Molecular docking studies on α-amylase inhibitory peptides from milk of different farm animals

Milk derived peptides have emerged as a popular mean to manage various lifestyle disorders such as diabetes. Fermentation is being explored as one of the faster and efficient way of producing peptides with antidiabetic potential. Therefore, in this study an attempt was made to comparatively investigate the α-amylase (AA) inhibitory properties of peptides derived from milk of different farm animals through probiotic fermentation. Peptide’s identification was carried out using LC-MS-QTOF and inhibition mechanism were characterized molecular docking. Results obtained showed an AA-IC 50 value between 2.39 and 36.1 µg protein equivalent for different fermented samples. Overall, Pediococcus pentosaceus MF000957 (PPe) derived fermented milk from all animals indicated higher AA inhibition than other probiotic derived fermented milk (AA-IC 50 values of 6.01, 3.53, 15.6 and 10.8 µg protein equivalent for bovine, camel, goat and sheep fermented milk). Further, molecular docking analysis indicated that camel milk derived peptide IMEQQQTEDEQQDK and goat milk derived peptide DQHQKAMKPWTQPK were the most potent AA inhibitory peptides. Overall, the study concluded that fermentation derived peptides may prove useful in for managing diabetes via inhibition of carbohydrate digesting enzyme AA.


INTRODUCTION
Milk and milk derived products not only have a substantial role in the human nutrition, but they are also considered a rich source of essential nutrients required by humans.These products serve as excellent source of proteins, lipids minerals, and vitamins (Silva et al., 2020).Milk can be fermented by different microbes, Lactobacillus spp. or Bifidobacteria bacteria, to produce a partially digested milk with varying properties (Salli et al., 2021).Upon fermentation of milk, lactose sugar and proteins are broken down into simpler sugar (glucose and galactose) and peptides, respectively.Moreover, the breakdown of proteins could result in bioactive peptides with diverse pharmaceutical and nutraceutical potentials, for example anti-oxidant, antidiabetic, anti-obesity, anti-inflammatory, anti-cancer, and anti-hypertension activities (Linares et al., 2017, Marco et al., 2017, Mathur et al., 2020).
Anti-diabetic peptides can reduce level of blood glucoses, improve insulin uptake, and restrain some important enzymes associated with carbohydrate metabolism and glucose absorption (Antony and Vijayan, 2021).Peptides with anti-diabetic activities have been mainly screened via their capability to inhibit major carbohydrases (α-amylase (AA), and α-glucosidase (AG)), and dipeptidyl peptidase IV (DPP-IV) an enzyme implicated in degradation of insulinotropic hormones.AA Inhibition can reduce the enzyme activity and thus decrease the digestion rate of starch and disaccharides into glucose, causing a slow secretion and reduced absorption of glucose in the gut (Karimi et al., 2020).Previous studies have shown the enhanced efficacy of fermented dairy products toward AA inhibition in comparison with their unfermented form.This is largely attributed to release of bioactive peptides from intact proteins via bacterial proteases during fermentation.For Molecular docking studies on α-amylase inhibitory peptides from milk of different farm animals example, cow and camel milk-based yogurt fermented by Allium sativum showed higher α-amylase inhibitory activity in comparison with unfermented samples (Shori and Baba, 2014).Besides, fermentation of soya milk by kefir has been reported to produce bioactive peptides with significant α-amylase inhibitory potential (Tiss et al., 2020).In fact, camel milk fermented with probiotic Lactobacillus spp.has shown significantly higher AA inhibitory capability in comparison to unfermented milk (Ayyash et al., 2018b).Moreover, significant differences between the AA inhibitory potential of camel and cow milk were reported as well.
The milk composition of different mammals differs significantly.The type of species, genes, physiological, nutritional, and environmental factors influence the composition of mammal milk and thus their protein content and composition (Pietrzak-Fiecko and Kamelska-Sadowska, 2020).Differences in protein composition and content between different mammals will result in the production of a wide range of peptides upon milk fermentation.Those peptides will vary in composition, sequence, molecular weight, and bioactivity.In fact, Microbial fermentation offers uncontested advantages over other methods of peptides production due to their broad range of microbial proteases can produce peptides varying sizes and sequences that may result in superior biological activity and their characteristics (Khan et al., 2018).
To the best of our knowledge, previous literature related to obtaining bioactive peptides with AA inhibitory activities via fermentation from major farm animals has been quite limited.Only few studies that have been conducted are majorly carried out on comparison between bovine and camel milk.However, looking at the increasing demand for alternative milk sources such as goat and sheep milk, a through investigation in this direction is necessitated.Therefore, in this study, bovine, camel, goat, and sheep milk were fermented by Lactiplantibacillus argentoratensis MF000943 (LA), Limosilactobacillus fermentum MF000944 (LiF), Lactiplantibacillus pentosus MF000946 (LPe), Pediococcus pentosaceus MF000957 (PPe), and Enterococcus hirae MF000958 (EH).The degree of proteolysis and AA inhibitory activity of fermented milk was measured on d 0, 7, and 14 of refrigerated storage.Finally, the peptides present in PPe fermented milk samples were identified and sequenced for in silico AA inhibitory activity.

