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Single and mixed starter cultures of lactic acid bacteria (LAB): Weissella confusa MNC20, Lactobacillus plantarum MNC21, Lactococcus lactis MNC24 and Lactobacillus fermentum MNC34 and yeasts: Issatchenkia orientalis MNC20Y and Saccharomyces cerevisiae MNC21Y were used to produce Obushera, a fermented sorghum beverage. Microbial counts, pH, sugars, organic acids, and volatile compounds in starter culture and spontaneous fermentations were monitored during 48 hrs. Maximum counts of LAB (8.4–9.4 log cfu g−1) and yeasts (7.5 ± 0.1 cfu g−1) starter cultures were attained in 6–48 hrs. Weissella confusa, Lc. lactis, and Lb. fermentum showed possible acid sensitivity while I. orientalis produced surface films. LAB starter cultures and their combinations with S. cerevisiae lowered pH from 5.83 to

Single and mixed starter cultures of lactic acid bacteria (LAB): Weissella confusa MNC20, Lactobacillus plantarum MNC21, Lactococcus lactis MNC24 and Lactobacillus fermentum MNC34 and yeasts: Issatchenkia orientalis MNC20Y and Saccharomyces cerevisiae MNC21Y were used to produce Obushera, a fermented sorghum beverage. Microbial counts, pH, sugars, organic acids, and volatile compounds in starter culture and spontaneous fermentations were monitored during 48 hrs. Maximum counts of LAB (8.4–9.4 log cfu g−1) and yeasts (7.5 ± 0.1 cfu g−1) starter cultures were attained in 6–48 hrs. Weissella confusa, Lc. lactis, and Lb. fermentum showed possible acid sensitivity while I. orientalis produced surface films. LAB starter cultures and their combinations with S. cerevisiae lowered pH from 5.83 to

Malted and un-malted sorghum ( Sorghum bicolor (L.) Moench) flour was gamma irradiated with a dose of 10 kGy and then re-irradiated with 25 kGy. The effects of irradiation on microbial decontamination, amylase activity, fermentability (using an amylolytic L. plantarum MNC 21 strain), starch granule structure and viscosity were determined. Standard methods were used during determinations. The 10 kGy dose had no effect on microbial load of un-malted flour but reduced that of malted flour by 3 log cycles. Re-irradiation resulted in complete decontamination. Irradiation of malt caused a significant ( p

Starter cultures of lactic acid bacteria (Lactobacillus fermentum MINF99, Weissella confusa MINF8, Lactobacillus plantarum MINF277, Lactobacillus brevis MINF226, and Lactobacillus paracasei subsp paracasei MINF98) were used to ferment Bushera during fermentation (96 h). Organic acids and volatile compounds produced during starter and natural fermentation were investigated. Microbial counts, pH, and sugars were also determined. LAB counts increased from 5.87 ± 0.00 to 8.32 ± 0.02 log cfu mL−1 while yeasts increased from 4.39 ± 0.02 to 7.10 ± 0.04 log cfu mL−1 during natural fermentation. The pH decreased from 6.5 to 3.55–4.0. W. confusa MINF8 attained similar final pH (3.55) as naturally fermented Bushera. Lactate was the dominant acid and varied between 0.34% and 0.66%. W. confusa MINF8 produced the highest amounts of lactate (0.66%). Lactate content in naturally fermented Bushera was 0.89%, 96 h. Glucose and maltose decreased from 8.64–9.27 g kg−1 to 0.13–2.11 gkg−1 and 7.95–8.42 g kg−1 to 0.06–2.66 g kg...

A tomato flavor enhancer, 2-isobutylthiazole (IBT), was added (5 mg/kg) to dressings emulsified with either a whey protein concentrate-80 (WPC-80), a WPC-80 hydrolysate or β-lactoglobulin at high pressure (70 MPa) at either 20 or 75 °C. The short (2-4 min), high-temperature treatment left the proteins essentially unchanged. IBT addition gave a dominant, green tomato flavor that masked the intrinsic odor of the WPC-80 hydrolysate but enhanced bitter flavor. The sensory IBT odor intensity was determined by oil level (5-30%) and pH; pH 4.0 gave higher IBT odor than pH 6.5. The green (IBT) odor release correlated with the sensory viscosity (p = 0.001) and with instrumentally determined complex modulus (p = 0.001), but not to the dressings’ microstructure. The presence of small (<

The classical propionibacteria produce genetically unique antimicrobial peptides, whose biological activities are without equivalents, and to which there are no homologous sequences in public databases. In this review, we summarize the genetics, biochemistry, biosynthesis, and biological activities of three extensively studied antimicrobial peptides from propionibacteria. The propionicin T1 peptide constitutes a bona fide example of an unmodified general secretory pathway (sec)-dependent bacteriocin, which is bactericidal towards all tested species of propionibacteria except Propionibacterium freudenreichii. The PAMP antimicrobial peptide represents a novel concept within bacterial antagonism, where an inactive precursor protein is secreted in large amounts, and which activation appears to rely on subsequent processing by proteases in its resident milieu. Propionicin F is a negatively charged bacteriocin that displays an intraspecies bactericidal inhibition spectrum. The biosynthesis of propionicin F appears to proceed through a series of unusual events requiring both N- and C-terminal processing of a precursor protein, which probably requires the radical SAM superfamily enzyme PcfB.

A method for kappa-casein genotyping in bulls has been developed. By analysis of DNA polymorphisms we are able to discriminate between the kappa-casein variant A and B in the bulls. This method will be an efficient tool in selection for the most desirable kappa-casein variant.

The aim of this study was to perform a screening of various milk protein samples of both cow and goat origin to study their in vitro immunomodulating properties on human peripheral blood mononuclear cells (PBMC). The protein content in the milk of the two different species varies most notably in the amount of αs1-casein. A high degree of genetic polymorphism is related to the goat αs1-casein genes resulting in a variable amount of total protein in the goat milk. The milk proteins were hydrolysed using human gastric and duodenal juice or commercial pig derived enzymes to simulate in vivo digestion. Although different immunomodulating effects caused by various milk protein components have been observed, the mechanisms underlying these effects are not always known. In addition, most studies on the immunomodulating properties of milk protein digests have used a wide variety of commercial enzymes to simulate in vivo digestion. Exploring the difference in immunomodulating properties of milk protein-derived peptides produced by the aid of enzymes from human gastric secretions, compared to those produced by commercial enzymes, is a novel approach that may be of great importance. It could help to explore which peptides are actually produced during in vivo early digestion of milk and how they influence the immune system. Especially the whey protein concentrates from goat and cow showed a dose-dependent inhibition of human PBMC proliferation in vitro. This effect could neither be explained by a toxic effect on the PBMCs as shown by a standard viability test, nor by induction of apoptosis caused by the same milk protein samples. We suggest that intact or hydrolysed components in the milk protein samples affect the production of activation signals thereby inhibiting lymphocyte proliferation.

