Characterization of enterococci isolated from homemade Bulgarian cheeses and katuk

A collection of 107 lactic acid bacteria (LAB) isolates was obtained from traditional Bulgarian dairy products??homemade cheeses and katuk samples, produced from heat-treated cow, goat, ewe and buffalo milk without the addition of any bacterial starter culture. The samples were collected from mountain region of Rhodope (south part of Bulgaria), Tracian valley and mountain region of Stara Planina (west part of Bulgaria). These LAB produced bacteriocin-like inhibitory substances (BLIS) and proteinases. Preliminary strain determination was performed according to their fermentation ability using API 50CHL and API 20 Strep. Most of the characterized strains (58) belong to genus Enterococcus; 21, 20 and 11 strains were identified as Lactobacillus sp., Streptococcus sp. and Lactococcus sp., respectively. Isolated enterococcal strains were characterized using phenotypic features as well as by DNA typing. The strains were identified as Enterococcus faecium (34), Enterococcus durans (22) and Enterococcus faecalis (2). The proteolytic activity varied from 0.094 to 0.455?mM/L Gly into the group of E. faecium and from 0.109 to 0.487?mM/L Gly into the group of E. durans strains, while both E. faecalis strains showed relatively high proteolytic activity. The samples obtained after 3?h of hydrolysis of ??-casein by E. faecalis B1 strain were further used for mass spectrometry analysis, and 31 peptides were identified.     

Lactic acid bacteria isolated from a hand-made blue cheese

Nearly 500 strains isolated from different media used to study the aerobic mesophilic and lactic acid flora of Valdeón cheese (a Spanish hand-made blue cheese) have been identified. Nearly 95% of aerobic mesophiles were lactic acid bacteria (LAB). From these, Enterococcus (40·4%) and Lactococcus (42·2%) were the dominant genera, with Lactobacillus (4·1%) and Leuconostoc (5·0%) being also found. The selectivity of the other media used was variable and this is discussed. Several species of Enterococcus were isolated from our samples (Ent. avium, Ent. faecium and Ent. durans), although one was outstanding (Ent. faecalis, 24·7% of the total of strains identified). The dominating LAB species found was Lact. lactis subsp. lactis (31·1%). Other LAB identified were Lact. raffinolactis, Lb. plantarum, Lb. casei, Leuc. mesenteroides subsp. dextranicum, Leuc. mesenteroides subsp. mesenteroides and Leuc. paramesenteroides. The evolution of the lactic-acid flora found during the manufacture and ripening of this variety of cheese showed a pattern marked by the dominance of lactococci and enterococci during the first stages and the substitution of lactococci by lactobacilli and leuconostoc from drying onwards which would be, along with enterococci, the major genera found in the cheese at the stage of consumption. From a technological point of view, the quantitative importance of certain species such as Lact. lactis and Ent. faecalis, as well as the presence of others, i.e. Lb. plantarum and Leuc. mesenteroides, suggest their possible use as starters in the industrial manufacture of this variety.     

Effect of coagulant type and level on the properties of half-salt,half-fat Cheddar cheese made with or without adjunct starter: Improving texture and functionality

The potential of increasing proteolysis as a means of enhancing the texture and heat-induced flow of half-fat, half-salt Cheddar cheese made with control culture (CL, Lactococcus lactis subsp. cremoris/lactis) or adjunct culture (AC, CL + Lactobacillus helveticus) was investigated. Proteolysis was altered by substituting bovine chymosin (BC) with camel chymosin (CC), or by a 2.5-fold increase in level of BC. In cheese with CL-culture, increasing BC led to a large increase in pH and more rapid degradation of α_S1-casein during maturation, and cheese that was less firm after 180 d. In contrast, substitution of BC with CC in cheeses made with CL-culture had an opposite effect. While chymosin type and level had a similar influence on α_S1-casein hydrolysis in the AC-culture cheeses, it did not affect texture or flowability. Grading indicated that cheese made with AC-culture and with a higher level of BC was the most appealing.     