Materials
Fresh raw milk samples from same breed animals i.e., (bovine (Holstein Friesian), camel (Omani), goat (Sa-lali) and sheep (Al-Nuaimi breed) (n = 3 for each milk type), were obtained under refrigerated conditions from local farms of Al Ain, an eastern region of Abu Dhabi Emirates, United Arab Emirates (UAE).Various probiotic microorganism used in this study for fermentation were isolated from camel milk.The probiotics obtained were identificatied using 16s rRNA sequencing and upon submission to NCBI database the accession number was obtained.The names for probiotics used are as follows; Lactiplantibacillus argentoratensis MF000943 (LA), Limosilactobacillus fermentum MF000944 (LiF), Lactiplantibacillus pentosus MF000946 (LPe), Pediococcus pentosaceus MF000957 (PPe), and Enterococcus hirae MF000958 (EH).α-amylase from porcine pancreas (AA), p-nitrophenyl-α-D-maltohexaoside as amylase chromogenic substrate, were obtained from Sigma-Aldrich (St. Louis, MO, USA).All chemicals used in this study were of analytical grade.

Protein hydrolysis via microbial fermentation
Fresh milk samples obtained above were skimmed 2 times by refrigerated centrifugation at 4,000 × g and 10,000 × g for 15 min, respectively.These skim milk samples were then heated at 90°C for 10 min causing whey protein denaturation and then cooled down to 37°C in ice-cold water.Probiotic inoculation was performed at a level of 10 5 cfu/mL from an 18 h old cultures in triplicate and inoculated milk samples were fermented under static conditions for 24 h at 37°C.The physicochemical changes related to bacterial cell count, pH and titratable acidity were evaluated at different time intervals during 24 h intervals.Following fermentation, fermented milk samples were stored at 4°C for 2 weeks and a weekly analysis was performed until 14 d.Briefly, at each sampling intervals, fermented milk samples were first neutralized to pH 7.0 using 1 M NaOH and clear water-soluble extracts (WSE) containing peptides were obtained via centrifugation at 10000 × g for 15 min at 4°C.

Determination of degree of proteolysis (DH%)
Based on the original method of (Nielsen et al., 2001), the degree of proteolysis was determined by ophthaldialdehyde (OPA) method as further modified by (Mudgil et al., 2019): where h and h tot represents is the amount of hydrolyzed and the total amount of peptide bonds per protein equivalent, respectively.The amount of hydrolyzed bonds was estimated using h = (SerineNH 2 − β)/α, where α, β, and h tot values were taken from (Nielsen et al., 2001) as 1.039, 0.383, and 8.2 mEq/g of protein, respectively.

