Exploiting tropical fruit processing coproducts as circular resources to promote the growth and maintain the culturability and functionality of probiotic lactobacilli.

This study evaluated the use of acerola (Malpighia glabra L., CACE), cashew (Anacardium occidentale L., CCAS), and guava (Psidium guayaba L., CGUA) fruit processing coproducts as substrates to promote the growth, metabolite production, and maintenance of the viability/metabolic activity of the probiotics Lactobacillus acidophilus LA-05 and Lacticaseibacillus paracasei L-10 during cultivation, freeze-drying, storage, and exposure to simulated gastrointestinal digestion. Probiotic lactobacilli presented high viable counts (≥8.8 log colony-forming units (CFU)/mL) and a short lag phase during 24 h of cultivation in CACE, CCAS, and CGUA. Cultivation of probiotic lactobacilli in fruit coproducts promoted sugar consumption, medium acidification, and production of organic acids over time, besides increasing the of several phenolic compounds and antioxidant activity. Probiotic lactobacilli cultivated in fruit coproducts had increased survival percentages after freeze-drying and during 120 days of refrigerated storage. Moreover, probiotic lactobacilli cultivated and freeze-dried in fruit coproducts had larger subpopulations of live and metabolically active cells when exposed to simulated gastrointestinal digestion. The results showed that fruit coproducts not only improved the growth and helped to maintain the viability and metabolic activity of probiotic strains but also enriched the final fermented products with bioactive compounds, being an innovative circular strategy for producing high-quality probiotic cultures.

Characterization and Identification of Probiotic Features in Lacticaseibacillus Paracasei Using a Comparative Genomic Analysis Approach.

Lacticaseibacillus paracasei species are widely used for their health-promoting properties in food and agricultural applications. These bacteria have been isolated from various habitats such as the oral cavity, cereals, vegetables, meats, and dairy products conferring them the ability to consume different carbohydrates. Two subspecies are recognized, Lacticaseibacillus paracasei subsp. paracasei and Lacticaseibacillus paracasei subsp. tolerans according to their acid production from carbohydrates. Some strains are currently used as probiotics. In this study, we performed a comparative genomic analysis of 181 genomes of the Lacticaseibacillus paracasei species to reveal genomic differences at the subspecies level and to reveal adaptive and probiotic features, and special emphasis is given to inulin consumption. No clear distinction at the subspecies level for L. paracasei was shown using a phylogenetic tree with orthologous genes from the core-genome set. In general, a good correlation was observed between genomic distance and isolation origin, suggesting that L. paracasei strains are adapted to their natural habitat, giving rise to genetic differences at the genomic level. A low frequency of undesirable characteristics such as plasmids, prophages, antibiotic resistance genes, absence of virulence factors, and frequent bacteriocin production supports these species being good candidates for use as probiotics. Lastly, we found that the inulin gene cluster in L. paracasei strains seems to differ slightly in the presence or absence of some genes but maintains a core defined by at least three fructose-PTS proteins, one hypothetical protein, and extracellular ß-fructosidase. Finally, we conclude that further work has to be done for L. paracasei subspecies classification. Improving outgroup selection criteria is a key factor for their correct subspecies assignation.

Protective Effect of the Intracellular Content from Potential Probiotic Bacteria against Oxidative Damage Induced by Acrylamide in Human Erythrocytes.

The aim of this study was to assess the protective effect of the intracellular content obtained from potential probiotic bacteria against acrylamide-induced oxidative damage in human erythrocytes. First, the antioxidant properties of 12 potential probiotic strains was evaluated. Two commercial probiotic bacteria were included as reference strains, namely, Lactobacillus casei Shirota and Lactobacillus paracasei 431. Data showed that the intracellular content from four strains, i.e., Lactobacillus fermentum J10, Lactobacillus pentosus J24 and J26, and Lactobacillus pentosus J27, showed higher (P < 0.05) antioxidant capacity in most methods used. Thereafter, the intracellular content of such pre-selected strains was able to prevent the disturbance of the antioxidant system of human erythrocytes exposed to acrylamide, thereby reducing cell disruption and eryptosis development (P < 0.05). Additionally, the degree of oxidative stress in erythrocytes exposed to acrylamide was significantly (P < 0.05) reduced to levels similar to the basal conditions when the intracellular content of Lact. fermentum J10, Lact. pentosus J27, and Lact. paracasei 431 were employed. Hence, our findings suggest that the intracellular contents of specific Lactobacillus strains represent a potential source of metabolites with antioxidant properties that may help reduce the oxidative stress induced by acrylamide in human erythrocytes.

Evaluation of autochthonous cultures to improve the cheese flavor: A case study in hard cheese model.