Screening for differences in the proteolytic systems of Lactococcus, Lactobacillus and Propionibacterium

The caseinolytic, and the endo- and aminopeptidase activities of the intracellular (IC) and cell-envelope-associated (CE) fractions of selected strains of Lactococcus, Lactobacillus and Propionibacterium have been compared. With the exception of one Lactococcus strain, most of the caseinolytic activity of the three genera was located in the IC fraction, as was the case for the amino- and endopeptidases. The lactococci showed low activity on Pro-pNA and high activity on Gly-Pro-pNA, the reverse was characteristic for the propionibacteria (PAB) while lactobacilli took an intermediary position. Lactococcus lactis ssp. cremoris INF-C12 was the strain with the highest total endopeptidase activity. The experiment with phosphoramidon and a peptide inhibitor, β-casein f58–72 (β-CN f58–72), indicated differences of IC endopeptidases of lactic acid bacteria (LAB) and PAB. In contrast to the LAB endopeptidases, the PAB endopeptidases were little inhibited by β-CN f58–72, and were not affected by phosphoramidon. Lysozyme was used to produce spheroplasts from whole cells; however, the susceptibility to such treatment varied. Intracellular material, ≥97%, was easily released from the lactococci strains, for other strains additional sonication was most often necessary for ≥93% lactate dehydrogenase release.     

Employment of autochthonous microflora in Pecorino Sardo cheese manufacturing and evolution of physicochemical parameters during ripening

The isolation and identification of lactic acid bacteria (LAB) from raw ewes’ milk and traditional Pecorino Sardo cheese made from this milk without the addition of starter culture was carried out to define the autochthonous lactic microflora present in milk and the evolution of LAB during cheese ripening. Isolation of 275 strains belonging to different Lactococcus, Lactobacillus, Streptococcus and Enterococcus species was achieved. Coccal-shaped LAB were found to predominate during cheese fermentation, while lactobacilli were preponderate during the latter phase of ripening. The technological selection of a total of 174 LAB strains belonging to the species Lactococcus lactis, Streptococcus thermophilus, Lactobacillus helveticus and Lb. casei allowed an experimental starter to be prepared, in which a potentially probiotic species, Lb. casei was used. The suitability of the autochthonous starter culture was tested in cheese-making trials, using thermised ewes’ milk, by comparing experimental Pecorino Sardo cheese with a control cheese produced at industrial scale using a whey starter culture from previous batches of manufacture. In particular, microbiological and physicochemical parameters were determined over 210 days of cheese ripening. Although sensory evaluation did not show any significant difference between experimental and control Pecorino Sardo cheeses, the use of the selected autochthonous starter allowed the production of experimental cheese with a significantly higher level of free amino acids, in particular essential amino acids, in comparison with the Pecorino Sardo control cheeses.     

Selected lactic acid bacteria as a hurdle to the microbial spoilage of cheese: Application on a traditional raw ewes' milk cheese

To evaluate the efficacy of lactic acid bacteria (LAB) to improve the hygienic safety of a traditional raw milk cheese, the raw ewes' milk protected denomination of origin (PDO) Pecorino Siciliano cheese was used as a model system. Different Pecorino Siciliano curds and cheeses were used as sources of autochthonous LAB subsequently used as starter and non-starter LAB. These were screened for their acidification capacity and autolysis. Starter LAB showing the best performance were genotypically differentiated and identified: two strains of Lactococcus lactis subsp. lactis were selected. From the non-starter LAB, Enterococcus faecalis, Lactococcus garvieae and Streptococcus macedonicus strains were selected. The five cultures were used in individual or dual inocula to produce experimental cheeses in a dairy factory for which production was characterised by high numbers of undesirable bacteria. At 5-month of ripening, the experimental cheeses produced with LAB were characterised by undetectable levels of enterobacteria and pseudomonads and the typical sensory attributes.     

Influence of proteolytic Lactococcus lactis subsp. cremoris on ripening of reduced-fat Cheddar cheese made with camel chymosin

This study investigated proteolysis in reduced-fat Cheddar cheese produced with camel chymosin and Lactococcus lactis subsp. cremoris with the ability to cleave the N-terminus of α_S1-casein. The aim was to match the activity of bovine chymosin, which leads to softer cheese structure than camel chymosin. Cheeses were analysed for gross composition, casein and peptide breakdown, release of free amino acids, structure parameters and sensory characteristics. Selected Lc. lactis subsp. cremoris increased the amount of peptides and, to a limited extent, the total amount of free amino acids in the cheeses. One group of experimental cheeses was found to have a significantly firmer structure, higher stress at fracture and modulus of deformability than the reference cheeses. The addition of the selected proteolytic dairy strains of Lc. lactis subsp. cremoris to the cheeses did not result in extended breakdown of α_S1-casein or a softer cheese structure.     

