TWI824684B - Bacillus subtilis LYS that can improve poultry intestinal health and promote growth - Google Patents
Bacillus subtilis LYS that can improve poultry intestinal health and promote growth Download PDFInfo
- Publication number
- TWI824684B TWI824684B TW111132429A TW111132429A TWI824684B TW I824684 B TWI824684 B TW I824684B TW 111132429 A TW111132429 A TW 111132429A TW 111132429 A TW111132429 A TW 111132429A TW I824684 B TWI824684 B TW I824684B
- Authority
- TW
- Taiwan
- Prior art keywords
- bacillus subtilis
- lys
- subtilis lys
- poultry
- strain
- Prior art date
Links
- 235000014469 Bacillus subtilis Nutrition 0.000 title claims abstract description 59
- 244000063299 Bacillus subtilis Species 0.000 title claims abstract description 58
- 244000144977 poultry Species 0.000 title claims abstract description 13
- 230000007413 intestinal health Effects 0.000 title claims description 5
- 239000000203 mixture Substances 0.000 claims abstract description 16
- 238000000855 fermentation Methods 0.000 claims description 38
- 230000004151 fermentation Effects 0.000 claims description 38
- 241000287828 Gallus gallus Species 0.000 claims description 19
- 238000010563 solid-state fermentation Methods 0.000 claims description 7
- 235000019764 Soybean Meal Nutrition 0.000 claims description 4
- 239000000049 pigment Substances 0.000 claims description 4
- 239000004455 soybean meal Substances 0.000 claims description 4
- 238000011161 development Methods 0.000 claims description 3
- 238000011160 research Methods 0.000 claims description 3
- 235000013305 food Nutrition 0.000 claims description 2
- 108090000765 processed proteins & peptides Proteins 0.000 abstract description 22
- 235000019733 Fish meal Nutrition 0.000 abstract description 14
- 239000004467 fishmeal Substances 0.000 abstract description 14
- 238000004519 manufacturing process Methods 0.000 abstract description 14
- 230000000968 intestinal effect Effects 0.000 abstract description 7
- 210000001198 duodenum Anatomy 0.000 abstract description 3
- 210000001630 jejunum Anatomy 0.000 abstract description 3
- 238000010521 absorption reaction Methods 0.000 abstract description 2
- 235000015097 nutrients Nutrition 0.000 abstract description 2
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 43
- 239000000047 product Substances 0.000 description 28
- AFWTZXXDGQBIKW-UHFFFAOYSA-N C14 surfactin Natural products CCCCCCCCCCCC1CC(=O)NC(CCC(O)=O)C(=O)NC(CC(C)C)C(=O)NC(CC(C)C)C(=O)NC(C(C)C)C(=O)NC(CC(O)=O)C(=O)NC(CC(C)C)C(=O)NC(CC(C)C)C(=O)O1 AFWTZXXDGQBIKW-UHFFFAOYSA-N 0.000 description 13
- NJGWOFRZMQRKHT-UHFFFAOYSA-N surfactin Natural products CC(C)CCCCCCCCCC1CC(=O)NC(CCC(O)=O)C(=O)NC(CC(C)C)C(=O)NC(CC(C)C)C(=O)NC(C(C)C)C(=O)NC(CC(O)=O)C(=O)NC(CC(C)C)C(=O)NC(CC(C)C)C(=O)O1 NJGWOFRZMQRKHT-UHFFFAOYSA-N 0.000 description 13
- NJGWOFRZMQRKHT-WGVNQGGSSA-N surfactin C Chemical compound CC(C)CCCCCCCCC[C@@H]1CC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)O1 NJGWOFRZMQRKHT-WGVNQGGSSA-N 0.000 description 13
- 108010052285 Membrane Proteins Proteins 0.000 description 10
- 102000018697 Membrane Proteins Human genes 0.000 description 9
- 235000013330 chicken meat Nutrition 0.000 description 7
- 239000007788 liquid Substances 0.000 description 7
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- ATJFFYVFTNAWJD-UHFFFAOYSA-N Tin Chemical compound [Sn] ATJFFYVFTNAWJD-UHFFFAOYSA-N 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- 235000013594 poultry meat Nutrition 0.000 description 6
- 241001465754 Metazoa Species 0.000 description 5
- 238000010586 diagram Methods 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 108020004465 16S ribosomal RNA Proteins 0.000 description 4
- 241000193830 Bacillus <bacterium> Species 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 4
- 102000004196 processed proteins & peptides Human genes 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 235000019786 weight gain Nutrition 0.000 description 4
- 241000276408 Bacillus subtilis subsp. subtilis str. 168 Species 0.000 description 3
- 244000068988 Glycine max Species 0.000 description 3
- 235000010469 Glycine max Nutrition 0.000 description 3