Protein degradation of caprine whey by human proteolytic enzymes was studied with regard to antibacterial effect on Listeria monocytogenes . The digestion was performed by a two-step degradation-assay, using human gastric juice (HGJ) at pH 2.5, and human duodenal juice (HDJ) at pH 8. Protein profiles were studied by SDS-PAGE after each step and compared with degradation performed by commercial enzymes. Both types of enzymes, both human and commercial, left most of β-LG intact. However, proteins like serum albumin, laktoferrin and immunoglobulins were rapidly degraded. Only minor parts of α-lactalbumin (α-LA) was degraded by human enzymes, while treatment with commercial enzymes gave full degradation of α-LA. The two types of enzymes resulted in different peptide profiles, where the commercial enzymes degraded whey into smaller peptides much more efficiently. The protein digests produced by HGJ and HDJ were screened for antibacterial effects against L. monocytogenes , a food born bacteria responsible for fatal and sometimes deadly infections. Cells of L. monocytogenes were strongly inhibited by caprine whey obtained after reaction with both HGJ and HDJ. Undigested caprine whey and the products from the first step of digestion with HGJ demonstrated no significant effect. This indicates that during digestion the antibacterial effect of caprine whey hydrolysates are most effective in the duodenum. This gives a promising opportunity to inhibit listeriosis in humans, and results are also useful for development of dietary supplement, nutraceuticals and functional foods.

Five strains of propionibacteria with 70-90% autolysis in sodium lactate broth (SLB) were studied by renaturing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Several lytic bands ranging in size between 25 and 143 kDa were detected by using propionibacteria cells or cell walls as substrate in the gel. Four Propionibacterium freudenreichii strains showed similar autolytic-enzyme profiles, consisting of two autolytic bands, one with molecular mass 162 kDa and one in the range 123-143 kDa. However, the Propionibacterium acidipropionici strain showed a completely different profile, consisting of 8 autolytic bands with molecular masses of 122, 97, 71, 55, 43, 39, 31, and 25 kDa. Lytic enzymes from P. freudenreichii INF-alpha, P. freudenreichii ISU P-59, P. freudenreichii ISU P-24, and P. freudenreichii ISU P-50 showed lytic activity against cells from all these four strains, but not against P. acidipropionici ATCC 4965. However, P. acidipropionici ATCC 4965 autolysed only its own cells. Effects of pH, temperature, and ions on autolytic activity were tested by renaturing SDS-PAGE and in buffer systems. Results from the SDS-PAGE electrophoresis showed optimal autolytic activity of P. acidipropionici ATCC 4965 at 37 degrees C and in the pH range 7 to 8.5 and of P. freudenreichii ISU P-59 at 20 degrees C and in the pH range 5 to 7. The autolytic activity of P. acidipropionici ATCC 4965 was extremely heat stable (100 degrees C, 2 h), in contrast to the lytic activity of P. freudenreichii ISU P-59, which was heat labile. The autolytic activities of P. acidipropionici ATCC 4965 were inhibited by divalent cations, however, the lytic activities of P. freudenreichii ISU P-59 were activated by Mn(2+), Ca(2+), and Co(2+). In buffer, optimum autolysis of P. acidipropionici ATCC 4965 was observed at pH 8.5 and at 40 degrees C. P. freudenreichii ISU P-59 showed optimum autolysis in buffer at pH 7.5 and at 30 degrees C.

Autolysis is self-degradation of the bacterial cell wall that results in the release of enzymes and DNA. Autolysis of starter bacteria, such as lactococci and propionibacteria, is essential for cheese ripening, but our understanding of this important process is limited. This is mainly because the current tools for measuring autolysis cannot readily be used for analysis of bacteria in mixed populations. We have now addressed this problem by species-specific detection and quantification of free DNA released during autolysis. This was done by use of 16S rRNA gene single-nucleotide extension probes in combination with competitive PCR. We analyzed pure and mixed populations of Lactococcus lactis subsp. lactis and three different species of Propionibacterium . Results showed that L. lactis subsp. lactis INF L2 autolyzed first, followed by Propionibacterium acidipropionici ATCC 4965, Propionibacterium freudenreichii ISU P59, and then Propionibacterium jensenii INF P303. We also investigated the autolytic effect of rennet (commonly used in cheese production). We found that the effect was highly strain specific, with all the strains responding differently. Finally, autolysis of L. lactis subsp. lactis INF L2 and P. freudenreichii ISU P59 was analyzed in a liquid cheese model. Autolysis was detected later in this cheese model system than in broth media. A challenge with DNA, however, is DNA degradation. We addressed this challenge by using a DNA degradation marker. We obtained a good correlation between the degradation of the marker and the target in a model experiment. We conclude that our DNA approach will be a valuable tool for use in future analyses and for understanding autolysis in mixed bacterial populations.

In vitro digestion was performed by human proteolytic enzymes on bovine and caprine individual milks. Two types of caprine milk were investigated: with high and low contents of α S1 -casein (CN). In addition the influence of heating of the milk on digestion was examined. The digestion was performed in two steps using human gastric and duodenal juice. Protein and peptide profiles were studied by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing (IEF). Caprine milk proteins were digested faster than bovine milk proteins. This was confirmed by the degradation profile obtained for both cows’ and goats’ milk, and was most evident for β -lactoglobulin. Comparing the digestion of milk protein from two groups of goats, high and low in α S1 -CN content, respectively, did not show significant differences. Heat treatment of milk had a strong and significant effect on the level of digestion. Raw milk was degraded faster than the heat-treated milk, and the effect of heating was different for bovine and caprine milk.

Dressings were produced according to a fractional factorial design with protein type, protein level, oil level, pH, addition of NaCl, CaCl2 and sucrose, and processing temperature as design variables. The dressings were produced by high-pressure, and were model-systems emulsified and stabilized with different whey protein types. The dressings were characterized by sensory analysis with regard to smell and taste, and texture properties. Analysis of variance revealed that oil level explained 56% of the variation in the sensory attribute viscosity. Protein type explained the largest part of the sensory attributes syneresis and smell. Addition of CaCl2 increased syneresis and gave a bitter taste The dressings' microstructures were captured by Scanning electron micrographs (SEM). The images were analysed using two different algorithms for feature extraction; the ABDF method, measuring absolute differences in greyness between pixels at fixed distances, and a local box-counting (fractal) method measuring the maximum differences in greyness within boxes of fixed sizes. After vectorization of the images by the algorithms, the vectors were analyzed to find which design variables influenced the vectors the most. The analysis of variance revealed that protein type was the design variable that could clarify the largest part of the variation that could be explained in the images, followed by addition of NaCl. The fat was not visible in the images, and was hardly recognized by the feature extraction algorithms. The correlation between viscosity and image analysis was fair, as the oil was not detected by the images or vectorized images. Sensory attributes explained by protein type or other design variables visible in the images were well explained by images.

