WO2023022662A2 - Culture pour l'amélioration de la qualité de sauce soja moromi - Google Patents

Culture pour l'amélioration de la qualité de sauce soja moromi Download PDF

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WO2023022662A2
WO2023022662A2 PCT/SG2022/050591 SG2022050591W WO2023022662A2 WO 2023022662 A2 WO2023022662 A2 WO 2023022662A2 SG 2022050591 W SG2022050591 W SG 2022050591W WO 2023022662 A2 WO2023022662 A2 WO 2023022662A2
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moromi
fermentation
lactobacillus
pobuzihii
lactobacillus pobuzihii
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PCT/SG2022/050591
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WO2023022662A9 (fr
WO2023022662A3 (fr
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Maxim SHELUDCHENKO
Xianning LAI
Huay Ee CHENG
Wei-Chung Chuang
Wei-Min CHUANG
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Wilmar International Limited
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
    • A23L27/50—Soya sauce
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/225—Lactobacillus

Definitions

  • the present disclosure relates to the field of food science.
  • the present disclosure relates to the use of microbes to manipulate flavour.
  • soy sauce being one of the primary condiments in Asian countries. China’s annual production of soy sauce was shown to have reached more than 8 million tons per annum, as reported in 2022. While there are range of various products on the market covering customers with specific flavour preferences, there is still a demand for natural food products with enhanced taste characteristic. This enhanced taste characteristic coincides with a higher content of peptides with rich umami (meaty) taste and kokumi mouthfeel experience, without employing artificial additives such as sodium glutamate. Since food allergies are rising within the growing, urbanized population, further concerns of potential customers would be being access gluten free foods and products with depleted histamine content.
  • the present disclosure refers to a culture comprising Lactobacillus pobuzihii WZ3 (DSM 33648), or Lactobacillus pobuzihii WZ5 (DSM 33658), or a combination thereof.
  • the present disclosure refers to a process of fermentation comprising a starter culture comprising at least one Lactobacillus pobuzihii strain.
  • the present disclosure refers to a soy sauce product made using the culture as disclosed herein or the process as disclosed herein.
  • the present disclosure refers to a method of reducing and/or suppressing growth of undesired bacteria during fermentation, the method comprising the use of the culture as disclosed herein.
  • the present disclosure refers to a method of lowering undesirable taste components in fermented black bean soy sauce, the method comprising use of the culture as disclosed herein.
  • the present disclosure refers to a method of increasing desirable taste components in fermented black bean soy sauce, the method comprising use of the culture as disclosed herein, wherein the increase in desirable taste components is characterised by the increase in taste conferring peptides and/or taste conferring amino acids in relation to total protein content of a sample.
  • the present disclosure refers to a method of reducing the concentration of Weissella sp. during fermentation, the method comprising the use of the culture disclosed herein.
  • the present disclosure refers to Lactobacillus pobuzihii WZ3 (DSM 33648).
  • the present disclosure refers to Lactobacillus pobuzihii WZ5 (DSM 33658).
  • Fig. 1 shows a histogram depicting the results of a comparison of taste grades between various soy sauce types and brands. Specifically, the quality of Taiwanese black bean Wuan Chuang soy sauce is shown compared to other products, thereby showing an overall quality of Taiwanese black bean Wuan Chuang soy sauce compared to other soy sauces available on the Chinese market.
  • Fig. 2 is a column graph showing the accumulation of taste 3-6 mer peptides over time during a black bean moromi fermentation.
  • Fig. 3 is a column graph showing the development of the level of tasty peptides over 4 months. This shows that the abundance of Lactobacillus pobuzihii correlates with level of tasty peptides over 4 months. Lactobacillus pobuzihii and Staphycoccus bacteriophages were the only ones to be positively associated with taste peptides.
  • Fig. 4 is a column graph showing the concentration of valine-proline-proline (VPP) in black bean (BB) compared to yellow bean (V) and wheat (YW) soy sauce moromi made with WZ.
  • VPP is known to have a blood pressure lowering effect by inhibiting angiotensin converting enzymes (ACS).
  • ACS angiotensin converting enzymes
  • the valine-proline-proline (VPP) peptide content is indicative of being an inhibitor of antiotension converting enzymes in Wuang Zhong (WZ) soy sauces.
  • Fig. 5 is a heatmap showing the results of a comparison of the volatile organic compounds obtained from traditional black bean moromi (BB) with yellow bean (YB), industrial yellow bean, and wheat (YW) moromi for the final 5 months of fermentation.
  • Black bean final moromi accumulated pyrazines, various organic compounds with butane chain, acetic acid, acetone, propanoic acid. Levels of octanoic acid are high at the first month, and disappeared at later stages of fermentation.
  • Fig. 6 is a heatmap showing the chemical product profile of fermentation with various bacterial strains. Lactobacillus pobuzihii produces 3 hydroxy-2-butanone, butanoic acid, pentyl octanoate and acetic acid. Weissella paramensteroides is responsible for octanoic acid, ethyl acetate, methylbutanols. Tetragenococcus halophilus releases benzaldehydes, pentanedione, toluene and butenal. Bacillus amyloliquefaciens produces pyrazines compounds. [0021] Fig.
  • FIG. 7 is a column graph showing that bacteria and bacteriophages are negatively associated with taste peptides. Shown are the bacteria Bacillus amyloliquefaciens , Escherichia coli, Weissella cibaria, Enterococcus casseliflavus, and Ochrobactrum.
  • Fig. 8 is a line graph depicting the optical density of various bacterial strains over time. Strains of Lactobacillus pobuzihii WZ3 and WZ5 are shown to have different growth characteristics, which affects characteristics, such as stress tolerance and the ability to grown under 17% salt during moromi fermentation.
  • Fig. 9 shows the relative abundances of microbial members responsible for the quality of koji and moromi of black bean soy sauce.
  • the sampling event had taken place in March 2016
  • Fig. 9A is a horizontal stacked bar chart showing the microbial composition based on shotgun metagenomics of black bean soy sauce fermentation during 4 months of fermentation. Samples were taken from vats at timepoints of 0, 0.5, 1, 2, 3 and 4 months in March 2016 (Spring) in three replicates (rep 1- vatl, rep2 - vat2, rep3 - vat3). Bioinformatics analysis was conducted with Metaphlan2 pipeline as input metagenomics reads. Viral composition is omitted.
  • Fig. 9A is a horizontal stacked bar chart showing the microbial composition based on shotgun metagenomics of black bean soy sauce fermentation during 4 months of fermentation. Samples were taken from vats at timepoints of 0, 0.5, 1, 2, 3 and 4 months in March 2016 (Spring) in three replicates (rep 1-
  • FIG. 9B is another stacked bar chart horizontal stacked bar chart showing the results of amplicon sequencing output on microbial composition of black bean soy sauce fermentation during 4 months of fermentation. Samples were taken from vats back to 0, 0.5, 1, 2, 3 and 4 months starting in March 2016 (Spring) in three replicates (rep 1- vatl, rep2 - vat2, rep3 - vat3).
  • Fig. 9C shows a line graph showing a summary of the growth dynamic of major bacterial species during koji maturation of black beans.
  • Fig. 9D shows a line graph depicting a summary of the growth dynamic of four most abundant bacterial species in black bean moromi.
  • Fig. 10 shows the relative abundances of microbial members together with the profiles showing the bacteriophage diversity in koji and moromi of black bean soy sauce.
  • the sampling event had taken place in March 2016.
  • Fig. 10A is a heatmap depicting the total population of bacterium present in the initial sampling of traditional black bean soy sauce. Bacteriophages of Weissella, Enterobacteria and Cronobacter had an abundance of almost 60%.
  • Fig. 10B shows a stacked bar chart showing the identity and relative amounts of DNA as determined by direct DNA extraction from 5g of koji and 500 mL of sample.
  • FIG. 10C shows a stacked bar chart depicting the relative abundance of archaea, eukaryotic, viral, and bacterial DNA present in a pellet obtained from the 500 mL of sample in a subsequent DNA extraction step.
  • Fig. 11 shows the dynamics of main microbial functions (based on shotgun metagenome reads) changing over period of soy sauce maturation from koji until end of 4 months of moromi maturation. The sampling event had taken place in March 2016.
  • Fig. 11A shows a heatmap of the metabolic pathways activated during koji and moromi fermentations, based on HUMANN2 output.
  • 11B shows a cladogramm depicting the pathway contribution of Lactobacillus pobuzihii, Bacillus amyloliquefaciens, Tetragenococcus halophilus, Weisella paramensteroides, and Staphylococcus scuiri at koji (time point 0) and during 4 months of black bean moromi fermentation in with following timepoints: 0.5 month - timepoint 1, 1 month - timepoint 2, 2 months - timepoint 3, 3 months - timepoint 4, 4 months - timepoint 5. Months of fermentation are coloured. Folate production happened in koji stage with help of Bacillus and Staphylococcus. Production of methionine, threonine at 2 months; isoleucine and arginine at 5 months. V pentose phospate cycle is solely shared between 4 species.
  • Fig. 12 shows the relative abundances of microbial members responsible for the quality of koji and moromi of black bean soy sauce (BB), yellow soy sauce (Yellow) and yellow wheat soy sauce (YW). These sampling events had taken place in October 2016 and February 2017.
  • Fig. 12A shows a stacked bar chart showing the microbial composition of black soy sauce fermentation during 5 months of fermentation. Samples were taken from vats back to 0, 0.5, 1, 2, 3, 4 and 5 months on October 2016 (Fall) in two replicates (A - vatl, B - vat2). Viral composition is included. Lactobacillus pobuzihii maximum abundance was 33% at month 2.
