[보고서]발효미생물을 이용한 안전 별미장 개발

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보고서 정보

주관연구기관

국립농업과학원
National Institute of Agricultural Sciences

연구책임자

조용식

참여연구자

김소영 , 박신영 , 최혜선 , 김재현 , 송진 , 김순희 , 엄정선

보고서유형

최종보고서

발행국가

대한민국

언어

한국어

발행년월

2018-02

과제시작연도

2017

주관부처

농촌진흥청
Rural Development Administration(RDA)

연구관리전문기관

농촌진흥청
Rural Development Administration

등록번호

TRKO201800043231

과제고유번호

1395050716

사업명

농업기초기반연구

DB 구축일자

2018-12-15

키워드

발효식품.발효미생물.안전.별미장.fermented food.fermented microorganism.safety.Byeolmijang.

DOI

https://doi.org/10.23000/TRKO201800043231

표 Bacillus cereus gene primer sequences used for qPCR
표 qPCR conditions for B. cereus
표 The ratio between Bacillus sp. and B. cereus KACC10004 innoculated into fermented soybeans product
표 List of selected bacteria for survival rate of B. cereus in antibacterial activity of Bacillus species isolated from fermented soybean pastes
표 Survival of B. cereus KACC10004 incubated with Bacillus spp. on selective media. (A) B. amyloliquefaciens HJ5-2 and B. amyloliquefaciens HJ25-1 (B) B. amyloliquefaciens HJ39-3 and B. amyloliquefaciens HJ39-5
표 Survival of B. cereus KACC10004 in co-culture depending on the concentration of (A) B. amyloliquefaciens HJ5-2 and (B) B. amyloliquefaciens HJ39-5. Data are presented as the mean bacterial survival for three independent experiments. The error bars correspond to the mean ± SD
표 Expression of B. cereus spore-related genes groEL, nheA, nheC, and entFM mRNA was determined by qPCR. Band intensities were quantified by densitometry and normalized to 16S rRNA. qPCR expression data were normalized to 16S rRNA and reported relative to the expression level in the B. cereus KACC10004 control. Graphs represent relative mRNA levels for the groEL, nheA and nheC genes. Each value is a mean ± SD of three replicate analysis
표 AtpB levels in the whole cell were determined by western blotting and quantified by densitometry. All experiments were performed at least three times, nd representative data are shown. Bars correspond to means ± SD
표 Appearance of the cheonggukjang fermented with starter
표 Growth inhibition of B. cereus KACC10004 in soybean products fermented with B. cereus KACC10004 and B. amyloliquefaciens HJ5-2. Data are presented as the mean bacterial survival for 3 independent experiments. Error bars indicate SD
표 Expression of the Bacillus cereus spore-related genes groEL, nheA and nheC was determined by qPCR of fermented soybean product samples. All qPCR data were normalized to 16S rRNA and reported relative to the control culture. Graphs indicate the relative mRNA levels for groEL, nheA and nheC
표 The AtpB levels in whole cell extracts from fermented soybean products were determined by immunoblotting. Expression of AtpB was quantified by densitometry. The data shown are representative of three independent experiments. Error bars indicate the mean ± SD. ND: not detected
표 Transmission electron microscopy (TEM) (A) and scanning electron microscopy (SEM) (B) images of Bacillus amyloliquefaciens RD7-7 grown on Luria-Bertani medium after 24 h at 30°C. Scale bars indicate 1μm and 5μm, respectively. Representative images of three independent experiments are shown
표 The antimicrobial activity of B. amyloliquefaciens RD7-7 was detected using an agar spot test. The diameters of the inhibition zones of various pathogenic bacteria were measured. The data shown are representative of at least three independent experiments. Diameter of the inhibition zone: (+) weak (≤ 1.3 cm), (++) intermediate (1.31-1.50 cm), (+++) strong (1.51-1.80 cm)
표 SDS-PAGE and detection of antibacterial activity of the partially purified bacteriocin from B. amyloliquefaciens RD7-7. (A) Coomassie blue stained SDS-PAGE gel. Lanes: 1, molecular weight standards; 2, purified B. amyloliquefaciens RD7-7. (B) Gel overlaid with 0.8% NB agar containing indicator organism, 1% of B. cereus KCTC 3624. (C) Proteomic analysis using LC-MS/MS
표 Primer sequences used for RT-PCR and qPCR in this study
표 Selection of biogenic amine-reducing strains in the decarboxylase broth. Color development on the synthetic medium containing histidine, tyrosine, and cresol red as pH indicator during incubation of biogenic amine-producing bacterial strains. Negative reaction: yellow, Positive reaction: purple. Strains: A, B. subtilis HJ53-3. Biogenic amine-non-producing Bacillus strains isolated in this study. B, B. subtilis HJ0-6; C, B. subtilis DJ53-4; D, B. idriensis RD13-10
