Development of a selective culture medium for bifidobacteria, Raffinose-Propionate Lithium Mupirocin (RP-MUP) and assessment of its usage with Petrifilm™ Aerobic Count plates

AbstractThis study aimed to develop a selective culture media to enumerate bifidobacteria in fermented milk and to assess this medium when used with Petrifilm™ AC plates. For this purpose, Bifidobacterium spp., Lactobacillus spp. and Streptococcus thermophilus strains were tested to verify their fermentation patterns for different carbohydrates. All bifidobacteria strains were able to use raffinose. Based on these characteristic, a selective culture medium was proposed (Raffinose-Propionate Lithium Mupirocin, RP-MUP), used with Petrifilm™ AC plates, and was used to enumerate bifidobacteria in fermented milk. RP-MUP performance was assessed by comparing the results with this medium to reference protocols and culture media for bifidobacteria enumeration. RP-MUP, whether used or not with Petrifilm™ AC, presented similar performance to TOS-MUP (ISO 29981), with no significant differences between the mean bifidobacteria counts (p < 0.05) and with high correlation indices (r = 0.99, p < 0.05). As an advantage, reliable results were obtained after just 48 h of incubation when RP-MUP was used with Petrifilm™ AC, instead of the 72 h described in the ISO 29981 protocol

Lactobacillus curvatus UFV-NPAC1 and other lactic acid bacteria isolated from calabresa, a fermented meat product, present high bacteriocinogenic activity against Listeria monocytogenes

Bacteriocins produced by lactic acid bacteria (LAB) can be considered as viable alternatives for food safety and quality, once these peptides present antimicrobial activity against foodborne pathogens and spoilage bacteria. Fermented foods, such as artisanal sausages and cured meats, are relevant sources of LAB strains capable of producing novel bacteriocins, with particular interest by the food industry.Three LAB strains (firstly named as Lactobacillus curvatus 12, L. curvatus 36 and Weissella viridescens 23) were obtained from calabresa by presenting promising bacteriocinogenic activity, distinct genetic profiles (rep-PCR, RAPD, bacteriocin-related genes) and wide inhibitory spectrum. Among these strains, L. curvatus 12 presented higher bacteriocin production, reaching 25,000 AU/mL after incubation at 25, 30 and 37 °C and 6, 9 and 12 h. Partially purified bacteriocins from L. curvatus 12 kept their inhibitory activity after elution with isopropanol at 60% (v/v). Bacteriocins produced by this strain were purified by HPLC and sequenced, resulting in four peptides with 3102.79, 2631.40, 1967.06 and 2588.31 Da, without homology to known bacteriocins.LAB isolates obtained from calabresa presented high inhibitory activity. Among these isolates, bacteriocins produced by L. curvatus 12, now named as L. curvatus UFV-NPAC1, presented the highest inhibitory performance and the purification procedures revealed four peptides with sequences not described for bacteriocins to date

BACTERIOCINOGENIC POTENTIAL OF LACTIC ACID BACTERIA ISOLATED FROM ARTISANAL COLONIAL TYPE - CHEESE

Autochthonous microbiota from artisanal cheeses is predominantly composed of lactic acid bacteria (LAB), which are able to produce antimicrobial compounds, such as bacteriocins, suggesting their application in food biopreservation. Knowledge about LAB growth and bacteriocin production during food production and conservation is essential to determine their use. In this way, the study aimed at isolating bacteriocinogenic LAB from twenty-one artisanal cheeses from the western region of Parana state, Brazil, determining the best conditions for growth and bacteriocin production (25°C, 30°C, and 37°C/24h); bacteriocin stability under different ranges of pH (2, 4, 6, 8, and 10 for 2h) and temperature (60oC/2h; 80oC/2h; 121oC/15min). Their activity against different target microorganisms was also evaluated. A total of 34 LAB strains presented characteristics compatible with bacteriocin production. Most of them presented better results for bacteriocin production when cultured at 25ºC and 30ºC. Bacteriocins remained active against L. monocytogenes when exposed from pH 4 to 8 and a wide temperature range; some bacteriocins were even resistant to sterilization temperatures. Bacteriocins produced were able to inhibit spoilage and pathogenic microorganisms, such as L. monocytogenes, B. cereus, and P. fluorescens. These results indicated that isolated bacteriocinogenic LAB present potential to be used as food biopreservatives

Destructive and nondestructive procedures to obtain chicken carcass samples for Escherichia coli and Salmonella spp. detection

Destructive and nondestructive sampling procedures were compared for Escherichia coli and Salmonella spp. detection in 60 fresh chicken carcasses, which were submitted to the following sampling procedures: rinsing, skin swabbing, tissue excision, and skin excision; the proximity or not to the cloacae region was also considered. The obtained results were compared to identify significant differences (p0.05), thus indicating equivalencies between these techniques. Skin swabbing produced a statistically significant lower frequency of positive results (p0.05), possibly due to the low overall frequency of positive carcasses. No significant differences in the number of positive samples (E. coli or Salmonella spp.) were observed between samples collected near or far from the cloacae region (p>0.05), regardless of the sampling technique. The obtained results demonstrate that the tested sampling techniques were equivalent for Salmonella spp. detection in chicken carcasses, as observed for E. coli with the exception of skin swabbing

Comparison of destructive and nondestructive sampling techniques of retail chicken carcasses for enumeration of hygiene indicator microorganisms