  • Fig. 12 shows the relative abundances of microbial members responsible for the quality of koji and moromi of black bean soy sauce (BB), yellow soy sauce (Yellow) and yellow wheat soy sauce (YW). These sampling events had taken place in October 2016 and February 2017.
  • Fig. 12A shows a stacked bar chart showing the
  • FIG. 12B shows a stacked bar chart showing the microbial composition of black soy sauce fermentation during 5 months of fermentation. In total, 19 samples were analysed. Samples were taken from vats with 0, 0.5, 1, 2, 3, 4 and 5 months starting in February 2017 (winter) in three replicates (A - vatl, B - vat2). Viral composition is included. Halococcus spp was only detected in the 5 th month vat 2 (BB-5-2m). Lactobacillus pobuzihii population was its highest at month 3 with 66% abundance.
  • Fig. 12C shows a stacked bar chart depicting the metagenomic profiles of industrial process of wheat soy sauce.
  • Fig. 12D is a stacked bar chart depicting the abundance and identity of various bacterial strains in yellow (Y) bean soy sauce, sampled in February 2017. Lactobacillus pobuzihii abundance was 7.2%. The amount of Klebsiella pneumoniae was shown to be increased up to 6.3% in contrast to black bean.
  • Fig. 13 shows two line graphs depicting the summary of dynamics of major microbial species in black bean (BB; top) versus wheat (YW; bottom). These figures show the comparison between microbial population of black bean moromi (top figure) and yellow and wheat soy sauce moromi (bottom picture).
  • Fig. 14 shows growth curves of microbial species isolated from black bean and yellow and wheat moromi at 0, 10, 14 and 18% NaCl in TSB broth.
  • Fig. 14A shows a line graph depicting the growth of Lactobacillus pobuzihii WZ3 under various concentration of NaCl in TSB broth.
  • Fig. 14B shows a line graph depicting the growth of Tetragenococcus halophilus #6 under various concentration of NaCl in TSB broth.
  • Fig. 14C shows a line graph depicting the growth of Bacillus amyloliquefaciens #8 under various concentration of NaCl in TSB broth.
  • Fig. 14A shows a line graph depicting the growth of Lactobacillus pobuzihii WZ3 under various concentration of NaCl in TSB broth.
  • Fig. 14B shows a line graph depicting the growth of Tetragenococcus halophilus #6 under various concentration of NaCl in TSB
  • FIG. 14D shows a line graph depicting the growth of Weissella paramenstroides under various concentration of NaCl in TSB broth.
  • Fig. 14E shows a line graph depicting the growth of Staphylococcus sciuri under various concentration of NaCl in TSB broth.
  • Fig. 14F shows a line graph depicting the growth of Enterococcus f aecium under various concentration of NaCl in TSB broth.
  • Fig. 15 shows a line graph depicting the concentrations of Weissella (top) and Enterococcus (bottom), both of which can maintain growth in up to 14% NaCl. At 18% NaCl, their growth is inhibited. Measurements taken after 61 days were affected, possibly due to cross contamination (possibly due to salt-resistant Staphylococcus) as cups started to crack due to CO2 pressure. The negative control was also contaminated after 2 months of sampling.
  • Fig. 16 shows primary metabolite concentrations and pH changes over 25 days growth of Lactobacillus pobuzihii WZ3, Tetragenococcus halophilus #6, Bacillus amyloliquefaciens and Staphylococcus scuiri under 18% NaCl in TSB medium.
  • Fig. 16A shows a line graph depicting Lactobacillus pobuzihii WZ3 metabolites under 18% NaCl in TSB.
  • Fig. 16B shows a line graph depicting Tetragenococcus halophilus #6 metabolites 18% NaCl in TSB.
  • FIG. 16C shows a line graph depicting Bacillus amyloliquefaciens metabolites 18% NaCl in TSB.
  • Fig. 16D shows a line graph depicting Staphylococcus scuiri metabolites 18% NaCl in TSB.
  • Fig. 17 shows the dynamics of microbial relative abundances in pilot trials.
  • Fig. 17A is a horizonal stacked bar chart showing the results of the pilot trial al.
  • Tank 1 was with crushed beans + oat bran.
  • Tank 2 was with crushed beans without bran.
  • Fig. 17B is a horizonal stacked bar chart showing the microbial composition of Trial 2 and Trial 3.
  • Weissella green coloured dominates up to 70% in moromi from 1 to 7 months of fermentation at 17.5% sat.
  • Tetragenococcus halophilus developed without need of addition of it as starter culture.
  • Pediococcus pentosaceus developed and took up 17% of total abundance.
  • Hl - 45 kg of 1 -month moromi was used as starter culture, H2 - 45 kg of 2-month moromi as starter, H3 - 45 kg of 3-month moromi as starter, H4 - 45 kg of 4 month moromi as starter, Gl-12, G2-12 - washed koji, E10 - Tetragenococcus halophilus no washed koji, E12 - Bacillus amyloliquefaciens no washed koji, F10 - Lactobacillus pobuzihii no washed koji, F12 - Lactobacillus pobuzihii no washed koji. Fig.
  • FIG. 17C is a horizonal stacked bar chart showing the results of trial 4a performed at Xiluo, Taiwan, according to experimental design of Table 8 from August 2018 - January 2019.
  • Fig. 17D is a horizonal stacked bar chart showing the results of the Quindao trial B conducted in February 2018. Vat inoculated with: 2 - Lactobacillus pobuzihii WZ3, 4 - Bacillus amyloliquefaciens # 8, 6 - Tetragenococcus halophilus # 6, 11126 - 6 month moromi inoculum, koji composition. Koji in Qingdao was also contained Weissella spp. as back in the Wuang Zhong (WZ) factory.
  • Fig. 17E is a horizonal stacked bar chart showing the results of Trial C. 16r RNA amplicon bacterial identification. Trial C in Tai Zhou.
  • the highest content of Lactobacillus was 0.5% in 3-4 months of fermentation. 16s rRNA amplicon sequencing picked up only a few hundred Lactobacillus pobuz.ihii at 1 month, accounting for less than 1% of the total bacteria.
  • Fig. 18 shows line graphs depicting the total nitrogen (TN) content and pH values taken during trial a3 over 5 months. Only vats with Bacillus inoculated into moromi together with Tetragenococcus and Lactobacillus. TN content was the highest in experiment with lowest drop in pH for AL
  • Fig. 19 shows a stacked bar chart showing the relative abundance of microbes in traditional BB moromi samples used for metatranscriptomic study. An abundance of 60% of Lactobacillus pobuz.ihii were observed at the first month versus 18% at fourth month of moromi fermentation.
  • Fig. 20 is a heatmap showing the top 20 most abundant peptides of soy sauce maturation in black bean (3 rd _0 - 3 rd _5), yellow bean only, trial al (tank 1 and Tank 2) and wheat (YW) starting from 6-hour koji until 5-month moromi.
  • Fig. 21 shows column graphs representing the profile of taste peptides. Left to right: black bean (BB) moromi (0-5) vs yellow bean (YB) (/ and //) and wheat (YW) (1-5B) over time of fermentation. Colours in (grey) green - umami, (black background) blue - kokumi, pink (white) - sweet, (slash line) yellow - sour, (white dots on grey background) grey - salty, (vertical lines) red - bitternesssuppressing taste.
  • black bean (BB) moromi (0-5) vs yellow bean (YB) (/ and //) and wheat (YW) (1-5B) over time of fermentation.
  • Fig. 22 shows the protease activity of Aspergillus oryzae in moromi and koji samples.
  • Fig. 22A is a column graph depicting the protease activity of WZ and DX starters in YPD medium at 117 hours. Laboratory experiment compared to Pediococcus acidilactici isolated from black soy sauce moromi. Protease activity on starter cultures, salt tolerant bacteria by NaCl. Casein as substrate according to the Folin method as described herein.
  • Fig. 22B shows a scatter plot depicting protease activity measured by FITC-casein express method in the field (Wuang Zhong factory, Silo, Taiwan). Assays were applied at the point of collection taking koji protease activity from bamboo trays.
  • Fig. 23 shows images of the appearance of bacterial isolates from black soy sauce moromi on solid agar plates.
  • Fig. 24 shows a schematic representation of traditional black bean moromi fermentation.
  • Fig. 25 is a line graph showing the concentration of valine -proline -proline (VPP) peptide in 3 types of moromi: black bean 3 rd (0-5 months), yellow bean only moromi, trials with black beans (Tank 1 and Tank 2) and wheat moromi (YW).
  • VPP valine -proline -proline
  • Fig. 26 is a heatmap showing the correlation of volatile organic compounds (VOC) obtained by microbial fermentation in moromi from black bean (BB), yellow bean (Y), and wheat.
  • VOC volatile organic compounds
  • Fig. 27 is a heatmap showing the distinctive volatile organic compounds found in black bean (BB) moromi moths of fermentation BB, yellow beans moromi (Y) and wheat moromi (YW).
  • BB black bean
  • Y yellow beans moromi
  • YW wheat moromi
  • Fig. 28 is a schematic plan of a koji maturation room for inoculation of Bacillus amyloliquefaciens starter culture by sprinkling.
  • Fig. 29 shows a horizontal column graph depicting the protease activity in an ex vivo laboratory trial 500 mL experiment. Timepoints taken are 1 day and 10 days.
  • Fig. 30 shows a horizontal column graph depicting the amylase activity in an ex vivo laboratory trial 500 mL experiment. Timepoints taken are 1 day and 10 days.
  • Fig. 31 shows a horizontal column graph depicting the lipase activity in an ex vivo laboratory trial 500 mL experiment. Timepoints taken are 1 day and 10 days.
  • Fig. 32 shows a line graph depicting the microbial abundance in Pilot 1 trial al.
