Production of organic flavor compounds by dominant lactic acid bacteria and yeasts from Obushera, a traditional sorghum malt fermented beverage.

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 <4.5 (3.50-4.13) in a shorter time (12 hrs) than spontaneous fermentations (24 hrs). Lactococcus lactis and W. confusa metabolized glucose the fastest (p < .05) during the first 6 hrs. Lactobacillus fermentum, Lb. plantarum, and S. cerevisiae utilized glucose and maltose concurrently. Lactobacillus plantarum and S. cerevisiae additionally utilized fructose. S. cerevisiae metabolized sugars the fastest (p < .05) during the first 12-24 hrs. Lactobacillus plantarum and W. confusa produced the highest (p < .05) amounts of lactate (5.43 g kg-1) and diacetyl (9.5 mg kg-1), respectively. LAB also produced acetate, ethanol, acetaldehyde, acetone, and acetoin. Coculturing LAB with S. cerevisiae reduced (p < .05) lactate and diacetyl yield. Yeasts produced high amounts of acetaldehyde and methyl alcohols. Issatchenkia orientalis produced higher (p < .05) amounts of 2-methy-1-propanol and 3-methyl-1-butanol than S. cerevisiae. Combinations of LAB with S. cerevisiae produced a profile flavor compounds close to that of spontaneously fermented Obushera. These combinations can be adopted for controlled fermentation of Obushera and related fermented cereal products.

The dominant microbial community associated with fermentation of Obushera (sorghum and millet beverages) determined by culture-dependent and culture-independent methods.

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 to <1 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.

Influence of cofermentation by amylolytic Lactobacillus plantarum and Lactococcus lactis strains on the fermentation process and rheology of sorghum porridge.

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 <4.5 within 12 h, while malt as a carrier of wild starter took about 20 h. Coculturing increased lactic acid yield by 46% and 76.8% compared to the yields of MNC 21 and MNC 24 monocultures, respectively. The coculture accumulated significantly larger (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.

Antibacterial peptides derived from caprine whey proteins, by digestion with human gastrointestinal juice.

Peptides in caprine whey were identified after in vitro digestion 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 against Escherichia coli K12, Bacillus cereus RT INF01 and Listeria monocytogenes, less activity against Staphylococcus aureus ATCC 25 923 and no effect on Lactobacillus rhamnosus GG. 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, ß-lactoglobulin f(92-100) and ß-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.

The unconventional antimicrobial peptides of the classical propionibacteria.

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.

Structural characteristic, pH and thermal stabilities of apo and holo forms of caprine and bovine lactoferrins.

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 ß 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.

Construction of a reporter vector system for in vivo analysis of promoter activity in Propionibacterium freudenreichii.

A beta-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.

Propionibacterium jensenii produces the polyene pigment granadaene and has hemolytic properties similar to those of Streptococcus agalactiae.

The red polyene pigment granadaene was purified and identified from Propionibacterium jensenii. Granadaene has previously been identified only in Streptococcus agalactiae, where the pigment correlates with the hemolytic activity of the bacterium. A connection between hemolytic activity and the production of the red pigment has also been observed in P. jensenii, as nonpigmented strains are nonhemolytic. The pigment and hemolytic activity from S. agalactiae can be extracted from the bacterium with a starch extraction solution, and this solution also extracts the pigment and hemolytic activity from P. jensenii. A partial purification of the hemolytic activity was achieved, but the requirement for starch to preserve its activity made the purification unsuccessful. Partially purified hemolytic fractions were pigmented, and the color intensity of the fractions coincided with the hemolytic titer. The pigment was produced in a soluble form when associated with starch, and the UV-visual spectrum of the extract gave absorption peaks of 463 nm, 492 nm, and 524 nm. The pigment could also be extracted from the cells by a low-salt buffer, but it was then aggregated. The purification of the pigment from P. jensenii was performed, and mass spectrometry and nuclear magnetic resonance analysis revealed that P. jensenii indeed produces granadaene as seen in S. agalactiae.

Autolysis of propionibacteria: detection of autolytic enzymes by renaturing SDS-PAGE and additional buffer studies.

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.

Use of DNA quantification to measure growth and autolysis of Lactococcus and Propionibacterium spp. in mixed populations.

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 studies of the digestion of caprine whey proteins by human gastric and duodenal juice and the effects on selected microorganisms.

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.

Influence of controlled lactic fermentation on growth and sporulation of Bacillus cereus in milk.

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.

Production of antimicrobial metabolites by strains of Lactobacillus or Lactococcus co-cultured with Bacillus cereus in milk.

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.

Prevalence of the genes encoding propionicin T1 and protease-activated antimicrobial peptide and their expression in classical propionibacteria.

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.

Inhibition of Bacillus cereus by strains of Lactobacillus and Lactococcus in milk.

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.

Cooperation between Lactococcus lactis and nonstarter lactobacilli in the formation of cheese aroma from amino acids.

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 alpha-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 alpha-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.

An antimicrobial peptide is produced by extracellular processing of a protein from Propionibacterium jensenii.

A protease-activated antimicrobial peptide (PAMP) and its inactive precursor were purified from the culture supernatant of Propionibacterium jensenii LMG 3032 and characterized at the molecular level. PAMP is a 64-amino-acid cationic peptide of 6,383 Da with physicochemical features similar to those of bacteriocins from gram-positive bacteria. This peptide displayed bactericidal activity against several propionibacteria and lactobacilli. DNA sequencing indicated that the PAMP-encoding gene (pamA) is translated as a proprotein of 198 amino acids with an N-terminal signal peptide of 27 amino acids and that PAMP constitutes the C-terminal part of this precursor. The amino acid sequence of pro-PAMP showed no similarity to those of other known proteins. By using activity tests and mass spectrometry, we showed that PAMP was formed upon protease treatment of the precursor protein. The propionibacteria produced the PAMP precursor constitutively during growth up to a level of approximately 4 mg/liter, but the producing bacteria were unable to activate the precursor. The requirement for an external protease represents a novel strategy for generating antimicrobial peptides.