Milk Protein Hydrolysates Research Articles

Sensory-directed flavor analysis of off-flavor compounds in infant formula with deeply hydrolyzed milk protein and their possible sources

Off-flavor compounds in IF-DHMP were identified using sensory-directed analysis. In total, 56 types of aroma-active compounds were identified in the off-flavor (OF) samples, positive control (PC) samples, and accelerated oxidation (AO) samples. The Aroma Extraction Dilution Analysis (AEDA) results showed that methional, generated via Maillard reaction, was the most important component with the highest dilution factor in OF. The results of aroma recombination, omission and spiking experiment revealed that compounds such as pentanal, methional, (E)-2-nonenal, 3-methylbutanal, (E)-2-heptenal, nonanal, (E,E)-2,4-heptadienal, 1-penten-3-one, 1-octen-3-one, and (E,E)-2,4-nonadienal were the major sources of off-flavor in OF. We observed that the off-flavor in OF samples originated due to proteolysis, Maillard reaction, or lipid oxidation during processing rather than due to lipid oxidation during storage.

Average molecular weight, degree of hydrolysis and dry-film FTIR fingerprint of milk protein hydrolysates: Intercorrelation and application in process monitoring

Fourier-transform infrared (FTIR) spectroscopy was applied to predict the degree of hydrolysis (DH%) and weight-average molecular weight (Mw) in milk protein hydrolysates. Both DH% and Mw are important quality parameters of protein hydrolysates. Measuring these parameters and following their development during proteolytic reactions is therefore essential for process control and optimization in industry. In the present study the intercorrelation and the complimentary nature of these parameters were investigated and a partial least squares regression (PLSR) model was developed for the prediction of DH% from molecular weight distributions. Finally, we developed PLSR models based on dry-film FTIR spectroscopy for the prediction of both DH% and Mw. Here spectral changes in the amide region were found to be important for the two calibration models, underlining the advantage of dry-film FTIR measurement. This shows that dry-film infrared spectroscopy is a promising tool for dual prediction of DH% and Mw.

Identification and molecular docking study of novel cholesterol esterase inhibitory peptides from camel milk proteins

Novel bioactive peptides from camel milk protein hydrolysates (CMPH) were identified and tested for inhibition of cholesterol esterase (CEase), and their possible binding mechanisms were elucidated by molecular docking. Papain-generated CMPH showed the highest degree of hydrolysis. All CMPH produced upon enzymatic degradation demonstrated a dramatic enhancement of CEase inhibition compared with intact camel milk proteins, with papain-generated hydrolysate P9 displaying the highest inhibition. Peptide identification and their modeling through PepSite 2 revealed that among 20 potential bioactive peptides in alcalase-generated hydrolysate A9, only 3 peptides, with sequences KFQWGY, SQDWSFY, and YWYPPQ, showed the highest binding toward CEase catalytic sites. Among 43 peptides in 9-h papain-generated hydrolysate P9, 4 peptides were found to be potent CEase inhibitors. Molecular docking revealed that WPMLQPKVM, CLSPLQMR, MYQQWKFL, and CLSPLQFR from P9 hydrolysates were able to bind to the active site of CEase with good docking scores and molecular mechanics-generalized born surface area binding energies. Overall, this is the first study reporting CEase inhibitory potential of peptides generated from milk proteins.