표 Expression of tdc, hdc, and yobN mRNA was determined by RT-PCR. Band intensities were quantified by densitometry and normalized to 16S rRNA. Graphs represent relative mRNA levels for tdc, hdc, and yobN. Each value is a mean ± SD of three replicate analyses, within each row means with different superscripts letters are statistically significantly different, P < 0.05 (one-way ANOVA, followed by Duncans multiple comparison test). The experiment was performed at least three times, and representative data are shown
표 Expression of tdc, hdc, and yobN mRNA was determined by qPCR. These expression data were normalized to 16S rRNA levels and reported relative to the expression level in the HJ53-3 positive control. Graphs represent relative mRNA levels for tdc, hdc, and yobN. Each value is a mean ± SD of three replicate analyses, within each row means with different superscripts letters are statistically significantly different, P < 0.05 (one-way ANOVA, followed by Duncans multiple comparison test). The experiment was performed at least three times, and representative data are shown
표 Contents of biogenic amines produced by selected strains using UPLC analysis. Determination of biogenic amines were performed after cultivation at 30 °C for 24h in the LB broth contained 0.25% histidine and tyrosine
표 Degradation of biogenic amines by selected strains using UPLC. Determination of biogenic amines were performed after cultivation at 30 °C for 24h in the LB broth contained 0.25% histamine and tyramine
표 The levels of histamine and tyramine produced by the selected bacterial strains. The expression levels of histamine in the whole cell from all strains were determined by western blotting using primary antibody specific for the histamine H3 Receptor. Data are representative of three independent experiments
표 Quantification of histamine and tyramine levels in cell supernatants using an ELISA assay. Histamine and tyramine concentration was increased in the B. subtilis HJ53-3 strain compared to that in other strains. All experiments were performed at least three times, and representative data are shown. Bars correspond to means ± SD. The sample means were compared by one-way ANOVA followed by Duncans multiple comparison tests. Mean of different samples labeled with different letters are statistically significantly different (P < 0.05)
표 Expression of yobN mRNA was determined by qPCR. These expression data were normalized to 16S rRNA levels and reported relative to the expression level in the HJ53-3. Graphs represent relative mRNA levels for yobN. Each value is a mean ± SD of three replicate analyses (**, p<0.01; ***, p<0.001)
표 Distribution of protease activity (A) and amino nitrogen (B) of HJ0-6. These figures were compared with KACC15935. Each value is a mean ± SD of three replicate analyses (p<0.05)
표 Primer sequences used for RT-PCR
표 Growth inhibition of B. cereus KACC10004 in LB broth with B. cereus and B. amyloliquefaciens HJ5-2 or Bacillus spp.. All experiments were performed at least double times, and representative data are shown. Bars correspond to means ± SD. The sample means were compared by one-way ANOVA followed by Duncans multiple comparison tests. Mean of different samples labeled with different letters are statistically significantly different (P < 0.05)
표 Expression of the Bacillus cereus toxin-related genes groEL, nheA, nheC and entFM was determined by RT-PCR of co-cultured starter with B. cereus. All RT-PCR data were normalized to 16S rRNA
표 Contents of biogenic amines produced by co-cultured strains using HPLC
표 Expression of the biogenic amine-related genes hdc, tdc, and YobN was determined by RT-PCR of co-cultured starter. Band intensities were quantified by densitometry and normalized to 16S rRNA
표 The AtpB levels with co-cultured strains. The AtpB levels were determined by immunoblotting from whole cell extracts
표 The H3 receptor levels with co-cultured strains. The H3 receptor levels were determined by immunoblotting from whole cell extracts
