Desenvolvimento de bebidas lácteas probióticas adicionadas de polpa de morango e suplementadas com L-triptofano elaboradas a partir de leite bubalino e bovino / Development of probiotic dairy beverages with strawberry pulp and supplemented with L-tryptophan from buffalo and bovine milk

O objetivo deste estudo foi desenvolver uma formulação de bebida láctea bubalina probiótica adicionada de polpa de morango, comparando os efeitos do uso do leite de búfala e de vaca na elaboração dos produtos e verificando a possibilidade de suplementação com triptofano nos produtos lácteos probióticos. Como primeira etapa do trabalho, bebidas lácteas probióticas foram elaboradas a partir de leite bubalino e bovino, fermentadas com Streptococcus thermophilus TA040, Lactobacillus bulgaricus LB340 e Lactobacillus acidophilus La5, e formuladas com 0, 25 e 50% de soro em sua formulação. As bebidas foram avaliadas quanto à cinética de fermentação das culturas láticas utilizadas, ao teor de proteína, gordura e sólidos totais não gordurosos, pós-acidificação, viabilidade das culturas fermentadoras e sua capacidade de sobrevivência ao estresse gastrointestinal in vitro. As bebidas lácteas bubalinas apresentaram resultados superiores as bebidas bovinas. O uso do leite de búfala na elaboração das bebidas lácteas promoveu benefícios quanto as culturas láticas presentes nos produtos, exercendo efeito protetivo e influindo na preservação da viabilidade das bactérias ao longo do armazenamento refrigerado e durante a simulação do estresse gastrointestinal in vitro. As bebidas lácteas elaboradas com 25% apresentaram os resultados mais próximos aos obtidos pelos produtos controle, sem adição de soro, sendo selecionadas para a segunda parte do estudo. Nesta etapa, as formulações de bebida láctea com 25% de soro, foram acrescidas de um preparado com polpa de morango e bebidas sem adição da fruta, utilizadas como controle. As bebidas lácteas bubalinas frutadas, apresentaram menor teor de gordura e melhores características reológicas, com maior viscosidade e consistência do que os produtos controle, sem afetar a pós-acidificação, o perfil de ácido graxo, assim como, a viabilidade e a resistência às condições de estresse gastrointestinal in vitro das culturas fermentadoras. A avaliação da possibilidade de suplementar lácteos probióticos com triptofano foi realizada em conjunto com a Universidade de Milão. Para isso, iogurtes probióticos receberam adição de triptofano antes ou após a fermentação, sendo avaliados com relação ao perfil de pós-acidificação, quantidade de triptofano nos produtos, número de células viáveis por plaqueamento e citometria de fluxo ao longo do armazenamento a 25° e 4°C. Complementarmente, a influência da presença do triptofano no crescimento e produção de compostos antimicrobianos pelas culturas láticas, também foi avaliada. A adição de triptofano após a fermentação dos iogurtes, que foram armazenados sob refrigeração (4°C), além de não afetar a pós-acidificação dos produtos, apresentou benefícios quanto a viabilidade L. acidophilus, redução do dano e aumento do número de células vivas, promovendo teor maior do aminoácido nos iogurtes. A presença do triptofano nos meios de cultivo, também influenciou de forma positiva o crescimento de S. thermophilus e L. acidophilus, melhorando o desenvolvimento das bactérias durante a fermentação e influindo em uma maior atividade antilistérica por parte do S. thermophilus. Diante da influência positiva da aplicação do leite de búfala na elaboração das bebidas lácteas, assim como, a adição do triptofano em iogurtes probióticos, a suplementação do aminoácido em bebidas lácteas bubalinas frutadas permitiria a obtenção de um produto funcional, onde seus benefícios estariam relacionados tanto ao consumo do probiótico presente no produto quanto a complementação de triptofano na dieta do consumidor

The aim of this study was to develop a formulation of probiotic buffalo dairy beverage added with strawberry pulp, comparing the effects of using buffalo and cow's milk in the preparation of products and verifying the possibility of tryptophan supplementation in probiotic dairy products. As a first stage of the work, probiotic dairy beverages were made from buffalo and bovine milk, fermented with Streptococcus thermophiles TA040, Lactobacillus bulgaricus LB340 and Lactobacillus acidophilus La5, and formulated with 0, 25 and 50% whey in their formulation. The beverages were evaluated for the fermentation kinetics of the used lactic cultures, the levels of protein, fat and total no fat solids, post-acidification, fermenting cultures viability and their ability to survive gastrointestinal stress in vitro. Buffalo milk use in dairy beverages production promoted benefits regarding the lactic cultures present in the products, exerting a protective effect and influencing the viability preservation of bacteria during the cold storage and simulation of gastrointestinal stress in vitro. Dairy beverages made with 25% whey addition showed results similar to those obtained by the control products, without whey addition, being selected for the second part of the study. In this part, the dairy beverages formulations with 25% whey, were added with a preparation were added with a strawberry pulp preparation and dairy beverages without added fruit, used as a control. Fruity bubaline dairy beverages had lower fat content and better rheological characteristics, with higher viscosity and consistency than control products, without affecting post-acidification, fatty acid profile, as well as viability and resistance to in vitro gastrointestinal condition of fermented cultures. The possibility of supplementing probiotic dairy products with tryptophan was evaluated in partnership with the University of Milan. For this, probiotic yogurts received the addition of tryptophan before or after fermentation, being evaluated in relation to the post-acidification profile, tryptophan amount in the products, viable cell number per plating and flow cytometry during storage at 25°C and 4°C. In addition, the influence of the tryptophan presence on the growth and production of antimicrobial compounds by lactic cultures was also evaluated. The addition of tryptophan after the yogurt fermentation, which were stored under refrigeration (4°C), in addition to not affecting the post-acidification of the products, showed benefits to the viability of L. acidophilus, reduced the damage and increased the number of cells promoting higher amino acid content in yogurts. Tryptophan presence in the culture media also positively influenced the growth of S. thermophiles and L. acidophilus, improving the development of bacteria during fermentation and influencing better antilisteric activity in the part of S. thermophiles. In view of the buffalo milk positive influence observed after the application in dairy beverage preparation, as well as the addition of tryptophan in probiotic yoghurts, amino acid supplementation in fruity buffalo dairy beverages would allow to obtain a functional product, where its benefits would be related both to the consumption of the probiotic present in the product as to the supplementation of tryptophan in the consumer's diet

