Use of glutaminase for soy sauce made by Koji or a preparation of proteases from Aspergillus oryzae

Although a small amount of glutamic acid was released in the hydrolysis of protein or soy sauce made by a preparation of proteases containing little glutaminase, a large amount of glutamic acid was formed in such hydrolyzate or soy sauce made by the addition of mycelia of black Aspergilli or glutaminase from Cryptococcus albidus. The former effect was caused mainly by glutaminase produced by black Aspergilli. The former crude enzyme showed an optimum pH of 5.0, broad pH stability and salt tolerance. The addition of glutaminase from C. albidus ATCC 20293 in soy sauce manufacture using a preparation of proteases resulted in a 42% increase in glutamic acid per total nitrogen content.     

Culture conditions of Aspergillus oryzae for production of enzyme preparation

Submerged culture was better than solid culture in the production of proteinase and peptidases from Aspergillus oryzae 460. On the contrary, solid culture was better than submerged culture in the production of α-amylase, carboxymethyl cellulase, and pectinlyase from the same fungus.The soy souce mash (moromi) made with the enzyme preparation from submerged culture was highly viscous and the soy sauce produced was characteristic in low contents of alcohol and reducing sugar, low pH value, and less aroma. Soy sauce made with the enzyme preparation from solid culture was superior on these points to that from submerged culture.Wheat bran was best as the raw material for the enzyme preparation in easy koji making, large amount produced, and low cost.In enzyme production from a solid culture, addition of urea (0.8% to wheat bran) nearly doubled the leucine aminopeptidase for Leu-Gly-Gly. The incubation period was reduced to 30 to 40 h from 50 to 60 h using germinated spores and moisture-controlled culture with forced aeration.     

Identification and Quantitation of New Glutamic Acid Derivatives in Soy Sauce by UPLC/MS/MS

Glutamic acid is an abundant amino acid that lends a characteristic umami taste to foods. In fermented foods, glutamic acid can be found as a free amino acid formed by proteolysis or as a non‐proteolytic derivative formed by microorganisms. The aim of the present study was to identify different structures of glutamic acid derivatives in a typical fermented protein‐based food product, soy sauce. An acidic fraction was prepared with anion‐exchange solid‐phase extraction (SPE) and analyzed by UPLC/MS/MS and UPLC/TOF‐MS. α‐Glutamyl, γ‐glutamyl, and pyroglutamyl dipeptides, as well as lactoyl amino acids, were identified in the acidic fraction of soy sauce. They were chemically synthesized for confirmation of their occurrence and quantified in the selected reaction monitoring (SRM) mode. Pyroglutamyl dipeptides accounted for 770 mg/kg of soy sauce, followed by lactoyl amino acids (135 mg/kg) and γ‐glutamyl dipeptides (70 mg/kg). In addition, N‐succinoylglutamic acid was identified for the first time in food as a minor compound in soy sauce (5 mg/kg).     

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.     

γ—射线对酱油中细菌总数及营养成分影响的初步研究

用~(60)Co—γ—射线以不同剂量(30,50,70,90万拉德)照射瓶装酱油,然后对酱油中细菌总数及总酸和氨基酸态氮进行分析。结果证明:γ—射线具有良好的杀菌作用,剂量30万拉德已能使细菌总数降低到标准含量以下。而各个剂量的γ—照射对总酸及氨基酸态氮的数值几乎没有影响。     

Changes in microflora and biochemical composition during the Baceman stage of traditional indonesian Kecap (soy sauce) production

Indonesian soy sauce (kecap) is made from black soybeans in a traditional way which involves two microbiological stages: a solid-state fermentation and a brine fermentation. This study is concerned with the brine fermentation, called baceman. Samples from different kecap producers were analyzed for (bio)chemical content and micro-organisms. It was found that the final composition of the baceman differed from manufacturer to manufacturer, and even within companies large differences were found in microflora and the amounts of fermentation products, formol nitrogen and salt concentration. The main fermentation products were lactate, acetate, glycerol and ethanol. Pediococcus halophilus, staphylococci, a coryneform bacterium and yeasts belonging to Candida, Debaromyces and Sterigmatomyces were isolated from the brines. Compared to Japanese soy sauce production, fermentation by yeasts does not play an important role in Indonesian kecap production. This is due to the fact that kecap is made from whole soybeans only, which are poor in sugars. After fermentation by P. halophilus no substrates are left for growth and ethanol production by yeasts. The presence of film forming yeasts can even lead to spoilage of the product.     

Characterization of Aspergillus oryzae aspartyl aminopeptidase expressed in Escherichia coli

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.     

