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.     

Synthesis of extracellular lipase by a strain ofPseudomonas fluorescensisolated from raw camel milk

Ten strains of psychrotrophic bacteria were isolated from raw camel milk and were arranged according to their lipase production. Lipolytic activities ranged between 0.26 to 3.43 meq of palmetic acid 100 g^-;1of bovine milk fat h^-;1.Pseudomonas fluorescensRM₄was the most active strain. This bacterium could grow and secrete lipase over a wide range of temperatures. The optimum temperature for growth was 37°C, and the maximum lipase production was at 25°C. Growth was maximal after 96 h of incubation at 25°C, and lipase activity was maximal at 72 h post-inoculation at 25°C (during the late logarithmic phase). Shaking of the cultures (100 rpm) led to an increase in both growth and enzyme activities. Pseudomonas fluorescensRM₄was able to grow and secrete lipase over a pH range of 5.5-;8.50. Synthesis of the enzyme appeared to be inducible because no enzyme was detected in the absence of organic nitrogen. Supplementation of the basal medium with milk proteins enhanced lipase production. Tryptophan and lysine induced enzyme synthesis most effectively. Addition of 4 g l^-;1of glucose to broth stimulated both growth and production of lipase. Beyond this level of supplementation, lipase activity was considerably depressed.     

Biochemical Characterization of an Extracellular Heat‐Stable Protease from Serratia liquefaciens Isolated from Raw Milk

The protease Ser2 secreted by the psychrotrophic strain Serratia liquefaciens L53, a highly proteolytic strain isolated from Brazilian raw milk was purified and characterized. Using azocasein as substrate, Ser2 exhibited activity in a wide range of pH (5 to 10) and temperature (4 to 60 °C). The optimal activity was detected at pH 8.0 and at a temperature of 37 °C. This protease, still active at 4, 7, and 10 °C, was strongly inhibited by chelating agents and by dithiothreitol, a reducing agent. These results confirmed that Ser2 belongs to the peptidase family M10 and requires Ca²⁺, Zn²⁺, and disulfide bridges for stability. This protease is able to hydrolyze three kinds of casein in the preferential order of κ→ β→ α‐casein. Highly heat‐stable in skimmed, semi‐skimmed, and whole milk at 140°C with D‐values of 2.8, 3.9, and 4.5 min, respectively, Ser2 showed a residual activity between 87 and 100 percent after heat‐treatment of 65 °C for 30 min, 72 °C for 20 s, and 140 °C for 4 s that are commonly used in dairy industries. As the protease AprX that is mainly secreted by Pseudomonas genus, Ser2 could be one of the main causes of UHT milk destabilization during storage.     

Differential behavior of Lactobacillus helveticus B1929 and ATCC 15009 on the hydrolysis and angiotensin-I-converting enzyme inhibition activity of fermented ultra-high-temperature milk and nonfat dried milk powder

Consumers' growing interest in fermented dairy foods necessitates research on a wide array of lactic acid bacterial strains to be explored and used. This study aimed to investigate the differences in the proteolytic capacity of Lactobacillus helveticus strains B1929 and ATCC 15009 on the fermentation of commercial ultra-pasteurized (UHT) skim milk and reconstituted nonfat dried milk powder (at a comparable protein concentration, 4%). The antihypertensive properties of the fermented milk, measured by angiotensin-I-converting enzyme inhibitory (ACE-I) activity, were compared. The B1929 strain lowered the pH of the milk to 4.13 ± 0.09 at 37°C after 24 h, whereas ATCC 15009 needed 48 h to drop the pH to 4.70 ± 0.18 at 37°C. Two soluble protein fractions, one (CFS1) obtained after fermentation (acidic conditions) and the other (CFS2) after the neutralization (pH 6.70) of the pellet from CFS1 separation, were analyzed for d-/l-lactic acid production, protein concentration, the degree of protein hydrolysis, and ACE-I activity. The CFS1 fractions, dominated by whey proteins, demonstrated a greater degree of protein hydrolysis (7.9%) than CFS2. On the other hand, CFS2, mainly casein proteins, showed a higher level of ACE-I activity (33.8%) than CFS1. Significant differences were also found in the d- and l-lactic acid produced by the UHT milk between the 2 strains. These results attest that milk casein proteins possessed more detectable ACE-I activity than whey fractions, even without a measurable degree of hydrolysis. Findings from this study suggest that careful consideration must be given when selecting the bacterial strain and milk substrate for fermentation.     