Aims: We describe a novel DNA-micro-array-based method that targets 16S rDNA to quantify changes in both the total bacterial DNA and the species-specific DNA composition. Methods and Results: Quantifications were achieved by combining competitive PCR for quantifying total bacterial DNA with quantification of species-specific DNA composition based on signature 16S rDNA sequences. We constructed 11 different probes, which were evaluated on 21 different strains, in addition to complex samples. The signals obtained with sequence-specific labelling of the probes corresponded well with what should be expected based on 16S rDNA phylogenetic reconstruction. The quantification of species-specific DNA composition showed that the micro-array approach could be used to accurately determine differential growth of bacteria in mixed samples. We analysed samples containing mixtures of Lactococcus lactis and different species of propionibacteria during a 2-week incubation period. Lactococcus lactis grew fast, reaching a maximum after 12 h, Propionibacterium acidipropionici and Propionibacterium freudenreichii reached a maximum after 48 h, whereas Propionibacterium jensenii showed a slow increase during the whole growth period. The 16S rDNA total bacterial DNA quantification was compared with real-time PCR, absorbance measurements (ABS600) and colony forming units (CFU). Conclusion: The accuracy of the array approach was in the same range or better than the alternative techniques. The potential of the 16S rDNA micro-array method was further demonstrated using a liquid cheese model. Significance and Impact of the Study: This is to our knowledge the first time quantification of the total bacterial DNA and the species-specific DNA compositions of mixed populations have been achieved in the same assay.

The growth and sporulation of Bacillus cereus NVH 45 in a fermentor with controlled pH or simulated pH conditions were investigated. The study was carried out in a fermentor to measure the influence of a rapid and a slow lactic acid production on the inhibition of B. cereus in a controlled environment during the initial part of fermentation and to observe if other factors than lactic acid influenced the inhibition. In the controlled pH experiments the pH was allowed to decrease to an end pH 5.0, 5.5 or 6.0 either by Lactobacillus casei 2756 (a fast acid producer) or Lactobacillus acidophilus NCFB 1748 (a slow acid producer). In co-cultures of Lb. casei 2756 and B. cereus NVH 45, low numbers (10-70 cfu/ml) of B. cereus NVH 45 were observed at end pH 5.5 (72 h) while at pH 5.0 no viable cells ( 10(7) cfu/ml) than with Lb. casei 2756 (

During co-culture of Lactobacillus (five strains) or Lactococcus (two strains) with Bacillus cereus, organic acids and other potentially antimicrobial metabolites are produced. Lactic acid was produced at very different rates by the lactic acid bacteria (LAB) and the final concentrations varied much, however, the crucial point of rapid pH reduction during the initial hours of fermentation coincides with lactic acid production. Moderate amounts of acetic acid were produced during fermentation and the final concentrations were much smaller compared to lactic acid. According to these experiments, production of diacetyl, carbon dioxide and ethanol was considered too small to contribute to inhibition of B. cereus. The inhibitory substance produced by the LAB strains was not sensitive to proteinase K, trypsin or pepsin, so it was not likely that the LAB strains produced bacteriocins antagonistic against B. cereus. The strains that produced lactic acid fastest inhibited B. cereus best. Increased concentrations of lactic and acetic acid and carbon dioxide were also observed after co-culture with B. cereus compared to growth of the LAB strains alone, which indicates that B. cereus stimulates the biosynthetic capacities of the LAB strains.

Thirty-eight model dressings were produced according to a fractional factorial design. Dressings prepared with hydrolysed whey protein concentrate (DH 1 with Corolase PN-L from RohmGmbH) were compared to dressings produced with whey protein concentrate (WPC) and purified β-lactoglobulin (β-LG) as emulsifiers. The dressings were produced in a high-pressure homogeniser at ∼70 kPa pressure. The factors varied were protein%, oil%, pH, process temperature, and in addition NaCl, CaCl2 and sucrose. The dressings were evaluated with regard to textural and structural properties by emulsion stability (ES), rheological measurements, scanning electron microscopy (SEM) and image analysis. The dressings produced spanned from thin milk-like liquids to thick pastes. WPC formed mainly thin dressings, while dressings produced by hydrolysate and β-LG were mainly creamy. None of the dressings showed separation of the oil phase, however some syneresis of water was observed. Variation in protein and oil content affected the stability of the dressings. When the protein content was increased from 2 to 4% and the oil content from 5 to 30%, the hydrolysate and β-LG dressings became more stable, in contrast to WPC dressings, which became less stable. Addition of sucrose increased the stability and the phase angle of all protein types. High processing temperature (75 °C) affected the dressings differently. A positive effect was observed on the stability of WPC and β-LG dressings, while hydrolysate dressings showed reduced stability, except for the combination of low pH (4.0), high protein (4%) and fat content (30%). This dressing was highly stable at high processing temperature. SEM-images revealed that the fat globule size was smallest for WPC (0.1–0.9 μm) and increased in the order WPC

Recently, we demonstrated that mesophilic lactobacilli exhibiting glutamate dehydrogenase (GDH) activity were capable of degrading aromatic amino acids (ArAAs) and branched-chain amino acids (BcAAs) in vitro to precursors of aroma compounds and to aroma compounds, when they were combined with Lactococcus lactis. Indeed, they produced via their GDH activity the α-ketoglutarate required for the amino acid transamination. In the present study, we compared the ability of two Lactobacillus strains, with and without GDH activity, to produce aroma compounds from amino acids in a cheese model. The GDH-positive Lactobacillus INF15D produced mainly diacetyl and acetoin from catabolism of aspartate (Asp). However, there were no aroma compounds produced from BcAAs, even when Lactobacillus was combined with L. lactis. In fact, Lactobacillus INF15D exhibited 5–10-fold higher aminotransferase (AT) activity towards Asp than towards BcAAs. We concluded that due to competition of ATs for the α-ketoglutarate produced by GDH, the aroma compounds produced in cheese depends on the relative AT activities towards Asp, BcAAs and ArAAs of the GDH-positive strain.

In Gouda and Cheddar type cheeses the amino acid conversion to aroma compounds, which is a major process for aroma formation, is essentially due to lactic acid bacteria (LAB). In order to evaluate the respective role of starter and nonstarter LAB and their interactions in cheese flavor formation, we compared the catabolism of phenylalanine, leucine, and methionine by single strains and strain mixtures of Lactococcus lactis subsp. cremoris NCDO763 and three mesophilic lactobacilli. Amino acid catabolism was studied in vitro at pH 5.5, by using radiolabeled amino acids as tracers. In the presence of α-ketoglutarate, which is essential for amino acid transamination, the lactobacillus strains degraded less amino acids than L. lactis subsp. cremoris NCDO763 , and produced mainly nonaromatic metabolites. L. lactis subsp. cremoris NCDO763 produced mainly the carboxylic acids, which are important compounds for cheese aroma. However, in the reaction mixture containing glutamate, only two lactobacillus strains degraded amino acids significantly. This was due to their glutamate dehydrogenase (GDH) activity, which produced α-ketoglutarate from glutamate. The combination of each of the GDH-positive lactobacilli with L. lactis subsp. cremoris NCDO763 had a beneficial effect on the aroma formation. Lactobacilli initiated the conversion of amino acids by transforming them mainly to keto and hydroxy acids, which subsequently were converted to carboxylic acids by the Lactococcus strain. Therefore, we think that such cooperation between starter L. lactis and GDH-positive lactobacilli can stimulate flavor development in cheese.