Probiotic properties of native Lactobacillus spp. strains for dairy calves.

The use of native microorganisms with probiotic capacity is an alternative tool for the treatment and prevention of several diseases that affect animals, such as neonatal calf diarrhoea. The selection of probiotic strains within a collection is based on different in vitro and in vivo assays, which predict their potential. The aim of this study was to characterise a group of native Lactobacillus spp. strains isolated from faeces of healthy calves using an in vitro approach and to assess their ability to colonise the gastrointestinal tract (GIT) of calves. Native Lactobacillus spp. strains were evaluated on their capacity to survive low pH conditions and bile salts presence, biofilm formation and adhesion to both mucus and Caco-2 cells. Based on the in vitro characterisation, four strains (Lactobacillus johnsonii TP1.1, Lactobacillus reuteri TP1.3B, L. johnsonii TP1.6 and Lactobacillus amylovorus TP8.7) were selected to evaluate their capacity to colonise and persist in the GIT of calves. The assessment of enteric persistence involved an in vivo assay with oral administration of probiotics and quantification in faeces of the administered bacterial species with real-time quantitative PCR (qPCR). The study was conducted using 15 calves (1-month-old) which were divided into five groups of three animals, four of which were treated with four different selected strains and one was the control group. Strains TP1.3B and TP1.6 managed to persist in treated animals until ten days after the end of the administration period, indicating that they could be promising candidates for the design of probiotics for calves.

Evaluation of Probiotic Potential of Bacteriocinogenic Lactic Acid Bacteria Strains Isolated from Meat Products.

In this study, the probiotic potential of five bacteriocin-producing lactic acid bacteria (LAB) strains, isolated from meat products, was investigated. They were presumptively identified as Lactococcus lactis subsp. cremoris CTC 204 and CTC 483, L. lactis subsp. hordinae CTC 484, and Lactobacillus plantarum CTC 368 and CTC 469 according to morphological, biochemical, and physiological characteristics. Analysis of genetic variability (random amplified polymorphic (RAPD)-PCR) and whole-cell proteins (SDS-PAGE) revealed similarity between Lactobacillus strains and variability among Lactococcus strains. The evaluation of the probiotic potential showed that the five LAB strains were tolerant to pH 2.0, and only strain CTC 469 was tolerant to the lowest concentration of the bile salts evaluated (0.1%). All strains showed survival or growth ability at 4, 25, and 37 °C, and tolerance at - 20 °C. Although strain CTC 204 in TSB Broth supplemented with MgSO4 showed the highest intensity of biofilm production, this compound was produced by all of them. The safety assessment showed that no thermonuclease, hemolytic, or gelatinase activities were detected. All strains were resistant to erythromycin and sensitive to amoxicillin and phenoxymethylpenicillin; furthermore, CTC 204 was resistant to chloramphenicol, CTC 368 and CTC 469 to chloramphenicol and vancomycin, CTC 483 to tetracycline and vancomycin, and CTC 484 to clindamycin and chloramphenicol. The evaluated strains showed biogenic amine production; the lowest levels were produced by CTC 204 and CTC 368 strains. It was concluded that CTC 204 and CTC 368 strains have the greatest potential for becoming probiotics.

In Vitro Characterization of Lactobacillus Strains Isolated from Fruit Processing By-Products as Potential Probiotics.

Nine wild Lactobacillus strains, namely Lactobacillus plantarum 53, Lactobacillus fermentum 56, L. fermentum 60, Lactobacillus paracasei 106, L. fermentum 250, L. fermentum 263, L. fermentum 139, L. fermentum 141, and L. fermentum 296, isolated from fruit processing by-products were evaluated in vitro for a series of safety, physiological functionality, and technological properties that could enable their use as probiotics. Considering the safety aspects, the resistance to antibiotics varied among the examined strains, and none of the strains presented hemolytic and mucinolytic activity. Regarding the physiological functionality properties, none of the strains were able to deconjugate bile salts; all of them presented low to moderate cell hydrophobicity and were able to autoaggregate, coaggregate with Listeria monocytogenes and Escherichia coli, and antagonize pathogenic bacteria. Exposure to pH 2 sharply decreased the survival of the examined strains after 1- or 2-h exposure; variable decreases were noted after 3-h exposure to pH 3. Overall, exposure to pH 5 and to bile salts (0.15, 0.3, and 1%) did not decrease the strains' survival. Examined strains presented better ability to survive from the exposure to simulated gastrointestinal conditions in laboratorial media and milk than in grape juice. Considering the technological properties, all the strains were positive for proteolytic activity and EPS and diacetyl production, and most of them had good tolerance to 1-4% NaCl. These results indicate that wild Lactobacillus strains isolated from fruit processing by-products could present performance compatible with probiotic properties and technological features that enable the development of probiotic foods with distinct characteristics.

