The impact of botanical fermented foods on metabolic syndrome and type 2 diabetes: a systematic review of randomised controlled trials.

Our systematic review assessed the impact of botanical fermented food (BFF) consumption on glucose, lipid, anthropometric, inflammatory and gut microbiota parameters, in adults with metabolic syndrome (MetS), MetS components or type 2 diabetes mellitus (T2DM). Embase, MEDLINE, Cochrane CENTRAL and Google Scholar were searched with no language limits, from inception to 31 August 2022, for eligible randomised controlled trials (RCTs). Two independent reviewers screened 6873 abstracts and extracted relevant data. Risk of bias (ROB) was assessed using the Cochrane Collaboration's ROB2 tool. The final review included twenty-six RCTs, with thirty-one reports published between 2001 and 2022. Significant (p < 0·05) within-group and between-group changes in cardiometabolic outcome means were reported in twenty-three and nineteen studies, respectively. Gut microbiota composition was assessed in four studies, with two finding significant between-group differences. No significant difference between groups of any measured outcomes was observed in five studies. There were fourteen studies at low ROB; ten were of some concern; and two were at high ROB. In 73% of included studies, BFF consumption by participants with obesity, MetS or T2DM led to significant between-group improvements in discrete cardiometabolic outcomes, including fasting blood glucose, lipid profile, blood pressure, waist circumference, body fat percentage and C-reactive protein. BFF consumption increased the abundance of beneficial gut bacteria, such as Bifidobacterium and LAB, whilst reducing potential pathogens such as Bacteroides. To determine the clinical significance of BFFs as therapeutic dietary adjuncts, their safety, tolerability and affordability must be balanced with the limited power and magnitude of these preliminary findings.

Age-related effects on the modulation of gut microbiota by pectins and their derivatives: an in vitro study.

Introduction: The present study investigates whether supplementation with pectin-type polysaccharides has potential to improve aging-associated dysbiosis of the gut microbiota. The influence of different types of pectins on the gut microbiota composition and short-chain fatty acids (SCFAs) profiles of elderly was compared to younger adults. Methods: Pectins studied included a pectin polysaccharide (PEC), a partially hydrolyzed pectin (PPH), and a pectin oligosaccharide (POS). Additionally, inulin was used as a reference prebiotic substrate. Individual fecal samples were collected from healthy elderly volunteers (70-75 years) and younger adults (30-35 years). In vitro fermentations were performed using the CoMiniGut model with controlled temperature and pH. Samples were withdrawn at baseline and after 24 h fermentation for measurement of SCFAs production and microbiota composition by 16S rRNA gene sequencing. Results and Discussion: The results showed that fermentations with PEC and PPH resulted in a specific stimulation of Faecalibacterium prausnitzii regardless of the age groups. Collinsella aerofaciens became a dominating species in the young adult group with fermentations of all three pectins, which was not observed in the elderly group. No significant differences in SCFAs production were found among the pectins, indicating a high level of functional redundancy. Pectins boosted various bacterial groups differently from the reference prebiotic substrate (inulin). We also found inulin had reduced butyrogenic and bifidogenic effects in the elderly group compared to the younger adult group. In conclusion, the in vitro modulating effects of pectins on elderly gut microbiota showed potential of using pectins to improve age-related dysbiosis.

Diversity and succession of contaminating yeasts in white-brined cheese during cold storage.

Contamination of white-brined cheeses (WBCs) with yeasts is of major concern in the dairy industry. This study aimed to identify yeast contaminants and characterize their succession in white-brined cheese during a shelf-life of 52 weeks. White-brined cheeses added herbs (WBC1) or sundried tomatoes (WBC2) were produced at a Danish dairy and incubated at 5 °C and 10 °C. An increase in yeast counts was observed for both products within the first 12-14 weeks of incubation and stabilized afterwards varying in a range of 4.19-7.08 log CFU/g. Interestingly, higher incubation temperature, especially in WBC2, led to lower yeast counts, concurrently with higher diversity of yeast species. Observed decrease in yeast counts was, most likely, due to negative interactions between yeast species leading to growth inhibition. In total, 469 yeast isolates from WBC1 and WBC2 were genotypically classified using the (GTG)5-rep-PCR technique. Out of them, 132 representative isolates were further identified by sequencing the D1/D2 domain of the 26 S rRNA gene. Predominant yeast species in WBCs were Candida zeylanoides and Debaryomyces hansenii, while Candida parapsilosis, Kazachstania bulderi, Kluyveromyces lactis, Pichia fermentans, Pichia kudriavzevii, Rhodotorula mucilaginosa, Torulaspora delbrueckii, and Wickerhamomyces anomalus were found in lower frequency. Heterogeneity of yeast species in WBC2 was generally larger compared to WBC1. This study indicated that, along with contamination levels, taxonomic heterogeneity of yeasts is an important factor influencing yeast cell counts, as well as product quality during storage.