One hundred and thirteen strains of lactic acid bacteria (LAB) were selected from 351 isolates from 15 samples of traditionally fermented household bushera from Uganda and also from laboratory-prepared bushera. Isolates were phenotypically characterised by their ability to ferment 49 carbohydrates using API 50 CHL kits and additional biochemical tests. Coliforms, yeasts and LAB were enumerated in bushera. The pH, volatile organic compounds and organic acids were also determined. The LAB counts in household bushera varied between 7.1 and 9.4 log cfu ml(-1). The coliform counts varied between < 1 and 5.2 log cfu ml(-1). The pH of bushera ranged from 3.7 to 4.5. Ethanol (max, 0.27%) was the major volatile organic compound while lactic acid (max, 0.52%) was identified as the dominant organic acid in household bushera. The initial numbers of LAB and coliforms in laboratory-fermented bushera were similar; however, the LAB numbers increased faster during the first 24 h. LAB counts increased from 5.5 to 9.0 log cfu ml(-1) during the laboratory fermentation. Coliform counts increased from 5.9 to 7.8 log cfu ml(-1) at 24 h, but after 48 h, counts were less 4 log cfu ml(-1). Yeasts increased from 4.3 to 7.7 log cfu ml(-1) at 48 h, but thereafter decreased slightly. The pH declined from 7.0 to around 4.0. Lactic acid and ethanol increased from zero to 0.75% and 0.20%, respectively. Lactic acid bacteria isolated from household bushera belonged to Lactobacillus, Streptococcus and Enterococcus genera. Tentatively, Lactobacillus isolates were identified as Lactobacillus plantarum, L. paracasei subsp. paracasei, L. fermentum, L. brevis and L. delbrueckii subsp. delbrueckii. Streptococcus thermophilus strains were also identified in household bushera. LAB isolated from bushera produced in the laboratory belonged to five genera (Lactococcus, Leuconostoc, Lactobacillus, Weissella and Enterococcus. Eight isolates were able to produce acid from starch and were identified as Lactococcus lactis subsp. lactis (four strains), Leuconostoc mesenteroides subsp. mesenteroides (one strain), Leuconostoc mesenteroides subsp. dextranicum (one strain), Weissella confusa (one strain) and L. plantarum (one strain).

Antimicrobial peptides are produced by all kinds of organisms, from bacteria to mammals. In higher organisms these compounds are produced as an innate host defense mechanism to protect against pathogenic attack, whereas microorganisms presumably use these compounds as weapons in the competition for limited resources. A large number of antimicrobial peptides have been isolated from amphibians (35), fish (2, 22), insects (32), mammals (13), plants (1), and different microorganisms (12). Antimicrobial proteins and peptides from bacteria include toxins like diphtheria and cholera toxins (24, 25), bacteriolytic enzymes like lysostaphin (30) and hemolysins (8), and bacteriocins and bacteriocin-like peptides (10, 12). Numerous bacteriocins have been characterized from gram-positive bacteria, and some of them show a relatively broad spectrum of inhibition. Antimicrobial peptides produced by food-grade organisms such as lactic acid bacteria and propionibacteria have received special interest due to their potential applications in food preservation (33). The classical propionibacteria have a long history of use in dairy fermentations, in particular the production of Swiss-type cheeses. A few antimicrobial peptides from these bacteria have been described so far (7, 9, 14, 15, 17, 21, 29), and only two bacteriocins have been characterized at the molecular level (7, 17). Bacteria use a number of different mechanisms to regulate and produce active peptides and proteins. Most conventional bacteriocins are produced as precursor peptides, which are modified posttranslationally inside the cell or at the cell exterior during export to generate their biologically active forms (12). However, it has been shown that antimicrobial peptides from both bacteria (27, 28) and higher organisms (23, 31) can be produced from the degradation of larger proteins. In this work we describe a novel antimicrobial peptide isolated from Propionibacterium jensenii LMG 3032. This compound is secreted from the cell as an inactive proprotein which is proteolytically activated by proteases in the environment. The mature peptide has several features in common with well-known antimicrobial peptides like class II bacteriocins and antimicrobial cationic peptides from higher organisms. This is to our knowledge the first bacteriocin-like peptide formed from an inactive extracellular protein by an external protease.

Dairy propionibacteria produce a number of inhibitory compounds like propionate, ace- tate and diacetyl. In addition, a number of strains produce bacteriocins. Unlike lactic acid bacteria, they often produce compounds that inhibit Gram (-) bacteria as well as yeasts and molds. These dairy propionibacteria are food-grade organisms and their bacteriocins may be of interest as food preserva- tives. Some of their bacteriocins have been characterized. Most are heat-stable peptides containing less than 100 amino acid residues. They are insensitive to large variations in pH, and lactic acid bacte- ria are often more sensitive to the bacteriocins than propionibacteria. However, at present only bacteriocins with inhibitory spectra restricted to propionibacteria have been fully sequenced. Most of the bacteriocins are produced in very low amounts when the cells are grown in liquid culture, which limits their usefulness in practical applications. propionibacteria / bacteriocin / biopreservative / food safety / food spoilage microorganism Resume — Bacteriocines des bacteries propioniques. Les bacteries propioniques laitieres produi- sent plusieurs composes inhibiteurs tels que propionate, acetate, diacetyle. De plus, certaines souches produisent des bacteriocines. Contrairement aux bacteries lactiques, elles produisent souvent des composes qui inhibent les bacteries Gram(-) aussi bien que les levures et moisissures. Ces bacteries propioniques laitieres sont des organismes « alimentaires » et leurs bacteriocines peuvent presenter un interet comme conservateur alimentaire. Certaines de leurs bacteriocines ont ete caracterisees. La plupart sont des peptides thermostables contenant moins de 100 acides amines. Elles sont resistantes a de larges variations de pH, et les bacteries lactiques sont souvent plus sensibles aux bacteriocines

Eleven strains of Bacillus cereus isolated from milk and meat products have been used to study growth and sporulation profiles in detail. Polymerase chain reaction (PCR) using primers detecting cold shock protein A gene signatures (cspA), showed that none of the strains were the newly suggested species in the B. cereus group, B. weihenstephanensis, comprising psychrotolerant cereus strains, although one of the strains grew at 4 degrees C, two at 6 degrees C and seven grew at 7 degrees C. One of the two strains that grew at 6 degrees C had a maximum growth temperature of 42 degrees C, while the remaining 10 strains all grew at temperature of 43 degrees C or higher. Only three strains grew at 48 degrees C. At 42 degrees C, the generation time varied between 11 and 34 min. Spore germination was much faster for the two strains that grew at 6 degrees C than for the other nine strains in milk at 7 degrees C and 10 degrees C. All strains were cytotoxic and contained the non-haemolytic enterotoxin gene (nhe), 10 strains contained the enterotoxin T gene (bceT), and only six had the gene (hbl) encoding haemolytic enterotoxin. Two strains showed some microheterogeneity in the nhe operon. but contained all three genes. We can conclude that true B. cereus strains can have growth profiles as expected for B. weihenstephanensis, and that nhe and bceT were not correlated with growth profiles. However, the two psychrotolerant strains with minimal growth temperature of 4 degrees C and 6 degrees C did not contain hbl, as judged from our PCR results.

