Preliminary characterization of lactic acid bacteria isolated from Zlatar cheese

AIMS: Isolation, characterization and identification of lactic acid bacteria (LAB) from artisanal Zlatar cheese during the ripening process and selection of strains with good technological characteristics. METHODS AND RESULTS: Characterization of LAB was performed based on morphological, physiological and biochemical assays, as well as, by determining proteolytic activity and plasmid profile. rep-polymerase chain reaction (PCR) analysis and 16S rDNA sequencing were used for the identification of LAB. PCR analysis was performed with specific primers for detection of the gene encoding nisin production. Strains Lactobacillus paracasei subsp. paracasei, Lactobacillus plantarum, Lactobacillus brevis, Lactococcus lactis subsp. lactis, Enterococcus faecium and Enterococcus faecalis were the main groups present in the Zlatar cheese during ripening. CONCLUSIONS: Temporal changes in the species were observed during the Zlatar cheese ripening. Mesophilic lactobacilli are predominant microflora in Zlatar cheese. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study we determined that Zlatar cheese up to 30 days old could be used as a source of strains for the preparation of potential starter cultures in the process of industrial cheese production. As the Serbian food market is adjusting to European Union regulations, the standardization of Zlatar cheese production by using starter culture(s) based on autochtonous well-characterized LAB will enable the industrial production of this popular cheese in the future.     

The spatial distribution of bacteria in Grana-cheese during ripening

The microbial composition and its spatial distribution of Grana Trentino, a hard Parmesan-like cheese, was determined, from vat milk to cheese. After cutting along the vertical axis of the cheese wheels, three layers were sampled diagonally across the cheese: under the cheese rind, an intermediate section and the cheese core. After two different ripening periods (9 and 18 months), the cheese samples were analysed using traditional culture dependent and culture independent methods. Milk samples were dominated by mesophilic and psychrophilic bacterial counts. Thermophilic bacteria (Lactobacillus helveticus) were found in high amounts in cooked whey and natural whey starter cultures. After 9 months of ripening, lactic acid bacteria (LAB) counts were higher than those after 18 months. Furthermore, the LAB numbers in the cheese core was lower than those under the rind or in the intermediate section. The main LAB species isolated from milk (Lactococcus lactis, Pediococcus pentosaceus, Streptococcus uberis and Lactococcus garvieae) were not found in the corresponding cheeses. Some differences were observed in the species composition among the three cheese sections. Microbiota under the rind and in the intermediate section was similar and dominated by Lactobacillus paracasei and Lactobacillus rhamnosus. The core, after 18 months of ripening, was characterized by a total absence of LAB. In each sample, all LAB were genotypically grouped and the different biotypes were subjected to several technological tests indicating that some non-starter LAB (NSLAB) displayed technological features that are favorable for the production of Grana Trentino cheese.     

Microbiota of the <Emphasis Type="Italic">Planalto de Bolona</Emphasis>: an artisanal cheese produced in uncommon environmental conditions in the Cape Verde Islands

The present study aimed to evaluate the dominant microbial community naturally present in the Planalto de Bolona cheese, produced in the Cape Verde Islands. Samples of milk, curd and cheese from two different producers were examined through culture-dependent and independent-methods. Traditional plating and genetic identification of lactic acid bacteria (LAB) and yeast isolates were carried out. Moreover, DNA and RNA extracted directly from samples were subjected to Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis (PCR-DGGE). Concerning the LAB population, a total of 278 isolates were identified: Lactococcus lactis subsp. lactis and Enterococcus faecium represented the most isolated species. Regarding yeasts, the analysis of isolates throughout the manufacturing period showed a consistent presence of the genus Candida. Divergences in species detection between culture-dependent and culture-independent methods were observed, as well as between DNA and RNA analysis. PCR-DGGE underlined high heterogeneity among bacterial species while yeast microbiota was dominated by Aureobasidium pullulans at DNA level. The obtained results represent a first approach in the understanding of the Planalto de Bolona cheese microbial ecology and consequently may constitute a first step towards the full comprehension of the microbiota of this artisanal cheese produced in unusual environmental conditions in the Cape Verde Islands.     