Alteration in propagating colonic contractions by dairy proteins in isolated rat large intestine

Gastrointestinal conditions in which the transit of contents is altered may benefit from nutritional approaches to influencing health outcomes. Milk proteins modulate the transit of contents along different regions, suggesting that they have varying effects on neuromuscular function to alter gastrointestinal motility. We tested the hypothesis that bovine whey and casein milk protein hydrolysates could have direct modulatory effects on colonic motility patterns in isolated rat large intestine. Casein protein hydrolysate (CPH), whey protein concentrate (WPC), whey protein hydrolysate (WPH), and a milk protein hydrolysate (MPH; a hydrolyzed blend of 60% whey to 40% casein) were compared for their effects on spontaneous contractile waves. These contractions propagate along the length of the isolated intact large intestine (22 cm) between the proximal colon and rectum and were detected by measuring activity at 4 locations. Milk proteins were perfused through the tissue bath, and differences in contraction amplitude and frequency were quantified relative to pretreatment controls. Propagation frequency was decreased by CPH, increased by MPH, and unaffected by intact whey proteins. The reduced motility with CPH and increased motility with MPH indicate a direct action of these milk proteins on colon tissue and provide evidence for differential modulation by hydrolysate type. These findings mirror actions on lower gastrointestinal transit reported in vivo, with the exception of WPH, suggesting that other factors are required.

Postprandial Effects of a Proprietary Milk Protein Hydrolysate Containing Bioactive Peptides in Prediabetic Subjects.

Milk proteins have been hypothesized to protect against type 2 diabetes (T2DM) by beneficially modulating glycemic response, predominantly in the postprandial status. This potential is, amongst others, attributed to the high content of whey proteins, which are commonly a product of cheese production. However, native whey has received substantial attention due to its higher leucine content, and its postprandial glycemic effect has not been assessed thus far in prediabetes. In the present study, the impact of a milk protein hydrolysate of native whey origin with alpha-glucosidase inhibiting properties was determined in prediabetics in a randomized, cross-over trial. Subjects received a single dose of placebo or low- or high-dosed milk protein hydrolysate prior to a challenge meal high in carbohydrates. Concentration–time curves of glucose and insulin were assessed. Incremental areas under the curve (iAUC) of glucose as the primary outcome were significantly reduced by low-dosed milk peptides compared to placebo (p = 0.0472), and a minor insulinotropic effect was seen. A longer intervention period with the low-dosed product did not strengthen glucose response but significantly reduced HbA1c values (p = 0.0244). In conclusion, the current milk protein hydrolysate of native whey origin has the potential to modulate postprandial hyperglycemia and hence may contribute in reducing the future risk of developing T2DM.

Production of highly active fungal milk-clotting enzyme by solid-state fermentation

Cheese production is projected to reach 20 million metric tons by 2020, of which 33% is being produced using calf rennet (EC 3.4.23.4). There is shortage of calf rennet, and use of plant and microbial rennets, hydrolyze milk proteins non-specifically resulting in low curd yields. This study reports fungal enzymes obtained from cost effective medium, with minimal down streaming, whose activity is comparable with calf and Mucor rennet. Of the fifteen fungi that were screened, Mucor thermohyalospora (MTCC 1384) and Rhizopus azygosporus (MTCC 10195) exhibited the highest milk-clotting activity (MCA) of 18,383 ± 486 U/ml and 16,373 ± 558 U/ml, respectively. Optimization exhibited a 33% increase in enzyme production (30 g wheat bran containing 6% defatted soy meal at 30 °C, pH 7) for M. thermohyalospora. The enzyme was active from pH 5–10 and temperature 45–55 °C. Rhizopus azygosporus exhibited 31% increase in enzyme production (30 g wheat bran containing 4% defatted soy meal at 30 °C, pH 6) and the enzyme was active from pH 6–9 at 50 °C. Curd yields prepared from fungal enzyme extract decreased (5–9%), when compared with calf rennet and Mucor rennet. This study describes the potential of fungal enzymes, hitherto unreported, as a viable alternative to calf rennet

ACE Inhibitory and Antioxidative Bioactive Peptides Derived from Hydrolyzed Soy Milk