표 Appearance of the cheonggukjang fermented with co-cultured strains
표 Growth inhibition of B. cereus KACC10004 in soybean products fermented with B. cereus and B. amyloliquefaciens HJ5-2 or Bacillus spp.. All experiments were performed at least double times, and representative data are shown. Bars correspond to means±SD. The sample means were compared by one-way ANOVA followed by Duncans multiple comparison tests. Mean of different samples labeled with different letters are statistically significantly different (P < 0.05)
표 Distribution of protease activity(A) and contents of amino 1nitrogen(B) in soybean products fermented with B. cereus KACC10004 and B. amyloliquefaciens HJ5-2 or Bacillus spp.. Data are presented as the mean for 3 independent experiments. Error bars indicate SD (p<0.05)
표 Distribution of dynamic viscosity in soybean products fermented with B. cereus KACC10004 and B. amyloliquefaciens HJ5-2 or Bacillus spp.. Data are presented as the mean for 3 independent experiments. Error bars indicate SD (p<0.05)
표 Inhibition of biogenic amines in soybean products fermented with B. subtilis HJ0-6 or Bacillus spp
표 GenBank homologs of Bacillus bacterial isolates from Gochujang samples
표 The sfp amplicons(616 bp) from different Bacullus isolates. Lanes of L6, L9, Y5, and Y8 are positive bacterial isolates for the sfp gene. M: 100 bp DNA ladder
표 Antibacterial activity from Bacillus species isolated from Gochujang. (A) lane shows the antibacterial activity against B. cereus on 1.5% top agar. (B) lane shows the antibacterial activity against staphylococcus or E. coli on 0.8% top agar
표 List of collected Byeolmijang from several region
표 Total aerobic bacteria counts and Lactic acid bacteria counts of collected Byeolmijang
표 Protease and α-amylase activity of collected Byeolmijang
표 Amino type nitrogen and Reducing sugar of collected Byeolmijang
표 Amino acid contents of collected Byeolmijang
표 Organic acid contents of collected Byeolmijang
표 Changes in microorganism of Bealmijang during fermentation. Microorganism Changes of deoduk-doragijang. Triangle, control; square, DD, Each value indicates the mean ± SD of 3 replicate analysis
표 Changes in microorganism of Bealmijang during fermentation (A) Microorganism Changes of wheat bran-soybean deonjang. Triangle, control; square, DD, (B) Microorganism Changes of wheat bran deonjang and wheat bran-soybean deonjang. Circle, wheat bran deonjang; triangle, control; square, wheat bran-soybean deonjang. Each value indicates the mean ± SD of 3 replicate analysis
표 Changes in Lactic acid bacteria of Bealmijang during fermentation. Lactic acid bacteria changes of deoduk-doragijang. Triangle, control; square, DD, Each value indicates the mean ± SD of 3 replicate analysis
표 Changes in Lactic acid bacteria of Bealmijang during fermentation (A) Lactic acid bacteria changes of wheat bran-soybean deonjang. Triangle, control; square, DD, (B) Lactic acid bacteria changes of wheat bran deonjang and wheat bran-soybean deonjang. Circle, wheat bran deonjang; triangle, control; square, wheat bran-soybean deonjang. Each value indicates the mean ± SD of 3 replicate analysis
표 Changes of Enzyme activities of Bealmijang during fermentation (A) Protease activity changes of deoduk-doragijang. Triangle, control; square, DD, (B) Amylase activities changes of deoduk-doragi danjang. Triangle, control; square, DD, Each value indicates the mean ± SD of 3 replicate analysis
표 Changes in Protease activities of Bealmijang during fermentation (A) Protease activity changes of wheat bran-soybean deonjang. Triangle, control; square, DD, (B) Amylase activity changes of wheat bran deonjang and wheat bran-soybean deonjang. Circle, wheat bran deonjang; control; square, wheat bran-soybean deonjang. Each valu e indicates the mean ± SD of 3 replicate analysis
표 Changes in aminotype nitrogen of Bealmijang during fermentation (A) Aminotype nitrogen changes of deoduk-doragijang. Triangle, control; square, DD, Each value indicates the mean ± SD of 3 replicate analysis
표 Changes in aminotype nitrogen of Bealmijang during fermentation (A) Aminotype nitrogen changes of wheat bran-soybean deonjang. Triangle, control; square, DD, (B) Aminotype nitrogen changes of wheat bran deonjang and wheat bran-soybean deonjang. Circle, wheat bran deonjang; triangle, control; square, wheat bran-soybean deonjang. Each value indicates the mean ± SD of 3 replicate analysis
