Milk Protease Research Articles

Establishment of a multiplex qPCR method for sensitive detection of proteolytic psychrotrophic bacteria in raw milk

Initial microbiological species and quantity of raw milk significantly affect dairy product quality. In particular, some psychrotrophic bacteria in raw milk can produce heat-stable hydrolases that reduce the stability of dairy products during their shelf life. The purpose of this study was to establish a multiplex real-time fluorescence quantitative polymerase chain reaction (qPCR) rapid detection method for Pseudomonas fluorescens and Lactococcus lactis subsp. lactis, which generally produce protease in raw milk. The primers and probes with good specificity, universality and high sensitivity to the target bacteria were screened by ordinary PCR and qPCR experiments. The quantitative linear relationship and sensitivity of the method for detecting pure DNA and artificially contaminated simulated milk samples were verified. The results showed that the limit of detection (LOD) of this method for pure DNA could reach 3.16×10-5 ng/μL. The detection of artificially contaminated samples showed that the LOD for P. fluorescens and L. lactis subsp. lactis could reach 250 and 22 CFU/mL, respectively. The multiplex qPCR method established in this study has good specificity and high sensitivity, which can be used to rapidly detect psychrotrophic bacteria in raw milk. It provides an effective reference strategy for the dairy industry to ensure the microbiological quality of raw milk from the source.

A comprehensive study of the instability of direct-steam-infusion UHT milk: Insights from metabolomics and proteomics

Direct-steam-infusion UHT (dUHT) milk retains more nutrients compared to traditional UHT milk but challenged by age gelation, impacting shelf life. To understand this instability, raw milk, dUHT milk, and age gelation milk were comprehensively studied using metabolomics and proteomics. 2496 metabolites were detected, including amino acids and derivatives, nucleotides and nucleotide derivatives, lipids and fatty acids, and organic acids and derivatives. The most significant change after age gelation was in amino acids, particularly L-lysine, which emerged as the most important indicator related to protein hydrolysis. Proteomics revealed increased calcium-binding proteins, protein fragments, and various proteases in age gelation milk. Ribonuclease A family, cathepsin D, carboxypeptidases, serine/threonine-protein kinase, PLK serine protease 23 contributed to metabolite changes and protein hydrolysis during age gelation. These findings suggested that the hydrolysis of various enzymes altered the overall composition of milk, leading to age gelation, which provided new insights into the instability of dUHT milk.

Human proinsulin production in the milk of transgenic cattle.

The worldwide growing demand for human insulin for treating diabetes could be supplied by transgenic animals producing insulin in their milk. Pseudo-lentivirus containing the bovine β-casein promoter and human insulin sequences was used to produce modified adult fibroblasts, and the cells were used for nuclear transfer. Transgenic embryos were transferred to recipient cows, and one pregnancy was produced. Recombinant protein in milk was evaluated using western blotting and mass spectrometry. One transgenic cow was generated, and in milk analysis, two bands were observed in western blotting with a molecular mass corresponding to the proinsulin and insulin. The mass spectrometry analysis showed the presence of human insulin more than proinsulin in the milk, and it identified proteases in the transgenic milk that could convert proinsulin into insulin and insulin-degrading enzyme that could degrade the recombinant protein. The methodologies used for generating the transgenic cow allowed the detection of the production of recombinant protein in the milk at low relative expression compared to milk proteins, using mass spectrometry, which was efficient for detecting recombinant protein with low expression in milk. Milk proteases could act on protein processing converting recombinant protein to functional protein. On the other hand, some milk proteases could act in degrading the recombinant protein.

Development of a real-time loop-mediated isothermal amplification method for monitoring Pseudomonas lurida in raw milk throughout the year of pasture.