α-Amylase (AA) inhibition assay
AA inhibitory activity potential was measured as per the method of (Baba et al., 2021b).Briefly, various concentrations of WSE from fermented milks were incubated with 100 µL of AA enzyme (2 mg/mL) in sodium phosphate buffer (0.02 M with 0.006 M of sodium chloride; pH 6.9), and preincubation at 37°C was carried out for 15 min.Thereafter, 50 µL of substrate p-nitrophenyl-α-maltohexaoside (PNPM; 5mM) was added for initiating the reaction and incubation was carried out at 37°C for 60 min.The absorbance of pnitrophenyl developed was monitored at 405 nm using a microplate reader (Epoch 2, BioTek, Winooski, 156 VT, USA).For each test reaction their reaction blanks with sample and buffer were run to minimize background noise.The percent inhibition of enzyme activity was calculated at each concentration of sample using the following equation:

%AA Inhibition
Abs Abs The inhibition values were plotted against the concentration of WSE as µg protein equivalent and IC 50 values were determined from the slope of the curve.

Molecular binding mechanism of identified peptides against AA
Molecular interactions of identified peptides with AA were performed to authenticate the mechanism of AA inhibition.For this, Crystal Structure of Human Salivary Enzyme (PDB; 1SMD) was downloaded from RCSB Protein Data Bank (RCSB PDB) database and pepsite 2 (http: / / pepsite2 .russelllab.org/ ) web server was used for understanding molecular binding of peptides with AA (Trabuco et al., 2012).The most potent AA inhibitory bioactive peptide was selected based on the significance of binding (p-value < 0.05) and total number of potential binding sites on target enzyme AA.

Statistical analysis
All the experiments were carried out in triplicate and the data analysis was performed via one-way ANOVA (ANOVA) using SPSS version 28.0 software (SPSS INC., Chicago, IL, USA).Mean significant difference among different fermented samples were separated using Tukey's New Multiple Range Test for establishing significance at P ≤ 0.05.

Degree of hydrolysis (DH%)
Extent of proteolysis from fermented milk samples obtained by different probiotics was determined on d 0, 7, and 14 of refrigerated storage and results obtained are shown in Table 1.The unfermented control had the lowest DH% value and varied significantly (P ≤ 0.05) throughout the study.This inherent DH in unfermented control milk sample on Day 0 and during the storage suggests that proteases from native microbial flora might have caused some degradation of proteins.