Soy milk is a soybean processed product rich in protein as well as sources of bioactive peptides. Bioactive peptides defined as specific protein fragments that have a positive impact on body functions and conditions and may ultimately influence health. This study was conducted to explore the potential of hydrolyzed soy milk as a source of antioxidative and antihypertensive bioactive peptides through enzymatic hydrolysis. The initial treatment of soy milk protein was acidic precipitation with hydrochloric acid. Furthermore, protein precipitate was hydrolyzed using pepsin proteolytic enzyme with an enzyme: substrate ratio (1:5, 1:10 and 1:20). Protein hydrolysis was carried out for 0–48 hours at 37 °C in an acetate buffer pH 4.5. The soy milk protein hydrolysates were subjected to determination of % DH (Degree of Hydrolysis) and protein profile by SDS-PAGE (sodium dodecyl sulphate polyacrylamide gel electrophoresis). The antihypertensive assay was carried out by in vitro inhibition of the ACE enzyme (Angiotensin Converting Enzyme) and antioxidative activity assay using the DPPH method. The results showed that the optimum conditions for hydrolysis of soy milk were obtained at 40 hours with a % DH value of 53.24% in enzyme ratio 1:20 and the highest antihypertensive activity was obtained from 48 hours hydrolysis with % ACE inhibition value of 79.31%. The highest antioxidative activity of bioactive peptide was obtained at hydrolysis 48 hours with IC50 69.1034 µg / ml. After fractionated and LCMS characterized it was obtained 2 bioactive peptides with molecular weights of 8.954 and 2,696 kDa. These bioactive peptides from hydrolyzed of soymilk might be potential as an antihypertensive agent and reduce oxidative stress.

Analysis of amino acid composition of skim milk and buttermilk for the production of dairy drink when introducing whey protein hydrolysate

The imbalance of macronutrient composition of people's diets determines the relevance of the functional foods development - sources of protein and essential amino acids. The aim of the work was to analyze the amino acid composition and determine the functional significance of the product based on skim milk and buttermilk byadding whey protein hydrolysate. Material and methods. The control options were samples of skimmed milk, buttermilk and their mixtures in a 1:1 ratio. From 1 to 3% of the whey protein hydrolysate with a degree of hydrolysis of about 60% of peptide bonds were added to the same milk base samplesin the experimental options. The amino acid composition was analyzed by high performance liquid chromatography using a cation exchange analyzer and subsequent post-column derivatization with ninhydrin. Results and discussion. The content of essential amino acids per 100 g of whey protein hydrolysate (WPH) is 50.6-168.0% from the adequate level of their daily intake, while there are only 3.0-22.3 and 2.6-19.9% in skimmed milk and buttermilk, respectively. Despite the practically equal protein content in skimmed milk and buttermilk 3.36±0.02 and 3.09±0.01%, respectively, the absolute content of all essential amino acids in buttermilk is lower than in skimmed milk. This can be explained by differences in the qualitative composition of proteins in these types of dairy raw materials. Conclusion. The amino acid composition of the milk base, consisting of buttermilk and skimmed milk mixture in a 1:1 ratio, can be presented as «functional» product in relation to Val, Ile, Leu, Lis, Trp and Tyr, due to the addition of 3% WPH, because the product portion of 200 g provides 15% of adequate daily intake of these essential amino acids. There is the reason to include additional information: low-fat, low-calorie, contains only natural sugars and a source of protein for people whose protein requirement does not exceed 99 grams per day in the labeling of the developed product, in addition to its nutritional and energy value.

Molecular binding mechanism and identification of novel anti-hypertensive and anti-inflammatory bioactive peptides from camel milk protein hydrolysates

