Carnobacterium maltaromaticum as bioprotective culture against spoilage bacteria in ground meat and cooked ham.

This study assessed the bioprotective effect of Carnobacterium maltaromaticum (CM) against Pseudomonas fluorescens (PF) and Brochothrix thermosphacta (BT) in ground beef and sliced cooked ham stored in high- and low-oxygen-modified atmospheres (66/4/30% O2/N2/CO2 and 70/30% N2/CO2, respectively). Both meat products were inoculated with CM, PF, and BT individually or in combination and stored for 7 days (3 days at 4 °C + 4 days at 8 °C) for ground beef and 28 days (10 days at 4 °C + 18 days at 8 °C) for sliced cooked ham. Each food matrix was assigned to 6 treatments: NC (no bacterial inoculation, representing the indigenous bacteria of meat), CM, BT, PF, CM + BT, and CM + PF. Bacterial growth, pH, instrumental color, and headspace gas composition were assessed during storage. CM counts remained stable from inoculation and throughout the shelf-life. CM reduced the population of inoculated and indigenous spoilage bacteria, including BT, PF, and enterobacteria, and showed a negligible impact on the physicochemical quality parameters of the products. Furthermore, upon simulating the shelf-life of ground beef and cooked ham, a remarkable extension could be observed with CM. Therefore, CM could be exploited as a biopreservative in meat products to enhance quality and shelf-life.

Novel immobilization method of Lactiplantibacillus plantarum in grains for dry food products.

This study aimed to assess the potential use of grains (amaranth, millet, and quinoa) as immobilizing matrices on the metabolic activity of Lactiplantibacillus plantarum and evaluate the viability of the probiotic immobilized in millet during simulated oro-gastrointestinal digestion (OGI) and storage. Firstly, different grains, sterilization and immobilization times were assessed. Secondly, Lp. plantarum was immobilized in millet and its viability and metabolic activity were assessed during non-refrigerated long-term storage and OGI. Metabolic activity was higher with 15 min. of grain sterilization and 72 h of immobilization. Lp. plantarum in millet showed the highest count. Millet grains were able to maintain the high population (>7 log CFU/g) and the metabolic activity of Lp. plantarum during up to 150 d of storage and conferred a protective effect on entrapped probiotic cells during OGI. Millet is a promising immobilizing matrix for the addition of probiotics in dry food products.

The influence of eugenol, benzocaine and ice water on the welfare of adult grass carp (Ctenopharyngodon idella).

The aim of this study was to evaluate the effects of eugenol, benzocaine, and ice water during the sedative, anesthetic or euthanasia processes on the welfare of adult grass carp (Ctenopharyngodon idella). The experimental design was randomized and the animals were divided into eight groups. Sixty-two animals underwent an acclimation period. The neutral group used to obtain basal data of grass carp were not subjected to treatments, but anesthetized to collect blood samples and euthanized by medullary section. The others seven groups were submitted to seven treatments with eight repetitions (control group; ethanol; eugenol 50 mgL-1, eugenol 250 mgL-1, benzocaine 100 mgL-1, benzocaine 300 mgL-1, and ice water 2:1), their behavior was observed. Blood samples was collected and then euthanized by medullary sectioning. Biometric data were measured and a part of the liver was collected for hepatic glycogen analysis. There was a statistically significant difference in the time required to reach the anesthetic stage between the groups (p < 0.01). Benzocaine and eugenol at the higher concentration provided the fastest responses to sedatives and anesthetics, respectively. The animals subjected to higher anesthetic concentrations reached stage five and did not return from anesthesia, therefore, benzocaine and eugenol were effective euthanizing agents. Benzocaine at the lowest concentration showed the highest concentrations of glucose and cortisol (p < 0.05). Although benzocaine at 100 mgL-1 concentrations is widely used as an anesthetic in fish, this study demonstrated its use as a stressor agent. Basal data of grass carp for stress parameters are presented for the first time.

Carnobacterium as a bioprotective and potential probiotic culture to improve food quality, food safety, and human health - a scoping review.

