Profiling of dynamic changes in the microbial community during the soy sauce fermentation process

Soy sauce is a traditional condiment manufactured by natural inoculation and mixed culture fermentation. As is well known, it is the microbial community that plays an important role in the formation of its flavors. However, to date, its dynamic changes during the long period of fermentation process are still unclear, intensively constraining the improvement and control of the soy sauce quality. In this work, we revealed the dynamic changes of the microbial community by combining a cultured dependent method and a cultured independent method of polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis. Results indicated that the two methods verified and complemented each other in profiling microbial community, and that significant dynamics of the microbial community existed during the fermentation process, especially the strong inhibition of the growth of most of the microbes when entering into the mash stage from the koji stage. In the analysis of bacterial community, Staphylococcus and Bacillus were found to be the dominant bacteria and detected in the whole fermentation process. Kurthia and Klebsiella began to appear in the koji stage and then fade away in the early stage of the mash fermentation. In the analysis of fungal community, Aspergillus sojae and Zygosaccharomyces rouxii were found to be the dominant fungi in the koji and mash fermentation, respectively. It was clearly shown that when A. sojae decreased and disappeared in the middle stage of the mash fermentation, Z. rouxii appeared and increased at the meantime. Aspergillus parasiticus, Trichosporon ovoides and Trichosporon asahii also appeared in the koji and the early period of the mash fermentation and disappeared thereafter. Similar to Z. rouxii, Millerozyma farinosa and Peronospora farinosa were also found spontaneously which appeared in the mid-late period of the mash fermentation. The principal component analysis suggested that the microbial community underwent significant changes in the early period of the fermentation and, thereafter, tended to the stabilization in the mid-late periods. This study gave us important clues to understand the fermentation process and can serve as a foundation for improving the quality of soy sauce in the future.     

Isolation and Analysis of Molds from Soy Sauce Koji in Thailand

Five different isolates of Aspergillus and one of Mucor were compared with a Japanese commercial strain of Aspergillus oryzae for proteolytic activity on wheat bran substrate. One isolate of Aspergillus with superior protease production, identified as Aspergillus flavus var. columnaris, showed no detectable aflatoxin production on glutinous rice or soybean substrate. Preliminary tests using this fungus as a koji mold in a traditionally operated factory resulted in a soy sauce superior in quality to that usually produced.     

Studies on Osmophillic Yeasts

Influences of concentrations of sodium chloride and pH values of soy mash on the changes of microflora during the ripening process were investigated. The proportions of various yeasts in the soy mash were proved to be changed with the above two environmental factors. And it was shown that growth sequence of yeasts in soy mash was controlled according to their pH sensitivities at the hypertonic condition. From young soy mash (3 days’ old), the following yeasts were isolated: salt-tolerant type.…Saccharomyces rouxii Boutrpux, Torulopsis famata (Harrison) Lodder et Kregervan Rij, Candida polymorpha Ohara et Nonomura, Pichia farinosa (Lindner) Hansen, and Trichosporon behrendii Lodder et Kreger-van Rij ; salt-sensitive type.…Candida tropicalis (de Beurm., Gougerot et Vaucher) Ota and Candida rugosa (Anderson) Diddens et Lodder.     

Hydrolysis of acid-treated protein by a preparation of proteases

Because of less glutaminase activity, soy sauce made with a preparation of proteases from yellow-green Aspergilli contains less glutamic acid than soy sauce made by the traditional shoyu koji method. Thus, an acid treatment was developed to increase this amino acid in enzyme-made shoyu. Amide bonds of glutamine and asparagine in protein molecules were hydrolyzed at 100°C for 30 min with 1.3 N HCl (acid treatment). Using this method, glutamic acid per total nitrogen freed from various proteins by the concerted action of proteinases and peptidases of yellow-green Aspergillus increased to 1.0 to 3.8 times that of control (no acid treatment). An increase of about 31% of glutamic acid per total nitrogen resulted from the acid treatment method in soy sauce made with an enzyme preparation of proteases.     

