Heterogeneity of proteolytic enzyme activities in milk samples of different somatic cell count

Milk contains the alkaline proteinase plasmin and lysosomal proteinases; the significance of the latter is ill-defined. The objective of this study was to investigate composition and activities of several different proteolytic enzymes in milk samples of varying somatic cell count (SCC). Increasing milk SCC was correlated with increased plasmin, cathepsin D and cysteine protease activities, with concomitant increases in proteolysis in milk. Addition of plasmin inhibitors confirmed the heterogeneity of proteinase activities in milk, as urea-PAGE analysis of milk samples showed casein hydrolysis in milk after 7 d storage even in samples with inhibitors added; extent and heterogeneity of proteolysis was correlated with milk SCC. Rennet coagulation properties were not significantly correlated with SCC, or activities of measured enzymes. Milk of increasing SCC also exhibited decreased physical stability during incubation of milk at 37 degrees C. Pasteurized milk was more stable than raw milk, suggesting that the enzyme(s) or mechanisms leading to such instability are impaired by pasteurization. Overall, milk has a very heterogeneous proteolytic enzyme population, with a higher significance of non-plasmin enzymes, such as cathepsin D and cysteine proteinases, than perhaps previously recognised.     

原生态与人工添加二十二碳六烯酸羊乳制品贮藏期脂肪酸稳定性比较

通过饲喂奶山羊富含二十二碳六烯酸（docosahexaenoic acid，DHA）的微藻粉，获得原生态DHA羊乳（DHA含量为30 mg/100 g原料乳），然后将其制作成超高温瞬时灭菌（ultra-high temperature instantaneous sterilization，UHT）乳及全脂乳粉，同时设立人工添加富含DHA微胶囊粉的UHT乳及全脂乳粉作为对照组，在常温（25 ℃）和高温（37 ℃）下进行为期28 d的贮藏实验，研究原生态与人工添加DHA羊乳制品贮藏期脂肪酸稳定性。结果表明，与人工添加组相比，贮藏期间原生态UHT乳及全脂乳粉的DHA含量下降速率明显减缓，在UHT乳中，人工添加组乳制品DHA含量降低率在37 ℃下最高达（40.92±3.52）%（贮藏第28天），此时原生态组DHA降低率为（36.70±4.84）%。贮藏期间，原生态与人工添加DHA的UHT乳及全脂乳粉中多不饱和脂肪酸相对含量总体均下降，且与人工添加DHA的乳制品相比，原生态组中多不饱和脂肪酸相对含量更高，更易氧化生成碳链更短的脂肪酸。此外，随着贮藏期的延长，原生态DHA乳制品组中的油脂氧化指标过氧化值和酸价上升速率明显低于人工添加DHA乳制品组。综上，本实验可为制备富含DHA的天然奶制品提供理论参考。     

Effect of Nitrogen Flushing on the Production of Proteinase by Psychrotrophic Bacteria in Raw Milk

Raw milk was flushed with 100 ml N₂ min ^-1 during storage at 4°C. Microflora (total psychrotrophs, proteolytic psychrotrophs, lactic acid bacteria) in nitrogen-flushed milk exhibited a longer lag phase and slower growth rates than those in milk stored aerobically at 4°C. Although proteolytic psychrotrophs grew in nitrogen-flushed milk, proteinases could not be detected in these milk samples. Proteinase assays and electrophoresis showed extensive proteolytic activity and hydrolysis of β-casein in control milk but no detectable casein degradation in nitrogen-flushed milk, even after storage for 18 days at 4°C. This study shows the potential of controlled atmosphere storage of raw milk for inhibition of the accumulation of proteolytic enzymes from psychrotrophic bacteria.     

New aspects of naturally occurring proteases in bovine milk

The scientific literature on milk proteases, along with recent findings in the author's laboratory, are summarized and reviewed comprehensively. Emphasis is on detection of proteolytic enzymes and their activity, purification and kinetic characterization of the isolated enzymes, and technological problems associated with proteolytic enzymes in milk and milk products. Two serine proteinases isolated from milk are compared with plasmin of bovine blood serum. Results from these comparisons strongly suggest that milk proteinase I and plasmin are identical. Proteolysis studies with cold stored milk indicate a direct relationship between gamma-casein formation and milk proteinase association with casein micelles.     