The Potential of Pectins to Modulate the Human Gut Microbiota Evaluated by In Vitro Fermentation: A Systematic Review.

Pectin is a dietary fiber, and its health effects have been described extensively. Although there are limited clinical studies, there is a growing body of evidence from in vitro studies investigating the effect of pectin on human gut microbiota. This comprehensive review summarizes the findings of gut microbiota modulation in vitro as assessed by 16S rRNA gene-based technologies and elucidates the potential structure-activity relationships. Generally, pectic substrates are slowly but completely fermented, with a greater production of acetate compared with other fibers. Their fermentation, either directly or by cross-feeding interactions, results in the increased abundances of gut bacterial communities such as the family of Ruminococcaceae, the Bacteroides and Lachnospira genera, and species such as Lachnospira eligens and Faecalibacterium prausnitzii, where the specific stimulation of Lachnospira and L. eligens is unique to pectic substrates. Furthermore, the degree of methyl esterification, the homogalacturonan-to-rhamnogalacturonan ratio, and the molecular weight are the most influential structural factors on the gut microbiota. The latter particularly influences the growth of Bifidobacterium spp. The prebiotic potential of pectin targeting specific gut bacteria beneficial for human health and well-being still needs to be confirmed in humans, including the relationship between its structural features and activity.

Occurrence and Identification of Yeasts in Production of White-Brined Cheese.

The aim of this study was to reveal the sites of yeast contamination in dairy production and perform taxonomic characterization of potential yeast spoilers in cheese making. Occurrence of spoilage yeasts was followed throughout the manufacture of white-brined cheese at a Danish dairy, including the areas of milk pasteurization, curd processing, and packaging (26 sites in total). Spoilage yeasts were isolated from whey, old cheese curd, and air samples in viable counts of 1.48-6.27 log CFU/mL, 5.44 log CFU/g, and 1.02 log CFU/m3, respectively. Yeast isolates were genotypically classified using (GTG)5-PCR fingerprinting and identified by sequencing of the D1/D2 region of the 26S rRNA gene. The largest yeast heterogeneity was found in old curd collected under the turning machine of molds, where 11 different yeast species were identified. The most frequently isolated yeast species were Candida intermedia, Kluyveromyces marxianus, and Pichia kudriavzevii. The less abundant yeast species included Candida auris, Candida parapsilosis, Candida pseudoglaebosa, Candida sojae, Cutaneotrichosporon curvatus, Cutaneotrichosporon moniliiforme, Papiliotrema flavescens, Rhodotorula mucilaginosa, Vanrija humicola, and Wickerhamiella sorbophila. The awareness on occurrence and taxonomy of spoilage yeasts in cheese production will contribute to a knowledge-based control of contaminating yeasts and quality management of cheese at the dairies.

Spoilage Potential of Contaminating Yeast Species Kluyveromyces marxianus, Pichia kudriavzevii and Torulaspora delbrueckii during Cold Storage of Skyr.

This study investigated the spoilage potential of yeast strains Kluyveromyces marxianus (Km1, Km2 and Km3), Pichia kudriavzevii Pk1 and Torulaspora delbrueckii Td1 grown in skyr in cold storage. Yeast strains were isolated from skyr and identified by sequencing of the 26S rRNA gene. K. marxianus yeasts were grown in skyr to high numbers, generating large amounts of volatile organic compounds (VOC) associated with off-flavours, among them were alcohols (3-methyl-1-butanol, 2-methyl-1-propanol and 1-hexanol), esters (ethyl acetate and 3-methylbutyl acetate) and aldehydes (hexanal, methylbutanal and methylpropanal). Growth of P. kudriavzevii Pk1 led to moderate increases in several alcohols and esters (mostly, 3-methyl-1-butanol and ethyl acetate), whereas only minor shifts in VOCs were associated with T. delbrueckii Td2. The levels of the key aroma compounds, diacetyl and acetoin, were significantly decreased by all K. marxianus strains and P. kudriavzevii Pk1. In contrast to the other yeast species, K. marxianus was able to utilize lactose, producing ethanol and carbon dioxide. Based on the overall results, K. marxianus was characterised by the highest spoilage potential. The study revealed the differences between the yeast species in fermentative and spoilage activities, and clarified the role of yeast metabolites for off-flavour formation and quality defects in skyr during cold storage.