Mudgil et al.: Molecular docking studies…
The DH% of different probiotic fermented bovine milks on Day 0 was similar, without any significant differences, apart from the LA fermented sample that had the lowest DH% (25.95 ± 0.13%).By Day 7, the DH% of the samples ranged between 37 and 43%.By Day 14, LPe fermented bovine milk showed the highest DH% (57.06 ± 1.34%), being significantly higher than the other probiotic fermented bovine milks (P ≤ 0.05).In fact, this DH% value was significantly higher compared with LPe fermented milk from other animals by Day 14.
Initiation of camel milk fermentation on Day 0 resulted in significantly varying DH% between the samples (P ≤ 0.05).LPe fermented camel milk sample had the highest DH% on Day 0 (33.73 ± 0.82%) compared with the other camel milk samples.Different fermented camel milk samples showed similar DH% by Day 7, however by Day 14, LiF showed the highest DH% value (47.80 ± 1.39%) significantly different than the other camel milk samples (P ≤ 0.05).The DH% values of other milk sources fermented by LiF were similar on Day 14 with no significant difference (P > 0.05).A study by (Moslehishad et al., 2013b) reported that L. fermentum PTCC 1638 and L. rhamnosus PTCC 1637 showed high protease activity in fermenting camel and cow's milk.
Goat milk samples had the highest DH% values since Day 0, this indicates the capability of goat proteins to undergo fermentation and production of peptides at a faster rate compared with the milk of other animals.This is also clear when the DH% values of goat milk samples of Day 7 and Day 14 are compared with the other animals.Goat milk samples always had the highest DH% values with 2 significant exceptions on Day 14 for LA and LPe.EH produced the significantly higher number of peptides as indicated by degree of hydrolysis on Day 7 (53.85 ± 0.46%) and Day 14 (55.62 ± 0.33%) in comparison to the other fermented goat milk samples (P ≤ 0.05).Besides, this DH% value was significantly higher compared with other milk sources fermented by EH by Day 14.
Among the fermented sheep milk samples of Day 0, an unexpected DH% value was determined for LPe fermented sheep milk (14.96 ± 1.21%).This was the lowest DH% value among all the other fermented sheep milk samples and the other animals' fermented milk of Day 0, the difference was significant (P ≤ 0.05).By Day 7, sheep milk fermented by LiF resulted in the highest value of DH% (42.58 ± 0.79%) which was significantly different that the other samples of that day and similar to that of goat milk on the same day.In fact, on Day 14, L. fermentum-fermented sheep milk also had the highest DH% value (44.67 ± 0.33%) with a significant difference in comparison to the other fermented sheep milk on Day 14.This value is significantly higher than the L. fermentum-fermented camel milk of Day 14, yet it is significantly lower than those for L. fermentumfermented goat and bovine milk (P ≤ 0.05).
The differences in DH% values of fermented milk of different sources by different bacteria can be attrib- uted to variations in protein by-products in the milk of different animals and differences between species (Soleymanzadeh et al., 2016).In addition, the release of proteases by the microorganisms for protein hydrolysis can vary and this influences the degree of hydrolysis of proteins into large peptides, and thus shorter peptides and free amino acids (Hou et al., 2017).Overall, the hydrolysis of proteins and peptides increased throughout the storage duration and this is in agreement with several studies as residual microbial exo-proteases even during refrigerated storage can cause further protein degradation (Moslehishad et al., 2013a).
Several previous studies that involved measurement of the breakdown of intact proteins to peptides or amino acids, through fermentation, reported their results in terms of proteolytic activity.Effect of incubation period on the proteolytic activity of camel milk fermented by Lactobacillus bulgaricus NCDC, Lactobacillus fermentum TDS030603 (incubation time: 0, 3, 6, 9 and 12 h) (Solanki et al., 2017), and Lactobacillus plantarum KGL3A (incubation time: 0, 6, 12, 24, and 48 h) (Dharmisthaben et al., 2021), showed a positive correlation between proteolytic activity in camel milk and incubation time.Besides, similar observations were also reported by (Moslehishad et al., 2013a), in Lactobacillus rhamnosus PTCC 1637 fermented bovine and camel milk during 21 d of storage.This effect is associated with the amount of amino acids needed by the bacteria during their growth through which releasing free NH 3 groups can varies with inoculation levels (Solanki et al., 2017).

