Productions and monomer compositions of exopolysaccharides by Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus strains isolated from traditional home-made yoghurts and raw milk

In this study, a total of forty‐five strains of lactobacilli and streptococci were determined exopolysaccharide (EPS) production in skim milk and Man Rogosa and Sharpe (MRS)/M17 medium, viscosity and proteolytic activity. The exopolysaccharide production by lactobacilli strains during growth in MRS medium was twenty‐one to 211 mg L^?1, while in skim milk was to thirty‐six to 315 mg L^?1. The EPS production by streptococci strains during growth in M17 medium was sixteen to 114 mg L^?1, while in skim milk was to twenty‐four to 140 mg L^?1. The EPS production of strains was lower in MRS/M17 medium than skim milk. Results showed that it was not clear correlation between the viscosity and EPS production of some strains. All strains were shown proteolytic activity. Positive correlations between exopolysaccharide production and proteolytic activity in skim milk were found some strains of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus. These results indicated that the high exocellular protease‐producing strains can produce high EPS in skim milk. The monomer compositions of the EPSs formed by selected five strains were analysed. Mannose dominated (99–100%) on the EPS produced by L. delbrueckii subsp. bulgaricus and S. thermophilusstrains (except L. delbrueckii subsp. bulgaricus 22) in skim milk and MRS/M17 medium. Besides, the EPSs of strains in skim milk contained small amount of lactose.     

Optimisation of bambara groundnut water extract and skim milk composition as cryoprotectant for increasing cell viability of Lactobacillus spp. using response surface methodology

Four legume water extracts, that is bambara groundnut, soya bean, red kidney bean and black bean as well as skim milk, were examined for their effectiveness in protecting Lactobacillus rhamnosus GG and Lactobacillus fermentum SK5 during the freeze‐drying and storage. Bambara groundnut water extract (BGWE) showed promising cryoprotective activity that was comparable to skim milk. BGWE and skim milk at 2–10% w/v and 5–20% w/v individually produced survival rates for both strains of 87–88%. To further optimise the synergistic cryoprotective medium, response surface methodology was employed. The optimal combination was 4.93% w/v BGWE and 11.68% w/v skim milk for L. rhamnosus GG and 5.17% w/v BGWE and 11.36% w/v skim milk for L. fermentum SK5 with survival rates of 95.17% and 94.36%, respectively. The storage life of freeze‐dried cells of both probiotics at 4 °C and 30 °C for 6 months was markedly improved when they were produced with these optimal combinations.     

Technological and flavour potential of cultures isolated from traditional Greek cheeses – A pool of novel species and starters

This study explored the microbiota of Formaella, Kopanisti, Feta and Mana cheeses. A total of 133 wild lactic acid bacteria were isolated and classified phenotypically. Mesophilic lactobacilli were the most abundant group. Thermophilic lactobacilli and thermophilic cocci were the best milk acidifiers, whereas thermophilic lactobacilli were the most proteolytic isolates. Higher peptidolytic and esterolytic activities were obtained with thermophilic cocci. Only five isolates were lipolytic, whereas none was able to catabolize citrate. Fast gas chromatography−mass spectrometry analysis of the metabolites produced and subsequent principal components analysis revealed segregated groups of isolates in accordance with the phenotypic ones. Electronic nose analysis revealed similar results. Lactobacillus rennini and Lactobacillus acidipiscis were found to be the sole microbial species in Kopanisti cheese and Mana. These isolates produced alcohols and aldehydes as major volatile compounds, as a result of secondary amino acid catabolism.     