Influence of a bacteriocin-producing lactic culture on proteolysis and texture of Hispánico cheese

Hispánico cheese was manufactured in duplicate experiments, each consisting of two 50-L vats, and ripened for 75 days. Lactic cultures for experimental cheese were 0.5% Lactococcus lactis subsp. lactis INIA 415 (Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Madrid, Spain), a bacteriocin-producing (Bac⁺) strain harbouring the structural genes of nisin Z and lacticin 481, 0.5% L. lactis subsp. lactis INIA 415-2, a Bac⁻ mutant and 2% TA052, a commercial Streptococcus thermophilus culture. Lactic cultures for control cheese were 1% L. lactis subsp. lactis INIA 415-2 and 2% TA052. S. thermophilus counts were lower, and levels of cell-free intracellular aminopeptidases higher, from day 1 in cheese made with the bacteriocin producer, indicating early lysis of the thermophilic culture. Experimental cheese showed reduced proteolysis of α_s-casein and lower levels of hydrophilic and hydrophobic peptides than control cheese. However, proteolysis as estimated by the o-phthaldialdehyde method and total level of free amino acids were in experimental cheese 1.38- and 2.47-fold, respectively, those in control cheese on day 25, and 1.49- and 2.34-fold, respectively, on day 75. Higher values of fracturability, elasticity and hardness were recorded from day 50 for cheese made with the bacteriocin producer, which were related to its higher residual α_s-casein content. The use of a bacteriocin-producing culture, though retarding α_s-casein proteolysis and softening of texture, enhanced considerably secondary proteolysis during cheese ripening.     

Selection of wild lactic acid bacteria isolated from traditional Egyptian dairy products according to production and technological criteria

A total of 755 wild lactic acid bacteria (LAB), belonging to Lactococcus, Lactobacillus, Enterococcus, Streptcoccus, Leuconostoc and Pediococcus isolated from Egyptian raw milk and its products were screened and selected according to their production and technological properties. Many strains showed high yield of biomass in fermentation and some strains were resistant to lyophilization conditions. Most strains showed a good separation after centrifugation, 2% of Lactococcus, 13% of Lactobacillus and 1% of Enterococcus were fast acidifying strains. Aminopeptidase and autolytic activity were generally higher for most lactobacilli compared to other strains. In milk cultures many strains were able to produce pleasant flavours. Antimicrobial activity was detected in 47% of Lactococcus, 21% of Lactobacillus and 41% of Enterococcus strains and some strains produced exopolysaccharides (slime and capsule). Several strains were able to maintain a high activity of two or three technological characteristics together. Detailed information about the characteristics of each strain is available in the culture collection of Faculty of Agriculture Alexandria University (FAAU). A wide variety of LAB from traditional Egyptian dairy products that showed potentially important properties are not only valuable for practical application but they may also provide an expanded gene pool for designing genetic modified strains with improved traits.     

The biodiversity and evolution of lactic flora during ripening of the Iranian semisoft Lighvan cheese

Ninety‐five isolated strains of Lactic acid bacteria (LAB) were identified from Lighvan cheese. The LAB evolution showed the dominance of lactococci and lactobacilli in the first stage and substitution of these genera by enterococci at the end of ripening. The most predominant strains were Enterococcus faecium (22.44%), Lactococcus lactis ssp. lactis (20.4%), Lactobacillus plantarum (18.36%) and E. faecalis (14.28%), respectively. Eleven and 51 different carbohydrate fermentation profiles were observed according to API 20 STREP and API 50 CH, respectively. API 20 STREP dendogram showed identical fermentation profiles of some E. faecalis and E. faecium strains, indicating that these strains might be well adapted to the whole cheese manufacture.     

