Adzuki and Mung Bean Sprouts Enriched with Probiotic Lactiplantibacillus plantarum 299v Improve Body Mass Gain and Antioxidant Status and Reduce the Undesirable Enzymatic Activity of Microbiota in Healthy Rats.

Introducing and establishing new food requires a detailed evaluation of its safety, nutritional value and functionality, thus the control and probiotic-rich adzuki and mung bean sprouts were studied in an in vivo rats model. However, the total feed intake did not differ significantly between the groups, the highest body weight gain and body weight change were recorded in the control AIN diet. At the same time, the addition of legume sprouts caused a reduction of these parameters (up to 25% in the variant with probiotic-rich adzuki bean sprouts). There was no significant effect on serum morphology, except white blood cells (ca. 20% reduction in the control sprout-supplemented diets). Serum and liver antiradical properties were significantly elevated by consuming mung bean sprouts (no effect of the probiotics). The faecal lactic acid bacteria were already increased by the control sprouts (a 2.8- and 2.1-fold increase for adzuki and mung bean sprouts, respectively). The probiotic-rich sprouts further improved this parameter. The diets enriched with mung bean sprouts significantly decreased the urease (by ca. 65%) and ß-glucuronidase activities (by ca. 30%). All the tested diets caused also a significant reduction of faecal tryptophanase activity (the effect was intensified by Lactiplantibacillus plantarum 299v). The functional components did not affect negatively the nutritional parameters and blood morphological characteristics. They improved also the antioxidant potential and significantly decreased the activities of colon cancer-related enzymes (urease and tryptophanase). The results confirmed that these new probiotic carriers may be a valuable, safe and functional element of a healthy diet.

Molecular Routes to Specific Identification of the Lactobacillus Casei Group at the Species, Subspecies and Strain Level.

The genus Lactobacillus includes, among others, Lactobacillus casei, Lactobacillus paracasei and Lactobacillus rhamnosus, species that are collectively referred to as the Lactobacillus casei group. Many studies have shown that strains belonging to this group may decrease lactose intolerance, the effects of inflammatory bowel disease, diarrhea, constipation, food allergies and even colon cancer. Moreover, evidences exists of positive effects of these bacteria on mucosal immunity and blood cholesterol level. Because of their beneficial influence on human health, many of them are used as food additives and probiotic pharmaceuticals. It should be stressed that health-promoting properties are not attributed at the species level, but to specific strains. Therefore, procedures are necessary to allow specific identification at each phylogenetic level-genus, species and strain. In this paper we present a practical overview of molecular methods for the identification and differentiation of L. casei bacteria. The research included 30 bacterial strains belonging to three species: L.casei, L. paracasei and L. rhamnosus. Among the tested procedures were genus- and species-specific PCR, multiplex-PCR, Real-Time HRM analysis, RFLP-PCR, rep-PCR, RAPD-PCR, AFLP-PCR, and proteomic methods such as MALDI-TOF MS typing and SDS-PAGE fingerprinting. The obtained results showed that multiplex-PCR and MALDI-TOF MS turned out to be the most useful methods to identify the tested bacteria at the species level. At the strain level, the AFLP-PCR method showed the highest discriminatory power. We hope that the presented results will allow for the easy selection of an appropriate procedure, depending on the experiment conducted and the equipment capabilities of any given laboratory.

Genomic and Proteomic Characterization of Bacteriophage BH1 Spontaneously Released from Probiotic Lactobacillus rhamnosus Pen.

Lactobacillus rhamnosus Pen is a human endogenous strain used for the production of probiotic formula, which is effective in the prevention of antibiotic-associated diarrhoea. Our study showed that this probiotic strain releases bacteriophage BH1 without the addition of any inducing agent. Our research revealed that phage BH1 has a circular genome with a length of 40721 nt and a GC content of 44.8%. The genome of phage BH1 possesses 57 open reading frames which could be divided into functional modules associated with DNA packaging, morphogenesis, lysis, integration, genetic switch, and replication. In spite of similarity in morphology and genomic organization, comparative analysis revealed substantial genetic diversity and mosaic genomic architecture among phages described for the Lactobacillus casei group. Additionally, qPCR and ddPCR analysis confirmed earlier microscopic observations indicating that L. rhamnosus Pen liberates bacteriophage particles during growth. This occurs spontaneously, and is not a result of external inducing factors. For samples collected after 4 and 24 h of L. rhamnosus Pen culture, the number of attB and attP copies increased 2.5 and 12 times, respectively. This phenomenon, by introducing resistance to other phages or enhancing the biofilm-forming capabilities, may increase the survivability of microorganisms in their natural ecological niche. Conversely, spontaneous phage induction may be an important virulence factor for bacteria, posing a potential threat for the human host.

Lactobacillus plantarum 299V improves the microbiological quality of legume sprouts and effectively survives in these carriers during cold storage and in vitro digestion.

Probiotics improve consumers' health and additionally may positively influence the microbiological and organoleptic quality of food. In the study, legume sprouts were inoculated with Lactobacilllus plantarum 299V to produce a new functional product ensuring the growth and survival of the probiotic and high microbiological quality of the final product. Legume sprouts, which are an excellent source of nutrients, were proposed as alternative carriers for the probiotic. The key factors influencing the production of probiotic-rich sprouts include the temperature (25°C) of sprouting and methods of inoculation (soaking seeds in a suspension of probiotics). Compared to the control sprouts, the sprouts enriched with the probiotic were characterized by lower mesophilic bacterial counts. In the case of fresh and stored probiotic-rich sprouts, lactic acid bacteria (LAB) accounted for a majority of total microorganisms. The Lb. plantarum population was also stable during the cold storage. The high count of LAB observed in the digest confirmed the fact that the studied sprouts are effective carriers for probiotics and ensure their survival in the harmful conditions of the digestive tract in an in vitro model. Enrichment of legume sprouts with probiotics is a successful attempt and yields products for a new branch of functional foods.

Pullulan degrading enzymes of bacterial origin.

Pullulan degrading enzymes belong to a group of glycosylhydrolases that are widely distributed in nature and are produced by an extremely wide variety of species. Among them the thermophilic and mesophilic bacteria are a rich source of these enzymes. There are many biotechnological applications for these enzymes and a rapidly growing amount of information about their diversity, genetic as well as biochemical and biophysical characteristics. The properties of these enzymes vary and are somewhat linked to the natural environment inhabited by the producing organisms. Genes for these enzymes have been cloned from several strains and their amino acid sequences show highly conserved regions common to the enzymes of the amylase family. Molecular studies have greatly extended our knowledge on pullulan degrading enzymes and their biosynthesis. However, enzyme production levels have usually not been as high as had been assumed possible, and the properties of some enzymes are less than optimal for their industrial applications. Some of these problems can be overcome with the use of good producer organisms, optimized expression/secretion vectors, and site-directed mutagenesis. The molecular biology of pullulan degrading enzymes has been and continues to be a valuable system for studying basic questions of cell biology, such as mechanisms of gene regulation and secretion, and the structure-function relationships of proteins.