RESUMO
Lactic acid bacteria (LAB) are pivotal in shaping the technological, sensory, and safety aspects of dairy products. The evaluation of proteolytic activity, citrate utilization, milk pH reduction, and the production of organic compounds, acetoin, and diacetyl by cheese associated LAB strains was carried out, followed by Principal Component Analysis (PCA). Citrate utilization was observed in all Leuconostoc (Le.) mesenteroides, Le. citreum, Lactococcus (Lc.) lactis, Lc. garvieae, and Limosilactobacillus (Lm.) fermentum strains, and in some Lacticaseibacillus (Lact.) casei strains. Most strains exhibited proteolytic activity, reduced pH, and generated organic compounds. Multivariate PCA revealed Le. mesenteroides as a prolific producer of acetic, lactic, formic, and pyruvic acids and acetoin at 30 °C. Enterococcus sp. was distinguished from Lact. casei based on acetic, formic, and pyruvic acid production, while Lact. casei primarily produced lactic acid at 37 °C. At 42 °C, Lactobacillus (L.) helveticus and some L. delbrueckii subsp. bulgaricus strains excelled in acetoin production, whereas L. delbrueckii subsp. bulgaricus and Streptococcus (S.) thermophilus strains primarily produced lactic acid. Lm. fermentum stood out with its production of acetic, formic, and pyruvic acids. Overall, cheese-associated LAB strains exhibited diverse metabolic capabilities which contribute to desirable aroma, flavor, and safety of dairy products.
RESUMO
The biodiversity and succession of lactic acid bacteria (LAB) involved in the production and storage of Brazilian buffalo mozzarella cheese were evaluated. The isolates were characterized by Gram staining and catalase test, by the ability to grow at different conditions: temperatures, pH, concentrations of NaCl, and production of CO2 from glucose. The biodiversity and succession of 152 LAB isolated during cheese production were evaluated by 16S rRNA gene sequencing, Random Amplified Polymorphic DNA (RAPD-PCR), and Restriction Fragment Length Polymorphism (RFLP-PCR) techniques. Most of the strains grow well at 30 °C and are tolerant to 6.5% of NaCl, and in general, the best pH for growing was 9.6. Leuconostoc mesenteroides, Lacticaseibacillus casei, Limosilactobacillus fermentum, and Enterococcus sp. were prevalent and present in almost all steps of production. The LAB strains are typically found in the traditional Italian cheese, except the Leuconostoc citreum species. Sixty clusters were obtained by RAPD-PCR with 85% of similarity (114 isolates) while most of the LAB was clustered with 100% of similarity by the RFLP-PCR technique. The applied techniques enabled a valuable elucidation of the LAB biodiversity and succession, contributing to a better understanding of the specific microbial cultures with a technological aptitude of this cheese.
Assuntos
Queijo , Microbiota , Animais , Biodiversidade , Búfalos , Queijo/microbiologia , Microbiologia de Alimentos , Leite/microbiologia , RNA Ribossômico 16S/genética , Técnica de Amplificação ao Acaso de DNA PolimórficoRESUMO
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.
Assuntos
Búfalos , Queijo/microbiologia , Microbiologia de Alimentos , Lactobacillus/genética , Animais , Brasil , Queijo/análise , DNA Bacteriano/análise , Enterococcus/genética , Humanos , Ácido Láctico , Lactobacillus/isolamento & purificação , Leuconostoc/genética , Reação em Cadeia da Polimerase/métodos , RNA Ribossômico 16S , Técnica de Amplificação ao Acaso de DNA Polimórfico , Streptococcus thermophilus/isolamento & purificaçãoRESUMO
This study evaluated the probiotic properties (stability at different pH values and bile salt concentration, auto-aggregation and co-aggregation, survival in the presence of antibiotics and commercial drugs, study of ß-galactosidase production, evaluation of the presence of genes encoding MapA and Mub adhesion proteins and EF-Tu elongation factor, and the presence of genes encoding virulence factor) of four LAB strains (Lactobacillus casei SJRP35, Leuconostoc citreum SJRP44, Lactobacillus delbrueckii subsp. bulgaricus SJRP57 and Leuconostoc mesenteroides subsp. mesenteroides SJRP58) which produced antimicrobial substances (antimicrobial peptides). The strains survived the simulated GIT modeled in MRS broth, whole and skim milk. In addition, auto-aggregation and the cell surface hydrophobicity of all strains were high, and various degrees of co-aggregation were observed with indicator strains. All strains presented low resistance to several antibiotics and survived in the presence of commercial drugs. Only the strain SJRP44 did not produce the ß-galactosidase enzyme. Moreover, the strain SJRP57 did not show the presence of any genes encoding virulence factors; however, the strain SJRP35 presented vancomycin resistance and adhesion of collagen genes, the strain SJRP44 harbored the ornithine decarboxylase gene and the strain SJRP58 generated positive results for aggregation substance and histidine decarboxylase genes. In conclusion, the strain SJRP57 was considered the best candidate as probiotic cultures for further in vivo studies and functional food products development.
Assuntos
Queijo/microbiologia , Lactobacillaceae/metabolismo , Animais , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bovinos , Fermentação , Lactobacillaceae/efeitos dos fármacos , Lactobacillaceae/genética , Lactobacillaceae/crescimento & desenvolvimento , Leite/microbiologia , Probióticos/química , Probióticos/metabolismoRESUMO
The production of bacteriocins by Leuconostoc mesenteroides represents an important opportunity for exploration of their potential use for industrial purpose. The antimicrobial compounds produced by L. mesenteroides subsp. mesenteroides SJRP55 strain were characterized and purified. Cell-free supernatant of Leuc. mesenteroides subsp. mesenteroides SJRP55 produced antibacterial compounds against Listeria spp. strains and not inhibiting against Lactobacillus spp. The antimicrobial substances were stable at high temperatures (100 °C for 2 h and 121 °C for 20 min) and low pH (pH 2-4) values, but sensitive to proteolytic enzymes and resistant to α-amylase, lipase and catalase enzymes. The optimal temperature for active peptides production was 25 °C. The antimicrobial compounds were purified by ammonium sulfate precipitation, affinity column and reverse-phase chromatography. Mass spectrometry and amino acids analyses showed that the bacteriocins were identical to mesentericin Y105 and B105. The producer strain's DNA analysis revealed presence of open reading frames possibly coding for virulence factors, such as enterococcal surface protein (esp), collagen adhesion (ace) and intrinsic vancomycin resistance (vanA); however, biogenic amines encoding genes were not observed. Leuc. mesenteroides subsp. mesenteroides SJRP55 is a promising biopreservative culture in fermented milk, and the purified bacteriocins can also be applied in food preservation.