초록 ▼

○ 연구결과
-HACCP 매뉴얼 3건 개발(고추장,된장,청국장)및 공급
-산야초 발효액 제조 및 항산화 특성 분석
-산야초 발효액 장류 소스 레시피 확립
-발효 당침지 검정콩 스낵 개발 및 영양학적 우수성 분석
-메주의 발효기간별 발효대사체를 분석을 통해 메주의 품질 특성에 관여하는 37개의 발효대사체 동정,이들 대사체들을 이용하여 메주 발효대사경로를 도출
- 우수균주 B.licheniformis67를 이용하여 표준제조 청국장을 제조하여 전통 청국장과 비교한 결과 PGA 함량과 품질특성이 우수하며 대

○ 연구결과
-HACCP 매뉴얼 3건 개발(고추장,된장,청국장)및 공급
-산야초 발효액 제조 및 항산화 특성 분석
-산야초 발효액 장류 소스 레시피 확립
-발효 당침지 검정콩 스낵 개발 및 영양학적 우수성 분석
-메주의 발효기간별 발효대사체를 분석을 통해 메주의 품질 특성에 관여하는 37개의 발효대사체 동정,이들 대사체들을 이용하여 메주 발효대사경로를 도출
- 우수균주 B.licheniformis67를 이용하여 표준제조 청국장을 제조하여 전통 청국장과 비교한 결과 PGA 함량과 품질특성이 우수하며 대사체 분석결과 발효에 따라 아미노산,펩타이드,isoflavone, soyasaponin,LPC 계열의 변화가 두들어지게 관찰,soyasaponin과 LPC의 함량은 크게 증가하는 결과를 보임
-5개월동안 발효된 고추장의 색소 대사체 분석결과 대부분의 발효는 1개월에서 3개월 사이에 일어났으며 zeaxanthine,cryptoxanthine,oxidizedcapsanthines함량은 발효가 진행될수록 증가하는 경향임
-표준제조 청국장의 추출물에서는 신경보호효과가 있음
-표준제조 청국장의 항비만 항당뇨 실험을 한 결과 isoflavone이 많이 들어있는 추출물에서 항비만과 항당뇨 효과가 확인되었으며 PGA도 항당뇨 효능이 있는 것이 확인됨
-동물실험결과 표준제조 메주는 장염예방활성을 나타낼 가능성이 있으며 표준제조 청국장의 PGA와 고추장의 색소에서도 항염증 효능이 있는 것으로 관찰됨
-국내 전통 발효식품으로 2,170종의 유용미생물 자원을 확보
-B.cereus등 병원성미생물에 대한 항균활성 균주 220점 확보
-발효용 미생물들의 단백질 분해능 782종,섬유소분해능 325종,탄수화물 분해능 미생물 210종 확보
-항균활성 우수 미생물의 특성 분석
-분리 미생물의 생리학적,발효학적 특성 분석을 통한 장류제조용 유용미생물의 확보
-확보된 유용미생물로부터 생육 저해 물질의 대량 생산 및 분리 정제 기술 개발
-확보된 유용미생물의 배양학적 특성 분석
-유용미생물의 대량생산 기술 개발
-유용미생물의 항생제 대체용 및 축산 사일리지 제조용 등 산업적 활용기술 개발 및 기술이전
- 전통 고추장 발효미생물의 Microflora의 확인을 위하여 각각의 시료로부터 전체 미생물의 cfu값을 확인하며 동시에 각 시료로부터 대표성이 있는 colony를 분리하여 동정하여 Microflora를 확인 함.
- 전통장류의 metagenome을 분석함으로 모든 미생물의 개체군을 확인함으로서 기존에 보고되어진 각종 장류 관련 미생물 및 일반 미생물 분리 실험에서 분리가 잘 안 되는 미생물의 유전자를 확인함으로서 각종 전통 장류에 포함되어있는 다양한 미생물 군집을 확인
-전통장류의 PCR-RAPD을 분석함으로 이들에 포함 되어 있는 모든 미생물의 개체군을 확인
-protease활성 및 amylase활성을 시험함으로서 각각의 장류에 포함된 생리활성도의 측정을 수행함.