Occurrence of Yeasts in White-Brined Cheeses: Methodologies for Identification, Spoilage Potential and Good Manufacturing Practices.

Yeasts are generally recognized as contaminants in the production of white-brined cheeses, such as Feta and Feta-type cheeses. The most predominant yeasts species are Debaryomyces hansenii, Geotrichum candidum, Kluyveromyces marxianus, Kluyveromyces lactis, Rhodotorula mucilaginosa, and Trichosporon spp. Although their spoilage potential varies at both species and strain levels, yeasts will, in case of excessive growth, present a microbiological hazard, effecting cheese quality. To evaluate the hazard and trace routes of contamination, the exact taxonomic classification of yeasts is required. Today, identification of dairy yeasts is mainly based on DNA sequencing, various genotyping techniques, and, to some extent, advanced phenotypic identification technologies. Even though these technologies are state of the art at the scientific level, they are only hardly implemented at the industrial level. Quality defects, caused by yeasts in white-brined cheese, are mainly linked to enzymatic activities and metabolism of fermentable carbohydrates, leading to production of metabolites (CO2, fatty acids, volatile compounds, amino acids, sulfur compounds, etc.) and resulting in off-flavors, texture softening, discoloration, and swelling of cheese packages. The proliferation of spoilage yeast depends on maturation and storage conditions at each specific dairy, product characteristics, nutrients availability, and interactions with the co-existing microorganisms. To prevent and control yeast contamination, different strategies based on the principles of HACCP and Good Manufacturing Practice (GMP) have been introduced in white-brined cheese production. These strategies include milk pasteurization, refrigeration, hygienic sanitation, air filtration, as well as aseptic and modified atmosphere packaging. Though a lot of research has been dedicated to yeasts in dairy products, the role of yeast contaminants, specifically in white-brined cheeses, is still insufficiently understood. This review aims to summarize the current knowledge on the identification of contaminant yeasts in white-brined cheeses, their occurrence and spoilage potential related to different varieties of white-brined cheeses, their interactions with other microorganisms, as well as guidelines used by dairies to prevent cheese contamination.

Probiotic potential of Saccharomyces cerevisiae and Kluyveromyces marxianus isolated from West African spontaneously fermented cereal and milk products.

The yeast species Saccharomyces cerevisiae and Kluyveromyces marxianus are associated with fermentation of West African indigenous foods. The aim of this study was to characterize potential probiotic properties of S. cerevisiae and K. marxianus isolates from the West African milk products lait caillé and nunu and a cereal-based product mawè. The strains (14 in total) were identified by 26S rRNA gene sequencing and characterized for survival at gastrointestinal stress (bile salts and low pH) and adhesion to Caco-2 intestinal epithelial cells. Selected yeast isolates were tested for their effect on the transepithelial electrical resistance (TEER), using the intestinal epithelial cell line Caco-2 and for maintenance of intracellular pH (pHi ) during perfusion with gastrointestinal pH (3.5 and 6.5). All tested yeasts were able to grow in bile salts in a strain-dependent manner, exhibiting a maximum specific growth rate (µmax ) of 0.58-1.50 h-1 . At pH 2.5, slow growth was observed for the isolates from mawè (µmax of 0.06-0.80 h-1 ), whereas growth of yeasts from other sources was mostly inhibited. Yeast adhesion to Caco-2 cells was strain specific and varied between 8.0% and 36.2%. Selected strains of S. cerevisiae and K. marxianus were able to maintain the pHi homeostasis at gastrointestinal pH and to increase TEER across the Caco-2 monolayers, indicating their potential to improve intestinal barrier functions. Based on overall results, strains of K. marxianus and S. cerevisiae from mawè exhibited the highest probiotic potential and might be recommended for further development as starter cultures in West African fermented products.

