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In vitro investigations on the impact of constituents on fermented dairy on fecal microbiota composition

ABSTRACT: The consumption of fermented food has been linked to positive health outcomes due to a variety of functional properties. Fermented dairy constitutes a major dietary source and contains lactoseas main carbohydrate and living starter cultures. To investigate whether nutritional and microbial modulation impacted intestinal microbiota composition and activity, we employed fecal microbiota fermentations and a dairy model system consisting of lactose and β-galactosidase positive and negative Streptococcus thermophilus. Based on 16S rRNA gene based microbial community analysis, we observed that lactose addition increased the abundance of Bifidobacteriaceae, and of Veillonellaceae and Enterobacteraceae in selected samples. The supplied lactose was hydrolysed within 24 h of fermentation and led to higher expression of community indigenous β-galactosidases. Targeted protein analysis confirmed that bifidobacteria contributed most β-galactosidases together with other taxa including Escherichia coli and Anaerobutyricum hallii. Lactose addition led to 1.1-1.8 fold higher levels of butyrate compared to controls likely due to (i) lactate-crossfeeding and (ii) direct lactose metabolism by butyrate producing Anaerobutyricum and Faecalibacterium spp. Representatives of both genera used lactose to produce butyrate in single cultures. When supplemented at around 5.5 log cells mL-1, S. thermophilus or its beta-galactosidase negative mutant outnumbered the indigenous Streptococcaceae population at the beginning of fermentation but had no impact on lactose utilisation and final SCFA profiles. This study brings forward new fundamental insight into interactions of major constituents of fermented dairy with the intestinal microbiota. We provide evidence that lactose addition increases fecal microbiota production of butyrate through cross-feeding and direct metabolism without contribution of starter cultures.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Bifidobacteriaceae

SUBMITTER: Carsten Scavenius

LAB HEAD: Carsten Scavenius

PROVIDER: PXD054534 | Pride | 2025-01-30

REPOSITORIES: pride

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Project description:The present study aims to explore chemostat-based transcriptome analysis of mixed cultures by investigating interactions between the yeast S. cerevisiae and the lactic acid bacterium L. bulgaricus . S. cerevisiae and L. bulgaricus are both frequently encountered in kefir, a fermented dairy product. In the context of this study, this binary culture serves as a model for the many traditional food and beverage fermentation processes in which yeasts and lactic acid bacteria occur together. The design of the cultivation conditions was based on the observation that L. bulgaricus, but not S. cerevisiae, can use lactose as a carbon source for growth and that S. cerevisiae, but not L. bulgaricus, can grow on galactose that is released upon hydrolysis of lactose by the bacterial β-galactosidase. Mixed populations of yeasts and lactic acid bacteria occur in many dairy, food and beverage fermentations, but knowledge about their interactions is incomplete. In the present study, interactions between Saccharomyces cerevisiae and Lactobacillus delbrueckii subsp. bulgaricus, two microorganisms that co-occur in kefir fermentations, were studied during anaerobic growth on lactose. By combining physiological and transcriptome analysis of the two strains in the co-cultures, five mechanisms of interaction were identified. 1. L. bulgaricus hydrolyses lactose, which cannot be metabolized by S. cerevisiae, to galactose and glucose. Subsequently, galactose, which cannot be metabolized by L. bulgaricus, is excreted and provides a carbon source for yeast. 2. In pure cultures, L. bulgaricus only grows at increased CO2 concentrations. In anaerobic mixed cultures, the yeast provides this CO2 via alcoholic fermentation. 3. Analysis of amino acid consumption from the defined medium indicated that S. cerevisiae supplied alanine to the bacteria. 4. A mild but significant low-iron response in the yeast transcriptome, identified by DNA microarray analysis, was consistent with the chelation of iron by the lactate produced by L. bulgaricus. 5. Transcriptome analysis of L. bulgaricus in mixed cultures showed an overrepresentation of transcripts involved in lipids metabolism suggesting either a competition of the two microorganisms for fatty acids, or a response to the ethanol produced by S. cerevisiae.

