Bitter Peptides Research Articles

BITTER TASTE PERCEPTION MECHANISM, REMOVAL OF BITTERNESS CAUSED BY PEPTIDE IN DAIRY PRODUCTS AND CURRENT APPROACHES

Protein bakımından zengin gıdaların hidrolizasyona uğraması sonucu acı tattan sorumlu peptitler oluşabilmektedir. Acılık algısı, acı tat reseptörlerinin uyarılması ve sinir sistemi ile beyne gönderilen sinyalin yorumlanması ile oluşmaktadır. Süt ürünleri çoğu zaman acı peptitlerle karakterize edilmektedir. Yapılan çalışmalarda peynirde tanımlanan acı peptitlerin genellikle αs1- ve β-kazein kaynaklı olduğu belirtilmiştir. Protein hidrolizatlarının acı tat yoğunluğu peptitin hidrofobisitesine, hidrofobik amino asit sayısına ve zincir uzunluğuna bağlı olarak değişmektedir. Peynirlerde olgunlaşmayı hızlandırırken, acı tat kusurlarının oluşumunu engellemek için son zamanlarda aminopeptidaz, karboksipeptidaz ve starter kültürle birlikte yardımcı kültürlerin yer aldığı metotlar kullanılmaktadır. Bu derlemede; acı peptitlerin oluşumu ve algılanma mekanizması açıklanmış ve acılık giderme yöntemleri ile ilgili güncel çalışmalar sunulmuştur.

ACE inhibitory peptides derived from de-fatted lemon basil seeds: optimization, purification, identification, structure-activity relationship and molecular docking analysis.

The oil processing industry generates significant quantities of lemon basil seed residue which is not currently used to any significant extent. However, this by-product has important potential as a source of bioactive peptides which may play a role as ingredients in functional foods. This study therefore sought to optimize the preparation techniques used to obtain the necessary protein hydrolysate from de-fatted lemon basil seeds (DLBS), and subsequently to examine the ACE inhibitory capabilities of the resulting hydrolysate. Response Surface Methodology (RSM) was used for the hydrolysis of DLBS by Alcalase®, with observation of the resulting ACE inhibitory activity and degree of hydrolysis (DH). The optimum conditions were 55 °C and 103 minutes with a ratio of enzyme to substrate of 7.0% w/v. The hydrolysate was fractionated by ultrafiltration and purified through RP-HPLC. The results reveal that the F2 sub-fraction demonstrated the highest ACE inhibitory activity. The amino acid sequence of this peak was identified by mass spectrometry as LGRNLPPI and GPAGPAGL with a molecular weight of 879.06 and 639.347 Dalton, respectively. These peptides were classified as non-toxic and bitter peptides. For the synthesized version of these peptides, the ACE inhibitory activity values, measured by IC50, were 0.124 ± 0.02 mM and 0.013 ± 0.001 mM, respectively. Analysis of the Lineweaver-Burk plot confirmed that these peptides served as non-competitive ones. The study of molecular docking showed that the ACE inhibitory behavior of both purified peptides was mainly due to the interactions of the hydrogen bonds between the peptides and ACE. It is therefore suggested that DLBS may be a useful raw material allowing the production of antihypertensive peptides which can offer therapeutic and commercial benefits as an ingredient in functional foods.

Enzymatic Production, Bioactivity, and Bitterness of Chickpea (Cicer arietinum) Peptides.

Chickpeas are inexpensive, protein rich (approximately 20% dry mass) pulses available worldwide whose consumption has been correlated with positive health outcomes. Dietary peptides are important molecules derived from dietary proteins, but a comprehensive analysis of the peptides that can be produced from chickpea proteins is missing in the literature. This review provides information from the past 20 years on the enzymatic production of peptides from chickpea proteins, the reported bioactivities of chickpea protein hydrolysates and peptides, and the potential bitterness of chickpea peptides in food products. Chickpea peptides have been enzymatically produced with pepsin, trypsin, chymotrypsin, alcalase, flavorzyme, and papain either alone or in combination, but the sequences of many of the peptides in chickpea protein hydrolysates remain unknown. In addition, a theoretical hydrolysis of chickpea legumin by stem bromelain and ficin was performed by the authors to highlight the potential use of these enzymes to produce bioactive chickpea peptides. Antioxidant activity, hypocholesterolemic, and angiotensin 1-converting enzyme inhibition are the most studied bioactivities of chickpea protein hydrolysates and peptides, but anticarcinogenic, antimicrobial, and anti-inflammatory effects have also been reported for chickpea protein hydrolysates and peptides. Chickpea bioactive peptides are not currently commercialized, but their bitterness could be a major impediment to their incorporation in food products. Use of flavorzyme in the production of chickpea protein hydrolysates has been proposed to decrease their bitterness. Future research should focus on the optimization of chickpea bioactive peptide enzymatic production, studying the bioactivity of chickpea peptides in humans, and systematically analyzing chickpea peptide bitterness.