Camel milk protein hydrolysates (CMPHs) were investigated for anti-hypertensive and anti-inflammatory activities using in-vitro assays. CMPHs with potent ACE inhibitory and anti-inflammatory activities were subjected to peptide identification and their possible binding mechanism to ACE were depicted via molecular docking. Papain generated CMPHs showed higher degradation of proteins compared to bromelain and alcalase generated CMPHs as depicted in SDS-PAGE profile. Dramatic increase in ACE inhibitory and anti-inflammatory activity was demonstrated in CMPHs with highest activity for papain and alcalase generated hydrolysates, respectively. Based on peptide ranking score, a total of 20, 3 and 43 peptides were potentially regarded as bioactive peptides in hydrolysates A9, B9 and P9, respectively. Binding studies based on pepsite-2 modelling suggested that among 20 potential bioactive peptides in A9 hydrolysate, only 1 (AEWLHDWKL) showed high binding towards three catalytic sites of ACE. While, among 43 bioactive peptides of P9, 14 peptides were found to be potent binder to ACE catalytic sites. Further insight into molecular mechanism of binding revealed that 4 peptides were able to bind to active site of ACE with good docking scores and MM-GBSA binding energies. Overall, the current report remains among very few reports on identification and molecular docking of ACE inhibitory peptides from camel milk.

Highly proteolytic bacteria from semi-ripened Chiapas cheese elicit angiotensin-I converting enzyme inhibition and antioxidant activity

Chiapas cream cheese (CCH) manufacturing process involves a long acid-enzymatic coagulation period of full-fat cow raw milk to achieve an acid and crumbly cheese. These sensorial aspects are related to lactic acid bacteria activity during ripening. Our main objective was to test the hypothesis that CCH contained highly proteolytic strains able to release bioactive compounds upon milk-protein hydrolysis. First, the proteolysis of CCH was evaluated considering the peptide and amino acid profiles of cheese samples collected from Veracruz (AVCH) and Tabasco (HTCH). The angiotensin-converting-enzyme (ACE) inhibitory activity in cheese water-soluble fractions was evaluated. Thereafter, strains from both CCH samples were isolated and selected based on their proteolytic capability, genetic fingerprint differentiation and growth conditions. Finally, a range of activities in vitro were tested in milk fractions fermented with selected strains. CCH showed ACE inhibitory activity: IC50 = 1.75–2.75 mg/mL. Interestingly, AVCH contains 0.78 g/kg of the antihypertensive γ-aminobutyric acid. Three highly proteolytic strains showed ACE and high antioxidant activities upon milk fermentation. In conclusion, CCH contain proteolytic strains able to release bioactive compounds from milk proteins and potentially useful to produce functional ingredients and foods.

Variable Glycemic Responses to Intact and Hydrolyzed Milk Proteins in Overweight and Obese Adults Reveal the Need for Precision Nutrition

Dietary modifications can contribute to improved pancreatic β cell function and enhance glycemic control. The objectives of this study were as follows: 1) to investigate the potential of milk protein hydrolysates to modulate postprandial glucose response; 2) to assess individual responses; and 3) to explore the inter- and intraindividual reproducibility of the response. A 14-d randomized crossover study investigated interstitial glucose levels of participants in response to 12% w/v milk protein drinks (intact caseinate and casein hydrolysate A and B) consumed in random order with a 2-d washout between treatments. Milk protein drinks were consumed immediately prior to study breakfast and evening meals. Twenty participants (11 men, 9 women) aged 50±8 y with a body mass index (in kg/m2) of 30.2±3.1 were recruited. Primary outcome was glucose levels assessed at 15-min intervals with the use of glucose monitors. Repeated-measures ANOVA revealed that for breakfast there was a significant difference across the 3 treatment groups (P=0.037). The ability to reduce postprandial glucose was specific to casein hydrolysate B in comparison with intact caseinate (P=0.039). However, despite this significant difference, further examination revealed that only 3 out of 18 individuals were classified as responders (P<0.05). High intraclass correlation coefficients were obtained for glucose response to study meals (intraclass correlation coefficient: 0.892 for breakfast with intact caseinate). The interindividual CVs were higher than the intraindividual CVs. Mean inter- and intraindividual CVs were 19.4% and 5.7%, respectively, for breakfast with intact caseinate. Ingestion of a specific casein hydrolysate successfully reduced the postprandial glucose response; however, at an individual level only 3 participants were classified as responders, highlighting the need for precision nutrition. Exploration of high interindividual responses to nutrition interventions is needed, in combination with the development of precision nutrition, potentially through an n-of-1 approach. This clinical trial was registered as ISRCTN61079365 (https://www.isrctn.com/).

