Title Page

Abstract

Contents

List of Abbreviations 22

I. Introduction 24

A. Background 24

1. Domestic livestock industry 24

2. Microbial preparation 25

3. Feed additive industry and market size 27

B. Study purpose 29

II. Health Promotion and livestock-house Environment Improvement by Using Functional Feed Additives 32

A. Introduction 32

B. Materials and methods 36

1. Manufacture of functional fermented feed additive(FFFA) and analysis of ingredients 36

2. Analysis of ingredients of functional feed additives 40

3. Measurement of physiological activity of fermented functional feed additives(FFFA) 42

4. Application of FFFA in poultry farm 45

C. Results and Discussion 46

1. Manufacture of FFFA and analysis of its component 46

2. Analysis of components in FFFA 54

3. Measurement of enzyme activities in FFFA 60

4. Application of FFFA in poultry farm 66

III. Enzyme activity dynamics in livestock-farm environment 70

A. Introduction 70

B. Materials and methods 73

1. Real-time enzyme reaction quality kit manufacturing 73

2. Measurement of RF(Relative Fluorescence) according to concentration of MUF 73

3. Development of enzyme substrate kit 74

4. Application of enzyme activity measurement 78

5. Development of apparatus for enzyme activity measurement 79

C. Results and Discussion 83

1. Real-time enzyme reaction quality kit manufacturing 83

2. Measurement of RF according to concentration of MUF 87

3. Development of enzyme reactive material kit 88

4. Application of enzyme activity measurement 110

5. Enzyme activity measurement terminal development 119

IV. Research on hygiene improvement in livestock-house environment 131

A. Introduction 131

B. Materials and methods 135

1. Surfactin's cleaning power efficacy test 135

2. Quantitative/Qualitative Analysis of Surfactin 136

3. Prototype of detergent containing Surfactin 142

C. Results and Discussion 146

1. Surfactin's cleaning power efficacy test 146

2. Quantitative/Qualitative Analysis of Surfactin 155

3. Prototype of detergent containing Surfactin 168

V. Development of microbial agents for feed additives ; Biofunctionality and bioconvertibility of oral microbes 172

A. Introduction 172

B. Materials and methods 174

1. Oral microbial community and population analysis 174

2. Total DNA extraction and analysis of oral microbes 175

3. Antibiotic-resistant bacteria (resistance gene) distribution analysis and virulence gene analysis 176

4. Research on physiological activity and bioconversion ability of oral microbes 181

5. NGS analysis of oral microbiome 187

C. Results and Discussion 190

1. Oral microbial community and population analysis 190

2. Total DNA extraction and analysis of oral microbes 196

3. Antibiotic-resistant bacteria (resistance gene) distribution analysis and virulence gene analysis 199

4. Research on physiological activity and bioconversion ability of oral microbes 212

5. Results of NGS analysis of oral microbiome 221

VI. Conclusion 233

REFERENCES 246

국문 초록 259

Table I-1. Current status of domestic veterinary drug market in Korea. 27

Table I-2. Global major feed additive market forecast. 29

Table II-1. Formulation medium conditions for functional feed additives. 39

Table II-2. Microorganisms used for bioconversion and fermentation. 39

Table II-3. List of measurable enzymes. 43

Table II-4. Inoculum(seed) size for fermentation 51

Table II-5. Microbial populations in powdered rice bran 51

Table II-6. Microbial populations after dry sterilization 52

Table II-7. Changes of seed populations(starters) during fermentation... 53

Table II-8. Microbial analysis of dry sterilized FFFA 54

Table II-9. Analysis of moisture, fat, protein, ash, dietary fiber, carbohydrates... 57

Table II-10. Changes of sugar, alcohol, and pH in FFFA 59

Table II-11. Analysis of extracellular enzyme activity in FFFA 60

Table II-12. Changes of intestinal microbial populations (CFU/g)... 67

Table II-13. Analysis of growth rate of broiler chickens according to the... 69

Table III-1. List of measured enzyme activity for the establishment of... 74

Table III-2. Measurement of enzyme activity according to reaction time 75

Table III-3. List of enzyme substrates used in measuring enzyme activity in... 77