Characterization of ginger proteases and their potential as a rennin replacement

BACKGROUND: Ginger rhizome (Zingiber officinale Roscoe) contains ginger proteases and has proteolytic activity. Ginger proteases have been used for tenderizing meat but rarely for milk clotting. The purpose of this study was to purify ginger proteases and to research their biochemical characteristics. RESULTS: The milk clotting activity (MCA) and proteolytic activity (PA) of the proteases was stable after storage at 4 °C for 24 h. The MCA and PA of fresh ginger juice with 0.2% L ‐ascorbic acid remained stable for 6 days at 4 °C. When under storage at ?80 °C for 2 months, the MCA and PA of the fresh ginger juice and acetone precipitate were still high. Two peaks with protease activity were purified from a DEAE FF ion‐exchange column; the specific activity (units mg^?1 protein) of the MCA (MCSA) and PA (PSA) for the first peak was significantly higher than the second peak (P < 0.05). The protease activity of the ginger proteases was significantly inhibited by E‐64, leupeptin, and iodoacetic acid. Zymography results showed that two protease fractions purified from ginger juice with 62 and 82 kDa had a higher PA against α‐ and β‐casein than against κ‐casein. CONCLUSION: The ascorbic acid addition significantly stabilized the MCA and PA of ginger proteases. The protease inhibition test suggested that ginger proteases belonged to the cysteine type. The biochemical characteristics of ginger protease described in this paper can provide useful information for making new milk curd products. Copyright © 2009 Society of Chemical Industry     

Survival of Lipase during Manufacture of Nonfat Dry Milk

Extracellular lipases from several strains of Pseudomonas fluorescens were active against milk fat. The activity of P. fluorescens strain B52 lipase in reconstituted NDM using β-naphthyl caprylate as substrate was 70% of that found with skim milk; however, hydration of the NDM at 4°C for 12 h resulted in 85% recovery of activity. As little as .0025% (vol/vol) of P. fluorescens spent media was detected in reconstituted skim milk powder using β-naphthyl caprylate. The process of freeze-drying or spray-drying without heating had little effect on either native or P. fluorescens B52 lipase. The bacterial lipase activity was reduced by 12 to 21% and 55 to 59% in NDM treated with low heat (72°C/16 s) and high heat (110°C/2 min), respectively, while the native lipases were completely inhibited by these treatments. Both native and bacterial enzymes were stable when stored in NDM for extended periods at 20°C. In a limited study of commercial NDM samples, microbial lipase was not detected, suggesting that the quality of the raw milk was sufficiently high to restrict the presence of heat-stable lipolytic enzymes.     

The effect of legume protease inhibitors on native milk and bacterial proteases

Protease inhibitors from legume seed extracts (soybean, cowpea and marama beans) and purified soybean protease inhibitor were evaluated with regards to their abilities to inhibit proteases produced by important milk contaminating bacteria, i.e. Bacillus spp. and Pseudomonas spp., and native milk protease, plasmin. Although heat treatment is the most common mean of inactivating enzymes, some heat-stable enzymes can survive the ultra-high temperature (UHT) processing of milk and cause sensory and consistency defects during storage at room temperature. The legume protease inhibitors reduced the activity of plasmin and proteases produced by Bacillus spp. by up to 94% and 97%, respectively, while it showed low inhibitory activity towards Pseudomonas fluorescens proteases (19%) in a buffer system. The protease inhibitors reduced the activity of plasmin (41%) and Bacillus proteases (50%) in UHT milk, however to a lesser extent as compared to inhibition in the buffer system; while it had little or no effect on proteases form Pseudomonas spp. Legume protease inhibitors show great potential in preventing or reducing proteolytic activity of Bacillus proteases and plasmin and may be exploited in various applications where these proteases cause sensory or consistency defects in the product.     