Bitter peptides increase engulf of phagocytes in vitro and inhibit oxidation of myofibrillar protein in peeled shrimp (Litopenaeus vannamei) during chilled storage

We have shown that bitter peptides derived from the hydrolysates of pepsin digested fish protein can increase lymphocytes and lysozyme activity and improve fish survival in aquaculture. In this study, we investigated how the immune function of bitter peptides increases the phagocytes’ engulf of latex beads, and analyzed the antioxidation of myofibrillar protein in peeled shrimp during chilled storage with /without bitter peptides by evaluating the activity of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals, and the effects of the total carbonyl and sulfhydryl. Also, the bitter peptides’ effect on the structural stability of muscles protein throughout oxidation was examined in vitro by both scanning electron microscope (SEM) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The engulf assay demonstrated phagocytosis increased from the control group to the highest bitter peptide concentration of 1.2 mg/ml; the bitter peptides had a significant DPPH radical-scavenging activity, reducing the power of ferric ions and metal chelating ability. Chemical analysis showed that myofibrillar protein in chilled storage was highly susceptible to oxidation, and the results indicate that treating peeled shrimp with bitter peptides before chilled storage significantly decreases the formation of carbonyl derivatives and reduces the loss of thiol groups when compared with the water treated sample. In addition, the results of both SEM and SDS-PAGE confirm that there was less distortion of tissue structure and less degradation of the protein in the samples treated with bitter peptides. Bitter peptides are both beneficial for fish disease control and preservation of marine food.

Correction to Identification of Bitter Peptides in Aged Cheddar Cheese.

Correction to Identification of Bitter Peptides in Aged Cheddar Cheese.

Proteomics-based analysis of functional proteins after fermentation of compound wheat embryo Chinese medicine

Abstract Compound probiotics were used to ferment compound wheat embryo Chinese medicine (CWECM) at 37 °C for a given period, and the fermentation end point was determined based on pH value. The full spectrum of the proteins within the fermentation supernatant was determined by ion mass spectrometry, and then the fermentation was analyzed by proteomics. The difference of functional protein of CWECM before and after fermentation was analyzed. Additionally, the changes in flavor peptides were examined. Our results indicated that when the fermentation was performed for 96 h, the pH value became stable over time, where it shifted from an initial 3.98 ± 0.04 to a stable 2.78 ± 0.04. Proteomic-based analysis of the fermentation supernatant indicated that the fermentation of the probiotics increased. The amount of functional proteome and protein within the supernatant increased from 60 proteomes and 136 proteins before fermentation to 70 proteomes and 149 proteins post fermentation. These proteins were primarily members of the Ubiquitin and Histone families. These families contain more specific peptides after fermentation, and the coverage was reduced. Analysis of the unique P0C512 protein after fermentation indicated that this protein is an unstable, 3D-effect columnar protein. We also found that probiotic fermentation can reduce the proportion of bitter peptides within fermentation broth from 47.49% to 36.98%, and the proportion of sweet peptides increased after fermentation from 35.29% to 51.01%. Based on our findings, we conclude that fermentation of CWECM by compound probiotics can reduce the pH of the CWECM solution, increase the number of functional proteomes and proteins, improve the taste of CWECM solutions, and improve the therapeutic efficacy of CWECM.

Quantitative Structure-Activity Relationship Study of Bitter Di-, Tri- and Tetrapeptides Using Integrated Descriptors.