-분리된 전통장류 미생물의 생리적 특성에 관한 기본 정보를 구축 함.
-분리된 전통장류 미생물의 관리 시스템을 구축하기 위한 database작성을 위한 기본 정보표준화 구축.
-전통 장류 표준미생물의 안전한 보존을 위하여 미생물의 적절한 보존 시스템을 구축함.
-다양한 전통장류에서 31개의 박테리오파지 분리
-분리된 파지 중 8개를 선택하여 특성분석
-BCP8-2의 genomesequencing및 분석
-B.subtilis를 이용한 청국장 발효 시 B.cereus증식을 박테리오파지를 이용하여 제어
-B.cereus가 제거된 볏짚유래 starterculture의 가능성 입증
-바실러스 세레우스,아플라톡신 저감화 균주 분리 및 이전
-바이오제닉 아민 분해능 높은 균주 6종 분리 및 이전
-청국장 활용 음료 및 쿠키제품 개발
-라면 또는 파우치 형태의 1회용 제품 개발 (과립고추장을 이용한 간편 비빔소재 개발)
-편의성 장류 컵제품 생산기술 개발
-통계적 처리방법을 이용한 starter고체배양 조건 최적화
-생물반응기에서의 균체 대량생산 및 분리 농축 시스템 확립
-전통장류 품질표준화 기술개발 :위생메주 생산 적용 및 방법 확립
-품질표준화 기술을 적용한 전통장류용 메주 대량생산기술 정립
-효소전극의 sensing효율의 제고
-DTNB수식전극(Carboncloth-DTNB)을 적용한 NAD재생형 electroenzymatic bioreactor의 확립
-수출용 장류(고추장,된장)소스 제품 개발
-우수발효미생물적용 기능성장류제품 개발 :양갱 개발
-복합기술 융합 장류제품 개발 :대두 기능성 물질 캡슐화 제품 개발

Abstract ▼

Ⅲ. The result of Research and Development
1. Study on the Safety of fermented foods(Development of HACCP Manual for Soybean Products & Expansion of Corporate HACCP Certification)
In order to secure the safety of traditional Soybean Products there were the works relating to hazard analyses of g

Ⅲ. The result of Research and Development
1. Study on the Safety of fermented foods(Development of HACCP Manual for Soybean Products & Expansion of Corporate HACCP Certification)
In order to secure the safety of traditional Soybean Products there were the works relating to hazard analyses of gochujang, doenjang, cheonggukgang, and CCP drawing up, Manual Makingup. This was for the purpose of development of process manual regarding gochujang, deonjang, which meets the standards of FDA and Traditional Quality Certification. As the result, the general model of gochujang, HACCP Plane was established with the sterilization temperature of85～100℃, and sterilization time 30～35 for the managementstandard in the sterilizing process. And operator should keep the establishment every shift. For the process of metal detection, there was checking the sensitivity of metal detection instrument every 30 minute, that is before and after works including the period of course.
In case of general model HACCP Plane of deonjang, the process of stone separator and cleaning were monitored by workers before and after the worksatimein operation as wellasin the course with the setting-up 6 RPM of cleaning speed in management standard. Sterilization Process was ordered to ethyl alcohol of 3%. Metal detection Process was checked before and after the works, and every 30 mins in operation for sensitivity of metal detection instrument.
The CCP of cheonggukjang manufacturing process was manifested in the course of picking up stone(P)and fermentation process(B), metaldetection process(P). The general model HACCP Plane was monitored by workers before and after the works as wellas1 time in operation with 6RPM setting-up ofcleaning speed. Fermentation Process was established 44hours for fermentation time. Metal detecting process was directed tocheck up the sensitivity of metal detecting instrument in the course of the operation with every 30mins. and before and after it.