Soy sauce increased the oxidative stress tolerance of nematode via p38 MAPK pathway

Soy sauce – a fermented food made from soybeans and wheat – is considered a healthy seasoning, but little scientific evidence is available to support this. In this study, physiological effects of soy sauce were analyzed using Caenorhabditis elegans. When soy sauce was fed to C. elegans together with Escherichia coli OP50, fat accumulation decreased, and resistance to oxidative stress by H₂O₂ was greatly increased in the nematodes. qRT-PCR revealed that mRNA expression of oxidative stress tolerance genes, including sod, ctl, and gpx, was markedly increased in soy sauce-fed nematodes. Worms ingesting soy sauce showed high mitochondrial membrane potential and reactive oxygen species (ROS) and low intracellular ROS, suggesting that soy sauce induced mitohormesis and decreased cytoplasmic ROS. Therefore, soy sauce ingestion affects the mitochondria and may alter the fat metabolism in C. elegans. Furthermore, the increase in oxidative stress tolerance is mediated through p38 MAPK pathway.     

酱油酿造高产菌株的筛选

根据种曲克孢子数、成曲蛋白酶活力及发酵酱醅的氨基酸态氮含量3个指标,对生产中使用菌种米曲霉进行了筛选,得到高产菌株米曲霉3号,蛋白酶活力提高42.69%,发酵酱醅氨基酸态氮含量提高了20.3%。     

ATP bioluminescence rapid detection of total viable count in soy sauce

The adenosine triphosphate (ATP) bioluminescence rapid determination method may be useful for enumerating the total viable count (TVC) in soy sauce, as it has been previously used in food and beverages for sanitation with good precision. However, many factors interfere with the correlation between total aerobic plate counts and ATP bioluminescence. This study investigated these interfering factors, including ingredients of soy sauce and bacteria at different physiological stages. Using the ATP bioluminescence method, TVC was obtained within 4 h, compared to 48 h required for the conventional aerobic plate count (APC) method. Our results also indicated a high correlation coefficient (r = 0.90) between total aerobic plate counts and ATP bioluminescence after filtration and resuscitation with special medium. The limit of quantification of the novel detection method is 100 CFU/mL; there is a good linear correlation between the bioluminescence intensity and TVC in soy sauce in the range 1 × 10²–3 × 10⁴ CFU/mL and even wider. The method employed a luminescence recorder (Tristar LB‐941) and 96‐well plates and could analyse 50–100 samples simultaneously at low cost. In this study, we evaluated and eliminated the interfering factors and made the ATP bioluminescence rapid method available for enumerating TVC in soy sauce. Copyright © 2011 John Wiley & Sons, Ltd.     

Anisakis physeteris: Amino acids in body tissues

The whole cuticle, perienteric fluid, and reproductive organs of Anisakis physeteris Baylis, 1923 from the sperm whale were analyzed for amino acid composition. Amino acid nitrogen per total nitrogen accounted for 96.2% in the cuticle. Corresponding values of TCA supernatant and precipitated fractions of perienteric fluid and reproductive organ were obtained. Proline, glycine, and arginine occurred abundantly in cuticle hydrolyzate. Lysine, glutamic acid, glycine, valine, leucine and histidine occounted for approximately 62% of total nitrogen in reproductive organ fractions. Differences were observed in the amounts of certain amino acids present in corresponding tissue of males and females as well as different tissue fractions from nematodes of the same sex, e.g., more serine in male cuticle than female, more proline in the protein of female reproductive organs than male; methionine present in cuticular protein and reproductive organ protein and nonprotein fractions but absent in perienteric fluid, and cystine in cuticular protein but not the other fractions. Untreated Anisakis hemolymph showed at least a 2-fold increase in ammonia in analyses made 2 and 10 days after bleeding. The relatively large ammonia content could have been in part the result of functional ornithine cycle and amino acid oxidase activity in vivo.     

Influence of prebrining treatments on microbial and biochemical changes during the baceman stage in Indonesian kecap (soy sauce) production