PROPERTIES OF YEAST PROTEINASES

The properties of yeast proteinases, present in the extract of brewer's yeast, were compared with corresponding properties of plant proteinases, pepsin and trypsin in order to find suitable conditions for their activities to be determined independently. The main differences between yeast and plant proteinases were found in their themostability and activity at pH 3·0. In contrast to plant proteinases, yeast proteinases are thermolabile and active at pH 3·0. The proteolytic activity of yeast proteinases in beer is low and may be detected with sensitive methods using radiolabelled protein substrate. Nevertheless, a very wide range of proteolytic activities (67–1082 nkat/litre) was found in samples of unpasteurized beers.     

Assessment of the production of Bacillus cereus protease and its effect on the quality of ultra-high temperature-sterilized whole milk

Bacillus cereus is one of the most important spoilage microorganisms in milk. The heat-resistant protease produced is the main factor that causes rotten, bitter off-flavors and age gelation during the shelf-life of milk. In this study, 55 strains of B. cereus were evaluated, of which 25 strains with protease production ability were used to investigate proteolytic activity and protease heat resistance. The results showed that B. cereus C58 had strong protease activity, and its protease also had the highest thermal stability after heat treatment of 70°C (30 min) and 100°C (10 min). The protease was identified as protease HhoA, with a molecular mass of 43.907 kDa. The protease activity of B. cereus C58 in UHT-sterilized whole milk (UHT milk) showed an increase with the growth of bacteria, especially during the logarithmic growth phase. In addition, the UHT milk incubated with protease from B. cereus C58 at 28°C (24 h) and 10°C (6 d) were used to evaluate the effects of protease on the quality of UHT milk, including protein hydrolysis and physical stability. The results showed that the hydrolysis of casein was κ-CN, β-CN, and α_S-CN successively, whereas whey protein was not hydrolyzed. The degree of protein hydrolysis, viscosity, and particle size of the UHT milk increased. The changes in protein and fat contents indicated that fat globules floated at 28°C and settled at 10°C, respectively. Meanwhile, confocal laser scanning microscopy images revealed that the protease caused the stability of UHT milk to decrease, thus forming age gelation.     

Profile of gelatinolytic capacity of raw goat milk and the implications for milk quality

Both endogenous and exogenous proteinases occur in milk, and they can have beneficial or detrimental effects on dairy production. Because the lactation length of dairy goats is shorter and the somatic cell count (SCC) of goat milk is generally greater compared with dairy cows, the objectives of the present study were to investigate the prevalence of major proteinases in raw goat milk, their association with SCC and production stage, and their effects on milk quality. Milk samples were collected from individual goats in consecutive weeks for different durations, covering regular lactation, late lactation, and post-milk stasis. Long-term (monthly) or short-term (weekly) fluctuations of milk fibrinolytic and gelatinolytic capacities of individual goats were revealed chronologically on fibrin and gelatin zymograms, respectively. In a separate trial involving milk samples from 23 goats at random production stages, the percentage of ultracentrifuge force-precipitable casein of total milk protein was calculated to represent milk quality and was assessed to evaluate its correlation with the corresponding proteolytic capacities. The results for regular milk indicate that gelatinase B was more abundant than gelatinase A when they first appeared at SCC of ∼1 × 10⁶/mL. During the last month before milk stasis, both gelatinases A and B were found to be prevalent and prominent in milk regardless of the broad SCC range recorded there. Fibrinolytic activity and the active form of gelatinase A were only regularly detected in post-stasis secretions and were scarce before stasis. The results of the milk quality trial indicate that milk of relatively high proteinase capacity tended to have a low casein ratio. Correlation analysis confirmed a significant relationship between gelatinase capacity of goat milk and production stage, SCC, or casein ratio. It is suggested that an elevation of gelatinolytic capacity of goat milk coincides with an increase in somatic cell number accompanying the extension of lactation length, which is unfavorable for the production of a more desirable quality of goat milk.     