The above outcome is expected to be utilized as manual through a participating enterprise. Through enhancing the process and manufacturing circumstance of every participant company, insects, metal foreign substance that were the main causes of claims diminished sharply. Besides, there was improvementofthe safety in regard of harmfulmicrobe and foreign substances. Apart from the mentioned merits, The Jang brand manufacturing firm of Jangbonga traditional foods acquired HACCP application business among participating companies. And Sunchang Jang brand is in the midst of applying for HACCP Certification.
To develop a new functional product, firstly wild plants(butterbur, water parsley, Angelica Utilis Makino, mugwort, Pimpinella brachycarpa, Chwinamul, Kale) was manufactured, and then analysed for the fermentation. The results of the testsforprotease activity and physiologicalactivation showed the increase. In the analysed antithrombotic outcome effect, Angelica Utilis Makino, waterparsley and mugwortappeared with high grades. Theoutcomeofsensorytest, theoverallpreferencewasthatfermentedextractsof mugwort4. 4±0. 5, water parsley 4. 2±1. 5, butterbur 3. 7±1. 2, Angelica 3. 2±1. 0, brachycarpa 3.0±0.9, Chwinamul 2.4±0.9, Kale 2.4±0.9 in the order. The fermented extracts of mugwort and waterparsley were significantly higher than the other extracts in the preferences.
The recipe has established by making bibimbap sauce, fried pork sauce, soy chicken sauce, soydressingfrom makingthepastesauceusingthefermentedbroth.
Fermented black soy product has made using by representative microzyme, Bacillus subtilis and Lactobacillus plantanum to develop export strategic product, and this increased the excellent soy product consumption, this lead to develop the soy product which is highly regarded upon economic side and functionality to make the customers access easier to the product which not only health-value but also convenience was added. It was established how to make the black soy snack by fermant, and technology has been transmit to the company-Hamok traditional food so traditional food has been modernized, and convenient, and secured the competitiveness than the various soy product of Japan.
2. Metabolic analysis of advanced fermented soy foods and determination of their functional properties
To improve the nuritional and functional qualities of fermented soy foods including meju, cheonggukjang, and kochujang, they were prepared using traditional methods and/or selected micro organism(s)and their metabolites produced during fermentation were analyzed using LC-MS. Various metabolites, including aminoacids, small peptides, nucleosides, urea cycle intermediates, and organic acids in meju, amino acids, smallpeptides, isoflavones, soyasaponins, and lypos in cheonggukjang, and pigments in kochujang, which are responsible forthe unique taste and nutritionaland functionalquality offermented soy foods, wereclearly alteredbyincreasing thefermentationperiod. Thesemetabolites, which were positively related to the sensory quality offermented soy foods, can be used as fermentation biomarkers for the production offermented soy foods. In particular, meju fermentation metabolic pathway was provided in this project. Therefore, ourresults indicate that monitoring the changes in metabolites during fermentation might be important for producing fermented soy foods with good nutritional and sensory quality.
Inaddition, it was observed that is of lavone-richcheonggukjang(BL67)extract had the effects of anti-obestiy and anti-diabetes. Especially, PGA had strong anti-diabetes effect.
Anti-inflammation effect was observed from PGA of BL67 cheonggukjang and pigments from kochujang. Additionally, we established an ischemicanimalmodelasan brain blood circulation obstacle model. Meju fermented by B. subtilis and A. oryzae and traditional meju samples were not shown neuro protective effect in the hippocampal CA1 region after ischemia/reperfusion, but the positive effect was observed from cheonggukjang fermented by BL67.