Table III-4. List of enzymes substrates used for anaerobic digester system 78

Table III-5. RF measurements according to MUF concentration 87

Table III-6. List of enzyme substrates used in measuring enzyme activity 88

Table III-7. Changes in Enzyme activities after reaction according to the... 89

Table III-8. Changes in Enzyme activities after reaction according to the... 90

Table III-9A. Enzyme activity measurement table according to the change in... 97

Table III-9B. Enzyme activity measurement table according to the change in... 97

Table III-9C. Enzyme activity measurement table according to the change in... 98

Table III-9D. Enzyme activity measurement table according to the change in... 98

Table III-10. Result table of correlation between RF value of... 99

Table III-11. The number of cells (CFU/ml) according... 99

Table III-12A. Anaerobic digester enzyme activity measurement -... 113

Table III-12B. Anaerobic digester enzyme activity measurement -... 113

Table III-12C. Anaerobic digester enzyme activity measurement -... 114

Table III-12D. Anaerobic digester enzyme activity measurement -... 114

Table III-13A. Anaerobic digester enzyme activity measurement -... 115

Table III-13B. Anaerobic digester enzyme activity measurement -... 115

Table III-13C. Anaerobic digester enzyme activity measurement -... 116

Table III-13D. Anaerobic digester enzyme activity measurement -... 116

Table III-14. Measurement of enzyme activity of Dongchimi broth. 118

Table III-15. Components of the enzyme activity measurement terminal 119

Table III-16. Size measurement test of real-time enzyme... 125

Table III-17. Results of terminal operating temperature 127

Table III-18. Terminal operating time test result values 129

Table III-19. Real-time enzyme activity measurement terminal power... 130

Table IV-1. Experimental conditions medium 137

Table IV-2. List of pathogenic microorganisms 137

Table IV-3. Analyzer and analysis conditions 140

Table IV-4. Prototype composition 143

Table IV-5. comparison of surfactant's cleaning power (Engine oil) 146

Table IV-6. Surfactant's cleaning power according to the bacterial strains 149

Table IV-7. Quantitative comparative analysis of washing power of culture... 150

Table IV-8. Antimicrobial activity of each sample of B. amyloliquefaciens KB3 152

Table IV-9. Antimicrobial activity of each sample of Bacillus subtilis EBM13 153

Table IV-10. Antimicrobial activity of each sample of Bacillus subtilis KL1114 154

Table IV-11. Investigation of dry yield of surfactin purified from culture 155

Table IV-12. Qualitative evaluation of surfactin of culture supernatant,... 162

Table IV-13. Quantitative evaluation of surfactin of culture... 163

Table IV-14. Emulsification activity and emulsion stability of surfactin 164

Table IV-15. Solubility of surfactin by Organic Solvent 167

Table IV-16. Consumer sensory test of detergent prototypes (5- types)... 169

Table IV-17. Daphnia test results by concentration for the detergent prototype 5 171

Table V-1. Antibiotic resistance gene Primer 177

Table V-2. Condition of real-time PCR(qPCR) 177

Table V-3. Virulence gene Primer 178

Table V-4. Condition of real-time PCR(qPCR) 178

Table V-5. Condition of real-time PCR(qPCR) by periodontitis diagnosis 179

Table V-6. Antimicrobial activity analysis strains 182

Table V-7. Medium composition 182

Table V-8. Conditional medium for TLC experiment 184

Table V-9. List of strains selected for bioconversion ability analysis 184

Table V-10. Peak time of haltosis substances (reactive gas) of Oral Chroma... 186

Table V-11. M9 minimal medium composition 186

Table V-12. Result of oral microbial community and population counts 190

Table V-13. Results of measuring physiological activity of microorganisms. 194

Table V-14. Selected strains for measuring extracellular enzyme activity and... 195

Table V-15. Identifications of isolated strains in these experiments on the basis... 198

Table V-16. DNA concentration 199

Table V-17. qPCR result table of antibiotic resistance gene (tet A) 200

Table V-18. Standard real-time PCR (qPCR) results of Set 1,2 204

Table V-19. Tooth corrosion analysis of oral microbes 210

Table V-20. Results of analysis of antimicrobial activity of oral microorganisms 213

Figure II-1. Domestic feed additive market scale 34

Figure II-2. Feed additive technology trend 35

Figure II-3. Viability of each strain according to the liquid formula... 49

Figure II-4. Viability of each strain according to the liquid formula... 49

Figure II-5. Viability of each strain according to the semi-solid... 50

Figure II-6. Viability of each strain according to the semi-solid... 50

Figure II-7. Changes of seed populations(starters) during fermentation for FFFA 53

