test this, the fractions (<10 kDa) were analyzed by LC-MS/MS to search for ACEIPs.We identified 29 and 11 peptides containing ACEIP sequences in FBM and FCM, respectively, based on the structural characteristics of ACEIPs (Figure 5) (Maruyama et al., 1987).The peptides RFFVAPFPEVFGK, FVAPFPE-VFGK, PIGSENSGKTTMPLW, NMAINPSK, RELEELNVPGEIVE, DELQDKIHPF, ELQD-KIHPF, DKIHPFAQ, and SQSKVLPVPQ, which we identified (Table 3), were known ACEIPs according to the BIOPEP database (http: / / www .uwm.edu.pl/biochemia/ index) and previous reports (Amorim et al., 2019;Rubak et al.,, 2020;Ekhtab et al., 2017;Fan et al., 2019;Otte et al., 2011;Robert et al., 2004;Pihlanto et al., 1998).The amino acid sequence at the C-terminus, plays a dominant role in the competitive binding of ACEIPs to the ACE active site, and when hydrophobic amino acid residues are present at the C-terminus of the peptide sequence, such as aromatic or branched side chains, or when lysine residues are present, the peptide exhibits strong ACE inhibitory activity (Hernánde-Ledesma B et al., 2005;Maruyama et al., 1987).In this study, 18 peptides with hydrophobic amino acid residues at the C-terminus (Table 3) are likely ACEIPs.Furthermore, the peptides containing ACEIP sequences may be further hydrolyzed in the gastrointestinal tract during digestion to produce ACEIPs (Ibrahim et al., 2017).The results showed that ACEIPs in FBM were mostly derived from β-CN, consistent with the β-CN dominance reported by Sandeep et al. for buffalo cheese protease hydrolysates (Sandeep et al., 2012).
Trypsin preferentially cleaves the C-terminus of lysine (K) and arginine (R), and α-chymotrypsin preferentially cleaves the C-terminus of tyrosine (Y), tryptophan (W), and phenylalanine (F) (Ibrahim et al., 2017).The number of ACEIPs was greater in FBM than in FCM, which explains the strong ACE inhibitory activity of FBM.It is worth noting that peptides that are highly hydrophobic or contain proline prevent hydrolysis by trypsinase enzymes (Meisel et al., 2004).Possible ACEIPs from FBM and FCM after gastrointestinal protease digestion are shown in Table 4. Theoretically, FBM digested by gastrointestinal proteases may form a richer variety of ACEIPs than FCM, optimizing the hypotensive effects in the human body, although this requires further investigation.

Anti-PIH effect of FBM
The treatment of hypertension with yogurt has been widely studied, but less research has been reported on the treatment of PIH.To analyze the effect of FBM on PIH, an animal model of PIH rat was used.As shown in Table 5, there was no significant difference in blood pressure within or between the 3 groups on d 0 and d 14 (P > 0.05).Modeling was started on d 15 of conception, and methylnitrosoarginine was injected subcutaneously daily.Blood pressure on d 21 in the negative control group was significantly increased compared with that on d 0 and 14, indicating successful model induction of hypertension.The blood pressure of the rats in the labetalol and FBM groups did not change significantly after modeling and there was no significant difference in blood pressure between the 2 groups (Table 5).This suggested that FBM could be effective for the treatment of PIH (P < 0.05) with similar effects to labetalol in lowering blood pressure.Furthermore, no significant change in blood pressure was observed between the FBM and labetalol group, before and after modeling, which indicated that FBM could be effective at preventing PIH.

CONCLUSION
In this study, LPB with high ACE inhibitory activity was isolated from breast milk and co-fermented with yogurt culture to produce FBM.The fermentation procedure was optimized by RSM, and a higher ACE inhibitory activity of 84.51 ± 1.11%, which was significantly higher than that of cow milk fermented under the same conditions (P < 0.05).The ACE inhibitory activity in fermented milk was mainly dependent on the ACEIPs produced from casein by the ferments.Amino      Data are expressed as mean values ± standard deviation (n = 6).One-way ANOVA and Tukey post-hoc tests were used for statistical analysis.a and b indicate significant differences (P < 0.05) at 0, 14, and 21 d within each group.α and β indicate significant differences (P < 0.05) between groups.
Figure 1.Schematic representation of the buffalo milk fermentation process and identification of angiotensin converting enzyme inhibitory peptides (ACEIPs).LPB: Lactobacillus plantarum B.

Figure 2 .
Figure 2. Angiotensin converting enzyme (ACE) inhibitory activity, acidity and pH of fermented buffalo milk (FBM) with different probiotics.A, B, R5, R7, and 299253 represent Lactobacillus griseus A, Lactobacillus plantarum B, R5, R7 and 299253, respectively.Data expressed as mean values ± standard deviation (n = 3).Differences between the groups were analyzed by One-way ANOVA and Tukey tests.Different letters indicate significant differences between the strains (P < 0.05).Error bars represent standard deviation.

Figure 3 .
Figure 3. Angiotensin converting enzyme (ACE) inhibitory activity of different products.Data expressed as mean values ± standard deviation (n = 3).One-way ANOVA and Tukey tests were used to analyze the differences between the products.Different letters indicate a significant difference (P < 0.05).Error bars represent standard deviation.

Figure 4 .
Figure 4. Angiotensin converting enzyme (ACE) inhibitory activities of different fractions of fermented buffalo milk (FBM) and fermented cow milk (FCM).One-way ANOVA and Tukey tests were used to analyze the differences between the groups and data were expressed as mean values ± standard deviation (n = 3).Different letters indicate significant differences in ACE inhibitory activity of different fractions (P < 0.05).Error bars represent standard deviation.

Figure 5 .
Figure 5. Distribution of polypeptides (<10 kDa) in (a) fermented buffalo milk (FBM) and (b) fermented cow milk (FCM).CN stands for casein.FBM II and FBM III represent fermented buffalo milk supernatant fractions with molecular weight cutoffs less than 3k Da and 3-10k Da, respectively.FCM II and FCM III represent fermented cow milk supernatant fractions with molecular mas cutoffs less than 3k Da and 3-10k Da, respectively.

FBM
II and FBM III represent fermented buffalo milk supernatant fractions with molecular weight cutoffs less than 3k Da and 3-10k Da, respectively.FCM II and FCM III represent fermented cow milk supernatant fractions with molecular weight cutoffs than 3k Da and 3-10k Da, respectively.

Table 1 .
Yi et al.:A buffalo yogurt fermented… The Box-Behnken design for the fermentation conditions and theangiotensin converting enzyme (ACE) inhibitory activity

Table 3 .
Known and potential angiotensin converting enzyme inhibitory activity peptides (ACEIPs) in fermented buffalo milk (FBM) and fermented cow milk (FCM) FBM III represent fermented buffalo milk supernatant fractions with molecular weight cutoffs less than 3k Da and 3-10k Da, respectively.FCM II and FCM III represent fermented cow milk supernatant fractions with molecular weight cutoffs less than 3k Da and 3-10k Da, respectively.no.Guike ZY22096025) and the Funding Scheme for High-Level Overseas Chinese Students' Return of Ministry of Human Resources and Social Security (No.RSTH [2019] 160).We thank Liwen Bianji (Edanz) (https: / / www .liwenbianji.cn)for editing the language of a draft of this manuscript.