The characterization of autochthonous cultures based on their contribution to cheese flavor is an additional selection criterion for their use in cheese making. The objective of the present work was to assess the ability of three strains of mesophilic lactobacilli: Lactobacillus casei 72 (Lc72), L. paracasei 90 (Lp90), and L. plantarum 91 (Lp91), one strain of thermophilic lactobacillus: L. helveticus 209 (Lh209), and the thermophilic-mesophilic combinations, to grow and produce aroma compounds in a hard cheese model. Microbiological counts, pH, and the profiles of carbohydrates, organic acids, and volatile compounds were analyzed during incubation for 14 days at 37 ℃. The population of mesophilic lactobacilli reached levels around 8.0 log CFU ml-1 at three days, but then decreased until ∼7.0 log CFU ml-1 toward 14 days. Thermophilic lactobacillus population reached and maintained levels around 7.7 log CFU ml-1 during incubation. Carbohydrates were absent in the hard cheese model, and so no change in the pH values and in the levels of lactic acid was detected. Mesophilic lactobacilli, inoculated individually or in association with Lh209, metabolized the citric acid and produced ethanoic acid. The profiles of volatile compounds of mesophilic lactobacilli (characterized mainly by butan-2-one, 3-hydroxybutan-2-one, 3-methylbutan-1-ol, hexan-1-ol, 2-phenylethanol, and ethanoic acid) were different from the profile of thermophilic lactobacillus Lh209 (characterized mainly by heptan-2-one, ethyl acetate, isoamyl hexanoate, pentan-1-ol, decanoic acid, and 2- and 3-methylbutanal). Cooperative effects in the production of compounds related to cheese flavor, such as 3-hydroxybutan-2-one, ethyl butanoate, ethanol, pentan-2-ol, hexan-1-ol, benzeneacetaldehyde, 2-phenylethanol, and heptanoic acid, were largely evidenced between Lh209 and Lp91; in a lesser extent, cooperative effects were also found for Lh209+Lp90 for the following compounds: 3-hydroxybutan-2-one, isoamyl acetate, and ethanoic acid. Of the mesophilic lactobacilli strains evaluated, Lp91 and Lp90 would be interesting candidates for its use as adjunct cultures in hard cheeses to improve and diversify the flavor.

"Physicochemical, immunomodulatory and safety aspects of milks fermented with Lactobacillus paracasei isolated from kefir".

The use of Lactobacillus paracasei strains isolated from kefir grains as starters for the development of functional dairy products was evaluated. The physicochemical and immunomodulatory properties of milks fermented with L. paracasei CIDCA8339, CIDCA83123 and CIDCA83124 were analyzed. The three strains produced bioactive metabolites during fermentation, since the fermented milk supernatants were able to downregulate >75% of the induced innate immune response in vitro. Although all strains presented absence of hemolytic activity and susceptibility to antibiotics, L. paracasei CIDCA8339 presented more attractive probiotic and technological properties. Mice consuming the fermented milk with L. paracasei CIDCA 8339 did not present significant modifications in sIgA levels or TNF-α, TGF-ß and IL-10 mRNA expression in ileum. Additionally, a decrease of INF-γ level in ileum and no microbiological translocation to liver and spleen was observed. These results demonstrate that L. paracasei CIDCA8339 represents a safe promising potential probiotic strain for the development of functional foods.

Influence of the culture preparation and the addition of an adjunct culture on the ripening profiles of hard cheese.

The aim of this work was to evaluate the impact of two factors on the ripening profiles of hard cooked cheeses: (F1) the growth medium for the primary and adjunct cultures, constituted by autochthonous strains: Lactobacillus helveticus 209 (Lh209) and Lactobacillus paracasei 90 (Lp90), respectively, and (F2) the addition of L. paracasei Lp90 as adjunct culture. Four types of cheeses were made: W and M cheeses in which only Lh209 was added after its growth in whey and MRS, respectively; Wa and Ma cheeses in which both strains (Lh209 and Lp90) were added after their growth in whey and MRS, respectively. Physicochemical and microbial composition, proteolysis and profiles of organic acids and volatile compounds were analyzed. According to the methodology of the cultures preparation, W and Wa cheeses showed a higher level of secondary proteolysis and lower level of primary proteolysis (P < 0·05), lower content of citric and acetic acids and higher amount of propionic acid (P < 0·05), in comparison with M and Ma cheeses. The incorporation of Lp90 increased the secondary proteolysis (P < 0·05), decreased the citric acid (P < 0·05), and increased the propionic acid only when was added after their growth in whey (P < 0·05). Both factors significantly modified the percentages of the volatile compounds grouped in chemical families; in addition, for the half of the compounds detected, significant differences were found. Based on the obtained results, the use of Lp90 as an adjunct in hard cooked cheeses, and the preincubation of the cultures in whey are strategies to accelerate the cheese ripening and to enhance the production of some characteristic compounds of this type of cheeses, such as propan-2-one, hexan-2-one, 2- and 3-methyl butanal, heptan-2-ol, acetic and 3-methylbutanoic acids and 3-hydroxy butan-2-one.