Determination of proteolytic activity in different milk systems

A method for the determination of proteolytic activity in whole milk, skim milk or in a solution similar to skim milk ultrafiltrate was optimized and validated. An artificial substrate (azocasein) was used to quantify the enzyme activity through the release of a chromogenic product that was measured spectrophotometrically after clarifying the samples by the addition of trichloracetic acid. The method gave linear results in the range of 0–50 mU/ml of added Bacillus subtilis protease and the least detection and quantification limits were 2.29 and 7.64 mU/ml of protease, respectively. The precision, measured as relative standard deviation, was between 1.14 and 7.99% and mean recovery ranged between 99 and 104%. Reliability of the method was satisfactory for all products evaluated.     

Proteolysis,protein distribution and stability of UHT milk during storage at room temperature

Proteolytic degradation and distribution of caseins and whey proteins between the soluble and colloidal phases were studied in six batches of commercial UHT milk (three skim and three whole milks) during storage at 25 ± 2 °C. For that purpose, at 30 day intervals, milk samples were ultracentrifuged and the pellets and supernatants analysed by capillary electrophoresis and SDS‐PAGE. Samples were also visually examined for signs of gelation. Extensive proteolytic degradation of the micellar fractions and severe changes in the electrophoretic pattern of the proteins present in the serum fractions were observed in all the batches. A higher proportion of denatured whey proteins not attached to the micelle surface was found in the skim milk samples as compared with the whole milk samples that could provide less resistance against gelation. In addition to β‐Lg, para‐κ‐casein was also found in the serum fraction. A high proteolytic activity against κ‐casein could be responsible for the hydrolysis of serum‐liberated κ‐casein or could have enhanced the liberation of β‐Lg–para‐κ‐casein complexes through proteolysis of micellar κ‐casein. © 1999 Society of Chemical Industry     

Effects of Some Chemical and Physical Treatments on Proteolysis in Milk

Addition of either a water-soluble or oil-soluble surfactant to milk enhanced proteolysis. This effect was attributed primarily to alteration in the availability of the casein and secondarily to a partial release of proteolytic enzymes from the milk fat globule membrane. Adding milk fat globule membrane to milk also increased proteolysis, presumably by contributing additional proteinase.Proteolysis increased after milk was subjected to Waring blendor treatment, ultrasonic treatment, or temperature fluctuations. These treatments presumably cause dissociation of proteinases from milk fat globule membranes or casein micelles.Addition of carrageenan to milk inhibited proteolysis. Carrageenans may inhibit proteolysis by preventing dissociation of casein micelles or by blocking access to the active sites on casein. Removing either calcium ions or colloidal phosphates from milk by dialysis enhanced proteolysis. This enhancement may be due to dissociation of casein micelles or milk proteinases originally adsorbed on the casein micelles. Removal of calcium ions increased proteolysis more than removal of colloidal phosphates.     

Molecular Characterization and Identification of Bioactive Peptides Producing Lactobacillus sps. Based on 16S rRNA Gene Sequencing

In the present study, 3 bacterial cultures were isolated from faecal samples of human infant. The biochemical traits showed similarity with Lactobacillus sps and 16S rRNA sequence analyses, confirmed as Lactobacillus plantarum, Lactobacillus casei, and Lactobacillus rhamnosus. The cultures were screened for their proteolytic activity and good ability to release peptides from milk proteins was found. Hence, these bacteria were used as a proteolytic starter culture for the fermentation of skim milk and whey for the liberation of small peptides. Bioactive nature of the peptides released from whey and skim milk was tested, and results demonstrated that peptides obtained after fermentation of whey and skim milk by Lactobacillus strains showed antimicrobial activity against all the pathogens causing food borne infections in humans. These peptides also indicated antioxidant as well as ACE (angiotensin-converting enzymes) inhibitory activity.     