Presence of halophilic and alkaliphilic lactic acid bacteria in various cheeses

AIM: We sought to confirm the presence of halophilic and alkaliphilic lactic acid bacteria (HALAB) of marine origin in cheeses and thus contribute to the understanding of the roles of LAB flora in cheese ripening. METHODS AND RESULTS: We used 7% NaCl glucose-yeast extract-peptone-fish extract broth and agar media (pH 9.5) for pour-plating and enrichment culture for 16 cheese samples produced in six European countries. HALAB were present in 9 of the 16 samples at < 20 --> 10(7) CFU g(-1). In three mould-ripened soft cheeses, HALAB counts ranged from 10(6) to 10(7) CFU g(-1) and were one order (two samples) and six orders (one sample) of magnitude greater than that of nonhaloalkaliphilic, common LAB, as enumerated on lactobacilli MRS agar. The 16S rRNA gene sequences (500 bp) of 51 of the 55 isolates examined were identical or similar to that of Marinilactibacillus psychrotolerans or Alkalibacterium olivapovliticus and related species, all of which are HALAB. CONCLUSIONS: HALAB of possible marine origin were present in various soft, semi-hard and semi-soft cheeses and were highly predominant in some mould-ripened cheeses. Significance AND IMPACT OF THE STUDY: HALAB of possible marine origin are members of the microflora of various cheeses and, when dominant, may play a role in the ripening of cheeses. Microbial analysis of LAB flora in cheeses should take into consideration the presence of HALAB.     

Glutamate dehydrogenase activity: a major criterion for the selection of flavour-producing lactic acid bacteria strains

Lactic acid bacteria (LAB) have the enzyme potential to transform amino acids into aroma compounds that contribute greatly to cheese flavour. Generally, amino acid conversion by LAB is limited by their low production of -ketoglutarate since this -ketoacid is essential for the first step of the conversion. Indeed, we have demonstrated that adding exogenous -ketoglutarate to cheese curd, as well as using a genetically modified L. lactis strain capable of producing -ketoglutarate from glutamate, greatly increased the conversion of amino acid to potent aroma compounds in cheese. Here we report the presence of glutamate dehydrogenase (GDH) activity required for the conversion of glutamate to -ketoglutarate in several natural LAB strains, commonly used in cheese manufacturing. Moreover, we show that the ability of LAB to produce aroma compounds from amino acids is closely related to their GDH activity. Therefore, GDH activity appears to be a major criterion for the selection of flavour-producing LAB strains, which could be used as a starter or as an adjunct to intensify flavour formation in some cheeses.     

Microbial Activation of Wooden Vats Used for Traditional Cheese Production and Evolution of Neoformed Biofilms

Three Lactococcus lactis subsp. cremoris strains were used to develop ad hoc biofilms on the surfaces of virgin wooden vats used for cheese production. Two vats (TZ) were tested under controlled conditions (pilot plant), and two vats (TA) were tested under uncontrolled conditions (industrial plant). In each plant, one vat (TA1 and TZ1) was used for the control, traditional production of PDO Vastedda della Valle del Belìce (Vastedda) cheese, and one (TA2 and TZ2) was used for experimental production performed after lactococcal biofilm activation and the daily addition of a natural whey starter culture (NWSC). Microbiological and scanning electron microscopy analyses showed differences in terms of microbial levels and composition of the neoformed biofilms. The levels of the microbial groups investigated during cheese production showed significant differences between the control trials and between the control and experimental trials, but the differences were not particularly marked between the TA2 and TZ2 productions, which showed the largest numbers of mesophilic lactic acid bacterium (LAB) cocci. LAB populations were characterized phenotypically and genotypically, and 44 dominant strains belonging to 10 species were identified. Direct comparison of the polymorphic profiles of the LAB collected during cheese making showed that the addition of the NWSC reduced their biodiversity. Sensory evaluation showed that the microbial activation of the wooden vats with the multistrain Lactococcus culture generated cheeses with sensory attributes comparable to those of commercial cheese. Thus, neoformed biofilms enable a reduction of microbial variability and stabilize the sensorial attributes of Vastedda cheese.     