Impact of the incorporation of probiotic strains and fruit by-products in fermented synbiotic soy product and on the composition and metabolic activity of the gut microbiota in vitro / Impacto da incorporação de cepas probióticas e de subprodutos de frutas em um produto fermentado de soja simbiótico e sobre a composição e a atividade metabólica da microbiota intestinal humana in vitro

The present study aimed to develop a fermented soy beverage containing fruit by-products and probiotics and to evaluate the impact of this product on the composition and metabolic activity of the human intestinal microbiota using an in vitro simulation model of the intestinal conditions (TIM-2). Therefore, the present study was divided into three stages. Stage I was based on obtaining, processing and physical-chemical, microbiological and functional characterization of fruit by-products (acerola, orange, mango, and passion fruit) and soybean (okara), as well as amaranth flour. Additionally, the ability to use these vegetable by-products and amaranth flour by probiotic and non-probiotic strains was evaluated. The results showed that the acerola byproduct presented the highest dietary fibre content (48.46 g/100 g) among the by-products tested, as well as amaranth flour. Orange and passion fruit by-products were the substrates that most promoted the growth of bacterial populations, including strains of Escherichia coli and Clostridium perfringens. On the other hand, the acerola by-product was the substrate that showed the highest selectivity for beneficial bacteria. Also, in this stage, ten probiotic strains (seven lactobacilli and three bifidobacteria) and three starter strains (Streptococcus thermophilus) were tested for their ability to deconjugate bile salts and for proteolytic activity against milk and soy proteins. The results showed that none of the tested strain showed proteolytic ability against milk and soybean proteins. In addition, the probiotic strains Lactobacillus acidophilus LA-5 and Bifidobacterium longum BB-46 deconjugated more types of bile acids tested, and the strains of S. thermophilus tested showed no ability to deconjugate bile salts. Next, the acerola by-product (ABP) and the probiotic strains LA-5 and BB-46 were selected to continue stage II of the study (development of a fermented soy beverage). For this purpose, a 23 factorial design was used, in a total of 8 trials with three replicates of each one, and the effects of the probiotic strains and the acerola by-product on the physical-chemical, microbiological, and sensory characteristics of these fermented soy beverages were evaluated. At the same time, probiotic viability and survival under in vitro gastrointestinal (GI) simulated conditions were evaluated in fermented soy beverage (FSB). The results showed that the presence of BB-46 and ABP affected the sensory acceptability of FSB negatively. ABP also led to significant differences in the texture profile of the FSB (P<0.05). Populations of probiotic strains ranged from 7.0 to 8.2 log CFU equivalent/mL during 28 days of refrigerated storage (4° C) of FBS, and the co-culture (LA-5+BB-46) and the ABP did not affect the viability of both microorganisms significantly (P> 0.05). However, ABP increased the survival of BB-46 under in vitro simulated GI conditions significantly. For stage III, a 22 experimental design was performed. To evaluate the impact of these FBS on the composition and metabolic activity of the intestinal microbiota of lean and obese humans, a validated in vitro model called TIM-2 was used, available at the Maastricht University (Venlo, The Netherlands), which simulates normal conditions of the lumen of the proximal colon, with all parameters controlled by a computer. Samples were collected from TIM-2 to quantify probiotic microorganisms (LA-5 and BB-46), Lactobacillus spp., Bifidobacterium spp., and total bacteria, using the quantitative PCR method (qPCR) and the intestinal microbiota profile was determined using an Illumina Mysec Next Generation Sequencing (NGS) method. Concentrations of shortchain fatty acids and branched-chain fatty acids and lactate produced by the different microbiotas during fermentation in TIM-2 were also determined. The results showed that the lean microbiota presented the high production of acetate and lactate than the microbiota of obese individuals. Significant reductions in Bifidobacterium populations in the lean microbiota were observed at 0 and 48 h of an assay for all experimental meals, except for the meal that had the probiotic combination (LA-5 and BB-46) and the ABP supplementation, which showed an increased total Bifidobacterium and Lactobacillus populations throughout the experimental period for both microbiotas tested. The FSB supplemented with ABP presented the best characteristics regarding the modulation of the obese microbiota, with an increase in Bifidobacterium spp. and Lactobacillus spp. Additionally, after 48 hours of intervention in TIM-2, the obese microbiota was apparently similar to the lean microbiota, showing a beneficial modulation of this microbiota. The results suggest that the fermented soy beverage supplemented with the acerola by-product and the probiotic strains may present beneficial health effects. However, clinical studies are required to complement and confirm the results observed in the in vitro assays