Effect of potato fiber on survival of Lactobacillus species at simulated gastric conditions and composition of the gut microbiota in vitro.

Potato fiber is a side product in starch manufacturing rich in dietary fibers such as pectin, cellulose, hemicellulose and resistant starch. So far, the beneficial properties of potato fiber have been poorly characterized. This study investigated the effect of FiberBind 400, a commercial potato fiber product, on survival of probiotic Lactobacillus strains at simulated gastric conditions and on the composition and metabolic activity of the gut microbiota, using the TIM-2 colon model. Resistant starch and native starch from potato were used as reference substrates. FiberBind 400 had an ability to improve survival of the four tested strains, Lactobacillus fermentum PCC®, L. rhamnosus LGG®, L. reuteri RC-14® and L. paracasei F-19® in a strain-dependent way. The highest effect was observed for L. fermentum PCC® and L. rhamnosus LGG®. The effect of starches on bacterial survival was insignificant. Composition of the fecal microbiota in TIM-2 fermentations was assessed by high-throughput sequencing of 16S rRNA gene amplicon. Fermentation of FiberBind 400 resulted in more diverse microbial communities compared to starches. Changes in microbial abundances specifically mediated by FiberBind 400, included increases in the genera Lachnospira, Butyrivibrio, Mogibacterium, Parabacteroides, Prevotella and Desulfovibrio, and the species B. ovatus, as well as decreases in Ruminococcus torques and unassigned Ruminococcus spp. Shifts in other bacterial populations, such as increased abundances of Oscillospira, Enterococcus, Bacteroidales, Citrobacter, along with reduction of Roseburia, Ruminococcus, and Faecalibacterium prausnitzii were not significantly different between the substrates. Cumulative production of individual short-chain fatty acids was similar between potato fiber and starches. The study demonstrated that FiberBind 400 had a potential to protect probiotic Lactobacillus strains during the passage through the gastrointestinal tract and selectively modulate the gut bacterial populations. This knowledge can support application of potato fiber as a functional food ingredient with added biological benefits.

Impact of botanical fermented foods on metabolic biomarkers and gut microbiota in adults with metabolic syndrome and type 2 diabetes: a systematic review protocol.

INTRODUCTION: Dysfunctional gut microbiota is a common finding in patients with metabolic syndrome (MetS) and type 2 diabetes mellitus (T2DM). Recent clinical trials have assessed whether botanical fermented foods (BFFs) have beneficial effects on metabolic biomarkers, inflammatory markers and gut microbiota. The aim of this review is to critically evaluate all randomised controlled trials (RCTs) of BFF for evidence of impact on the outcome measures of these disease states. METHODS AND ANALYSIS: Four electronic databases (Embase, MEDLINE, CENTRAL and Google Scholar) as well as the grey literature will be searched from inception to present without language or publication status restrictions applied. Eligible RCTs which have enrolled adult participants with T2DM, any MetS components or combinations of these components, treated prophylactically or therapeutically with any botanical fermented food intervention, compared with a control group (no intervention, placebo or active control) will be assessed. Primary outcomes are related to the target conditions, including metabolic biomarkers, inflammatory markers and gut microbiota composition/function. Using Covidence, two independent investigators will conduct title and abstract screening, followed by full-text screening to identify appropriate studies. Methodological quality of the trials will be assessed using the Cochrane risk of bias assessment tool. Findings will be summarised with a narrative synthesis of the differences between included studies. A meta-analysis will be conducted if sufficient data are obtained. ETHICS AND DISSEMINATION: Ethical approval is not required as primary data will not be collected. Results will be disseminated through peer-reviewed publication, conference presentations and press. PROSPERO REGISTRATION NUMBER: CRD42018117766.

In vitro modulation of human gut microbiota composition and metabolites by Bifidobacterium longum BB-46 and a citric pectin.