Determination of staphylococcal enterotoxin A in cheddar cheese produced without starter activity.

Three variants of the chloramine-T radioiodination method were used to iodinate staphylococcal enterotoxin A with 125I. Only one method consistently produced usable labels for radioimmunoassay. The iodine incorporation was 55 to 76%; the specific activity was 3.5 to 5.5 muCi/microgram of enterotoxin, and the label was extremely stable on storage at -20 degrees C. Determinations of the enterotoxin in extracts of cheddar cheese produced without starter activity were carried out with the radioimmunoassay system and protein A as antibody immunoadsorbent. The assay buffer used in this system significantly influenced the detected levels of enterotoxin in the cheese extracts. Phosphate buffer, but not tris(hydroxymethyl)aminomethane (Tris) buffer, caused gelling of cheese extract proteins, thus resulting in an incomplete separation of free from antibody-bound 125I enterotoxin. When Tris buffer was used, the results indicated a high degree of accuracy and precision for this radioimmunoassay. The lowest detectable enterotoxin concentration in cheese extract was 0.5 ng/ml.     

Cooperation between Lactococcus lactis and nonstarter lactobacilli in the formation of cheese aroma from amino acids

In Gouda and Cheddar type cheeses the amino acid conversion to aroma compounds, which is a major process for aroma formation, is essentially due to lactic acid bacteria (LAB). In order to evaluate the respective role of starter and nonstarter LAB and their interactions in cheese flavor formation, we compared the catabolism of phenylalanine, leucine, and methionine by single strains and strain mixtures of Lactococcus lactis subsp. cremoris NCDO763 and three mesophilic lactobacilli. Amino acid catabolism was studied in vitro at pH 5.5, by using radiolabeled amino acids as tracers. In the presence of alpha-ketoglutarate, which is essential for amino acid transamination, the lactobacillus strains degraded less amino acids than L. lactis subsp. cremoris NCDO763, and produced mainly nonaromatic metabolites. L. lactis subsp. cremoris NCDO763 produced mainly the carboxylic acids, which are important compounds for cheese aroma. However, in the reaction mixture containing glutamate, only two lactobacillus strains degraded amino acids significantly. This was due to their glutamate dehydrogenase (GDH) activity, which produced alpha-ketoglutarate from glutamate. The combination of each of the GDH-positive lactobacilli with L. lactis subsp. cremoris NCDO763 had a beneficial effect on the aroma formation. Lactobacilli initiated the conversion of amino acids by transforming them mainly to keto and hydroxy acids, which subsequently were converted to carboxylic acids by the Lactococcus strain. Therefore, we think that such cooperation between starter L. lactis and GDH-positive lactobacilli can stimulate flavor development in cheese.     

Resident lactic acid bacteria in raw milk Canestrato Pugliese cheese

AIMS: Investigation of the autochthonous lactic acid bacteria (LAB) population of the raw milk protected designation of origin Canestrato Pugliese cheese using phenotypic and genotypic methodologies. METHODS AND RESULTS: Thirty phenotypic assays and three molecular techniques (restriction fragment length polymorphism, partial sequencing of the 16S rRNA gene and recA multiplex PCR assay) were applied to the identification of 304 isolates from raw milk Canestrato Pugliese cheese. As a result, 168 of 207 isolates identified were ascribed to genus Enterococcus, 25 to Lactobacillus, 13 to Lactococcus and one to Leuconostoc. More in details among the lactobacilli, the species Lactobacillus brevis and Lactobacillus plantarum were predominant, including 13 and 10 isolates respectively, whereas among the lactococci, Lactococcus lactis subsp.cremoris [corrected] was the species more frequently detected (seven isolates). CONCLUSIONS: Except for the enterococci, phenotypic tests were not reliable enough for the identification of the isolates, if not combined to the genotype-based molecular techniques. The polyphasic approach utilized allowed 10 different LAB species to be detected; thus suggesting the appreciable LAB diversity of the autochthonous microbial population of the Canestrato Pugliese cheese. SIGNIFICANCE AND IMPACT OF THE STUDY: A comprehensive study of the resident raw milk Canestrato Pugliese cheese microbial population has been undertaken.     

