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최종연구보고서 Preparation of Culture Media from Agricultural Byproducts for Soil Microbial Inoculants and Encapsulation of the Inoculant Organisms

SUMMARY Ⅰ. Tittle of the Project Preparation of Culture Media from Agricultural Byproducts for Soil Microbial Inoculants and Encapsulation of the Inoculant Organisms Ⅱ. Objectives of the Research and Development These studies were carried out primarily to develop the relatively simple processes for hydrolysis of natural protein substances, which could be obtained as byproducts of food and dairy industries, and to utilize the hydrolysates and extracts as microbiological medium ingredients to produce the soil inoculant preparations. Secondarily, the studies were aiming to develop the inoculant preparations with longer shelf life that is, microencapsulation of the microbial cells with sodium alginate and/or granulation of the microbial cells with inorganic materials. Ⅲ. Contents and Scope of the Research and Development 1. Processes for hydrolysis of agricultural byproducts were investigated and optimized. A. Soybean meals and isolated soy proteins

B. Malts and molasses C. Others such as silk pupae D. Analysis for the physicochemical properties of the hydrolysates and extracts E. Application as cultivation media ingredients 2. Processes for hydrolysis of byproducts from dairy and meat industries were investigated and optimized. A. Casein and whey B. Low-grade meats C. Blood meals D. Analysis for the physicochemical properties of the hydrolysates and extracts E. Application as cultivation media ingredients 3. Materials and processes for microencapsulation of the microbial cells were selected and optimized. A. Cultivation of production of the microbial cell mass B. Selection of materials for immobilization and encapsulation C. Conitons and processes for microencapsulation D. Cell viability during the process for microencapsulation E. Shelf life of the immobilized capsules 4. Processes for granulation of the microbial cells with inorganic materials were developed.

A. Inorganic materials and mixing ratio B. Compatibility of the microbial cells to the materials C. Conditions and methods for granulation D. Shelf life of the inorganics-microbe granules 5. Field tests for the inorganics-microbe granules according to the regulations for the specifications of fertilizers by ORD (Dec. 31, 2002) were carried out. A. Zeolite-microbe granules on lettuce B. Inorganics-microbe granules <L> on jasmin trees C. Inorganics-microbe granules <S> on lettuce 6. Economic analysis for production of the media ingradients from agricultural byproducts were done. A. Soybean peptone or Soytone B. Tryptone or Casitone C. Whey hydrolysates D. Blood peptone E. Malt extracts Ⅳ. Results and Suggestions for Application of the Results 1. Processes for hydrolysis of agricultural byproducts A. Acid-enzymatic processes for hydrolysis of soybean meals, ISP, and pupae were established to produce SM-, ISP-hydrolysates, and Pupae hydrolysates.

The processes consisted of the acid hydrolysis by 0.2 N hydrolchloric acid and the enzymatic hydrolysis by commercial proteolytic enzymes such as Protamex and Flavourzyme. B. Process for malt extracts preparation was developed. The yields were 52.0 and 53.9% of the malt powders. C. The hydrolysates extracts were analyzed for their physicochemical properties such as ph, contents for moisture, nitrogens (T-N, A-N), carbohydrates, ashes and inorganics, and compositions of amino acids. It was found that their properties were similar to those of Difco medium ingredients such as Bacto soytone, peptone and malt extracts. D. Standard test organisms were cultivated in a variety of media, broths and agar plates, which prepared with the hydrolysates and extracts as substitutions for Difco medium ingredients. The levels of growth and colony morphologies of the test strains were similar or superior to those prepared with Difco medium ingredients. 2. Processes for hydrolysis of byproducts from dairy and meat industries A. Enzymatic processes for hydrolysis of milk proteins such as casein and whey were established to produce caseinⅠ,Ⅱ- and wheyⅠ,Ⅱ- hydrolysates. The hydrolysates Ⅰ were those which digested by Protamex and Flavourzyme in combination, and Ⅱ by Pancreatin alone. B. Enzymatic processes for hydrolysis of meats, sirloin and shank, were established to produce Sirloin- and Shank-hydrolysates, employing Protamex and Flavourzyme in combination. Alkali-enzymatic process for hydrolysis of blood meals were established to produce Serum-hydrolysates: the process