Biogenic amine content and proteolysis in Manchego cheese manufactured with Lactobacillus paracasei subsp. paracasei as adjunct and other autochthonous strains as starters

Two different autochthonous strain starter cultures, in which the acidifying starter was composed of strains of Lactococcus lactis, were used for the manufacture of pasteurised milk Manchego cheese. Proteolysis parameters, biogenic amines and sensory characteristics were evaluated and compared with those of commercial starter Manchego cheese and raw milk Manchego cheese manufactured without starter. Autochthonous starter cheeses, and especially those including Lactobacillus paracasei subsp. paracasei as adjunct, presented higher levels of proteolysis than in commercial starter cheese. The concentrations of total biogenic amines in autochthonous starter cheeses were much lower than in raw milk cheese and even lower than in commercial starter cheese. Cheese manufactured with the adjunct strain gave the best results for both flavour intensity and flavour quality, and was the most preferred by panellists. The results suggest that the culture containing Lb. paracasei subsp. paracasei as adjunct could be used for the manufacture of industrial Manchego cheese.     

Enzymatic activity of lactic acid bacteria (with antimicrobial properties) isolated from a traditional Spanish cheese

Twenty-four strains of lactic acid bacteria (LAB) isolated from a traditional Spanish cheese (Genestoso cheese) were evaluated for their enzymatic activities (acidifying and proteolytic abilities and carboxypeptidase, aminopeptidase, dipeptidase, caseinolytic and esterase activities), in order to select indigenous strains of technical interest for the manufacture of cheese. These strains were selected on the basis of their antimicrobial activity relative to five reference strains and were identified as Lactococcus lactis subsp. lactis (thirteen strains), Leuconostoc mesenteroides (two strains), Leuconostoc pseudomesenteroides (one strain), Lactobacillus paracasei (two strains), Lactobacillus plantarum (one strain) and Enterococcus faecalis (five strains).     

In vitro evaluation of physiological probiotic properties of different lactic acid bacteria strains of dairy and human origin

Eleven lactic acid bacteria strains of importance to the dairy industry were subjected to in vitro analyses to determine their probiotic potential. Seven strains were isolated from ewe’s and cow’s milk (Enterococcus faecalis – five –, Lactococcus lactis and Lactobacillus paracasei). Four were obtained from American Type Culture Collection (ATCC), isolated from cheese (Lactobacillus casei 393), human feces (L. paracasei 27092 and Lactobacillus rhamnosus 53103) and used in cheese making (L. lactis 54104). Although none of the strains was able to degrade mucin, all E. faecalis showed, at least, one transferable antibiotic resistance, which excluded them as candidates for addition to foods. Of the remaining six safe strains, L. lactis strains were more tolerant to low pH than Lactobacillus spp.; all were tolerant to pancreatin and bile salts and showed antibacterial activity. The highest level of adhesion to Caco-2 cells was observed with L. lactis 660, even higher than L. rhamnosus ATCC 53103 (recognized probiotic and used as control). The physiological probiotic properties of these strains, mainly isolated from dairy sources, are interesting in view of their use in cheese productions as starter and non starter cultures. The five LAB safe strains studied may have potential as novel probiotics in the dairy foods.     

Differentiation of intracellular peptidases of starter and adjunct cultures using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Selection of starter and adjunct cultures is important to minimize bitterness of Cheddar and Gouda cheeses. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry may be useful for rapid screening of cheese cultures for propensity to produce bitter cheese. The objective of this study was to demonstrate the application of MALDI-TOF for differentiating intracellular peptidase activities of starter and adjunct cultures on β-CN f193-209 under simulated cheese condition. Bovine β-casein was incubated with chymosin in 9.55 g/l citrate buffer (pH 5.4, 40 g/l sodium chloride) at 30°C for 24 h, followed by incubation with cell-free extract (CFE) of starter or adjunct culture. Mixed strains of Lactococcus lactis ssp. lactis and L. lactis ssp. cremoris designated as 56 and 105 were the sources of nonbitter and bitter starter cultures, respectively. Lactobacillus helveticus WSU-19 and W900R represented adjunct cultures having high and low debittering activities, respectively. The degradation pattern of β-CN f193-209 by CFE of WSU-19 indicates general aminopeptidase and endopeptidase activities, while degradation of the peptide by CFE of W900R, 56, and 105 are mainly from endopeptidase activity. The rates of β-CN f193-209 hydrolysis by CFE of WSU-19, W900R, 56, and 105 are 6.90, 0.38, 0.39, and 0.23 mg/l per h, respectively.     

