Studies on Improvement of Feed Efficiency and Fermentation in Gastrointestinal Tract using Benefical Microorganisms in Livestock

국문 초록 (Abstract)

사료첨가제의 사용목적은 사료의 품질의 저하를 방지하기 위하여 첨가하는 용도와 가축의 효용 증대를 위하여 사료에 첨가하는 것으로 구분할 수 있다. 사료의 품질의 저하를 방지하기 위해서 결착제, 유화제 및 보존제가 사용되며, 가축의 효용 증대를 위해서 아미노산제, 비타민제, 효소제, 생균제, 향미제, 비단백태질소화합물, 규산염제, 완충제, 착색제, 추출제, 올리고당 등을 이용한다. 본 연구에서는 사료 첨가제 사용에 따른 효과 및 특성을 파악하고자, 1) 유산균 첨가에 따른 미역부산물 사료의 발효성상 및 미생물 군집 분석, 2) 유산균 첨가에 따른 섬유질배합사료의 발효성상 및 반추위 발효특성 연구, 3) 인위적인 대장균 접종에 따른 박테리오파지 효과 및 반추위 발효특성 연구, 4) 발효 유황사료첨가제 급여가 육계의 생산성 및 장내 미생물에 미치는 영향을 조사하였다.
염 저항성, 유기산 생성, 항균활성이 뛰어난 Lactobacillus brevis RNAL14, Leuconostoc citreum SROD-3 을 선발하여 미역부산물 사료의 발효제로 이용한 실험에서는 미역부산물 사료를 100 일 동안 상온에 보관하면서 발효성상 및 미생물 군집분석을 분석하였다. 그 결과, 유산균 첨가구에서는 발효기간 경과에 따라 유산, 초산 생성량이 증가하였고, 총 호기성세균, 유산균 및 효모는 감소하였다. 특히, 발효 15 일차에 T1 처리구에서 L.brevis 가 우점균주로 나타나 적어도 15 일까지는 접종균주의 효과를 확인 할 수 있었고, Bacteria 종 다양성 또한 가장 낮았다. 반면에 미생물 대신 물을 첨가한 대조구에서는 발효기간 경과에 따라 pH 감소폭이 적었고 발효 100 일차에 pH 증가, 유산 생성량 감소, 호기성세균 및 살모넬라 증가, Penicillum 검출되었으며, Bacteria 종 다양성이 가장 높았다. 위의 결과를 종합하면, 미역부산물 사료의 저장성 개선을 위해서는 풍부한 유기산생성능, 접종균주의 우점, 유해미생물 저감을 나타낸 L.brevis 사용이 효과적이며, 미생물 무첨가는 사료의 장기적인 보존에 부정적인 영향을 미치는 것으로 나타났다.
이상발효 유산균인 Lactobacillus plantarum KACC11451, Lactobacillus brevis ATCC14869T, Lactobacillus mucosae S32T 을 섬유질배합사료 발효제 이용한 실험에서 섬유질배합사료는 농후사료와 조사료의 비율을 8 : 2 로하여 60℃에서 2 일간 살균하여 제조하였다. 유산, 초산 및 프로피온산 생성을 보인 이상발효 유산균 3 종을 (1x108 cfu/mL) 각 10% 접종하였으며, 수분함량을 조절하기 위해 멸균수를 30%를 추가하였고, 대조구는 멸균수 40%를 혼합하여 37℃에서 7 일간 배양하였다. 그 결과, 섬유질배합사료에 Lactobacillus plantarum 처리는 pH 저감 및 유산생성, 총 호기성세균, 유산균수가 가장 높았다. Lactobacillus brevis 처리는 총 유기산생성이 우수하였고, 곰팡이를 억제하였다. Lactobacillus mucosae 처리는 초산생성과 사료의 조단백질 함량이 가장 높았다. In vitro 반추위 발효성상을 평가한 결과, 유산균을 첨가하여 발효한 TMR 사료는 pH, 총 가스발생량 및 암모니아태 질소농도에 통계적인 유의차는 없었지만, 대조구에 비해 총 휘발성지방산 농도가 높았다. 위의 결과를 종합하면, 섬유질배합사료의 유산균 발효는 사료의 발효성상과 반추위 발효에 있어 개선되는 효과를 보였다. 또한, 처리구간에는 큰 차이를 보이지 않았지만, 섬유질배합사료의 총 유기산생성과 곰팡이 억제 및 in vitro 반추위 발효에서 총 휘발성지방산 생성량이 높았던 L.brevis ATCC14869T 균주 사용이 사료의 저장성을 개선하고 반추위 발효 개선에 보다 효과적 일 것으로 사료된다.