It is well-known that some bacteria can promote human and animal health. Bacteria of the genus Carnobacterium, while underexplored, have demonstrated significant probiotic and bioprotective potential. In this review, the recent scientific advances in this area are discussed. There are several requirements for a strain to be considered a probiotic or bioprotective agent, including the absence of antimicrobial resistance and the ability to colonize the gastrointestinal tract. Several researchers have reported such features in Carnobacterium bacteria, especially with regard to the production of antimicrobial substances. Research into animal production has advanced, especially in the aquaculture field, wherein inhibitory activity has been demonstrated against several important pathogens (for example Vibrio), and improvement in zootechnical indexes is evident. With respect to human health-related applications, research is still in the early stages. However, excellent in vitro results against pathogens, such as Candida albicans and Pseudomonas aeruginosa, have been reported. Carnobacterium bacteria have been assessed for a variety of applications in food, including direct application to the matrix and application to smart packaging, with proven effectiveness against Listeria monocytogenes. However, there is a lack of in vivo studies on Carnobacterium applications, which hinders its applications in various industries despite its high potential.

Escherichia coli ST224 and IncF/blaCTX-M-55 plasmids drive resistance to extended-spectrum cephalosporins in poultry flocks in Parana, Brazil.

Resistant Enterobacterales of avian intestinal origin can contaminate carcasses during broiler processing and thereby spread through the human food chain. This study aimed at assessing the prevalence, diversity and genomic characteristics of ESBL/AmpC Enterobacterales in poultry flocks from different farms and cities in the state of Paraná, Brazil. Enterobacterales isolated from cloacal samples were subjected to antimicrobial susceptibility testing (AST). ESBL/AmpC isolates were whole-genome sequenced and subjected to S1-nuclease pulsed-field gel electrophoresis (S1-PFGE) followed by Southern blotting to determine the location of resistant genes on plasmids. A surprisingly high proportion of E. coli (40.6 %) collected on non-selective plates presented an ESBL/AmpC phenotype. Multidrug resistance was statistically not higher in ESBL/AmpC E. coli having the potential to be Avian Pathogenic (APEC-like) compared to non-APEC-like ESBL/AmpC E. coli isolates. Resistance to antibiotics not authorized for use in poultry in the State of Paraná was observed, suggesting that antimicrobial resistance (AMR) is co-selected by the use of veterinary-licensed antibiotics. Phylogenetic analyzes revealed the presence of identical or highly similar ESBL/AmpC E. coli clones on farms distant up to 100 km of each other; this strongly suggests that the centralization and verticalization of the poultry industry can facilitate the spread of resistant bacteria among different farms, companies, and cities. The molecular characterization of clones and plasmids proved the dominance of the ST224 E. coli lineage and the IncF/blaCTX-M-55 plasmid, possibly indicating the emergence of successful clones and plasmids adapted to the chicken host. Our data contribute to the epidemiological tracking of resistance mechanisms in Enterobacterales from poultry and to knowledge for further One Health studies to control the spread of resistant bacteria from food animals to humans.

Intake and performance of growing lambs supplemented with quebracho tannins.

Tannins can modulate rumen fermentation and increase the bypass protein; however, the use of different doses and sources of condensed tannins (CT) in animal diets has divergent results. The aim of this study was to evaluate the effect of different levels of dietary quebracho extract inclusion, as a CT source, in the performance and carcass characteristics of growing lambs. Forty-eight weaned lambs (Texel, Suffolk, and Hampshire Down crossbred) were homogenously blocked into four groups and supplemented at 0, 10, 30, and 60 g tannins/kg dry matter intake (DMI) (CONT, T1%, T3%, and T6%, respectively). The DMI, the roughage to concentrate ratio, the feed conversion rate (FCR), the average daily gain (ADG), and the body condition score (BCS) were evaluated. After the trial, animals were slaughtered, and the cold carcass weight, carcass yield, and subcutaneous fat thickness (SFT) were determined. Higher levels of CT in T3% and T6% reduced (P < 0.001) the total DMI and intake of concentrate without impacting (P = 0.071) the roughage intake (% body weight). ADG was the lowest for T6%, although the total weight gain and BCS were the lowest also for T3% (P < 0.001). FCR was worst for T6% (P < 0.001). Regarding carcass characteristics, T6% had the lowest weight of carcass (P = 0.003) and SFT (P = 0.021) compared to CONT. Carcass yields were similar among all the groups (P = 0.247). T1% did not differ from CONT to any parameter (P > 0.05). Supplementing quebracho CT at a low dose did not show beneficial effects; moreover, higher doses of CT led to detrimental effects in intake and animal performance.