Figure II-8. Results of analysis of moisture, fat, protein, ash,... 56

Figure II-9. Analysis results of 16 constituent amino acids in FFFA 56

Figure II-10. Analysis of protein in FFFA M; Maker,... 58

Figure II-11. Changes in extracellular enzyme activity in FFFA according... 61

Figure II-12. TLC of amino acids in FFFA. 64

Figure II-13. TLC analtsis of lipids in FFFA during fermentation 65

Figure III-1. Operating temperature test configuration diagram. 81

Figure III-2. Real-time enzyme activity measurement; terminal... 81

Figure III-3. Real-time enzyme activity measurement; terminal... 82

Figure III-4. Measurement of fluorescence activity of MUF according to visible... 84

Figure III-5. Spectrum of 365nm wavelength 85

Figure III-6. UV spectrum according to temperature 85

Figure III-7. UV pattern according to wide angle 85

Figure III-8. Photosensitcity measurement at room temperature 86

Figure III-9. Receiving sensor specification 86

Figure III-10. RF measurements according to MUF concentration 87

Figure III-11. Reaction stop in ice 91

Figure III-12A. Enzyme activity measurement graph according to the change in... 93

Figure III-12B. Enzyme activity measurement graph according to the change in... 94

Figure III-12C. Enzyme activity measurement graph according to the change in... 95

Figure III-12D. Enzyme activity measurement graph according to the change in... 96

Figure III-13. Relationship between cell... 100

Figure III-14. Correlation chart of... 100

Figure III-15A. Enzyme activity measurement graph according to substrate... 102

Figure III-15B. Enzyme activity measurement graph according to substrate... 104

Figure III-15C. Enzyme activity measurement graph according to substrate... 106

Figure III-15D. Enzyme activity measurement graph according to substrate... 108

Figure III-16A. Graph of measuring enzyme activity in anaerobic digester - 10 minutes 111

Figure III-16B. Graph of measuring enzyme activity in anaerobic digester - 20 minutes 111

Figure III-16C. Graph of measuring enzyme activity in anaerobic digester - 30 minutes 112

Figure III-16D. Graph of measuring enzyme activity in anaerobic digester - 60 minutes 112

Figure III-17. Measurement of enzyme activity of Dongchimi broth. 118

Figure III-18. System Block Diagram 120

Figure III-19. Micro-Processor Unit circuit diagram 120

Figure III-20. Circuit diagram 120

Figure III-21. Current measurement module board artwork 121

Figure III-22. Power supply circuit diagram 121

Figure III-23. Power and terminal measurement process 122

Figure III-24. Display configuration 123

Figure III-25. Display circuit diagram 123

Figure III-26. Real-time enzyme activity measurement results of... 125

Figure III-27. Internal structure diagram of a prototype of a terminal for... 126

Figure III-28. Design of a prototype of a terminal for measuring enzyme... 126

Figure III-29. Operating temperature... 128

Figure III-30. Graph of measured... 128

Figure III-31. Changes in current according to operation of... 130

Figure IV-1. Small and Medium Business Technology Roadmap... 132

Figure IV-2. Structure of biosurfactants 133

Figure IV-3. Surface tension measurement method using Du Nouy surface... 145

Figure IV-4. Equation for calculating the ecotoxicity value (TU) 145

Figure IV-5. Detergency verification result of various surfactins... 147

Figure IV-6. Comparison of surfactant generation according to the... 149

Figure IV-7. Quantitative comparative analysis of washing power of culture... 150

Figure IV-8. Surfactin TLC results of the supernatant for each sample of... 152

Figure IV-9. Surfactin TLC results of the supernatant for each sample of... 153

Figure IV-10. Surfactin TLC results of the supernatant for each sample of... 154

Figure IV-11. Surfactin TLC results of the supernatant for each sample... 157

Figure IV-12. Surfactin TLC results of the supernatant for each sample... 158

Figure IV-13. Surfactin TLC results of the supernatant for each sample... 159

Figure IV-14. Surfactin TLC result of concentrate of each sample of... 160

Figure IV-15. Surfactin calibration curve 161

Figure IV-16. Emulsifying activity and emulsion stability of surfactin by... 165

Figure IV-17. Surface tension depending on the concentration of surfactin 166

Figure IV-18. Solubility of each organic solvent according to the... 167

Figure IV-19. Cleansing power of contaminated cloths of surfactin prototype 169

Figure IV-20. Stability under severe aging for 5 prototypes and commercial... 170