New quantitative structure–activity relationship (QSAR) models for bitter peptides were built with integrated amino acid descriptors. Datasets contained 48 dipeptides, 52 tripeptides and 23 tetrapeptides with their reported bitter taste thresholds. Independent variables consisted of 14 amino acid descriptor sets. A bootstrapping soft shrinkage approach was utilized for variable selection. The importance of a variable was evaluated by both variable selecting frequency and standardized regression coefficient. Results indicated model qualities for di-, tri- and tetrapeptides with R2 and Q2 at 0.950 ± 0.002, 0.941 ± 0.001; 0.770 ± 0.006, 0.742 ± 0.004; and 0.972 ± 0.002, 0.956 ± 0.002, respectively. The hydrophobic C-terminal amino acid was the key determinant for bitterness in dipeptides, followed by the contribution of bulky hydrophobic N-terminal amino acids. For tripeptides, hydrophobicity of C-terminal amino acids and the electronic properties of the amino acids at the second position were important. For tetrapeptides, bulky hydrophobic amino acids at N-terminus, hydrophobicity and partial specific volume of amino acids at the second position, and the electronic properties of amino acids of the remaining two positions were critical. In summary, this study not only constructs reliable models for predicting the bitterness in different groups of peptides, but also facilitates better understanding of their structure-bitterness relationships and provides insights for their future studies.

Enzymatic hydrolysis of lupin protein isolates-Changes in the molecular weight distribution, technofunctional characteristics, and sensory attributes.

Enzymatic hydrolysis of lupin protein isolates (LPI; Lupinus angustifolius L.) was performed with nine different protease preparations to investigate their effect on technofunctionality, sensory properties, and the integrity of the proteins to estimate the reduction of the immunoreactivity. Alcalase 2.4 L, papain, and pepsin were most effective in the degradation of the α‐ and β‐conglutin examined by SDS–PAGE analysis, although the degree of hydrolysis only slightly increased. The technofunctional properties of LPI—solubility, emulsifying, and foaming activity—were improved by most of the proteolytic enzymes with the most impressive increase from 980% foam activity for LPI up to 3,614% foam activity for pepsin hydrolysate. The formation of bitterness, most likely linked to generation of bitter peptides, was pronounced in the Alcalase hydrolysate, while the other hydrolysates did not show an extensive increase in bitterness compared to the LPI. Other sensory attributes of the hydrolysates—with the exception of Alcalase treatment—were also very similar to the LPI. The results of this study show the potential of enzymatic degradation of LPI to modify the IgE‐reacting polypeptides and to improve the technofunctionality of the isolates and therefore their use as food ingredients.

Bitter‐tasting hydrophobic peptides prepared from soy sauce using aqueous ethanol solutions influence taste sensation

SummaryIn this study, bitter‐tasting hydrophobic peptides were prepared and purified from soy sauce using aqueous ethanol solutions and ion exchange resin, and analysed using RP‐HPLC and UPLC‐Q‐TOF‐MS/MS. Most of bitter fraction from soy sauce was obtained by treatments involving 80% ethanol and subsequent chromatographic separation. This fraction contained sixteen small peptides (molecular weight < 1000 Da; rich in hydrophobic amino acids with Lys‐Pro (KP), Glu‐Phe (EF), Glu‐Val (EV), Asp‐Leu (DL), Leu‐Trp (LW) and Asp‐Pro (DP)) capable of enhancing significantly (P < 0.05) umaminess and/or imparting kokumi effect in taste reconstitution experiments. Thus, the bitter peptides affected bitterness as well as other taste sensations like umaminess, kokuminess and saltiness. This was the first study to report the impacts of the bitter‐tasting hydrophobic peptides on soy sauce and solutions of salt, monosodium l‐glutamate.

The variation of two extracellular enzymes and soybean meal bitterness during solid-state fermentation of Bacillus subtilis

The debittering effect of extracellular enzymes from Bacillus subtilis ACCC 01746 was studied using soybean meal as a substrate for solid-state fermentation (SSF). Results showed that B. subtilis produces proteases and carboxypeptidase in the early stage of SSF (0–8 h). Proteases are dominant and can hydrolyze the soybean protein into long-chain peptides with mild bitterness. Carboxypeptidase production is dominant at 8–16 h SSF, at which point soybean protein is further hydrolyzed and bitterness is enhanced. The strain then produces additional carboxypeptidase after 16 h, and bitterness is reduced. We compared the amino acid composition of the hydrolysates from soybean protein isolates to that of the fermented liquid of SSF. In the hydrolysates from soybean protein isolates that exhibit strong bitterness, 62.81% of amino acids are hydrophobic and occur in the form of peptides. In the fermented liquid from soybean meal, 16.22% of amino acids are hydrophobic and are mainly present in the form of free amino acids. The bitterness of fermented soybean hydrolysate is reduced from 5 to 0 when fermented for 24 h, suggesting that B. subtilis can effectively reduce bitterness, possibly due to the carboxypeptidase. Enzyme analysis shows that B. subtilis excretes carboxypeptidase during growth. The amino acids phenylalanine, alanine, tyrosine, and leucine at the C-terminal of the soybean bitter peptides in hydrolysates are cleaved in the presence of carboxypeptidase, resulting in complete debitterness.

