Proteolytic activities in togwa, a Tanzanian fermented food.

Proteolytic activities were investigated in sorghum-based togwa prepared by natural fermentation and using starter cultures previously isolated from the native product, i.e., Lactobacillus brevis, Lactobacillus cellobiosus, Lactobacillus fermentum, Lactobacillus plantarum, Pediococcus pentosaceus, and Issatchenkia orientalis in coculture with either L. brevis or L. plantarum. Both proteinase and aminopeptidase activities were substantially higher in naturally fermented togwa than in those with starters (14-30%, 12-70%, respectively). A variable but substantial part of the proteinase activity followed the particulate fraction of togwa; aminopeptidase activity was mainly in that fraction. The breakdown of relatively high molecular mass protein (64 kDa) in togwa was detected by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE); the products were mainly in the 14-30 kDa range. Reversed-phase fast-protein liquid chromatography (RP-FPLC)-protein/peptide patterns changed during fermentation with some variation between togwa of different cultures. Supplementation of gruel with malt increased the concentration of total protein [from 9.5% to 11.0% (w/w) on dry weight basis)] and of most of the free amino acids. Fermentation had no effect on total protein content; however, the concentration of most of the amino acids was reduced, except for the proline content that increased. Natural fermentation also increased the concentration of glutamic acid and ornithine. Fermentation by P. pentosaceus increased aspartic acid, while L. cellobiosus, L. fermentum, and L. brevis in coculture with I. orientalis increased the concentration of citrulline.

Use of starter cultures of lactic acid bacteria and yeasts in the preparation of togwa, a Tanzanian fermented food.

Starter cultures of lactic acid bacteria (Lactobacillus brevis, Lactobacillus cellobiosus, Lactobacillus fermentum, Lactobacillus plantarum and Pediococcus pentosaceus) and yeasts (Candida pelliculosa, Candida tropicalis, Issatchenkia orientalis and Saccharomyes cerevisiae) isolated from native togwa were tested singly or in combination for their ability to ferment maize-sorghum gruel to produce togwa. All species of bacteria showed an ability to ferment the gruel as judged by lowering the pH from 5.87 to 3.24-3.49 and increasing the titratable acidity from 0.08% to 0.30-0.44% (w/w, lactic acid) in 24 h. Yeasts used singly showed little activity within 12 h, but lowered the pH to 3.57-4.81 and increased the acidity to 0.11-0.21% in 24 h. Yeasts in co-culture with lactic acid bacteria (LAB) had a modest effect on the final acidity (P<0.05). The number of lactic acid bacteria and yeasts increased while the Enterobacteriaceae decreased with fermentation time. The pH was lowered and lactic acid produced significantly (P<0.05) fastest in natural togwa fermentation and in samples fermented by L. plantarum or L. plantarum in co-culture with I. orientalis. The content of fermentable sugars was reduced during fermentation. Most volatile flavour compounds were produced in samples from fermentation by P. pentosaceus and I. orientalis in co-culture with either L. plantarum or L. brevis.

Microbiological and fermentation characteristics of togwa, a Tanzanian fermented food.

Selected microbiological and metabolic characteristics of sorghum, maize, millet and maize-sorghum togwa were investigated during natural fermentation for 24 h. The process was predominated by lactic acid bacteria (LAB) and yeasts. The mesophiles, lactic acid bacteria, and yeasts increased and the Enterobacteriaceae decreased to undetectable levels within 24 h. The isolated microorganisms were tentatively identified as Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus fermentum, Lactobacillus cellobiosus, Pediococcus pentosaceus, Weissella confusa, Issatchenkia orientalis, Saccharomyces cerevisiae, Candida pelliculosa and Candida tropicalis. The pH decreased from 5.24-5.52 to 3.10-3.34. Maltose increased initially and then decreased, fructose decreased and glucose levels increased during the first 12 h of fermentation. The organic acids detected during fermentation included DL-lactic, succinic, formic, pyruvic, citric, pyroglutamic and uric acid. Lactate was the predominant acid and increased significantly with time. The volatile organic compounds (VOC) detected included acetaldehyde, 2-methyl-propanal, 2-methyl-butanal, 3-methyl-butanal, ethanol, 2-methyl-1-propanol, 2-methyl-1-butanol, 3-methyl-1-butanol, diacetyl and acetoin. Ethanol was the predominant VOC and it increased significantly with time.