The psychrophilic bacterium Pseudomonas lurida (P. lurida) and its thermostable alkaline proteases can seriously damage raw milk quality. In this study, specific primers were designed for P. lurida's gyrB and aprX genes, and a real-time loop-mediated isothermal amplification (RealAmp) rapid detection method was developed for the early monitoring of P. lurida and its proteases in raw milk. A phylogenetic tree of the gyrB and aprX genes of P. lurida was constructed to analyze the homology of the design sequence of the RealAmp primer. The DNA of 2 strains of P. lurida and 44 strains of non-P. lurida were detected via RealAmp to analyze the specificity of the primer. It was found that aprX-positive proteases were produced by P. lurida-positive strains only when Pseudomonas fluorescens was negative. The dissociation temperatures of gyrB and aprX in the RealAmp-amplified products were approximately 85.0°C and 90.0°C, respectively. Moreover, DNA was detected through a 10-fold dilution of P. lurida in a pure bacterial solution and artificially contaminated skimmed milk. The limit of detection of P. lurida DNA copy number in the pure bacterial solution was 8.6 copies/μL and that in the 10% skimmed milk was 5.5 copies/μL. Further, 144 raw milk samples throughout the year from three farms in Hebei province were analyzed using RealAmp. The highest detection rate of P. lurida was 56% in the first and third quarters, and that of proteases was 36% in the second quarter. The detection rates of P. lurida and its proteases were the highest in samples collected from pasture 2 (52 and 46%, respectively), and the ability of P. lurida to produce proteases reached 88%. In conclusion, RealAmp established an early and rapid method for the detection of P. lurida and its proteases in raw milk samples, allowing the identification and control of contamination sources in a timely manner to ensure the quality of milk and dairy products.

Activities of indigenous proteases in cow, buffalo and goat milk of Indian subcontinent and their correlation with somatic cell count

Activities of indigenous proteases such as plasmin, plasminogen-derived and cathepsin D in milk from different milch species (Murrah buffalo, Sahiwal and Gir cow and Alpine × Beetal and Sanen × Beetal goat) varied significantly (P <0.05) with the stage of lactation. Plasmin and plasminogen-derived activities were highest in goat and cow milk, respectively. Cathepsin D activity was found to be highest in goat milk and lowest in buffalo milk. The plasminogen-derived activity was 5–6 times higher in buffalo and cow milk vis-à-vis plasmin activity while goat milk exhibited a higher plasmin activity than plasminogen-derived activity. Somatic cell count showed a direct influence on protease activity with the highest being in goat milk. The correlation coefficients of the protease(s) activity with the stage of lactation and somatic cell count were positive indicating a direct impact of somatic cells and lactation on it.

Activities of Indigenous Proteases in Cow, Buffalo and Goat Milk of Indian Subcontinent and Their Correlation with Somatic Cell Count

Activities of Indigenous Proteases in Cow, Buffalo and Goat Milk of Indian Subcontinent and Their Correlation with Somatic Cell Count

Expression profile of different classes of proteases in milk derived somatic cells across different lactation stages of indigenous cows (Bos indicus) and riverine buffaloes (Bubalus bubalis)

Proteases play a significant role in milk and its products by affecting flavor, texture and longevity. The expression of endogenous proteases varies across different stages of lactation. The study was conducted to understand the transcriptional pattern of different classes of protease-pathways associated genes (CTSB, CTSD, CTSH, CTSL, CTSK, CTSS, CTSZ, PLAU, PLAT) and potential protease inhibitors (SERPIN E2 and SERPIN F2) in 40 milk somatic cells (MSC) samples isolated during early, peak, mid and late lactation stages of Sahiwal cows and Murrah buffaloes – the two most important dairy breeds of India. In Sahiwal cows, except CTSK and PLAU, the expression of other proteases class was not affected significantly (p > 0.05) across lactation stages. However, in Murrah buffaloes, the expression of different proteases increased as the lactation progressed. Most of the proteases showed lower expression during early and peak lactation stages while their expression tends to increase during mid to late lactation stages. The overall trend was somewhat similar in both the dairy species albeit the level of expression was higher in buffalo MSC as compared to cow MSC. The study has provided valuable information on expression kinetics of different proteases in milk somatic cells of two major dairy breeds of India.