O presente trabalho visou desenvolver uma bebida fermentada de soja adicionada de resíduos de frutas e suplementada com cepas probióticas e avaliar o impacto desse produto sobre a composição e a atividade metabólica da microbiota intestinal humana, utilizando um modelo de simulação in vitro das condições intestinais (TIM-2). Para tanto, o presente trabalho foi dividido em três etapas. A etapa I foi baseada na obtenção, processamento e caracterização físico-química, microbiológica e funcional de subprodutos de frutas (acerola, laranja, manga e maracujá) e soja (okara), bem como da farinha de amaranto. Adicionalmente, a capacidade de utilização desses subprodutos vegetais e da farinha de amaranto por cepas probióticas e não probióticas foi avaliada. Os resultados mostraram que o subproduto de acerola apresentou o maior conteúdo de fibras alimentares totais (48,46 g/100 g) dentre os subprodutos testados, bem como a farinha de amaranto. Os subprodutos de laranja e maracujá foram os substratos que mais promoveram a multiplicação das populações bacterianas, incluindo das cepas de Escherichia coli e Clostridium perfringens. Por outro lado, o subproduto de acerola foi o substrato que apresentou a maior seletividade para bactérias benéficas. Ainda nessa etapa, dez cepas probióticas (sete lactobacilos e três bifidobacterias) e três cepas starter (Streptococcus thermophilus) foram testadas quanto à sua capacidade de desconjugação de sais biliares e atividade proteolítica frente às proteínas do leite e da soja. Os resultados revelaram que nenhuma cepa testada apresentou capacidade de proteólise das proteínas do leite e da soja. Adicionalmente, as cepas probióticas Lactobacillus acidophilus LA-5 e Bifidobacterium longum BB-46 desconjugaram a maior quantidade de ácidos biliares testados e as cepas de S. thermophilus testadas não apresentaram capacidade de desconjugação de sais biliares. Após a análise dos resultados da etapa I, o resíduo de acerola (ABP) e as cepas probióticas LA-5 e BB-46 foram selecionadas para dar continuidade à etapa II do estudo(desenvolvimento de uma bebida fermentada a base de soja). Para esse fim, foi utilizado um delineamento experimental do tipo fatorial 23, totalizando 8 ensaios com três repetições de cada, e foram avaliados os efeitos das cepas probióticas e do subproduto de acerola sobre as características físico-químicas, microbiológicas e sensoriais dessas bebidas fermentadas de soja. Paralelamente, foram realizadas análises da sobrevivência das cepas probióticas frente às condições gastrintestinais simuladas in vitro nas bebidas fermentadas de soja (FSB). Os resultados mostraram que a presença de BB-46 e ABP afetaram negativamente a aceitabilidade sensorial das FSB. O ABP também levou a diferenças significativas no perfil de textura das FSB (P<0,05). As populações das cepas probióticas nas diferentes formulações de FSB variaram de 7,0 a 8,2 log de UFC equivalente/mL durante os 28 dias de armazenamento (4 ºC) e a co-cultura (LA-5+BB-46) e o ABP não afetaram (P>0,05) a viabilidade de ambos os microrganismos. No entanto, ABP aumentou significativamente a sobrevivência de BB-46 frente às condições gastrintestinais sumuladas in vitro. Para a etapa III do presente estudo, um delineamento experimental fatorial 22 foi realizado. Para a avaliação do impacto dessas FSB sobre a composição e atividade metabólica da microbiota intestinal de humanos eutróficos e obesos, foi utilizado um modelo in vitro TIM-2 na Maastricht University (Venlo, Holanda), que simula as condições normais do lúmen do cólon proximal, com todos os parâmetros controlados por um computador. Amostras foram coletadas do TIM-2 para a quantificação dos microrganismos probióticos (LA-5 e BB-46), Lactobacillus spp., Bifidobacterium spp. e bactérias totais, utilizando o método de PCR quantitativo (qPCR), e o perfil da microbiota intestinal foi determinado utilizando Next-Generation Sequencing (NGS) Illumina Mysec. A concentração de ácidos graxos de cadeia curta e de cadeia ramificada e lactato produzidos pelas diferentes microbiotas durante a fermentação no TIM-2 também foi determinada. Os resultados mostraram que a microbiota de humanos eutróficos apresentou uma alta produção de acetato e lactato em comparação com a microbiota de obesos. Reduções significativas das populações de Bifidobacterium na microbiota de eutróficos foram observadas entre 0 e 48 h de ensaio para todas as refeições experimentais, exceto para a refeição que apresentou a combinação probiótica (LA-5 e BB-46) e a suplementação com ABP, que apresentou aumento de Bifidobacterium e Lactobacillus totais durante todo o período de análise para ambas as microbiotas testadas. As FSB suplementadas com ABP apresentaram os melhores resultados em relação à modulação da microbiota de humanos obesos, com o aumento Bifidobacterium spp. e Lactobacillus spp. Adicionalmente, após 48 horas de intervenção no TIM-2, a microbiota de obesos foi aparentemente similar à microbiota de eutróficos, mostrando uma modulação benéfica dessa microbiota. Os resultados sugerem que as bebidas fermentadas de soja suplementadas com o subproduto de acerola e cepas probióticas podem apresentar efeitos benéficos à saúde. No entanto, estudos clínicos são necessários para complementar e confirmar os resultados observados nos ensaios in vitro

Potential mechanisms linking probiotics to diabetes: a narrative review of the literature.