Non-conventional cultures and metabolism-derived compounds for bioprotection of meat and meat products: a review.

Meat and meat products are important nutritional sources for humans; however, they are highly susceptible to pathogenic or spoilage bacteria, which leads to economic losses for the industry and has become a public health threat. Several biological methods have been developed to promote meat safety and to increase shelf life, mostly focusing on lactic acid bacteria from the genera Lactobacillus and Pediococcus. However, little attention has been paid to other groups of microorganisms with potential use as bioprotective cultures. This review aims to discuss the potential use of non-conventional microorganisms and biometabolites for bioprotection in meat and meat products. This review covers recent research involving the use of non-conventional microorganisms to improve the quality and safety of meat products. Bacteria that are often overlooked also have the potential for use in the food industry, either by direct application or by using their metabolites in the product itself or the development of intelligent packaging systems. Hopefully, this review will instigate new studies on emerging biological methods to ensure the safety of food and protect the health of consumers.

Simultaneous feeding of calcium butyrate and tannin extract decreased the incidence of diarrhea and proinflammatory markers in weaned piglets.

OBJECTIVE: This study was conducted to investigate the effect of associating calcium butyrate with tannin extract, compared to an antimicrobial on the growth performance, incidence of diarrhea, intestinal histology, immune-expression of cyclooxygenase-2 (COX-2) and tumor necrosis factor α (TNF-α) in piglets. METHODS: Seventy-two piglets (36 barrows and 36 gilts) weaned at 28±2 d and initial body weight of 7.17±1.07 kg were allocated to 3 treatments in a randomized complete block design with 8 replicates per treatment and 3 animals per experimental unit. Treatments were composed of NC, negative control: basal diet without additives; PC, positive control: basal diet + 40 mg/kg of colistin sulfate; or BT, basal diet + calcium butyrate + tannin extract. The butyrate and tannin inclusion levels were 0.15% in the pre-starter phase and 0.075% in the starter phase. Incidence of diarrhea was monitored daily, and on d 14 and 35 of experiment, 1 animal from each experimental unit was slaughtered to collect intestinal samples. RESULTS: No significant differences were observed for growth performance. The butyrate-and tannin-based additive resulted in reduced (p<0.05) incidence of diarrhea in piglets during d 1 to 14 and d 1 to 35 in comparison with the other treatments. Piglets that consumed the diet containing the calcium-butyrate and tannin showed a lower (p<0.05) crypt depth in the duodenum than those receiving the NC treatment at 14 d of experimentation. The BT treatment provided a lower (p<0.05) immune-expression of COX-2 at 14 d and TNF-α at 35 d in the duodenum. CONCLUSION: Association between calcium butyrate and tannin extract resulted in a significant decrease in the incidence of diarrhea and inflammatory process in the duodenum of piglets. Therefore, calcium-butyrate combined with tannin could be a part of an alternative program to reduce the use of antimicrobials in the diet of weaned piglets.

Nisin and ε-poly-L-lysine as natural antimicrobials towards spoilage-associated Lactobacillus plantarum / Combinação de nisina e ε-poly-L-lysina como antimicrobianos naturais contra Lactobacillus plantarum associado à deterioração

