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

Genetic diversity, antimicrobial activity range and bacteriocinogenic potential of Lactic Acid Bacteria isolated from raw goat milk

O leite de cabra, devido a diversidade de sua microbiota autóctone, é considerado uma boa fonte de novas cepas de Bactérias Ácido Láticas (BAL) com potencial para exploração como alternativas à biopreservação de alimentos. A crescente demanda dos consumidores por essas alternativas justifica estudos que investiguem o potencial antimicrobiano desses micro-organismos. O presente estudo teve como objetivo avaliar a diversidade genética e o espectro de ação de cepas de BAL antagonistas autóctones de leite de cabra que podem ser potencialmente utilizadas como biopreservantes em alimentos. A atividade bacteriocinogênica contra Listeria monocytogenes e estabilidade das bacteriocinas em diferentes condições ambientais também foram alvo deste trabalho. Cinquenta e sete isolados de BAL (33 isolados de Enterococcus spp. e 24 isolados de Lactococcus spp.), previamente caracterizados como bacteriocinogênicos por metodologias genotípicas e fenotípicas, foram submetidos à macrorrestrição com a enzima SmaI e PFGE e seus perfis foram comparados aos resultados de genes bacteriocinogênicos previamente pesquisados. Alta variabilidade genética foi observada para ambos os gêneros. Embora o PFGE tenha apresentado poder discriminatório suficiente, isolados com diferentes resultados para genes de bacteriocinas foram agrupados como idênticos em ambos os gêneros. Doze isolados de Lactococcus spp. e dezoito isolados de Enterococcus spp., representativos de diferentes pulsotipos, foram selecionados para avaliação do espectro de ação antimicrobiana contra 46 micro- organismos indicadores (incluindo BAL, micro-organismos deteriorantes e patógenos). Os isolados de Lactococcus spp. demonstraram amplo espectro de ação sobre os micro-organismos alvo, com destaque para a atividade contra os patógenos L. monocytogenes e Clostridium spp.. Enterococcus spp., de modo similar, foi capaz de inibir diversos micro-organismos alvo, apresentando atividade principalmente contra Listeria spp. Cepas Gram-negativas também apresentaram sensibilidade aos isolados de ambos os gêneros. Seis isolados (quatro Enterococcus spp. e dois Lactococcus spp.) foram avaliados quanto ao potencial bacteriocinogênico contra cepas de L. monocytogenes de diferentes sorotipos e todos os sorotipos foram inibidos pelas bacteriocinas produzidas pelas BAL. Adicionalmente, as bacteriocinas produzidas por estes isolados apresentaram ampla faixa de estabilidade em diferentes valores de pH e temperaturas. Os dados obtidos demonstraram que o leite de cabra pode conter uma microbiota bacteriocinogênica diversificada, capaz de inibir micro-organismos de interesse à indústria de alimentos, podendo ser potencialmente empregadas na bioconservação de alimentos produzidos em diferentes condições de processamento.Because of the diversity of its indigenous microbiota, goat milk is considered a good source of new strains of lactic acid bacteria (LAB) with potential for exploitation as alternatives to food biopreservation. The increasing consumer demand for these alternatives justifies studies to investigate the antimicrobial potential of these microorganisms. The present study aimed to evaluate the genetic diversity and the antimicrobial action range of autochthonous strains of BAL isolated from goat milk that can be potentially used as biopreservatives in food. The bacteriocinogenic activity against Listeria monocytogenes and stability in different environmental conditions were also the target of this study. Fifty-seven isolates of BAL (33 isolates of Enterococcus spp., and 24 isolates of Lactococcus spp.), previously characterized as bacteriocinogenic by genotypic and phenotypic methods, were subjected to macrorestriction with SmaI and PFGE and their profiles were compared with the results of bacteriocinogenic genes previously searched. High genetic diversity was observed for both genders. Although PFGE showed sufficient discriminatory power, isolates with different results for bacteriocinogenic genes were grouped as identical in both genders. Twelve isolates from Lactococcus spp. and eighteen isolates of Enterococcus spp., representing different pulsotypes, were selected for evaluation of the antimicrobial activity range against 46 target microorganisms (including BAL, pathogens and spoilage microorganisms). Lactococcus strains showed a wide spectrum against the targets, especially against L. monocytogenes and Clostridium spp.. Enterococcus spp., similarly, was able to inhibit various targets, acting mainly against Listeria spp. Gram- negative microorganisms also showed sensitivity to isolates of both genders. Six isolates (four Enterococcus spp., and two Lactococcus spp.) were evaluated for bacteriocinogenic potential against L. monocytogenes strains from different serotypes and all of them were inhibited by bacteriocins produced by these LAB. Additionally, bacteriocins produced by these isolates showed a wide range of stability at different pH values and temperatures. The data showed that goat milk can contain a diverse microbiota able to inhibit microorganisms of interest to the food industry and can be potentially employed in biopreservation of foods produced at different processing conditions.Coordenação de Aperfeiçoamento de Pessoal de Nível Superio