Project description:Analysis of non-differentiated Caco-2 intestinal epithelial cell line treated with polydextrose fermentation metabolites fermented for 48 hours in 4-stage in vitro colon simulator, in which the conditions mimic the human proximal, ascending, transverse and distal colon in sequence , as well as with medium, 100 mM NaCl and 5 mM butyrate. Polydextrose, a soluble fiber fermented in colon, was fermented with the in vitro colon simulator in three amounts of 0%, 1% and 2%. Results provide insight into the mechanisms underlying colon cancer cells and a comparison of a complex fiber metabolome to 5 mM butyrate and 100 mM NaCl. Furthermore, the results give insight of dosage effect of increasing the concentration of fiber. High level of dietary fiber has been epidemiologically linked to protection against the risk for developing colon cancer. The mechanisms of this protection are not clear. Fermentation of dietary fiber in the colon results in production of for example butyrate that has drawn attention as a chemopreventive agent. Polydextrose, a soluble fiber that is only partially fermented in colon, was fermented in an in vitro colon simulator, in which the conditions mimic the human proximal, ascending, transverse and distal colon in sequence. The subsequent fermentation metabolome were applied on colon cancer cells, and the gene expression changes studied. Polydextrose fermentation down-regulated classes linked with cell cycle, and affected number of metabolically active cells. Further, up-regulated effects on classes linked with apoptosis implicate that polydextrose fermentation plays a role in induction of apoptosis in colon cancer cells. The up-regulated genes involved also key regulators of lipid metabolism, such as PPARg and PGC-1α. These results offer hypotheses for the mechanisms of two health benefits linked with consumption of dietary fiber, reducing risk of development of colon cancer, and dyslipidemia. Non-differentiated Caco-2 cells were treated with polydextrose fermentation metabolites from the vessels representing different parts of the colon, or with 100 mM NaCl or with 5 mM butyrate for 24 hours. For polydextrose fermentation three concentrations of polydextrose were used: 0%, 1% and 2% for a simulation that lasted for 48 hours. Polydextrose fermentation samples from total of 12 vessels, as well as from medium sample, 5 mM butyrate and 100 mM NaCl were analysed as single replica.

Project description:The present study aims to explore chemostat-based transcriptome analysis of mixed cultures by investigating interactions between the yeast S. cerevisiae and the lactic acid bacterium Lb. bulgaricus . S. cerevisiae and Lb. bulgaricus are both frequently encountered in kefir, a fermented dairy product (25). In the context of this study, this binary culture serves as a model for the many traditional food and beverage fermentation processes in which yeasts and lactic acid bacteria occur together (19,26-30). The design of the cultivation conditions was based on the observation that Lb. bulgaricus, but not S. cerevisiae, can use lactose as a carbon source for growth and that S. cerevisiae, but not Lb. bulgaricus, can grow on galactose that is released upon hydrolysis of lactose by the bacterial M-NM-2-galactosidase. Mixed populations of yeasts and lactic acid bacteria occur in many dairy, food and beverage fermentations, but knowledge about their interactions is incomplete. In the present study, interactions between Saccharomyces cerevisiae and Lactobacillus delbrueckii subsp. bulgaricus, two microorganisms that co-occur in kefir fermentations, were studied during anaerobic growth on lactose. By combining physiological and transcriptome analysis of the two strains in the co-cultures, five mechanisms of interaction were identified. 1. Lb. bulgaricus hydrolyses lactose, which cannot be metabolized by S. cerevisiae, to galactose and glucose. Subsequently, galactose, which cannot be metabolized by Lb. bulgaricus, is excreted and provides a carbon source for yeast. 2. In pure cultures, Lb. bulgaricus only grows at increased CO2 concentrations. In anaerobic mixed cultures, the yeast provides this CO2 via alcoholic fermentation. 3. Analysis of amino acid consumption from the defined medium indicated that S. cerevisiae supplied alanine to the bacteria. 4. A mild but significant low-iron response in the yeast transcriptome, identified by DNA microarray analysis, was consistent with the chelation of iron by the lactate produced by Lb. bulgaricus. 5. Transcriptome analysis of Lb. bulgaricus in mixed cultures showed an overrepresentation of transcripts involved in lipids metabolism suggesting either a competition of the two microorganisms for fatty acids, or a response to the ethanol produced by S. cerevisiae. To our knowledge, this is the first transcriptome study of a cross-kingdom binary mixed culture that analyses responses of both microorganisms. This study demonstrates that chemostat-based transcriptome analysis is a powerful tool to investigated microbial interaction in mixed populations. To investigate the impact of of co-cultivation with Lb. bulgaricus on S. cerevisiae, a DNA microarray-based transcriptome analysis of S. cerevisiae's response was performed on anaerobic, lactose-limited chemostat cultures grown in the presence and absence of L. bulgaricus.