  • Fig. 33 shows a LefSe analysis of pilot 1 large scale trial. 1A - traditional black bean moromi at month 1. In contrast, tank with pilot 1 showed the list of wild strains playing the main role in moromi. Most of species belong to Weissella sp strains.
  • Fig. 34 shows the results of adaptation of Lactobacillus pobuzihii strains at various concentration NaCl in De Man, Rogosa and Sharpe broth (MRS) and with supplementation of soy lecithin and tween-80.
  • Fig. 34A shows line graphs depicting growth curves of WZ3 and WZ5 under 10-18% NaCl (v/w) in De Man, Rogosa and Sharpe broth and with additional of choline.
  • Fig. 34B shows line graphs depicting ranges of soy lecithin concentrations under 0, 16, 17% of NaCl (w/v) at De Man, Rogosa and Sharpe broth.
  • Fig. 34C shows line graphs depicting the cell concentration after addition of tween 80 to lecithin in 16 % and 17 % NaCl in De Man, Rogosa and Sharpe broth.
  • Fig. 35 shows the lag phase reduction of Lactobacillus pobuzihii live cells supplied with soy lecithin at 16 -17% of NaCl (concentration of salt used at factory manufactures) and down/upregulated genes at 17% of NaCl vs MRS and with/without soy lecithin.
  • Fig. 35A shows line graphs depicting ranges of soy lecithin concentrations under 0, 16, 17% NaCl (w/v) at De Man, Rogosa and Sharpe broth.
  • Fig. 35B shows column graphs depicting cell counts taken of Lactobacillus pobuzihii WZ3 during growth on De Man, Rogosa and Sharpe broth with soy lecithin (SL).
  • Fig. 35A shows line graphs depicting ranges of soy lecithin concentrations under 0, 16, 17% NaCl (w/v) at De Man, Rogosa and Sharpe broth.
  • Fig. 35B shows column graphs depicting cell counts taken of Lac
  • 35C shows a volcano plot of Lactobacillus pobuzihii WZ3 gene expression at 2 mM lecithin with salt vs Salt without lecithin added.
  • Four genes were significantly upregulated (red) with addition of lecithin: manX_2, manX_3, manZ_4 and sorA.
  • First three genes are associated with mannose transport and sorA is responsible for sorbose utilization.
  • Fig. 35D shows a volcano plot of up-regulated genes under salt stress - argF Ornithine carbamoyltransferase responsible for production of citrulline.
  • Fig. 35E is a schematic showing the pathway of terminal reactions of degradation of amino acid L-citrulline upregulated in Lactobacillus pobuzihii WZ3 under salt conditions.
  • Fig. 36 shows a column graph depicting the relative abundance of 3- to 6-mer peptides.
  • the relative abundance of 3- to 6-mer peptides shown in Tank 1 and Tank 2 were obtained after inappropriate fermentation (after 3.5 months of brewing). It is shown that Tanks 1 and 2 have same amount of taste peptides as present in black bean moromi at the beginning of moromi fermentation (at one month). This data indicates that the presence of L. pobuzihii in the black moromi is necessary to enrich amount of taste peptides during the time of brewing.
  • Fig. 37 shows a schematic of pilot trial with a Bacillus amyloliquefaciens strain having been introduced at beginning of koji stage.
  • Koji room was split into 3 zones which were planned for conducting of experiment for assessment of impact of introduced B. amyloliquefaciens on the inhibition of Weissella spp. population.
  • the left zone contained only A. oryzae spores without any additional bacterial strain (negative control);
  • the middle zone served as a buffer zone containing trace amounts of B. amyloliquefaciens', and the right zone of koji was sprayed with B. amyloliquefaciens culture.
  • Table 4 Summary of most prevalent microbes in high-grade black bean soy sauce moromi vs generic wheat soy sauce moromi. Both soy sauces are produced in Wuang Zhong (WZ) factory, Silo, Taiwan. [0056] Table 5. Fermentation products produced with pure cultures on De Man, Rogosa and Sharpe (MRS) broth with 14% NaCl.
  • Table 8 Scheme started cultures addition for trial a4 with aim to enhance taste of industrial black bean (BB), yellow bean (YB), and wheat (YW) soy sauces.
  • BB black bean
  • YB yellow bean
  • YW wheat
  • Table 9 Metatranscriptome output of moromi fermentation at 1 and 4 months. The most abundant species including Lactobacillus pobuzihii and Tetragenococcus halophilus are shown at 1 month (1A, IB, 1C) and 4 month (4A, 4B, 4C) of moromi fermentation.
  • Table 10 Microorganisms which had a high abundance in Wuang Zhong (WZ) soy sauce moromi with some have been already isolated on the agar plate. Metagenome results provide guidance for key microorganisms responsible for moromi quality. Isolates were identified by 16s rRNA i
  • Table 12 Experimental design for laboratory trial with 600 mL moromi with various combinations of starter wild cultures.
  • B Bacillus amyloliquefaciens #8, W - Weisella paramensteroides #1, Ec - Enterobacter cloacea, Ef - Enterococcus faecalis, Sc - Staphylococcus sciuri, Sg - Streptococcus gangivalis, M - Micrococcus luteus.
  • B1-B5 Bacillus amyloliquefaciens added with aim to test Bacillus amylolysin for Weissella inhibition; C1-C2 only addition of Tetragenococcus halophilus and Lactobacillus pobuzihii WZ3, C3-C4: Weissella starter, C5: no starter cultures added, A1-A2: Bacillus amyloliquefaciens was added into moromi together with Tetragenococcus halophilus and
  • Table 15 Diffusion disk assay results of bacteriocins screening from wild soy moromi strains Bacillus amyloliquefaciens #8 (B), Weissella paramensteroides #1 (W), and nisin producing type strains Lactobacillus lactis B-978 and Lactobacillus lactis B-1948. No culture - negative control without addition any bacteriocins, x - indicates no growth, number indicates diameter of halo spread around disk.
  • Table 16 Table of correlations (Pearson and R2) between microbial abundances (Metaphlan2 relative numbers) of 3 types of soy sauce moromi (BB, Y and YW) and concentrations of main volatile organic compounds (VOC) measured in the same samples. Bacterial species and related VOC are ranked by the strongest correlations from top to the bottom. Microbial values are given at left hand side and VOC values are given at the right-hand side of the table.
  • Table 17 -List of class Ila bacteriocines found in the public NCBI database based on original weissellin A bacteriocin amino acid sequence from Weissella paramensteroides
  • Table 18 Bacteriocins identified from whole genome sequences of various microbial species deposited in NCBI, Pfam and InterProScan protein databases, which were found to be similar to Bacillus amyloliquefaciens bacteriocin obtained from Wuang Zhong (WZ) samples.
  • Table 19 Diffusion disk assay of potential bacteriocins produced by soy sauce isolates Weissella paramensteroides and Bacillus amiloliquefaciens and their impact (halo spread size, in mm) on other members of soy sauce microbial community.
  • 6 Bacillus amyloliquefaciens #S; 16 - Weissella paramensteroides #1; N - bacteriocin nisin; 43 - Lactococcus lactis B-978 (nisin producer); 44 - Lactococcus lactis B-1948 (nisin producer) ; ? - unclear; x - no effect.
  • Table 20 List of volatile organic compounds (VOC) their flavour and taste profiles, as well as their detection threshold. Also shown is the concentration found in black bean (BB), yellow bean (Y), and yellow wheat (YW) moromi at 5, or 5.5, months fermentation.
  • VOC volatile organic compounds
  • Table 21 Comparative analysis of proteins from whole genome sequencing (WGS) of L. pobuzihii WZ3 and WZ5 indicating the number of proteases, peptidase and amylases related to cleavage of protein resulting in release of taste peptides and sugars from the starch.
  • Table 22 -List of upregulated genes under salt stress for L. pobuzihii WZ3. Output was generated as results of RNA-seq on De Man, Rogosa and Sharpe broth (MRS) versus MRS + 17 % NaCl (SALT). Id - gene abbreviation; log2FC - fold change in log2 value; p-adj - FDR > 0.01; description - functions of upregulated genes.
  • the term “umami” is defined as the taste of the amino acid L-glutamate and 5’- ribonucleotides, such as glutamates (salts of glutamic acid), the amino acid L-glutamate, guanosine monophosphate (GMP) and inosine monophosphate (IMP). It can be described as a pleasant "brothy” or “meaty” taste with a long-lasting, mouth-watering and coating sensation over the tongue.
  • kokumi which translates as “heartiness”, “full flavour” or “rich”, describes compounds in food that do not have their own taste but enhance the characteristics when combined. Alongside the five basic tastes of sweet, sour, salty, bitter and savoury, kokumi has been described as something that may enhance the other five tastes by magnifying and lengthening the other tastes, or “mouthfulness”. Garlic is a common ingredient to add flavour used to help define the characteristic kokumi flavours.
  • Calcium-sensing receptors are receptors for "kokumi" substances. Kokumi substances, applied around taste pores, induce an increase in the intracellular Ca concentration in a subset of cells.
  • CaSR-expressing taste cells is independent from the influenced basic taste receptor cells.
  • CaSR agonists directly activate the CaSR on the surface of taste cells and integrated in the brain via the central nervous system.
  • a basal level of calcium corresponding to the physiological concentration, is necessary for activation of the CaSR to develop the kokumi sensation.
  • 3- to 6-mer peptides refers to oligomers consisting of 3, 4, 5, or 6 amino acids in length.
  • high salt fermentation condition refers to fermentation conditions using a salt content of up to 17%.
  • Lactobacillus strains disclosed herein are able to tolerate conditions including up to 17% salt content.
  • culture refers to a cell culture or a bacterial culture.