Caracterização dos isolados bacteriocinogênicos Enterococcus hirae e Pediococcus pentosaceus obtidos de queijo artesanal e suas bacteriocinas

Dairy products present a rich and diverse autochthonous microbiota, in which Lactic Acid Bacteria (LAB) are relevant, due to their beneficial, technological and biopreservative features, attracting the interest for their biotechnological application, in food industry, pharmaceutic area and human and veterinary medicine fields. The aim of this study was to isolate and to identify bacteriocinogenic LAB from artisanal cheeses, characterizing some aspects linked to bacteriocin production and purification, safety and beneficial potential of the isolates, as well as their inhibitory properties against Listeria spp. Bacteriocinogenic strains Enterococcus hirae ST57ACC and Pediococcus pentosaceus ST65ACC were isolated by using the triple- layer technique and identified by phenotypical and molecular methods. Bacteriocins produced by E. hirae ST57ACC and P. pentosaceus ST65ACC were stable in a wide range of pH and temperature, losing their activity after treatment with proteolytic enzymes, confirming their proteinaceous nature. Treatments with EDTA, SDS, NaCl and Tween 80 did not affect bacteriocin activity. Cell-free supernatants from both isolates were able to inhibit Listeria innocua and several L. monocytogenes strains, from different serogroups obtained from diverse sources, eliminating L. monocytogenes after 12 h. In co-culture experiments conducted in skimmed milk with the bacteriocinogenic isolates and the target strain L. monocytogenes 422, E. hirae ST57ACC controlled the target strain growth after 48 h. E. hirae ST57ACC and P. pentosaceus ST65ACC did not present positive results for 25 known bacteriocin related genes, indicating that they might express new bacteriocins. E. hirae ST57ACC e P. pentosaceus ST65ACC were also evaluated for their beneficial and safety features: both isolates remained viable after treatment replicating gastrointestinal conditions, showing high levels of auto and co-aggregation with L. monocytogenes and diverse levels of hydrophobicity, demonstrating that E. hirae ST57ACC and P. pentosaceus ST65ACC might prevent the establishment of infections caused by this pathogen. Interference of 33 commercial drugs from different groups on growth of E. hirae ST57ACC and P. pentosaceus ST65ACC was tested by agar-spot method, revealing that only anti-inflammatories and drugs containing loratadine and propranolol hydrochloride influenced the growth of bacteriocinogenic strains. Phenotypical tests employed to determine antibiotic susceptibility have shown that E. hirae ST57ACC and P. pentosaceus ST65ACC were resistant to vancomycin, oxacillin and sulfa/trimethoprim out of 11 antibiotics tested by disk-diffusion test, nonetheless low number of antibiotic resistance genes was observed by PCR analysis. None of the isolates amplified biogenic amines encoding genes neither presented phenotypical evidence of their production. Expression of different ABC transporters linked to bacteriocin export and sugar metabolism was detected, for both isolates. Changes in inoculum size did not influenced the growth of E. hirae ST57ACC and P. pentosaceus ST65ACC; however, bacteriocin production was affected, and bacteriocins were detected only after 9 h with inoculation at 5% and 10% of bacteriocinogenic strains. Additionally, it was observed that cell density of both bacteriocinogenic strains was linked to bacteriocin production in traditional and pH at 5.5 and agitation controlled fermentation continuous. E. hirae ST57ACC and P. pentosaceus ST65ACC were capable to grow and produce bacteriocins in the presence of xylo-oligossacharides after 6 h of incubation, but in lower levels than those obtained with cultivation in MRS broth. Finally, E. hirae ST57ACC and P. pentosaceus ST65ACC were purified from different methods. The bacteriocin produced by P. pentosaceus ST65ACC was purified in two-steps, with final yield of 101.33, recognized as a 3.5 to 8.5 kDa peptide, determined by Tricine-SDS-PAGE. In contrast, a three-step-protocol was used to purify the bacteriocin produced by E. hirae ST57ACC, with final yield of 3.05. Moreover, a semi-purified fraction of E. hirae ST57ACC bacteriocin was tested in HT-29 cell-line, demonstrating no-cytotoxic effects in human cells, which means the bacteriocin can be considered safe in this aspect. Obtained data from this study indicate that E. hirae ST57ACC and P. pentosaceus ST57ACC may be considered as important biotechnological tools for bacteriocin production to control L. monocytogenes and as biopreservatives in food.Os produtos lácteos possuem uma microbiota autóctone bastante diversificada, na qual o grupo das Bactérias Ácido Lácticas (BAL) é de