Avaliação da interação entre leveduras isoladas de kombucha e bactérias probióticas / Evaluation of the interaction between isolated kombucha yeasts and probiotic bacteria

Um dos maiores desafios no desenvolvimento de produtos probióticos é entender como os microrganismos interagem entre si e com o hospedeiro. Quando falamos em alimentos fermentados tradicionais, este obstáculo aumenta porque a matriz alimentar já possui um microbioma intrínseco. No entanto, também é conhecido que muitos microrganismos podem interagir e cooperar para sobreviver quando condições de estresse são encontradas. Assim, o objetivo deste trabalho foi isolar leveduras de quatro diferentes kombuchas em distintos momentos fermentativos e verificar a influência que leveduras isoladas de kombucha têm na manutenção da viabilidade da bactéria probiótica Bifidobacterium animalis subsp. lactis HN019 em condições de aerobiose. Meyerozyma guilliermondii, Candida albicans, Rhodotorula mucilaginosa e Pichia membranifaciens foram leveduras encontradas nas kombuchas, das quais as duas últimas favoreceram a manutenção da alta viabilidade de HN019 em cocultura por 14 dias. Observou-se a viabilidade da bactéria acima de 9 log ao longo de todo o experimento, o que não foi observado em monocultura. Ademais, utilizou-se de análise de autoagregação, hidrofobicidade, atividade enzimática de proteases e fosfolipases das leveuras para analisar seu potencial patogênico. Observou-se que R. mucilaginosa demonstrou características semelhantes à Saccharomyces cerevisiae subsp. boulardii, e sua interação benéfica com HN019 reforça a possibilidade de que esta levedura seja uma chave para a inserção da bactéria em uma kombucha probiótica. Análises metabólicas foram realizadas e encontrou-se uma vasta diversidade de dipeptídeos, principalmente os compostos de prolina, durante a cocultura da bactéria com as leveduras. Tais dipeptídeos apresentam importantes mecanismos de ação no controle biológico e quorum sensing de bactérias e leveduras, e supostamente regulam a manutenção das relações mutualísticas entre ambos microrganismo

One of the biggest challenges in the development of probiotic products is to understand how microorganisms interact with each other and with the host. When we talk about traditional fermented foods, this obstacle increases because the food matrix already has an intrinsic microbiome. However, it is also known that many microorganisms can interact and cooperate to survive when stressful situations are encountered. Thus, the objective of this work was to isolate yeasts from four different kombuchas at different fermentation times and to verify the influence that yeasts isolated from kombucha have on maintaining the viability of the probiotic bacterium Bifidobacterium animalis subsp. lactis HN019 under aerobic conditions. Meyerozyma guilliermondii, Candida albicans, Rhodotorula mucilaginosa and Pichia membranifaciens were yeasts found in kombuchas, of which the last two favored the maintenance of HN019 high viability in co-culture for 14 days. Bacteria viability above 9 log was observed throughout the experiment, which was not observed in monoculture. In addition, analysis of autoaggregation, hydrophobicity, enzyme activity of proteases and phospholipases of yeasts was used to analyze their pathogenic potential. It was observed that R. mucilaginosa demonstrated characteristics similar to Saccharomyces cerevisiae subsp. boulardii, and its beneficial interaction with HN019 reinforces the possibility that this yeast is a key to the insertion of the bacterium in a probiotic kombucha. Metabolic analysis were performed and a wide diversity of dipeptides, mainly proline-based, was found during the co-culture of the bacteria with the yeasts. Such dipeptides have important mechanisms of action in the biological control and quorum sensing of bacteria and yeast, and supposedly regulate the maintenance of mutualistic relationships between both microorganism

Viabilidade de culturas probióticas em queijos tipo Minas Frescal / Viability of probiotic cultures in cheese Minas Frescal type

Os consumidores estão mais exigentes com a qualidade dos alimentos, e diante deste cenário, a adição de bactérias probióticas com apelo na saúde é uma tendência promissora, principalmente em produtos lácteos como queijo Minas Frescal. Objetivou-se verificar a viabilidade da adição de L. acidophilus LA-5 e B. animalis subsp. lactis BB-12 em queijo Minas Frescal, sob refrigeração a 4ºC, no 0, 7, 14 e 21 dias de armazenamento. As amostras foram inoculadas em ágar MRS e incubadas em anaerobiose a 35°C ± 2ºC por 48 horas. A população de células viáveis de bactérias probióticas se manteve nos níveis mínimos exigido pela legislação (10(8)-10(9) UFC g-1) durante todo o tempo de prateleira, o que evidencia a qualidade do queijo Minas Frescal como matriz alimentícia adequada e como um alimento potencialmente probiótico.