Evaluation of the antihypertensive angiotensin-converting enzyme inhibitory (ACE-I) activity and other probiotic properties of lactic acid bacteria isolated from traditional Greek dairy products

The technological and probiotic potential of lactic acid bacteria isolated from artisanal Greek yoghurt and fermented milks were evaluated. Fifty-three strains were identified by rep-PCR and 16S rDNA sequencing to belong to different Lactobacillus or Enterococcus spp., as well as to Streptococcus thermophilus and Lactococcus lactis. Several strains exhibited promising technological and probiotic properties. Among them, we focused on the production of bioactive peptides with angiotensin-converting enzyme inhibitory (ACE-I) activity during milk fermentation. The majority of strains produced ACE-I peptides when grown in skimmed milk. ACE-I peptides were sometimes sequestered in the original fermented milk sample, but were released and detected following high performance liquid chromatography (HPLC) purification. Mass spectrometry analysis of major peptide peaks in HPLC fractions with ACE-I activity revealed that they derived from the N- or C-terminal of the isracidin peptide region of α_S1-casein and two internal peptide fragments, one from β-casein and one from κ-casein.     

Dynamics of starter,adjunct non-starter lactic acid bacteria and propionic acid bacteria in low-fat and full-fat Dutch-type cheese

The microbial dynamics of Dutch-type cheeses differing in starter (commercial DL starter or single strain of Lactococcus lactis ssp. cremoris), adjunct (Lactobacillus or Propionibacterium) and fat contents (10% or 28% fat) were investigated by culture-dependent and culture-independent analysis. The cheese microbiota was dominated by the adjunct Lactobacillus after 4 weeks of ripening and the fat content did not influence the microbial diversity. The Leuconostoc sp., presumably from the DL starter, was detected in cheeses made with added Lactobacillus plantarum and Lactobacillus rhamnosus and was not detected in cheese made with added Lactobacillus paracasei after 4 and 7 weeks. No Lactobacillus spp. were detected in cheese with added Propionibacterium, while Leuconostoc was the only species detected. In cheeses made with Lc. lactis ssp. cremoris as starter, the Lactobacillus microbiota was similar to the cheese milk microbiota after 24 h while after 4 weeks different species of Lactobacillus and Leuconostoc were detected.     

Investigation of the ability of a purified protease from Pseudomonas fluorescens RO98 to hydrolyze bitter peptides from cheese

The ability of a purified protease from Pseudomonas fluorescens RO98 to hydrolyze bitter peptides found in Cheddar cheese was investigated. The purified protease was incubated with α_s1-casein f1–9 and β-casein f193–209 in a model system (pH 6.8, 30°C) to determine hydrolysis. Residual substrate and hydrolysis products were determined by capillary electrophoresis. Both peptides were hydrolyzed by the protease during the 90-min assay. α_s1-Casein f1–9 was hydrolyzed into two products and β-casein f193–209 was degraded completely in 90 min to three hydrolytic products. This protease hydrolyzed bitter peptides that are known to accumulate in Cheddar and Gouda cheese during aging, suggesting a possibility to debitter Cheddar cheese.     

Diversity of Lactic Acid Bacteria Isolated from Brazilian Water Buffalo Mozzarella Cheese

The water buffalo mozzarella cheese is a typical Italian cheese which has been introduced in the thriving Brazilian market in the last 10 y, with good acceptance by its consumers. Lactic acid bacteria (LAB) play an important role in the technological and sensory quality of mozzarella cheese. In this study, the aim was to evaluate the diversity of the autochthones viable LAB isolated from water buffalo mozzarella cheese under storage. Samples were collected in 3 independent trials in a dairy industry located in the southeast region of Brazil, on the 28th day of storage, at 4 ºC. The LAB were characterized by Gram staining, catalase test, capacity to assimilate citrate, and production of CO₂ from glucose. The diversity of LAB was evaluated by RAPD‐PCR (randomly amplified polymorphic DNA‐polymerase chain reaction), 16S rRNA gene sequencing, and by Vitek 2 system. Twenty LAB strains were isolated and clustered into 12 different clusters, and identified as Streptococcus thermophilus, Enterococcus faecium, Enterococcus durans, Leuconostoc mesenteroides subsp. mesenteroides, Lactobacillus fermentum, Lactobacillus casei, Lactobacillus delbrueckii subsp. bulgaricus, and Lactobacillus helveticus. Enterococcus species were dominant and citrate‐positive. Only the strains of L. mesenteroides subsp. mesenteroides and L. fermentum produced CO₂ from glucose and were citrate‐positive, while L. casei was only citrate positive. This is the first report which elucidates the LAB diversity involved in Brazilian water buffalo mozzarella cheese. Furthermore, the results show that despite the absence of natural whey cultures as starters in production, the LAB species identified are the ones typically found in mozzarella cheese.     