Evaluation of in vitro antidiabetic activity via AA inhibition
The antidiabetic activity of different types of fermented milk was evaluated by measuring the AA inhibitory properties of the samples and their AA-IC 50 (µg protein equivalent/mL) values are shown in Table 2.The AA-IC 50 of all the unfermented control milk samples was always significantly higher (P ≤ 0.05) than all the types of fermented milk throughout the storage period.This clearly indicates the capability of AA inhibition of peptides produced through microbial fermentation in comparison to intact proteins of milk.In fact, a clear trend is noticed with respect to AA inhibitory activity of unfermented milk throughout the storage.Camel milk showed the highest AA inhibitory activity throughout the 14 d period, with the maximum activity on Day 14 (AA-IC 50 17.30± 2.11 µg protein/mL), that were significantly higher throughout this period.Moreover, unfermented camel milk had lower AA-IC 50 values in comparison with several samples of fermented milk of other animals.Again, this evidently reveals the anti-diabetic potential of camel milk in comparison to the milk and peptides obtained from other animals, and this has been highlighted in previous studies (Agrawal et al., 2005, Mudgil andMaqsood, 2023).
It was observed that AA inhibitory activity of fermented milk samples increased during refrigerated storage and could be attributed to release of newer peptides with α-amylase inhibitory activity across all the type of fermented milks i.e., bovine, camel, and sheep milk.PPe was the most effective in producing bovine milk peptides with the highest AA inhibitory activity, the IC 50 value of its fermented bovine milk was 12.00 ± 2.38 µg protein/mL on Day 0, which decreased further to 9.08 ± 0.68 and 6.01 ± 0.10 µg protein/mL on Day 7 and Day 14, respectively.Nevertheless, LA and for LPe fermented bovine milk produced peptides with a promising AA inhibitory activity that was not significantly different to the activity of PPe fermented bovine milk (P > 0.05).
Fermented camel milk showed the most promising α-amylase inhibitory activity compared with all the other milk provided from different sources and fermented by different microorganisms.Out of all the 60 fermented milk samples of different storage time, LPe -fermented camel milk of Day 14 had the lowest AA-IC 50 value (2.39 ± 0.51 µg protein/mL), followed closely by PPe fermented camel milk samples with an AA-IC 50 value (3.53 ± 0.39 µg protein/mL) (P ≤ 0.05), suggesting that LPe and PPe were effective in producing the peptides from camel milk with higher AA inhibitory potential.In fact, it also derived potent α-amylase inhibitory peptides from bovine and sheep milk.It was expected that fermented camel milk will show higher anti-diabetic activity compared with other types of milk.Several studies have underlined the effectiveness of camel milk-derived peptides in inhibiting diabetesrelated enzymes such as α-amylase, α-glucosidase, and dipeptidyl peptidase IV (Baba et al., 2021a, Ali Redha et al., 2022, Khakhariya et al., 2023, Mudgil and Maqsood, 2023).
Various studies in past few years have explored the antidiabetic functionality of fermented camel and bovine milk through AA inhibition.In one such study, fermentation of camel and bovine milk was carried out by Lactococcus lactis KX881782 (Lc-KX), camel milk probiotic strain and Lactobacillus acidophilus DSM9126 (La-DSM), and the effect of storage time (0, 7, 14, and 21 d) was explored (Ayyash et al., 2018a).Bovine milk fermented by La-DSM and camel milk fermented by Lc-KX showed an α-amylase inhibition percentage of > 40% throughout the storage periods.In addition, prolonged storage positively and significantly influenced the AA inhibitory activity of fermented camel milk produced by both bacteria, which agrees with our current findings.Yet, this effect was insignificant in fermented bovine samples.Comparing the AA inhibitory activity of fermented bovine and camel milk, authors reported that camel milk fermented by Lc-KX had significantly greater inhibitory activity (P < 0.05).Another study used camel milk probiotic strains L. reuteri KX881777, L. plantarum KX881772, and L. plantarum KX881779 along with a reference strain L. plantarum DSM2468 to ferment bovine and camel milk for evaluation of their AA and AG inhibitory activity (Ayyash et al., 2018b).The authors reported that all fermented samples (except camel milk fermented by L. plantarum KX881772) showed an α-amylase inhibition percentage of > 34% throughout the storage periods.Moreover, prolonged storage also positively and significantly influenced the AA inhibitory activity (with the exception of fermented bovine milk by L. plantarum KX881772).
Further, unfermented sheep milk had the highest AA-IC 50 values among the other unfermented milk samples, however, upon fermentation, it showed a similar AA inhibitory potential to fermented bovine milk.The main difference between the AA inhibitory activity of fermented sheep milk in comparison to the other samples is related to the effect of storage duration.Increasing the storage time, and thus fermentation duration, had a positive impact on the formation of AA inhibitory peptides from bovine, camel, and goat proteins; that resulted in samples with lower AA-IC 50 values by Day 14.Nevertheless, sheep milk did not follow this trend.The lowest AA-IC 50 values of fermented sheep milk were determined on Day 7, those values increased by Day 14 suggesting a decrement in AA activity.This could be due to an increase of proteolysis of peptides into amino acids after Day 14 that resulted in a decrease of AA inhibitory peptides.Overall, a clear trend was not observed to decide the most efficient bacteria in producing fermented goat milk with AA inhibitory potential.EH fermented goat milk of Day 7 showed the lowest AA-IC 50 value (5.30 ± 0.04 µg protein/mL) that was significantly lower (P ≤ 0.05) than the other fermented sheep samples of Day 7.
Among the 4 types of milk, fermented goat milk showed the least AA inhibitory activity.This can be associated with the type and quantities of goat milk proteins.The lowest AA-IC 50 value among the fermented goat milk samples was for LA fermented sample on Day 7 (12.90 ± 2.04 µg protein/mL), yet this was significantly higher than the values of fermented camel and sheep milk of the same bacteria and storage duration.Nevertheless, a study reported the effectiveness of Lactobacillus plantarum strains in producing ferment goat milk with considerable α-amylase inhibitory activity reaching above 40% in some of the samples (Hashemi and Gholamhosseinpour, 2020).The fermentation process in this study involved ultrasonication treatment that significantly affected the α-amylase inhibitory activity of fermented goat milk and was attributed to further degradation of remaining intact proteins and larger peptides to smaller peptides due to ultrasonic vibrations (Hashemi and Gholamhosseinpour, 2020).Overall, no clear correlation between degree of hydrolysis and AA inhibitory activity was established.This is attributed to the fact that during the degradation of proteins peptides with various sequences and length are produced which vary considerably in their biological activity (Mudgil et al., 2023).