notável relevância devido às suas características benéficas, tecnológicas e bioconservantes, atraindo o interesse para sua utilização em diversos segmentos biotecnológicos, em especial na indústria de alimentos. O objetivo deste trabalho foi isolar e identificar BAL bacteriocinogênicas de queijos artesanais, caracterizando aspectos ligados à produção e purificação das bacteriocinas, inocuidade, potencial benéfico dos isolados e propriedades inibitórias contra Listeria spp. As cepas bacteriocinogênicas Enterococcus hirae ST57ACC e Pediococcus pentosaceus ST65ACC foram isoladas a partir da técnica de tripla camada e identificadas por metodologias fenotípicas e moleculares. As bacteriocinas produzidas por E. hirae ST57ACC e P. pentosaceus ST65ACC demostraram estabilidade em ampla faixa de pH e temperatura, e foram inativadas após tratamento com enzimas proteolíticas, comprovando sua natureza proteica. Tratamentos com EDTA, SDS, NaCl e Tween 80 não afetaram a atividade das bacteriocinas. Os sobrenadantes de ambos os isolados foram capazes de inibir Listeria innocua e diversas cepas de L. monocytogenes pertencentes à diferentes sorogrupos e obtidas de fontes distintas, inibindo completamente o desenvolvimento de L. monocytogenes após 12 h. Em co-culturas das cepas bacteriocinogênicas com a cepa indicadora L. monocytogenes 422 em leite desnatado, observou-se que E. hirae ST57ACC foi capaz de controlar a multiplicação do patógeno após 48 h. E. hirae ST57ACC e P. pentosaceus não apresentaram resultados positivos para 25 genes relacionados a bacteriocinas conhecidas, indicando que podem produzir novas bacteriocinas. As cepas de E. hirae ST57ACC e P. pentosaceus ST65ACC foram também avaliadas quanto ao seu potencial benéfico e segurança: ambos os isolados permaneceram viáveis após tratamento em condições gastrointestinais simuladas, exibindo altos níveis de auto e co-agregação com L. monocytogenes e níveis variados de hidrofobicidade, demonstrando que E. hirae ST57ACC e P. pentosaceus ST65ACC podem prevenir potencialmente o estabelecimento de infecções pelo patógeno. Por meio da metodologia de agar-spot, avaliou-se a possibilidade de interferência de 33 medicamentos comerciais, de diferentes grupos sobre a multiplicação de E. hirae ST57ACC e P. pentosaceus ST65ACC, revelando que apenas antiinflamatórios e medicamentos contendo loratadina e cloridrato de propranolol apresentaram atividade inibitória sobre as cepas. Testes fenotípicos para determinação da susceptibilidade antimicrobiana demonstraram que E. hirae ST57ACC e P. pentosaceus ST65ACC foram resistentes à vancomicina, oxacilina e sulfa/trimetoprim dentre os 11 antibióticos testados pelo método de disco difusão. Com relação à PCR, poucos genes relacionados à resistência a antibióticos foi foram identificados. Nenhum dos isolados amplificou genes de produção de aminas biogênicas e nem apresentou produção das mesmas. A expressão de diferentes elementos do sistema de transporte ABC e metabolismo de açúcares foi identificada para ambos os isolados. Variações na proporção de inóculo não influenciaram a taxa de multiplicação de E. hirae ST57ACC nem de P. pentosaceus ST65ACC, no entanto, a produção de bacteriocinas foi detectada apenas 9 horas após a inoculação das cepas, quando inoculadas nas proporções de 5% e 10%. Adicionalmente, verificou-se que a densidade celular das cepas bacteriocinogênicas esteve correlacionada à produção de bacteriocinas em sistemas de fermentação tradicional e fermentação com controle de pH a 5,5 e agitação. E. hirae ST57ACC e P. pentosaceus ST65ACC foram capazes de se multiplicar e produzir bacteriocinas na presença de xilo-oligossacarídeos após 6 horas de incubação, porém em níveis reduzidos quando comparados ao cultivo em meio MRS. Por fim, as bacteriocinas produzidas por E. hirae ST57ACC e P. pentosaceus ST65ACC foram purificadas a partir de diferentes metodologias. A bacteriocina produzida por P. pentosaceus ST65ACC foi purificada em duas etapas, com rendimento final de 101,33 revelando- se um peptídeo com massa molecular de 3,5 a 8,5 kDa, determinado por SDS-PAGE. Em contrapartida, um protocolo de três etapas foi empregado na purificação da bacteriocina produzida por E. hirae ST57ACC, com rendimento final de 3,05. Adicionalmente, uma fração semi-purificada foi testada com a linhagem celular HT- 29, demonstrando que a bacteriocina não apresenta efeitos citotóxicos contra células humanas, sendo considerada segura neste aspecto. Os dados obtidos neste trabalho indicam que os isolados E. hirae ST57ACC e P. pentosaceus ST57ACC podem ser considerados importantes ferramentas biotecnológicas na produção de bacteriocinas de interesse ao controle de L. monocytogenes e na biopreservação de alimentos.Coordenação de Aperfeiçoamento de Pessoal de Nível Superio