Amino acid metabolism by one strain each of Lactobacillus casei and Lactobacillus paracasei subsp. paracasei in resting cell suspensions was studied. The experiment was performed under cheese like conditions in terms of pH, salt concentration, temperature and carbohydrate starvation using a mixture of l-amino acids as substrate. The effect of supplementing the amino acids mixture with α-ketoglutarate was estimated. Asparagine, serine and glutamine were utilised in the suspension with only amino acids. In the suspension with both amino acids and α-ketoglutarate, the degradation of leucine and lysine was observed. Production of metabolites that could be important for cheese flavour such as carbon dioxide, ammonia, organic acids and volatiles was measured. The suspensions with amino acids were characterised by high production of acetoin and ammonia and both increased even more when α-ketoglutarate was added. Carbon dioxide was produced in high amounts in the suspension with both amino acids and α-ketoglutaric acid.

A collection of propionibacteria was screened for bacteriocin production. A new bacteriocin named propionicin T1 was isolated from two strains of Propionibacterium thoenii . This bacteriocin shows no sequence similarity to other bacteriocins. Propionicin T1 was active against all strains of Propionibacterium acidipropionici , Propionibacterium thoenii , and Propionibacterium jensenii tested and also against Lactobacillus sake NCDO 2714 but showed no activity against Propionibacterium freudenreichii . The bacteriocin was purified, and the N-terminal part of the peptide was determined with amino acid sequencing. The corresponding gene pctA was sequenced, and this revealed that propionicin T1 is produced as a prebacteriocin of 96 amino acids with a typical sec leader, which is processed to give a mature bacteriocin of 65 amino acids. An open reading frame encoding a protein of 424 amino acids was found 68 nucleotides downstream the stop codon of pctA . The N-terminal part of this putative protein shows strong similarity with the ATP-binding cassette of prokaryotic and eukaryotic ABC transporters, and this protein may be involved in self-protection against propionicin T1. Propionicin T1 is the first bacteriocin from propionibacteria that has been isolated and further characterized at the molecular level.

Milk samples of 59 cows of the Norwegian Red Cattle breed receiving three different supplementary concentrates, were analysed for genotypes of caseins and whey proteins, the content of different milk salts (Ca 2+ , Ca, Mg and citrate), the content of total protein, casein and whey protein and the mean micellar size of native and heated casein micelles. The genotype of α s1 -casein had a statistically significant effect on the content of protein and casein, and the content of whey protein and the casein number were significantly influenced by different feeding regimes, and the content of citrate. The mean size of native and heated casein micelles was significantly influenced by the feeding regimes, genotype of α s1 -casein (native mean size only) and κ -casein, pH and the content of casein, whey protein and casein number. The heat-induced changes in mean micellar size were significantly affected by the calcium ion activity which accounted for approximately 40% of the total variation.

Limited enzymatic hydrolysis trials of WPC-80 and β -lactoglobulin AA, AB and BB were performed using specific enzymes (trypsin, Neutrase, Corolase PP, Corolase PS) in a pH-stat under controlled conditions. The hydrolysates of WPC-80 and β -lactoglobulin were fractionated into high and low molecular fractions. Residual antibody binding activity of the peptides was dependent on the degree of hydrolysis (DH 2-20), but also on the enzyme used. Heat treatment affected the solubility and thereby the antigenic response. Dialysis influenced the antibody binding activity of the peptides. Tryptic hydrolysis of β -lactoglobulin AA was slower than for β -lactoglobulin BB and AB. Antigenic responses of the hydrolysates and fractions were measured by SLOT-BLOT and ELISA. SLOT-BLOT, a rapid screening method, was not able to differentiate the hydrolysates. The ELISA, being a more sensitive method, differentiated between the genetic variants, but was more time consuming. The lowest antigenicity was observed in the 1000–5000 Da fraction and β -lactoglobulin AA showed the lowest response.

Lactococcal strains were screened for bacteriolytic activity against Micrococcus luteus cells, lactococcal cells, and cell walls. Thirty strains were screened for bacteriolytic activity against cells and cell walls incorporated into agar medium. Enzymes from all strains hydrolyzed the substrates; however, the activity against Micrococcus cells was much higher than against Lactococcus cells or cell walls. Electrophoretic profiles of bacteriolytic activities of culture supernatants, sodium dodecyl sulfate-treated cell extracts, cell wall fractions, and cell extracts were analyzed in sodium dodecyl sulfate-polyacrylamide gels containing M. luteus cells or lactococcal cell walls as the substrate. The 22 strains tested contained two to five lytic bands in the culture supernatant, ranging in size between 32 and 53 kDa. The cell extracts, the sodium dodecyl sulfate-treated cell extracts, and the cell wall fractions revealed two lytic bands of 47 and 53 kDa. Effects of external factors on autolysis of some strains were determined in buffer systems. Optimal autolysis was observed in the exponential growth phase at pH 6.0 to 7.5 and at a temperature of 30(deg)C. Two of three strains tested seemed to contain a glycosidase, and all three strains contained an N-acetylmuramyl-l-alanine amidase or an endopeptidase.

Cet article fait l'etat des connaissances actuelles sur le systeme proteolytique des bacteries propioniques, et sur sa capacite a degrader des acides amines. Propionibacterium contient au moins deux proteinases, l'une associee a la paroi cellulaire et l'autre cytoplasmique ou membranaire. Une vaste variete de peptidases ont ete decrites et caracterisees, telles que aminopeptidases, proline iminopeptidase, proline imidopeptidase, X-prolyl-dipeptidyl-aminopeptidase, endopeptidases et carboxypeptidase. Une grande variete d'acides amines, en particulier l'acide aspartique, l'alanine, la serine et la glycine, pouvaient etre facilement degrades par Propionibacterium, mais de grandes variations dues a la souche et a l'espece etudiee ont ete observees.