3. Development of Advanced Technology for Production of Fermented Soybean Products
○ Development of convenient food using fermented soy bean products Development of beverage and cookies using cheonggukjang
Development of preparation techniques for block- and stew-type products using fermented soybean products
Development of convenient cup-type products using do enjangandcheonggukjang
○ Quality standardization for preparation of fermented soybean products
Optimization ofsolid-fermentation condition forthe starterby statisticalexperimental method Establishment of mass production techniques, isolation and concentration system of starterinsolid-fermenter Establishment of application methods for production of hygienic meju
○ Development of production techniques applied with standardization techniques for fermented soybean products
Establishment of mass production methods applied with standardization techniques for traditional fermented soybean products
Production of fermented soybean products by usings uperiormicrobial strain Development of enzyme-based controlsystem for soybean fermentation
○ Development of new fermented foods
Development of fermented soybean products-based sauces for export (gochujang, deonjang, cheonggukjang)
Development of functional fermented soybean products using superior strain
Development of fermented soybean products-based capsule products added with functional ingredients
4. Development of industrial application of food fermented microorganisms
We were isolated useful lactic acid bacteria(LAB), yeast and fungi from Korean tradition fermented food. Among the isolated LAB, Useful lactic acid bacteria having antibacterial activity against Bacilluscereus KCTC 3624 was isolated from Jeotgalusinga MRS selectiveplate. StrainSY99 finally selected was identified as Lactobacillusplantarum based on sugar fermentation pattern test using API50 CHL system. The 16S rDNA sequence of strain SY99 showed 99% identity to those of reference strain of L. plantarum.
And also Bacteriocin-producing lactic acid bacterium having antagonistic activity against Bacillus cereus, was isolated from Kimchi. The selected strain was identified as Lactococcuslactis by the Bergey's manual and 16S rDNA analysis, and named as L.lactis ET45. The bacteriocin was stable in the pH range 3. 0-11. 0. The bacteriocin was active overa wide temperaturerange from 40°C to 121°C. The molecular weight of bacteriocin was estimated to be about 4.5 kDa by tricine sodium dodecylsulfate polyacryamide gel electrophoresis(TSDS-PAGE).
A new zymogram method, silver-stained fibrin zymography, for separation of protease bandsandactivity detection using as inglesubstrategel, was developed. The method takes advantage of the nanoscale sensitivity of both zymography and silver staining. After SDS-PAGE inagelcontainingfibrin, the gel was incubatedinenzyme reaction buffer and thezymogram wassilver-stained. Bandswith proteaseactivity were stained with silver in clear areas where the protein substrate had been degraded. The molecular sizes of proteases were accurately determined.
5. Identification and isolation of food fermented micro organisms and their deposit of patent and preservation
This research aim to the availability of micro organisms on the technology development asatraditional fermented food availability for microbial separation and micro floramap for conducting research and traditional fermented source of micro organisms in the long run safely preserved by the traditional fermented food of the efficient maintenance of goal of eachother, and each isusefulconserved microbial fermentation for the efficient use of the system of permanent preservation of micro organisms for the development of information management systems.
As a functional food in Korea for along time traditional fermented soybean meals, hot pepper and Cheonggukjang and these traditional fermented foods and the health of the world known as functional despite the fact that the manufacturing process for the scientific, safety, health, and functionalstrengthening the comprehensive study lack of fermented foods, especially microorganisms play an important role inresearchon the place many years and remained in the study state that the individual affects of the traditional fermentation process that unidentified yet to change of microorganisms.
In case of commercial gochujang, the viable cell numbers of total bacteria, halophilic bacteria, and ther mophilicbacteriawereintherangeof 4~6 log CFU/g, and yeasts were not detected. Lacticacid bacteria and fungi were not detected in both type sofgochujang after aging.
The sensory evaluation of both types of gochujang showed that color, flavor, savory, pungency, sweety, salty, and overall preference scores were higher in commercial gochujang than in traditional gochujang, but the sensory evaluation scores were not so different between both types of gochujang.
Five genera and 17 species of microorganisms were identified in commercial gochujang, of which 69% was B. subtilis subsp. subtilis, 10% was B. licheniformis, 9% was B. velezensis, and 4% was B. amyloliquefaciens. In halophilic condition, Oceanobacillusspp. suchasO. iheyensis, O. oncorhynchi, andO. soya were newly found.
