Bioactive components of fermented soya beans effective against diarrhoea-associated bacteria

Tempe is a fermented food, obtained by fungal fermentation of soya beans. During the processing specific flavour, texture and nutritional properties are achieved. Previous research has indicated that tempe reduced the incidence and severity of diarrhoea. In this thesis the bioactive effects of tempe on diarrhoea-associated bacteria are described. Tempe appeared to be antibacterial against Bacillus cereus cells and spores, but not against enterotoxigenic Escherichia coli (ETEC). Nevertheless, tempe was found to inhibit the adhesion of ETEC to intestinal epithelial cells. Experiments showed that the inhibition of adhesion was caused by an interaction between ETEC and tempe extracts. A range of ETEC strains was shown to be sensitive for the anti-adhesive component, making the bioactivity of broader interest for applications in feed matrices. Furthermore, several substrates (legumes and cereals) were fermented and all fermented legumes tested were equally bioactive as the fermented soya beans, whereas the fermented cereals were not active. The use of different starter cultures showed that Bacillus spp., moulds and some yeasts were capable to release or form the bioactive component during fermentation, thus the bioactivity was not specific for one microbial species. After heating, defatting and protease treatment of the bioactive tempe extracts, they remained bioactive. On the contrary, after treatment with polysaccharide degrading enzyme mixtures the bioactivity was lost. This suggests that the bioactive component contains carbohydrates, and explains the interaction between ETEC and tempe extracts, which could indeed be established by carbohydrates of the tempe extract. Ultra-filtration revealed the bioactive component to have molecular masses >30 kDa. Further purification yielded an active fraction with an increased carbohydrate content. Monosaccharide analysis showed the importance of arabinose in the bioactive components. In conclusion, the bioactive component is released or formed during fermentation by enzymatic degradation of legumes. The bioactive component is of carbohydrate nature and contains arabinose, which originates from arabinan or arabinogalactan chains of the pectic cell wall polysaccharides of legumes. <br/

Soya bean tempe extracts show antibacterial activity against Bacillus cereus cells and spores

Aims: Tempe, a Rhizopus ssp.-fermented soya bean food product, was investigated for bacteriostatic and/or bactericidal effects against cells and spores of the food-borne pathogen Bacillus cereus. Methods and results: Tempe extract showed a high antibacterial activity against B. cereus ATCC 14579 based on optical density and viable count measurements. This growth inhibition was manifested by a 4 log CFU ml-1 reduction, within the first 15 min of exposure. Tempe extracts also rapidly inactivated B. cereus spores upon germination. Viability and membrane permeability assessments using fluorescence probes showed rapid inactivation and permeabilization of the cytoplasmic membrane confirming the bactericidal mode of action. Cooked beans and Rhizopus grown on different media did not show antibacterial activity, indicating the unique association of the antibacterial activity with tempe. Subsequent characterization of the antibacterial activity revealed that heat treatment and protease addition nullified the bactericidal effect, indicating the proteinaceous nature of the bioactive compound. Conclusions: During fermentation of soya beans with Rhizopus, compounds are released with extensive antibacterial activity against B. cereus cells and spores. Significance and Impact of Study: The results show the potential of producing natural antibacterial compounds that could be used as ingredients in food preservation and pathogen contro

Bioactivity of tempe by inhibiting adhesion of ETEC to intestinal cells, as influenced by fermentation substrates and starter pure cultures

Soya bean tempe is known for its bioactivity in reducing the severity of diarrhoea in piglets. This bioactivity is caused by an inhibition of the adhesion of enterotoxigenic Escherichia coli (ETEC) to intestinal cells. In this paper, we assessed the bioactive effect of soya tempe on a range of ETEC target strains, as well as the effect of a range of cereal and leguminous substrates and starter pure cultures. Soya bean tempe extracts strongly inhibited the adhesion of ETEC strains tested. All tempe made from other leguminous seeds were as bioactive as soya bean tempe, whereas tempe made from cereals showed no bioactivity. Using soya beans as substrate, fermentation with several fungi (Mucor, Rhizopus spp. and yeasts) as well as Bacillus spp. resulted in bioactive tempe, whereas fermentation with lactobacilli showed no bioactivity. The active component is releasedor formed during the fermentation and is not present in microbial biomass and only partly in unfermented substrates. The bioactivity being not specific for a single ETEC strain, makes the bioactive tempe relevant for applications in animal husbandry