Development and characterization of an innovative synbiotic fermented beverage based on vegetable soybean

Abstract Soymilk was produced from vegetable soybean and fermented by probiotics (Lactobacillus acidophilus La-5, Bifidobacterium animalis Bb-12) in co-culture with Streptococcus thermophilus. The composition of the fermented beverage and oligosaccharides content were determined. The effect of fructooligosaccharides and inulin on the fermentation time and viability of probiotic microorganisms throughout 28 days of storage at 5 °C were evaluated. The soymilk from vegetable soybeans was fermented in just 3.2 h, when pH reached 4.8. Fermentation reduced the contents of stachyose and raffinose in soymilk. Prebiotics had no effect on acidification rate and on viability of B. animalis and S. thermophilus in the fermented beverage. The viable counts of B. animalis Bb-12 remained above 108 CFU mL-1 in the fermented soymilk during 28 days of storage at 5 °C while L. acidophilus La-5 was decreased by 1 log CFU mL-1. The fermented soymilk from vegetable soybeans showed to be a good food matrix to deliver probiotic bacteria, as well as a soy product with a lower content of non-digestible oligosaccharides.

A influência de bebida láctea com cultura probiótica (Bifidobacterium animalis) no tratamento dos sintomas de constipação / Influence of drinking a probiotic fermented milk beverage containing bifidobacterium animalis on the symptoms of constipation

ABSTRACT BACKGROUND Constipation is a chronic problem in many patients all over the world. OBJECTIVE - To evaluate the effect of consumption of a probiotic fermented milk beverage containing Bifidobacterium animalis on the symptoms of constipation. METHODS - This randomized, double-blind controlled trial included 49 female patients aged 20 to 50 years and diagnosed with constipation according to the ROME III criteria (Diagnostic Criteria for Functional Gastrointestinal Disorders) and the Bristol Stool Form Scale. The patients were randomized into two groups: the intervention group received the probiotic fermented milk beverage and the control group received non-probiotic milk. Participants were instructed to ingest 150 mL of the beverages during 60 days. At the end of this period, patients were assessed again by the ROME III criteria and Bristol scale. The Wilcoxon test was used to evaluate pre and post-intervention results of the ROME III criteria and Bristol scale. The statistical significance level was considered as 5% ( P ≤0.05). RESULTS - The intervention group showed improvement in the following criteria: straining during a bowel movement ( P <0.001), feeling of incomplete evacuation ( P <0.001) and difficulty in passing stool ( P <0.014), in addition to Bristol scale results ( P <0.001). In the control group, improvements were observed in the following criteria: straining during a bowel movement ( P <0.001), feeling of incomplete evacuation ( P <0.001) and difficulty in passing stool ( P <0.025), in addition to Bristol scale results ( P <0.001). No statistically significant post-intervention differences were observed between the two groups for the Rome III criteria and Bristol scale. CONCLUSION - The results show that the consumption of milk resulted in the improvement of constipation symptoms, regardless of the probiotic culture.

RESUMO CONTEXTO - Constipação é um sintoma crônico que acomete grande parte da população mundial. OBJETIVO - Avaliar o efeito do consumo de bebida láctea com cultura probiótica ( Bifidobacterium animalis ) nos sintomas de constipação intestinal. MÉTODOS - Ensaio clínico randomizado, duplo cego e controlado. A amostra foi de 49 pacientes, do gênero feminino, com idade entre 20 a 50 anos e com diagnóstico de constipação intestinal conforme os critérios de ROMA III ( Diagnostic Criteria for Functional Gastrointestinal Disorders ) e escala de Bristol. As pacientes foram divididas em dois grupos através de randomização, o grupo intervenção recebeu bebida láctea com cultura probiótica e o grupo controle recebeu bebida láctea. Estas foram orientadas a consumir 150 mL diariamente durante 60 dias. Ao final deste período, aplicaram-se novamente os critérios de ROMA III e escala de Bristol. Para avaliação dos critérios de ROMA III e escala de Bristol pré e pós-intervenção, o teste de Wilcoxon foi aplicado. O nível de significância estatística considerado foi de 5% ( P ≤0,05). RESULTADOS - No grupo intervenção houve melhora nos seguintes critérios: esforço para evacuar ( P <0,001), sensação de evacuação incompleta ( P <0,001) e dificuldade na passagem das fezes ( P =0,014), além da escala de Bristol ( P =<0,001). No grupo controle houve melhora nos seguintes critérios: esforço para evacuar ( P <0,001), sensação de evacuação incompleta ( P <0,001) e dificuldade na passagem das fezes ( P =0,025), além da escala de Bristol ( P =<0,001). Verificou-se que os critérios de Roma III e escala de Bristol não houve diferenças estatisticamente significativas entre os grupos pós-intervenção. CONCLUSÃO - Concluiu-se que o consumo de bebida láctea auxiliou na melhora dos sintomas de constipação intestinal, independentemente da cultura probiótica.