ABSTRACT: Sausages are highly susceptible to microbial spoilage. Lactic acid bacteria (LAB) is the main group of spoilage bacteria in vacuum packed cooked sausages. To control microbial growth natural antimicrobials have been used as food preservatives. The aim of this study was to identify strains of lactic acid bacteria isolated from spoiled commercial Calabresa sausages and use them in an in vitro challenge with the natural antimicrobials, nisin (NI) and ε-poly-L-lysine (ε-PL). Mass spectrometry identification of LAB isolated from sausages using MALDI-TOF revealed a predominance of L. plantarum in the LAB population. RAPD-PCR of L. plantarum strains showed four different genetic profiles. Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of NI and ε-PL, alone and in combination, against a pool of different profiles L. plantarum were determined. MIC of NI and ε-PL were 0.468 mg/ L and 75 mg/ L; respectively, whereas MBC of NI and ε-PL were 12.48 mg/L and 150 mg/L, respectively. The combined effect of NI and ε-PL was determined using concentrations at 1/4 and 1/8 of individual MICs. Synergistic effect was confirmed at both concentrations showing a fractional inhibitory concentration index of 0.5 and 0.2, respectively. The combination of NI and ε-PL at a small concentration of 0.05 mg/L and 9.375 mg/L, respectively, showed inhibitory effect towards spoilage L. plantarum Results show the potential of the combined use of NI and ε-PL to control sausage spoilage-associated with lactobacilli.

RESUMO: Linguiças são altamente susceptíveis à deterioração microbiana. As bactérias ácido lácticas (BAL) são o principal grupo de bactérias deteriorantes em linguiças cozidas embaladas a vácuo. Para controlar o crescimento microbiano, antimicrobianos naturais têm sido utilizados como conservantes de alimentos. O objetivo deste estudo foi identificar cepas de bactérias ácido lácticas isoladas de linguiças Calabresa em deterioração e utilizá-las como modelo in vitro com os antimicrobianos naturais nisina (NI) e ε-poly-L-lysina (ε-PL). A identificação por espectrometria de massa das BAL isoladas das linguiças usando a técnica de MALDI-TOF revelou predominância de L. plantarum. A PCR-RAPD revelou quatro perfis genéticos diferentes. A concentração inibitória mínima (CIM) e a concentração bactericida mínima (CBM) de NI e ε-PL, individualmente e em combinação, contra o pool de L. plantarum foram determinadas. O valor de CIM de NI e ε-PL foi 0,468 mg/L e 75 mg/L, respectivamente, e de CBM de NI e ε-PL foi 12,48 mg/L e 150 mg/L, respectivamente. O efeito da combinação de NI e ε-PL foi determinada usando concentrações de 1/4 e 1/8 dos CIMs individuais. O efeito sinérgico foi confirmado em ambas concentrações mostrando um índice de concentração inibitório fracionado de 0,5 e 0,2, respectivamente. A combinação de NI e ε-PL em baixa concentração, 0,05 mg/L e 9,375 mg/L, respectivamente, mostrou efeito inibitório contra L. plantarum. Os resultados demonstram o potencial do uso combinado de NI e ε-PL para o controle de lactobacilos associados à deterioração em linguiça.

Carnobacterium maltaromaticum as bioprotective culture in vitro and in cooked ham.

The bioprotective effects of Carnobacterium maltaromaticum (CM) strains were assessed in vitro and in sliced cooked ham. CM strains were tested in vitro against Listeria monocytogenes (LM), Escherichia coli O157:H7 (EC) and Salmonella Typhimurium (ST). In vitro effect was evaluated using co-culture (with and without EDTA) and cell-free supernatant (CFS). CFS was tested by agar well diffusion and minimum inhibitory concentration. In cooked ham, the inhibitory effect of CM on L. innocua (LI) and on the physicochemical parameters were evaluated for 7 days at 4 °C. In co-cultures at -1 °C and 4 °C, all CM isolates inhibited LM. A slight inhibition was observed against the Gram-negative bacteria with the addition of EDTA. CFS did not show inhibitory effect under the studied conditions. In cooked ham, CM inhibited LI growth and did not affect the physicochemical parameters of the product during storage. CM strains show potential to be used as bioprotective cultures in cold-stored cooked ham and improve its safety.

Combination of garlic essential oil, allyl isothiocyanate, and nisin Z as bio-preservatives in fresh sausage.