Probiotic and Synbiotic Sorbets Produced with Jussara (Euterpe edulis) Pulp: Evaluation Throughout the Storage Period and Effect of the Matrix on Probiotics Exposed to Simulated Gastrointestinal Fluids.

The aims of the present study were to develop and evaluate different formulations of probiotic and synbiotic sorbets produced with jussara (Euterpe edulis) pulp, polydextrose, Lactobacillus acidophilus LA3, and Lactobacillus paracasei BGP1. The pasteurized jussara pulp presented high content of phenolic compounds, especially anthocyanins, which were not inhibitory to the probiotics used in this study. The levels of polyphenols and anthocyanins present in the sorbets were also high and kept stable for 120 days, as well as the populations of both probiotics. On the other hand, probiotic populations reduced ca. 4 log CFU/g when exposed to simulated gastrointestinal fluids. Altogether, the sorbets produced in this study showed interesting results, indicating the viability on producing functional foods with probiotics, prebiotics, and other components that are rich in polyphenols, such as jussara pulp. The combination of these elements can improve the health beneficial effects of these compounds and provide important advantages to the intestinal microbiota of consumers.

Simulated gastrointestinal conditions increase adhesion ability of Lactobacillus paracasei strains isolated from kefir to Caco-2 cells and mucin.

Gastrointestinal conditions along the digestive tract are the main stress to which probiotics administrated orally are exposed because they must survive these adverse conditions and arrive alive to the intestine. Adhesion to epithelium has been considered one of the key criteria for the characterization of probiotics because it extends their residence time in the intestine and as a consequence, can influence the health of the host by modifying the local microbiota or modulating the immune response. Nevertheless, there are very few reports on the adhesion properties to epithelium and mucus of microorganisms after passing through the gastrointestinal tract. In the present work, we evaluate the adhesion ability in vitro of L. paracasei strains isolated from kefir grains after acid and bile stress and we observed that they survive simulated gastrointestinal passage in different levels depending on the strain. L. paracasei CIDCA 8339, 83120 and 83123 were more resistant than L. paracasei CIDCA 83121 and 83124, with a higher susceptibility to simulated gastric conditions. Proteomic analysis of L. paracasei subjected to acid and bile stress revealed that most of the proteins that were positively regulated correspond to the glycolytic pathway enzymes, with an overall effect of stress on the activation of the energy source. Moreover, it is worth to remark that after gastrointestinal passage, L. paracasei strains have increased their ability to adhere to mucin and epithelial cells in vitro being this factor of relevance for maintenance of the strain in the gut environment to exert its probiotic action.

Impact of growth temperature on exopolysaccharide production and probiotic properties of Lactobacillus paracasei strains isolated from kefir grains.

EPS-producing LAB are widely used in the dairy industry since these polymers improve the viscosity and texture of the products. Besides, EPS might be responsible for several health benefits attributed to probiotic strains. However, growth conditions (culture media, temperature, pH) could modify EPS production affecting both technological and probiotic properties. In this work, the influence of growth temperature on EPS production was evaluated, as well as the consequences of these changes in the probiotic properties of the strains. All Lactobacillus paracasei strains used in the study showed changes in EPS production caused by growth temperature, evidenced by the appearance of a high molecular weight fraction and an increment in the total amount of produced EPS at lower temperature. Nevertheless, these changes do not affect the probiotic properties of the strains; L. paracasei strains grown at 20 °C, 30 °C and 37 °C were able to survive in simulated gastrointestinal conditions, to adhere to Caco-2 cells after that treatment and to modulate the epithelial innate immune response. The results suggest that selected L. paracasei strains are new probiotic candidates that can be used in a wide range of functional foods in which temperature could be used as a tool to improve the technological properties of the product.

Efeitos de formulações probióticas contendo Lactobacillus paracasei 28. 4 sobre o crescimento, produção de polissacarídeos e formação de biofilmes de Streptococcus mutans / Effects of different probiotics formulations containing Lactobacillus paracasei 28. 4 on Streptococcus mutans growth, polysaccharides production and biofilms formation