Milk Bioactive Peptides: Antioxidant, Antimicrobial and Anti-Diabetic Activities

The main milk proteins are caseins and whey proteins, some other minor proteins and peptides, are also found in milk. Proteins are vital ingredients for human because they provide all the essential amino acids needed for body and human health. Milk proteins are very important sources of bioactive peptides. The bioactive peptides are inactive within the sequence of the parent protein and can be released by proteolytic enzymes, during gastrointestinal digestion or during milk processing, for example the adding coagulation enzymes and starter culture. Once bioactive peptides are present in the body, these peptides may act as regulatory compounds with hormone-like activity. Furthermore, Bioactive peptides from milk proteins have many biological activities such as antimicrobial, antihypertensive, antithrombotic, antioxidant, mineral binding, and anti-diabetic. Bioactive peptides have potential health and have pharmaceutical applications. Antimicrobial peptides are recognized as an important component of innate immunity, particularly at mucosal surfaces such as the lungs and small intestine that are constantly exposed to a range of potential pathogens. The ability of protein hydrolysates to inhibit deleterious changes caused by lipid oxidation appears to be related to the nature and composition of the different peptide fractions. Milk protein hydrolysate possesses free-radical-scavenging and anti-inflammatory activities have many beneficial effects on the increase of the glucose-induced insulin secretion and reduction in postprandial glycemia. This article is tried through exposure in some detail to review characteristics of some milk protein peptides and its positive effects on human health.

Effect of Milk Protein Hydrolysate on Some Hematological Parameters of Type II Diabetic Rats

Milk protein-derived bioactive peptides could be used in human nutrition in both newborns and adults, since they are claimed to be health-improving components that can be used to reduce the risk of disease or to enhance certain physiological functions. This study investigated the effect of oral intake of milk protein concentrate (MPC) and milk protein concentrate hydrolysate (MPCH) on body weight, organ relative weight, and hematological parameters in alloxan-induced diabetes rats. The animals were divided into six major groups after stabilization of diabetes for one week. Diabetic and non-diabetic rats groups were received intra-gastric dose of MPC or MPCH (150mg/kg B.W) for six weeks. Feed treatment by MPC and MPCH significantly (<i>P</i><0.05) increased the final body weight of normal rats and improved the body weight of diabetic rats and significantly protective (<i>P</i><0.05) the liver, kidney, heart and lung of normal rats and reduced the fallers of these organs in diabetic rats. The diabetes groups treated by MPCH showed improvement in RBC, Hb, Ht and PLT values. Meanwhile WBC not affected. The oral intake of MPCH protected the normal range of Lymphocytes and S. Nutrophils in healthy and diabetic rats. The oral intake of MPCH caused significant (<i>P</i><0.05) decrease of Monocytes in diabetic and non-diabetic rats, while the oral intake of MPC had no affected on Monocytes of diabetic and non-diabetic rats. MPC and MPCH reduced the harmful effect of T2D on body weight, organs weights of rats, and improvement most hematological parameters of normal and diabetes rats.

Identification of antihypertensive peptides in nutraceuticals by capillary electrophoresis-mass spectrometry

We present capillary electrophoresis-mass spectrometry (CE-MS) in combination with advanced chemometric tools for the analysis of bioactive compounds in food, in particular for the identification of antihypertensive peptides in a nutraceutical derived from a bovine milk protein hydrolysate. Different extracts of the nutraceutical were analyzed by CE-MS, and the electropherograms were processed using a novel data analysis workflow that included regions of interest (ROIs) compression and multivariate curve resolution alternating least squares (MCR-ALS). MCR-ALS permitted the description of the nutraceutical extract as ten characteristic components with their electrophoretic profiles and mass spectra. Twenty-two compounds were tentatively identified as antihypertensive bovine casein fragments through a mass search in a database of bioactive peptides. The identity of 17 antihypertensive peptides was reliably confirmed by capillary electrophoresis-tandem mass spectrometry. The proposed analytical approach demonstrated the potential to obtain a characteristic and activity-related fingerprint for quality control and authentication of the antihypertensive nutraceutical.

