Microbiota of Minas cheese as influenced by the nisin producer Lactococcus lactis subsp. lactis GLc05

Minas cheese is a popular dairy product in Brazil that is traditionally produced using raw or pasteurized cow milk. This study proposed an alternative production of Minas cheese using raw goat milk added of a nisin producer Lactococcus lactis subsp. lactis GLc05. An in situ investigation was carried on to evaluate the interactions between the L. lactis subsp. lactis GLc05 and the autochthonous microbiota of a Minas cheese during the ripening; production of biogenic amines (BA) was assessed as a safety aspect. Minas cheese was produced in two treatments (A, by adding L. lactis subsp. lactis GLc05, and B, without adding this strain), in three independent repetitions (R1, R2, and R3). Culture dependent (direct plating) and independent (rep-PCR and PCR-DGGE) methods were employed to characterize the microbiota and to assess the possible interferences caused by L. lactis subsp. lactis GLc05. BA amounts were measured using HPLC. A significant decrease in coagulase-positive cocci was observed in the cheeses produced by adding L. lactis subsp. lactis GLc05 (cheese A). The rep-PCR and PCR-DGGE highlighted the differences in the microbiota of both cheeses, separating them into two different clusters. Lactococcus sp. was found as the main microorganism in both cheeses, and the microbiota of cheese A presented a higher number of species. High concentrations of tyramine were found in both cheeses and, at specific ripening times, the BA amounts in cheese B were significantly higher than in cheese A (p < 0.05). The interaction of nisin producer L. lactis subsp. lactis GLc05 was demonstrated in situ, by demonstration of its influence in the complex microbiota naturally present in a raw goat milk cheese and by controlling the growth of coagulase-positive cocci. L. lactis subsp. lactis GLc05 influenced also the production of BA determining their amounts in the cheeses were maintained at acceptable levels for human consumption

In vitro evaluation of the safety and probiotic and technological potential of Pediococcus pentosaceus isolated from sheep milk

Six isolates (Ac1Pd, Ac3Pd, Ac4Pd, Ac5Pd, Ac7Pd, and Ac22Pd) of Pediococcus pentosaceus from sheep milk were tested for safety and for probiotic and technological potential. The results showed that none of the isolates were able to produce biogenic amines or virulence factors. The isolates tested showed low hydrophobicities, high auto-aggregation capacities and co-aggregation with L. monocytogenes ATCC 7644, L. sakei ATCC 15521 and E. faecalis ATCC 19444, but none produced ?-galactosidase and bacteriocins. The isolates did not show growth at pH values 3 and 12, while in a pH range from 4 to 10 the growth was variable. In the absence of bile, all the isolates showed growth, with suppression at bile concentrations of 0.1%, 0.3%, 0.6% and 1.0%. In the disc-diffusion test, the isolates tested were resistant to oxacillin, sulfatrimethoprim and vancomycin but were sensitive to chloramphenicol and tetracycline. The isolates showed variable responses to penicillin G and were resistant to most of the drugs tested, except for amoxicillin trihydrate and ibuprofen. All cultures showed a high milk-acidification capacity after 24 hours and none produced exopolysaccharides. The isolates of P. pentosaceus were able to produce diacetyl; however, no culture showed extracellular proteolytic activity and the autolysis varied from 21.3% to 30.5% after 24 h. The isolates grew at NaCl concentrations of 4.0 and 6.0%, but the growth was lower at 10.0%. Finally, all the isolates were found to be safe but had limited application as probiotics and in some technological uses

Characterization of interfering factors in the bacteriocin production by lactic acid bacteria isolated from raw milk and cheese