표 Changes in Reducing sugar of Bealmijang during fermentation (A) Reducing sugar changes of deoduk-doragijang. Triangle, control; square, DD, Each value indicates the mean ± SD of 3 replicate analysis
표 Changes in Reducing sugar of Bealmijang during fermentation (A) Reducing sugar changes of wheat bran-soybean deonjang. Triangle, control; square, DD, (B) Protease activity changes of wheat bran deonjang and wheat bran-soybean deonjang. Circle, wheat bran deonjang; triangle, control; square, wheat bran-soybean deonjang. Each value indicates the mean ± SD of 3 replicate analysis
표 Organic acid composition of Bealmijang during fermentation period. (A) deoduk-doragijang, (B) wheat bran deonjang, (C) wheat bran-soybean deonjang
표 Proximate composition of Bealmijang during fermentation
표 Free sugar contents of Bealmijang during fermentation
표 Organic acid composition of Bealmijang during fermentation
표 Changes in total bacteria, fungi and lactic acid bacteria of Bealmijang during fermentation
표 Contents of biogenic amines produced by Bealmijangs using HPLC analysis. Determination of biogenic amines were performed after cultivation at 30 °C in Bealmijangs contained 0.05% histamine and tyramine
표 Changes in amino nitrogen of Bealmijang during fermentation. (A) Amino nitrogen changes of appling of HJ5-2. (B) Amino nitrogen changes of appling of HJ0-6. S1: HJ5-2, S2: 15935, S3: B. cereus, S4: HJ0-6, H: Histamine, T: Tyramine, (* p<0.05)
표 Proximate composition in freeze-dried soybean powders (unit: %)
표 SDS-PAGE analysis for freeze-dried soybean powders with pre-treatments. M, molecular weight marker; NT, no treatment soybean; S, steamed soybean; P, puffed soybean; AF, air-fried soybean
표 Protein solubility of isolated soy protein different treatment methods at various pH. ● no treatment; ○ steamed soybean; ▲ puffed soybean; △ air-fried soybean
표 Water and oil absorption capacities of freeze-dried soybean powders with pre-treatments (unit: mL/g)
표 DSC characteristics of freeze-dried soybean powders with pre-treatments
표 Fluorescence spectra (excitation = 360 nm) in organic phase of chloroform/methanol extractions(***p<0.0001). ● no treatment; ○ steamed soybean; ▲ puffed soybean; △ air-fried soybean
표 Appearance of the cheonggukjang fermented with soybean treted by different heat mothods
표 Distribution of protease activity(A) and contents of amino nitrogen(B) in cheonggukjang fermented with soybean treted by different heat mothods. Data are presented as the mean for 3 independent experiments. Error bars indicate SD (p<0.05)
표 Distribution of protease activity(A) and contents of amino nitrogen(B) in Bealmijang fermented with soybean treted by different heat mothods. Data are presented as the mean for 3 independent experiments. Error bars indicate SD(*, p<0.05)
표 The primer sequences used for gas producing genes
표 Gochujang samples and gas production
표 GenBank homologs of Bacillus bacterial isolates from Gochujang samples
표 The expression of target genes from 4 samples named by S5, S11, S17, and S26 with gas expantion. The star markers show correct bands. Primer names: 1, Hog1 (621 bp); 2, Gpp1 (708 bp); 3, GPD1 (870 bp); 4, PBS2 (960 bp)
표 The expression of target genes from raw materials of Gochujang. 1, DIY Gochjang final product; 2, DIY Puffed rice; 3, DIY Meju flour; 4, DIY Chili powder; 5, DIY Rice flour; M, 100 bp DNA ladder. The star marker correct band at Meju flour
표 The expression of target genes from raw materials of commercial Gochujang. 1, Cho Meju flour1; 2, Cho Meju flour2; 3, Han Meju flour; 4, Sun Meju flour; 5, Yang Meju flour; M, 100 bp DNA ladder. The rectangle boxes show the correct band at Meju flour

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[국가R&D연구보고서] 발효미생물을 이용한 안전 별미장 개발
Development of safe Bealmijang using functional microorganisms from fermented foods 원문보기

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[국가R&D연구보고서] 발효미생물을 이용한 안전 별미장 개발 Development of safe Bealmijang using functional microorganisms from fermented foods 원문보기

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