Potential application of non-thermal atmospheric plasma in reducing the activity of Pseudomonas-secreted proteases in milk

Application of non-thermal atmospheric plasma was examined against the activity of Pseudomonas-secreted proteases in milk. UHT milk samples were inoculated with crude extract of Pseudomonas-secreted proteases and treated in atmospheric plasma discharge system. Evaluation of the progress in protein hydrolysis of plasma-treated and non-treated milk samples revealed that the degree of hydrolysis was lower in plasma-treated milk samples. The longer the plasma treatment time, the lower the degree of hydrolysis during storage. Furthermore, plasma-induced changes in milk proteins and volatile compounds were also evaluated. Caseins, whey proteins, aldehydes, ketones, free fatty acids and hydrocarbons were all affected by plasma discharge. The amount of increase or decrease in these components was dependent on the duration of plasma treatment. Overall, this method is a promising approach that could be used to inactivate bacterial hydrolytic enzymes in milk.

Oxidative Modification of the Proteins of Breast and Cow Milk

One of the important modern characteristics of the nutritional and biological value of milk and dairy products is the antioxidant properties. The high stability and sensitivity of the determination of carbonyl derivatives of proteins, as well as the informative value of the action of antioxidants, allow using them as the markers of oxidative damage. The purpose of this paper was to compare the level of carbonyl derivatives of proteins in breast and cow milk. The determination of the oxidative modification of proteins was based on the reaction of carbonyl derivatives of amino acid residues with 2, 4-dinitrophenylhydrazine. The content of the products was determined during spontaneous and metal-catalyzed oxidative modification of the proteins. During the determination of the spontaneously formed carbonyl derivatives of the proteins, their significantly higher content in cow milk compared to breast milk was established. This increase ranged from 46% to 83% at different wavelengths. Thus, the determination of carbonyl derivatives of amino acid residues of the proteins made it possible to reveal significant differences in the antioxidant properties of breast and cow milk, manifested in a lower level of carbonyl derivatives in breast milk. The lower level of carbonyl derivatives in the composition of breast milk proteins is likely associated with the increased activity of the antioxidant system of breast milk or the increased rate of removal of damaged proteins upon activation of milk proteases.&#x0D; Keywords: carbonyl derivatives, oxidative modification, proteins, human milk, breast milk, metal-catalyzed

Heat-Treatments Affect Protease Activities and Peptide Profiles of Ruminants' Milk.

Proteases present in milk are heat-sensitive, and their activities increase or decrease depending on the intensity of the thermal treatment applied. The thermal effects on the protease activity are well-known for bovine milk but poorly understood for ovine and caprine milk. This study aimed to determine the non-specific and specific protease activities in casein and whey fractions isolated from raw bovine, ovine, and caprine milk collected in early lactation, and to determine the effects of low-temperature, long-time (63°C for 30 min) and high-temperature, short-time (85°C for 5 min) treatments on protease activities within each milk fraction. The non-specific protease activities in raw and heat-treated milk samples were determined using the substrate azocasein. Plasmin (the main protease in milk) and plasminogen-derived activities were determined using the chromogenic substrate S-2251 (D-Val-Leu-Lys-pNA dihydrochloride). Peptides were characterized using high-resolution liquid chromatography coupled with tandem mass spectrometry. The activity of all native proteases, shown as non-specific proteases, was similar between raw bovine and caprine milk samples, but lower (P < 0.05) than raw ovine milk in the whey fraction. There was no difference (P > 0.05) between the non-specific protease activity of the casein fraction of raw bovine and caprine milk samples; both had higher activity than ovine milk. After 63°C/30 min, the non-specific protease activity decreased (44%; P > 0.05) for the bovine casein fraction only. In contrast, the protease activity of the milk heated at 85°C/5 min changed depending on the species and fraction. For instance, the activity decreased by 49% for ovine whey fraction, but it increased by 68% for ovine casein fraction. Plasmin and plasminogen were in general inactivated (P > 0.05) when all milk fractions were heated at 85°C/5 min. Most of the peptides present in heat-treated milk were derived from β-casein and αS1-casein, and they matched the hydrolysis profile of cathepsin D and plasmin. Identified peptides in ruminant milk samples had purported immunomodulatory and inhibitory functions. These findings indicate that the non-specific protease activity in whey and casein fractions differed between ruminant milk species, and specific thermal treatments could be used to retain better protease activity for all ruminant milk species.