- 239000003242 anti bacterial agent Substances 0.000 description 3
- 229940088710 antibiotic agent Drugs 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 230000037396 body weight Effects 0.000 description 3
- 235000013312 flour Nutrition 0.000 description 3
- 230000002209 hydrophobic effect Effects 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 2
- 241000193744 Bacillus amyloliquefaciens Species 0.000 description 2
- 235000017934 Bacillus subtilis subsp subtilis str 168 Nutrition 0.000 description 2
- 241000193468 Clostridium perfringens Species 0.000 description 2
- 235000019750 Crude protein Nutrition 0.000 description 2
- 241000233866 Fungi Species 0.000 description 2
- 108010028921 Lipopeptides Proteins 0.000 description 2
- 230000000844 anti-bacterial effect Effects 0.000 description 2
- 239000006030 antibiotic growth promoter Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 235000019253 formic acid Nutrition 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 229930000044 secondary metabolite Natural products 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 241000194108 Bacillus licheniformis Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 208000004232 Enteritis Diseases 0.000 description 1
- 241000442132 Lactarius lactarius Species 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 101710089165 Protein white Proteins 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 102400000368 Surface protein Human genes 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 238000011088 calibration curve Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 239000003797 essential amino acid Substances 0.000 description 1
- 235000020776 essential amino acid Nutrition 0.000 description 1
- 235000021050 feed intake Nutrition 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 244000000010 microbial pathogen Species 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 230000001338 necrotic effect Effects 0.000 description 1
- 235000008935 nutritious Nutrition 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 238000007363 ring formation reaction Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 238000013207 serial dilution Methods 0.000 description 1
- 101150056746 sfp gene Proteins 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000010902 straw Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 229910052717 sulfur Inorganic materials 0.000 description 1
- 239000011593 sulfur Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 108010041266 surfactin peptide Proteins 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 230000004584 weight gain Effects 0.000 description 1
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Fodder In General (AREA)
Abstract
本發明係將由自然界中分離純化之一種枯草芽孢桿菌LYS（Bacillus subtilis LYS）菌株應用於家禽飼料，可改善家禽十二指腸及空腸之腸道絨毛高度，以及絨毛高度與腺窩深度之比率，以促進家禽對營養分之吸收，使家禽有較佳的生長表現，並可於飼料中完全取代魚粉。且本發明之枯草芽孢桿菌LYS菌株之表面素產量顯著優於目前已知菌株，且其表面素組成比率與現有已知菌株亦顯著不同，為一新穎菌株。The present invention applies Bacillus subtilis LYS (Bacillus subtilis LYS) strain isolated and purified from nature to poultry feed, which can improve the intestinal villus height of poultry duodenum and jejunum, as well as the ratio of villus height to crypt depth, so as to promote the growth of poultry. The absorption of nutrients enables better growth performance of poultry and can completely replace fish meal in feed. Moreover, the surface peptide production of the Bacillus subtilis LYS strain of the present invention is significantly better than that of currently known strains, and its surface peptide composition ratio is also significantly different from that of currently known strains, making it a novel strain.
Description
本發明係有關由自然界中分離純化之一種生產顯著高量且含有特定組成比率表面素之枯草芽孢桿菌LYS（Bacillus subtilis LYS）菌株。更特別地，本發明係關於一種枯草芽孢桿菌LYS（Bacillus subtilis LYS）菌株用於改善家禽腸道健康以促進生長。The present invention relates to a Bacillus subtilis LYS (Bacillus subtilis LYS) strain that is isolated and purified from nature and produces a significantly high amount of surfactin and contains a specific composition ratio. More specifically, the present invention relates to a Bacillus subtilis LYS strain for improving intestinal health in poultry to promote growth.