consisted of alkali hydrolysis by 1.8 N potassium hydroxide and enzymatic hydrolysis by trpsin. 3. Microencapsulation of the microbial cells A. Three types of microcapsules were prepared by: Sodium alginate-microbe beads were formed in calcium chloride solution, Then the sodium alginate-microbe beads were entrapped in a chitosan layer, Polysaccharide and microbe mixtures were spray-dried. B. Cell viability during the process for microencapsulation of the sodium alginate-microbe beads was not dropped markedly, however the spray dried capsules lost viability 80.1%. C. Shelf life of the sodium alginate-microbe beads entrapped in a chitosan layer was 30 months at 25 in case of limit viability, 1 10 8 c.f.u./g. 4. Granulation of the microbial cells with inorganic materials A. Inorganic materials such as serpentine, sericite, pyrophyllite, rock phosphate and zeolite were tested for the granulation. B. A pilot-scale granulation apparatus with the mixer capacity of 500 kg was designed and constructed. The powdered inorganic materials were mixed thoroughly in the mixer. and then the microbial culture broth, 20% (v/w), was mixed to form a dough. The dough was sent to the primary and secondary granulator to produce the granules. These were dried on wired vats in a convection oven at 45.

C. Shelf life of the granules calculated from the data of vial counts: Those of inorganics-microbe granules were 12 months at 25 in case of limit viability, 1 10 8 c.f.u./g. 5. Field tests for the inorganics-microbe granules A. Field tests were carried out for the zeolite-microbe granules on lettuce, inorganics-microbe granules <L> on jasmin trees, and inorganics-microbe granules <S> on lettuce, according to the regulations for the specifications of fertilizers by ORD (Dec. 31, 2002) were carried out. B. Positive effects on the growth of the lettuce and jasmin trees were recognised, however harmful effects were not found. 6. Economic analysis for production of the media ingradients from agricultural byproducts A. Production costs of Soybean peptone or Soytone, Tryptone or Casitone, Whey hydrolysates, Blood peptone, and Malt extracts were calculated in case of those produced by a batch process. B. The production costs were markedly low as compared to the commercial products. 7. Suggestions for application Many manufacturers of the microbial preparations for agricultural purpose

or feed additives are operated without the microbiologist and the proper equipments. It is considered that a regulation to inspect the commercial products on the shelves of the supplier is necessary, and that the consumer and/or farmer should not purchase the microbial inoculants of unreliable qualities, paying high prices.

CONTENTS Chapter 1. Introduction 21 Section 1. Necessities of the Research and Development 21 1. Scientific and Technological Aspects 22 2. Economic and Industrial Aspects 22 3. Social and Cultural Aspects 22 Section 2. Objectives and Scope of the Research and Development 23 Section 3. Periods of the Research and Development 29 Chapter 2. Current Status of the Research and Development 30 Section 1. Domestic Status 30 Section 1. Foreign Status 31 Chapter 3. Contents and Results of the Research and Development 32 Section 1. Contents and Methods 32 1. Protein and Sugar Substances 32 2. Hydrolysis of Protein Substances 32 A. Enzymes for Protein Hydrolysis 32 B. Process for Hydrolysis 34 1) Soybean Proteins 35 2) Whey 36 3) Casein 37 4) Low-grade Meat 38 5) Blood Meal 39 6) Silkworm pupae 40 3. Preparation of Malt Extracts 41 4. Analytical Methods 41 A. ph 41 B. Moisture 41 C. Nitrogen 41 D. Degree of Hydrolysis 42 E. Nitrogen Solubility Index 42