Detection of Chymotrypsin by Optical and Acoustic Methods.

Chymotrypsin is an important proteolytic enzyme in the human digestive system that cleaves milk proteins through the hydrolysis reaction, making it an interesting subject to study the activity of milk proteases. In this work, we compared detection of chymotrypsin by spectrophotometric dynamic light scattering (DLS) and quartz crystal microbalance (QCM) methods and determined the limit of chymotrypsin detection (LOD), 0.15 ± 0.01 nM for spectrophotometric, 0.67 ± 0.05 nM for DLS and 1.40 ± 0.30 nM for QCM methods, respectively. The sensors are relatively cheap and are able to detect chymotrypsin in 3035 min. While the optical detection methods are simple to implement, the QCM method is more robust for sample preparation, and allows detection of chymotrypsin in non-transparent samples. We give an overview on methods and instruments for detection of chymotrypsin and other milk proteases.

Food grade silica nanoparticles cause non‐competitive type inhibition of human salivary α‐amylase because of surface interaction

AbstractGiven the potential of ingested particles to interact with enzymes in oral cavity, we compared different grades of SiO2 particles (food‐grade and non‐food grade nanoparticles (FG‐SiO2‐NP, NFG‐SiO2‐NP), and food grade microparticles (FG‐SiO2‐MP)) for their interaction with human salivary α‐amylase (HSA). There were differences in the agglomeration behavior and relative abundance of silanol and siloxane groups among different grades of SiO2 particles where FG‐SiO2‐NPs contained less cyclic siloxane groups but more silanol groups. Secondary structure and function of HSA were negatively impacted by FG‐SiO2‐NPs. In order to verify if this inhibition is mediated through surface interactions, pristine particles were compared with those interacted with pure protein (bovine serum albumin‐BSA) and with food matrix (milk) for HSA inhibition. BSA coating of SiO2 particles ameliorated HSA inhibition, but milk interacted ones showed an enhanced the HSA inhibition because of the presence of milk protease suggesting the relevance of surface interactions in manifesting potential negative impacts of silica particles used in food.

Comprehensive Profiling of the Native and Modified Peptidomes of Raw Bovine Milk and Processed Milk Products.

Bovine milk contains a variety of endogenous peptides, partially formed by milk proteases that may exert diverse bioactive functions. Milk storage allows further protease activities altering the milk peptidome, while processing, e.g., heat treatment can trigger diverse chemical reactions, such as Maillard reactions and oxidations, leading to different posttranslational modifications (PTMs). The influence of processing on the native and modified peptidome was studied by analyzing peptides extracted from raw milk (RM), ultra-high temperature (UHT) milk, and powdered infant formula (IF) by nano reversed-phase liquid chromatography coupled online to electrospray ionization (ESI) tandem mass spectrometry. Only unmodified peptides proposed by two independent software tools were considered as identified. Thus, 801 identified peptides mainly originated from αS- and β-caseins, but also from milk fat globular membrane proteins, such as glycosylation-dependent cell adhesion molecule 1. RM and UHT milk showed comparable unmodified peptide profiles, whereas IF differed mainly due to a higher number of β-casein peptides. When 26 non-enzymatic posttranslational modifications (PTMs) were targeted in the milk peptidomes, 175 modified peptides were identified, i.e., mostly lactosylated and a few hexosylated or oxidized peptides. Most modified peptides originated from αS-caseins. The numbers of lactosylated peptides increased with harsher processing.

Advances in Analysis of Milk Proteases Activity at Surfaces and in a Volume by Acoustic Methods.