The type of sampling technique used to obtain food samples is fundamental to the success of microbiological analysis. Destructive and nondestructive techniques, such as tissue excision and rinsing, respectively, are widely employed in obtaining samples from chicken carcasses. In this study, four sampling techniques used for chicken carcasses were compared to evaluate their performances in the enumeration of hygiene indicator microorganisms. Sixty fresh chicken carcasses were sampled by rinsing, tissue excision, superficial swabbing, and skin excision. All samples were submitted for enumeration of mesophilic aerobes, Enterobacteriaceae, coliforms, and Escherichia coli. The results were compared to determine the statistical significance of differences and correlation (P < 0.05). Tissue excision provided the highest microbial counts compared with the other procedures, with significant differences obtained only for coliforms and E. coli (P < 0.05). Significant correlations (P < 0.05) were observed for all the sampling techniques evaluated for most of the hygiene indicators. Despite presenting a higher recovery ability, tissue excision did not present significant differences for microorganism enumeration compared with other nondestructive techniques, such as rinsing, indicating its adequacy for microbiological analysis of chicken carcasses

Identificação dos principais pontos de contaminação por microrganismos indicadores de higiene em plantas de processamento de carnes

The microbiological quality of beef and meat products is strongly influenced by the conditions of hygiene prevailing during their production and handling. Without proper hygienic control, the environment in slaughterhouses and butcher shops can act as an important source of microbiological contamination. To identify the main points of microbiological contamination in the beef processing chain, 443 samples of equipment, installations and products were collected from 11 establishments (1 slaughterhouse and 10 butcher shops) located in the state of Paraná, Brazil. The microbiological quality of all the samples was evaluated using Petri dishes to obtain counts of mesophilic aerobes (AC), total coliforms, Escherichia coli (EC), yeasts and molds (YM). The main contamination points identified in butcher shops, in decreasing order, were stainless steel boxes, beef tenderizers, grinders, knives, mixers, sausage stuffers, plastic boxes, floors and drains. In the slaughterhouse, these points were sausage stuffers, platforms, floors and drains. The most severely contaminated products were fresh sausages and ground beef. This information about the main points of microbiological contamination in the beef processing chain is expected to aid professionals responsible for hygiene in similar establishments to set up proper hygienic procedures to prevent or reduce microbiological contamination of beef and meat products.A qualidade microbiológica de carne e derivados é altamente influenciada pelas condições higiênicas durante sua produção e manipulação. Sem um controle higiênico adequado, o ambiente de abatedouros e açougues pode representar um importante ponto de contaminação. Com o objetivo de identificar os principais pontos de contaminação microbiológica na linha de processamento de carne, 443 amostras de equipamentos, instalações e produtos foram coletados em 11 estabelecimentos (1 abatedouro e 10 açougues) localizados no Estado do Paraná, Brasil. A qualidade microbiológica das amostras foi determinada utilizando-se placas Petrifilm&trade; para contagem de aeróbios mesófilos (AC), coliformes totais e Escherichia coli (EC) e bolores e leveduras (YM). Nos açougues os principais pontos de contaminação identificados, em ordem decrescente, foram caixas de aço inoxidável, amaciadores de carnes, moedores, facas, misturadores, embutideiras, caixas plásticas, pisos e ralos. No abatedouro, os principais pontos foram embutideiras, superfícies da plataforma de abate, pisos e ralos. Os produtos cárneos que apresentaram maiores níveis de contaminação foram lingüiças frescas e carne moída. Esse trabalho identificou os principais pontos de contaminação microbiológica na linha de processamento de carne, podendo direcionar profissionais responsáveis pelas condições higiênicas na obtenção desses produtos na determinação de procedimentos higiênicos adequados, para evitar ou reduzir a contaminação microbiológica em carne e seus derivados

Selective enumeration of propionibacteria in Emmental-type cheese using PetrifilmTM Aerobic count plates added to lithium glycerol broth

Propionibacteria derived from dairy products are relevant starter cultures for the production of Swiss and Emmental-type cheeses, and the monitoring of which is mandatory for proper quality control. This study aimed to evaluate an alternative procedure to enumerate propionibacteria, in order to develop a reliable and practical methodology to be employed by dairy industries. 2,3,5-triphenyltetrazolium chloride (TTC) inhibitory activity was tested against five reference strains (CIRM 09, 38, 39, 40 and 116); TTC at 0·0025% (w/v) was not inhibitory, with the exception of one strain (CIRM 116). Subsequently, the four TTC-resistant strains, three commercial starter cultures (PS-1, PB-I, and CHOO) and twelve Emmental-type cheese samples were subjected to propionibacteria enumeration using Lithium Glycerol (LG) agar, and Petrifilm™ Aerobic Count (AC) plates added to LG broth (anaerobic incubation at 30 °C for 7 d). Petrifilm™ AC added to LG broth presented high counts than LG agar (P<0·05) for only two reference strains (CIRM 39, and 40) and for all commercial starter cultures. Cheese sample counts obtained by both procedures did not show significant differences (P<0·05). Significant correlation indexes were observed between the counts recorded by both methods (P<0·05). These results demonstrate the reliability of Petrifilm™ AC plates added to LG broth in enumerating select Propionibacterium spp., despite some limitations observed for specific commercial starter cultures

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