A feasibility study on the pilot scale manufacture of fresh cheese from skim milk retentates without acid whey production: Effect of calcium content on bitterness and texture

The sensory and structural properties of fresh cheeses (8.0%, w/w, protein) made from fermented skim milk retentates [concentrated-fermented (CoF) fresh cheese] with different calcium-protein ratios (Ca:Prot; 15.9–36.6 mg g−1) by means of pilot plant scale microfiltration (MF) and MF in the diafiltration (DF) mode without acid whey (AW) production were characterised. Calcium reduction reduced the bitterness level significantly (by 53.4%) and the bitter peptide content (by ≈33%) of the CoF fresh cheese after ≤ 1 week of storage; however, storage for ≥ 2 weeks resulted in an increase of both bitterness and bitter peptide content, and quantitatively changed the peptide pattern, possibly due to lack of thermisation. Fresh cheese with a lower bitterness level can be successfully manufactured, while avoiding AW production, if the Ca:Prot of the milk retentate is decreased to ≤15.9 mg g−1 (calcium content = 1240 mg kg−1 at 7.81%, w/w, protein) prior to fermentation.

Selective Elimination of Bitter Peptides by Adsorption to Heat-treated Porous Silica Gel

Selective Elimination of Bitter Peptides by Adsorption to Heat-treated Porous Silica Gel

The umami intensity enhancement of peanut protein isolate hydrolysate and its derived factions and peptides by Maillard reaction and the analysis of peptide (EP) Maillard products

This study aimed to examine the feasibility of improving umami taste of peanut protein isolate hydrolysate (PPIH) and its fractions (obtained via ethanol precipitation and gel filtration chromatography) and peptide constituents, through their reactions with glucose at 98 °C for 70 min (Maillard reaction). To get insights into the characteristics of the peptide contributors with high umami intensity and/or umami-enhancing activity, Maillard reaction-guided and sensory-guided fractionation and characterization using ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) and eXpose algorithm calculation method were subsequently involved. Three umami and umami-enhancing peptides, three umami-enhancing peptides with other tastes, three bitter peptides and one astringent peptide were found. For the first time, peptide TP was reported to suppress umami intensity of monosodium glutamate (MSG) aqueous solution. Combined treatments of enzymatic hydrolysis and Maillard reaction on PPI facilitated umami enhancement. Maillard products of identified peptides had higher umami intensities and/or greater umami-enhancing abilities. Pyroglutamylproline (pyroEP) and glucosylated EP were discovered as the major compounds of EP Maillard product.

Valorisation of protein hydrolysates from animal by‐products: perspectives on bitter taste and debittering methods: a review

SummaryConversion of animal by‐products to high value‐added food ingredients is one of the top trends in the slaughter industry. Enzymatic hydrolysis of animal by‐products can generate protein hydrolysates, which provides an opportunity for effective utilisation. However, bitterness of protein hydrolysate is a major undesirable aspect for various applications. In this review, the current knowledge on protein hydrolysates from animal by‐products is briefly reviewed. The structural features of bitter peptides and bitter taste receptors are summarised. Moreover, the potential approaches for debittering protein hydrolysates are highlighted, including exopeptidase treatment, Maillard reaction, plastein reaction and encapsulation. In addition, the current debittering strategies and challenges are also discussed. This article presents some opportunities to utilise protein hydrolysates from animal by‐products and their debittering methods.

Sensoproteomics: A New Approach for the Identification of Taste-Active Peptides in Fermented Foods.