Antimicrobial activity of lactic acid bacteria isolated from Tenerife cheese: initial characterization of plantaricin TF711, a bacteriocin-like substance produced by Lactobacillus plantarum TF711

AIMS: The screening and initial characterization of bacteriocins produced by lactic acid bacteria (LAB) from raw Tenerife goats' cheese with possible application as biopreservatives or ripening accelerators for Tenerife cheese. METHODS AND RESULTS: One hundred and eighty LAB of the genera Lactobacillus (95), Leuconostoc (64) and Lactococcus (21) isolated from raw Tenerife goats' cheese were screened for the production of antimicrobial substances. Lactobacillus plantarum TF711, which had the broadest spectrum of antimicrobial activity, was selected for further characterization. The antimicrobial compound was determined as a proteinaceous substance, as it was sensitive to proteases. The bacteriocin-like substance, which we called plantaricin TF711, was active against the Gram-positive bacteria Bacillus cereus, Clostridium sporogenes and Staphylococcus aureus; and against the Enterobacteriaceae Shigella sonnei and Klebsiella pneumoniae. It was stable to heat and to treatment with surfactants and organic solvents. Highest antimicrobial activity was found between pH 1 and 9. Plantaricin TF711 exhibited primary metabolite kinetics, a bacteriostatic mode of action and a molecular mass of c. 2.5 kDa as determined by tricine SDS-PAGE. CONCLUSIONS: Lact. plantarum TF711 produces a low molecular mass bacteriocin-like compound with a wide spectrum of activity and interesting technological properties (thermostability, good pH stability and stability against surfactants and organic solvents). SIGNIFICANCE AND IMPACT OF THE STUDY: Plantaricin TF711 was found to have potential for use as a biopreservative in the food industry.     

Phenotypic, genotypic and technological characterization of predominant lactic acid bacteria in Pecorino cheese from central Italy

AIMS: Identification and biotyping of lactic acid bacteria (LAB) isolated from raw-milk Pecorino cheese manufactured in the Marche region (central Italy) for selection of suitable starter cultures or adjuncts. METHODS AND RESULTS: Preliminary characterization with morphological and biochemical assays were undertaken for 112 Gram-positive and catalase-negative isolates. Unequivocal identification of the isolates was obtained through restriction analysis of the amplified 16S rRNA gene and sequencing of 360-380 bp amplicons. Fifty-nine isolates belonging to LAB species generally recognized as safe and potentially utilized as starters or flavour-producing adjuncts were preselected and tested for their acidifying, proteolitic and autolytic activities. Fifty-five of these isolates were also subject to RAPD (randomly amplified polymorphic DNA) fingerprinting and unweighted pair-group method with arithmetic averages (UPGMA) cluster analysis for the estimation of genotypic intra-species variation. As a result, in Pecorino cheese, a heterogeneous lactic acid bacteria population, which includes strains with metabolic characteristics of technological interest, was characterized. CONCLUSIONS: The polyphasic approach proposed allows the bacterial ecology of Pecorino cheese to be investigated and allows to assess the potential role of autochthonous LAB strains for the dairy industry. SIGNIFICANCE AND IMPACT OF THE STUDY: The great economic importance of Pecorino cheese encouraged a deeper knowledge of its microbiota, which is known to influence the peculiar sensory properties of this cheese, also in view of its exploitation.     

Quantitative analysis of histidine decarboxylase gene (hdcA) transcription and histamine production by Streptococcus thermophilus PRI60 under conditions relevant to cheese making

This study evaluated the influence of parameters relevant for cheese making on histamine formation by Streptococcus thermophilus. Strains possessing a histidine decarboxylase (hdcA) gene represented 6% of the dairy isolates screened. The most histaminogenic, S. thermophilus PRI60, exhibited in skim milk a high basal level of expression of hdcA, upregulation in the presence of free histidine and salt, and repression after thermization. HdcA activity persisted in cell extracts, indicating that histamine might accumulate after cell lysis in cheese.     