Survival of probiotic adjunct cultures in cheese and challenges in their enumeration using selective media

Various selective media for enumerating probiotic and cheese cultures were screened, with 6 media then used to study survival of probiotic bacteria in full-fat and low-fat Cheddar cheese. Commercial strains of Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus paracasei, or Bifidobacterium lactis were added as probiotic adjuncts. The selective media, designed to promote growth of certain lactic acid bacteria (LAB) over others or to differentiate between LAB, were used to detect individual LAB types during cheese storage. Commercial strains of Lactococcus, Lactobacillus, and Bifidobacterium spp. were initially screened on the 6 selective media along with nonstarter LAB (NSLAB) isolates. The microbial flora of the cheeses was analyzed during 9 mo of storage at 6°C. Many NSLAB were able to grow on media presumed selective for Lactococcus, Bifidobacterium spp., or Lb. acidophilus, which became apparent after 90 d of cheese storage, Between 90 and 120 d of storage, bacterial counts changed on media selective for Bifidobacterium spp., suggesting growth of NSLAB. Appearance of NSLAB on Lb. casei selective media [de man, Rogosa, and Sharpe (MRS) + vancomycin] occurred sooner (30 d) in low-fat cheese than in full-fat control cheeses. Differentiation between NSLAB and Lactococcus was achieved by counting after 18 to 24 h when the NSLAB colonies were only pinpoint in size. Growth of NSLAB on the various selective media during aging means that probiotic adjunct cultures added during cheesemaking can only be enumerated with confidence on selective media for up to 3 or 4 mo. After this time, growth of NSLAB obfuscates enumeration of probiotic adjuncts. When adjunct Lb. casei or Lb. paracasei cultures are added during cheesemaking, they appear to remain at high numbers for a long time (9 mo) when counted on MRS + vancomycin medium, but a reasonable probability exists that they have been overtaken by NSLAB, which also grow readily on this medium. Enumeration using multiple selective media can provide insight into whether it is the actual adjunct culture or a NSLAB strain that is being enumerated.     

FTIR-based polyphasic identification of lactic acid bacteria isolated from traditional Greek Graviera cheese

This study used a combination of phenotypic, physical (Fourier Transformed Infra-Red [FTIR] spectroscopy) and molecular (RFLP and SSCP analysis of 16S rRNA genes) methods to identify the lactic acid bacteria (LAB) flora present in traditional Greek Graviera cheese after five weeks of ripening. A total of 300 isolates collected from high dilution plates of TSAYE (incubated at 30 °C), M-17 (22 °C) and M-17 (42 °C) agar media were clustered by FTIR and then representative strains of each cluster were cross-identified blindly by all methods. Based on their FTIR spectra, 282 isolates were LAB grouped in 28 clusters. The LAB species identified and their prevalence in the cheese samples were: Lactobacillus casei/paracasei (68.8%), Lactobacillus plantarum (19.5%), Streptococcus thermophilus (8.9%), Enterococcus faecium (2.1%), and Lactococcus lactis (0.7%). Also, Staphylococcus equorum (11 isolates), Corynebacterium sp. (5 isolates) and Brevibacterium sp. (1 isolate) were recovered from TSAYE. Comparative identification results showed that phenotypic and molecular methods were in mutual agreement as regards the LAB species identified. The present polyphasic identification approach based on rapid FTIR screening of 10-fold more isolates than a previous classical identification approach allowed or improved detection of few sub-dominant species; however the predominant LAB species in the cheese samples were the same with both approaches.