Aiming at the identification of the key bitter peptides in fermented foods, a new approach, coined "sensoproteomics", was developed and applied to fresh cheese samples differing in bitter taste intensity. By means of MPLC fractionation of the water-soluble cheese extracts in combination with taste dilution analysis, complex fractions with intense bitter taste were located and then screened by UPLC-MS/MS for the entire repertoire of ∼1600 candidate peptides, extracted from a literature meta-analysis on dairy products, by using a total of 120 selected reaction monitoring methods computed in silico. A total of 340 out of the 1600 peptides were found in the cheese samples, among which 17 peptides were identified as candidate bitter peptides by considering only peptides that were located in the bitter-tasting MPLC fractions (signal-to-noise ratio: ≥10) with a fold-change of ≥3 when comparing the less bitter to the more bitter cheese sample and that were validated by comparison with the synthetic reference peptides. While EIVPNS[phos]VEQK (αs1-CN70-78) and INTIASGEPT (κ-CN122-131) did not exhibit any bitter taste up to 2000 μmol/L, 15 of the 17 target peptides showed bitter taste thresholds ranging from 30 (ARHPHPHLSFM, κ-CN96-106) to 690 μmol/L (IQKEDVPS, αs1-CN81-88). Finally, quantitative peptide analysis followed by calculation of dose-overthreshold factors revealed a primary contribution of MAPKHKEMPFPKYPVEPF (β-CN102-119) and ARHPHPHLSFM (κ-CN96-106) to the perceived bitter taste of the fresh cheese samples. Finally, the evolution of the bitter peptides throughout two different fresh cheese manufacturing processes was quantitatively recorded.

Bitter peptides from enzymatically hydrolyzed protein increase the number of leucocytes and lysozyme activity of large yellow croaker (Larimichthys crocea)

The large yellow croakers (Larimichthys crocea) are mainly present in the Chinese coast and near seas with high economic importance, but vulnerable to many diseases, especially in the breeding and aquaculture. The purpose of this research was to boost the innate immune system of the large yellow croaker by administering bitter peptides into their peritoneal cavity. Total 120 Juvenile of large yellow croakers in very even weight of 60 g were divided into 4 different groups in 200/300 L of water tank, respectively. Fish growth were observed for 3 months before and after different treatments. The bitter peptides from pepsin hydrolysis were applied because they possess the highest bitter sensory scores. The blood of fish from the different groups was collected and tested for different immune parameters to evaluate the effectiveness of bitter peptides as immune stimulants after administration for 8 weeks. The average ratio of leukocytes/total blood cells (%) for control was found at 14.6%, for the low dose of bitter peptides 0.6 mg/fish was at 29.3%, for middle dose of 1.2 mg/fish was at 35%, and high dose of 2.4 mg/fish was at 30%. The lysozyme assay showed that the OD (optical density) units of relative progress lysis activity at 60 min were 0.17, 0.101, 0.307 and 0.198, respectively. Similarly in the same order as in phagocyte assay, most importantly the middle dose (1.2mg/fish) gave the highest survival rate throughout the assay. The results showed that bitter peptides can be used as immune boosters for the yellow croakers and the optimum dose was 1.2 mg/fish due to both leukocytes and lysozyme activity in the treated samples increased significantly compared with the control group. According to the results obtained, we suggest that the incorporation of middle dose of bitter peptides into fish feeds may reduce the fish diseases in aquaculture, at least for large yellow croakers.

Structural characteristics of food protein-originating di- and tripeptides using principal component analysis

This paper presents the use of principal component analysis (PCA) to study the effect of specific physicochemical attributes on bitterness of di- and tripeptides originating from food proteins. Peptide sequences were derived from the BIOPEP-UWM database of sensory peptides and amino acids. Descriptors defining the physicochemical properties of amino acids forming the analyzed peptides were study variables. They were derived from ProtScale program and Biological Magnetic Resonance Data Bank. Finally, PCA was carried out for 51 dipeptides/12 variables, and 51 tripeptides/18 variables using STATISTICA®13.1 software. PCA allowed reducing the input datasets to 4 principal components (PCs) for dipeptides and to 5 PCs for tripeptides. The impact of the following properties on the bitterness of peptides was observed: relatively high molecular weight, bulkiness, increasing number of carbon and hydrogen atoms of amino acids forming the sequences. These properties characterized the N- (negative correlations) and C-terminal residue (positive correlations) of both di- and tripeptides. An additional property affecting peptide bitterness was amino acids’ hydrophobicity. Our results were consistent with scientific reports on structure–bitterness of peptides. Thus, we find PCA a chemometric approach helpful in broadening the knowledge about the function of peptides resulting from their chemical nature.