Detection of resistance of lactic acid bacteria to a mixture of the hop analogue compounds tetrahydroiso-alpha-acids by noninvasive measurement of intracellular pH

AIMS: To examine the resistance of beer isolates of lactic acid bacteria (LAB) towards a mixture of tetrahydroiso-alpha-acids (Tetra) by growth experiments as well as by measurement of intracellular pH. METHODS AND RESULTS: Beer LAB isolates were identified to species level by SDS-PAGE of whole-cell proteins. Beer isolates of Lactobacillus brevis showed better ability for growth in the presence of Tetra than nonbeer isolates of the L. brevis or other species of LAB including beer and nonbeer isolates. The antimicrobial effect of Tetra was also examined by noninvasive measurement of intracellular pH by fluorescence ratio imaging microscopy for selected beer isolates of L. brevis and Pediococcus inopinatus. Strains of L. brevis showing limited decrease of intracellular pH during exposure to Tetra also showed better ability for growth in the presence of these compounds as well as in commercial beer products. CONCLUSIONS: It was possible to apply a method for noninvasive measurement of intracellular pH to predict the resistance of beer spoilage LAB towards the Tetra hop analogue compounds. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated the usability of a new rapid method for detecting hop-resistant variants of known beer spoilage LAB species.     

Microbiological profile in Serra ewes' cheese during ripening

The microflora of Serra cheese was monitored during a 35 d ripening period at three different periods within the ewe's lactation season. After 7 d ripening, the numbers of micro-organisms reached their maximum, and lactic acid bacteria (LAB) and coliforms were the predominant groups. Pseudomonads were not detected after 1 week of ripening. At all stages of ripening, cheeses manufactured in spring exhibited the lowest numbers of LAB and yeasts, whereas cheeses manufactured in winter showed the lowest numbers of coliforms and staphylococci.
Leuconostoc lactis was the most abundant LAB found in Serra cheese whereas Enterococcus faecium and Lactococcus lactis spp. lactis exhibited the highest decrease in percentage composition. Numbers of both Leuc. mesenteroides and Lactobacillus paracasei tended to increase throughout ripening. The most abundant coliform was Hafnia alvei. Klebsiella oxytoca was found in curd but declined in number during ripening. Staphylococcal flora of curd was mainly composed of Staphylococcus xylosus, Staph. aureus and Staph. epidermidis. Staphylococcus xylosus was the major species found at the end of ripening. Pseudomonas fluorescens , was the only Pseudomonas species isolated from the curd. Although a broad spectrum of yeasts were found in Serra cheese, Sporobolomyces roseus was the most abundant yeast isolated.     

Assessment of the immunomodulatory activity of cheese extracts by a complete and easy to handle <Emphasis Type="Italic">in</Emphasis><Emphasis Type="Italic">vitro</Emphasis> screening methodology

A simple and rapid screening methodology based on the in vitro culture of murine hybridoma and human T-lymphocytes was developed to assess the potential immunomodulatory activity of water-soluble extracts (WSE) from cheese. The two immune cell lines were cultured in microplates with or without cheese WSE. The proliferation and the metabolic activity of cells were monitored at their different growth phases by the BrdU (5-bromo-2′-deoxyuridine) and MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylterazolium bromide] assays, respectively. WSE from Abondance cheese enhanced DNA synthesis and the metabolic activity of the hybridoma cells (1.2-and 1.3-to 1.6-fold of the control, respectively) and also of the T lymphocytes (1.5-fold of the control) for almost all of the dilutions tested. To evaluate the validity of the results obtained following this screening methodology, hybridoma and T-lymphocyte cells were cultivated in 50 ml flasks: the maximal cell density was increased by about 10–16% in presence of cheese WSE for both cell lines and the antibody production by the hybridoma cells was increased by 50%.     