Potential Control of Listeria monocytogenes by Bacteriocinogenic Enterococcus hirae ST57ACC and Pediococcus pentosaceus ST65ACC Strains Isolated From Artisanal Cheese

Bacteriocinogenic Enterococcus hirae ST57ACC and Pediococcus pentosaceus ST65ACC strains, previously isolated from artisanal cheese, were evaluated for their safety with the aim to determine whether they could be used as beneficial strains, especially in the control of Listeria monocytogenes. Both isolates survived simulated gastrointestinal conditions and showed high levels of auto- and co-aggregation with L. monocytogenes, although the hydrophobicity of cells varied. Using the agar-spot test with 33 commercial drugs from different groups, only anti-inflammatory drugs and drugs containing loratadine and propranolol hydrochloride were able to affect the growth of the tested strains. Both strains were resistant to 3 out of 11 antibiotics tested by the disc diffusion method, and low frequencies of antibiotic resistance-encoding genes were observed by PCR analysis. Tested strains neither presented biogenic amine-related genes nor produced these substances. Aside from some antibiotic resistance characteristics, the tested strains were considered safe as they lack other virulence-related genes. E. hirae ST57ACC and P. pentosaceus ST65ACC both presented beneficial properties, particularly their ability to survive gastrointestinal conditions and to aggregate with L. monocytogenes, which can facilitate the elimination of this pathogen. Further studies should be conducted to better understand these interactions

Physiological and molecular insights of bacteriocin production by <em>Enterococcus hirae</em> ST57ACC from brazilian artisanal cheese