Characterization of the angiotensin-converting enzyme inhibitory activity of fermented milks produced with Lactobacillus casei

Our study assayed angiotensin-converting enzyme (ACE) inhibitory activity and fermentation characteristics of 41 food-originated Lactobacillus casei strains in fermented milk production. Twenty-two of the tested strains produced fermented milks with a high ACE inhibitory activity of over 60%. Two strains (IMAU10408 and IMAU20411) expressing the highest ACE inhibitory activity were selected for further characterization. The heat stability (pasteurization at 63°C for 30 min, 75°C for 25 s, and 85°C for 20 s) and resistance to gastrointestinal proteases (pepsin, trypsinase, and sequential pepsin/trypsinase treatments) of the ACE inhibitory activity in the fermented milks produced with IMAU10408 and IMAU20411 were determined. Interestingly, such activity increased significantly after the heat or protease treatment. Because of the shorter milk coagulation time of L. casei IMAU20411 (vs. IMAU10408), it was selected for optimization experiments for ACE inhibitory activity production. Our results show that fermentation temperature of 37°C, inoculum density of 1 × 10⁶ cfu/g, and fermentation time of 12 h were optimal for maximizing ACE inhibitory activity. Finally, the metabolite profiles of L. casei IMAU20411 after 2 and 42 h of milk fermentation were analyzed by ultra-HPLC electron spray ionization coupled with time-of-flight mass spectrometry. Nine differential abundant metabolites were identified, and 2 of them showed a strong and positive correlation with fermented milk ACE inhibitory activity. To conclude, we have identified a novel ACE inhibitory L. casei strain, which has potential for use as a probiotic in fermented milk production.     

Isolation and characterisation of a heat-resistant peptidase from Pseudomonas panacis withstanding general UHT processes

A secreted peptidase from Pseudomonas panacis was identified and purified. Genome sequencing of the producer strain allowed identification of the peptidase as AprA based on a comparison to peptide sequences of mass spectra obtained from the purified enzyme. The amino acid sequence of the 49.4 kDa peptidase was 98% similar to the metallopeptidase AprX from a Pseudomonas fluorescens strain. The peptidase showed maximum activity at pH 8 and 40 °C and withstood general ultra-high temperature (UHT) processing (138 °C for 18 s) in skim milk, with 88.0 ± 7.7% of the initial enzyme activity remaining after heating. The peptidase showed considerable enzyme activity under storage conditions of UHT milk. The potential for spoilage of milk might during storage was verified by adding very low enzyme activities to UHT-treated milk. The addition of 1 pkat mL⁻¹ peptidase activity resulted in a destabilisation of the milk during four weeks storage.     

Growth of a rifampicin-resistant strain of a proteolytic psychrotroph,Pseudomonas fluorescens,in raw and ultra heat-treated milk

Pseudomonas fluorescens UQM2490, resistant to 250 μg rifampicin/ml, was derived from P. fluorescens JC1, a proteolytic psychrotroph isolated from raw milk. Growth of UQM2490 was followed in raw and ultra heat-treated milk, by viable counting on rifampicin-containing agar medium. The growth curves obtained demonstrate slower growth in raw milk than in treated milk and the variation in growth with change in inoculum level. Generation times in ultra heat-treated milk ranged from 9.5 to 14.1 h compared with 9.6 to 33 h in raw milk.     

Characterization and Cheese-Making Properties of Rennet-Like Enzyme Produced by a Local Algerian Isolate of Aspergillus niger

An ochratoxin free extracellular acid protease was produced by solid state cultivation of Aspergillus niger FFB1. The purified enzyme (48.7 kDa) showed an optimal milk clotting activity at pH 5.5 and 45°C in the presence of 0.01 M CaCl₂. The enzyme was stable at least 24 h at 35°C in the pH range of 5.5–7.0. Thermal denaturation started above 45°C. Fresh cheese manufactured with reconstituted cow milk and the purified enzyme showed similar basic characteristics (pH 4.5, acid taste, white color) as marketed cheeses obtained with calf rennet. This emphasizes the value of exploiting local biological resources for value added food processing in developing countries.     