El papel de las bifidobacterias en el funcionamiento del organismo humano / Role of bifidobacteria in the function of the human body

Bifidobacterium animalis ssp lactis Bb12 is a probiotic strain that has received considerable attention from the scientific community. It has tolerance to higher temperatures and lower, acidic pH than other bacteria. When administered for 12 months to infants and children together with S. thermophilus this was associated with lower incidences of acute diarrhea. Studies in different countries and with different experimental designs confirmed these results. It was also shown that its administration did not interfere the growth or the normal weight gain of the children. Bifidobacterium animalis ssp lactis Bb12 was associated with decreases of the fecal excretion of rotavirus during episodes of diarrhea, a fact that represents an epidemiological benefit. Bifidobacterium animalis ssp lactis Bb12 exerts positive effects on manifestations of atopy/eczema, with decreases of the parameters of inflammation such as CD4 in blood serum and eosinophil protein X in urine. This was associated with changes in the extension and severity of the skin lesions. No modifications of the normal growth parameters were observed when Bifidobacterium animalis ssp lactis CNCM I-3446 was given. Administration of Bifidobacterium animalis ssp lactis Bb12 is associated with higher levels of fecal sIgA and calprotectin, which are considered parameters of immune responses and ofthe capacity to launch inflammatory responses. The statements made by some groups that infants under 4 months of age who are not breastfed should not receive probiotics have weak support if it is considered that maternal milk contains a large number and variety of strains of bacteria which may be considered as probiotics. These may not only protect from acute diarrhea but also from upper respiratory infections. Although cases of septicemia due to probiotic have been reported these represent an infinitely small proportion of the total numbers of consumers. No outbreaks have been reported that would point to invasive properties in a strain. It is not advisable to administer any living bacteria to individuals in shock or with innate or severe defects of immunity. However, carriers of HIV or patients with AIDS benefit from probiotic agents. A study carried out in Chile showed that although without evident clinical benefits L. rhamnosus HN001, significantly increased fecal sIgA as a manifestation of improved mucosal defense in the digestive tract.

Bifidobacterium animalis ssp lactis Bb12 es una de las cepaBifidobacterium animalis ssp lactis Bb12 es una de las cepas probióticas más estudiadas. Posee ventajas que la hacen útil para el uso industrial: tolerancia a ambientes ácidos y a temperaturas superiores a las de los demás probióticos. En un estudio de 12 meses Bifidobacterium animalis ssp lactis Bb12 fue administrada por un año asociada con S. thermophilus y se observó que los niños experimentaron menos episodios de diarrea aguda. Estudios con seguimientos de distinta duración y diseño refrendaron estos resultados y su administración no ejercía influencias negativas en el crecimiento. Esta bifido-bacteria inducía disminuciones de la excreción de rotavirus durante episodios de diarrea, lo que disminuye la posibilidad de contagios. Bifidobacterium animalis ssp lactis Bb12 ejerce efectos positivos sobre las manifestaciones de atopia como el eccema con el descenso de marcadores de inflamación tales como CD4 en el suero y de la proteína X de los eosinófilos en la orina; y mejorías de las alteraciones de la permeabilidad. Estudios con Bifidobacterium animalis ssp lactis CNCM I-3446 administrada con ARA y DHA mostraron alteraciones de los parámetros de crecimiento y bioquímicos en lactantes y preescolares en seguimientos entre 14 y 119 días. La administración de Bifidobacterium animalis ssp lactis Bb12 junto con inmunizaciones se asociaba con niveles más elevados de sIgA específica anti-poliovirus y de calprotectina, cuyos niveles estarían asociados con los de esta inmunoglobulina. Se ha sostenido que no existe justificación para administrar probióticos a menores de 4-6 meses no amamantados, el hecho que la leche materna los proporciona en cantidad y variedad apoya las posibles ventajas de su administración. Los probióticos protegerían no sólo de infecciones del tracto gastrointestinal sino también de las respiratorias altas. Hay casos aislados de sepsis en individuos con diversas afecciones, pero la proporción de afectados es mínima. No se han comunicado brotes epidémicos que indicarían cepas probióticas con capacidades invasoras. Probablemente no es aconsejable administrarlos a sujetos en shock o con defectos de la función de barrera intestinal. Los portadores de VIH y los pacientes con SIDA experimentan mejorías al recibir probióticos. En un estudio en Chile se demostró que aunque sin efectos clínicamente evidentes, administrar L. rhamnosus HN001 produjo aumentos del la IgA secretoria fecal, que representa un aumento de las defensas del tubo digestivo.