Amylolytic lactic acid bacteria (ALAB) can potentially replace malt in reducing the viscosity of starchy porridges. However, the drawback of using ALAB is their low and delayed amylolytic activity. This necessitates searching for efficient ALAB and strategies to improve their amylolytic activity. Two ALAB, Lactobacillus plantarum MNC 21 and Lactococcus lactis MNC 24, isolated from Obushera , were used to ferment starches in MRS broth: sorghum, millet, sweet potato, and commercial soluble starch. The amylolytic activity of MNC 21 was comparable to that of the ALAB collection strain Lb. plantarum A6, while that of MNC 24 was extremely low. MNC 21, MNC 24, and their coculture were compared to A6 and sorghum malt for ability to ferment and reduce the viscosity of sorghum porridge (11.6% dry matter). ALAB and the coculture lowered the pH from 6.2 to P < 0.05) amounts of maltose and diacetyl than the monocultures. Sorghum malt control and the coculture hydrolyzed more starch in sorghum porridge than the monocultures. The coculture initiated changes in the rheological parameters storage modulus ( G ′), loss modulus ( G ″), phase angle (δ), and complex viscosity (η*) earlier than its constituent monocultures. The shear viscosity of sorghum porridge was reduced significantly ( P < 0.05) from 1950 cP to 110 cP (malt), 281 cP (coculture), 382 cP (MNC 21), 713 cP (MNC 24), and 722 cP (A6). Coculturing strong ALAB with weak ALAB or non-ALAB can be exploited for preparation of nutrient-dense weaning foods and increasing lactic acid yield from starchy materials.

Obushera includes four fermented cereal beverages from Uganda namely: Obutoko, Enturire, Ekitiribita and Obuteire, whose microbial diversity has not hitherto been fully investigated. Knowledge of the microbial diversity and dynamics in these products is crucial for understanding their safety and development of appropriate starter cultures for controlled industrial processing. Culture-dependent and culture-independent techniques including denaturating gradient gel electrophoresis (DGGE) and mixed DNA sequencing of polymerase chain reaction (PCR) amplified ribosomal RNA genes were used to study the bacteria and yeast diversity of Obushera. The pH dropped from 6.0-4.6 to 3.5-4.0 within 1-2 days for Obutoko, Enturire and Obuteire whereas that of Ekitiribita decreased to 4.4 after 4 days. Counts of lactic acid bacteria (LAB) increased from 5.0 to 11.0 log cfug(-1) and yeasts increased from 3.4 to 7.1 log cfug(-1) while coliform counts decreased from 2.0 to1 log cfug(-1) during four days of fermentation. LAB and yeast isolates were identified by rRNA gene sequence analysis. LAB isolates included: Enterococcus spp., Lactobacillus (Lb.) plantarum, Lb. fermentum, Lb. delbrueckii, Lactococcus lactis, Leuconostoc lactis, Streptococcus (S.) infantarius subsp. infantarius, Pediococcus pentosaceus and Weisella (W.) confusa. DGGE indicated predominance of S. gallolyticus, S. infantarius subsp. infantarius, Lb. fermentum, Lb. delbrueckii, W. confusa, Lb. reuteri, Fructobacillus spp., L. lactis and L. lactis. Yeast isolates included Clavispora lusitaniae, Cyberlindnera fabianii, Issatchenkia orientalis and Saccharomyces cerevisiae. DGGE indicated predominance of S. cerevisiae in Obutoko, Enturire and Obuteire and also detected Pichia spp. and I. orientalis in Obutoko. Obushera produced in the laboratory was initially dominated by Enterobacteriaceae and later by Lactococcus spp. Enterobacteriaceae and Bacillus spp. were also detected in Ekitiribita. Development of starters for Obushera may require combinations of LAB and S. cerevisiae for Obutoko, Enturire and Obuteire and LAB for Ekitiribita.

Peptides in caprine whey were identified afterin vitrodigestion with human gastrointestinal enzymes in order to determine their antibacterial effect. The digestion was performed in two continuing steps using human gastric juice (pH 2·5) and human duodenal juice (pH 8) at 37°C. After digestion the hydrolysate was fractionated and 106 peptides were identified. From these results, twenty-two peptides, located in the protein molecules, were synthesised and antibacterial activity examined. Strong activity of the hydrolysates was detected againstEscherichia coliK12,Bacillus cereusRT INF01 andListeria monocytogenes, less activity againstStaphylococcus aureusATCC 25 923 and no effect onLactobacillus rhamnosusGG. The pure peptides showed less antibacterial effect than the hydrolysates. When comparing the peptide sequences from human gastrointestinal enzymes with previously identified peptides from non-human enzymes, only two peptides, β-lactoglobulin f(92–100) and β-casein f(191–205) matched. No peptides corresponded to the antibacterial caprine lactoferricin f(14–42) or lactoferrampin C f(268–284). Human gastrointestinal enzymes seem to be more complex and have different cleavage points in their protein chains compared with purified non-human enzymes. Multiple sequence alignment of nineteen peptides showed proline-rich sequences, neighbouring leucines, resulting in a consensus sequence LTPVPELK. In such a way proline and leucine may restrict further proteolytic processing. The present study showed that human gastrointestinal enzymes generated different peptides from caprine whey compared with non-human enzymes and a stronger antibacterial effect of the hydrolysates than the pure peptides was shown. Antimicrobial activity against pathogens but not against probiotics indicate a possible host-protective activity of whey.

Caprine αs1-casein polymorphism is one of the key factors which determines important technological properties of milk, such as rennetability and cheese yield. The indigenous Norwegian goat breed is characterized by a remarkably high frequency (>70%) of a “null” variant of αs1-casein unique to this population. This mutation, referred to as the exon 12 D allele, involves a single base deletion in exon 12 of the αs1-casein gene. The aim of this study was to provide more detailed information on how αs1-casein polymorphism affects composition and rennet coagulation properties of milk from Norwegian dairy goats. Class of αs1-casein significantly affected milk composition; crude protein, casein and pH. The mean casein micelle size was larger in milks containing αs1-casein “null” variant when compared to “strong” milks; in contrast, the heat-induced changes in micelle size appeared to be least pronounced in this group. Compositional differences between “strong” and “null” milks may explain these differences. αs1-Casein class had a significant effect on gel strength recorded 30 min after the addition of rennet, with the “strong” milks giving the greatest firmness. However, a considerable fraction of the milk samples in this study were unable to form a strong clot, regardless of the αs1-casein variant. These results indicate that environmental and compositional factors not considered in this study may be important in the curd formation process.

Apo and holo forms of lactoferrin (LF) from caprine and bovine species have been characterized and compared with regard to the structural stability determined by thermal denaturation temperature values (T (m)), at pH 2.0-8.0. The bovine lactoferrin (bLF) showed highest thermal stability with a T (m) of 90 ± 1°C at pH 7.0 whereas caprine lactoferrin (cLF) showed a lower T (m) value 68 ± 1°C. The holo form was much more stable than the apo form for the bLF as compared to cLF. When pH was gradually reduced to 3.0, the T (m) values of both holo bLF and holo cLF were reduced showing T (m) values of 49 ± 1 and 40 ± 1°C, respectively. Both apo and holo forms of cLF and bLF were found to be most stable at pH 7.0. A significant loss in the iron content of both holo and apo forms of the cLF and bLF was observed when pH was decreased from 7.0 to 2.0. At the same time a gradual unfolding of the apo and holo forms of both cLF and bLF was shown by maximum exposure of hydrophobic regions at pH 3.0. This was supported with a loss in α-helix structure together with an increase in the content of unordered (aperiodic) structure, while β structure seemed unchanged at all pH values. Since LF is used today as fortifier in many products, like infant formulas and exerts many biological functions in human, the structural changes, iron binding and release affected by pH and thermal denaturation temperature are important factors to be clarified for more than the bovine species.