F. Carbohydrate 43 G. Ash 43 H. Inorganics 44 I. Amino Acid Composition 44 5. Microbiological Methods 45 A. Standard Organism 45 B. Growth Rate 45 6. Microencapsulation of Microbial Cells 48 A. Materials for Encapsulation 48 B. Conditions and Processes 48 C. Calculation of Shelf Life 48 7. Granulation of Inorganics and Microbes 52 A. Inorganic Materials 52 B. Lab-scale Granulator 52 C. Pilot-scale Granulator 52 D. Processes 52 8. Field Tests 56 A. Experimental Groups 56 B. Investigation and Analysis 56 Section 2. Results and Discussion 59 1. Hydrolysis of Soybean Proteins 59 A. HCl 59 B. Enzymes 59 C. HCl-Enzyme 60 2. Hydrolysis of Milk Proteins 67 A. Whey 67 B. Casein 67 3. Hydrolysis of Meats and Blood 68 4. Hydrolysis of Silkworm Pupae 68 5. Yields and DH of the Hydrolysates 69 6. Physicochemical Properties of the Hydrolysates 69 A. General Composition 69 B. Inorganics 70 C. Amino Acid Composition 71 7. Microbial Growth on the Substitution Media 83

A. Soybean Protein Hydrolysates 83 B. Milk Protein Hydrolysates 83 C. Meat and Blood Hydrolysates 91 D. Pupae Hydrolysates 91 E. Malt Extracts 91 8. Microencapsulation of Microbial Cells 102 A. Processes for Encapsulation 102 B. Properties of the Capsules 102 C. Changes in Viability during Encapsulation 102 D. Shelf Life of the Capsules 102 9. Granulation of Inorganics and Microbes 109 A. Inorganics-Microbe Mixed Granules 109 B. Properties of the Mixed Granules 109 C. Shelf Life of the Mixed Granules 110 10. Field Experiments 115 A. Zeolite-Microbe Granules 115 B. Inorganics-Microbe Granules <Ⅰ> 119 C. Inorganics-Microbe Granules <Ⅱ> 123 11. Economic Analysis 127 Chapter 4. Achievements and Contributions 130 Section 1. Achievements of the Objectives 130 Section 2. Contributions to the Related Field of Research and Development 131 Chapter 5. Application of the Results 134 Chapter 6. Nobel Informations from Foreign Researches 135 Chapter 7. References 136 Acknowledgements 142

Air atomizing device Sodium alginate solution Air compressor (2 kgr/cm 3 ) (a) Chitosan solution CaCl 2 solution Permanent capsule (b)

2 1 4 14 10 3 stirer 5 11 6 9 12 13 15 7 8

A B E C D

1. 가.

40 30 DH(%) 20 10 0 0.15 0.3 0.6 1.1 2 3.4 Concentration of HCl (N)

40 China Brazil 30 DH(%) 20 10 0 0.2 0.4 0.8 1.6 Concentration of enzymes (%)

100 80 Soybean meal Isolated soy protein DH(%) 60 40 20 0 A B Hydrolysate C

100 90 Soybean meal Isolated soy protein 80 NSI(%) 70 60 50 40 30 A B C Hydrolysate

10.0 10.0 9.5 9.5 Log CFU/ml 9.0 8.5 Log CFU/ml 9.0 8.5 8.0 7.5 E. coli S. aureus P. vulgaris K. pneumoniae 8.0 7.5 E. coli S. aureus P. vulgaris K. pneumoniae 7.0 0 10 20 30 40 50 7.0 0 10 20 30 40 50 Time(h) Time(h) <A> 10.0 10.0 9.5 9.5 Log CFU/ml 9.0 8.5 Log CFU/ml 9.0 8.5 8.0 7.5 E. coli S. aureus P. vulgaris K. pneumoniae 8.0 7.5 E. coli S. aureus P. vulgaris K. pneumoniae 7.0 0 10 20 30 40 50 7.0 0 10 20 30 40 50 Time(h) Time(h) <C> <D>