CONTEXT AND OBJECTIVE:: Some studies have suggested a wide range of possible mechanisms through which probiotics may play a role in diabetes prevention and treatment. However, the underlying mechanisms are not fully understood. We conducted this study to review the potential mechanisms suggested for the effect of probiotics in diabetes. DESIGN AND SETTING:: Narrative review conducted at the Food Security Research Center of Isfahan. METHODS:: A search in the electronic databases MEDLINE (PubMed), Cochrane Library, Web of Science and Google scholar was performed up to October 2016. RESULTS:: The initial search yielded 1214 reports. After removing duplicates, 704 titles and abstracts were screened. Finally, out of 83 full-text articles that were reviewed for eligibility, 30 articles were included in the final analysis. The anti-diabetic mechanisms for probiotics reported encompass intraluminal and direct effects on the intestinal mucosa and microbiota (n = 13), anti-inflammatory and immunomodulatory effects (n = 10), antioxidative effects (n = 5), effects on endoplasmic reticulum (ER) stress and expression of genes involved in glucose homeostasis and insulin resistance (n = 6), with some studies pointing to more than one mechanism. CONCLUSION:: The results may throw some light on the capacity of probiotics as a novel approach towards controlling diabetes. However, further human studies are warranted to elucidate and confirm the potential role of probiotics in diabetes prevention and treatment. Also, it needs to be ascertained whether the effectiveness of probiotics in diabetes prevention and treatment is dependent on the strain of the microorganisms.

Potential mechanisms linking probiotics to diabetes: a narrative review of the literature / Mecanismos potenciais ligando probióticos a diabetes: uma revisão narrativa da literatura

ABSTRACT CONTEXT AND OBJECTIVE: Some studies have suggested a wide range of possible mechanisms through which probiotics may play a role in diabetes prevention and treatment. However, the underlying mechanisms are not fully understood. We conducted this study to review the potential mechanisms suggested for the effect of probiotics in diabetes. DESIGN AND SETTING: Narrative review conducted at the Food Security Research Center of Isfahan. METHODS: A search in the electronic databases MEDLINE (PubMed), Cochrane Library, Web of Science and Google scholar was performed up to October 2016. RESULTS: The initial search yielded 1214 reports. After removing duplicates, 704 titles and abstracts were screened. Finally, out of 83 full-text articles that were reviewed for eligibility, 30 articles were included in the final analysis. The anti-diabetic mechanisms for probiotics reported encompass intraluminal and direct effects on the intestinal mucosa and microbiota (n = 13), anti-inflammatory and immunomodulatory effects (n = 10), antioxidative effects (n = 5), effects on endoplasmic reticulum (ER) stress and expression of genes involved in glucose homeostasis and insulin resistance (n = 6), with some studies pointing to more than one mechanism. CONCLUSION: The results may throw some light on the capacity of probiotics as a novel approach towards controlling diabetes. However, further human studies are warranted to elucidate and confirm the potential role of probiotics in diabetes prevention and treatment. Also, it needs to be ascertained whether the effectiveness of probiotics in diabetes prevention and treatment is dependent on the strain of the microorganisms.

RESUMO CONTEXTO E OBJETIVO: Alguns estudos têm sugerido ampla gama de possíveis mecanismos, pelos quais os probióticos podem desempenhar um papel na prevenção e tratamento do diabetes. No entanto, os mecanismos subjacentes não são totalmente compreendidos. Realizamos este estudo para revisar os possíveis mecanismos sugeridos para o efeito dos probióticos na diabetes. TIPO DE ESTUDO E LOCAL: Revisão narrativa conduzida no Food Security Research Centro de Isfahan. MÉTODOS: Busca sistemática nas bases de dados eletrônicas MEDLINE (PubMed), Cochrane Library, Web of Science e Google scholar até outubro de 2016. RESULTADOS: A busca inicial resultou em 1.214 artigos. Após a remoção de duplicatas, foram pesquisados 704 títulos e resumos. Finalmente, de 83 artigos completos revisados para elegibilidade, 30 foram incluídos na análise final. Os mecanismos antidiabéticos relatados dos probióticos abrangem efeitos intraluminais e diretos na mucosa e microbiota intestinal (n = 13), efeitos anti-inflamatórios e imunomoduladores (n = 10), efeitos antioxidativos (n = 5), efeitos sobre o estresse de retículo endoplasmático (RE) e expressão de genes envolvidos na homeostase da glicose e resistência à insulina (n = 6), com alguns estudos apontando para mais de um mecanismo. CONCLUSÃO: Os resultados podem lançar alguma luz sobre os probióticos como uma nova abordagem no controle do diabetes, no entanto, mais estudos em humanos são justificados para elucidar e confirmar o papel potencial dos probióticos na prevenção e tratamento do diabetes. Além disso, deverá ser determinado se a eficácia dos probióticos na prevenção e tratamento do diabetes é dependente da cepa dos microrganismos.

Gram-Positive Bacteria with Probiotic Potential for the Apis mellifera L. Honey Bee: The Experience in the Northwest of Argentina.