表面素（Surfactin）為一種脂肽（lipopeptide），其為芽孢桿菌（Bacillus）菌株特有的次級代謝產物，能夠降低表面張力或使水中的疏水性物質溶解（Soberón-Chávez, 2010）。表面素由一親水性的七肽環及疏水性的β羥基脂肪酸鏈（β-hydroxyl fatty acids）所構成之脂肽結構，如下圖所示。 Surfactin is a lipopeptide, which is a secondary metabolite unique to Bacillus strains and can reduce surface tension or dissolve hydrophobic substances in water (Soberón-Chávez, 2010). Surfactin is a lipopeptide structure composed of a hydrophilic heptapeptide ring and hydrophobic β-hydroxyl fatty acids chain, as shown in the figure below.
表面素肽環序列為：L-Glu-L-Leu-D-Leu-L-Val-L-Asp-D-Leu-L-Leu，而β羥基脂肪酸鏈之長度為12-17個碳數（C 12-17）（Théatre et al., 2021）。表面素之物理化學性質會受到其分子結構種類影響，如越長的β羥基脂肪酸鏈使表面素有越強的疏水作用力。而芽孢桿菌所生產表面素之β羥基脂肪酸鏈長度組成種類及比率會因菌種特性而有所不同，如Bacillus subtilis TD7 所生產表面素之組成比率為C 13：6.7%，C 14：42.6%，C 15：46.2%；C 16：4.5%（Liu et al., 2012），Bacillus subtilis BS-37所生產表面素之組成比率為C 13：13.6%，C 14：11.85%，C 15：74.55%（Zhu et al., 2014）。 The sequence of the surfactin peptide ring is: L-Glu-L-Leu-D-Leu-L-Val-L-Asp-D-Leu-L-Leu, and the length of the β-hydroxy fatty acid chain is 12-17 carbons ( C 12-17 ) (Théatre et al., 2021). The physical and chemical properties of surface proteins are affected by their molecular structure. For example, the longer the β-hydroxy fatty acid chain, the stronger the hydrophobic force of surface proteins. The type and ratio of β-hydroxy fatty acid chain lengths of surfactins produced by Bacillus vary depending on the characteristics of the bacterial species. For example, the composition ratios of surface peptides produced by Bacillus subtilis TD7 are C 13 : 6.7% and C 14 : 42.6%. , C 15 : 46.2%; C 16 : 4.5% (Liu et al., 2012). The composition ratio of surface pigment produced by Bacillus subtilis BS-37 is C 13 : 13.6%, C 14 : 11.85%, C 15 : 74.55 % (Zhu et al., 2014).
目前商業生產表面素以液態發酵方式為主流，然而其高生產成本及低產率，大幅限制其產業應用範圍（Makkar and Cameotra, 2002; Chtioui et al., 2010）。因此，以液態發酵所生產之表面素並不適合直接應用於禽畜生產中。藉由固態發酵技術可較液態發酵減少生產成本、縮短製程及提高次級代謝產物等優點（Beltran-Gracia et al., 2017）。已有多項研究成功利用固態發酵技術將低成本之農工副產品作為生產表面素之發酵基質，並可以大幅降低生產成本（Das and Mukherjee, 2007; Mizumoto and Shoda, 2007）。Zhu et al.（2013a）將大豆粉分別以液態及固態發酵進行表面素之生產，表面素產量以固態發酵明顯高於液態發酵（11.61 VS 4.97 mg/g）。Zhu et al.（2013b）將大豆粉搭配稻稈以液化澱粉芽孢桿菌（Bacillus amyloliquefaciens XZ-173）配合反應曲面法生產表面素，最佳固態發酵條件為接菌量15.96%、水分62.8%及溫度26.9℃，經發酵48小時後，表面素產量可達15.03 mg/g，為目前發表已知的最高產量。Currently, liquid fermentation is the mainstream method for commercial production of surfactins. However, its high production cost and low yield greatly limit its industrial application scope (Makkar and Cameotra, 2002; Chtioui et al., 2010). Therefore, surface proteins produced by liquid fermentation are not suitable for direct application in livestock production. Solid-state fermentation technology can reduce production costs, shorten the process, and increase secondary metabolites compared with liquid fermentation (Beltran-Gracia et al., 2017). Several studies have successfully used solid-state fermentation technology to use low-cost agricultural and industrial by-products as fermentation substrates for the production of surface proteins, which can significantly reduce production costs (Das and Mukherjee, 2007; Mizumoto and Shoda, 2007). Zhu et al. (2013a) used liquid and solid fermentation to produce surface peptides from soybean flour. The surface peptide yield in solid-state fermentation was significantly higher than that in liquid fermentation (11.61 VS 4.97 mg/g). Zhu et al. (2013b) used soybean flour and rice straw to produce surface pigment using Bacillus amyloliquefaciens After 48 hours of fermentation at 26.9°C, the surfactin yield can reach 15.03 mg/g, which is the highest yield known so far.