Proteolytic patterns and plasmin activity in ewes' milk as affected by somatic cell count and stage of lactation

A total of 120 milk samples were collected from Comisana ewes throughout lactation. The ewes were ranked into two somatic cell count (SCC) categories: normal milk (N Milk) with SCC lower than 5.00x 10(5)/ml and high somatic cell milk (HSC Milk) with SCC higher than 1.00 x 10(6)/ml. Milk samples were analysed in triplicate for pH, fat and protein contents, renneting parameters, and plasmin and plasminogen activities. The peptide profile due to total proteolytic activity (endogenous and exogenous enzymes) on alpha- and beta-CNs were determined using urea-PAGE on sodium caseinate (pH 8.0 and pH 5.0) incubated at 37 degrees C for 4 d after sampling. The peptide profile due to non-plasmin enzyme activities at pH 5.0 was also determined using urea-PAGE. Plasmin activity was higher in the HSC milk than in the N milk throughout the study period. A decrease in plasmin activity was observed in the N milk during mid-lactation, which was probably related to decrease in pH, and in the HSC milk during late lactation, which may be ascribed to an enhanced influx of plasmin inhibitors from the blood stream. Proteolytic patterns in Comisana ewe milk were mainly affected by plasmin activity that increased with the SCC in milk. Also non-plasmin proteolytic activity was strongly enhanced by elevated SCC and resulted in a higher degradation of alpha-casein than of beta-casein. In general, plasmin activity did not increase with the advancement of lactation and exhibited a different trend in HSC and N milk, suggesting that physiological factors did not play a key role in regulating the plasminogen-plasmin system in ewes' milk. Plasmin activity, detected with the colorimetric assay was consistent with proteolytic activity on sodium caseinate shown in urea-PAGE electrophoregram.     

ELECTROPHORETIC STUDY OF SUBSTRATE AND pH DEPENDENCE OF ENDOPROTEOLYTIC ENZYMES IN GREEN MALT*

The endoproteolytic enzymes of malt influence several different aspects of malt and beer quality. For this reason, we are extracting and characterizing these enzymes from green malt. The proteolytic activity of a Morex green malt extract was highest at pH 3.8 with haemoglobin substrate but gelatin hydrolysis was maximal from pH 4.7 to 6.0. Endoproteolytic hydrolysis of a 55% isopropanol-soluble reduced hordein fraction was about three times slower than gelatin hydrolysis but was relatively constant over the pH range from 3.8 to 6.5, although the activity did decrease at more acidic (3.0) or basic (7.0) pH values. To study the green malt proteinases in detail, a non-denaturing electrophoretic system was developed in which substrate proteins—either gelatin, edestin or hordein—were incorporated into an electrophoretic gel. After electrophoresis and incubation of the gels at pH 3.8, 4.7, 5.5, or 6.5 to allow enzymatic hydrolysis, the separated activities were determined by using protein staining to determine where the incorporated substrate had been hydrolysed. Using this system, seven proteolytic activity bands were detected. Five of the bands were maximally active at pH 3.8 and their activities dropped quickly as the pH increased. The other two bands, which migrated more slowly, hydrolysed gelatin more rapidly than they did the other substrates tested. Their gelatinolytic activities increased as the pH was raised (by 3- to 6-fold in the pH range tested). The electrophoretic system described has proven very useful for studying the proteinases of germinating barley seeds. The results indicate that much past research on malt proteinases may not be particularly relevant to the malting and brewing industries because it was conducted under pH conditions and with substrates that are likely quite different from those in the seed during the barley germination process. By using electrophoresis to separate proteinases before analysis, we can now study their individual characteristics and thus can conduct studies more relevant to malting and brewing.     