Isolation and characterization of Shiga toxin-producing Escherichia coli strains from raw milk cheese with a trial for controlling them by some milk protein hydrolysates.

Isolation and characterization of Shiga toxin-producing Escherichia coli strains from raw milk cheese with a trial for controlling them by some milk protein hydrolysates.

Artificial neuronal networks (ANN) to model the hydrolysis of goat milk protein by subtilisin and trypsin.

The enzymatic hydrolysis of milk proteins yield final products with improved properties and reduced allergenicity. The degree of hydrolysis (DH) influences both technological (e.g., solubility, water binding capacity) and biological (e.g., angiotensin-converting enzyme (ACE) inhibition, antioxidation) properties of the resulting hydrolysate. Phenomenological models are unable to reproduce the complexity of enzymatic reactions in dairy systems. However, empirical approaches offer high predictability and can be easily transposed to different substrates and enzymes. In this work, the DH of goat milk protein by subtilisin and trypsin was modelled by feedforward artificial neural networks (ANN). To this end, we produced a set of protein hydrolysates, employing various reaction temperatures and enzyme/substrate ratios, based on an experimental design. The time evolution of the DH was monitored and processed to generate the ANN models. Extensive hydrolysis is desirable because a high DH enhances some bioactivities in the final hydrolysate, such as antioxidant or antihypertensive. The optimization of both ANN models led to a maximal DH of 23·47% at 56·4 °C and enzyme-substrate ratio of 5% for subtilisin, while hydrolysis with trypsin reached a maximum of 21·3% at 35 °C and an enzyme-substrate ratio of 4%.

Characterization and identification of novel antidiabetic and anti-obesity peptides from camel milk protein hydrolysates

In-vitro inhibitory properties of peptides released from camel milk proteins against dipeptidyl peptidase-IV (DPP-IV), porcine pancreatic α-amylase (PPA), and porcine pancreatic lipase (PPL) were studied. Results revealed that upon hydrolysis by different enzymes, camel milk proteins displayed dramatic increase in inhibition of DPP-IV and PPL, but slight improvement in PPA inhibition was noticed. Peptide sequencing revealed a total of 20 and 3 peptides for A9 and B9 hydrolysates respectively, obtained the score of 0.8 or more on peptide ranker and were categorized as potential DPP-IV inhibitory peptides. KDLWDDFKGL in A9 and MPSKPPLL in B9 were identified as most potent PPA inhibitory peptide. For PPL inhibition only 7 and 2 peptides qualified as PPL inhibitory peptides from hydrolysates A9 and B9, respectively. The present study report for the first time PPA and PPL inhibitory and only second for DPP-IV inhibitory potential of protein hydrolysates from camel milk.

Gut peptidases from a specialist herbivore of latex plants are capable of milk protein hydrolysis: Inputs for hypoallergenic milk formulas

Transitory allergies to cow milk proteins in infants or adults have become a public health problem. Although extensively or partially hydrolyzed cow milk protein formulas are available, these products are costly. Therefore, studies into innovative enzymes to digest cow milk proteins are needed. Danaus plexippus gut peptidases were purified and examined with regard to cow milk protein hydrolysis. The peptidases hydrolyzed caseins and whey proteins. However, after heat treatment, there was a significant improvement in β-lactoglobulin hydrolysis. The hydrolyzed cow milk proteins were not recognized by anti-casein antibodies and only reacted slightly with antibodies against whey proteins. This performance was better than that of partially hydrolyzed formulas and similar to that of an extensively hydrolyzed formula. These results suggest that D. plexippus gut peptidases are suitable and innovative enzymes to produce hypoallergenic cow milk protein formulas.