Microbial Protease and Phospholipase C Stimulate Lipolysis of Washed Cream

The effect of extracellular protease from a Pseudomonas sp. and phospholipase C from Bacillus cereus on lipolysis of washed cream incubated at 37 and 7°C was determined. Preincubation of the washed cream with protease at pH 9.0 and 37°C increased the subsequent rate of lipolysis. Rate of lipolysis was associated with the extent of proteolysis of the washed cream (r² = .89). Preincubation of washed cream with phospholipase C at pH 7.35 and 37°C did not affect subsequent lipolysis. Incubation of washed cream with protease and lipase as well as with phospholipase and lipase at pH 6.65 and 37°C resulted in an increase in the initial rate of lipolysis. However, rate of lipolysis decreased after 2 h of incubation when microbial protease was present; this probably resulted from proteolytic inactivation of the lipase. Microbial protease and phospholipase C exhibited a synergistic effect in enhancing lipolysis for up to 4 h of incubation at 37°C, but this effect decreased with additional incubation. When cream was incubated at 7°C, protease and phospholipase decreased the rate of lipolysis.     

The main spoilage-related psychrotrophic bacteria in refrigerated raw milk

Refrigerated raw milk may contain psychrotrophic microorganisms that produce thermoresistant exoproteases and lipases, which may compromise the quality of processed fluid milk and dairy products during storage. The aim of this work was to quantify and identify the deteriorating psychrotrophic microbiota in Brazilian refrigerated raw milk using genetic diversity analysis. The mean psychrotrophic count was 1.1 × 10⁴ cfu/mL. Of the total isolates, 47.8 and 29.8% showed deteriorating activity at 35°C within 48 h and 7°C within 10 d, respectively. Among the proteolytic species, more isolated by this study were Lactococcus lactis (27.3%), Enterobacter kobei (14.8%), Serratia ureilytica (8%), Aerococcus urinaeequi (6.8%), and Bacillus licheniformis (6.8%). Observed among lipolytics were E. kobei (17.7%), L. lactis (15.6%), A. urinaeequi (12.5%), and Acinetobacter lwoffii (9.4%). The isolates S. ureilytica, E. kobei, Pseudomonas spp., and Yersinia enterocolitica potentially produced alkaline metalloprotease (aprX). Despite the low counts, a considerable portion of the psychrotrophic microbiota presented spoilage potential, which reaffirms the need for rigor in the control of contamination and the importance of rapid processing as factors that maintain the quality of milk and dairy products.     

Characterization of Gram‐Negative Psychrotrophic Bacteria isolated from Italian Bulk Tank Milk

Eighty psychrotrophic bacterial strains, isolated from different northwest Italian bulk tank milks destined for Grana Padano cheese production, were identified by 16S rRNA gene amplification and partial sequence analysis of the rpoB gene. Pseudomonas spp. were the most commonly occurring contaminants, P. fluorescens being the predominant isolated species, along with Enterobacteriaceae, primarily Serratia marcescens. RAPD‐PCR was used to study genetic variability and distinguish closely related strains; a high degree of genetic heterogeneity among the strains was highlighted. All the strains were characterized for their ability to produce proteases, lipases and lecithinases at different temperatures (7, 22, and 30 °C). Forty‐one of the psychrotrophic strains were positive for all the enzymatic activities. The highest number of positive strains for all the incubation temperatures was found for lipolytic activity (59), followed by proteolytic (31) and lecithinase (28) activities, and the enzymatic traits varied among the Pseudomonas and Enterobacteriaceae strains. The proteolytic psychrotrophic strains were screened for the presence of the aprX gene, coding for a heat‐resistant metalloprotease in Pseudomonas spp. The aprX gene was detected in 19 of 63 Pseudomonas strains, and was widespread in the P. fluorescens strains (14/19).     