Effect of refrigerated storage on the probiotic survival and sensory properties of milk/carrot juice mix drink

Background: There is a genuine interest in the development of probiotic milk and juice based beverages because they are a good-vehicle to deliver probiotic microorganisms to consumers. For this purpose, the viability and metabolism of four probiotic strains (Lactobacillus acidophilus LA5, Bifidobacterium lactis BB12, L. rhamnosus and L. plantarum) were studied in non-fermented milk and carrot juice mix drink. The drinks were evaluated in 5 days interval for viable cell count, pH, acidity, sedimentation and sensory quality during refrigerated storage at 4 ± 2ºC for up to 20 days. Results: The results showed that all strains had good viability in milk/carrot juice drink (88-98%), but L. acidophilus LA5 seemed more stable than three other strains. The levels of pH and acidity were ranged 5.33-6.6 and 0.13-0.31%, respectively. The drinks inoculated with L. rhamnosus and control (non-probiotic) showed more variation in pH and acidity. The most sedimentation was detected in drinks inoculated with L. rhamnosus, reaching 3.73 mL/10 mL sample. Sensory assessment indicated lowest acceptability in control and milk/carrot juice drink inoculated with L. rhamnosus, respectively. Conclusion: This study indicated that some probiotic bacteria can be applied by food producers to produce functional drinks with an increased shelf-life.

Efeito do leite probiótico fermentado na resposta imune celular em cólon de camundongos BALB/c / Effect of probiotic fermented milk in immune cellular response of BALB/c mice colon

O principal crescimento na indústria de alimentos funcionais corresponde ao dos produtos probióticos e prebióticos. A literatura mostra efeitos imunomoduladores de certas cepas probióticas, contudo, os resultados são às vezes controversos e os mecanismos implicados ainda são pouco elucidados. Sabe-se, no entanto que algumas cepas de probióticos aumentam significantemente a liberação de IL-10 e γ-INF modulando a resposta imune, além destas respostas serem de forma mais branda relacionada às bactérias Gram-positivas probióticas do que às Gram-positivas patogênicas. O presente trabalho teve como objetivo geral estudar o efeito do leite probiótico fermentado na resposta imune celular em cólon de camundongos BALB/c. Os objetivos específicos foram: (i) determinar o efeito imunomodulador do leite adicionado de probiótico em camundongos normais, (ii) identificar os tipos celulares implicados na resposta imune específica por citometria de fluxo e, (iii) colocalizá-los nos cortes histológicos. Simultaneamente, a análise e a comparação da resistência do probiótico à digestão gastrintestinal in vitro e a produção de metabólitos bioativos de acordo com os deferentes produtos foi realizada. Foram preparados leites nos quais as variáveis estudadas foram a tecnologia empregada para a produção das formulações (a) leite; (b) água, (c) leite não fermentado; (d) leite fermentado; (e) leite fermentado seguido de pasteurização, usando a mesma concentração da cepa comercial Bifidobacterium animalis subsp. lactis HOWARU HN019. O leite desnatado e a água foram usados como controles

Functional food industry is in expansion mainly due to probiotic and prebiotic products. Studies have shown some probiotic strains develop immune modulation effect, however, these results are controversial and the mechanisms are not been well understood. Although, some probiotic strains increase IL-10 and γ-INF release modulating immune response, this response is weaker in probiotic strains when compared to pathogenic Gram-positive bacteria. The major aim of the present study was to assess the effect of probiotic fermented milk in cellular immune response of Balb/c mice colon. The specific objectives were: (i) to determine the immunomodulation of the milk added of probiotic in normal mice; (ii) to identify the cellular types implied in immune specific response and, (iii) to colocalize them in histological sections. Besides, the analyze and comparation of the probiotic resistance upon in vitro gastrointestinal and bioactive metabolites release in fermented or unfermented bifido milk using the same matrix, probiotic strain and probiotic dose in CFU. mL-1 were conducted. Dairy products were prepared in which variable form of technological appliance were: (i) milk, (ii) water, (iii) unfermented milk, (iv) fermented milk, and (v) fermented and heat treatment milk, all using Bifidobacterium subsp. lactis HOWARU HN019 strain in the same concentration. The skimmed milk and water were used as controls. The immune effects were evaluated by histological sections and the lymphocytic infiltrated was analyzed by flow citometry and histology