Nonstarter Lactobacillus strains as adjunct cultures for cheese making: in vitro characterization and performance in two model cheeses

Nonstarter lactic acid bacteria are the main uncontrolled factor in today's industrial cheese making and may be the cause of quality inconsistencies and defects in cheeses. In this context, adjunct cultures of selected lactobacilli from nonstarter lactic acid bacteria origin appear as the best alternative to indirectly control cheese biota. The objective of the present work was to study the technological properties of Lactobacillus strains isolated from cheese by in vitro and in situ assays. Milk acidification kinetics and proteolytic and acidifying activities were assessed, and peptide mapping of trichloroacetic acid 8% soluble fraction of milk cultures was performed by liquid chromatography. In addition, the tolerance to salts (NaCl and KCl) and the phage-resistance were investigated. Four strains were selected for testing as adjunct cultures in cheese making experiments at pilot plant scale. In in vitro assays, most strains acidified milk slowly and showed weak to moderate proteolytic activity. Fast strains decreased milk pH to 4.5 in 8 h, and continued acidification to 3.5 in 12 h or more. This group consisted mostly of Lactobacillus plantarum and Lactobacillus rhamnosus strains. Approximately one-third of the slow strains, which comprised mainly Lactobacillus casei, Lactobacillus fermentum, and Lactobacillus curvatus, were capable to grow when milk was supplemented with glucose and casein hydrolysate. Peptide maps were similar to those of lactic acid bacteria considered to have a moderate proteolytic activity. Most strains showed salt tolerance and resistance to specific phages. The Lactobacillus strains selected as adjunct cultures for cheese making experiments reached 10⁸ cfu/g in soft cheeses at 7 d of ripening, whereas they reached 10⁹ cfu/g in semihard cheeses after 15 d of ripening. In both cheese varieties, the adjunct culture population remained at high counts during all ripening, in some cases overcoming or equaling primary starter. Overall, proximate composition of cheeses with and without added lactobacilli did not differ; however, some of the tested strains continued acidifying during ripening, which was mainly noticed in soft cheeses and affected overall quality of the products. The lactobacilli strains with low acidifying activity showed appropriate technological characteristics for their use as adjunct cultures in soft and semihard cheeses.     

Influence of microfiltration and adjunct culture on quality of Domiati cheese

The effects of microfiltration and pasteurization processes on proteolysis, lipolysis, and flavor development in Domiati cheese during 2 mo of pickling were studied. Cultures of starter lactic acid bacteria isolated from Egyptian dairy products were evaluated in experimental Domiati cheese for flavor development capabilities. In the first trial, raw skim milk was microfiltered and then the protein:fat ratio was standardized using pasteurized cream. Pasteurized milk with same protein:fat ratio was also used in the second trial. The chemical composition of cheeses seemed to be affected by milk treatment—microfiltration or pasteurization—rather than by the culture types. The moisture content was higher and the pH was lower in pasteurized milk cheeses than in microfiltered milk cheeses at d 1 of manufacture. Chemical composition of experimental cheeses was within the legal limits for Domiati cheese in Egypt. Proteolysis and lipolysis during cheese pickling were lower in microfiltered milk cheeses compared with pasteurized milk cheeses. Highly significant variations in free amino acids, free fatty acids, and sensory evaluation were found among the cultures used in Domiati cheesemaking. The cheese made using adjunct culture containing Lactobacillus delbrueckii ssp. lactis, Lactobacillus paracasei ssp. paracasei, Lactobacillus casei, Lactobacillus plantarum, and Enterococcus faecium received high scores in flavor acceptability. Cheeses made from microfiltered milk received a higher score in body and texture compared with cheeses made from pasteurized milk.     

Influence of lamb rennet paste on composition and proteolysis during ripening of Pecorino foggiano cheese

Rennet pastes produced by lambs subjected to three different feeding systems (mother suckling [MS], artificial rearing [AR], and artificial rearing with Lactobacillus acidophilus supplementation [ARLB]) and slaughtered at two different ages (20 and 40 d) were used for the manufacture of Pecorino foggiano cheese. Composition and proteolysis during ripening of Pecorino foggiano cheese (four replicates batches) were analyzed. Proteolysis was greater in cheeses made with rennet pastes from lambs slaughtered at 20 d, as shown by analysis of nitrogen fractions (water-soluble N and proteose peptones). Supplementation of milk substitute with L. acidophilus may have influenced the growth dynamics of lactic acid bacteria in the rennet pastes, with positive effects on levels of lactobacilli in cheese at the beginning of the ripening time. Lower pH values in ARLB cheese during ripening, together with higher cell loads, suggest that supplementation of milk replacer with L. acidophilus resulted in higher proteolytic activity, as also confirmed by the composition of the pH 4.6—insoluble nitrogen fraction. No differences were found in total concentration of free amino acids among the experimental cheeses; phenylalanine, isoleucine, leucine, lysine were found at the highest levels. The addition of probiotic bacteria to milk substitute in lamb rearing appears to give good-quality lamb rennet paste.     