Apis mellifera L. is one of the most important natural pollinators of significant crops and flowers around the world. It can be affected by different types of illnesses: american foulbrood, nosemosis, varroasis, viruses, among others. Such infections mainly cause a reduction in honey production and in extreme situations, the death of the colony. Argentina is the world's second largest honey exporter and the third largest honey producer, after China and Turkey. Given both the prominence of the honey bee in nature and the economic importance of apiculture in Argentina and the world, it is crucial to develop efficient and sustainable strategies to control honey bee diseases and to improve bee colony health. Gram-positive bacteria, such as lactic acid bacteria, mainly Lactobacillus, and Bacillus spp. are promising options. In the Northwest of Argentina, several Lactobacillus and Bacillus strains from the honey bee gut and honey were isolated by our research group and characterized by using in vitro tests. Two strains were selected because of their potential probiotic properties: Lactobacillus johnsonii CRL1647 and Bacillus subtilis subsp. subtilis Mori2. Under independent trials with both experimental and commercial hives, it was determined that each strain was able to elicit probiotic effects on bee colonies reared in the northwestern region of Argentina. One result was the increase in egg-laying by the queen which therefore produced an increase in bee number and, consequently, a higher honey yield. Moreover, the beneficial bacteria reduced the incidence of two important bee diseases: nosemosis and varroosis. These results are promising and extend the horizon of probiotic bacteria to the insect world, serving beekeepers worldwide as a natural tool that they can administer as is, or combine with other disease-controlling methods.

Feeding Programs for Broiler Breeders in the Start Phase

The fast-growing Brazilian aviculture requires studies to improve zootechnical performance indexes for broiler breeders. The purpose of this study was to assess different feeding programs for broiler breeders on performance and development of digestive organs. A total of 48,000, 1d-old, Cobb 500 broiler breeders were divided into two sheds with 24,000 birds each. The experiment was randomized in block design, considering each shed a block, with 4 treatments and 6 replications per treatment with 2,000 birds in each. Treatments consisted of: Shed 1) T1 = starter feed; T2 = T1 + probiotics; Shed 2) T3 = pre-starter feed; T4 = T3 + probiotics. The productive performance characteristics (bird weight, weight gain, feed intake and feed conversion), the development of digestive organs (gizzard, proventriculus, spleen, bursa of Fabricius and small bowel) as well as the small bowel length were assessed weekly for all experimental groups, in samples of 10 birds per treatment. The best feed conversion and weight gain were observed with pre-starter feed in the first 7 days of age, with or without probiotic. Small bowel, gizzard and proventriculus development at 28 days was better for birds on pre-starter feed compared to those on starter feed alone. It is possible to conclude that broiler breeders on pre-starter feed during the first 7 days of age are likely to show better physical and productive performances in the adult phase.

Feeding Programs for Broiler Breeders in the Start Phase

The fast-growing Brazilian aviculture requires studies to improve zootechnical performance indexes for broiler breeders. The purpose of this study was to assess different feeding programs for broiler breeders on performance and development of digestive organs. A total of 48,000, 1d-old, Cobb 500 broiler breeders were divided into two sheds with 24,000 birds each. The experiment was randomized in block design, considering each shed a block, with 4 treatments and 6 replications per treatment with 2,000 birds in each. Treatments consisted of: Shed 1) T1 = starter feed; T2 = T1 + probiotics; Shed 2) T3 = pre-starter feed; T4 = T3 + probiotics. The productive performance characteristics (bird weight, weight gain, feed intake and feed conversion), the development of digestive organs (gizzard, proventriculus, spleen, bursa of Fabricius and small bowel) as well as the small bowel length were assessed weekly for all experimental groups, in samples of 10 birds per treatment. The best feed conversion and weight gain were observed with pre-starter feed in the first 7 days of age, with or without probiotic. Small bowel, gizzard and proventriculus development at 28 days was better for birds on pre-starter feed compared to those on starter feed alone. It is possible to conclude that broiler breeders on pre-starter feed during the first 7 days of age are likely to show better physical and productive performances in the adult phase.(AU)

Development of a potential probiotic yoghurt using selected anti-inflammatory lactic acid bacteria for prevention of colitis and carcinogenesis in mice.

AIMS: To evaluate the beneficial properties of a potentially probiotic yoghurt obtained by the fermentation of two selected anti-inflammatory bacterial strains using in vivo mouse models of intestinal inflammation and colon carcinogenesis. METHODS AND RESULTS: Yoghurt was administered to mice suffering chemically induced intestinal inflammation or colon carcinogenesis. It was shown that this novel yoghurt was able to prevent local inflammation in the intestines of mice through a regulation of the immune response, prevent macroscopic and histological damages, and prevent colon carcinogenesis through an anti-inflammatory response. CONCLUSIONS: The developed yoghurt showed in vivo anti-inflammatory properties by modulation of the host immune response for the prevention of colon inflammation and carcinogenesis. SIGNIFICANCE AND IMPACT OF THE STUDY: This new yoghurt could thus be considered a probiotic food and be useful as a complement to current treatment protocols for inflammatory bowel diseases and colon cancer, a first since there are no current functional foods specifically oriented for these patients.

Probiotic properties of lactic acid bacteria isolated from human milk.

AIM: The objective of this study was to identify and characterize lactic acid bacteria isolated from human milk, with an emphasis on their probiotic properties. METHODS AND RESULTS: The strains were tested for their ability to inhibit growth of Enterococcus faecalis, Salmonella enterica subsp. enterica serotype Enteritidis, Listeria monocytogenes, Staphylococcus aureus and Escherichia coli, as well as for susceptibility to antimicrobial agents and for acid pH and bile salt tolerance. Gram-positive and catalase-negative were selected and identified as Enterococcus (83·3%) after sequencing the 16S rDNA gene. All the isolates inhibited growth of Ent. faecalis and S. serotype Enteritidis, 97% inhibited growth of L. monocytogenes and Staph. aureus and 78·8% inhibited growth of E. coli. Most of the isolates were resistant to gentamicin (50%) and vancomycin (47%). Twelve isolates grew when subjected to pH 3·0 and 0·1% bile salts. At lower pH (2·5-2·0), Ent. faecalis F1 and Weissella confusa F8 were more efficient. CONCLUSION: It was possible to isolate from human milk the lactic acid bacteria with potential for use as probiotics. SIGNIFICANCE AND IMPACT OF THE STUDY: Lactic acid bacteria isolated of nursing mothers have probiotic properties.