The gut microbiota composition and its metabolites have high impact on human health. Exploitation of prebiotics and probiotics for modulation of gut microbiota can lead to promising outcomes. This study aimed to evaluate the effects of the probiotic strain Bifidobacterium longum BB-46 alone and in combination with a citric pectin from lemon on the gut microbiota from healthy adults using the Simulator of Human Intestinal Microbial Ecosystem (SHIME®). Changes in microbiota composition and in metabolic activity were assessed by the 16S rRNA gene sequencing and by analyses of short-chain fatty acids (SCFAs) and ammonium ions (NH4+). An increase in the relative abundances of Firmicutes (especially the members of Lachnospiraceae and Lactobacillaceae families) and Bacteroidetes was observed during treatment with B. longum BB-46 alone in all compartments of the colon. Treatment with B. longum BB-46 and pectin stimulated an increase in the proportions of genera Faecalibacterium, Eubacterium and Lactobacillus, as well as in the Ruminococcaceae family in the transverse and descending colons. Concurrently, the butyrate levels increased in these two compartments. Additionally, the combination of B. longum BB-46 and pectin reduced the abundance of proteolytic bacteria Bacteroides, Clostridium, Peptoniphilus, and Streptococcus, along with decreased NH4+ production. No significant changes could be observed on NH4+ production by treatment with B. longum BB-46, nor did it increase the amount of SCFAs. In this study, we observed that although each treatment was able to modulate the microbiota, the combination of B. longum BB-46 and pectin was more efficient in decreasing the intestinal NH4+ levels and in increasing butyric acid-producing bacteria. These findings indicate that B. longum BB-46, especially when combined with the specific citric pectin, might have beneficial impact on human health.

Potential of Pectins to Beneficially Modulate the Gut Microbiota Depends on Their Structural Properties.

Pectins are plant cell-wall polysaccharides which can be utilized by commensal bacteria in the gut, exhibiting beneficial properties for the host. Knowledge of the impact of pectins on intestinal bacterial communities is insufficient and limited to a few types of pectins. This study characterized the relationship between the structural properties of pectins and their potential to modulate composition and activity of the gut microbiota in a beneficial way. For this purpose we performed in vitro fermentations of nine structurally diverse pectins from citrus fruits and sugar beet, and a pectic derivative, rhamnogalacturonan I (RGI), using a TIM-2 colon model. The composition of microbiota during TIM-2 fermentations was assessed by 16S rRNA gene amplicon sequencing. Both general and pectin-specific changes were observed in relative abundances of numerous bacterial taxa in a time-dependent way. Bacterial populations associated with human health, such as Faecalibacterium prausnitzii, Coprococcus, Ruminococcus, Dorea, Blautia, Oscillospira, Sutterella, Bifidobacterium, Christensenellaceae, Prevotella copri, and Bacteroides spp. were either increased or decreased depending on the substrate, suggesting that these bacteria can be controlled using structurally different pectins. The main structural features linked to the pectin-mediated shifts in microbiota included degree of esterification, composition of neutral sugars, distribution of homogalacturonan and rhamnogalacturonan fractions, degree of branching, and the presence of amide groups. Cumulative production of the total short chain fatty acids and propionate was largest in fermentations of the high methoxyl pectins. Thus, this study indicates that microbial communities in the gut can be specifically modulated by pectins and identifies the features in pectin molecules linked to microbial alterations. This knowledge can be used to define preferred dietary pectins, targeting beneficial bacteria, and favoring more balanced microbiota communities in the gut.

Modulation of gut microbiota from obese individuals by in vitro fermentation of citrus pectin in combination with Bifidobacterium longum BB-46.

This study aimed to evaluate the effects of three treatments, i.e., Bifidobacterium longum BB-46 (T1), B. longum BB-46 combined with the pectin (T2), and harsh extracted pectin from lemon (T3) on obesity-related microbiota using the Simulator of the Human Intestinal Microbial Ecosystem (SHIME®). The effects of the treatments were assessed by the analysis of the intestinal microbial composition (using 16S rRNA gene amplicon sequencing) and the levels of short-chain fatty acids (SCFAs) and ammonium ions (NH4+). Treatments T2 and T3 stimulated members of the Ruminococcaceae and Succinivibrionaceae families, which were positively correlated with an increase in butyric and acetic acids. Proteolytic bacteria were reduced by the two treatments, concurrently with a decrease in NH4+. Treatment T1 stimulated the production of butyric acid in the simulated transverse and descending colon, reduction of NH4+ as well as the growth of genera Lactobacillus, Megamonas, and members of Lachnospiracea. The results indicate that both B. longum BB-46 and pectin can modulate the obesity-related microbiota; however, when the pectin is combined with B. longum BB-46, the predominant effect of the pectin can be observed. This study showed that the citric pectin is able to stimulate butyrate-producing bacteria as well as genera related with anti-inflammatory effects. However, prospective clinical studies are necessary to evaluate the anti/pro-obesogenic and inflammatory effects of this pectin for future prevention of obesity.