  • Such cell or bacterial culture also include amounts of bacteria used to inoculate clean substrate, such as, but not limited to, starter cultures for inoculating fermentation.
  • microaerophilic refers to an environment containing lower levels of dioxygen than that are present in the atmosphere (such as, less than 21% O2; this can range between 2 to 10% O2) for bacterial growth.
  • Japanese-style liquid fermentation requires addition of a specific starter culture of Zygosaccharomyces rouxii, which in the presence of 55% wheat will produce a smell and alcohol content that is characteristic of this type of moromi.
  • the starter culture Tetragenococcus halophilus can be added as well. Wild species Candida etchellsii and Candida versatilis have been reported for their specific flavour formation with 4-ethylphenol, along with main bacterial species such as Weissella cibaria, Bacillus spp., Lactobacillus fermentum, Streptococcus gallinarum, and Staphylococcus saprophyticus.
  • Korean-style soy sauce fermentation can be considered as a modification of Japanese-style soy sauce fermentation, which includes addition of Zygosacchramyces spp.
  • Co-abundant wild yeasts such as, but not limited to, Candida temnochilae, Pichia guilliermondii, Pichia sorbitophila, Pichia triangularis, Absidia corymbifera, Rhodotorula mucilaginosa, and bacteria, such as, but not limited to, Enterococcus durans, Bacillus subtilis, and Enterococcus faecium had been previously reported as part of a moromi flavour formation.
  • Chinese-style soy sauce moromi is characterised by presence of wild species of Candida spp., Kluyveromyces marxianis, Pichia fabianii, Weissella cibaria, Weissella confusa, Bacillus spp., Corynebacterium qlutamicum, Staphylococcus gallinarum, Staphylococcus saccharolyticus, and Enterococcus faecalis, without addition of specific Zygosacchramyces rouxii.
  • Taiwanese soy sauce characterised by a lack of wheat and yeasts in liquid fermentation, can be considered a modification of Chinese-style soy sauce.
  • Few wild bacterial species are involved at the moromi stage, such as, but not limited to, Staphylococcus sciuri, Klebsiella pneumoniae, Enterobacter cloacae, Salmonella enterica, Enterococcus faecium, Weissella confusa, Bacillus amyloliquefaciens , and Staphylococcus gallinarum.
  • yeast Zygosaccharomyces rouxii and some lactic acid bacteria are widely used for mass-production of wheat-based soy sauce
  • other fermenting species are wild origin which bring very generic taste characteristics to the final product.
  • Lactobacillus pobuzihii also referred to as L. pobuzihii
  • Lactobacillus pobuzihii as a starter culture for the production of Taiwanese-style, gluten-free black bean soy sauce with shows an enriched umami and kokumi taste and has an increased content of valine-proline-proline (VPP) peptide.
  • VPP valine-proline-proline
  • Lactobacillus pobuzihii can be used in gluten-free soy sauce to obtain a “meaty taste”.
  • a culture or starter culture comprising Lactobacillus pobuzihii WZ3 (accession number DSM 33648), or Lactobacillus pobuzihii WZ5 (DSM 33658), or a combination thereof.
  • the culture or starter culture disclosed herein further comprises wild-type Lactobacillus pobuzihii.
  • the Lactobacillus pobuzihii strain is selected from the group consisting of wild-type Lactobacillus pobuzihii; Lactobacillus pobuzihii WZ3 (DSM 33648); Lactobacillus pobuzihii WZ5 (DSM 33658); a combination of Lactobacillus pobuzihii WZ3 (DSM 33648) and Lactobacillus pobuzihii WZ5 (DSM 33658); a combination of wild-type Lactobacillus pobuzihii and Lactobacillus pobuzihii WZ3 (DSM 33648); a combination of wild-type Lactobacillus pobuzihii, Lactobacillus pobuzihii WZ5 (DSM 33658); and combination of a wild-type Lactobacillus pobuzihii, Lactobacillus pobuzihii WZ3 (DSM 33648), and Lactobacillus
  • the culture disclosed herein is for use in fermentation.
  • the fermentation is soybean fermentation, or moromi fermentation.
  • Exemplary steps of such a fermentation process include, but are not limited to, the steps of a koji maturation, one or more optional washing steps, moromi fermentation and maturation, moromi pressing, and pasteurisation.
  • the process disclosed herein comprises all of the steps described above.
  • soymilk fermentation involves aqueous extraction of soybeans, which is completely different processing compared to soy sauce fermentation.
  • soy sauce fermentation mash of whole soybeans gets immersed into 18% salt bran solution. Therefore, bacteria which are responsible for formation of taste peptides have to be extremely salt tolerant.
  • common Lactobacillus species found in milk cannot survive and grow under salt concentration higher than 5%.
  • the starter culture disclosed herein is a moromi fermentation starter culture.
  • the method disclosed herein applies to the strains disclosed herein which are used as starter cultures for fermentation.
  • Each batch of moromi is to be inoculated with IxlO 7 of salt tolerant Lactobacillus pobuzihii cells.
  • These Lactobacillus pobuzihii cells had been prepared in a fermenter with supplementation of 2 mM soy lecithin in MRS (De Man, Rogosa and Sharpe broth) medium or equivalent as osmoprotector, prior to application in fermentation. Addition of soy lecithin is essential for faster adaptation of Lactobacillus pobuzihii to high salt stress.
  • taste is characterized by relative abundance of taste peptides.
  • a heatmap of taste peptides (as shown herein, for example) reflects as quality (name of short peptides), as well as the relative quantity (relative numbers) of such peptides. It has been shown herein that the amount of taste peptides accumulates within maturation of moromi, as can be seen, for example, in case of black bean Wuang Zhong (WZ) soy sauce, while Lactobacillus pobuzihii abundance increase over the time.
  • taste peptides of yellow and wheat soy sauce formed mainly during the koji stage due to the contribution of Aspergillus oryzae present in the standard culture. Once the koji is immersed in salt brine, the activity of fungal protease (thought to be responsible for cleavage protein) is inhibited. Thus, only osmotolerant bacterial cultures will contribute to the number of unique taste peptides present in the resulting product.
  • flavour is the result of the presence of volatile organic compounds (VOC).
  • volatile organic compounds usually refers to classes of volatile organic compounds, such as, but not limited to, pyrazines, phenols, acetate, ethanol, and the like, present in soy sauce.
  • GC/MS analysis gas chromatography coupled mass spectrometry analysis.
  • concentration of these compounds was measured in moromi samples of three different types of soy sauces.
  • the resulting heatmap shows the concentrations of all volatile organic compounds.
  • the concentration of each measured compound is divergent by nature (e.g., pyrazines can be smelled by a human at a concentration of less than 0.001 mg/mL, while ethanol can only be detected/smelled by humans at a concentration of 1 mg/mL), thus the resulting values had been normalised in order to be able to build the heatmap. Due to this normalisation, all volatile organic compounds concentrations can be visualized at the same figure. Examples of absolute concentrations for each sample are provided in Table 20.
  • the purpose of the heatmap disclosed herein is to provide a representation of district features of black bean moromi compared to standard yellow and wheat moromi. As known in the art, overall flavour characteristics can be subjective (e.g., flavour preferences for soy sauce are formed and influenced based on geographical locations within China). Thus, focus was concentrated on the improvement of taste and flavour characteristics of the soy sauce disclosed herein.
  • Also disclosed herein is a method for providing taste and flavour characteristic of a traditional Taiwanese black bean soy sauce by using an inoculating mix of Lactobacillus pobuzihii during moromi fermentation.
  • the addition of Lactobacillus pobuzihii has been shown to reduce the number of undesirable wild species of Weissella spp. and Staphylococci spp., thus lowering ethanol and acetaldehyde content, and resulting in the production of valine -proline -proline (VPP) peptide.
  • the lowering of alcohol and acetaldehyde has been shown to improve kokumi and umami taste profiles.
  • Valine -proline-proline (VPP) peptide has been shown to possess angiotensin inhibiting properties.
  • undesirable taste components are, but are not limited to, ethanol, aldehydes, octanoic acid, ethyl acetate, methyl-butanol, benzene acetaldehyde, 4-ethyl-2-methoxy phenol, benzaldehyde, acetaldehyde, octanoic acid ethyl ester, 2-furanmethanol, propanoic acid, 2-hydroxyethyl ester, 3-methyl-l -butanol, and combinations thereof.
  • the undesirable taste components as disclosed herein can be considered to undesirable taste components only if a respective odour threshold is passed.
  • undesirable taste components are, but are not limited to, benzeneacetaldehyde, 4-ethyl-2-methoxy phenol, benzaldehyde, acetaldehyde, octanoic acid ethyl ester, 2-furanmethanol, 2-hydroxyethyl ester, 3-methyl-l -butanol, ethanol, octanoic acid, ethyl acetate, and methyl-butanol.
  • taste components for example, those that impart bitter or sour tastes
  • these may only be considered undesirable if a certain odour threshold is exceeded.
  • the increase in desirable taste components is characterised by the increase in taste conferring peptides and/or taste conferring amino acids in relation to total protein content of a sample.
  • Examples of taste conferring peptides and/or taste conferring amino acids are, but are not limited to, VPP, EV, EE, DES, ED, K, EEDGK, KGSLADEE, D, DE, DD, DEE, VE, VD, KGDEE, ADE, EGS, ES, EDD, EED, DDE, DED, DDD, EEE, EDE, E, SLAKGDEE, SLADEEKG, KGDEESLA, DA, VG, VV, LE, EL, EG, EY, V, P, SPE, EEN, EPAD, VGV, FFRPFFRPFF, GP, RRPFF, VYPFGGGINH, PR, LK, WP, FFPG, RGPPF, GGP, RKE, RPGGFF, YGY, RGPPGGF, RGPPGIG, PK, RGPPFIVRGPPFIV EM, GGFFGG, VF, DLL, YGG
  • the taste conferring peptides and/or taste conferring amino acids is/are, but are not limited to, EV, EE, DES, ED, EEDGK, KGSLADEE, D, DE, ECG, LT, EH, EQ, ECA, LA, LQ, VGV, AAA, AA, GGA, GAA, KGD, AGA, VD, KGDEE, ADE, E, SLAKGDEE, SLADEEKG, KGDEESLA, SPE, DD, DEE, EDD, VE, DA, VG, VV, LE, and RPGGFF.