10 9 Log CFU/ml 8 7 <A> 6 5 Lactobacillus delbrukii subsp. lactis Lactobacillus fermentum 0 10 20 30 40 50 Time(h) 10 9 Log CFU/ml 8 7 6 5 Lactobacillus delbrukii subsp. lactis Lactobacillus fermentum 0 10 20 30 40 50 Time(h) 10 9 Log CFU/ml 8 7 <C> 6 5 0 10 20 30 40 50 Time(h) Lactobacillus delbrukii subsp. lactis Lactobacillus fermentum

B.P WⅠ WⅡ <A> B.P WⅠ WⅡ

10 9 Log CFU/ml 8 7 <A> 6 5 0 10 20 30 40 50 Time(h) E. coli Bacillus subtilis 10 9 Log CFU/ml 8 7 6 5 0 10 20 30 40 50 Time(h) E. coli Bacillus subtilis 10 9 Log CFU/ml 8 7 <C> 6 5 0 10 20 30 40 50 Time(h) E. coli Bacillus subtilis

B.C CⅠ CⅡ <A> B.C CⅠ CⅡ

10.0 10.0 9.5 9.5 9.0 9.0 Log CFU/ml 8.5 8.0 Log CFU/ml 8.5 8.0 7.5 7.5 7.0 Salmonella typhimurium Staphylococcus aureus 7.0 Salmonella typhimurium Staphylococcus aureus 6.5 0 10 20 30 40 50 6.5 0 10 20 30 40 50 Time(h) Time(h) <A> 10.0 10.0 9.5 9.5 9.0 9.0 Log CFU/ml 8.5 8.0 Log CFU/ml 8.5 8.0 7.5 7.5 7.0 Salmonella typhimurium Staphylococcus aureus 7.0 Salmonella typhimurium Staphylococcus aureus 6.5 0 10 20 30 40 50 6.5 0 10 20 30 40 50 Time(h) Time(h) <C> <D>

B.B Si <A> Sh Se B.B Si Sh SeSe

<A> 5 0 6 0 0 5 0 6 0 <C> 5 0 6 0

<A> c o c c u s a u r e u s l l a t y p i m u r i u m 5 0 6 0 s t a p h y l o c o c c u s a u r e u s s a l m o n e l l a t y p i m u r i u m 4 0 5 0 6 0 <C> h l y o c o c c u s a u r e u s o n e l l a t y p i m u r i u m 5 0 6 0

<A>

Size in diam. ( ) Hardness (g) Remarks

<A>

<A> <C>

<A> 700 600 500 y = -20.833x + 575 R 2 = 0.9586 균수 ( 백만 ) 400 300 200 100 y = -33.333x + 533.33 R 2 = 0.9494 0 0 1 2 3 4 5 6 7 8 9 10 11 12 보존기간 ( 월 ) 500 450 400 y = -10.833x + 425 R 2 = 0.9826 균수 ( 백만 ) 350 300 250 200 150 100 50 0 y = -20.833x + 395 R 2 = 0.8096 0 1 2 3 4 5 6 7 8 9 10 11 12 보존기간 ( 월 ) <C> 120 100 y = -4.6667x + 113.67 R 2 = 0.9511 균수 ( 백만 ) 80 60 40 20 0 y = -8.4583x + 110.92 R 2 = 0.999 0 1 2 3 4 5 6 7 8 9 10 11 12 보존기간 ( 월 )