Characterization of lactic acid bacteria isolated from Bukuljac, a homemade goat's milk cheese

The Bukuljac cheese is traditionally homemade cheese, produced from heat-treated goat's milk without the addition of any bacterial starter culture. The presence of lactic acid bacteria (LAB) in Bukuljac cheese has been analyzed by using a polyphasic approach including microbiological and molecular methods such as rep-PCR with (GTG)5 primer. Lactobacillus paracasei subsp. paracasei represents a dominant strain in the microflora of analyzed cheese. Out of 55 Gram-positive and catalase-negative isolates, 48 belonged to L. paracasei subsp. paracasei species. Besides lactobacilli, five Lactococcus lactis subsp. lactis and two Enterococcus faecalis were found. Results of PCR-denaturing gradient gel electrophoresis (DGGE) of DNA extracted directly from the fresh cheese revealed the presence of Leuconostoc mesenteroides. Only lactobacilli showed a high proteolytic activity and hydrolyzed alpha(s1)- and beta-caseins. They are also producers of diacetyl. In addition, 34 out of 55 isolates, all determined as lactobacilli, showed the ability of auto-aggregation. Among 55 isolates, 50 also exhibited antimicrobial activity.     

Characterization of microflora in homemade semi-hard white Zlatar cheese

The Zlatar cheese belongs to the group of traditionally homemade cheeses, which are produced from nonpasteurized cow's milk, without adding of any bacterial starter culture. Changes were followed in lactic acid bacteria population and chemical composition during the ripening period of cheese up to 60 days. Results showed that the percentage of lactic acid cocci was higher in raw milk and one day old cheese and their percentage was gradually decreasing, whereas the number of lactobacilli was increasing. After 30 days of cheese ripening the number of cocci increased again, reaching the number of lactobacilli. The results of API 50 CH system and rep-PCR analysis showed that Lactobacillus paracasei subsp. paracasei, Lactobacillus brevis, Lactococcus lactis subsp. lactis, Enterococcuus faecium and Enterococcus faecalis were the main groups present during the ripening of Zlatar cheese. Results revealed that in older cheeses (45 and 60 days old) enterococci were the main group present. It was also demonstrated that 57 isolates showed antimicrobial activity. The number of bacteria showing antimicrobial activity slowly decreased during the ripening period and in samples of 60 days old cheese producers of antimicrobial activities were not detected.     

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.     

Quality Attributes of Cheddar Cheese Containing Added Lactobacilli

Various strains of homo- and heterofermentative lactobacilli isolated from cheddar cheese were added to milk with a commercial streptococci culture to produce cheddar cheese. The heterofermatative lactobacilli L. brevis and L. fermentum almost always led to the development of fruity flavors, openness and late-gassing within 10 months of aging. Cheddar cheese produced using combined cultures of heterofermentative lactobacilli and L. casei-casei or L. casei-pseudoplantarum did not exhibit gas formation and openness. The overall grading scores of cheese containing added lactobacilli were not higher than those for the control cheese (without lactobacilli). A definite correlation was found between the lactobacilli used and the flavors of the cheese. The controlled acidity development during cheese making, the fat and the salt in moisture content of the cheeses were not affected.     