Identification of α-amylase inhibitory peptides: in silico
α-amylase is made up of 3 distinct parts, known as A, B, and C; in which the most significant active sites are located in part A and between parts A and B. The main active sites of the enzyme are Asp197, Glu233, and Asp300; with a calcium-binding domain comprising of Asp100, Arg158, Asp167, and His201; and a chloridebinding domain involving Arg195, Asn298, and Arg337 (Esfandi et al., 2021).Yet, there are other residues that comprise in the enzyme's vital sites such as Trp58, Trp59, Arg61, Tyr62, His101, Pro163, Asp165, Lys200, Ile235, Asp236, Tyr258, His299, His305, and Ala307 that are considered as hotspot residues that play a definitive role in inhibition (Nadeem et al., 2020).
Camel milk is considered a functional dairy product with the most remarkable anti-diabetic potential.The fermented sample had 37 bioactive peptides with α-amylase inhibitory potential.MMPY peptide showed the highest HPEP dock score (−218.031)with all the residues bound to the enzyme to 9 different key hotspot residues: Trp58*, Trp59*, Tyr62*, Asp96*, Arg195*, Asp197*, His299*, Asp300*, and His305*.Two peptides, YDLY and YLDY, were common in fermented bovine and camel milk.Although both peptides have the same composition, the difference in their sequence has remarkably influenced their HPEP dock score.The HPEP dock score of YDLY and YLDY is −162.496and −212.217,respectively (from fermented camel milk).This suggests the significance of the effect of peptide sequence on the AA inhibitory potential.Similar observation was marked by (Baba et al., 2021b) for AA inhibitory peptides derived via pepsin hydrolysis of camel whey protein where peptides LRPFL and LR-FPL though has same number of bound residues but had variable peptide ranking score.
Fermented sheep milk sample comprised of 45 bioactive peptides (with 3 peptides of statistically insignificant activity, P > 0.05), that showed α-amylase inhibitory activity.The peptides MAQY and MSQF with HPEP dock scores of −219.983 and −218.251,respectively, showed the highest inhibitory potential with all the residues participating in binding to α-amylase.MAQY and MSQF had the capability of binding to 5 hotspot enzyme residues: Trp58*, Trp59*, Tyr62*, His299*, and Asp300*.In fact, MSQF was capable of binding to His305* as well.Overall, some common peptides between some milk samples.For instance, MMLF was observed between fermented bovine, goat and sheep milk samples.Whereas YDLY, YLDY and YPALVY were common to fermented bovine and camel milk samples, respectively FMLM was found to be common between fermented goat and camel Milk samples.Similarly, MMLM peptide was common to fermented bovine and sheep milk.
Peptides involving leucine, lysine, cysteine, methionine, glycine, and phenylalanine residues have been reported for their α-amylase inhibitory activity (Baba et al., 2021b).Peptides with residues proline, leucine, methionine, and cysteine at their N and C terminal ends were common in peptides with α-amylase inhibitory activity.Among the key peptides discussed earlier, 3 peptides (MMPY, MAQY and MSQF) have a methionine residue and one peptide (LLYQEPVLGPVRGPFPIIV) has leucine at their N terminus.Besides, leucine, lysine, methionine, glycine, and phenylalanine residues were REQEELNVVGETVESLSSSEESITHINK 0.01656 Arg-1, Phe17,Glu18,Gln41,Val42,Ser43,Pro44,Trp58   present in those key peptides.Over 20 peptides with tyrosine at their N terminus were identified.Residues with aromatic amino acids can form aromatic-aromatic interactions with α-amylase by hydrogen bonds, van der Waals and electrostatic interactions (Ali Redha et al., 2022).