Prior to the baceman stage or brine fermentation in Indonesian kecap (soy sauce) production, the soybeans undergo a number of treatments, such as soaking, cooking and a fungal solid state fermentation (bungkil stage). The influence of these prebrining steps on the baceman stage was investigated. Acidification during soaking inhibited bacterial growth during the solid state fermentation, causing lower initial numbers of bacteria in and a slower acidification of the baceman. Cooking time influenced the softness of the beans and thus fungal growth during the bungkil stage. Increasing the cooking time from 1 to 3 h resulted in a better solubility of macromolecules and gave higher final concentrations of fermentation products and formol nitrogen in baceman. When cooking time was increased from 3 to 5 h only slight differences could be observed. Length of the bungkil stage had some minor influences; the amount of fermentation products and residual monosaccharides decreased by 16% when the length was increased from 2 to 7 d. The highest amounts of formol nitrogen were found with 5 d of bungkil fermentation. The addition of rice flour showed that the absence of yeast fermentation in baceman is related to the lack of sugars after the previous lactic acid fermentation. In addition to yeast fermentation, the growth of fungi during the bungkil stage was also positively influenced by the addition of rice flour. Sun drying and crumbling bungkil caused lower enzyme activities and resulted in lower amounts of formol nitrogen and glycerol. Growth of Pediococcus halophilus during the baceman stage was faster when bungkil was sun dried, although the final concentrations of fermentation products were lower due to a lower content of amino acids which resulted in less buffering. Increasing the amount of bungkil added to salty water caused a similar increase in formol nitrogen and fermentation products.     

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.     

Characterization of salt-tolerant glutaminase from Stenotrophomonas maltophilia NYW-81 and its application in Japanese soy sauce fermentation

Glutaminase from Stenotrophomonas maltophilia NYW-81 was purified to homogeneity with a final specific activity of 325 U/mg. The molecular mass of the native enzyme was estimated to be 41 kDa by gel filtration. A subunit molecular mass of 36 kDa was measured with SDS-PAGE, thus indicating that the native enzyme is a monomer. The N-terminal amino acid sequence of the enzyme was determined to be KEAETQQKLANVVILATGGTIA. Besides l-glutamine, which was hydrolyzed with the highest specific activity (100%), l-asparagine (74%), d-glutamine (75%), and d-asparagine (67%) were also hydrolyzed. The pH and temperature optima were 9.0 and approximately 60°C, respectively. The enzyme was most stable at pH 8.0 and was highly stable (relative activities from 60 to 80%) over a wide pH range (5.0–10.0). About 70 and 50% of enzyme activity was retained even after treatment at 60 and 70°C, respectively, for 10 min. The enzyme showed high activity (86% of the original activity) in the presence of 16% NaCl. These results indicate that this enzyme has a higher salt tolerance and thermal stability than bacterial glutaminases that have been reported so far. In a model reaction of Japanese soy sauce fermentation, glutaminase from S. maltophilia exhibited high ability in the production of glutamic acid compared with glutaminases from Aspergillus oryzae, Escherichia coli, Pseudomonas citronellolis, and Micrococcus luteus, indicating that this enzyme is suitable for application in Japanese soy sauce fermentation.     

Enzymatic Modification of Proteins in Foodstuffs

Peptic hydrolyzate of soy protein was submitted to the plastein reaction with α-chymotrypsin under the following condition: substrate concentration, 20%; enzyme-substrate ratio by weight, 1/100; reaction pH, 7.0; and reaction temperature, 37°C. The plastein yield resulting from the plastein reaction for 24 hr was found to depend on the degree of hydrolysis of the substrate (per cent ratio between nitrogen amount in 10% trichloroacetic acid soluble nitrogen and that in whole hydrolyzate); the optimum degree of hydrolysis for the highest plastein yield seemed to lie around 80%. A turbidity appeared in the process of the plastein reaction, whose intensity was correlative to the plastein yield. The peptic hydrolyzate of soy protein per se had bitterness and its magnitude decreased with increasing plastein yield.

As a result of the plastein reaction applied for 24 hr to the hydrolyzate whose degree of hydrolysis was 80%, the average molecular weight estimated by the change in amino nitrogen content increased by approximately three times. The molecular weight distribution pattern obtained by gel filtration supported the above result. The total amount of amino acids liberated from the plastein reaction product by its treatment with either leucine aminopeptidase or carboxypeptidase A was significantly less than that liberated from the original hydrolyzate by its similar treatment. This result also supports the formation of higher-molecular protein-like substances by the plastein reaction. Deuteration study followed by IR spectrometry showed the occurrence of peptide bond formation, i.e. decrease in ionized carboxyl group at 1575 cm^?1 and increase in deuterated amide at 1450 cm^?1, even at the earlier stages of the plastein reaction.     