This review is focused on the application of surface and volume-sensitive acoustic methods for the detection of milk proteases such as trypsin and plasmin. While trypsin is an important protein of human milk, plasmin is a protease that plays an important role in the quality of bovine, sheep and goat milks. The increased activity of plasmin can cause an extensive cleavage of β-casein and, thus, affect the milk gelation and taste. The basic principles of surface-sensitive acoustic methods, as well as high-resolution ultrasonic spectroscopy (HR-US), are presented. The current state-of-the-art examples of the application of acoustic sensors for protease detection in real time are discussed. The application of the HR-US method for studying the kinetics of the enzyme reaction is demonstrated. The sensitivity of the acoustics biosensors and HR-US methods for protease detection are compared.

Differences and Similarities in the Peptide Profile of Preterm and Term Mother's Milk, and Preterm and Term Infant Gastric Samples.

Our previous studies revealed that milk proteases begin to hydrolyze proteins in the mammary gland and that proteolytic digestion continues within the infant stomach. No research has measured how the release of milk peptides differs between the gastric aspirates of term and premature infants. This study examined the presence of milk peptides in milk and gastric samples from term and preterm infants using an Orbitrap Fusion Lumos mass spectrometer. Samples were collected from nine preterm-delivering and four term-delivering mother–infant pairs. Our study reveals an increased count and ion abundance of peptides and decreased peptide length from mother’s milk to the infant stomach, confirming that additional break-down of the milk proteins occurred in both preterm and term infants’ stomachs. Protein digestion occurred at a higher level in the gastric contents of term infants than in gastric contents of preterm infants. An amino acid cleavage site-based enzyme analysis suggested that the observed higher proteolysis in the term infants was due to higher pepsin/cathepsin D activity in the stomach. Additionally, there was a higher quantity of antimicrobial peptides in term infant gastric contents than in those of preterm infants, which could indicate that preterm infants benefit less from bioactive peptides in the gut.

Stability of fat globules in UHT milk during proteolysis by the AprX protease from Pseudomonas fluorescens and by plasmin

Fat separation is a limiting factor for the shelf life of UHT milk. It may be promoted by the proteolysis of fat surface-adsorbed proteins (FSAP) by proteases that remain active after UHT treatment. The aim of this research was to explore the relationship between the proteolysis of FSAP and fat destabilization. In this study, we developed a full-fat UHT milk-based model system and added either the major bacterial protease AprX from Pseudomonas fluorescens or the major native milk protease plasmin at high levels to induce fast destabilization of the milk fat globules. We monitored changes in physical properties and FSAP composition, and structural changes in fat globules, over 24 h. Our results showed that AprX-induced sedimentation as a result of the flocculation of fat globules, and plasmin induced cream to float as a result of the coalescence of fat globules. This study confirmed that AprX and plasmin can both lead to fat destabilization in full-fat UHT milk, and it provides insights in the underlying mechanisms.

Ultrasound-assisted preparation of ACE inhibitory peptide from milk protein and establishment of its in-situ real-time infrared monitoring model.

A scheme for preparing milk ACE inhibitory peptides by in vitro proteolysis and simulated gastrointestinal digestion was constructed. The ultrasonic assisted pretreatment was used to improve the enzymolysis of milk protein. The in-situ real-time infrared was used to establish monitoring model of enzymatic process. Results showed that under the conditions of single frequency 28 kHz, ultrasound time 40 min, ultrasound power density 20 W/L, milk protein concentration 34 g/L, batch ratio 2:4 and initial temperature 30 °C, the ACE inhibition rate of gastric digestion of enzymatic hydrolysate reached 67.20%, which was 22.87% higher than that of non-ultrasound samples. The results of secondary structure studies of proteins showed that after the ultrasonic treatment, the content of α-helix and β-corner reduced, and the content of β-folding and random coil increased. Compared with the control group, the ultrasonic treatment increased surface hydrophobicity and the content of SH while reduced the content of SS in milk protein, thus improving the ACE inhibitory activity of enzymatic hydrolysates. Furthermore, three quantitative prediction models of PLS, iPLS and Si-PLS for ACE inhibition rate of milk protease hydrolysates were established. And all these three different in-situ real-time prediction models had good predictive effect on the ACE inhibition rate of milk proteolysis products and gastrointestinal simulated digestion products.