O uso de probióticos como método de prevenção para a cárie dental tem sido amplamente investigado com resultados promissores. Portanto, torna-se necessário o desenvolvimento de formulações, para aplicação na cavidade bucal, que contenham cepas probióticas com atividade contra Streptococcus mutans. Assim, o objetivo desse projeto foi avaliar os efeitos antimicrobianos de formulações probióticas de Lactobacillus paracasei 28.4 sobre a atividade antibacteriana em crescimento planctônico, produção de polissacarídeos e formação de biofilmes de S. mutans. Inicialmente, a atividade antibacteriana de 3 diferentes formulações probióticas (concentrações de 0,5, 0,75 e 1% p/v de gellan gum em CaCl2) foram testadas em culturas planctônicas sobre cepas de S. mutans (cepa padrão UA 159 e 5 cepas clínicas). Os resultados demonstraram inibição total do crescimento das cepas de S. mutans quando tratadas com todas as formulações probióticas testadas. Nos estudos subsequentes, foram avaliados os efeitos profiláticos da formulação probiótica na concentração de 1% p/v nos biofilmes de 4 e 24 horas de S. mutans, utilizando-se a cepa UA 159 e 2 cepas clínicas. Por meio do método sulfúrico-antrona, foi observado que a aplicação da formulação probiótica reduziu a capacidade de produção de polissacarídeos extracelulares pelas cepas de S. mutans em aproximadamente 80% nos dois tempos dos biofilmes testados. A formulação probiótica também foi capaz de reduzir significativamente a biomassa total dos biofilmes de S. mutans nos testes de absorbância do Cristal Violeta, com redução média na leitura de densidade óptica de 2,15 ± 0,10 no tempo de 4 horas e 2,67 ± 0,25 no tempo de 24 horas. A seguir, os efeitos antimicrobianos da formulação probiótica foram avaliados em biofilmes de S. mutans formados sobre a superfície de discos de hidroxiapatita, encontrando-se reduções de 0,57 a 1,54 UFC (Log) na contagem de células viáveis de S. mutans nos grupos tratados em relação aos grupos controle. Também foi possível observar redução na quantidade de células de S. mutans aderidas aos discos de hidroxiapatita pela Microscopia Eletrônica de Varredura (MEV). Por fim, utilizando-se análise de Realtime PCR, foram testados os efeitos das formulações probióticas sobre a expressão de genes de virulência de S. mutans relacionados à adesão e formação do biofilme. Os resultados mostraram que na presença da formulação probiótica, os genes luxS, brpA, gbpB e gtfB de S. mutans foram negativamente regulados. Concluiu-se que a formulação probiótica contendo L. paracasei 28.4 em gellan gum apresentou efeitos inibitórios sobre o crescimento planctônico e biofilme de S. mutans, interferindo na produção de polissacarídeos extracelulares, quantidade de biomassa total, viabilidade celular, aderência de células à hidroxiapatita e transcrição de genes de virulência(AU)

The use of probiotics as a prevention method for dental caries has been extensively investigated with promising results. Therefore, it becomes necessary to develop formulations, for application in the oral cavity, containing probiotic strains with activity against Streptococcus mutans. Thus, the objective of this project was to evaluate the antimicrobial effects of probiotic formulations of Lactobacillus paracasei 28.4 on the antibacterial activity in planktonic growth, polysaccharide production and biofilms formation of S. mutans using reference and clinical strains. Firstly, antibacterial activity of 3 different probiotic formulations (concentrations of 0.5, 0.75 and 1% w/v of gellan gum in CaCl 2) were tested in planktonic cultures on S. mutans strains (standard strain UA 159 and 5 clinical strains). The results demonstrated complete inhibition of S. mutans strains growth when treated with all probiotic formulations tested. In the subsequent studies, the prophylactic effects of the 1% w/v probiotic formulation on 4 and 24 hours S. mutans biofilms were evaluated using the UA 159 strain and 2 clinical strains. Using the sulfuric-anthrone method, it was observed that the application of the probiotic formulation reduced the production capacity of extracellular polysaccharides by S. mutans strains in approximately 80% in both times of the biofilms tested. The probiotic formulation was also able to significantly reduce the total biomass of S. mutans biofilms in the Crystal Violet absorbance tests, with a mean reduction in the optical density reading of 2.15 ± 0.10 in 4 hours and 2.67 ± 0.25 in 24 hours. Next, the antimicrobial effects of the probiotic formulation were evaluated in S. mutans biofilms formed on the surface of hydroxyapatite discs, with reductions of 0.57 to 1.54 CFU (Log) in the viable cells count of S. mutans in the treated groups compared to the control groups. It was also possible to observe a reduction in the amount of S. mutans cells adhered to the hydroxyapatite disks by Scanning Electron Microscopy (SEM). Finally, using Realtime PCR analysis, the effects of probiotic formulations on the expression of S. mutans virulence genes related to adhesion and biofilm formation were tested. The results showed that in the presence of the probiotic formulation, the luxS, brpA, gbpB and gtfB genes of S. mutans were down regulated. It was concluded that the probiotic formulation containing L. paracasei 28.4 in gellan gum presented inhibitory effects on planktonic growth and S. mutans biofilm, interfering in the production of extracellular polysaccharides, amount of total biomass, cell viability, cell adhesion to hydroxyapatite and transcription of virulence genes(AU)