Bactérias ácido láticas (BAL) são constituintes naturais da microbiota do leite cru, exercendo importante papel na segurança alimentar, devido ação antimicrobiana sobre microrganismos deteriorantes e patógenos, como Listeria monocytogenes e Staphylococcus aureus. Várias espécies de BAL já foram isoladas e identificadas como produtoras de diversas bacteriocinas. Porém, pesquisas devem ser conduzidas a fim de otimizar a utilização e a produção dessas substâncias antimicrobianas, visando o controle de patógenos e deteriorantes. Considerando a viabilidade de aplicação das bacteriocinas em indústrias de alimentos, este trabalho teve como objetivo verificar a natureza protéica e a interferência de condições de incubação na produção de substâncias antimicrobianas por isolados de Bactérias Ácido Láticas (BAL) de leite cru e queijo. As substâncias antimicrobianas produzidas foram caracterizadas quanto à sensibilidade enzimática, atividade antimicrobiana contra diferentes cepas de Listeria spp., Staphylococcus spp. e BAL, e avaliadas quanto à interferência de oito variações de meios de cultura (MRS, MRS modificado - MRSm, M17, e BHI, adicionados ou não de solução de cisteína) e oito combinações de tempo e temperatura de incubação (25 e 35 °C por 12, 24, 48 e 72h). As substâncias antimicrobianas produzidas por 80 isolados de BAL apresentaram natureza protéica, as caracterizando como bacteriocinas. Lactococcus spp. foi o gênero que apresentou melhor desempenho de atividade antimicrobiana e Listeria spp. foi o gênero mais sensível. Os meios de cultura MRS e MRSm e a combinação de 25 °C por 24h, e a 35 °C por 12h de incubação, foram as condições que apresentaram melhor performance de atividade antimicrobiana. Assim, foi possível conhecer alguns aspectos relevantes em relação à produção de bacteriocinas por alguns gêneros de BAL, direcionando estudos complementares para sua futura aplicação em produtos lácteos.Lactic acid bacteria (LAB) occur naturally as indigenous microbiota in raw milk, and play an important role in the microbial ecology of this product due to their antimicrobial activity against spoilage and pathogenic microorganisms, such as Listeria monocytogenes and Staphylococcus aureus. Several LAB species were already isolated and identified as producers of some bacteriocins. However, researches should be conducted to optimize the antimicrobial substances production to control the development of foodborne pathogens and spoilage microorganisms. Considering the bacteriocins application viability in the food industry, the aim of this work was to verify the proteinaceus nature and the culture conditions interference on production of antimicrobial substances by Lactic Acid Bacteria (LAB) cultures isolated from raw milk and cheese. The antimicrobial substance produced were characterized for sensibility of different enzymes, for antimicrobial action against Listeria spp., Staphylococcus spp. and LAB cultures, and the effect of eight variations of culture media (MRS, modified MRS - mMRS, M17, and BHI, added or not with cistein solution) and eight combinations of time and temperature of incubation (25 and 35 °C for 12, 24, 48 and 72h) were evaluated. The antimicrobial substances produced by 80 LAB cultures presented a proteinaceous nature, indicating the bacteriocin production. Lactococcus spp. was the genera with better antimicrobial activity and Listeria spp. was the most sensitive genera. The culture media MRS and mMRS and the incubation conditions of 25 °C for 24h, and to 35 °C for 12h presented better antimicrobial activity performance. Considering the obtained results, it was possible to understand some important aspects in relation to the bacteriocin production by some genera of LAB, leading further studies to their future application in dairy products.Fundação de Amparo a Pesquisa do Estado de Minas Gerai

Molecular diversity of bacteriocinogenic lactic microbiota from goat milk and characterization of its bioconservative potential for producing a Minas cheese