Structure-Activity Relationship (SAR) Analysis using molecular docking
Inhibitors of carbohydrate digesting enzymes i.e., α-amylase and α-glucosidase are the primary target for diabetes management.Although not completely known, it is believed that inhibitors of these enzymes interact within the enzyme's active sites and inhibit the enzyme-substrate complex formation causing disruption of enzymatic functions.Therefore, research aimed toward identifying compounds making strong interactions with catalytic binding sites of these enzymes is gaining particular interest from drug developers.In silico analysis via molecular docking has emerged as an efficient approach for prediction of binding affinity of various compounds with drug targets and therefore is being used extensively drug development.Therefore, to get deeper insight into the enzyme binding affinity of fermented milk derived peptides molecular docking studies were conducted on selected peptides with strongest binding affinities toward different binding sites of AA as predicted by Pepsite2 and HPEPDock scores.Following structure and activity (SAR) analysis of peptides, a total of 8 peptides i.e., YQEPVLG-PVRGPFPIIV and RELEELNVPGE from fermented bovine milk, IMEQQQTEDEQQDK and MMPY from fermented camel milk, DQHQKAMKPWTQPK and CPAALS from fermented goat milk and VKET-MVPKHK and HANAGAAGH from fermented sheep milk were selected for molecular docking.The interaction between peptides and enzyme are presented in Table 4 and Figure 1a-e.Ability of various peptides to make hydrogen bonds with α-amylase indicate their high potential for inhibition via creation of a sliding barrier causing hindrance with substrate binding sites (Siow and Gan, 2016).
As shown in Table 4 all the selected peptides could make hydrogen bonds with the target enzyme.Peptide YQEPVLGPVRGPFPIIV from fermented bovine milk formed hydrophobic interactions, hydrogen bonding and salt bridges with the catalytic binding residue using Asp300 in addition hydrophobic interaction were seen with catalytic triad of α-amylase using Trp58, Trp59, and His305 with a binding energy of −10.5 kcal/mol which is comparatively higher than commercial drug Acarbose.Similarly, peptide RELEELN-VPGE with a binding energy of −10.5 kcal/mol showed hydrophobic interaction with α-amylase using Trp59, Glu233, Asp300 and His305.This peptide also showed salt bridge formation using His305 of catalytic triad but no hydrogen bonding with any hotspot.Overall, maximum number of hydrogen bonds (9) were shown by fermented camel milk derived peptide IMEQQQT-EDEQQDK with maximum binding energy of −11.4 kcal/mol among all peptides analyzed.This peptide interacted hydrophobically with catalytic pockets using Asp300, and His305.Further, peptide MMPY showed hydrophobic interactions with α-amylase hotspots using Trp59, Arg195, Asp197, Glu233, Asp300, and His305.Interestingly both selected peptides from fermented camel milk showed no interaction with α-amylase using salt bridges.Furthermore, second to IMEQQQTEDEQQDK fermented goat milk derived peptide DQHQKAMKPWTQPK indicated maximum binding energy of −11.3 kcal/mol and hydrogen bonding with 8 different residues on enzyme.This peptide also showed second most hydrophobic interactions with catalytic hotspots on α-amylase via interactions with Trp58, Trp59, Tyr62, Asp300 and His305.Another peptide CPAALS from fermented goat milk showed no interaction using salt bridge but interacted with enzyme using maximum hydrophobic number interactions in the catalytic hotspots involving Trp58, Trp59, Tyr62, Asp197, Glu233, His299, Asp300, His305.Hydrogen bonding using His201, Gly233 and Glu233 was also observed for the following peptide with a binding energy of −8.7 kcal/mol.Both the peptides i.e., VKET-MVPKHK and HANAGAAGH from fermented sheep milk showed a binding energy of −9.6 and −10.3 kcal/ mol, respectively.Both the peptides showed all 3 types of interactions i.e., hydrophobic, hydrogen bonds and salt bridges with α-amylase (Table 4).These results are comparable to those obtained by (Fadimu et al., 2022), where various peptides obtained from alcalase and flavorzyme hydrolyzed lupin hydrolysates indicated binding energy with −6.9 to −9.1 kcal/mol.Peptides obtained in our study has indicated significantly higher binding energy and more interaction in comparison to theirs.