Growth and toxin profiles of Bacillus cereus isolated from different food sources.

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.

Effect of protective solutes on leakage from and survival of immobilized lactobacillus subjected to drying, storage and rehydration

When lactic acid bacteria are used industrially as fermentation starters it is important to obtain stable and highly viable bacterial cultures. Six strains of Lactobacillus encapsulated in Ca-alginate gel beads were investigated to determine whether dehydration, storage and rehydration may inflict injury. A negative relationship between leakage of lactate dehydrogenase and survival rates was found. Mesophilic lactobacilli showed only negligible leakage compared with thermophilic strains when dehydrated at 30 degrees C to a level of 0.11 g H20 (g dry wt)-1. The choice of an appropriate suspending medium to be introduced before drying was therefore very important for thermophilic lactobacilli in order to increase the survival rates during dehydration, storage and rehydration. The osmoregulatory solutes tested were adonitol, betaine, glycerol and reconstituted non-fat milk solids (NFMS). Less injury was inflected during dehydration for Lactobacillus helveticus with adonitol, glycerol and NFMS. Survival rates for the strains subjected to immobilization, dehydration, storage and rehydration varied with the strain and the protective solute when fluidized-bed drying was used at 5 degrees C to a level as high as 0.34 g H20 (g dry wt)-1. Non-fat milk solids gave the best protection for thermophilic lactobacilli, while adonitol and NFMS were best for mesophilic lactobacilli.

Survival of Lactobacillus helveticus entrapped in Ca-alginate in relation to water content, storage and rehydration.

Lactobacillus helveticus CNRZ 303 entrapped in Ca-alginate gel beads was investigated for improved survival and stability during fluidized-bed drying, storage and rehydration. Addition of protective solutes was very important. Studies of the conditions showed that inactivation of entrapped L. helveticus started when the water content exceeded 0.3-0.4 g H2O (g dry wt)-1 for adonitol, glycerol and reconstituted non fat milk solids (NFSM). With Ringer's solution (control) and betaine, the fall in viability was evident above 1 g H2O (g dry wt)-1. Drying down to 0.2 g H2O (g dry wt)-1 required the removal of 98.5-98.9% of the water. The best survival rate with the least injured cells among survivors was experienced with adonitol and NFMS, respectively, 71% and 57% (compared to the initial) immediately after dehydration. Adonitol and NFMS were also best for survival during storage. The highest cell recovery was obtained by rehydrating the cells in cheese whey permeate between 20-30 degrees C done at pH 6.0-7.0, satisfying the demands for cell survival, repair and slow swelling (adaptions).

Characterization of an intracellular oligopeptidase from Lactobacillus paracasei.

An intracellular oligopeptidase from Lactobacillus paracasei Lc-01 has been purified to homogeneity by Fast Flow Q Sepharose, hydroxyapatite, and Mono Q chromatography. The molecular mass of the enzyme was determined to be 140 kDa by gel filtration and approximately 30 kDa by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and SDS-capillary electrophoresis. The pI of the enzyme was at pH 4.5. The enzyme expressed maximum activity at pH 8.0 and 40 degrees C. Oligopeptidase activity on bradykinin was inhibited strongly by 1,10-phenantroline and EDTA and partly by p-chloromercuribenzoic acid but not by phosphoramidon or phenylmethylsulfonyl fluoride. Marked inhibition by beta-casein fragment 58 to 72 was demonstrated. The enzyme showed neither general aminopeptidase nor caseinolytic activity, and it degraded only oligopeptides between 8 and 13 amino acids. The enzyme readily hydrolyzed the Phe-Ser and Pro-Phe bonds of bradykinin; the Phe-His bond of angiotensin I; the Pro-Gln, Gln-Phe, and Phe-Gly bonds of substance P; and the Pro-Tyr bond of neurotensin. Weak activity toward the Ala-Tyr and Pro-Ser bonds of alpha(s1)-casein fragment 157 to 164, was observed. The N-terminal amino acid sequence of the oligopeptidase showed a high degree of homology to the lactacin B inducer from Lactobacillus acidophilus.