Understanding the nature of bitter-taste di- and tripeptides derived from food proteins based on chemometric analysis.

Multiple linear regression (MLR) models were constructed to explain the bitter taste of di- and tripeptides based on their chemical nature (structure). Sequences (51 di- and 51 tripeptides) were derived from the BIOPEP-UWM database of sensory peptides and amino acids. The measure of their bitterness was Rcaf. , that is, bitterness relative to that of 1 mM caffeine solution (Rcaf. = 1.0). The variables were the indices describing properties of a single residue forming a peptide structure taken from ProtScale and Biological Magnetic Resonance Data Bank. MLR was made for two separate data sets by use of Statistica 13.1. We found that the presence of branched side residues or ring in a di- or tripeptide sequence (as in L, I, V, Y, F) affected its bitterness. Another variable affecting the bitter taste of di- and tripeptides was the hydrophobicity of amino acids. Using the commonly available statistical tools as well as chemical information reflecting the nature of peptides may be helpful in understanding the structure-taste relationship in food peptides. PRACTICAL APPLICATIONS: Our approach takes account of bioinformatic and cheminformatic techniques of data mining to analyze structure-bitterness of di- and tripeptides derived from food protein sources. Data on bitter peptides available in databases of biological and chemical information can be useful in creating models which help understanding the relationship between the role of structural properties of a molecule (e.g., peptide) and its function (e.g., taste). The bitterness of a peptide resulting from the presence of specific residues in its sequence, which represent different physicochemical properties may contribute to extending the knowledge about their taste-forming role in food systems. Such knowledge may be useful in designing food products with improved properties like taste which can be either enhanced or masked (considered as unwanted when thinking about the sensory value of foods). Our research strategy is universal and can also be applied to study structure-function relationships of peptides with other activities.

In silico approaches towards the exploration of rice bran proteins-derived angiotensin-I-converting enzyme inhibitory peptides

ABSTRACTRice bran is an important underutilized by-product of rice-processing industries, and a huge amount of bran is released during rice processing. It can be used as a prime source of bioactive peptides towards the development of functional food ingredients. The aim of the study was to evaluate the potential of rice bran proteins such as glutelin, globulin, and prolamin as a precursor of bioactive peptides using various in silico approaches (BLAST, BIOPEP, PeptideRanker, PepDraw, Pepcalc, and ToxinPred). Rice bran proteins majorly release angiotensin-I-converting enzyme (ACE) and dipeptidyl peptidase IV (DPP-IV) inhibitory peptides. Papain protease can cleave the rice bran proteins effectively compared with other proteases to release ACE-inhibitory peptides. Physicochemical studies have shown that ACE-inhibitory peptides have low molecular weight profiles. These peptides have been classified as bitter peptides with a non-toxic profile. The result of this study demonstrates the usefulness of in silico approaches and provides a theoretical basis for the development of rice bran proteins as a source of bioactive peptides. The predicted prominent ACE-inhibitory peptides could be used for the development of functional food products or nutraceuticals.

Effect of Storage on Lactase-Treated β-Casein and β-Lactoglobulin with Respect to Bitter Peptide Formation and Subsequent in Vitro Digestibility.

Using active lactose to hydrolyze lactose during storage is a common process to produce lactose-hydrolyzed (LH) milk. Proteolysis induced by residual proteases in commercial lactase was studied in a system using purified β-casein or β-lactoglobulin during a 60-day storage period at 22 or 38 °C. The proteolysis of β-casein by residual proteases occurred more extensively than that of β-lactoglobulin. Peptidomic analysis by LC-ESI-MS/MS revealed that Ile, Leu, Tyr, and Phe residues near the C-terminus of β-casein were the main sites of cleavage by the residual proteases, generating assumed bitter peptides. In the subsequent in vitro digestion study, proteolysis during storage was shown to greatly affect the subsequent digestibility of β-casein, leading to an elevated degree of hydrolysis and the formation of new digested peptides. This study highlights the potential influence of residual proteases in commercial lactase on the storage quality and digestibility of LH milk containing active lactase.