인위적인 E.coli KACC14818 접종에 따른 Bacteriophage 첨가 효과 및 반추위 발효특성 조사는 칵테일 형태의 Bacteriophage 사료를 CTC bio 업체에서 제공받아 본 연구에 이용하였다. Bacteriophage 사료가 병원성미생물에 미치는 영향을 알아보기 위해 9mL 의 병원성 미생물에 1.0g 의 Bacteriophage 사료를 첨가하여 24 시간동안 37℃에서 배양한 후 생균수를 측정하였다. 그 결과, E.coli KACC14818 은 70%, S.enterica KACC10763 은 93% 억제되었으나, E.coli O157:H7 아무런 영향을 미치지 못했다. 반추가축의 질병은 송아지 설사가 대표적이며, 질병유발병원균은 대표적으로 E.coli 이다. 따라서, E.coli 를 반추위에 인위적으로 접종하고 Bacteriophage 사료를 첨가한 후 in vitro 반추위 발효성상을 조사하였다.
E.coli 오염에 따른 Bacteriophage 무첨가구는 발효 24 시간 총가스발생량은 가장 낮은 경향을 보였고(P>0.05), pH 는 가장 높았으며 (P<0.05) 더불어 총휘발성지방산은 가장 낮았다 (P<0.05). 반면에 대장균 오염에 따른 Bacteriophage 첨가구는 positive control 과 유사한 결과를 나타내었다. 또한 미생물 정량분석결과 Bacteriophage 사료첨가제는 general bacteria 와
Lactobacillus spp. 의 영향을 미치지 않고 E.coli 을 억제하는 효과를 나타내었다. 결론적으로, E.coli 의 인위적인 오염에 따른 Bacteriophage 첨가는 반추위발효에서 일반적인 미생물에 영향을 미치지 않으면서 선택적으로 E.coli 을 억제하였으며, 정상적인 반추위 발효형태를 보였다.
발효 유황사료첨가제 급여가 육계의 생산성 및 장내 미생물에 미치는 영향 조사는 배합사료에 유황을 500ppm 수준으로 급여하였고, 육계 (Ross 308) 180 수를 대조구 및 처리구로 2 처리구, 3 반복, 반복당 30 수로 하여 평사에서 사육하였다. 물과 사료는 자유 급여하였으며, 총 사육기간은 35 일로 전기는 1 ~ 21 일, 후기는 22 ~ 35 일로 하였다. 시험 결과, 유황의 500ppm 수준의 급여는 육계의 폐사율을 감소시키고, 사양후기에 사료섭취량 증가하는 경향을 보여 증체량이 증가되었고, 사료요구율이 개선됨으로써 생산성 향상에 긍정적인 영향을 미쳤다. 또한 사양 4 주차 육계사의 암모니아 농도는 대조구에 비해 약 17% 억제하는 효과를 보였고, 계육의 oleic acid 와 alpha-linolenic acid 지방산 조성을 높였다. 혈청 ALT 분석결과, 대조구 및 처리구의 유의성이 관찰되지 않아 유해성분에 대한 영향은 없었다고 사료되며, 중성지방이 감소하여 지질대사가 개선되는 효과를 나타냈으며, 혈청 IgA 및 IgG 함량이 일반사료 급여구에 비해 높게 나타나 면역을 활성화 하는 효과가 있음을 확인하였다. 발효유황첨가제 급여에 따라 생산성이 향상된 닭과 일반사료급여 닭의 소화기관의 미생물 다양성과 미생물 함량을 비교 분석한 결과, Crop 을 제외한 소화기관에서 발효유황첨가제를 급여한 닭의 미생물 다양성과 미생물 함량이 낮은 경향을 보였다. 또한 닭의 소화기관 미생물의 다양성이 낮음과 증체량의 증가에 있어 상관관계가 있음을 확인하였다.