Cathepsin D activity in quarg

Quarg cheese was produced from raw skim milk, pasteurised skim milk, raw skim milk with rennet added and ultrafiltrated raw skim milk. Quarg was also produced from raw skim milk with pepstatin added at curd cutting and from ultrafiltration retentate of raw milk with added pepstatin. No starter bacteria were used in this model system, with the reduction of pH being achieved by addition of glucono- δ-lactone. Yields ranged between 20.25 and 23.5%, with protein levels of 13.6–15.7%. Proteolysis occurred during storage of all experimental cheese samples for 3 m at 8°C. By immunoblotting using antibodies against bovine cathepsin D, immunoreactive procathepsin D was identified in all cheese samples. Presence of cathepsin D or procathepsin D-derived activity was confirmed by a specific enzyme assay in all samples, except those which contained pepstatin. Inhibition of cathepsin D-catalysed proteolysis by pepstatin was observed in chromatograms of water-soluble extracts analysed by reverse-phase HPLC. Peptides thought to be produced as a result of cathepsin D activity were observed in cheese made from both raw and pasteurised milk, suggesting that the activity at least partially survived pasteurisation.     

Thermal inactivation of chymosin during cheese manufacture

The aspartic proteinase, chymosin (EC 3.4.23.4) is the principal milk clotting enzyme used in cheese production and is one of the principal proteolytic agents involved in cheese ripening. Varietal differences in chymosin activity, due to factors such as cheese cooking temperature, fundamentally influence cheese characteristics. Furthermore, much chymosin is lost in whey, and further processing of this by-product may require efficient inactivation of this enzyme, with minimal effects on whey proteins. In the first part of this study, the thermal inactivation kinetics of Maxiren 15 (a recombinant chymosin preparation) were studied in skim milk ultrafiltration permeate, whole milk whey and skim milk whey. Inactivation of chymosin in these systems (at pH 6.64) followed first order kinetics with a D45.5 value of 100 +/- 21 min and a z-value of 5.9 +/- 0.3 degrees C. D-Values increased linearly with decreasing pH from 6.64 to 6.2, while z-values decreased as pH decreased from 6.64 to 6.4, but were similar at pH 6.4 and 6.2. Subsequent determination of chymosin activity during manufacture of Cheddar and Swiss-type cheese showed good correlations between predicted and experimental values for thermal inactivation of chymosin in whey. However, both types of cheese curd exhibited relatively constant residual chymosin activity throughout manufacture, despite the higher cooking temperature applied in the manufacture of Swiss cheese. Electrophoretic analysis of slurries made from Cheddar and Swiss cheese indicated decreased proteolysis due to chymosin activity during storage of the Swiss cheese slurry, but hydrolysis of sodium caseinate by coagulant extracted from both cheese types indicated similar levels of residual chymosin activity. This may suggest that some form of conformational change other than irreversible thermal denaturation of chymisin takes place in cheese curd during cooking, or that some other physico-chemical difference between Swiss and Cheddar cheese controls the activity of chymosin during ripening.     

Production of novel dairy products using actinidin and high pressure as enzyme activity regulator

Milk clotting for the production of novel dairy products, alternative or complementary to cheese and yogurt type products can be achieved using plant sulfhydryl proteases. The objective was to apply the protease actinidin, from Actinidia chinensis, as the milk clotting agent, and High pressure (HP) technology to control excessive proteolysis. The effect of the dairy substrate and the process parameters on the coagulation rate and the texture and sensory properties of the end product, were studied. Selected values of design parameters were 25% total solids, 6.49 adjusted pH, 0.35 U activity of the clotting agent actinidin, 40 ºC process temperature and 2 h time. The selected pressure-temperature conditions, 600 MPa at 40 ºC, were applied to stop the potentially detrimental further proteolytic action of the enzyme. Results indicated that use of actinidin for milk clotting and HP to stop the enzyme activity in the final product, leads to a “fresh cheese” type dairy product.Industrial relevance: Alternative clotting methods for novel dairy products, complementary to cheese and yogurt type products, are of interest to the industry. Plant proteases can be a viable approach, provided that excessive proteolysis after structure formation is regulated. High hydrostatic pressure can be used for controlling proteolytic activity in the final products without affecting their texture and sensory characteristics.     