Identification of peptides reflecting the storage of UHT milk by MALDI-TOF-MS peptide profiling

Proteolysis during the storage of UHT milk is associated with major technological problems, particularly bitter off-flavors and age gelation limiting the shelf life of milk. In this study, untargeted peptide profiling by MALDI-TOF-MS identified peptides that were formed by proteolysis and reflected the storage of UHT milk. Analysis of nine different commercial UHT samples recorded peptide profiles during and at the end of their shelf life. Relative quantification and sequencing of the peptides revealed that the concentrations of 22 peptides increased significantly during the storage of UHT milk due to the activity of endogenous milk proteases and microbial proteases as well as other unidentified proteolytic mechanisms. Based on highly discriminative AUC values from receiver operator characteristic (ROC) curve analysis, we selected ten peptides as marker candidates. Among those, the peptide β-casein192–206 (m/z 1668.9) was the most suitable marker differentiating expired-UHT from regular-UHT samples with 100% accuracy. Additionally, β-casein191–206 (m/z 1782.0) showed 100% specificity and β-casein139–161 (m/z 2696.4) 100% sensitivity. Thus, β-casein192–206, either by itself or in combination with β-casein191–206 and β-casein139–161, presents a reliable marker to monitor the storage of UHT milk based on proteolytic mechanisms. SignificanceEnzymatic hydrolysis is the main reason why processed milk spoils during storage. The present study recorded peptide profiles to monitor the release or degradation of peptides in stored UHT milk. Among the detected peptides, statistical analysis revealed that the relative concentration of β-casein192–206 reflected those proteolytic processes most precisely.Food authorities can now refer to β-casein192–206 as a reliable marker to differentiate between freshly processed milk and products at the end of their shelf life. Furthermore, the food industry can use this marker peptide to improve production processes by monitoring the proteolysis during storage. The recorded peptide profile helps to explain the basic mechanisms leading to storage-induced proteolysis.

Peptidomics Analysis of Milk Protein-Derived Peptides Released over Time in the Preterm Infant Stomach.

Over the course of milk digestion, native milk proteases and infant digestive proteases fragment intact proteins into peptides with potential bioactivity. This study investigated the release of peptides over 3 h of gastric digestion in 14 preterm infant sample sets. The peptide content was extracted and analyzed from milk and gastric samples via Orbitrap tandem mass spectrometry. The relative ion intensity (abundance) and count of peptides in each sample were compared over time and between infants fed milk fortified with bovine milk fortifier and infants fed unfortified milk. Bioactivity of the identified peptides was predicted by sequence homology to known bioactive milk peptides. Both total and bioactive peptide abundance and count continuously increased over 3 h of gastric digestion. After accounting for infant weight, length, and postconceptual age, fortification of milk limited the release of peptides from human milk proteins. Peptides that survived further gastric digestion after their initial release were structurally more similar to bioactive peptides than nonsurviving peptides. This work is the first to provide a comprehensive profile of milk peptides released during gastric digestion over time, which is an essential step in determining which peptides are most likely to be biologically relevant in the infant. Data are available via ProteomeXchange with identifier PXD012192.

Human milk protein vs. formula protein and their use in preterm infants.

We review the current available evidence on the metabolic fate of human milk proteins and their potential clinical implications for growth and body composition development vs. those of formula proteins in preterm infants. The decreased content of human milk protein in preterm mothers throughout lactation might contribute to the reduced growth reported in exclusively human milk-fed infants compared with that of formula-fed infants. Recent studies have demonstrated that preterm infants are capable of degrading human milk proteins regardless of their degree of prematurity or postnatal age, with limited contribution from milk proteases to protein digestion. The nitrogen balance of fortified human milk-fed preterm infants is higher than that of formula-fed preterm infants. Moreover, the growth of human milk-fed preterm infants appears to be accompanied by fat-free mass deposition. Provided that adequate protein and energy intakes are delivered, human milk enhances protein use rather than oxidation as well as promotes tissue growth, leading to preferential fat-free mass deposition and contributing to the recovery of the body composition in preterm infants. Human milk feeding should be supported and promoted for all preterm mother-infant pairs.