  • CDSs coding sequences
  • xtmB phage terminase large subunit
  • gspA-1 universalal stress protein A
  • uxaC glucuronate isomerase
  • uxuB mannitol dehydrogenase
  • uxuA mannonate dehydratase
  • uxaA altronate hydrolase
  • iclR IclR family transcriptional regulator
  • bglG transcriptional antiterminator
  • yorL putative DNA polymerase YorL
  • yxaB general stress protein 30
  • ggaB minor teichoic acid biosynthesis protein GgaB
  • arcR ArcR family transcriptional regulator
  • malA maltodextrose utilization protein malA
  • wcaJ UDP-phosphate galactose phosphotransferase
  • vapl toxin-antitoxin system antitoxin subunit
  • sacA sucrose-6- phosphate hydrolase
  • relB relB
  • the moromi of wheat soy sauce did not contain any traceable Lactobacillus pobuzihii present in tanks examined with same shotgun metagenomics method.
  • the wheat soy sauce (YY) microbial population consisted of Weissella spp., Bacillus amyloliquefaciens, Staphylococcus spp., and Tetragenococcus halophilus species dominated in liquid fermentation (moromi). It was also observed that only the amount of B. amyloliquefaciens bacteriophages correlated with the population of bacterial B. amyloliquefaciens present in moromi (data not shown).
  • the method or fermentation process disclosed herein includes the introduction/addition of bacteriophages.
  • the bacteriophages introduced into the method are bacteriophages that infect Bacillus amyloliquefaciens.
  • the bacteriophages do not infect Staphyloccus scuiri and Weisseilla par noirroides strains.
  • the bacteriophages infect species that compete with Weissella spp. and Staphylococcus spp.
  • Bacteriophages can be introduced, for example, by inoculation.
  • bacteriophages are present in the starter culture.
  • bacteriophages are introduced at a concentration of at least 1 x 10 6 PFU/L.
  • the bacteriophages are introduced during the first month of moromi fermentation.
  • the first upregulated pathway clusters during koji maturation included branch chained amino acid formation with L-valine and L-isoleucine biosynthesis, sulphur containing essential amino acids such as L-methionine and L-homoserine biosynthesis, aromatic amino acids anabolism with L-tryptophan degradation pyruvate fermentation of isobutanol, and leukotriene biosynthesis during aerobic respiration related to cytochrome c.
  • Sulphate reduction, peptidoglycan maturation, fatty acid biosynthesis, palmitoleate biosynthesis, fatty acid elongation oleate biosynthesis formed another cluster which was shown to be upregulated during koji maturation. Polysaccharide degradation was detected based on stachyose degradation.
  • Moromi fermentation pathways showed few specific upregulated clusters with nucleotide synthesis: adenosine, guanosine and pyrimidine biosynthesis, GDP-mannose biosynthesis.
  • Presence of lactic acid bacteria (LAB) provided upregulation of oligosaccharides involving sucrose, lactose and galactose degradation.
  • the pentose phosphate pathway was also upregulated, indicating that lignin fractions of soybean bran (for example, arabinoxylan, xylan) can be actively involved in fermentation.
  • Lactobacillus pobuzihii was chosen as primary culture for the isolation, as this lactic acid bacterium (LAB) was found to play a role in unique taste and flavour formation of black bean moromi.
  • Lactobacillus pobuzihii in 4-month of fermentation, two cultures were isolated from vat 1A and IB of the 1-month, WZ3 and WZ4 together with Bacillus amyloliquefaciens. While Bacillus amyloliquefaciens abundance shown to be around 5%, Lactobacillus pobuzihii abundance shown to be less than 1%. Nevertheless, the presence of live Lactobacillus pobuzihii in the moromi indicated that wild strains had been present at the beginning of fermentation, and which can be used as strategy to inoculate this bacterium right after the beginning of moromi stage.
  • Lactobacillus pobuzihii WZ5 (DSM 33658) was isolated from the 4A vat which showed the highest abundance of Lactobacillus pobuzihii. Two strains of Tetragenococcus halophilus were also isolated from month 4 old moromi. It was noted that Lactobacillus pobuzihii WZ5 (DSM 33658) showed less resistance to salt solution compared to Lactobacillus pobuzihii WZ3 (DSM 33648). However, the growth rate of Lactobacillus pobuzihii WZ5 was shown to be faster than Lactobacillus pobuzihii WZ3 on De Man, Rogosa and Sharpe broth medium without salt. Lactobacillus pobuzihii strain WZ3 was resistant up to 17% of salt while growing on De Man, Rogosa and Sharpe broth and was chosen as starter culture for laboratory and pilot trials at industrial scale.
  • the at least one Lactobacillus pobuzihii strain is adapted to tolerate high salt concentrations in an adaptation step.
  • Lactobacillus pobuzihii cells Prior inoculation into moromi, Lactobacillus pobuzihii cells were inoculated into growth media (1 m 3 ) with 2 mM soy lecithin at 17% salt for 5 days until OD600 of 0.6 was reached. Thereafter, the adapted starter culture was added to a tank (capacity of about 100 tons).
  • the Lactobacillus pobuzihii strain is adapted to tolerate high salt concentrations prior to its use in fermentation.
  • the Lactobacillus pobuzihii strain is made to tolerate high salt concentrations. In one example, this is done prior to inoculation and/or fermentation using the Lactobacillus pobuzihii strain disclosed herein.
  • Such an adaptation step can include use of an osmoprotector, for example, soy lecithin.
  • Lactobacillus pobuzihii WZ3 and Tetragenococcus halophilus #6 were found to reduce pH to 6.0 in De Man, Rogosa and Sharpe broth, but glutamic acid (0.027 g/L) was only detected in the presence of Lactobacillus pobuzihii WZ3. Thus, Lactobacillus pobuzihii WZ3 should not be considered as spoilage agent of soy sauce.
  • the process disclosed herein includes use of a starter culture further comprises bacteria selected from the group consisting of Tetragenococcus halophilus, Bacillus amyloliquifaciens, and combinations thereof.
  • inoculation at 1% of inoculum (for example, with IxlO 7 cells) into 17% salt solution with koji immersed in it. Temperature of water is 25- 28°C and initial pH range from 5.6-6.0. Mixing with air should be avoided, otherwise Weissella and Staphylococcus will overgrow.
  • the fermentation process as disclosed herein is performed under microaerophilic conditions, or under conditions which are sufficiently anaerobic to enable preferential growth of Lactobacillus pobuzihii over Weissella and Staphylococcus.
  • Lactobacillus pobuzihii WZ3 and WZ5 strains were sequenced, assembled, and compared to the strain Lactobacillus pobuzihii E100301 which had been deposited as KCTC 13174.
  • Lactobacillus pobuzihii El 00301 has been described as homofermentative lactobacilli, which were unable to utilize mannose, mannitol, xylose, sorbose, dulcitol, inositol, sorbitol, fucose, arabitol, raffinose and inulin.
  • Lactobacillus pobuzihii WZ3 strain One of feature of the Lactobacillus pobuzihii WZ3 strain is its ability to utilize pentose sugars by altronate hydrolase (catalyzing the conversion of D-altronate into 2-dehydro-3-deoxy-D-gluconate) and glucuronate isomerase, in contrast to common hexose sugar fermentation.
  • Another feature distinguishing Lactobacillus pobuzihii WZ3 from KCTC 13174 Another feature distinguishing Lactobacillus pobuzihii WZ3 from KCTC 13174 is the presence of mannitol dehydrogenase and mannonate dehydratase, which results in the classification of Lactobacillus pobuzihii WZ3 to obligatory heterofermentative lactobacilli group type 3.
  • 6-PG/PK 6- phosphogluconate/phosphoketolase
  • one ATP molecule can be generated and only one molecule of acetate is formed from acetyl-CoA, which can explain the reduced acetate formation in Lactobacillus pobuzihii WZ3 compared to the Lactobacillus pobuzihii KCTC13174 strain.
  • This is metabolic function is specific to Lactobacillus, as only few species were described before with this metabolic function.
  • Aspergillus oryzae DX can be used as complete substitute of Aspergillus oryzae ⁇ NA strain.
  • the solid fermentation (koji) will not affect the quality of black bean moromi.
  • Koji fermentation is controlled by Aspergillus oryzae. Since koji maturation is stable and fixed, it is thought that the improvement of taste is based on addition of the Lactobacillus pobuzihii WZ3 and/or WZ5 strains disclosed herein to the liquid fermentation stage or to moromi.
  • protease activity during black bean koji making in traditional process the time of koji making could thereby be shortened from 7 days to 4 days.
  • protease activity of Aspergillus oryzae was fully inhibited under 17% salt, leaving salt-resistant bacteria as the primary peptide contributors during a 5-month moromi fermentation.
  • the moromi maturation can be potentially shortened from 6 months to 4 months with use of the Lactobacillus pobuzihii strains disclosed herein.
  • the number of tasty peptides in the sample reached a maximum. It was shown that moromi fermentation for longer than 4 months did not provide further enrichment in taste peptides.