Screening of strains of Lactococci isolated from Egyptian dairy products for their proteolytic activity

A collection of cocci isolates (293) obtained from traditional Egyptian dairy products collected from four Egyptian regions yielded 151 lactic acid bacteria (LAB) cocci isolates. Among them, 24 isolates were characterised as highly proteolytic. SDS–PAGE showed that 6 isolates were the most proteolytically active, which were classified into Enterococcus faecalis HH22 (4 isolates) and Enterococcus faecium DO623 (2 isolates). The proteolytic activity of E. faecalis was higher than that of E. faecium (particularly on β-casein). The maximal degradation of milk proteins was achieved at pH 6.5–7.2 (E. faecalis) or pH 6.5 (E. faecium) and at 42 °C for both strains. The proteolytic activities of the two strains were inhibited mostly by the presence of EDTA, showing that their proteases belong mainly to metalloproteases. A slight inhibition of proteolysis by PMSF in the case of E. faecalis HH22 suggests a limited inclusion of serine proteases in its protease system.     

Sourdough-isolated<Emphasis Type="Italic"> Lactobacillus fermentum</Emphasis> as a potent anti-mould preservative of a traditional Iranian bread

Sourdough bread is an ancient method for making long microbial shelf life and strongly flavoured breads. Traditional sourdough bread-making has been practised for centuries in Iran. Although rural bread-making is still highly reliant on sourdough, urban bakeries usually use bakers yeast and also sodium bicarbonate instead. In this study the anti-mould preservative effects of three lactic acid bacteria isolates were investigated on one of the popular traditional Iranian bread (Lavash). Starter cultures were prepared using 20 h cultures of three sourdough-isolated strains, Lactobacillus casei, Lactobacillus plantarum and Lactobacillus fermentum. Different concentrations (0.5%, 1%, 2%, 4% and 8%) of lactobacillus starter cultures were used to prepare a variety of sourdoughs. Lavash doughs were inoculated with 20% of each lactobacilli-fermented sourdough. After a 20 h incubation at 30 °C, a decrease in pH was observed in the different lactobacilli sourdoughs. However an 8% inoculum of L. fermentum resulted in a significant decrease in pH (3.87 to 2.70). Concentrations of 2% and higher of the three lactobacilli used in Lavash sourdough delayed mould growth during storage. However this preservative effect was more significant when an 8% inoculum of L. fermentum was used. These results tend to suggest that selected strains of lactobacilli have a beneficial role in prevention of bread waste by mould spoilage, and hence could extend bread shelf life.     

Impact of chymosin- and plasmin-mediated primary proteolysis on the growth and biochemical activities of lactobacilli in miniature Cheddar-type cheeses

Strongly proteolytic starters seem to improve the growth of nonstarter lactobacilli during cheese ripening, but no information is available on the impact of the nonmicrobial proteases usually active in cheese on their development. In the current study, the influence of chymosin- and plasmin-mediated proteolysis on the growth and biochemical activities of lactobacilli during ripening of miniature Cheddar-type cheeses, manufactured under controlled microbiological conditions, was studied. Two experiments were performed; in the first, residual chymosin activity was inhibited by the addition of pepstatin, and in the second, plasmin activity was increased by adding more enzyme, obtained in vitro through the activation of plasminogen induced by urokinase. Cheeses with or without a Lactobacillus plantarum I91 adjunct culture and with or without added pepstatin or plasmin solution were manufactured and ripened for 60 d. The addition of the adjunct culture resulted in enhancement of secondary proteolysis, evidenced by an increase in the total content of free amino acids (FAA) and modifications of the individual FAA profiles. Reduction in residual chymosin activity caused a decrease in primary and secondary proteolysis, characterized by the absence of α_s1-casein hydrolysis and reduced production of peptides and FAA, respectively. The increase in plasmin activity accelerated primary proteolysis but no enhancement of secondary proteolysis was observed. Chymosin- and plasmin-mediated proteolysis did not influence the growth and biochemical activities of adventitious or adjunct lactobacilli, indicating that it is not a limiting factor for the development and proteolytic-peptidolytic activities of lactobacilli in the cheese model studied.     