First characterization of bioactive components in soybean tempe that protect human and animal intestinal cells against enterotoxigenic Escherichia coli (ETEC) infection

Tempe extracts can inhibit the adhesion of enterotoxigenic Escherichia coli (ETEC) to intestinal cells and thereby can play a role in controlling ETEC-induced diarrhea. The component responsible for this adhesion inhibition activity is still unknown. This research describes the purification and partial characterization of this bioactive component of tempe. After heating, defatting, and protease treatment, the extracts were found to remain active. However, after treatment with polysaccharide-degrading enzyme mixtures the bioactivity was lost. Ultrafiltration revealed the active component to be >30 kDa. Further purification of the bioactive tempe extracts yielded an active fraction with an increased carbohydrate content of higher arabinose content than the nonactive fractions. In conclusion, the bioactive component contains arabinose and originates from the arabinan or arabinogalactan side chain of the pectic cell wall polysaccharides of the soybeans, which is probably released or formed during fermentation by enzymatic modification

Validation of growth as measurand for bacterial adhesion to food and feed ingredients

Aims: A miniaturized adhesion test was designed to study the binding capacity of food and feed ingredients for bacterial cells. Methods and Results: Bacteria were allowed to adhere to different fibrous materials supplied as well coatings in microtitration plates. The amount of bacteria retained on the materials was determined in an automated way as growth after addition of liquid medium. The test principle was based on an inverse relationship between initial cell densities and the appearance of growth: The higher adhering cell numbers are, the shorter are the detection times of growth. The growth curves obtained were fitted by nonlinear regression analysis employing a sigmoidal curve model. Growth parameters as (i) the time after incubation at which half of the maximum growth yield was reached; (ii) the time-coordinate of the point of inflection; (iii) the detection time calculated as x-axis intercept of the maximum specific growth rate in the point of inflection; and (iv) the time-coordinate of a growth detection threshold at OD = 0·05 were highly separating for the binding capacity of different food and feed ingredients for bacteria. Significance and Impact of the Study: With growth as measurand for adhesion, a simple, high-throughput method was developed for the screening of huge numbers of different binding matrices and bacteria

Bioactivity of tempe by inhibiting adhesion of ETEC to intestinal cells, as influenced by fermentation substrates and starter pure cultures

Soya bean tempe is known for its bioactivity in reducing the severity of diarrhoea in piglets. This bioactivity is caused by an inhibition of the adhesion of enterotoxigenic Escherichia coli (ETEC) to intestinal cells. In this paper, we assessed the bioactive effect of soya tempe on a range of ETEC target strains, as well as the effect of a range of cereal and leguminous substrates and starter pure cultures. Soya bean tempe extracts strongly inhibited the adhesion of ETEC strains tested. All tempe made from other leguminous seeds were as bioactive as soya bean tempe, whereas tempe made from cereals showed no bioactivity. Using soya beans as substrate, fermentation with several fungi (Mucor, Rhizopus spp. and yeasts) as well as Bacillus spp. resulted in bioactive tempe, whereas fermentation with lactobacilli showed no bioactivity. The active component is releasedor formed during the fermentation and is not present in microbial biomass and only partly in unfermented substrates. The bioactivity being not specific for a single ETEC strain, makes the bioactive tempe relevant for applications in animal husbandry

Effects of heat on physicochemical properties of whey protein-stabilised emulsions