International audienceThe bacteriocinogenic Enterococcus hirae ST57ACC recently isolated from a Brazilian artisanal cheese was subjected here to additional analyses in order to evaluate its bacteriocin production and the potential influence of ABC transporter system in its expression. Besides these physiological and molecular aspects, the bacteriocin was evaluated for its cytotoxicity against HT-29. Differences in the inoculum size had no impact on the growth of E. hirae ST57ACC; however, the bacteriocin was only produced after 9h of growth when the strain was inoculated at 5% or 10% (v/v), with similar levels of bacteriocin production obtained by both conventional growth and batch fermentation. Furthermore, potential expression of ABC transporters corresponding to the bacteriocin transport and sugar metabolism was identified. In terms of adverse effects, when a semi-purified fraction of the bacteriocin and the cell-free supernatant were tested against HT-29, total cell viability was similar to observed on untreated cells, indicating the absence of cytotoxic effect. Based on the obtained results, E. hirae ST57ACC can produce its bacteriocin at industrial level by using bioreactors, its bacteriocin expression is potentially influenced by the ABC transporter system, and no cytotoxic effects were observed on HT-29 cells, indicating its potential use as a bio-preservative

Long cold storage influences the microbiological quality of raw goat milk

Storage of goat milk production in cold temperatures is a current Brazilian legislation request, however there is no specification of a limit period for this. The present work aimed to characterize the microbiological characteristics of raw goat milk produced in a specific region of Brazil, as well as the influence of the storage system and period on its quality. Sixty-one samples from 12 goat farms were collected and subjected to analysis to enumerate hygiene indicator microorganisms, psychrotrophics and proteolytic psychrotrophics. The obtained counts were described and compared considering the system and period of storage (ANOVA, Tukey). Despite presenting low counts of mesophiles, the samples presented high counts of other groups and a relevant presence of proteolytics. Samples collected from bulk tanks presented higher counts of mesophiles and psychrotrophics when compared to immersion tanks and freezers (p < 0.05). When stored for a period of 48 h or longer, the counts of mesophiles, coliforms, Escherichia coli and psychrotrophics were also significantly higher when compared to a storage period of 24 h or less (p < 0.05). The results indicate specific problems in goat milk production in the studied area and the need of establishing a period limit for raw goat milk collection in Brazil

Inhibition of herpes simplex virus 1 (HSV-1) and poliovirus (PV-1) by bacteriocins from lactococcus lactis subsp. lactis and enterococcus durans strains isolated from goat milk

Bacteriocins have unusual inhibitory activity, including antiviral properties, and this can be exploited to give alternative applications. Semi–purified bacteriocins of six lactic acid bacteria (LAB) strains isolated from goat milk (two Lactococcus lactis: GLc03 and GLc05, and four Enterococcus durans: GEn09, GEn12, GEn14 and GEn17) were tested for cytotoxicity in Vero cells (50% Cytotoxicity Concentration: CC50), and for their antiviral activities against herpes simplex virus 1 (HVS-1) and poliovirus (PV-1). Semi-purified bacteriocins presented low cytotoxicity, with CC50 varying from 256.2 µg/mL (GLc05) to 1084.5 µg/mL (GEn14). CC10 was determined for all isolates (GLc03: 36.9 µg/mL; GLc05: 51.2 µg/mL; GEn09: 88.1 µg/mL; GEn12: 99.9 µg/mL; GEn14: 275 µg/mL; and GEn17: 62.2 µg/mL) and considered for antiviral activity assays. Antiviral activity before virus adsorption was recorded against PV-1 for GLc05 (4.9%), GEn09 (3.4%), GEn12 (24.7%) and GEn17 (23.5%), and against HSV-1 for GEn12 (27.9%), GEn14 (58.7%) and GEn17 (39.2%). Antiviral activity after virus adsorption was identified against PV-1 for GLc05 (32.7%), GEn09 (91.0%), GEn12 (93.7%) and GEn17 (57.2%), and against HSV-1 for GEn17 (71.6%). The results obtained indicate the potential of some bacteriocins, particularly those produced by E. durans strains investigated in the present study, in viral inhibition and their application as new antiviral agents

Genetic diversity and some aspects of antimicrobial activity of lactic acid bacteria isolated from goat milk