Project description:The present study aims to explore chemostat-based transcriptome analysis of mixed cultures by investigating interactions between the yeast S. cerevisiae and the lactic acid bacterium Lb. bulgaricus . S. cerevisiae and Lb. bulgaricus are both frequently encountered in kefir, a fermented dairy product (25). In the context of this study, this binary culture serves as a model for the many traditional food and beverage fermentation processes in which yeasts and lactic acid bacteria occur together (19,26-30). The design of the cultivation conditions was based on the observation that Lb. bulgaricus, but not S. cerevisiae, can use lactose as a carbon source for growth and that S. cerevisiae, but not Lb. bulgaricus, can grow on galactose that is released upon hydrolysis of lactose by the bacterial β-galactosidase. Mixed populations of yeasts and lactic acid bacteria occur in many dairy, food and beverage fermentations, but knowledge about their interactions is incomplete. In the present study, interactions between Saccharomyces cerevisiae and Lactobacillus delbrueckii subsp. bulgaricus, two microorganisms that co-occur in kefir fermentations, were studied during anaerobic growth on lactose. By combining physiological and transcriptome analysis of the two strains in the co-cultures, five mechanisms of interaction were identified. 1. Lb. bulgaricus hydrolyses lactose, which cannot be metabolized by S. cerevisiae, to galactose and glucose. Subsequently, galactose, which cannot be metabolized by Lb. bulgaricus, is excreted and provides a carbon source for yeast. 2. In pure cultures, Lb. bulgaricus only grows at increased CO2 concentrations. In anaerobic mixed cultures, the yeast provides this CO2 via alcoholic fermentation. 3. Analysis of amino acid consumption from the defined medium indicated that S. cerevisiae supplied alanine to the bacteria. 4. A mild but significant low-iron response in the yeast transcriptome, identified by DNA microarray analysis, was consistent with the chelation of iron by the lactate produced by Lb. bulgaricus. 5. Transcriptome analysis of Lb. bulgaricus in mixed cultures showed an overrepresentation of transcripts involved in lipids metabolism suggesting either a competition of the two microorganisms for fatty acids, or a response to the ethanol produced by S. cerevisiae. To our knowledge, this is the first transcriptome study of a cross-kingdom binary mixed culture that analyses responses of both microorganisms. This study demonstrates that chemostat-based transcriptome analysis is a powerful tool to investigated microbial interaction in mixed populations.

Project description:Roseburia inulinivorans is a recently identified motile representative of the Firmicutes that contributes to butyrate formation from a variety of dietary polysaccharide substrates in the human large intestine. Microarray analysis was used here to investigate substrate-driven gene expression changes in R. inulinivorans A2-194. A cluster of fructo-oligosaccharide (FOS)/inulin utilisation genes induced during growth on inulin included one encoding a b-fructofuranosidase protein that was prominent in the proteome of inulin-grown cells. This cluster also included a 6-phosphofructokinase and an ABC transport system, while a distinct inulin-induced 1-phosphofructokinase was linked to a fructose-specific phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS II transport enzyme). Real-time PCR analysis demonstrated that the b-fructofuranosidase and adjacent ABC transport protein showed greatest induction during growth on inulin, whereas the 1-phosphofructokinase enzyme and linked PTS II transport system were most strongly up-regulated during growth on fructose, indicating that these two clusters play distinct roles in the utilization of inulin. The R. inulinivorans B-fructofuranosidase was over-expressed in E. coli and shown to hydrolyse fructans ranging from inulin down to sucrose, with greatest activity on fructo-oligosacharides. Genes induced on starch included the major extra-cellular a-amylase and two distinct a-glucanotransferases together with a gene encoding a flagellin protein. The latter response may be concerned with improving bacterial access to insoluble starch particles. RNA was purified from mid-exponential phase (OD650 = 0.4) cultures of R. inulinivorans grown on basal YCFA supplemented with a single substrate of either starch or inulin using the RNeasy RNA purification kit (Qiagen), and the mRNA component enriched using the MICROBExpress system (Ambion). The purified RNA (1 ug) was labelled by reverse transcription (Amersham), employing random nonamer extension incorporating either dCTP-Cy3 or dCTP-Cy5 dyes. In order to ensure reproducibility, and to obtain statistically significant results, the dye labelling was swapped for a second hybridisation. RNA purified from a separate biological replicate was labelled and hybridised twice in the same way.

			Action	DRS
	202400528_MSRI054_11.raw	Raw
	202400528_MSRI054_12.raw	Raw
	202400528_MSRI054_13.raw	Raw
	202400528_MSRI054_14.raw	Raw
	202400528_MSRI054_15.raw	Raw

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In vitro investigations on the impact of constituents on fermented dairy on fecal microbiota composition

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