From these results, It is found thatB. velezensisaswellasB. licheniformisand B. subtilis subsp. subtilis were major bacteria affecting the fermentation of gochujang. B. subtilissubsp. subtilis was major bacteria in commercial gochujang, whereas B. velezensis, B. licheniformis, and B. subtilissubsp. subtilis were major bacteria in traditional gochujang with similar distribution rate each other. There is no notable correlation between the microflora and taste of gochujang.
Many studies on gochujang reported that B. licheniformis and B. subtilis subsp. subtilis were major microogarnisms especially in commercial gochujang, and these bacteria could affect the taste and quality of kochujang. In this study, It was revealed that B. velezensis was a major microorganism in gochujang, and it is expected that these results can be used for the manufacturing high quality gochujang and various Korea traditional fermentation foods.
6. Use of Bacteriophage to Control Harmful Microorganisms
Bacillus cereus is widely distributed in the environment and responsible for foodborne illnesses, causing diarrhea and vomiting. In this study, prevalence of B. cereus bacteriophages in Korean fermented food, jangryu, was studied using 6 reference strains. In a total of 47 samples, 19 (40. 4%) contained more than one kind of B. cereus bacteriophages. Fourteen phages were purified and determined for host lysis profile. All of them were specific for B. cereus group strains not able to lyse other tested gram positive and gram negative strains including B. subtilis. TEM, restriction enzyme digestion pattern analysis and one step growth study found that they are a member of Myoviridae. Furthermore, bacterial lysis studies revealed that isolated phages could be used to control the growth of B. cereus in various growth conditions. Complete genome sequencing and bioinformatic analysis showed that BCP901 and BCP8-2 do not contain any harmful genes such as allergen-, virulence- or toxin-related genes. In addition, the usefulness of bacteriophages to eradicate B. cereus from fermented food was investigated. Purified phages were used to inhibit B. cereus growth in a model fermented food system Cheonggukjang (fast-fermented soybean product). BCP1-1 and BCP8-2 were able to eradicateB. cereusfrom the food effectively only if divalent cations(Ca²⁺, Mg²⁺, or Mn²⁺)were added in themedium. Further studies reveal that divalent cations are essential for phage adsorption while mono valent cation (Na+)is for post adsorption phase of phage infection. Taken together, our finding simply that phage could be an ideal anti-bacterial agent in fermented food (requiring presence of beneficial microflora) and during phage application, optimization of phage reaction conditions is critical for success of phage biocontrol. This is the first report of studying prevalence of bacteriophages in fermented foods and our study suggest that the fermented jangryu is an useful source for B. cereus phage isolation and B. cereus bacterio phages could provide an additional tool to tackle the problems associated with B. cereus contamination.
7. Isolation of Bacillus licheniformis Producing Antimicrobial Agents against Bacillus cereus and Its Properties
Fermented soybean products are essential food stuff in Korean cooking. Sunchang county iswell-known Gochujang-producing district and has an industrial complex for the production of traditional soybean products. As the production is mainly relied on the Master’s skilled experiences, concepts or manuals for the microbial safety were not established during the traditional manufacturing processes We have focused on the screening of microbes for the production of fermented soybean products with premium quality in this region. Among microbes, Bacillus and Aspergillus species are major microbes that involved in the fermentation of soybean and areunder consideration on the safety problems. In order to avoid food poisoning caused by toxins of Bacilluscereus in Cheongkookjang, we have designed ascreening strategy using PCR and cultural properties of B. cereus. Eighty colonies from 30collections of traditionally prepared Cheongkookjang had first been screened with B. cereus selective media and then excluded isolates harboring toxin genes responsible for vomiting and diarrhoea by PCR. As a result, 10 types of morphologically different isolates with no toxin genes were separated and confirmed as B. licheniforminis and B. subtilis when analyzed by the sequence analysis of 16S rRNA genes. Among them, 3 strains with high production of polyglutamate were suitable for the production of Cheongkookjang. This screening procedure can also be applicable to the high throughput screening of safe Bacillus strains.