The effect of pectins on survival of probiotic Lactobacillus spp. in gastrointestinal juices is related to their structure and physical properties.

Pectins are plant polysaccharides used in food industry as gelling and stabilizing agents. This study investigated the ability of pectins to improve survival of probiotic species Lactobacillus fermentum PCC, L. reuteri RC-14, L. rhamnosus LGG and L. paracasei F-19 in simulated gastric solution in relationship to their structural and physical properties. Electrostatic interactions between pectins and bacteria were evaluated by the Zeta-potential approach. Bacterial survival was assessed by flow cytometry and plate counting. L. fermentum PCC and L. reuteri RC-14 were more resistant to gastric conditions; their survival rate was further improved in the presence of five out of ten tested pectins. Additionally, two of the pectins had a positive effect on viability of the less resistant L. rhamnosus LGG and L. paracasei F-19. The beneficial effect was generally observed for the high-methoxylated pectins, indicating that substituted polygalacturonic acid in the backbone is essential for bacterial protection. Other pectin features associated with improved survival, included less negative Zeta-potential, higher molecular weight, as well as lower values of hydrodynamic sizes, viscosity and degree of branching. The study indicates that pectins have a potential to protect probiotic bacteria through the gastro-intestinal transit and identifies the features linked to their functionality.

Transcriptome analysis of Lactococcus lactis subsp. lactis during milk acidification as affected by dissolved oxygen and the redox potential.

Performance of Lactococcus lactis as a starter culture in dairy fermentations depends on the levels of dissolved oxygen and the redox state of milk. In this study the microarray analysis was used to investigate the global gene expression of L. lactis subsp. lactis DSM20481(T) during milk acidification as affected by oxygen depletion and the decrease of redox potential. Fermentations were carried out at different initial levels of dissolved oxygen (dO2) obtained by milk sparging with oxygen (high dO2, 63%) or nitrogen (low dO2, 6%). Bacterial exposure to high initial oxygen resulted in overexpression of genes involved in detoxification of reactive oxygen species (ROS), oxidation-reduction processes, biosynthesis of trehalose and down-regulation of genes involved in purine nucleotide biosynthesis, indicating that several factors, among them trehalose and GTP, were implicated in bacterial adaptation to oxidative stress. Generally, transcriptional changes were more pronounced during fermentation of oxygen sparged milk. Genes up-regulated in response to oxygen depletion were implicated in biosynthesis and transport of pyrimidine nucleotides, branched chain amino acids and in arginine catabolic pathways; whereas genes involved in salvage of nucleotides and cysteine pathways were repressed. Expression pattern of genes involved in pyruvate metabolism indicated shifts towards mixed acid fermentation after oxygen depletion with production of specific end-products, depending on milk treatment. Differential expression of genes, involved in amino acid and pyruvate pathways, suggested that initial oxygen might influence the release of flavor compounds and, thereby, flavor development in dairy fermentations. The knowledge of molecular responses involved in adaptation of L. lactis to the shifts of redox state and pH during milk fermentations is important for the dairy industry to ensure better control of cheese production.

Production of autoinducer-2 by aerobic endospore-forming bacteria isolated from the West African fermented foods.