Oxygen tension measurement as a means of detecting incipient spoilage of raw milk by psychrotrophic bacteria?

A large fall (89% to 10% saturation) in the oxygen tension of raw milk incubated with constant aeration and agitation at 7°C in a fermenter was observed to occur prior to the onset of extracellular protease and lipase production by microorganisms. This same pattern with respect to oxygen tension and lipase production was also observed when raw milk samples from creamery silos were incubated at 7°C in an orbital incubator. The results are in agreement with earlier fermenter studies in which strains of Pseudomonas fluorescens were grown in a simulated milk medium at 7°C. Measurement of oxygen tension may be able to be used to detect incipient enzyme-mediated spoilage of raw milk by psychrotrophs.     

Effect of exopolysaccharides and proteolytic activity of Lactococcus lactis subsp. cremoris strains on the viscosity and structure of fermented milks

Two exopolysaccharide (EPS)-producing Lactococcus lactis subsp. cremoris strains (B35 and B891) were used to study the effect of the kinetics of EPS production and bacterial proteolytic activity on the structure of milk gels and the viscosity of stirred milk gels. Strains were grown at 20 °C in milk containing either yeast extract or casitone and at 30 °C in either milk alone or milk containing casitone. Lactococcal counts, pH decrease and production and molecular characteristics (molar mass and radius of gyration) of both EPSs were followed during milk fermentation. The level of proteolysis in the fermented milks was determined after 24 h of incubation. The results obtained showed that the yield of EPS and the timing of EPS production during milk-gel formation were the most important factors that influenced the structure of the milk gels and the viscosity of the stirred product. The proteolytic activity of the strains did not seem to play any significant role.     

Purification and properties of a heat-stable enzyme of <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> Rm12 from raw milk

Pseudomonas fluorescens Rm12 is a kind of Psychrotrophic bacteria growing in cold raw milk. It produced an extracellular heat resistant protease with an estimated molecular weight of 45 kDa by size exclusion chromatography and SDS-PAGE under both reducing and non-reducing conditions. The enzyme, designated Ht13, was purified to electrophoretic homogeneity from the culture supernatant by sequentially using ammonium sulfate precipitation, ion-exchange chromatography, hydrophobic chromatography and size exclusion chromatography. The specific activity of the enzyme increased 115.5-folds. The optimum pH value and temperature of Ht13 were 7.5 and 40 °C, respectively. Based on its biochemical characteristics, Ht13 can be included in the group of metalloproteases, which was inhibited by 1, 10-phenanthroline and EDTA but not by pepstatin A, chymostatin, STI, E-64, BBI, PMSF and pAPMSF. Mn²⁺ has positive effect on activity and can increased the heat resistance capability, while Ca²⁺ had a negligible effect. For the hydrolysis of azocasein, the Km was 0.012 mg mL⁻¹. The enzyme showed typical heat-stable behavior. After treatment of 160 °C 20 s, the residual activity was 9%. The half-life of the enzyme at 160 °C in buffer with Mn²⁺ was approximately 12 s. Among several main milk proteins, Ht13 can cleave α_s-casein, β-casein and κ-casein. The sequence of 1st–16th amino acids of N-terminal was MSKVKDKAIVSAAQAS, which was same as those proteases excreted from some other P. fluorescens. However, their molecular weights, the activation ion and amino acid composition were different, suggesting Ht13 from P. fluorescens Rm12 is a novel protease.     