Problematic Occurrence of Tyrosine Crystals in the Thai Soybean Paste Tao Chieo

In a Bangkok soy sauce factory that had recently converted to controlled inoculum and incubation for the koji stage of fermentation, a problem arose with unsightly white particles in the soybean paste condiment called tao chieo. This problem had not arisen during the previous history of the factory where the koji stage of the fermentation was uncontrolled. Microscopic examination of the particles showed that they were crystalline. Physical separation of the crystals followed by solvent extraction, recrystallization, chemical characterization, and spectroscopy showed that they consisted of tyrosine. Tao chieo prepared by using low- or high-protease strains of Aspergillus sp. indicated that the tyrosine crystals resulted from mold proteolysis of the soybean substrate. Addition of polyethylene glycol at the beginning of the moromi fermentation reduced the severity of crystal formation.     

Diversity of <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> genotypes (RFLP) isolated from traditional soy sauce production within Malaysia and Southeast Asia

DNA fingerprinting was performed on 64 strains of Aspergillus oryzae and 1 strain of Aspergillus sojae isolated from soy sauce factories within Malaysia and Southeast Asia that use traditional methods in producing “tamari-type” Cantonese soy sauce. PstI digests of total genomic DNA from each isolate were probed using the pAF28 repetitive sequence. Strains of A. oryzae were distributed among 32 genotypes (30 DNA fingerprint groups). Ten genotypes were recorded among 17 A. oryzae isolates from a single soy sauce factory. Genotypes Ao-46 and GTAo-47, represented by 8 and 5 strains, respectively, were isolated from a soy sauce factory in Kuala Lumpur and factories in two Malaysian states. Four strains of GTAo-49, isolated from three soy sauce factories in Malaysia; each produced sclerotia. Two strains were found to be naturally occurring color mutants of NRRL 32623 (GTAo-49) and NRRL 32668 (GTAo-52). Only two fingerprint matches were produced with the 43 DNA fingerprint groups in our database, representing A. oryzae genotypes from Japan, China, and Taiwan. Aspergillus sojae NRRL 32650 produced a fingerprint matching GTAo-9, the only known genotype representing koji strains of A. sojae. No aflatoxin was detected in broth cultures of these koji strains as determined by TLC.     

Involvement of meso-α,∊-Diamino-pimelate D-Dehydrogenase in Lysine Biosynthesis in Corynebacterium glutamicum

To characterize aspartyl aminopeptidase from Aspergillus oryzae, the recombinant enzyme was expressed in Escherichia coli. The enzyme cleaves N-terminal acidic amino acids. About 30% activity was retained in 20% NaCl. Digestion of defatted soybean by the enzyme resulted in an increase in the glutamic acid content, suggesting that the enzyme is potentially responsible for the release of glutamic acid in soy sauce mash.     

Occurrence of proteolytic inhibitors in various tissues of barley

Summary The three groups of proteolytic inhibitors present in resting barley grains, namely, trypsin inhibitors, Aspergillus-proteinase inhibitors, and inhibitors of endogenous proteinases, occur in both the embryo and the two endosperm tissues. There are pronounced quantitative differences, however. The three inhibitor activities in the embryo are, respectively, 6-, 0.1-, and 6-fold of those in the endosperm.During germination at 20° all inhibitor activities disappear from the endosperms in 4–5 days. Young rootlets and coleoptiles contain inhibitors of trypsin and Aspergillus proteinase, but these disappear after 4–5 days' germination. However, the trypsin inhibitor content per seedlings remains roughly constant through the whole period. The Aspergillus-proteinase inhibitors, in contrast, exhibit a pronounced increase of activity per seedling.No inhibitor activities were detected in leaves and roots at later stages of growth.The trypsin inhibitor which we have earlier purified from resting grains occurs exclusively in the two endospermal tissues and is immunologically entirely different from the trypsin inhibitors present in embryos and young seedlings.     