Autoinducer-2 (AI-2) is a quorum-sensing (QS) molecule which mediates interspecies signaling and affects various bacterial behaviors in food fermentation. Biosynthesis of AI-2 is controlled by S-ribosylhomocysteine lyase encoded by the luxS gene. The objective of this study was to investigate production of AI-2 by aerobic endospore-forming bacteria (AEB) isolated from the West African alkaline fermented seed products Mantchoua and Maari. The study included 13 AEB strains of Bacillus subtilis, B. cereus, B. altitudinis, B. amyloliquefaciens, B. licheniformis, B. aryabhattai, B. safensis, Lysinibacillus macroides and Paenibacillus polymyxa. All the tested strains harbored the luxS gene and all strains except for P. polymyxa B314 were able to produce AI-2 during incubation in laboratory medium. Production of AI-2 by AEB was growth phase dependent, showing maximum activity at the late exponential phase. AI-2 was depleted from the culture medium at the beginning of the stationary growth phase, indicating that the tested AEB possess a functional AI-2 receptor that internalizes AI-2. This study provides the evidences of QS system in Bacillus spp. and L. macroides and new knowledge of AI-2 production by AEB. This knowledge contributes to the development of QS-based strategies for better control of alkaline fermentation.

Expression of Virulence-Related Genes in Listeria monocytogenes Grown on Danish Hard Cheese as Affected by NaCl Content.

Expression of virulence-related genes in Listeria monocytogenes incubated on cheese was assessed by real-time quantitative polymerase chain reaction. The objective of the study was to investigate the impact of sodium chloride concentration in cheese on transcription of virulence genes and, thereby, virulence potential of L. monocytogenes. The expression studies were performed with L. monocytogenes strains characterized by different tolerance to salt stress. Strains ATCC(®) 51779 and DSMZ 15675 were incubated on the Danish hard-cheese type Samsoe, with low (<0.15% [wt/wt]) and high (3.6% [wt/wt]) content of NaCl. Genes differentially expressed (p<0.05) through the 48-h incubation were transcriptional regulators prfA and agrA, genes of the main virulence cluster inlA, hly, actA, involved in invasion of the epithelial cells, and genes bsh, opuC, gadC, clpP, and ami, associated with osmotic stress responses in L. monocytogenes. The more sensitive strain ATCC(®) 51779 was most responsive, showing significant upregulation of prfA, actA, hly, and bsh both at low and high NaCl. Strain DSMZ 15675 was less responsive to NaCl stress, showing reduced or consistent gene transcription at all conditions. Decreased transcription of agrA, ami, gadC, and opuC in both strains was promoted by low NaCl content. The study indicated that virulence gene expression of L. monocytogenes grown in cheese was affected by NaCl content and that effect was more significant in strains sensitive to both hypo- and hyperosmotic stresses.

Effect of dissolved oxygen on redox potential and milk acidification by lactic acid bacteria isolated from a DL-starter culture.

Milk acidification by DL-starter cultures [cultures containing Lactococcus lactis diacetylactis (D) and Leuconostoc (L) species] depends on the oxidation-reduction (redox) potential in milk; however, the mechanisms behind this effect are not completely clear. The objective of this study was to investigate the effect of dissolved oxygen on acidification kinetics and redox potential during milk fermentation by lactic acid bacteria (LAB). Fermentations were conducted by single strains isolated from mixed DL-starter culture, including Lactococcus lactis ssp. lactis, Lactococcus lactis ssp. cremoris, and Leuconostoc mesenteroides ssp. cremoris, by the DL-starter culture, and by the type strains. High and low levels of oxygen were produced by flushing milk with oxygen or nitrogen, respectively. The kinetics of milk acidification was characterized by the maximum rate and time of acidification (Vamax and Tamax), the maximum rate and time of reduction (Vrmax and Trmax), the minimum redox potential (Eh7 final), and time of reaching Eh7 final (Trfinal). Variations in kinetic parameters were observed at both the species and strain levels. Two of the Lc. lactis ssp. lactis strains were not able to lower redox potential to negative values. Kinetic parameters of the DL-starter culture were comparable with the best acidifying and reducing strains, indicating their additive effects. Acidification curves were mostly diauxic at all oxygen levels, displaying 2 maxima of acidification rate: before (aerobic maximum) and after (anaerobic maximum) oxygen depletion. The redox potential decreased concurrently with oxygen consumption and continued to decrease at slower rate until reaching the final values, indicating involvement of both oxygen and microbiological activity in the redox state of milk. Oxygen flushing had a negative effect on reduction and acidification capacity of tested LAB. Reduction was significantly delayed at high initial oxygen, exhibiting longer Trmax, Trfinal, or both. Concurrently, anaerobic acidification rate maximum Vamax was decreased and Tamax was extended. Fermentation kinetics in nitrogen-flushed milk was not statistically different from that in untreated milk except for Lc. lactis ssp. lactis CHCC D2, which showed faster reduction time after nitrogen flushing. This study clarifies the relationship between the redox state in milk and acidification kinetics of the predominant subspecies in DL-starter cultures. This knowledge is important for dairies to ensure optimized, fast, and controlled milk fermentations, leading to greater standardization of dairy products.