Figure V-1. TruSeq Index Plate Fixture 189

Figure V-2. Results of the investigation of oral microbial community and cell count 191

Figure V-3. Results of extracellular enzyme activity spectrum of oral microbes 195

Figure V-4. Phylogenetic tree of the isolated strains on NA and MRS media in... 197

Figure V-5. Amplicon size of gene tet A (Agarose gel) 200

Figure V-6. Antibiotic resistance gene qPCR result graph(NC : DEPC Water) 201

Figure V-7. Virulence gene qPCR result graph(NC : DEPC Water) 202

Figure V-8. Standard graph result of Set 1,2 203

Figure V-9. Results of detection and analysis of the pathogens causing... 207

Figure V-10. Tooth corrosion analysis of oral microbes 211

Figure V-11. Analysis spectrum of antimicrobial activity of oral microorganisms 214

Figure V-12A. Analysis of bioconversion ability of oral microbes 216

Figure V-12B. Analysis of bioconversion ability of oral microbes 216

Figure V-13. Oral halitosis analysis 218

Figure V-14A. Haltosis reduction analysis 219

Figure V-14B. Haltosis reduction analysis 220

Figure V-15. Analysis of Miseq NGS;16S metagenomic sequencing of oral... 221

Figure V-16. Analysis of Miseq NGS;16S metagenomic sequencing of oral... 223

Figure V-17. Analysis of Miseq NGS;16S metagenomic sequencing of oral... 225

Figure V-18. Analysis of Miseq NGS;16S metagenomic sequencing of oral... 227

Figure V-19. Analysis of Miseq NGS;16S metagenomic sequencing of oral... 229

Figure V-20. Analysis of Miseq NGS;16S metagenomic sequencing of oral... 231

본 논문에서는 기능성 미생물을 이용하여 양계장 환경을 제어하며, 육계의 성장 증진에 기여하는 기능성 사료첨가제를 개발하고자 연구를 진행하였다. 본 연구는 총 4파트로, 첫 번째는 기능성 사료 첨가제 사용에 따른 건강증진 및 축사 환경 개선의 연구를 진행하였다. 두 번째는 축사 환경내 효소 활성도 동태 파악 연구, 세번째는 축사 환경 내 위생 증진의 연구, 네 번째는 사료 첨가제용 미생물 제제의 개발 응용연구로 구성되어 있다.

첫 번째로 기능성 사료 첨가제 사용에 따른 건강증진 및 축사 환경 개선의 연구에서는 기능성 발효 사료 첨가제의 제조를 위한 공정도를 확립하였다. 생물기능성이 향상된 발효 사료첨가제의 연구 결과 생물전환 능력(bioconversion)이 우수하며, 구성아미노산의 양적 변화들은 다음과 같다. (Glutamic acid 267.2mg으로 발효 과정을 통해 변화 된 구성아미노산 중 가장 많은 변화를 가져왔다. Threonine은 50.3mg, Serine은 52mg, Proline은 96.9mg, Glycine은 132.6mg, Alanine은 247.8mg, Valine은 156.9mg으로) 발효 전후 비교하였을 때 전 후 모두 증가하였다. 기능성 사료 첨가제의 양계 현장에서 적용하여 양계의 장내 미생물 군집을 분석한 결과 유산균과, 고초균은 실험군에서 더 많이 증가하였고, 살모넬라, 대장균속은 대조군에서 더 많이 군집하고 있음을 확인하였다. 따라서 기능성 사료첨가제의 사용으로 장내 미생물 군집에서 유용미생물군이 우점적으로 군집하고 있으며, 이로 인해 육계의 증체량 역시 약 13% 이상 성장을 보였다. 더욱이 장내 유용미생물로 인해 양계장 바닥 환경이 좋게 변화 할 것이라 사료된다.