  • the moromi fermentation and maturation as disclosed herein is shortened due to the use of Lactobacillus pobuzihii compared to a moromi maturation without the use of Lactobacillus pobuzihii.
  • the fermentation process disclosed herein results in an increase in umami, and/or an increase in taste-conferring components. Also disclosed herein is the use of Lactobacillus pobuzihii for taste enhancement of moromi. In one example, as Lactobacillus pobuzihii is used as a main culture component.
  • VPP Valine-proline-proline
  • VPP valine-proline-proline
  • soy sauce product comprising at least one peptide comprising the sequence of Val-Pro-Pro (VPP). That is to say, at least one of enriched peptides valineproline -proline (VPP) disclosed herein has anti-angiotensin properties.
  • anti-angiotensin properties such as, an increase is vasodilation
  • Black bean moromi had distinct volatile organic compound (VOC) profiles and associated with lack of Weis sella, Bacillus, Kurthia, and Enterococcus, and the presence of Enterobacteria bacteriophages [00140] Gas chromatography/mass spectrometry (GC/MS) analysis found over 150 various volatile organic compounds (VOCs) when examining three types of moromi. A comparison of volatile organic compounds is presented in Fig. 5, Fig. 6, and Fig. 27.
  • VOC volatile organic compound
  • Black bean (BB) 5-month moromi (that is, moromi just before pasteurisation) had following distinctive flavour characteristics: benzene acetaldehyde - honey, floral rose, sweet; 4-ethyl phenol - smoky, phenolic, creosote and savoury; 2-methyl-propanoic acid - acidic sour cheese dairy; 2-hydrooxy- ethyl ester propanoic acid,- sweet, fruity, acidic; 2-methyl-, methyl ester propanoic acid - sweet, fruity;
  • the flavour compounds which had distinct profiles for black bean moromi included but were not limited to, hydroxy-2-butanone, butanoic acid, pentyl octanoate and ace
  • VOCs volatile organic compounds
  • stringent flavours such as, but not limited to, ethanol (phenyl ethanones, benzyne alcohol), aldehydes (2-methyl butanal), ethyl acetate.
  • Lactobacillus pobuzihii was correlated to higher concentration of isopropyl alcohol, isopropyl octanoate, 4-ethyl phenol, 2-methoxy phenol and 2,6-dimethyl pyrazine (Fig. 26). Bacillus amyloliquefacies had similar correlation values as shown for Lactobacillus pobuzihii.
  • VOCs Distinctive volatile organic compounds
  • BB moromi black bean
  • BB moromi black bean
  • 4-ethyl phenol and benzeneacetaldehyde were associated with an abnormal increase of Halococcus unclassified in the final month of moromi fermentation at 13% relative abundance (to the overall microbial population), with Halococcus thailandensis and Halococcus morrhuae present in very low abundance (0.005 - 0.26%).
  • a caramel flavour found in the second month of black bean (BB) moromi was represented by the presence of maltol, 3-methyl-3-buten-l-ol, 3-octanone, butyrolactone, n-Decanoic acid and ethyl ester of decanoic acid.
  • These fragrances were found to be lacking in other types of moromi analysed, and were associated with an abundance of 1.5% of Cronobacter bacteriophage vB CsaP GAP52 and 1.5% Enterobacteria bacteriophage CC31. Since the population of Enterobacteria was high in yellow bean (YB) moromi, the high abundance of bacteriophages was thought to contribute to Enterobacteria lysis, thereby contributing to the specific flavour profile.
  • YB yellow bean
  • Bacillus amyloliquefaciens bacteriocins including amylolisin
  • 25 genes were found to have a similarity to bacteriocins of Bacillus subtilis, Enterococcus faecium, Staphylococcus sciuri and Staphylococcus gallinarum (Table 18). Indeed, these bacteria were found to be major species present in wheat (YW) fermentation and represented a minority in black bean (BB) moromi.
  • undesired bacteria can be, but are not limited to, bacteria known to contribute to product spoilage, as well as bacteria which result in an unpalatable flavour of the product.
  • the undesired bacterium is, but is not limited to, Weissella sp., Weissella cibaria, Weissella paramenesteroides, and Enterococcus casseliflavus.
  • Also disclosed herein is a method of reducing the concentration of Weissella sp. during fermentation, the method comprising the use of the culture disclosed herein.
  • Lytic bacteriophages of wild strains of Weissellla paramensteroides, Bacillus spp and Staphiloccus scuiri were not obtainable from soy sauce wastewaters or soy sauce brine, compared to positive T2 proliferation on an E. coli lawn.
  • Weissellla cibaria bacteriophages phi YS60 had been reported in kimchi spoilage and were found to be important in controlling the dynamic of lactic acid bacteria (LAB) in kimchi fermentation.
  • Traditional black bean (BB) moromi contains markers of a similar Weissella bacteriophage phi YS65 and presence of such phage perhaps is thought to protect fermentation from Weissella spoilage.
  • Staphyloccocus xylosus Staphyloccocus scuiri and Staphylococcus saprophiticus are the second major group found during the ripping of salami in France, Spain and Greece.
  • Weissella spp caused a poor quality of crushed black beans moromi in the pilot tank 1 and tank 2. Usage of oat bran did not protect Weissella overgrowth during koji maturation stage. Overgrowth of Weissella spp could be also controlled by the Weissella bacteriophage phiYS61, which was detected in a high abundance in vats at month 4 of traditional black bean (BB) moromi.
  • BB black bean
  • Staphylococcus spp and Enterococcus spp were presented only in Tank 1 with oat bran, where Weissella was reduced by 10%.
  • Staphylcoccus. saprophyticus presented one third of total microbial population in wheat (YW) moromi during initial month under controlled 15 °C temperature. However, after 5 months of fermentation, population of staphylococci decreased down to 3%, together with an increase of Tetragenococcus halophilus (40%), Bacillus amyloliquefaciens (27%) and Weissella spp. (20%; Fig. 12C).
  • Lactic acid bacteria including but not limited to, Weissella spp., Tetragenoccocus spp., Lactobacillus spp., can produce antimicrobials against Staphylococcus spp. and others. Therefore, it was thought to keep a number of these lactobacteria in the moromi to prevent staphylococci overgrowth and other potential foodborne pathogenic bacteria.
  • staphylococci bacteriophages GH15 were observed for several months. Therefore, high titres of these bacteriophages is thought to be another type of biocontrol of staphylococci in the fermentation tanks.
  • usage of bacteriophages in soy sauce is not regulated by food regulatory standards.
  • protease activity of Bacillus amyloliquefaciens together with Tetragenoccocus halophilus and Pediococcus spp. are known in the art, and such protease activity can contribute to taste formation of the final product during month 5 of the moromi fermentation.
  • Bacillus amyloliquefaciens addition into the moromi did not improve the taste of the product.
  • Lactobacillus pobuzihii inoculation improved the taste and flavour reported by sensory panel (Table 13).
  • Shotgun metagenomics of trial a2 and trial a3 demonstrated the prevalence of Weissella, as in trial al, and showed a lack of Lactobacillus in all vats (Fig. 17B).
  • Weissella was present for up to 70% in moromi from 1 to 7 months of fermentation under 17.5% salt conditions.
  • Tetragenoccocus halophilus developed up to 20%, without need of its addition of as starter culture.
  • Pediococcus pentosaceus which was not sampled at the Xiluo factory moromi, was shown to be present in an abundance of up to 17% of the total amounts.
  • Trial b was started in February 2018 in Qingdao (China) and was conducted under more moderate weather conditions (ambient temperature in February in Quindao was around 5°C, compared to 22°C in Taiwan) than the trials in Taiwan. Vats were inoculated with monocultures of Lactobacillus pobuzihii WZ3, Bacillus amyloliquefaciens #8, or Tetragenoccocus halophilus #6 at the beginning of the moromi stage and kept at temperatures not exceeding 25°C. In addition, 6-month moromi of black beans (BB) from the Xiluo factory was used as starter culture.
  • BB black beans
  • a wild Lactobacillus lactis was observed to be present at around 1 % abundance, which was previously reported to be resistant to up to 7% salt and ethanol.
  • a vat with Lactobacillus pobuzihii was shown to contain 7% of Methanosaeta concilii, a methanogen usually found in low acetate environment with strict anaerobic conditions. It is of note that these archaea Methanosaeta concilii (with an abundance of 5%) were also found in one of the traditional Xiluo vats, together with Lactobacillus pobuzihii (at 62.4% abundance; Fig. 19).
  • trial C was conducted in Tai Zhou, China, with 1.3 m 3 inoculum of Lactobacillus pobuzihii WZ3 (DSM 33648) in large tanks.
  • Trial C revealed an abundance of Lactobacillus spp. of 0.5- 1% at 4 months of moromi fermentation, while wild Tetragenoccocus halophilus was present in up to 70% abundance. Since this trial was analysed using amplicon sequencing (which is a type of targeted next generation sequencing that uses PCR to create sequences of DNA called amplicons, and thereby allowing for analysis of specific genomic regions), it was understood that this lactobacilli species belonged to Lactobacillus pobuzihii.
  • glycine betaine was tested as a primary compound, together with choline, and other chemicals containing choline molecules, such as, but not limited to, raw soy lecithin containing phosphatidyl choline (which is a by-product of defatting of soybean of the company’s factories).
  • choline and other chemicals containing choline molecules, such as, but not limited to, raw soy lecithin containing phosphatidyl choline (which is a by-product of defatting of soybean of the company’s factories).
  • Ability of Tetragenoccocus halophilus to metabolise choline and accumulate the choline metabolite, glycine betaine enabled Tetragenoccocus halophilus to be adapted to high salt conditions in soy moromi.