The Impact of NaCl Substitution with KCl on Proteinase Activities Cell-Free Extract and Cell-Free Supernatant at Different pH Levels and Salt Concentrations: Lactobacillus delbrueckii ssp. bulgaricus and Streptococcus thermophilus

The effect of NaCl substitution with KCl at different pH levels (6.0, 5.5, and 5.0) and salt concentrations on proteinase activities of cell-free and supernatant of Lactobacillus delbrueckii ssp. bulgaricus 11824 (L. bulgaricus) and Streptococcus thermophilus MS (ST) was investigated. MRS broths were separately mixed with 4 salt treatments (NaCl only, 1NaCl:1KCl, 1NaCl:3KCl, and KCl only) at 2 different concentrations (5% and 10%) and incubated at 37 °C for 22 h. The cell pellets were used to prepare proteinase of cell-free extract and the cell-free supernatants were used as source of extracellular proteinases. The proteolytic activities and protein contents of both fractions were determined. The supernatants after incubation of both fractions with 3 milk caseins (α-, β-, κ-casein) were subjected to angiotensin-converting-enzyme inhibitory (ACE-inhibitory) activity and proteolytic activity by ortho-phthalaldehyde (OPA) method. Significant differences were observed in ACE-inhibitory activities and proteolytic (OPA) between salt treatments of cell-free extract and cell-free supernatant of L. bulgaricus and S. thermophilus at same salt concentration and same pH level. There was a significant effect of pH level and salt treatments interaction on ACE-inhibitory activity, OPA activity and azocasein activity. Practical Application: To reduce sodium concentration in cheese by substituting of NaCl with KCl, it was important to study the effect on starter culture proteinases which play a vital role in ripening and texture profile of cheese.     

Barley and Malt Proteins and Proteinases: I. Highly Degradable Barley Protein Fraction (HDBPF), a Suitable Substrate for Malt Endoprotease Assay

Barley grain proteins were extracted and fractionated, based on their solubility, to investigate their proteolytic digestibility and suitability to be used as a substrate for the assay of malt proteinases. The fraction extracted with alkaline buffer (Tris‐HCl or Tris‐glycine pH 8.8–9.5), at the end of the sequence, exhibited remarkably high degradability by malt proteases compared to other fractions or any known protein substrate. Gel filtration chromatographic analysis of this fraction revealed that it is composed of three different molecular weight components. Further investigation, after proteolytic treatment, demonstrated that the third and the low molecular weight component is the highly degradable protein(s) (HDP). We designated the whole fraction, the mixture of the three components, as the highly degradable barley protein fraction (HDBPF) and used it (and recommend it) as the substrate for the assay of malt endoproteases activity.     

Characterization of natural isolates of Lactobacillus strains to be used as starter cultures in dairy fermentation

The technological relevant characteristics of five homofermentative lactobacilli strains, isolated from natural fermented hard cheeses, were studied. Isolates CRL 581 and CRL 654, from Argentinian artesanal hard cheeses, and isolates CRL 1177, CRL 1178, and CRL 1179, from Italian Grana cheeses, were identified as Lactobacillus delbrueckii subsp. lactis and Lactobacillus helveticus, respectively, by physiological and biochemical tests, SDS-PAGE of whole-cell proteins and sequencing of the variable (V1) region of the 16S ribosomal DNA. All strains showed high levels of beta-galactosidase activity. However, proteolytic activity varied widely among isolates. Strains CRL 581, CRL 654, and CRL 1177 hydrolyzed alpha- and beta-caseins and were able to coagulate reconstituted skim milk in less than 16 h at 42 degrees C. According to the substrate specificity, these proteinases have a caseinolytic activity comparable to that of the P(III)-type of lactococcal proteinases. No strains produced inhibitor substances (bacteriocin) and all were insensitive to attack by 14 L. helveticus- and L. delbrueckii subsp. lactis-specific bacteriophages.     

Growth of Pseudomonas weihenstephanensis,Pseudomonas proteolytica and Pseudomonas sp. in raw milk: Impact of residual heat-stable enzyme activity on stability of UHT milk during shelf-life

One of the reasons for spoilage of UHT milk during shelf-life is the presence of residual proteolytic activity produced from Pseudomonas spp. during storage of raw milk. The aim of this study was to describe the product defects occurring in indirectly heated UHT milk during shelf-life, and to establish a correlation between proteolytic activity and onset of product spoilage. UHT milk was produced from raw milk incubated with different Pseudomonas strains, and examined over four months during storage at 20 °C. Inactivation kinetics of the peptidases were determined. In UHT milk, product defects occurred in the order: bitterness – particles – creaming – sediment – gelation in all the samples containing peptidases (apparent enzyme activity ≥ 0.03 pkat mL⁻¹). A linear correlation was found between proteolytic activity and onset of product defects, apart from onset of gelation.     