Diversity of amino acid converting enzymes in wild lactic acid bacteria

A total of 156 lactic acid bacteria isolates belonging to the genera Lactococcus, Lactobacillus and Leuconostoc were analysed for the amino acid converting enzymes aminotransferases, glutamate dehydrogenase, and α-ketoisovalerate decarboxylase. All isolates showed aminotransferase activity towards phenylalanine (substrate for the aromatic aminotransferase AraT) and isoleucine (substrate for the branched-chain aminotransferase BcaT). Although there was a high variability inter- and intra-species, the lactococcal strains showed the highest values for both aminotransferase activities. Moreover, α-ketoisovalerate decarboxylase (Kivd) activity was only found in lactococcal isolates, although at low relative numbers (16%). On the other hand, glutamate dehydrogenase (Gdh) activity values were highest in facultative heterofermentative lactobacilli (FHL) and the activity was found at high relative numbers (50%) in leuconostocs. Results showed a high variability in amino acid convertase activities within the wild LAB isolates assayed, therefore the utilisation in the dairy industry of new strains with high flavour-forming abilities could be a powerful tool to enhance cheese aroma development.     

Identification by fluorescence spectroscopy of lactic acid bacteria isolated from a small-scale facility producing traditional dry sausages

Three different fluorescence spectra were recorded following excitation at 250 nm (aromatic amino acids+nucleic acids, AAA+NA), 316 nm (NADH) and 380 nm (FAD) for 20 type strain collections of lactic acid bacteria (LAB). Evaluation of the data using principal component analysis and factorial discriminant analysis showed a good discrimination of considered LAB at the genus, species and genus-species level. AAA+NA fluorophores showed the highest percentage of good classification. From AAA+NA spectra recorded on LAB isolated from a small-scale facility producing traditional dry sausages, we succeeded to identify 28 of 29 wild strains. This method allowed us to discriminate between Lactobacillus sakei subsp. carnosus and Lactobacillus sakei subsp. sakei. Thus, intrinsic fluorescence is an economical and powerful tool for the identification of wild LAB isolated from meat and meat products.     

TASTE COMPONENTS OF CHEDDAR CHEESE: FRACTIONATION AND OPTIMIZATION OF CHEDDAR CHEESE TASTE IN WATER

To determine the taste components of Cheddar cheese, we fractionated one mild Cheddar cheese and one aged Cheddar cheese by water extraction, freeze‐drying and gel filtration. Salty, sour and umami were the three predominant tastes present in the fractions. Neither trigeminal sensations nor astringency was perceived. We used response surface methodology to reconstruct a mild Cheddar cheese taste and an aged Cheddar cheese taste in water. Less salt and less acid were needed to simulate the taste of mild Cheddar cheese, compared with aged Cheddar cheese. Our optimized water solutions (containing sodium chloride, lactic acid and monosodium glutamate) were as similar to the real cheese samples as were the water extracts of the standard cheeses. However, neither our optimized solutions nor the water extracts matched the taste of the actual cheeses.     

Technological properties of Enterococcus faecium isolated from ewe's milk and cheese with importance for flavour development

Eight Enterococcus faecium strains isolated from ewe milk and artisanal cheese from northwest Argentina were screened for biotechnological properties relevant to flavour development. The API ZYM test showed absence of proteases, presence of high amounts of peptidases, and high esterase-lipase activities. Low extracellular proteolytic activity was observed. Most strains produced diacetyl in milk, with E. faecium OvL 214 and OvL 254 being the best producers. Biomass and growth rate increased when citrate was added to the medium, suggesting that these strains could use citrate as a main energy source. After 24 h of incubation, citrate was completely consumed in complex medium supplemented with glucose and citrate. An average of 17% residual citrate was detected in complex media supplemented with citrate. For all strains, esterase activity was detected up to alpha-naphthyl-caproate. They hydrolyzed alpha-naphthyl derivatives of fatty acids in this order: C3 > C6 > C4 > C8 > C2. Post-electrophoretic detection of esterase activities revealed the presence of multiple esterases. Hydrolysis of tributyrin, tricaprylin, and milk fat was observed in cell-free extracts. Enterococcus faecium strains isolated from ewe milk and artisanal cheese from northwest Argentina present the metabolic potential to contribute to cheese flavour development.