  • Lactobacillus pobuzihii WZ3 (DSM 33648) actively utilises glycerol with gldA (COG0371 Glycerol dehydrogenase and related enzymes) and the accumulation of glycerol can help Lactobacillus pobuzihii to survive under salt stress.
  • gldA COG0371 Glycerol dehydrogenase and related enzymes
  • an adaptation step is carried out prior to inoculation.
  • the adaptation step comprises use of an osmoprotector.
  • the culture disclosed herein is adapted to tolerate high salt fermentation conditions.
  • the osmoprotector disclosed herein is selected from soy lecithin, glycerol, mannose, and mannitol or combinations thereof.
  • the osmoprotector is soy lecithin.
  • the osmoprotector is glycerol.
  • the concentration of soy lecithin is 0.5 to 4 mM. In another example, the concentration of soy lecithin is between 1 to 2 mM, between 1.5 to 2.5 mM, between 2 to 3 mM, between
  • the concentration of soy lecithin is 1 mM, 1.25 mM, 1.5 mM, 1.75 mM, 2 mM, 2.25 mM, 2.5 mM, 2.75 mM, 3 mM, 3.25 mM,
  • the first three genes are associated with mannose transport, while sorA is responsible for sorbose transport into cell for further utilization (for example, the Pts system, sorbose-specific iic component; Sorbose-permease PTS system IIC component).
  • Active mannose transport inside of the cell can lead to the production of mannitol, a polyol which serves as solute capable of accumulating water inside the cell during osmotic shock.
  • Whole genome sequencing (WGS) analyses identified that the genes of mannitol dehydrogenase specifically for this strain support mannitol’s role in water accumulation during the salt stress. Soy lecithin can therefore help to facilitate mannose transport and mannitol can serve as water absorbent during salt stress.
  • L-citrulline is known to accumulate during soy sauce fermentation by Pediococcus acidilactici and Weissella confusa and can react with ethanol under formation of carcinogenic ethyl-carbamate during pasteurization of the final product.
  • Lactobacillus pobuzihii WZ3 (DSM 33648) was shown to be complete since arcC carbamate kinase co-expressed with the transcription regulator arcR. It has been reported that carbamate kinase is a terminal enzyme of the citrulline pathway ending with release CO2 and ammonia (Fig. 35H). Thus, addition of Lactobacillus pobuzihii into fermentation can contribute to utilising the pre-cursor molecule L-citrulline, and therefore deplete concentrations of cancerogenic ethylcarbamate.
  • Lactobacillus pobuzihii WZ3 (DSM 33648) was shown to express the antilisterial bacteriocin subtilisin biosynthesis protein AlbC, which is thought to inhibit other salt tolerant bacteria such as, but not limited to, Weissella and Staphylococcus.
  • Another advantage of this bacteriocin can be the prevention of an overgrowth of common food pathogen Listeria monocytogens during the storage of soy sauce.
  • the Lactobacillus pobuzihii disclosed herein is capable of synthesising an antimicrobial compound.
  • Monosugar sorbose is actively transported with sorA (as seen with soy lecithin as well) and converted into sorbitol with sorbitol dehydrogenase gutB showing as upregulated.
  • sorA as seen with soy lecithin as well
  • sorbitol dehydrogenase gutB showing as upregulated.
  • a 3.3 log2 FC change in fumarate reductase was shown, and fumarate hydratase is indicative of production L-malate from fumarate.
  • Lactobacillus pobuzihii WZ3 (DSM 33648) is thought to actively integrate free amino acid histidine into its proteins with hisS (Histidine— tRNA ligase; histidyl-tRNA synthetase). This metabolism of Lactobacillus pobuzihii WZ3 (DSM 33648) can contribute to consumption of the histamine which is a known precursor of potentially allergenic histamine in moromi.
  • Taiwanese study reported negative role of Lactobacillus pobuzihii, which was one of few lactic acid bacteria (LAB) associated with spoilage of canned soy sauce.
  • the microbial community of normal and swollen canned soy sauce was investigated using PCR-denaturing gradient gel electrophoresis (DGGE), whose profiles showed that four lactic acid bacteria, including Lactobacillus pobuzihii, Lactobacillus acidipiscis, Lactobacillus piscium and Lactobacillus sp. were involved in the swollen canned samples.
  • DGGE PCR-denaturing gradient gel electrophoresis
  • isolation on solid agar showed that three other diverse species of Bacillus (B. subtilis, B. oleronius and B.
  • Lactobacillus pobuzihii reported as one of major bacterial species involved into fermentation of traditional non-salted fish sauce or ngari.
  • Ngari is the most popular traditionally processed non-salted fish product, prepared from sun-dried small cyprinid fish Puntius sophore (Ham.) in Manipur state of Northeast India.
  • PCR-denaturing gradient gel electrophoresis (DGGE) analysis was used for microbial profiling identified few bands of Tetragenoccocus halophilus, Lactobacillus pobuzihii, Staphylococcus carnosus, and Bacillus indicus, which are thought to be the main agents of ngari fermentation.
  • Lactobacillus pobuzihii DNA marker band on the DGGE was only detected once, as a spike at 6 th month of fermentation, where total bacterial load was estimated to be its highest (10 6 CFU/mL) of all 9 months of fermentation.
  • the pH of studied ngari was between 6.2-6.7 during all fermentation period, which supports the indication that Lactobacillus pobuzihii is a non-spoilage bacterium.
  • Lactobacillus pobuzihii was isolated only with addition of 5% NaCl into De Man, Rogosa and Sharpe (MRS) broth agar, despite the ngari sample coming from nonsalted environment, but required incubation for 15 days.
  • Lactobacillus pobuzihii as disclosed herein is more adaptive to salt stress than previously reported.
  • the present disclosure shows that the introduction of Lactobacillus pobuzihii required specific adaptation to a high salt environment with addition of osmoprotectors prior inoculation into large tanks. It has been demonstrated in the present disclosure that in large-scale trials with aim for industrialization, utilisation of a traditional fermented product enriched with wild Lactobacillus pobuz.ihii as surrogate for starter culture failed, and that only a pure starter culture stock can serve as the initial stock of Lactobacillus pobuz.ihii. It has further been shown that the lack of Lactobacillus pobuzihii in the trials leads to a significant decrease in accumulation of taste peptides and generic volatile organic compound (VOC) profiles similar to conventional middle quality wheat soy sauce.
  • VOC volatile organic compound
  • soy sauce product made using the culture disclosed herein or the process as disclosed herein.
  • the soy sauce product is Taiwanese-style black bean soy sauce.
  • the soy sauce product is gluten-free.
  • a genetic marker includes a plurality of genetic markers, including mixtures and combinations thereof.
  • the term “about”, in the context of concentrations of components of the formulations, typically means +/- 5% of the stated value, more typically +/- 4% of the stated value, more typically +/- 3% of the stated value, more typically, +/- 2% of the stated value, even more typically +/- 1% of the stated value, and even more typically +/- 0.5% of the stated value.
  • range format may be disclosed in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
  • Liquid fermentation or moromi lasts for 4-6 months depending on the weather seasons and characterized by declining pH from 5.8 down to 4.8 during first two months of fermentation.
  • salt layer is discarded and at first, moromi is collected using perforated plastic tube with steel mesh filter allowing raw liquid moromi to be concentrated.
  • This first filtration is marketed as a premium grade soy sauce.
  • Remaining soy mash is distributed manually on the cotton sheets (1x1 m) forming stacks of 300 sheets and moromi is filtered by applying industry press to get the 70% of liquid and sold out as a second-grade black bean soy sauce.
  • Remaining solid content is removed from sheets and used as animal feed or fertilizer on local farms.
  • Filtered moromi is kept at 16°C up to 7 days before following two stage pasteurization at 90°C for 30 minutes. Upon pasteurization, necessary sugar and seasonings are added to form the final product.
  • YW wheat soy sauce
  • koji koji room
  • Aspergillus oryzae spores added.
  • koji is slowly mixed by dipping cold brine solution with 25% NaCl (w/v).
  • Obtained semisolid slurry is further bypassed into a tank kept at a temperature of 17-20°C for the first month of fermentation.
  • Salt-resistant Zygosaccharomyces rouxii is added to the cold tank to reduce the pH value to combat undesirable growth of Bacillus and Micrococcus.
  • moromi is transferred into 85-ton tank, where it is mixed weekly by pressured air for 2 minutes until moromi fully matured at month 5.
  • moromi is filtered using same press as described above, heat pasteurized while adding seasoning.
  • Koji stage simulation was conducted in 800 mL Falcon tissue culture flasks with vented cap (#353138, Corning) and moromi stage was carried out in 500 mL Scott bottles with screw cap with two hose connectors (GL45, Duran) to purge air for mixing in order to reproduce factory conditions for industry scale soy sauce.
  • Trial a2 was conducted from 28 July until November 2017 and involved isolated cultures from moromi as starters (see Table 6 for details). Crushed black beans were socked in NK unit, steamed and mixed with Aspergillus oryzae spores. After, koji was divided into 250 kg batches one part was sprayed with B. amyloliquefaciens culture, the second part left untreated according to scheme (Fig. 37). After 3 days of koji maturation, 72 kg of koji was transferred to individual vats. After, starter cultures Lactobacillus pobuzihii WZ3, Weissella paramentsteroides #1, Tetragenococcus halophiles #6 were added according to scheme on Table 13.
  • Trial a3 was initiated in parallel of trial a2, as excess of koji was remaining for preparation of a2. Forty-five kg of each moromi from previous batches with various months of fermentation was used as seed. Following abbreviations used: F10, F12 - Lactobacillus pobuzihii WZ3 added without koji washing; Hl - 45 kg of 1 month moromi was used as starter culture, H2- 45 kg of 2 month moromi as starter, H3 - 45 kg of 3 month moromi as starter, H4 - 45 kg of 4 month moromi as starter, Gl-12, G2-12 - washed koji, E10 - Tetragenococcus halophiles #6 no washed koji, E12 - B.