Antioxidant capacity of egg, milk and soy protein hydrolysates and biopeptides produced by Bromelia pinguin and Bromelia karatas-derived proteases

Given the lack of knowledge in the generation and biological activity of biopeptides (BP) produced by Bromelia pinguin and Bromelia karatas-derived proteases, the objective of this work was to evaluate the antioxidant capacity (AOX) of protein hydrolysates and peptide fractions from hydrolysis of egg, milk and soy proteins with enzymes extracted from Bromelia pinguin and Bromelia karatas fruits. The degree of hydrolysis (DH) of the mentioned proteins was measured. The hydrolysates were sequentially fractionated by ultrafiltration using different membrane pore sizes (≤30, ≤10, ≤5 and ≤1 kDa) to obtain biopeptide fractions (BPF). The antioxidant capacity (AOX) of protein hydrolysates and BPF were evaluated. AOX was performed using 2,2´-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS•+), ferric reducing antioxidant power (FRAP) and oxygen radical absorbance capacity (ORAC) assays. after the enzymatic action of B. pinguin and B. karatas proteases, the highest DH values for egg, milk and soy protein hydrolysates ranged from 50-61%, 55-68% and 73-81%, respectively. The AOX was higher for soy hydrolysates than for egg and milk hydrolysates. Additionally, the highest AOX from ABTS and FRAP assays was observed for BPF ≤30 kDa, PF ≤5 kDa and ≤1 kDa, respectively. These results highlight the potential of B. pinguin and B. karatas proteases to hydrolyze food proteins and release bioactive peptides. Moreover, it has been shown that the hydrolysis products are a mixture of BP of different molecular weights, which could present AOX through different mechanisms.

Bioactivities and analysis of peptides from Sumbawa horse milk generated by Bacillus thuringiensis protease

&lt;p class="abstrak2"&gt;Sumbawa horse milk is claimed to cure some diseases such as asthma, hypertension, diabetes and gastrointestinal disorder but its potential bioactive peptide has not been explored. The aims of this study are to evaluate bioactivities peptides from Sumbawa horse milk protein hydrolysate and to analyzethe physio-chemical properties of selected peptides. The milk protein was hydrolyzed by Bacillus thuringiensis protease, the peptide produced were sequential fractionated and then analyzed for antibacterial and antioxidant activities. The peptide fraction &amp;lt;3 kDa was then sequenced using LCMS-MS and the physio-chemical properties of the peptides were analyzed. The result showed that peptides fraction &amp;lt;3 kDa from the 30 min hydrolysis was the most active as antibacterial and more active to Gram negative bacteria. For antioxidant, scavenging activity of the fraction per µg protein/mL were 83% to ABTS and 31% to DPPH radicals. The values were similar with vitamin C 12.5 µg/mL for ABTS and 14.5 µg/mL for DPPH. Peptide &lt;tt&gt;HPYFYAPELLYYANK with molecular weight prediction &lt;/tt&gt;1887.92 Da and isoelectric point 7.47 has high therapeutic index prediction (64.75). The result showed that peptides from Sumbawa horse milk hydrolyzed by Bacillus thuringiensis protease was active as antibacterial and antioxidant. Peptide &lt;tt&gt;HPYFYAPELLYYANK from fraction &amp;lt;3 kDa was potential as antibacterial.&lt;/tt&gt;&lt;/p&gt;&lt;p&gt;&lt;strong&gt;Key Words:&lt;/strong&gt; &lt;em&gt;Bacillus thuringiensis&lt;/em&gt;, Bioactive Peptide, Horse Milk&lt;/p&gt;