충전재 - 미생물혼합과립

Size in diam. ( mm ) Hardness (g) Remarks

<A> <C>

<A> 30 35 25 30 균수 ( 백만 ) 20 15 y = -1.5917x + 27.183 R 2 = 0.9989 균수 ( 백만 ) 25 20 15 y = -2.075x + 30.15 R 2 = 0.8641 10 y = -1.7x + 24.567 10 5 R 2 = 0.8542 5 y = -2.4x + 30.467 R 2 = 0.915 0 0 0 1 2 3 4 5 6 7 8 9 10 11 12 보존기간 ( 월 ) 0 1 2 3 4 5 6 7 8 9 10 11 12 보존기간 ( 월 ) <C> 100 <D> 100 90 90 80 80 70 70 균수 ( 백만 ) 60 50 40 30 y = -5.3333x + 78 R 2 = 0.7734 균수 ( 백만 ) 60 50 40 30 y = -6.1667x + 83.667 R 2 = 0.8946 20 10 0 y = -6.0833x + 78.167 R 2 = 0.8212 20 10 0 y = -6.7667x + 80.533 R 2 = 0.8338 0 1 2 3 4 5 6 7 8 9 10 11 12 보존기간 ( 월 ) 0 1 2 3 4 5 6 7 8 9 10 11 12 보존기간 ( 월 ) <E> 90 80 70 60 균수 ( 백만 ) 50 40 y = -5.1667x + 74.667 R 2 = 0.8218 30 20 10 y = -5.25x + 74.167 R 2 = 0.8091 0 0 1 2 3 4 5 6 7 8 9 10 11 12 보존기간 ( 월 )

충전재 - 미생물혼합과립의비해 비효 가. 제올라이트 - 미생물혼합과립

인공배양토를사용하여폿트시험하였다.

인공배양토에서의 폿트재배

다. 무기재료 - 미생물혼합과립 <Ⅱ>

항목별연구개발목표와수행내용을아래에기술하였으며. 그목표달성도를 <Table 4.1.> 에요약하여나타내었다. 1. 농산가공부산물의배지자원화기술개발 농산가공부산물중에서두부 두유부산물, 맥주양조부산물, 맥아및당밀을가수분해하거나추출하여토양미생물의배양을위한배지성분으로사용하기위한기술을개발하였다. 가수분해및추출공정의최적화, 가수분해물의영양성분평가, 분해물을첨가한배지에서의미생물의생육을비교하였다. 2. 축산가공부산물의배지자원화기술개발 축산가공부산물중에서도축부산물로서혈액및저급육, 유가공부산물로서카제인및유청, 또는기타잠업부산물로서번데기등을가수분해하여배지성분으로사용하기위한기술을개발하였다. 가수분해및추출공정의최적화, 가수분해물의영양성분평가, 분해물을첨가한배지에서의미생물의생육을비교하였다. 3. 생균체의고정화 미세캡슐화기술개발 미생물을배양하여얻은균체를다당류등의바이오폴리머로피복및고정하 기위한폴리머재료의선발, 조건및방법의최적화, 캡슐화공정이미생물의생 존율에미치는영향, 제품의장기보존효과등을구명하였다.

4. 충전재 - 미생물혼합제의과립화기술개발 충전재로서무기재료와미생물을혼합시켜서과립화하는기술을개발하고저 각재료의적정혼합비율을선정하고과립화장치를설계제작하였으며, 공정을 최적화하고과립제품의건조및보관조건을구명하였다. 5. 충전재 - 미생물과립화제제의비해 비효시험 주요과립화제제에대하여는비료공정규격 < 농촌진흥청고시 02. 12. 31.> 에 따라비해 비효시험으로서미생물제제의품목허가를위한채소 ( 상추 ), 수목 ( 재스민 ) 등에대하여작물재배시험을수행하였다. 6. 농축산부산물의배지자원화기술에대한경제성분석 주요단백질분해물및맥아추출물에대하여는단식공정 (batch process) 의 경우그생산원가를추산하고시판품과의가격을비교하였다. 1. 농축산가공부산물의배지자원화기술 농축산가공부산물즉두부 두유부산물, 맥주양조부산물, 맥아및당밀, 도축부산물, 우유가공부산물, 잠업부산물등을가수분해또는추출하여토양미생물의배양을위한배지성분으로사용하기위한기술은관련생산업계에활용될수있으며보조사료로서생균제생균제생산업계에도확대적용될수있다.