본 연구는 사료 첨가제 중 유산균, Bacteriophage 및 발효유황을 이용하였다. 유산균은 미역부산물 및 섬유질배합사료의 발효촉진제 목적으로 평가하였고, Bacteriophage 는 질병예방 목적으로 평가하였으며, 발효유황은 생산성 향상을 목적으로 조사되었다. 각 사료첨가제의 기능과 특성에 맞게 적용하면 사료의 품질의 저하를 방지하고 가축의 효용 증대에 도움이 될 것이다.

다국어 초록 (Multilingual Abstract)

CHAPTER I.
Lactic acid bacteria had been widely used as fermented sources. Meanwhile, seaweeds contain potential nutrients and bioactive phytochemicals, which can contribute to the protein and energy requirements of livestock animals. During seaweed processing in Korea, 90% of Undaria pinnatifida becomes by-products and are usually wasted. Seaweed by-products (SWBP) is quickly to spoil at room temperature due to high moisture concentrations. Hence, we isolated a strain of lactic acid bacteria that exhibits high levels of organic acid production, salt tolerance ability and antimicrobial activity. Also, organism was subsequently identified and characterized as Lactobacillus brevis RNAL14, Leuconostoc citreum SROD-3 by 16S rRNA gene sequencing. Utilization and preservation were done in this study through fermentation of seaweed by-products using two kind of lactic acid bacteria Lactobacillus brevis RNAL14 and Leuconostoc citreum SROD-3. Moreover, the effect of adding LAB on fermentation, microbial communities, and metabolites were evaluated. Seaweed by-products were collected from Goheung, South Korea, mixed with wheat bran at 1:1 ratio, and added with molasses at 2% final concentration. The mixed substrate were separated and inoculated either without (water; control), 5% L. brevis RNAL14 (1x108cfu/mL), or 5% Le. citreum SROD-3 (1x108cfu/mL). Then, it was sealed and incubated for 15 d, 50 d and 100 d at room temperature, and determined the fermentation characteristics, predominant LAB, and bacterial communities using bar-coded pyrosequencing. In the added-LAB group, lactic acid and acetic acid production increased depending with increasing storage duration, while pH, total aerobic bacteria, LAB, and yeast decreased. Coliform and Salmonella were not detected in the T1 treatment for the entire period. In addition, as L. brevis was the dominant strain at the 15 day of fermentation T1 treatment, the inoculated strain continued to be effective until at least 15 days. L. brevis was confirmed to dominate early fermentation by strong production of organic acids.
Analysis of microbial diversity analysis revealed that Streptophyta was the dominant on the 0 day of SWBP feed storage, while at the genus level, Lactobacillus was dominant throughout the storage period. The T1 treatment, in particular, showed the highest Lactobacillus dominance rate over the whole period. Fungal diversity analysis exposed that, while unclassified fungi was dominant from 0 to 15 days, Pichia became dominant from 50 days. In addition, the T1 treatment group showed the highest Pichia dominance rate over the whole period. Control feed, coliform bacteria showed their highest level of 5.02 log cfu/g on the 50 day, and the pH also increased rapidly to 5.32 by the 100 day. In particular, Penicillium was detected in the control after 100 days of storage, and OTU was 18, indicating very low fungal diversity. By the 100 day of storage in the control, a rapid increase in pH, decreased lactic acid content, increased aerobic bacteria and Salmonella spp., and detection of Penicillium are considered indicators of feed decay. Therefore L. brevis, which produces abundant organic acids, lowers the pH, is a dominant strain, and reduces the level of harmful microorganisms, can be used effectively to improve the storability of SWBP feed. Meanwhile, not adding any microorganisms were found to have a negative effect on feed storage, making an environment that is susceptible to feed decay.