  • Lactobacillus pobuzihii WZ3 were grown as adapted on 10% salt in De Man, Rogosa and Sharpe (MRS) broth and introduced into moromi and kept under anaerobic conditions for the moromi stage.
  • MCS De Man, Rogosa and Sharpe
  • Lactobacillus pobuzihii WZ3 were prepared as follows: 9 L of batch culture of Lactobacillus pobuzihii was prepared a week prior trial, max OD600 with 0.6 was reached after 10 days.
  • Koji were collected aseptically from each site at the sterile plastic bags containing 100 g of beans, stored at -20°C at the factory overnight, delivered next day on ice to airport, and transferred within 12 hours to the laboratory in Singapore. On delivery, bags were immediately stored at -80°C.
  • Colonies were picked up with a sterile bacteriological loop into 1 mL sterile water and DNA templates were prepared according InstaGene Matrix protocol (Bio-Rad, USA). Bacteria were further identified by 16s rRNA Sanger sequencing of PCR products with 27F and 1492R primers at Axil Scientific, Singapore and further identified with 16s rRNA gene matching with use of blastN online tool (https://blast.ncbi.nlm.nih.gov/).
  • strains of Lactobacillus disclosed herein are distinct from, for example, Lactobacillus pobuzihii strain E100301, based on 16s rRNA analysis.
  • pellet was mixed with 480 pL ice-cold lysis buffer, 48 pL of 10% SDS and 480 pL ice-cold phenol, chloroform, isoamyl Alcohol (25:24:1), vortexed for 20 seconds and homogenised with 750 pL of ice-cold Zirconia beads at 6.0 m/sec (Fast-Prep 24 5G, MP Biomedicals), placed on ice for Iminute between homogenization steps with total of 5 cycles. After, foamy material was settled at 15 000 x g for 5 minutes, aqueous phase was collected and mixed with 1.9 mL of ice-cold binding buffer.
  • RNA quality was assessed with RNA Screen tape with use of 4200 TapeStation (Agilent). Samples with RIN > 5 and total RNA amount more than 100 ng were processed for library generation for RNA-seq.
  • Taxonomic and functional profiles were processed with use of BioBakery tools. Briefly, all sequenced samples were trimmed and mapped to the human genome with filtering relevant microbial reads using KneadData (https://github.com/biobakery/kneaddata). Samples with less than 20 mln reads were excluded from further analysis. Taxonomic profiles of shotgun metagenomes were produced with MetaPhlan2 pipeline using a recent library of species with clade-specific markers. Functional profiling of metagenomes reads were generated with Humann2, where Humann2 builds a sample-specific reference database from pangenomes on species identified in the same samples by MetaPhlan2.
  • RNA libraries from moromi RNA extracts samples were prepared according to protocol for Universal Prokaryotic RNA-Seq with Prokaryotic AnyDeplete (Tecan, USA).
  • RNA was used to generate the first and second cDNA consequently following acoustic fragmentation under Covaris E220 focused-ultrasonicator (Covaris Inc, USA) and corresponding conditions targeting 200 bp size (Duty Cycle 10%, intensity 5%, cycles/burst 200, time 180 seconds, temperature of water bath 6-8°C).
  • Covaris Inc, USA Covaris Inc, USA
  • Duty Cycle 10%, intensity 5%, cycles/burst 200, time 180 seconds, temperature of water bath 6-8°C After subsequent steps of End-Repair and Adapter ligation, samples were stranded by Strand Selection I and II steps followed by ribosomal depletion with RD1 enzyme mix (Tecan).
  • the final library was amplified according to recommended protocol with initial denaturation at 95°C for 2 minutes, initial 2 cycles of amplification (95°C for 30 seconds, 60°C for 90 seconds) following 18 cycles (95°C for 30 seconds, 65°C for 90 seconds) with final extension at 65°C for 5 minutes.
  • Libraries were submitted to Macrogen, Korea for sequencing at NovaSeq platform 150PE (Illumina) to get 6Gb data per library which corresponded ⁇ 40 mln reads. Functional profiling was generated using Humann2 (Franzosa, McIver et al. 2018).
  • Salt was measured using Mohr’s method. Briefly, 5 mL of sample was dissolved with 100 mF water and mixed with 2 mL of 5% potassium chromate indicator solution (Sigma). Content was titrated with 0.1N silver nitrate (Sigma).
  • Soy sauce moromi (1 ml) was passed through 0.2 pm syringe filter to remove particles, diluted lOx with mobile phase A and centrifuged for 10 minutes at 14,000 rpm. The supernatant of 20 pl was injected directly into the LC-MS. Analysis was done on Vanquish LC coupled to Q Exactive Plus MS (Thermo Scientific).
  • LC parameters are as followed: mobile phase A is 0.1% formic acid in water; mobile phase B is 0.1% formic acid in methanol, flow rate is 50 pl/min, gradient is 2% B at 0-3 min, 2-30% B at 3-20 minutes, 30-99% B at 20-21 min, 99% B at 21-25 minutes, 99-2% B at 25-26 minutes and hold at 2% B until 30 minutes.
  • the column was ACE AQ 1.0 x 150 mm, 3.0 pm (ACE, UK), column temperature was 45°C. Sampler temperature was 4°C.
  • MS was set in fullMS-ddMS2 mode which performs a full scan followed by data-dependent MS/MS for top 10 most abundant masses. Each sample was run in triplicates. Data was analysed using PEAKS software (Bioinformatics Solutions Inc., Canada) de novo sequencing to generate peptide lists.
  • taste-conferring components taste-conferring peptides, and/or taste-conferring amino acids: 3-6 amino acid peptides are categorised into umami, kokumi, sweet, salty, sour and bitter groups, which are reflected in BIOPEP database http://www.uwm.edu.pl/biochemia/index.php/en/biopep. Thus, depending on what type of peptides are prevalent in a specific moromi, one can expect the taste characteristics defined by these groups of peptides.
  • the taste-conferring components are, but are not limited to, glutamate, inosine monophosphate (IMP), guanosine monophosphate (GMP), hydroxy-2-butanone, butanoic acid, pentyl octanoate and acetic acid, as well as the compound listed in the heatmap figure presented herewith.
  • VOC Volatile organic compounds
  • the volatiles were separated in GC (Agilent 7890B) on a DB-FFAP column (Agilent 60 m x 250 pm x 0.25 pm). The column was held at 40°C for 2 minutes, temperature was increased at 5°C/min to 150°C, followed by 10°C/min to 240°C and held for 10 minutes. Helium was used as carrier gas at a constant rate of 1.8 mL/min.
  • the MS (Agilent 5977B) scans from 40 - 350 amu with ionization energy 70eV and transfer line temperature of 250°C. Protease, amylase and lipase activity
  • Amylase activity in the supernatant was measured according to the EnzChekTM Amylase Assay Kit (E33651, Invitrogen, Carlsbad, US). The assay is based on the detection of highly fluorescent BIODIPY FL dye-labelled peptides released by amylase-catalysed hydrolysis. Amylase activities in the culture were expressed in fluorescence intensity units measured with an Infinite M Nano-i- plate reader (Tecan, Zurich, Switzerland) with a filter fluorometer (excitation wavelength -505 nm, emission wavelength 512 nm). During the second round of screening, protease activity for each of the analysed secretion tags was evaluated in triplicate. Lipase activity was evaluated with 405 nm with p-nitrophenol palmitate according to standard method.
  • MRS agar 1.5% MRS agar mixed with 100 pL of 0.22 pm cellulose acetate membrane (Millipore) filtrate of sample free of bacteria was mixed with 0.5% MRS agar included potential host. Plaques were examined in parallel with reference positive control lytic phage T4 mixed with E. coli K12as a host.
  • Nisin Sigma- Aldrich
  • a standard stock solution of nisin containing 1x105 lU/mL was prepared, by dissolving 100 mg of nisin in 0.02 M HC1 (1 mL) and adding 9 mL of distilled water. Nisin was added at concentrations of 100 lU/g and 500 lU/g, respectively to the bacterial solution. Diffusion disk (6 mm in diameter) assays was used was to test colonies of interest on solid agar MRS.

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Abstract

L'invention concerne des cultures comprenant des souches bactériennes et leurs utilisations. L'invention concerne également des procédés de fermentation, ainsi que des produits de sauce soja, des procédés utilisant les souches bactériennes décrites ici.
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CN117137086A (zh) * 2023-09-01 2023-12-01 佛山市海天(高明)调味食品有限公司 豆类风味肽基料、豆酱产品以及它们的制备方法
CN117229980A (zh) * 2023-11-08 2023-12-15 北京市农林科学院 一种食窦魏斯氏菌发酵饲料及其应用与除臭效果

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CN116284220A (zh) * 2023-02-24 2023-06-23 浙江兴业集团有限公司 一种胆固醇酯酶抑制多肽组合及其制备方法、应用
CN117137086A (zh) * 2023-09-01 2023-12-01 佛山市海天(高明)调味食品有限公司 豆类风味肽基料、豆酱产品以及它们的制备方法
CN117137086B (zh) * 2023-09-01 2024-06-04 佛山市海天(高明)调味食品有限公司 豆类风味肽基料、豆酱产品以及它们的制备方法
CN117229980A (zh) * 2023-11-08 2023-12-15 北京市农林科学院 一种食窦魏斯氏菌发酵饲料及其应用与除臭效果
CN117229980B (zh) * 2023-11-08 2024-01-30 北京市农林科学院 一种食窦魏斯氏菌发酵饲料及其应用与除臭效果

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