O leite de cabra cru é uma importante fonte de novos isolados de bactérias ácido láticas (BAL) bacteriocinogênicos. A constante demanda de consumidores por alimentos sem aditivos químicos justifica o estudo de alternativas para sua bioconservação. Este trabalho teve como objetivos isolar e identificar BAL bacteriocinogênicos presentes na microbiota autóctone do leite de cabra cru, caracterizar suas bacteriocinas produzidas, seu potencial de virulência e avaliar seu potencial bioconservador na produção de queijos Minas produzido com leite de cabra cru. Como aspecto adicional de segurança, aminas biogênicas (AB) presentes nos queijos produzidos foram quantificadas. BAL foram isoladas do leite de cabra cru usando meios de cultura seletivos e submetidas à avaliação de seu potencial bacteriocinogênico a partir de testes moleculares e fenotípicos usando como indicador Listeria monocytogenes ATCC 7644. O gene codificador de nisina dos isolados positivos foi submetido a sequenciamento para identificação de possíveis variações em sua composição de aminoácidos. Isolados caracterizados como bacteriocinogênicos foram identificados a partir do sequenciamento dos genes 16S rRNA e adicionalmente do gene pheS para identificação das espécies de Enterococcus. Esses isolados foram agrupados de acordo com sua similaridade genética por rep-PCR e a partir dos perfis gerados, alguns isolados foram selecionados e submetidos a testes fenotípicos e moleculares para identificação de seu potencial de virulência. O isolado Lactococcus lactis subsp. lactis GLc05 foi escolhido pelo seu interessante potencial bacteriocinogênico e ausência de fatores de virulência para produção de queijo Minas utilizando leite de cabra cru. A microbiota dos queijos com (A) e sem (B) adição de L. lactis subsp. lactis GLc05 foi analisada usando métodos cultura-dependentes (meios de cultura seletivos) e -independentes (rep-PCR e DGGE). A quantificação de AB nos queijos Minas foi realizada por HPLC. Produtores de bacteriocinas foram identificados como Lactococcus spp. (24) e Enterococcus spp. (33). Entre os isolados, 9 Lactococcus lactis foram identificados como produtores de uma nova variante de nisina ainda não descrita e com amplo espectro de ação. Testes de caracterização do potencial de virulência demonstraram a presença de genes relacionados a patogenicidade e expressão de alguns desses fatores em alguns isolados identificados como Lactococcus spp. Por outro lado, alguns isolados identificados como Enterococcus spp., usualmente considerados patógenos oportunistas, não apresentaram esses fatores. A análise da microbiota dos queijos A e B usando métodos cultura- dependentes e -independentes mostrou que L. lactis subsp. lactis GLc05 foi capaz de controlar a população de cocos coagulase-positivo e causar mudanças na microbiota autóctone em queijo Minas produzido com leite de cabra cru. Altas concentrações de AB foram encontradas em queijo Minas produzido com leite de cabra cru, revelando importância de garantir a qualidade higiênico-sanitária do leite utilizado, porém foram significativamente menores nos queijos Minas adicionados de L. lactis subsp. lactis GLc05. Os resultados indicam que este isolado pode ser utilizado para produção de queijos produzidos com leite cru, pois foi capaz de controlar populações de micro- organismos patogênicos e as concentrações de AB.The raw goat milk is an important source of new bacteriocinogenic lactic acid bacteria (LAB) strains. The constant demand of consumers for foods without chemical additives justifies the study of alternatives for biopreservation. This study aimed to isolate and identify bacteriocinogenic LAB strains present in the autochthonous microbiota of raw goat milk, characterize their bacteriocins, virulence potential and evaluate its bioconservative potential in Minas cheese manufactured with raw goat milk. As an additional preservative aspect, biogenic amines (BA) present in the produced cheeses were quantified. LAB were isolated from raw goat milk using selective media and subjected to evaluation of their bacteriocinogenic potential from molecular and phenotypic tests using Listeria monocytogenes ATCC 7644 as target. Nisin positive strains were submitted to gene sequencing in order to identify possible variations in amino acid composition of the peptide codified. Strains characterized as bacteriocinogenic were identified by 16S rRNA with additional pheS sequencing to identify Enterecoccus species. These strains were grouped according to their genetic similarity by rep-PCR and some isolates were selected and submitted to further phenotypic and molecular tests for identification of their virulence potential. Lactococcus lactis subsp. lactis GLc05 was selected based on its interesting bacteriocinogenic potential and absence of virulence factors for producing Minas cheese manufactureted with raw goat milk. The cheeses microbiota with (A) and without (B) addition of GLc05 was analyzed using culture-dependent (selective media) and - independent (rep-PCR and DGGE) methods. The BA quantification in the Minas cheeses was performed by HPLC. Bacteriocinogenic strains were identified as Lactococcus spp. (24) and Enterococcus spp. (33). Among them, 9 Lactococcus lactis were identified as producing a new variant of nisin with broad antimicrobial activity spectrum. Characterization of virulence potential showed the presence of genes related to pathogenicity and their expression in some isolates identified as Lactococcus spp. Moreover, some isolates identified as Enterococcus spp. usually considered opportunistic pathogens, did not presented these genes. Analysis of the cheeses A and B using culture-dependent and -independent methods showed that L. lactis subsp. lactis GLc05 was able to control the coagulase-positive cocci population and was capable to cause changes in the microbiota composition in Minas cheese manufactured with raw goat milk. High concentrations of BA were found in the cheeses, revealing the importance of ensuring the sanitary quality of the milk used. However BA concentrations were significantly lower in cheeses A. The results indicate that L. lactis subsp. lactis GLc05 can be used for the production of cheeses manufactured with raw milk as it is capable of controlling pathogenic microorganisms populations and also the concentrations of BA.Fundação de Amparo a Pesquisa do Estado de Minas Gerai

Antagonistic lactic acid bacteria isolated from goat milk and identification of a novel nisin variant Lactococcus lactis