The effects of natural antimicrobial compounds (garlic essential oil [GO], allyl isothiocyanate [AITC], and nisin Z [NI]) on microbiological, physicochemical and sensory characteristics of fresh sausage were assessed. The minimum inhibitory concentrations (MICs) and the minimum bactericidal concentrations (MBCs) towards Escherichia coli O157:H7 and Lactobacillus plantarum were determined in vitro. Sausages inoculated with E. coli O157:H7, were treated with different combinations of antimicrobials and assessed for microbiological and physicochemical parameters during storage (6C for 20 d). Treatments that presented the greatest antimicrobial effects were subjected to sensory evaluation. Combinations of 20 mg/kg NI + 125 µL/kg GO + 62.5 µL/kg AITC or 20 mg/kg NI + 62.5 µL/kg GO + 125 µL/kg AITC were effective in reducing E. coli O157H7 and spoilage lactic acid bacteria, and maintained the physicochemical characteristics of fresh sausage. Combinations of NI, GO and AITC were effective to improve the safety and the shelf life of fresh sausage, with no impact on its sensory acceptance.

Combined effects of garlic essential oil and allyl isothiocyanate against Escherichia coli O157:H7 in vitro and in pork sausage / Efeito combinado de óleo essencial de alho e isotiocianato de alila contra Escherichia coliO157:H7 in vitro e em linguiça suína

ABSTRACT: Escherichia coli O157:H7 is a toxigenic serotype of E. coli and has been associated with foodborne outbreaks involving meat products, vegetables and fresh produces worldwide. Salts for curing are usually employed as antimicrobials in the production of pork sausages. However, salts do not have a significant inhibitory effect on enterobacteria. Due to the growing demand for natural foods, the use of essential oils has been proposed as natural antimicrobials in food. This study aimed to evaluate the effects of garlic essential oil (GO) and allyl isothiocyanate (AITC) against E. coli O157:H7 in vitro and in pork sausage. The Minimal Inhibitory Concentration (MIC) and Minimal Bactericidal Concentration (MBC) of these oils, alone and in combination, against E. coli O157:H7 were determined. Pork sausage was inoculated with 8log CFU/g E. coli O157:H7 and different combinations of GO and AITC. A control group was performed without essential oils. Sausages were packaged and stored at 6°C for 20 days. E. coli O157:H7 population and instrumental color (L*, a*, b*, C* and hue) determinations were performed at 5-day intervals. AITC showed lower MIC and MBC than GO. When combined, AITC and GO showed a synergistic effect. Treatments T3 and T4 showed 1,01log CFU and 1,87log CFU reduction of E. coli O157:H7 compared to control. The redness and the chroma of sausages treated with AITC and GO increased during storage. Together, GO and AITC caused minor changes in taste and flavor of sausages, and were able to reduce the population of E. coli O157:H7 and to maintain the red color of sausage during storage.

RESUMO: Escherichia coli O157:H7 é um sorotipo toxigênico de E. coli associado mundialmente a surtos de doenças alimentares causadas por produtos cárneos. Os sais de cura são normalmente empregados como antimicrobianos na fabricação de linguiças, entretanto, possuem pouco efeito inibitório sobre enterobactérias como E. coli. Devido à crescente demanda por produtos naturais, o uso de óleos essenciais tem sido proposto como antimicrobiano natural em alimentos. O objetivo deste estudo foi avaliar os efeitos do óleo essencial de alho (GO) e isotiocianato de alila (AITC) contra E. coli O157:H7 in vitro e em linguiça. As concentrações inibitória mínima (MIC) e bactericida mínima (MBC) dos óleos, isolados e em combinação, contra E. coli O157:H7 foram determinadas in vitro. Lotes de linguiça foram inoculados com 8log UFC/g E. coli O157:H7 e diferentes concentrações de GO + AITC e estocados a 6°C por 20 dias. Contagens de E. coli O157:H7 e determinação de cor instrumental (L*, a*, b*, C* e hue) foram realizadas a cada 5 dias. Nos testes in vitro, AITC mostrou maior ação inibitória que GO, havendo efeito sinérgico dos óleos quando combinados. Os tratamentos T3 e T4 mostraram redução de 1,01log UFC e 1,87log UFC de E. coli O157:H7 comparados ao controle. A intensidade de cor vermelha e a saturação de cor aumentaram durante a estocagem nas linguiças adicionadas de óleos essenciais. A adição dos óleos GO + AITC causou mínima alteração no sabor e na aparência das linguiças e foi capaz de reduzir a população de E. coli O157:H7 no produto cárneo, mostrando potencial para uso como conservante natural nesse tipo de alimento.