Probiotic screening and safety evaluation of Lactobacillus strains from plants, artisanal goat cheese, human stools, and breast milk.

The aim of this study was to select autochthonous strains of Lactobacillus from stools of healthy infants and adults, human milk, artisanal goat cheese, and fruits and vegetables according to their probiotic properties and safety. From 421 strains of Lactobacillus isolated, 102 (24.2%) were shown to be tolerant to gastric pH and bile salts; they were used to determine their anti-Helicobacter pylori (agar diffusion assay), antioxidant (oxygen radical absorption capacity), and anti-inflammatory (inhibition of interleukin-8 release by tumor necrosis factor-α-stimulated HT-29 cells) activities as well as their ability to adhere to intestinal (Caco-2) and gastric (AGS) epithelial cells. Results obtained were compared with three commercial probiotic Lactobacillus rhamnosus GG, L. plantarum 299v, and L. johnsonii NCC533. The five strains most efficient according to these activities were subsequently identified by sequencing their 16S rRNA gene, their susceptibility to antibiotics was determined, and their safety evaluated in mice. One strain of L. plantarum was discarded due to the higher prevalence of liver bacterial translocation observed in the animals fed this strain. In conclusion, four autochthonous strains of L. rhamnosus were finally selected with probiotic properties and safety allowing their eventual use in human studies. These results contribute to increase the diversity of probiotic strains available for the development of nutraceuticals and functional foods.

Strain-specific probiotics properties of Lactobacillus fermentum, Lactobacillus plantarum and Lactobacillus brevis isolates from Brazilian food products.

A total of 234 LAB isolates from Brazilian food products were initially screened for their ability to survive at pH 2.0. Fifty one of the isolates survived and were selected. They were characterized by phenotypic methods, rep-PCR and identified using 16S rRNA gene sequencing as Lactobacillus fermentum (34 isolates), Lactobacillus plantarum (10) and Lactobacillus brevis (7). Based on being either highly tolerant to bile, showing an ability for auto-aggregation and/or hydrophobic properties, one L. fermentum (CH58), three L. plantarum (CH3, CH41 and SAU96) and two L. brevis (SAU105 and FFC199) were selected. The highest co-aggregation ability with Escherichia coli was observed to L. plantarum CH41. L. brevis SAU105 and FFC199 and L. fermentum CH58 exhibited antagonistic activity towards the pathogens Listeria monocytogenes and Staphylococcus aureus. L. plantarum CH3 and CH41 and L. brevis FFC199 showed adhesion ability to Caco-2 cells (1.6, 1.1 and 0.9%, respectively) similar to the commercial probiotic, Lactobacillus rhamnosus GG (1.5%). They were able to increase the transepithelial electrical resistance (TEER) of Caco-2 cells over 24 h (p < 0.05). The present work showed that the probiotic characteristics were strain-specific and that the isolates L. plantarum CH3 and CH41 (cocoa) and L. brevis FFC199 (cauim) exhibited potential probiotics properties.

Identification and evaluation of the probiotic potential of Lactobacillus paraplantarum FT259, a bacteriocinogenic strain isolated from Brazilian semi-hard artisanal cheese.

This study aimed to identify a bacteriocinogenic Lactobacillus isolate (FT259) obtained from Brazilian semi-hard Minas type cheese and to evaluate its probiotic and antimicrobial potentials. The strain was identified by biochemical tests (at genus level), and by 16S rDNA sequencing combined with recA gene amplification (for species). To determine the inhibitory spectrum towards food borne pathogens and lactic acid bacteria, the spot-on-the-lawn assay was carried out. Moreover, the proteinaceous nature of the antimicrobial compound produced was evaluated by susceptibility to degradation by proteolytic enzymes. The isolated strain was tested for survival in acidified culture media (pH 2.0, 2.5 and 3.5), in vitro tolerance to bile salts and viability under gastric conditions. Adhesion of Lactobacillus paraplantarum FT259 to Caco-2 cells was evaluated by surface plate count on De Man, Rogosa, and Sharpe (MRS) agar and also by FISH method (fluorescent in situ hybridization) with the aid of Eub338 probe for fluorescence microscopy analysis. The isolate was identified as L. paraplantarum FT259 and it produced bacteriocins that inhibited the growth of Listeria monocytogenes, Listeria innocua and several lactic acid bacteria. It was also observed that L. paraplantarum FT259 tolerated exposure to pH 3.5, and bile salts 0.3% for up to 180 min. In experiments with simulated gastric juice, viable cells of L. paraplantarum FT259 decreased from 8.6 log CFU/mL to 3.5 log CFU/mL after 180 min. For the same strain, in studies with Caco-2 cells, 74% of adhesion was observed through plate count and FISH assays. It was also demonstrated isolated FT259 was susceptible to the majority the antibiotics tested. Overall, the results indicated L. paraplantarum FT259 is a potential probiotic and the production of bacteriocin may be an interesting feature for food applications.