Metabolic footprint analysis of volatile metabolites by gas chromatography-ion mobility spectrometry to discriminate between different fermentation temperatures during Streptococcus thermophilus milk fermentation

Streptococcus thermophilus is widely used in the dairy industry to produce fermented milk. Gas chromatography-ion mobility spectrometry–based metabolomics was used to discriminate different fermentation temperatures (37°C and 42°C) at 3 time points (F0: pH = 6.50 ± 0.02; F1: pH = 5.20 ± 0.02; F2: pH = 4.60 ± 0.02) during S. thermophilus milk fermentation, and differences of fermentation physical properties and growth curves were also evaluated. Fermentation was completed (pH 4.60) after 6 h at 42°C and after 8 h at 37°C; there were no significant differences in viable cell counts and titratable acidity; water-holding capacity and viscosity were higher at 37°C than at 42°C. Different fermentation temperatures affected volatile metabolic profiles. After the fermentation was completed, the volatile metabolites that could be used to distinguish the fermentation temperature were hexanal, butyraldehyde, ethyl acetate, ethanol, 3-methylbutanal, 3-methylbutanoic acid, and 2-methylpropionic acid. Specifically, at 37°C of milk fermentation, branched-chain AA had higher levels, and leucine, isoleucine, and valine were involved in growth and metabolism, which promoted accumulation of some short-chain fatty acids such as 3-methylbutanoic acid and 2-methylpanprooic acid. At 42°C, at 3 different time points during fermentation, ethanol from glycolysis all presented higher levels, including acetone and 3-methylbutanal, producing a more pleasant flavor in the fermented milk. This work provides detailed insight into S. thermophilus fermented milk metabolites that differed between incubation temperatures; these data can be used for understanding and eventually predicting metabolic changes during milk fermentation.     

Characterization of a new isolate of Lactobacillus fermentum IFO 3956 from Egyptian Ras cheese with proteolytic activity

More than 200 isolates were obtained from 15 Egyptian traditional dairy products (Domiatti cheese, Ras cheese and Rayeb milk) collected from local markets of Alexandria, Tanta and Kafr El-Sheikh. Examination with optical microscope of these dairy samples allowed to classify 92 bacilli, 64 of which were identified as lactobacilli. The proteolytic activity of lactobacilli isolates was tested on skim milk agar. Eight isolates showing a high proteolytic activity were further tested on UHT skim milk. The strain showing the highest proteolytic activity was purified and identified as Lactobacillus fermentum IFO 3656. The specific proteolytic activity of this strain and the factors affecting it (pH, temperature and presence of inhibitors) were studied. The proteolysis targeted mainly caseins (73% of whole casein), especially β-casein (85%). Smaller portions of whey proteins were proteolyzed (20%) essentially β-lactoglobulin. The proteolysis process gave rise to medium-sized peptide populations. The optimum conditions for the proteolysis activity of the studied strains were pH 6.5 and 37 °C. Proteolytic activities were very slightly affected by the increase of the temperature to 42 °C or the pH to 8.2. The protease system of Lactobacillus fermentum IFO 3956 is most probably composed from a high amount of metalloproteases and small amount of cysteine and serine proteases.     

Purification and Characterization of Four Extracellular Proteases Isolated from Raw Milk Psychrotrophs

Extracellular proteases from psychrotrophic strains of Bacillus coagulans (LY 9), Bacillus sp. (LY 10), Bacillus subtilis (LY 11), and Pseudomonas fluorescens (LY 13) were purified and characterized. The molecular weight of the purified protease from Pseudomonas fluorescens LY 13 was 4.50 × 10⁴, and from the three Bacillus species ranged from 3.35 × 10⁴ to 3.90 × 10⁴. The proteases from LY 10 and LY 13 were monomeric proteins, whereas the protease from LY 9 was in a polymeric form that contained up to 14 subunits. Only the protease from Pseudomonas fluorescens LY 13 showed trypsin-like activity. All four proteases were inhibited by ethylenediaminetetraacetate and would be classified as metallo proteases. Casein was the preferred substrate for these proteases. Susceptibility of casein fractions to attack by these proteases varied with the enzyme source. Maximum enzyme activity was between pH 6.5 and 7.5. The protease from Pseudomonas fluorescens LY 13 retained more activity after heating at 63°C for 30 min than the proteases from the three Bacillus species. Calcium ion showed a protective effect by decreasing heat denaturation of the proteases from LY 9 and LY 11. This protective effect tended to be greater in the presence of Tris-HC1 buffer (.05 M, pH 7.5) plus 10% skim milk than in buffer only.