Metabolism of the Soy Isoflavones Daidzein and Genistein by Fungi Used in the Preparation of Various Fermented Soybean Foods

The ability of fungi used in the preparation of fermented soybean foods to metabolize the soy isoflavones daidzein and genistein was investigated. A total of 21 fungal strains from dou-chi, miso, sake, soy sauce, and sufu were screened. The genera of the tested fungi included Actinomucor, Aspergillus, Candida, Debaryomyces, Monascus, Mucor, Rhizopus, Saccharomyces, and Zygosaccharomyces. The results were that all tested Aspergillus strains from these soybean foods, including five A. oryzae strains, one A. sojae strain, and one A. tamarii strain, metabolized both daidzein and genistein. In contrast, no other tested fungi from the fermented soybean foods metabolized either daidzein or genistein. The metabolites of daidzein and genistein by Aspergillus strains were identified as 8-hydroxydaidzein and 8-hydroxygenistein, respectively, based on their mass, ¹H-, and ¹³C-NMR spectra.     

P-Factor,a New Peptidic Growth Factor for Pediococcus soyae

The P-factor a new growth factor for Ped. soyae, is found in partial acid and enzymic hydrolysates of Hammersten’s milk casein, and is therefore, supposed to be a kind of peptide. The well-known peptidic growth promoting substance for Lact. casei, strepogenin and its relating substances, are quite ineffective to the author’s organism. P-factor exists along with the S-factor which is another growth promoting-factor for this lactic acid bacterium, in soy mash juice and in the water extract of Aspergillus sojae mycelium. The former substance is also found in various kinds of commercial peptones. The factor was not substitutable with all of the 19 synthetic peptides tested. S-factor is effective, only in the presence of the P-factor.     

Identification of the putative N-acetylglucosaminidase CseA associated with daughter cell separation in Tetragenococcus halophilus

ABSTRACT

The lactic acid bacterium Tetragenococcus halophilus, which is used as a starter to brew soy sauce, comprises both cluster-forming strains and dispersed strains. The cluster-forming strains are industrially useful for obtaining clear soy sauce, because the cell clusters are trapped by filter cloth when the soy sauce mash is pressed. However, the molecular mechanism underlying cell cluster formation is unknown. Whole genome sequence analysis and subsequent target sequence analysis revealed that the cluster-forming strains commonly have functional defects in N-acetylglucosaminidase CseA, a peptidoglycan hydrolase. CseA is a multimodular protein that harbors a GH73 domain and six peptidoglycan-binding LysM domains. Recombinant CseA hydrolyzed peptidoglycan and promoted cell separation. Functional analysis of truncated CseA derivatives revealed that the LysM domains play an important role in efficient peptidoglycan degradation and cell separation. Taken together, the results of this study identify CseA as a factor that greatly affects the cluster formation in T. halophilus.     

Function of the High Concentration Alcohol-producing Factor

By use of the chemically defined synthetic medium, the formation of high concentration of alcohol, reaching 20–21 percent, was accomplished by Saccharomyces sake, at 20°C within 20 days, under a gradual addition of sucrose. Unsaturated fatty acid-containing phospholipid- macromolecule (albumin or methylcellulose) complex was the essential structure for the high concentration alcohol-producing factor. Unsaturated fatty acids, especially linoleic acid, incorporated to the yeast cells grown anaerobically in the statical fermentation test from the koji mold phosphatidylcholine-methylcellulose complex. These data show that the formation of a high concentration of alcohol in sake mash is related to the lipid metabolism of the yeasts.     