Lactic acid bacteria profiles and tyramine and tryptamine contents of Turkish tulum cheeses

Tulum cheese, commonly produced in Turkey, is a traditional Turkish dairy product made from raw milk. In this research, 20 samples of tulum cheese were purchased from different markets in Isparta and Ankara (Turkey) and the populations of lactic acid bacteria (LAB) and contents of biogenic amines of Turkish tulum cheeses were investigated. The mean counts of LAB (as log10 cfu/g) were 7.22±0.67 on MRS agar, 6.93±1.77 on M17 agar, 7.28±0.72 on APT agar and 5.98±2.20 on ADA agar. Isolates of LAB obtained from 20 tulum cheeses were characterised as lactobacilli (48.5%), enterococci (32.7%), lactococci (15.8%) and streptococci (3.0%). Lactobacillus paracasei ssp. paracasei, Lactobacillus bifermentans and Enterococcus faecium predominated among the LAB in the samples of tulum cheeses; these organisms represented 30.2% of the isolates. As amino acids, tyrosine and tryptophan were determined in tulum cheeses. Tyramine and tryptamine, biogenic amines, were found at 0.00–329.00 and 0.32–40.44 mg/kg, respectively.     

Proteolytic activities and safety of use of <Emphasis Type="Italic">Enterococci</Emphasis> strains isolated from traditional Azerbaijani dairy products

A collection of 147 isolates obtained from 23 samples of traditional Azerbaijani dairy products was screened for the presence of proteolytic enzymes. Six Enterococcus faecalis strains obtained from three cheese samples have been identified as proteinase-producing strains, according to their ability to hydrolyze caseins. RAPD–PCR profiles of their total DNA showed different patterns for strains isolated from different cheese samples. The proteolytic activities of these strains were studied during their growth in milk and in non-proliferative cells system. Isolated strains were able to hydrolyze α_S1-, α_S2-, β-caseins and BLG albeit to different extents, at optimal pH in the range 6.0–7.2 and optimal temperature in the range 37–45 °C, depending on the strain. Proteolysis was strongly inhibited in the presence of EDTA—specific inhibitor of metalloproteases—but the presence of other types of proteases cannot be excluded. The potential pathogenicity of the strains was evaluated investigating the presence of the genes coding different virulence factors and their resistance to antibiotics. The obtained results yield new information about technological characteristics and safety of studied Enterococci strains from Azerbaijani artisanal dairy products. Many from the isolated strains contribute certainly to the differences in flavor, texture, and taste of Azerbaijani traditional cheeses and could represent new adjunct cultures for the dairy industry.     

Production of angiotensin-converting enzyme inhibitory peptides in Iranian ultrafiltered white cheese prepared with Lactobacillus brevis KX572382

In this study, ultrafiltered (UF) Iranian white cheese made with adjunct cultures including six Lactobacillus isolates (Lactobacillus brevis, L. casei and L. plantarum) from traditional Iranian Motal cheese. The peptide extract (<5 kDa) of cheese samples were assessed for angiotensin-converting enzyme (ACE)-inhibitory activity during ripening (5 °C). Among the strains used, L. brevis KX572382 (M8) was selected because of the greater increase in (ACE)-inhibitory activity in the cheese (P < 0.05). The highest activity of M8 extract was observed on the 28th (71.72%) day of ripening (P < 0.05). Proteolytic activity assessment and RP-HPLC peptide profile of M8 water-soluble extracts (WSEs) indicated the effect of M8 on further protein degradation due to secondary proteolysis. A total of 7 different peptide sequences, previously known in the literature for their ACE-inhibitory activity, were tentatively identified by LC/ESI-MS in 28-day M8 peptide extract. Although the effect of M8 on pH and the proteolysis development in cheese was significant, no adverse effect was observed on the sensory properties. In conclusion, M8 strain can enhance the functional properties of Iranian UF white cheese.