CHAPTER II.
This study was conducted to evaluate the effects of adding LAB additives in total mixed ration (TMR) on fermentation quality and in vitro ruminal fermentation. Lactobacillus plantarum, Lactobacillus brevis and Lactobacillus mucosae were from KACC, isolated from Korean native goat and Korean native cattle, respectively and used for this study. The experimental treatments were control (TMR + water), treatment 1 (TMR + L. plantarum), treatment 2 (TMR + L. brevis), treatment 3 (TMR + L. mucosae). The rumen fluids were collected from Holstein Friesian cattle and mixed with buffer solution (3:1), after which 100 mL of buffered rumen fluid was transferred into 160 mL serum bottles
containing one g dry matter (DM) of TMR added with or without LAB under a stream of O2-free N2. The samples were then incubated for 0, 3, 6, 12, 24, or 48h at 39°C and 100 rpm. The chemical composition of TMR contained 129.10 g/kg DM of crude protein, 25.98 g/kg DM of ether extract, 44.33 g/kg DM of crude ash, 356.28 g/kg DM of neutral detergent fiber and 161.25 g/kg DM of acid detergent fiber. The fermented TMR feed with added LAB showed lower pH and higher concentrations of lactic acid, acetic acid and total organic acid compared to control, in which only water was added (P<0.05). In addition, the numbers of aerobic bacteria and LAB were higher, but E. coli counts were lower compared to the control (P<0.05). Notably, the T1 treatment, which had the lowest pH, highest lactic acid content and the highest numbers of aerobic bacteria and LAB, T2 treatment had the highest concentration of total organic acid and fungus was not detected. The fermented TMR feed with added LAB showed a lower pH than the control group at 0 hours, due to the abundant production of lactic acid and acetic acid, but the pH became similar level to the control by 3 hours of fermentation. The concentration of ammonia nitrogen was higher in the LAB group at 0 hours fermentation (P<0.05) and it was maintained with higher trend level throughout the whole fermentation period, even though this was not statistically significant (P>0.05). No differences in acetate and butyrate concentration after 48 h of incubation. However, a tendency towards higher acetate and butyrate concentration in LAB groups compared to the control was observed after 48 h. Nevertheless, at 48 h of incubation, total VFA concentration was higher (P<0.05) in the LAB groups than the control.
Especially, total VFA concentration differed significantly among the treatments with the highest value observed in the T2 treatment. Overall, the results of this study suggest that addition of LAB especially L. brevis in TMR increased the volatile fatty acid concentrations.

CHAPTER III.
Bacteriophages are viruses that infect and destroy a bacterial host. They have recently been the subject of much research seeking alternatives to antibiotics.