Proteinases in normal bovine milk and their action on caseins

Native proteolytic enzymes in good quality normal bovine milk readily hydrolysed the caseins during incubation or storage, producing the gamma-caseins, proteose-peptone components 5 (PP5) and 8-fast (PP8F) and a considerable number of other unidentified fragments, many of which were also subsequently found in the proteose-peptone fraction. The rate of casein hydrolysis was greater in pasteurized than in raw milk, with beta-casein being slightly more susceptible to attack than alpha S1-casein. Measurements of gamma-casein and proteose-peptone formation have been made and it was found that PP5 was an intermediate product that was subject to further proteolysis while PP8F was a stable end-product. With the exception of component 3 (PP3), virtually all constituents of the proteose-peptone fraction increased during storage and appeared to be products of the action of proteolytic enzymes. Further evidence was obtained from the effects of various inhibitors that the principal proteinase of normal milk is plasmin, but slight differences were apparent between the protein breakdown patterns induced by storage and by added plasmin, which was consistent with the presence of more than one proteinase. Incubations in the presence of soya bean trypsin inhibitor to prevent plasmin action clearly revealed that another enzyme(s) was also involved.     

Indigenous proteolytic enzymes in milk: A brief overview of the present state of knowledge

Key characteristics of dairy products, including texture, flavour and functionality, depend on the level and behaviour of milk proteins, especially the caseins. Hence, it is not surprising that the activity of indigenous proteinases in milk has been studied for many years. Milk contains two proteinase systems, both derived of from blood, one involved in dissolving blood clots (plasmin) and the other in defence against invasive micro-organisms (lysosomal proteinases of somatic cells). Both systems hydrolyse the caseins, are complex in their regulation and sensitivity to processing, and vary in activity due to factors such as stage of lactation and mastitis. While plasmin is the principal proteinase in good-quality milk, other proteinases, including cathepsins and elastase, are probably also active, particularly as the somatic cell count of milk increases. In this review, recent research on indigenous milk proteinases is discussed, and some questions raised about their level and significance.     

Effect of storage and separation of milk at udder quarter level on milk composition, proteolysis, and coagulation properties in relation to somatic cell count

Coagulation properties of milk are altered by elevated somatic cell count (SCC), partly due to increased proteolytic and lipolytic activity in the milk and, thereby, degradation of protein and fat during storage. Milk is commonly stored on the farm at cooling conditions for up to 2 d before transport to the dairy for processing. This study evaluated the effects of storage on milk with altered composition due to high SCC and the effects of exclusion of milk from individual udder quarters with high SCC on milk composition, proteolysis, and coagulation properties. Udder-quarter milk and cow-composite milk samples from 13 cows having at least 1 quarter with SCC above 100,000 cells/mL were collected on 1 occasion. In addition, commingled milk from only healthy quarters (<100,000 cells/mL) of each cow was collected, representing a cow sample where milk with elevated SCC was excluded. The milk samples were analyzed for total protein content; protein content in the whey fraction; casein, fat, and lactose contents; SCC; proteolysis; curd yield; coagulation time; and total bacterial count, on the day of sampling and after 2 and 5 d of storage at +4°C. In addition to SCC, duration of storage and total bacterial count had an effect on milk quality. The content of total protein, fat and protein contents in the whey fraction, and curd yield were found to have different storage characteristics depending on the level of SCC at udder-quarter level. The exclusion of milk from udder quarters with elevated SCC decreased the content of total protein and protein content in the whey fraction and increased the content of lactose at cow level. However, the effect of separating milk at udder-quarter level needs to be further studied at bulk tank level to evaluate the effect on overall total milk quality.