CONCLUSIONS
Use of food derived bioactive peptides for management of lifestyle related disorders is in great demand owing to their superior health benefits without any side effects.Peptides derived from milk proteins via enzymatic hydrolysis or fermentation has been widely investigated for their antihypertensive, antidiabetic and antioxidant properties.In the present investigation, a comparative analysis of peptides derived via probiotic fermentation from 4 different milk types was performed.The AA-IC 50 data obtained suggested that AA inhibition was significantly higher in fermented camel milk than other species after 7 D of refrigerated storage.Among probiotics LA and PPe were found to produce peptides with most potent AA inhibition.These characteristics of LA and PPe previously isolated from camel milk suggest their strong potential for use as starter culture for making functional foods with antidiabetic properties.Molecular docking studies via in silico approach, revealed that peptides derived from camel and goat milk proteins have very strong affinity toward catalytic site of the AA.The study also recognized that probiotic fermentation derived bioactive peptides could be used for large scale production of nutraceuticals or functional foods for diabetic patients.However, further studies on IMEQQQTEDEQQDK and DQHQKAMKPWTQPK in their synthetic form needs to be conducted for validation using in vitro, cell lines and in vivo approached.

Figure 1 .
Figure 1.Binding interactions between α-amylase and (a) acarbose as well as peptides from (b) bovine, (c) camel, (d) goat and (e) sheep.Note: Red circle, oxygen atom; blue circle, nitrogen atom; black circle, carbon atom; yellow circle, sulfur atom; purple line, peptide; brown line, α-amylase; red dotted line, salt bridge; green dotted line with number, hydrogen bond and bond length (in Armstrong, Å) between the proton donor and acceptor; brick red eyelashes, hydrophobic interaction.
Figure 1 (Continued).Binding interactions between α-amylase and (a) acarbose as well as peptides from (b) bovine, (c) camel, (d) goat and (e) sheep.Note: Red circle, oxygen atom; blue circle, nitrogen atom; black circle, carbon atom; yellow circle, sulfur atom; purple line, peptide; brown line, α-amylase; red dotted line, salt bridge; green dotted line with number, hydrogen bond and bond length (in Armstrong, Å) between the proton donor and acceptor; brick red eyelashes, hydrophobic interaction.

Table 4 : Summary of binding interactions between α-amylase (PDB ID: 1SMD) and selected peptides from fermented milk of different farm animals
* Potential hotspots for α-amylase inhibition if bounded by the peptide.