Genes involved in lactose catabolism and organic acid production during growth of Lactobacillus delbrueckii UFV H2b20 in skimmed milk.

There are three main reasons for using lactic acid bacteria (LAB) as starter cultures in industrial food fermentation processes: food preservation due to lactic acid production; flavour formation due to a range of organic molecules derived from sugar, lipid and protein catabolism; and probiotic properties attributed to some strains of LAB, mainly of lactobacilli. The aim of this study was to identify some genes involved in lactose metabolism of the probiotic Lactobacillus delbrueckii UFV H2b20, and analyse its organic acid production during growth in skimmed milk. The following genes were identified, encoding the respective enzymes: ldh - lactate dehydrogenase, adhE - Ldb1707 acetaldehyde dehydrogenase, and ccpA-pepR1 - catabolite control protein A. It was observed that L. delbrueckii UFV H2b20 cultivated in different media has the unexpected ability to catabolyse galactose, and to produce high amounts of succinic acid, which was absent in the beginning, raising doubts about the subspecies in question. The phylogenetic analyses showed that this strain can be compared physiologically to L. delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis, which are able to degrade lactose and can grow in milk. L. delbrueckii UFV H2b20 sequences have grouped with L. delbrueckii subsp. bulgaricus ATCC 11842 and L. delbrueckii subsp. bulgaricus ATCC BAA-365, strengthening the classification of this probiotic strain in the NCFM group proposed by a previous study. Additionally, L. delbrueckii UFV H2b20 presented an evolutionary pattern closer to that of probiotic Lactobacillus acidophilus NCFM, corroborating the suggestion that this strain might be considered as a new and unusual subspecies among L. delbrueckii subspecies, the first one identified as a probiotic. In addition, its unusual ability to metabolise galactose, which was significantly consumed in the fermentation medium, might be exploited to produce low-browning probiotic Mozzarella cheeses, a desirable property for pizza cheeses.

Identification and in vitro screening of avian yeasts for use as probiotic.

UNLABELLED: The objective of this study was to isolate and identify yeast strains from broilers excreta and to evaluate in vitro their potential for probiotic use in animal production. METHODS AND RESULTS: Nine yeast strains were isolated and presumptively pre-identified by biochemical assays. These isolates were grouped in six different molecular profiles using PCR-fingerprinting technique. Each profile was identified by sequencing of the D1/D2 domains of the large subunit of the 26S rRNA gene. These yeasts were identified as: Trichosporon sp. (LV-2), Wickerhamomyces anomalus (LV-6), Pichia kudriavzevii (LV-8), Kodamaea ohmeri (LV-9) and Trichosporon asahii (LV-10). A pre-screening of the strains for probiotic use was based on their ability to agglutinate pathogenic micro-organisms. These yeast strains were characterized for specific growth rate, duplication time, their cell surface hydrophobicity, medium acidification, resistance to low pH (2.0, 2.5 and 3.0) and concentrations of bile salts (0.3% and 0.6%). The isolate of W. anomalus (LV-6) had the highest agglutinating and adherence capacity, a growth rate of 2.07×10(8) cfu/mL in 24 h at 30 °C, decreasing the medium pH from 6.5 to 5.23, a 25% hydrophobicity and an elevated capacity to grow under stress conditions. CONCLUSIONS: W. anomalus strain LV-6 showed the best characteristics for use as a probiotic candidate. SIGNIFICANCE AND IMPACT OF THE STUDY: The data from this study helped in choosing a probiotic candidate from yeast to use in broiler production.

Urogenital pathogen inhibition and compatibility between vaginal Lactobacillus strains to be considered as probiotic candidates.

OBJECTIVES: To evaluate the production of inhibitory substances against several urogenital pathogens and to determine the compatibility between selected vaginal lactobacilli to be considered as probiotic candidates. STUDY DESIGN: The inhibitory activity of 38 vaginal Lactobacillus strains against urogenital pathogens was assessed using the agar plate diffusion. METHOD: The production of hydrogen peroxide was determined by the semi-qualitative tetramethyl-benzidine-peroxidase assay and that of lactic acid by high performance liquid chromatography. Pre-selected lactobacilli were genetically identified by ribosomal 16S-DNA sequencing and used to determine the compatibility of strains. Correlation values between some inhibitory properties were calculated. RESULTS: 45% of the lactobacilli assayed inhibited the growth of various urogenital pathogens, except Candida albicans, mainly by the effect of organic acids. Even though 71% of the Lactobacillus strains produced hydrogen peroxide in the semi-qualitative test, the antimicrobial effect of the oxidative metabolite on pathogens was not detected under the experimental conditions assayed. On the other hand, the inhibition spectrum of salivaricin CRL 1328 was widened with respect to the one previously reported by our research team. With respect to the degree of interaction between the pre-selected lactobacilli, most of them showed inhibition of at least one strain, mainly due to the effects of both organic acids and hydrogen peroxide. CONCLUSION: Several lactobacilli that showed inhibitory activity against urogenital pathogens or produced hydrogen peroxide are compatible between them. Therefore, different potential combinations of strains are proposed for their inclusion in the design of probiotic products to be vaginally administered for the prevention of urogenital infections in women.