Lactic acid bacteria (LAB, n = 57) were previously obtained from raw goat milk, identified as Lactococcus spp. (n = 24) and Enterococcus spp. (n = 33), and characterized as bacteriocinogenic. Fingerprinting by pulsed field gel electrophoresis (PFGE) demonstrated high genetic diversity, and 30 strains were selected and exhibited strong antimicrobial activity against 46 target strains (LAB, spoilage, and foodborne pathogens). Six strains (Lactococcus lactis: GLc03 and GLc05; and Enterococcus durans: GEn09, GEn12, GEn14, and GEn17) were selected to characterize their bacteriocinogenic features, using Listeria monocytogenes ATCC 7644 as the target. The six strains produced bacteriocins at higher titer when incubated in MRS at 37 °C up to 12 h, when compared to growth at 25 and 30 °C. The produced bacteriocins kept their antimicrobial activity after exposure to 100 °C for 2 h and 121 °C for 20 min; the antimicrobial activity was also observed after treatment at pH 2.0 to 10.0, except for GLc03. L. monocytogenes populations were reduced approximately two logs after treatment with cell-free supernatants from the selected strains. These data show that goat milk can contain a diverse microbiota able to inhibit L. monocytogenes, a common pathogen found in dairy products, and can be potentially employed in biopreservation of food produced under different processing conditions

Molecular tracking of Salmonella spp. in chicken meat chain: from slaughterhouse reception to end cuts

Due to the importance of Salmonella spp. in poultry products, this study aimed to track its main contamination routes since slaughtering reception to processing of chicken end cuts. Samples from different steps of slaughtering and processing (n = 277) were collected from two chicken slaughterhouses (Sl1 and Sl2) located in Minas Gerais state, Brazil, and subjected to Salmonella spp. detection. The obtained isolates were subjected to serological identification and tested by PCR for specific Salmonella spp. genes (ompC and sifB). Also, Salmonella spp. isolates were subjected to XbaI macrorestriction and pulsed-field gel electrophoresis (PFGE). Sixty-eight samples were positive for Salmonella spp. and 172 isolates were obtained. Sl1 and Sl2 presented similar frequencies of Salmonella spp. positive samples during reception, slaughtering and processing (p > 0.05), except for higher frequencies in Sl1 for chicken carcasses after de-feathering and evisceration (p < 0.05). PFGE allowed the identification of cross contamination and persistence of Salmonella spp. strains in Sl1. The results highlighted the relevance of the initial steps of chicken slaughtering for Salmonella spp. contamination, and the pre-chilling of carcasses as an important controlling tool. In addition, the presence of Salmonella spp. in chicken end cuts samples represents a public health concern

Influence of Emulsifying Salts on the Growth of Bacillus thuringiensis CFBP 3476 and Clostridium perfringens ATCC 13124 in Processed Cheese

Processed cheese is a dairy product with multiple end-use applications, where emulsifying salts play a fundamental role in physicochemical changes during production. Moreover, some of these salts may be a strategy to control spoilage and pathogenic microorganisms, contributing to safety and shelf life extension. This study aimed to evaluate the in vitro inhibitory activity of two emulsifying salts (ESSP = short polyP and BSLP = long polyP) against Bacillus thuringiensis CFBP 3476 and Clostridium perfringens ATCC 13124, and to compare the in situ effects of two emulsifying salts treatments (T1 = 1.5% ESSP and T2 = 1.0% ESSP + 0.5% BSLP) in processed cheeses obtained by two different methods (laboratory- and pilot-scales), during 45-day storage at 6 &deg;C. C. perfringens ATCC 13124 growth was not affected in vitro or in situ (p &gt; 0.05), but both of the treatments reduced B. thuringiensis CFBP 4376 counts in the tested condition. Counts of the treatments with B. thuringiensis CFBP 3476 presented a higher and faster reduction in cheeses produced by the laboratory-scale method (1.6 log cfu/g) when compared to the pilot-scale method (1.8 log cfu/g) (p &lt; 0.05). For the first time, the inhibitory effect of emulsifying salts in processed cheeses obtained by two different methods was confirmed, and changes promoted by laboratory-scale equipment influenced important interactions between the processed cheese matrix and emulsifying salts, resulting in B. thuringiensis CFBP 4376 growth reduction