Common genotypes (RFLP) within a diverse collection of yellow-green aspergilli used to produce traditional Oriental fermented foods

 DNA fingerprinting was performed on 72 strains of Aspergillus oryzae and 9 strains of Aspergillus sojae isolated from chu (China) or koji (Japan) mold inoculum used in the production of traditional Oriental fermented beverages or foods including soy sauce, miso, and sake. The cultures were deposited with the ARS Culture Collection (NRRL) between 1909 and 2001. PstI digests of total genomic DNA from each isolate were probed using the pAF28 repetitive sequence. All strains of A. sojae that we examined produced an identical DNA fingerprint and belong to the same DNA fingerprint group (GTAo-9). Strains of A. oryzae were distributed among 41 DNA fingerprint groups, including GTAo-12 represented by 11 strains, GTAo-19 represented by 5 strains, GTAo-5 and GTAo-15 each represented by 4 strains, and GTAo-8, GTAo-17, and GTAo-24 each represented by 3 strains. Thirty-three single strain isolates of A. oryzae produced unique fingerprints. Our data offer evidence suggesting that (1) the successful domestication of A. parasiticus genotypes yielding A. sojae occurred far less frequently than among genotypes of A. flavus var. oryzae; and (2) some Aspergillus genotypes employed in different fermentations and regions were derived from a common ancestral clonal population. Received: February 18, 2002 / Accepted: April 22, 2002     

Comparative studies on the in vitro properties of phytases from various microbial origins

The physical and chemical properties of six crude phytase preparations were compared. Four of these enzymes (Aspergillus A, Aspergillus R, Peniophora and Aspergillus T) were produced at commercial scale for the use as feed additives while the other two (E. coli and Bacillus) were produced at laboratory scale. The encoding genes of the enzymes were from different microbial origins (4 of fungal origin and 2 of bacterial origin, i.e., E. coli and Bacillus phytases). One of the fungal phytases (Aspergillus R) was expressed in transgenic rape. The enzymes were studied for their pH behaviour, temperature optimum and stability and resistance to protease inactivation. The phytases were found to exhibit different properties depending on source of the phytase gene and the production organism. The pH profiles of the enzymes showed that the fungal phytases had their pH optima ranging from 4.5 to 5.5. The bacterial E. coli phytase had also its pH optimum in the acidic range at pH 4.5 while the pH optimum for the Bacillus enzyme was identified at pH 7.0. Temperature optima were at 50 and 60°C for the fungal and bacterial phytases, respectively. The Bacillus phytase was more thermostable in aqueous solutions than all other enzymes. In pelleting experiments performed at 60, 70 and 80°C in the conditioner, Aspergillus A, Peniophora (measurement at pH 5.5) and E. coli phytases were more heat stable compared to other enzymes (Bacillus enzyme was not included). At a temperature of 70°C in the conditioner, these enzymes maintained a residual activity of approximately 70% after pelleting compared to approximately 30% determined for the other enzymes. Incubation of enzyme preparations with porcine proteases revealed that only E. coli phytase was insensitive against pepsin and pancreatin. Incubation of the enzymes in digesta supernatants from various segments of the digestive tract of hens revealed that digesta from stomach inactivated the enzymes most efficiently except E. coli phytase which had a residual activity of 93% after 60 min incubation at 40°C. It can be concluded that phytases of various microbial origins behave differently with respect to their in vitro properties which could be of importance for future developments of phytase preparations. Especially bacterial phytases contain properties like high temperature stability (Bacillus phytase) and high proteolytic stability (E. coli phytase) which make them favourable for future applications as feed additives.     

Production of Soy Sauce Koji Mold Spore Inoculum in Plastic Bags

An innovation is described for producing soy sauce koji mold spore inoculum by using inexpensive autoclavable plastic bags and reuseable plastic enclosures to make culture vessels. After growth, the spore mass could be dried and packaged in the same bag after removing the enclosure. Broken rice was used as the substrate for mold cultivation. Viable spore counts of 10⁹ spores per g were obtained under optimal conditions. After drying at 50°C for 6 h, the moisture content of the spore mass decreased from 35.22 to 6.32% with no significant effect on spore viability. The dry spores could be stored in the refrigerator or at room temperature for at least 3 months.     