2. 생균체의고정화 미세캡슐화기술개발 미생물균체를다당류등의바이오폴리머로피복및고정하여미세캡슐화하 는기술은미생물제제의유통기간을현저하게연장하고농가사용에는편리성을 향상시킬것이다. 3. 충전재 - 미생물혼합제의과립화기술개발 각종무기재료와미생물을혼합시켜서과립화하는기술및과립화장치는전 문학계의주요자료로서활용될수있으며, 미생물제제의유통업계및사용농가에 대하여크게편리성을증진시킬것이다.

Table 4.1. Objetives of the study and their achievements 연구개발목표달성도주요연구개발내용 농산가공부산물의배지자원화기술개발 100% 두부 두유부산물 맥주양조부산물 폐당밀및맥아 축산가공부산물의배지자원화기술개발 100% 도축부산물 : 혈액및저급육 유가공부산물 : 카제인및유청 기타잠업부산물 : 번데기 농축산부산물로부터얻은배지성분의영양성분평가 대채배지에서의표준미생물균주의생육비교 생균체의고정화및미세캡슐화 100% 캡슐화재료의특성비교및선발조건및방법의개발 캡슐화공정이균체에미치는영향 균체의장기보존효과 충전재 - 미생물혼합제의과립화기술개발 100% 무기재료의성발 과립화를위한재료의혼합비율 다공질무기재료에서의정착성 과립화방법및조건의구명 과립화제제의보존성및유통기간 충전재 - 미생물과립화제제의비해 비효시험 100% 제올라이트 - 미생물혼합제제 무기충진제 <Ⅰ>- 미생물혼합제제 무기충진제 <Ⅱ>- 미생물혼합제제 농축산부산물의배지자원화기술에대한경제성분석 100% 단식공정에의한생산원가의계산 시판품과의가격비교

1. 지적재산권의확보 농축산부산물로부터미생물배지성분의생산공정에관한특허 토양미생물의미세캡슐화및충전재혼합과립제조에관한특허 2. 참여기업등에기술이전을통하여산업화또는기업화에기여 농축산부산물로부터단백질및탄소원의가수분해및추출기술 토양미생물의대량배양기술 토양미생물의캡슐화기술 미생물-충전재혼합과립제조기술 토양미생물제제의장기보관기술 3. 미생물제제생산업체에배지생산기술의보급 교육및홍보 미생물전문인력을보유하고있지않은일반생산업체에서용이하게수용할 수있도록위 2 항의기술을보급하거나교육하며, 농업및축산관련잡지에자주 투고하고농수산박람회에출품하여적극홍보하고자한다.

1. 배지자원화기술 실험용배지성분은고가의것이므로최근에는산업용의미생물배지성분을 비교적염가로대량생산하여공급하는회사가늘고있다. Kosher Pareve, Netherland : 중국현지공장에서 Yeast extract 생산공급 NZP, New Zealand : Meat + Gelatin으로부터염가의 Peptone생산 Hubei Angel Yeast, China : 식품첨가물규격의각종 Yeast extract 생산 Unison(-Tianjin) Int'l Trading Co., Ltd, China : Soytone S20의공급 2. 미생물의캡슐화기술 1980 년이래프랑스의국립농업연구소 (INRA) 에서근권미생물 (PGPR) 의캪 슐화연구를진행해오고있다. 근권미생물의마크로캡슐화 (macroencapsulation) 에의한작물의생육촉진 (Jung, 1980) 약 10 8 의세균을함유한직경 6 mm의 alginate구의분사기술 (Digat, 1988) 근권미생물을 calcium alginate 지지체로캡슐화한비료의제조 (Fages et al., 1988) 길항성세균 Pseudomonas fluorecens-putida의미세캡슐화연구 (Amiet- Charpentier, 1998).