The raw goat milk microbiota is considered a good source of novel bacteriocinogenic lactic acid bacteria (LAB) strains that can be exploited as an alternative for use as biopreservatives in foods. The constant demand for such alternative tools justifies studies that investigate the antimicrobial potential of such strains. The obtained data identified a predominance of Lactococcus and Enterococcus strains in raw goat milk microbiota with antimicrobial activity against Listeria monocytogenes ATCC 7644. Enzymatic assays confirmed the bacteriocinogenic nature of the antimicrobial substances produced by the isolated strains, and PCR reactions detected a variety of bacteriocin-related genes in their genomes. Rep-PCR identified broad genetic variability among the Enterococcus isolates, and close relations between the Lactococcus strains. The sequencing of PCR products from nis-positive Lactococcus allowed the identification of a predicted nisin variant not previously described and possessing a wide inhibitory spectrum. Raw goat milk was confirmed as a good source of novel bacteriocinogenic LAB strains, having identified Lactococcus isolates possessing variations in their genomes that suggest the production of a nisin variant not yet described and with potential for use as biopreservatives in food due to its broad spectrum of action

Investigation of genes involved in nisin production in Enterococcus spp. strains isolated from raw goat milk

Different strains of Lactococcus lactis are capable of producing the bacteriocin nisin. However, genetic transfer mechanisms allow the natural occurrence of genes involved in nisin production in members of other bacterial genera, such as Enterococcus spp. In a previous study, nisA was identified in eight enterococci capable of producing antimicrobial substances. The aim of this study was to verify the presence of genes involved in nisin production in Enterococcus spp. strains, as well as nisin expression. The nisA genes from eight Enterococcus spp. strains were sequenced and the translated amino acid sequences were compared to nisin amino-acid sequences previously described in databases. Although containing nisin structural and maturation related genes, the enterococci strains tested in the present study did not present the immunity related genes (nisFEG and nisI). The translated sequences of nisA showed some point mutations, identical to those presented by Lactococcus strains isolated from goat milk. All enterococci were inhibited by nisin, indicating the absence of immunity and thus that nisin cannot be expressed. This study demonstrated for the first time the natural occurrence of nisin structural genes in Enterococcus strains and highlights the importance of providing evidence of a link between the presence of bacteriocin genes and their expression

Lantibiotics biosynthesis genes and bacteriocinogenic activity of Lactobacillus spp. isolated from raw milk and cheese

Lactobacillus species are usually used as starters for the production of fermented products, and some strains are capable of producing antimicrobial substances, such as bacteriocins. Because these characteristics are highly desirable, research are continually being performed for novel Lactobacillus strains with bacteriocinogenic potential for use by food industries. The aim of this study was to characterise the bacteriocinogenic potential and activity of Lactobacillus isolates. From a lactic acid bacteria culture collection obtained from raw milk and cheese, 27 isolates were identified by 16S rDNA as Lactobacillus spp. and selected for the detection of lantibiotics biosynthesis genes, bacteriocin production, antimicrobial spectra, and ideal incubation conditions for bacteriocin production. Based on the obtained results, 21 isolates presented at least one of the three lantibiotics biosynthesis genes (lanB, lanC or lamM), and 23 isolates also produced antimicrobial substances with sensitivity to at least one proteinase, indicating their bacteriocinogenic activity. In general, the isolates had broad inhibitory activity, mainly against Listeria spp. and Staphylococcus spp. strains, and the best antimicrobial performance of the isolates occurred when they were cultivated at 25 °C for 24 or 48 h or at 35 °C for 12 h. The present study identified the bacteriocinogenic potential of Lactobacillus isolates obtained from raw milk and cheese, suggesting their potential use as biopreservatives in foods

Comparison of phenotypic and molecular tests to identify lactic acid bacteria

Twenty-nine lactic acid bacteria (LAB) isolates were submitted for identification using Biolog, API50CHL, 16S rDNA sequencing, and species-specific PCR reactions. The identification results were compared, and it was concluded that a polyphasic approach is necessary for proper LAB identification, being the molecular analyzes the most reliable

Advancement in LH-PCR methodology for multiple microbial species detections in fermented foods

The length-heterogeneity PCR is a low throughput molecular biology methods explored to monitor bacteria populations in different environments. It could be more used in food microbiology analysis, not only for fingerprinting analysis, but it has been hampered until now by a limiting factor which relates to the high percentage of secondary peaks. With the aim to overcome this problem, different experiments were performed focusing on changing PCR parameters in order to obtain more specific amplicon patterns and also to reduce the complexity of community patterns. With this purpose, different annealing temperatures were tested on complex fermented food matrices taken from both animal and vegetable origin and also on the bacteria isolated from the same food source. In particular, the optimal annealing temperature identified for the fermented food samples is 59 C and the optimal for bacterial strains varied between 63 C and 65 C. The approach allowed the modification of the LH-PCR protocol increasing the amplification efficiency and therefore the bacteria species discrimination. These temperatures also allowed the implementation of the previous LH-PCR published database. The modification in the level of accuracy of the LH-PCR technique could also allow an improvement in the relative species quantification by the peak area evaluation