The effect of heating has been studied for whey protein-stabilised oil-in-water emulsions (25.0% (w/w) soybean oil, 3.0% (w/w) whey protein isolate, pH 7.0). These emulsions were heated between 55 and 95 degreesC as a function of time and the effect on particle size distribution, adsorbed protein amount, protein conformation and rheological properties was determined. Heating the emulsions as a function of temperature for 25 min resulted in an increase of the mean diameter (d(32)) and shear viscosity with a maximum at 75 degreesC. Heating of the emulsions at different temperatures as a function of time in all cases resulted in a curve with a maximum for d(32). A maximum increase of d(32) was observed after about 45 min at 75 degreesC and after 6-8 min at 90 degreesC. Similar trends were observed with viscosity measurements. Confocal scanning laser micrographs showed that after 8 min of heating at 90 degreesC large, loose aggregates of oil droplets were formed, while after 20 min of heating compact aggregates of two or three emulsion droplets remained. An increase of the adsorbed amount of protein was found with increasing heating temperature. Plateau values were reached after 10 min of heating at 75 degreesC and after 5 min of heating at 90 degreesC. Based on these results we concluded that in the whole process of aggregation of whey protein-stabilised emulsions an essential role is played by the non-adsorbed protein fraction, that the kinetics of the aggregation of whey protein-stabilised emulsions follow similar trends as those for heated whey protein solutions and that upon prolonged heating rearrangements take place leading to deaggregation of initially formed large, loose aggregates of emulsion droplets into smaller, more compact ones. (C) 2003 Elsevier B.V. All rights reserved

Fermented soya bean (tempe) extracts reduce adhesion of enterotoxigenic Escherichia coli to intestinal epithelial cells

Aims: This study aimed to investigate the effect of processed soya bean, during the successive stages of tempe fermentation and different fermentation times, on adhesion of enterotoxigenic Escherichia coli (ETEC) K88 to intestinal brush border cells as well as Caco-2 intestinal epithelial cells; and to clarify the mechanism of action. Methods and Results: Tempe was prepared at controlled laboratory scale using Rhizopus microsporus var. microsporus as the inoculum. Extracts of raw, soaked and cooked soya beans reduced ETEC adhesion to brush border cells by 40%. Tempe extracts reduced adhesion by 80% or more. ETEC adhesion to Caco-2 cells reduced by 50% in the presence of tempe extracts. ETEC K88 bacteria were found to interact with soya bean extracts, and this may contribute to the observed decrease of ETEC adhesion to intestinal epithelial cells. Conclusions: Fermented soya beans (tempe) reduce the adhesion of ETEC to intestinal epithelial cells of pig and human origin. This reduced adhesion is caused by an interaction between ETEC K88 bacteria and soya bean compounds. Significance and Impact of the Study: The results strengthen previous observations on the anti-diarrhoeal effect of tempe. This effect indicates that soya-derived compounds may reduce adhesion of ETEC to intestinal cells in pigs as well as in humans and prevent against diarrhoeal diseases

Fermented soya bean (tempe) extracts reduce adhesion of enterotoxigenic Escherichia coli to intestinal epithelial cells

Aims: This study aimed to investigate the effect of processed soya bean, during the successive stages of tempe fermentation and different fermentation times, on adhesion of enterotoxigenic Escherichia coli (ETEC) K88 to intestinal brush border cells as well as Caco-2 intestinal epithelial cells; and to clarify the mechanism of action. Methods and Results: Tempe was prepared at controlled laboratory scale using Rhizopus microsporus var. microsporus as the inoculum. Extracts of raw, soaked and cooked soya beans reduced ETEC adhesion to brush border cells by 40%. Tempe extracts reduced adhesion by 80% or more. ETEC adhesion to Caco-2 cells reduced by 50% in the presence of tempe extracts. ETEC K88 bacteria were found to interact with soya bean extracts, and this may contribute to the observed decrease of ETEC adhesion to intestinal epithelial cells. Conclusions: Fermented soya beans (tempe) reduce the adhesion of ETEC to intestinal epithelial cells of pig and human origin. This reduced adhesion is caused by an interaction between ETEC K88 bacteria and soya bean compounds. Significance and Impact of the Study: The results strengthen previous observations on the anti-diarrhoeal effect of tempe. This effect indicates that soya-derived compounds may reduce adhesion of ETEC to intestinal cells in pigs as well as in humans and prevent against diarrhoeal diseases