Peptides inhibitory to endopeptidase and aminopeptidase from Lactococcus lactis ssp. lactis MG1363, released from bovine beta-casein by chymosin, trypsin or chymotrypsin.

Peptides inhibitory to the 70-kDa endopeptidase (PepO) from the cytoplasm of Lactococcus lactis ssp. lactis MG1363 were isolated from the supernatant (pH 4.6) of chymosin, tryptic and alpha-chymotryptic hydrolysates of beta-casein (beta-CN) by reversed-phase HPLC and identified by sequencing and mass spectrometry. Chymosin released beta-CN f193-209, kinetic constant (Ki) of which for inhibition of PepO was 60 microM. This peptide also inhibited (Ki = 1700 microM) the 95-kDa aminopeptidase (PepN) from L. lactis ssp. lactis MG 1363. Trypsin released two PepO-inhibitory peptides: one, beta-CN f69-97, was not degradable by PepO (Ki = 4.7 microM), while the other, beta-CN f141-163, was degradable by PepO but competitively inhibited hydrolysis of methionine enkephalin by PepO. A peptide, beta-CN f69-84, which inhibited PepO with a Ki of 8.1 microM, was isolated from the alpha-chymotryptic hydrolysate. Peptides released from beta-CN by trypsin or chymotrypsin had very little inhibitory activity against PepN. PepO degraded beta-CN f193-209 very slowly compared with the hydrolysis of methionine enkephalin. All four inhibitory peptides (beta-CN f193-209, f69-97, f69-84, f141-163) were readily degraded by thermolysin.

Purification and characterization of a cell wall peptidase from Lactococcus lactis subsp. cremoris IMN-C12.

A peptidase from the cell wall fraction of Lactococcus lactis subsp. cremoris IMN-C12 has been purified to homogeneity by hydrophobic interaction chromatography, two steps of anion-exchange chromatography, and gel filtration. The molecular mass of the purified enzyme was estimated to be 72 kDa by gel filtration and 23 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme has a pI of 4.0, and it has the following N-terminal sequence from the 2nd to the 17th amino acid residues: -Arg-Leu-Arg-Arg-Leu-?-Val-Pro-Gly-Glu-Ileu-Val-Glu-Glu-Leu-Leu. The peptidase is most active at pH 5.8 and at 33 degrees C with trileucine as the substrate. Reducing agents such as dithiothreitol, beta-mercaptoethanol, and cysteine strongly stimulated enzyme activity, while p-chloromercuribenzoate had an inhibitory effect. Also, metal chelators lowered the peptidase activity, which could not be restored with Ca2+ and Mg2+. The divalent cations Cu2+, Zn2+, Fe2+, and Hg2+ completely inhibited peptidase activity. The peptidase is capable of hydrolyzing tripeptides and some dipeptides, with a preference for peptides containing leucine and with the highest activity towards the tripeptides Leu-Leu-Leu, Leu-Trp-Leu, and Ala-Leu-Leu, which were hydrolyzed with Kms of 0.37, 0.18, and 0.61 mM, respectively.

Quantitative studies of heat-stable proteinase from Pseudomonas fluorescens P1 by the enzyme-linked immunosorbent assay.