The present study aimed to investigate the effects of bacteriophage feed on rumen fermentation following artificial E. coli inoculation. The bacteriophage cocktail were developed to target 4 strains of Salmonella, 3 strains of E. coli, 2 strains of Clostridium perfringens, and Staphylococcus aureus. Basal feed(without bacteriophage) additive Escherchia coli (Negative Control), Bacteriophage feed additive Escherchia coli supernatant (Positive Control), and Bacteriophage feed additive Escherchia coli (EC Treatment) was used as substrates. Although we did not clearly demonstrate inhibition of the E. coli, E. coli O157:H7, and Salmonella enterica used in this experiment, there was a reduction in viable count for E.coli KACC14818 and Salmonella enterica KACC10763, while there was no effect on E. coli O157:H7. Some of the most common diseases occurring in ruminant cattle are diarrhea. Hence, in this study, we artificially inoculated the rumen with E. coli, and evaluated the effect on ruminal microorganisms when bacteriophage feed was added. One gram dry matter of substrate was put in triplicated serum bottles then filled with 100 mL of mixed rumen fluid and buffer (1:3), followed by incubation at 0, 6, 12 and 24h (39oC, 100 rpm). In each incubation time, fermentation parameters such as pH, gas production, ammonia-nitrogen (NH3-N), volatile fatty acids (VFA) and quantitative real time PCR were measured. In all treatment, the total gas production increased with increasing incubation time, while the pH decreased.
NC treatment showed a trend for having the lowest total gas production at 24 hours of fermentation (P>0.05), and had the highest pH (P<0.05). Moreover, it also had the lowest total VFA concentration (P<0.05). Conversely, the EC treatment showed similar results to the PC treatment. In a QPCR analysis, we found that general bacteria and Lactobacillus spp. did not show a statistically significant difference. However, E. coli was highest in the NC treatment after 24 hours of incubation, followed by the EC treatment, then the PC treatment group.
Hence, bacteriophage feed has no effect on general bacteria and Lactobacillus spp., which are involved in ruminant fermentation, but is able to suppress E. coli. Addition of bacteriophage is a potential strategy in in increasing total VFA as well as reducing the incidence or treat specific bacterial diseases caused by E. coli.

CHAPTER IV.
Sulfur is highly toxic when directly ingested and causes side effects and thus often physical or chemical methods are used to eliminate harmful ingredients.
Nevertheless, insufficient elimination of toxins leads to diseases in animals and thus special care is required. This study showed that cadmium, arsenic, lead, and mercury contents were lower than the permitted thresholds in fermented sulfur additives produced using fermentation method. A total of 180 broilers one-dayold Ross broiler chicken were used to investigate the effects of fermented sulfur on growth performance and gut microbial community. The two dietary treatments, each with 3 replicates were basal diet and base diet plus the 0.2% fermented sulfur. All broilers were reared in floor pens under typical commercial husbandry conditions. Each of the three replicates in each group was housed in one pen, and a total of 6 pens were housed in the same room at the Sunchon National University breeding farm of Korea. Chicken was provided free access to feed and water. The trial was run for 35 days. Providing of 500 ppm sulfur decreased mortality rate and increased feed intake in finisher stage, resulting in weight gain and improving feed conversion rate to positively affect productivity enhancement. Further, at the 4 week of feeding, ammonia concentration in broiler house was inhibited by 17% and the component ratios of oleic acid and alpha-linolenic acid increased. Blood ALT analysis results showed no significance between treatment and control groups, suggesting the lack of harmful ingredient, decreased triglyceride, showing improved lipid metabolism and increased blood IgA and IgG contents, showing enhanced immunity compared to the control group. Compare analysis of microbial diversity and contents in GIT of chickens with enhanced productivity from fermented sulfur additive and chickens fed basal feed showed reduced microbial diversity and contents in all GIT excluding crop in chickens fed fermented sulfur additive.
Further, a correlation between reduced microbial diversity in chicken GITsystem and weight gain was recognized.