Evaluation of commercial soy sauce <Emphasis Type="Italic">koji</Emphasis> strains of <Emphasis Type="Italic">Aspergillus oryzae</Emphasis> for γ-aminobutyric acid (GABA) production

In this study, four selected commercial strains of Aspergillus oryzae were collected from soy sauce koji. These A. oryzae strains designated as NSK, NSZ, NSJ and NST shared similar morphological characteristics with the reference strain (A. oryzae FRR 1675) which confirmed them as A. oryzae species. They were further evaluated for their ability to produce γ-aminobutyric acid (GABA) by cultivating the spore suspension in a broth medium containing 0.4 % (w/v) of glutamic acid as a substrate for GABA production. The results showed that these strains were capable of producing GABA; however, the concentrations differed significantly (P < 0.05) among themselves. Based on the A. oryzae strains, highest GABA concentration was obtained from NSK (194 mg/L) followed by NSZ (63 mg/L), NSJ (51.53 mg/L) and NST (31.66 mg/L). Therefore, A. oryzae NSK was characterized and the sequence was found to be similar to A. oryzae and A. flavus with 99 % similarity. The evolutionary distance (K _nuc) between sequences of identical fungal species was calculated and a phylogenetic tree prepared from the K _nuc data showed that the isolate belonged to the A. oryzae species. This finding may allow the development of GABA-rich ingredients using A. oryzae NSK as a starter culture for soy sauce production.     

Stimulation of protease production byAspergillus oryzae with oils in continuous culture

Summary The effect of soy sauce oil and various other oils on protease production by Aspergillus oryzae NISL 1913 was studied in chemostat cultures (dilution rate=0.02 h^–1). Soy sauce oil was consumed as a carbon source by the cells and also accelerated protease production. When soy sauce oil was used as sole carbon source, the specific protease production rate was 2.89 protease units·(mg dry weight of mycelium)^–1·h^–1, which was threefold higher than that with starch. The specific protease production rate with linoleic acid, oleic acid, Tween 80 and soybean oil exhibited similar values to that with soy sauce oil but the fatty acids with carbon chains shorter than six, such as caproic acid and acetic acid, did not stimulate protease production. The oils did not cause an increase in other exocellular enzymes such as -amylase, indicating that the protease production was selectively stimulated by the oils. Offprint requests to: Y. Fukushima     

Characterization of a <Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis> strain for reduction of citrulline accumulation during soy sauce fermentation

Objectives

To reduce the amount of citrulline produced by arginine-consuming bacteria in the moromi mash during soy sauce production.

Results

Bacillus amyloliquefaciens JY06, a salt-tolerant strain with high arginine consumption ability and low citrulline accumulation capacity, was isolated from moromi mash. The concentration of citrulline was decreased from 26.8 to 5.1 mM and ethyl carbamate in soy sauce, after sterilization, decreased from 97 to 17 μg kg^?1 when B. amyloliquefaciens JY06 was added during fermentation. The aroma of the sauce was improved by increasing the ester content.

Conclusions

B. amyloliquefaciens JY06 is a beneficial bacterium that can be used in soy sauce fermentation to eliminate ethyl carbonate and enhance the flavor of the sauce.

     

Antihypertensive and natriuretic effects of less-sodium soy sauce containing gamma-aminobutyric acid in spontaneously hypertensive rats

We investigated the mechanism of the antihypertensive effect of less-sodium soy sauce containing gamma-aminobutyric acid (GABA) in spontaneously hypertensive rats (SHRs). When SHRs were given a diet with less-sodium soy sauce containing GABA (GABA-rich soy sauce group) for 6 weeks, the systolic blood pressure decreased as compared with that in rats fed diets with less-sodium soy sauce or a solution of salt. Renal sympathetic nerve activity (RSNA) and positive Na balance were reduced, and the urinary Na excretion tended to increase in the GABA-rich soy sauce group. Vascular hypertrophy of the thoracic aorta and the coronary and renal interlobular arteries tended to reduce in the GABA-rich soy sauce group. These results suggest that inhibition of Na retention by natriuresis, as a result of inhibition of RSNA by the GABA in the soy sauce contributed to the antihypertensive effect of GABA in the SHRs. Intake of less-sodium soy sauce containing GABA might help to reduce overall cardiovascular risk.     

Cytokinin activity of some derivatives of n- n- butyl- n′ - aryl thiourea and their effect on the nitrogen metabolism in barley seedlings

The cytokinin activity of a group of derivatives of N-n-butyl-N′-arylthiourea and their effect on the uptake of amino acids and the accumulation of total nitrogen in seven-day barley seedlings have been investigated. It was established that N-n-butyl-N″-2,4-dinitrophenyl-thiosemicarbazide, N-n-butyl-N′-o-,m- andp-chlorophonylthioureas, N-n-butyl-N′-3,4-dichlorophenylthiourea and N-n-butyl-N′-2-methyl-4-ehlorophenylthiourea possess a high activity with respect to the retardation of the chlorophyll breakdown. Substances with a high cytokinin activity cause an increase in the amount of total nitrogen and amino acids, mainly of glutamic acid, aspartic acid and serine in the roots and loaves of seven-day barley seedlings.