Occurrence and moisture requirements of microbial growth in building materials

Microbial growth was studied in six damp buildings. Mesophilic fungi, especially Penicillium spp., yeasts, and species of Cladosporium and Aspergillus, occurred most abundantly on building constructions. Thermophilic fungi and mesophilic actinomycetes were occasionally found. A toxigenic fungus, Stachybotrys sp., was also detected on cellulose-based materials. In a cytotoxicity test, 23% of samples were positive. Spore counts varied considerably on materials, but no correlation between counts and the substrate or its water activity (a_w) was observed. In experiments a rapid increase in CO₂ production and spore propagule count was observed in all materials incubated at a relative humidity (RH) (RH=0·01^*water activity) of 96–98°. Some differences were noted between materials in CO₂ evolved, but not in propagule counts.     

Conidia of Aspergillus oryzae as Naturally Immobilized Phosphatases

The following enzymes were detected in conidia of Aspergillus oryzae var. No. 13 that were collected from a soy sauce factory: phosphatases acting nonspecifically on nucleotides; ribosidase(s) acting on adenosine, inosine, guanosine and xanthosine; and deaminases acting on guanine, adenosine, guanosine, cytidine, AMP and GMP. Most phosphatase activity was found to be bound firmly to the conidia. Thus, when a nucleotide solution was passed through a conidia-containing column, the corresponding nucleoside was recovered as a clear and colorless solution. The bound phosphatases were rather stable at pH 4 to 9 and at temperatures below 50°C. The optimum conditions for activity were at about 45°C and at pH about 5 and 8. When UMP·Na₂ solution was passed through a column containing 5 g of the conidia, about 200 mg of substrate was hydrolyzed per 1 hr. Conidia of molds belonging to the genus Aspergillus may be industrially employed as naturally immobilized phosphatases.     

Improvement of Gene Targeting in Aspergillus nidulans with Excess Non-Homologous Fragments

An α-glucosidase was purified in an electrophoretically pure state from an extract of koji culture of Aspergillus sp. KT-11. This enzyme was found to have a transferring activity when the reaction was done in a high concentration of leucrose at pH 4.5. Two kinds of transfer products, fractions I and II, were obtained from leucrose by the enzyme and they were identified as [(α-D-glucopyranosyl-(1 →6)-α-D-glucopyranosyl-(1 →6)- α -D-glucopyranosyl-(1→5)-D-fructopyranose] and [α-D-glucopyranosyl-(1 →6)-α-D-glucopyranosyl-(1→5)-D- fructopyranose], respectively. These are considered to be novel oligosaccharides     

Thermophilic Bacilli growing with carbon monoxide

Four strains of obligately thermophilic Bacilli capable of growing with carbon monoxide as a sole carbon and energy source were isolated from settling ponds of a sugar factory. Most of them could be identified as strains of Bacillus schlegelii on the basis of cell wall composition, DNA homology menaquinone and DNA base content. Growth with CO was very fast (t _d =3 h) and was optimal at 65°C. No growth occurred below 50°C. As with the mesophilic carboxydotrophs, hydrogen plus carbon dioxide could also serve as autotrophic substrates. Growth of the isolates with CO depended on the presence of molybdenum in the growth medium. This suggested CO oxidase in the newly isolated Bacilli being a molybdenum hydroxylase similar to the enzymes from the mesophilic carboxydotrophs. Some data characterizing the CO-oxidizing activity in extracts of the thermophilic isolates are also provided.This paper is respectively dedicated to Professor Dr. H. G. Schlegel on the occasion of his 60th birthday     

Studies on the Bacterial Formation of a Peptide Antibiotic,Colistin

Colistinase, an enzyme inactivating colistin, which appeared in a fermented broth of Bacillus colistinus attending with decay of colistin production was studied. The enzyme was partially purified and also chromatographed by DEAE-cellulose column. It was a heat labile, mostly basic protein, had optimum pH at 9.0 and was active against peptide antibiotics such as colistin and polymyxin and also against casein. Bacterial proteinase, Nagarse, exhibited colistinase activity exclusively among tested proteinases and immunochemical studies revealed that colistinase was identical with bacterial proteinase Nagarse.