The purpose of this study was to describe the sensory characteristics of traditionally produced Obushera and their influence on consumer acceptability using focus group discussions, a descriptive panel and a consumer acceptability panel. Four types of Obushera of commercial importance including: Obutoko and Enturire (sorghum based) plus Obuteire and Ekitiribita (millet-based) were identified. Descriptive profiles of the four types showed that they are sensorially distinct products. Preliminary studies indicated that their acceptability was significantly (P

SummaryLactose-negative mutants ofLactococcus lactissubsp.lactisandLacto-coccus lactissubsp.cremoriswith good autolytic properties were used at two different levels in addition to the normal starter in Gouda-type cheesemaking experiments. Increased numbers of bacteria were observed in fresh cheese, and the pH changes during ripening were as normal. A more rapid development of soluble N compounds was observed in cheeses with mutant addition, especially withLc. lactissubsp.lactisIMN-L2–3 andLc.lactissubsp.cremorisIMN-C12–1, compared with the control cheese. Cheese with addedLc. lactissubsp.lactisIMN-L2–3 andLc.lactissubsp.cremorisIMN-C12–1 showed the most extensive ripening and the highest product quality of the mutants tested. The experimental cheeses were graded higher than the control cheese. The ripening time was significantly reduced, and the quality was retained at an acceptable level during storage.

A β-galactosidase reporter system for the analysis of promoter elements in Propionibacterium freudenreichii was designed. The pTD210 in vivo reporter vector was constructed using a promoterless lacZ gene from Bifidobacterium longum cloned into the pAMT1 plasmid. The utility of the pTD210 reporter vector was demonstrated by an investigation of six predicted promoters in P. freudenreichii . The system produced accurate and reproducible measurements that facilitated both promoter identification and the quantification of promoter activities.

A method for β-lactoglobulin (β-lg) genotyping has been developed. By analysis of DNA polymorphisms (restriction fragment length polymorphism, RFLP) we are able to discriminate between the variant A and B in cattle. This method should be an efficient tool for use in breeding to increase the frequency of the most desirable β-lg variant.

The in vitro digestion of caprine whey proteins was investigated by a two-step degradation assay, using human gastric juice (HGJ) at pH 2.5 and human duodenal juice (HDJ) at pH 7.5. Different protein and peptide profiles were observed after the first (HGJ) and second (HDJ) enzymatic degradation. The minor whey proteins serum albumin, lactoferrin and Ig were rapidly degraded by HGJ, while alpha-lactalbumin (alpha-LA) and beta-lactoglobulin (beta-LG) were more resistant and survived both 30 and 45 min of the enzymatic treatment. Further digestion with HDJ still showed intact beta-LG, and the main part of alpha-LA also remained unchanged. The protein degradation by HGJ and HDJ was also compared with treatment by commercial enzymes, by using pepsin at pH 2.5, and a mixture of trypsin and chymotrypsin at pH 7.5. The two methods resulted in different caprine protein and peptide profiles. The digests after treatment with HGJ and HDJ were screened for antibacterial effects on some selected microorganisms, Escherichia coli, Bacillus cereus, Lactobacillus rhamnosus GG and Streptococcus mutans. Active growing cells of E. coli were inhibited by the digestion products from caprine whey obtained after treatment with HGJ and HDJ. Cells of B. cereus were inhibited only by whey proteins obtained after reaction with HGJ, while the products after further degradation with HDJ demonstrated no significant effect. Screenings performed on cells of Lb. rhamnosus GG and S. mutans all showed no signs of inhibition.

The growth and sporulation of Bacillus cereus NVH 45 in a fermentor with controlled pH or simulated pH conditions were investigated. The study was carried out in a fermentor to measure the influence of a rapid and a slow lactic acid production on the inhibition of B. cereus in a controlled environment during the initial part of fermentation and to observe if other factors than lactic acid influenced the inhibition. In the controlled pH experiments the pH was allowed to decrease to an end pH 5.0, 5.5 or 6.0 either by Lactobacillus casei 2756 (a fast acid producer) or Lactobacillus acidophilus NCFB 1748 (a slow acid producer). In co-cultures of Lb. casei 2756 and B. cereus NVH 45, low numbers (10-70 cfu/ml) of B. cereus NVH 45 were observed at end pH 5.5 (72 h) while at pH 5.0 no viable cells (10 cfu/ml) were detected (48-72 h). B. cereus NVH 45 did not sporulate in co-culture with Lb. casei 2756. In co-culture with Lb. acidophilus NCFB 1748, B. cereus NVH 45 sporulated and survived as spores. In these co-cultures B. cereus NVH 45 grew to higher maximum counts (10(7) cfu/ml) than with Lb. casei 2756 (10(7) cfu/ml). Significantly different amounts of lactic acid were observed between the two co-cultures after 7 and 12 h. A rapid decrease of pH appears to prevent B. cereus from sporulating and it seems that it is enough to just reach pH 5.0 rapidly and keep that pH to achieve the desirable inhibition of B. cereus. In the simulated pH experiments B. cereus NVH 45 was inoculated in the fermentor and the different pH developments from different LAB strains were monitored by addition of lactic acid. These experiments showed the same tendency: a fast pH reduction during the initial hours of fermentation, simulating lactococci, resulted in complete inhibition of B. cereus NVH 45 (10 cfu/ml). However, when simulating the pH development of the two different Lactobacillus strains, complete inhibition of B. cereus NVH 45 was not seen. In co-cultures competition for nutrients with consequences for cell density appears to be important. Based on these results it seems that B. cereus must reach a certain density to induce sporulation.

The purpose of this study was to investigate the frequency of production of the bacteriocin propionicin T1 and the protease-activated antimicrobial peptide (PAMP) and their corresponding genes in 64 isolates of classical propionibacteria. This study revealed that these genes are widespread in Propionibacterium jensenii and Propionibacterium thoenii but absent from the remaining species of classical propionibacteria that were studied. The pro-PAMP-encoding gene ( pamA ) was found in 63% of the P. jensenii strains and 61% of the P. thoenii strains, and all of these strains displayed PAMP activity. The propionicin T1-encoding gene ( pctA ) was present in 89% of the P. thoenii strains and 54% of the P. jensenii strains. All P. thoenii strains containing the pctA gene exhibited antimicrobial activity corresponding to propionicin T1 activity, whereas only 38% of the pctA -containing P. jensenii strains displayed this activity. Sequencing of the pctA genes revealed the existence of two allelic variants that differed in a single nucleotide in six strains of P. jensenii ; in these strains the glycine at position 55 of propionicin T1 was replaced by an aspartate residue (A variant). No strains harboring the A variant showed any antimicrobial activity against propionicin T1-sensitive bacteria. An open reading frame ( orf2 ) located immediately downstream from the pctA gene was absent in three strains containing the G variant of propionicin T1. Two of these strains showed low antimicrobial activity, while the third strain showed no antimicrobial activity at all. The protein encoded by orf2 showed strong homology to ABC transporters, and it has been proposed previously that this protein is involved in the producer immunity against propionicin T1. The limited antimicrobial activity exhibited by the strains lacking orf2 further suggests that this putative ABC transporter plays an important role in propionicin T1 activity.