목차 (Table of Contents)

• GENERAL INTRODUCTION ………………………………………….... 1
• REVIEW OF LITERATURES ….………………………………………... 3
• Feed Additives…………………………………………………………… 3
• Probiotics………………………………………………………………… 3
• Lactic acid bacteria………………………………………………………. 7
• Bacteriophage……………………………………………………………. 10
• Gut Microbiota ………………………………………………………….. 12
• Microbial Community Analysis……………..…………………………... 15
• REFERENCES……………………………………………………………... 17
• CHAPTER I. Effect of Fermenting Seaweed (Undaria pinnatifida) byproduct
• with Lactic Acid Bacteria on Microbial Flora and Metabolites… 21
• ABSTRACT……………………………………………………………... 22
• I. INTRODUCTION…………………………………………………….. 25
• II. MATERIALS AND METHODS……………………………………... 28
• Selective medium and isolation of lactic acid bacteria…………………… 28
• Salt ranges for growth……………………………………………………. 29
• Metabolite production…………………………………………………… 30
• Antibacterial activities…………………………………………………… 30
• Identification……………………………………………………….......... 31
• Undaria pinnatifida by-product fermentation…………………………… 32
• Fermentation quality analysis……………………………………………. 32
• Predominant LAB…………………………………………………........... 33
• Barcoded pyrosequencing, sequence processing and data analysis……… 34
• Statistical methods……………………………………………………….. 36
• III. RESULTS…………………………………………………………… 37
• IV. DISCUSSION……………………………………………………….. 58
• V. CONCLUSIONS…..…………………………………………………. 62
• VI. REFERENCES……………………………………………………… 63
• CHAPTER II. Effect of Lactic Acid Bacteria Additives in Total Mixed
• Ration (TMR) on Fermentation Quality and in vitro Rumen
• Fermentation……………………………………………………………….. 67
• ABSTRACT……………………………………………………………... 68
• I. INTRODUCTION…………………………………………………….. 70
• II. MATERIALS AND METHODS…………………………………….. 72
• Isolation, identification and metabolite production of microbes………… 72
• Fermentation of TMR………………………………………………......... 73
• Fermentation quality analysis………………………………………......... 74
• In vitro rumen fermentation……………………………………………… 75
• Parameters for analysis of in vitro rumen fermentation………………….. 77
• Statistical Method………………………………………………………... 78
• III. RESULTS…………………………………………………………… 79
• IV. DISCUSSION……………………………………………………….. 86
• V. CONCLUSIONS……………...……………………………………… 89
• VI. REFERENCES……………………………………………………… 90
• CHAPTER III. Effect of Bacteriophage Feed on Inhibition of
• Escherichia coli on in vitro Rumen Fermentation……………………........ 93
• ABSTRACT……………………………………………………………... 94
• I. INTRODUCTION…………………………………………………….. 96
• II. MATERIALS AND METHODS…………………………………….. 98
• Bacteriophage……………………………………………………………. 98
• Inhibition of bacterial growth by bacteriophage…………………………. 99
• In vitro rumen fermentation…………………………………………........ 99
• Analysis of rumen fermentation in vitro parameters…………………….. 101
• Quantitative real-time PCR……………………………………………… 103
• Statistical Method………………………………………………………... 104
• III. RESULTS……………………………………………………………. 105
• IV. DISCUSSION……………………………………………………….. 112
• V. CONCLUSIONS…………..…………………………………………. 115
• VI. REFERENCES…….………………………………………………… 116
• CHAPTER IV. Effect of Dietary Fermented Sulfur Feed on Growth
• Performance and Gut Microbiota in Broiler Chickens…………………... 120
• ABSTRACT……………………………………………………………... 121
• I. INTRODUCTION…………………………………………………….. 123
• II. MATERIALS AND METHODS…………………………………….. 125
• Fermented sulfur…………………………………………………………. 125
• Broilers management and experimental design………………………….. 126
• Odorous compounds analysis………………………….………………… 128
• Blood parameters and immunoglobulin………………………………….. 128
• Proximate composition, fatty acids composition of broier meat…………. 129
• Sampling and DNA extraction………………………………………........ 130
• Quantitative real-time PCR……………………………………………… 130
• Pyrosequencing analysis of gut microbiota ……………………………… 132
• Statistical Method………………………………………………………... 134
• III. RESULTS…………………………………………….……………… 135
• IV. DISCUSSION……………………………………………………….. 154
• V. CONCLUSIONS…………..…………………………………………. 164