Methods for the evaluation of antibiotic resistance in Lactobacillus isolated from fermented sausages / Comparação de métodos para avaliação de resistência microbiana em Lactobacillus isolados de embutidos fermentados

ABSTRACT: The present study aimed to assess the antibiotic resistance in 54 indigenous Lactobacillus plantarum isolated from artisanal fermented sausages. The confirmation of the strain species was performed by multiplex-PCR assay. Antibiotic resistance was assessed by disk diffusion (DD) and Minimum Inhibitory Concentration (MIC) methods. Of 54 L. plantarum, 44 strains were genotypically confirmed as L. plantarum and 3 as Lactobacillus pentosus. The highest resistance rates were to ampicillin and streptomycin. The highest susceptibility rates were shown to tetracycline, chloramphenicol and penicillin G. None of the strains showed multidrug resistance. Resistance rates by DD and MIC were not different (P>0.05) for ampicillin, chloramphenicol, erythromycin and penicillin G. Future research should assess the genetic mechanisms underlying the phenotypic resistance in Lactobacillus strains to screen the potential probiotic strains for the development of functional meat products.

RESUMO: O presente estudo teve como objetivo avaliar a resistência a antibióticos, de 54 cepas nativas de Lactobacillus plantarum isoladas de salames artesanais. A confirmação genotípica da espécie foi realizada por ensaio de PCR multiplex. A resistência aos antibióticos foi avaliada pelos métodos de disco difusão e concentração inibitória mínima. Das 54 cepas, 44 foram confirmadas genotipicamente como L. plantarum e 3 como Lactobacillus pentosus. As maiores frequências de resistência foram para ampicilina e estreptomicina, e as maiores frequências de sensibilidade para tetraciclina, cloranfenicol e penicilina G. Nenhuma das cepas apresentou multirresistência. As frequências de resistência para ampicilina, cloranfenicol, eritromicina e penicilina G foram semelhantes pelos métodos testados (P>0,05). Pesquisas futuras devem ser realizadas para avaliar os mecanismos genéticos envolvidos na resistência fenotípica das cepas de Lactobacillus, no intuito de selecionar as potenciais cepas probióticas para aplicação em produtos cárneos funcionais.

Avaliação de diferentes alíquotas de enxaguadura para a contagem pelo método direto de Campylobacter spp. em carcaças de frango resfriadas / Evaluation of the different rinse volumes for performing the direct technique for Campylobacter spp. enumeration in chilled broiler carcasses

O método direto de detecção e contagem de Campylobacter spp. em carne de frango é de fácil execução, porém os volumes de 100 μL e de 400 μL, preconizados em algumas metodologias, muitas vezes impossibilitam a contagem de colônias pela ocorrência de crescimento confluente ou de microbiota contaminante. O objetivo do presente estudo foi de avaliar os diferentes volumes de enxaguadura de carne de frango com a finalidade de minimizar a interferência da microbiota contaminante, sem comprometer a sensibilidade do método. Os volumes de enxaguadura de 5, 10,50, 100 e 400 μL foram testados utilizando-se os meios seletivo diferenciais ágar carvão cefoperazona desoxicolato modificado (mCCDA) e ágar Bolton modificado. A presença de Campylobacter spp. foi confirmada por métodos fenotípicos e por PCR. No entanto, a estratégia de utilização de volumes menores do que 100 μL de enxaguadura não melhoraram o isolamento e a contagem de colônias de Campylobacter, porque houve diminuição da sensibilidade do ensaio. A provável solução para minimizar a interferência da microbiota contaminante seria desenvolver novos meios seletivos ou incorporá-los aos antimicrobianos já existentes...