Characteristics and phylogeny of Bacillus cereus strains isolated from Maari, a traditional West African food condiment.

Maari is a spontaneously fermented food condiment made from baobab tree seeds in West African countries. This type of product is considered to be safe, being consumed by millions of people on a daily basis. However, due to the spontaneous nature of the fermentation the human pathogen Bacillus cereus occasionally occurs in Maari. This study characterizes succession patterns and pathogenic potential of B. cereus isolated from the raw materials (ash, water from a drilled well (DW) and potash), seed mash throughout fermentation (0-96h), after steam cooking and sun drying (final product) from two production sites of Maari. Aerobic mesophilic bacterial (AMB) counts in raw materials were of 10(5)cfu/ml in DW, and ranged between 6.5×10(3) and 1.2×10(4)cfu/g in potash, 10(9)-10(10)cfu/g in seed mash during fermentation and 10(7) - 10(9) after sun drying. Fifty three out of total 290 AMB isolates were identified as B. cereus sensu lato by use of ITS-PCR and grouped into 3 groups using PCR fingerprinting based on Escherichia coli phage-M13 primer (M13-PCR). As determined by panC gene sequencing, the isolates of B. cereus belonged to PanC types III and IV with potential for high cytotoxicity. Phylogenetic analysis of concatenated sequences of glpF, gmk, ilvD, pta, pur, pycA and tpi revealed that the M13-PCR group 1 isolates were related to B. cereus biovar anthracis CI, while the M13-PCR group 2 isolates were identical to cereulide (emetic toxin) producing B. cereus strains. The M13-PCR group 1 isolates harboured poly-γ-D-glutamic acid capsule biosynthesis genes capA, capB and capC showing 99-100% identity with the environmental B. cereus isolate 03BB108. Presence of cesB of the cereulide synthetase gene cluster was confirmed by PCR in M13-PCR group 2 isolates. The B. cereus harbouring the cap genes were found in potash, DW, cooking water and at 8h fermentation. The "emetic" type B. cereus were present in DW, the seed mash at 48-72h of fermentation and in the final product, while the remaining isolates (PanC type IV) were detected in ash, at 48-72h fermentation and in the final product. This work sheds light on the succession and pathogenic potential of B. cereus species in traditional West African food condiment and clarifies their phylogenetic relatedness to B. cereus biovar anthracis. Future implementation of GMP and HACCP and development of starter cultures for controlled Maari fermentations will help to ensure a safe product.

Characterization of Bacillus spp. strains for use as probiotic additives in pig feed.

Bacillus spp. are commonly used as probiotic species in the feed industry, however, their benefits need to be confirmed. This study describes a high throughput screening combined with the detailed characterization of endospore-forming bacteria with the aim to identify new Bacillus spp. strains for use as probiotic additives in pig feed. A total of 245 bacterial isolates derived from African fermented food, feces and soil were identified by 16S rRNA gene sequencing and screened for antimicrobial activity and growth in the presence of antibiotics, bile salts and at pH 4.0. Thirty-three Bacillus spp. isolates with the best characteristics were identified by gyrB and rpoB gene sequencing as B. amyloliquefaciens subsp. plantarum, B. amyloliquefaciens subsp. amyloliquefaciens, B. subtilis subsp. subtilis, B. licheniformis, B. mojavensis, B. pumilus and B. megaterium. These isolates were further investigated for their activity against the pathogenic bacteria, antibiotic susceptibility, sporulation rates, biofilm formation and production of glycosyl hydrolytic enzymes. Additionally, ten selected isolates were assessed for heat resistance of spores and the effect on porcine epithelial cells IPEC-J2. Isolates of B. amyloliquefaciens, B. subtilis and B. mojavensis, showed the best overall characteristics and, therefore, potential for usage as probiotic additives in feed. A large number of taxonomically diverse strains made it possible to reveal species and subspecies-specific trends, contributing to our understanding of the probiotic potential of Bacillus species.