Identification of virulent B. cereus strains was examined by PCR using primers specific for the detection of plc R-pap R, which encode regulatory proteins controlling the transcription of virulence factors in B. cereus. Total 96 strains of B. cereus that carriedat least one of diarrheal toxin genes including hblACD, nheABC, cytK showed all positive PCR products, while other 48 Bacillus strains that lacked the toxin genes were plcR-papR-negative. This PCR method targeting the plcR-papR genes appears to be simpleand effectivein identifying theentero toxin genes-harboring B. cereusstrains. The PCR primers were designed from the internal sequences ofplcR-papR genes, which is transcriptional regulators in expression of diarrheal enterotoxins, and from cereulide synthetaseA and B. Competition among microbialspeciestogetnutrientsforsurvivalis generalphenomenon in nature. We found thatfermented soybean sauces collected from some regions showed low levels of B. cereus cell number. In order to manufacture Bacillus cereus-free fermented soybean products, anantimicrobial agent-producing isolate against B. cereus was obtained from 150 traditionally fermented soybean products. Some strains isolated from these sauces strongly inhibited the growth of B. cereusin the mixed culture.
After step wise isolation procedures, one strain named as SCK 121057 inhibited the growth of 12 different strains of B. cereus. This strain was identified as B. icheniformis by 16S rRNA gene analysis and caused the collapse of cell shape of B. cereus during co-culture when judged from scanning electron microscope. The B. licheniformis isolate began to produce the antimicrobial agent after 48h of incubation. The agent was non-proteinaceous and in sensitive to heat, long term storage and protease K. Electron microscopic observation indicated that the agent attacked them embrane of B. cereus, leaving the ghost cell. The isolate inhibited growth of B. subtilis, L. brevis and various types of pathogenic strains including E. coli, E. faecalis, M. luteus, S. aureus, A. flavus, A. ochraceus, and A. parasiticus as wellas B. cereus. After inoculation of two strains, B. licheniformis SCK1210⁵7 and B. cereus JBE 008, intheratio of 10 to 1, respectively, on the surface of cooked soybeans, the cell numbers of JBE008 were reduced from 4x10⁵ to 200 CFU/sample after 30 days of incubation, of which cell numbers satisfied the KFDA’s safety guideline.
The isolates were further screened for the production of low levels of tryptamine and histamine. For fungal safety, nontoxic Aspergilus strains were isolated from traditional soybean sauces by using coconutmilk agar medium, on which the colonies that produced a flatoxinemitted fluore scence when exposed with UV light.
Isolation and identification of Biogenicamine-degrading microbes The ability of micro organisms to decarboxylate amino acids is highly variable, often beings trainspecific, not being specific specific. The detection of bacteria possessing amino acid decarboxylase activity is important to estimate the likehood that foods contain biogenic amines and to prevent the iraccumulation in food products
Assessment of amino acid decarboxylase activity was conducted using plate agar containing 1% histidine and tyrosine as biogenic amine precursor. The broths containing bromocresol purpleas indicator of biogenicamine formation were in oculated with selected strains isolated from fermented soybean products and colonies show in gpurp lecolours were discarded from selection because of their ability of biogenicamine production. The current method had the potential to beused for screening forimmediate producers of aminoacid decarboxylase. Selected strains(106 strains)were further screened to evaluate the degrading ability of biogenic amines using minimalmedia with histamine and tyramine as only carbon and energy sources. Among 67 strains grown in minimal agarmedia, 26 strains with high growth were selected and further examined the antibacterial capacity against B. cereus. The biogenic amine-degrading ability of the finally selected strain was evaluated by RP-HPLC in culture media. Although at different extent, all strains were able to degrade histamineand tyramine, which indicated that the primary selection method evaluating the growth in the minimal media was effective. The greast biogenic amine-degrading ability was exhibited by 6 strains belonging to the B. licheniformis, and all of them were able to simultaneously degrade two types of biogenic amines. Our results proved the potentialof fermented soybean products-associated B. licheniformis as a promising strategy to reduce biogenic amine in fermented soybean products.