Pseudomonas fluorescens P1 is a psychrotrophic bacterium isolated from milk. Proteinase P1, the main extracellular heat-stable proteinase fraction of P. fluorescens P1, has been purified to homogeneity. A procedure with a sandwich enzyme-linked immunosorbent assay, using microplates and alkaline phosphatase conjugate was shown to detect 0.25 ng of proteinase P1 in 1 ml of reconstituted skim milk or defatted cream. The method offers the combination of sensitivity and specificity for the detection of these enzymes in milk and dairy products. In reconstituted skim milk cultures proteinase P1 was detectable when the CFU approached 10(7)/ml. Cultures in milk diluted 1:10, 1:30, or 1:100 with water showed detectable proteinase at population densities close to 10(6) CFU/ml. Aeration stimulated proteinase production; thus, a skim milk culture with shaking at 5 degrees C had a proteinase level of 36,000 ng/ml after 7 days as compared to 80 ng/ml in a stationary culture. The rate of inactivation of proteinase P1 at 150 and 55 degrees C as expressed by residual antigenic activity determined by the enzyme-linked immunosorbent assay was somewhat different from the rate determined on the basis of residual proteolytic activity. The specificity of the enzyme-linked immunosorbent assay with proteinase P1 antibodies was identical for proteinase P1 and for enzymes from six other strains of P. fluorescens, one Chromobacterium strain, and one Flavobacterium strain. Some psychrotrophic strains produced immunologically unrelated proteinase(s). These preliminary observations indicate that proteinase P1-related enzymes are common among psychrotrophs appearing as spoilage bacteria in milk.

Cell-bound lipase and esterase of Brevibacterium linens.

The activities of glycerol ester hydrolase, lipase (EC 3.1.1.3) and carboxylesterase, and esterase (EC 3.1.1.1) were determined for whole cell preparations of Brevibacterium linens by using the pH-stat assay. The culture growth liquors were inactive against the three substrates, tributyrin emulsion, triacetin, and methyl butyrate. Cells washed in water had less activity than cells washed in 5% NaCl; the ratio of activities was close to 1:2 for all strains using tributyrin emulsion as the substrate. For the esterase substrates, this relationship varied widely and was strain dependent. The ability to hydrolyze the two esterase substrates varied independently of the level of lipase activity.

Fractionation of dipeptidase activities of Streptococcus lactis and dipeptidase specificity of some lactic acid bacteria.

Proteins in sonic extracts of Streptococcus lactis were separated by starch-gel electrophoresis at high voltage. Each slab was sliced longitudinally, and half was stained for peptidases in a mixture containing a peptide, L-amino acid oxidase (snake venom), peroxidase, and o-dianisdine; the other half was stained in amido black for protein. In addition to sonic treatment, trypsin also released enzyme from acetone-treated cells. Glycyl-L-phenylalanine, L-phenylalanyl-glycine, L-alanyl-L-phenylalanine, and L-phenylalanyl-L-alanine served as substrates in characterizing the enzymes. Five different fractions of various specificities appeared in the gels. Broad-range substrate specificities were found for sonic extracts of S. lactis, S. cremoris, S. durans, and Lactobacillus acidophilus.

Glycerol Ester Hydrolase Activity of Microbacterium thermosphactum.

Microbacterium thermosphactum possesses a significant glycerol ester hydrolase (lipase, EC 3.1.1.3) activity and a weak but definite carboxylic ester hydrolase (esterase, EC 3.1.1.1) activity. Harvested whole cell preparations contained 53 units of lipase activity with tripropionin as the substrate. This activity decreased with an increasing chain length of fatty acid in the triglyceride to 13 units with trilaurin as the substrate and no activity with tripalmitin. Maximum lipase activity was found at a temperature of 35 to 37 C and at a pH of 7.1 to 7.3. Lipase activity was associated with three different protein peaks when the protein of cell-free extract was fractionated by polyacrylamide gel electrophoresis.