두 번째로 축사 환경 내 효소 활성도 동태 파악 연구에서는 MUF(methylumbelliferyl) 형광기질을 이용하였다. 또한 MUF는 365nm에서 최대로 발광하는 것을 확인하여 발광부는 365nm의 파장대역을 방출 할 수 있는 램프를 탐색하였다. Estrase를 비롯한 14가지의 측정 가능 효소키트를 개발하였다. 많은 양의 시료를 측정하기 위해 효소기질 반응후 얼음에서 10분, 20분, 30분, 1시간, 2시간 보관하여 측정한 결과 측정값(RF값) 1768~1790으로 일정하게 유지되었다. 여러 시료를 한번에 측정할 때 기질을 넣은 후 측정 전 얼음에 넣어 보관한 후 측정하여도 결과값이 변하지 않으므로 단 시간 내 많은 측정이 용이할 것으로 사료된다. 효소 활성도를 산업현장에서 응용하기 위하여 혐기성 소화조 모형과 김치의 발효과정 또는 과숙성 과정중 효소활성도를 측정하였다. 음식물의 분해가 많이 될수록 효소 활성도 역시 높아짐을 확인하였다. 특히 estrases의 효소활성도 RF값 변화 양상으로, 동치미의 발효정도를 알 수 있을 것으로 사료된다. MUF기질의 효소 활성을 측정하는 단말기를 휴대형으로 개발하였다. 전원부는 3V로 구성하였다. 효소활성 측정 단말기는 산업현장에서 바로 응용하고자 휴대성이 높게 가로＝179.9 mm, 세로＝106.2mm, 높이＝71.8mm로 개발하였다.

세 번째로 축사환경 내 위생 증진의 연구에서는 생물계면활성제 surfactin을 이용한 축사 세척제를 개발하고자 하였다. 이를 위해 생물계면활성능이 뛰어난 균주(Bacillus amyloliquefaciens KB3, B. subtilis EBM13, B. subtilis KL1114)를 발굴하였다. 엔진오일을 대상으로 미생물 배양액과 상등액으로 세척력을 시험결과 상등액에서 생물계면활성 물질이 생합성 되는 것을 확인하였다. Surfactin의 농도에 따른 배양액 세척력 정량 비교 결과 KB3은 surfactin(0.0312%)정도의 확산을 보였다. EBM13은 surfactin(0.062%)정도의 확산을 보였다. KL1114은 surfactin(0.25%)정도의 매우 높은 확산을 보였다. 배양액에서 서펙틴의 건조수율은 51.2%를 보였다. 서펙틴의 표준물질과 배양 상등액, 거품포집액, 부분정제한 서펙틴 분말, 시제품에서 모두 동일한 peak pattern이 확인되었으며, 정량한 물질이 동일한 서펙틴임을 확인하였다. 또한 서펙틴의 함량을 달리하여 세척제 시제품을 제작하여 물벼룩 급성독성 시험을 실시하였다. 그 결과 서펙틴이 함유된 계면활성제 시제품은 물벼룩(Daphnia magna)에 대한 급성독성시험을 실시한 결과, 24시간 및 48시간 반수영향 농도 (EC50)는 모두 제품기준으로 100.000 mg/L 초과로 나와 독성이 없는 것으로 보였다.

네 번째로 사료첨가제용 미생물제제 개발연구에서는 본 연구진이 보유한 6종의 유산균과 선별된 구강 미생물들이 치주염 유발 미생물 3종(KCTC 2581, KCTC 5365, KCTC5666)에 대한 강력한 항균활성능을 보였다. 생리활성이 뛰어난 구강 미생물 15종을 대상으로 생물전환능 연구 결과 BL6-4(Lactobacillus fermentum), BL16-2(Lactobacillus fermentum) 균주에서 아르기닌을 오르니틴으로 100% 전환하는 것을 볼 수 있었다. BL16-2균주는 시트룰린까지 전환하는 것을 볼 수 있었다. BL16-5(Lactobacillus curvatus) 균주와 KA35(Lactobacillus casei)균주를 처리하고 구취 저감능력을 확인 결과 황화수소(H2S)는 BL16-5를 처리 했을 때에는 12.33ng/10㎖농도의 감소를 보였고, KA35를 처리 하였을 때에는 11.90ng/10㎖ 농도의 감소를 보였다. methyl mercaptan (CH3SH)는 BL16-5를 처리 했을 때 7.69ng/10㎖농도의 감소를 보였다. 구강 마이크로비옴을 차세대 염기서열 분석법으로 분석 결과 species수준에서는 parainfluenzae, pseudopneumoniae, flavescens, caviae, melaninogenica, mucosa, parvula, nanceiensis, dispar, pallens, mirabilis, mucilaginosa, histicola, atypica, parasanguinis, azotoformans, lundensis, fragi, tigurinus, infantis, sanguinis 등을 볼 수 있었다.