The growth and death or survival of Bacillus cereus in sterile skimmed milk fermented with 18 different lactic acid bacteria (LAB) were investigated. B. cereus alone in milk reached about 10(7)-10(8) colony-forming units (cfu)/ml. When B. cereus was cultivated together with different Lactobacillus or Lactococcus cultures at 30 or 37 degrees C, the B. cereus counts after 72 h of fermentation ranged between < 10 cfu/ml and about 10(6) cfu/ml. The inhibition patterns for the different Lactobacillus and Lactococcus cultures varied. All the Lactococcus cultures (with one exception) reduced pH to 5.3 or lower in 7 h. After 24 h, B. cereus was not detected in any of the fast Lactococcus-fermented milk samples. After 48 h, B. cereus was not detected for 4 of the 12 Lactobacillus cultures. These cultures reduced pH to below 5.0 in 24 h. The other Lactobacillus cultures also inhibited B. cereus, but the counts of B. cereus were still 10(4)-10(6) cfu/ml after 72 h. They also reduced pH at a slower rate. Survival of B. cereus was to a variable extent linked with formation of endospores. Proteinase K did not affect the antimicrobial activity observed. Acid production with decreasing pH, particularly the initial rate of pH decrease, appears to be most important for control of B. cereus with LAB.

A survey was conducted using a questionnaire to document the production methods of Bushera, a Ugandan traditional fermented cereal beverage, in the districts of Kabale and Rukungiri in the South Western region of Uganda. The chemical composition of raw materials and Bushera was determined using standard methods. Similarities in the production of Bushera in Kabale and Rukungiri districts were observed. In both districts, sorghum grains are usually (80% of respondents) soaked in water overnight (12 h), some households (20%) indicated a soaking period of 24-48 h. Eighty seven percent of the households soaked the grains in streams, rivers and ponds. The germination period for sorghum grains varied between two and four days. Sixty five percent of the households germinated the grains for two-three days. The duration of fermentation of Bushera ranged from one to six days. Most of the households (90%) consumed Bushera after two-four days of fermentation. The moisture, fat, protein and carbohydrate contents of germinated and non-germinated sorghum grains ranged from 8.8-12.4 %, 1.8-3.0 %, 7.2-10.8 % and 77.7-85.7%, respectively. Germinated sorghum flour had lower fat, protein and carbohydrate contents but higher ash and fibre than non-germinated sorghum flour. Germinated millet flour had higher moisture, protein and fibre compared to the non-germinated flour while the latter had higher ash and carbohydrate contents. Germination resulted in an increase in the concentration of sugars in both sorghum and millet grains. Great variations were observed in the proximate composition of Bushera obtained from the households. Under laboratory conditions, the protein content of Bushera produced from germinated grains was higher than Bushera from non-germinated grains (12.2% vs. 10.6%), on dry matter basis. Higher levels of iron, magnesium and zinc were observed in germinated grains due to addition of wood ash during germination. Germinated grains had lower phenol and tannin content compared to non-germinated grains. Resume Une etude basee sur les reponses a un questionnaire a ete menee pour documenter les methodes de production du Bushera, une boisson de cereales fermentees traditionnelle en Ouganda, dans les districts de Kabale et Rukungiri situes dans la region sud-ouest de l'Ouganda. La composition chimique des matieres premieres et du Bushera a ete etablie par methodes standard. On a observe des similarites dans la production du Bushera dans les districts de Kabale et de Rukungiri. Dans les deux districts, les graines de sorgho sont habituellement (80% des reponses) trempees dans de l'eau pendant une nuit (12h), certains menages (20%) signalant une periode de trempage de 24-48h.Les graines sont trempees dans des ruisseaux, rivieres et etangs par 87% des menages. La periode de germination des graines de sorgho varie de 2 a 4 jours. Soixante-cinq pour cent des menages font germer les graines pendant 2-3 jours. La duree de fermentation du Bushera va de 1 a 6 jours. La majorite des menages (90%) consomment le Bushera apres 2-4 jours de fermentation. Le contenu des graines de sorgho germees et non germees en humidite, graisses, proteines et feculents etait de 8,8-12,4%; 1,8-3.0%; 7,7-10,8%; et 77,7-80,2% respectivement. La farine de sorgho germe a une moindre teneur en graisses, proteines et feculents mais une plus forte teneur en cendres et fibres que la farine de sorgho non germe. La farine de millet germe contient plus d'humidite, proteines et fibres que la farine non germee, tandis que cette derniere a une plus forte teneur en cendres et feculents. La germination entraine une augmentation de la concentration des sucres dans les graines de sorgho et de millet. On a constate d'importantes variations dans la composition approximative du Bushera obtenu aupres des menages. En laboratoire, le contenu en proteines du Bushera fait de graines germees etait plus eleve que dans le Bushera de graines non germees (12,2% compare a 10,6%), sur base de matieres seches. Des niveaux plus eleves de fer, magnesium et zinc ont ete observes dans les graines germees en raison de l'apport de cendres de bois pendant la germination. Les graines germees avaient une plus basse teneur en phenol et en tanin compare aux graines non germees. (Af. J. Food Agriculture, Nutrition and Development: 2003 3(1): 10-19)

Samples of borde from open markets at five localities in southern Ethiopia showed average aerobic mesophilic count (AMC), lactic acid bacteria (LAB) and yeast counts of 9.9, 10.1, and 8.1 log CFU g-1 respectively. Enterobacteriaceae (EB) were Keywords: traditional technology; cereal fermentation; LAB; beverage; borde; Ethiopia J Food Tech in Africa (2002) 7, 85-92

A study of village-level processing techniques and raw materials used for the production of borde was carried out using open-ended questionnaires and on the spot interviews with producers at six localities in southern Ethiopia. The major focus of the study was on indigenous processing methods, types and proportions of ingredients, sources of energy, shelf life, sensory properties and the importance of borde for household food security. From results of the study, borde was characterized as an opaque, effervescent, whitish-grey to brown coloured beverage with a thick consistency and sweet-sour taste. It may be prepared from grits/flour of unmalted maize, barley, wheat, sorghum and/or finger millet and their malts using locally available earthenware and metal equipment. The type of unmalted cereal ingredients and amount of malt used for borde preparation varied within and between localities and were selected according to availability, price and preferences. A flow chart of borde production was constructed showing four major processing stages. The short shelf life of borde and the seasonal variations in production volume were identified as major problems for the vendors in the study areas. Keywords: indigenous methods; cereal fermentation; borde; beverage; Ethiopia J Food Tech in Africa (2002) 7, 59-64