表面素在極端的溫度、pH及鹽度條件下穩定，且可利用再生原料作為其發酵生產基質（Yeh et al., 2005; Cameotra et al., 2010），具有抗細菌、病毒、真菌及黴漿菌等特性（Meena et al., 2016）。近年由於抗生素過度使用，使部分歐洲國家及韓國已禁止抗生素生長促進劑（antibiotic growth promoter）添加於動物飼料中（Castanon, 2007; Alloui et al., 2013）。表面素特殊的殺菌機制、較廣的抗菌活性及不易產生抗藥性（Zhai et al., 2015），使其極具取代抗生素生長促進劑之潛力。近年已有研究表示，於飼料中添加可生產表面素之枯草芽孢桿菌（Bacillus subtilis）或其發酵產物可改善產氣莢膜梭菌（Clostridium perfringens）對白肉雞壞死性腸炎（necrotic enteritis, NE）之負面影響，並具有取代抗生素以促進雞隻生長之功效。添加0.3% B. subtilis NIU068發酵四天產物於白肉雞飼料中，可較NE組改善雞隻體增重及採食量，並可減緩產氣莢膜梭菌引起之腸道損傷（Cheng et al., 2018）。臺灣專利I552682揭露一種於飼料中添加含有特定指標量之表面素的地衣芽孢桿菌固態發酵產物，可控制家禽腸道病原微生物生長及感染以促進家禽生長。Surface proteins are stable under extreme temperature, pH and salinity conditions, and can use renewable raw materials as their fermentation production substrate (Yeh et al., 2005; Cameotra et al., 2010). They are resistant to bacteria, viruses, fungi and mildew. Plasma fungus and other characteristics (Meena et al., 2016). In recent years, due to the overuse of antibiotics, some European countries and South Korea have banned the addition of antibiotic growth promoters to animal feed (Castanon, 2007; Alloui et al., 2013). Surfactant's special bactericidal mechanism, broad antibacterial activity, and resistance to antibiotics (Zhai et al., 2015) make it highly potential to replace antibiotic growth promoters. In recent years, studies have shown that adding surfactin-producing Bacillus subtilis or its fermentation products to feed can improve the effects of Clostridium perfringens on necrotic enteritis (NE) in white broilers. Negative effects, and has the effect of replacing antibiotics to promote chicken growth. Adding 0.3% of the four-day fermentation product of B. subtilis NIU068 to white broiler chicken feed can improve the body weight gain and feed intake of chickens compared with the NE group, and can slow down the intestinal damage caused by Clostridium perfringens (Cheng et al. , 2018). Taiwan Patent I552682 discloses a solid-state fermentation product of Bacillus licheniformis containing a specific amount of surface pigment added to feed, which can control the growth and infection of poultry intestinal pathogenic microorganisms and promote the growth of poultry.
然而，目前尚未有前案或有研究文獻報告，以含有特定指標量表面素及其組成比率之發酵產物添加於飼料中，可完全取代優質動物性蛋白質-白魚粉之使用。However, there is currently no previous case or research literature report that adding fermentation products containing specific amounts of surface proteins and their composition ratios to feed can completely replace the use of high-quality animal protein-white fish meal.
而本發明嘗試從不同的來源及不同特性之芽孢桿菌中，分離並純化出一株枯草芽孢桿菌LYS（Bacillus subtilis LYS）新菌株，其以固態發酵所生產之表面素產量顯著高於其他已知菌株，為產量最高者，且其表面素組成比率與現有已知菌株顯著不同，為一新穎菌株。當其發酵產物使用於家禽飼料中，可以完全取代魚粉之使用，並可促進家禽生長及腸道健康。The present invention attempts to isolate and purify a new strain of Bacillus subtilis LYS (Bacillus subtilis LYS) from Bacillus from different sources and with different characteristics. The surface peptide yield produced by it through solid-state fermentation is significantly higher than that of other known strains. The strain has the highest yield, and its surface peptide composition ratio is significantly different from existing known strains, making it a novel strain. When its fermentation product is used in poultry feed, it can completely replace the use of fish meal, and can promote the growth and intestinal health of poultry.
本發明為由自然界經分離且純化出一種芽孢桿菌新菌株Bacillus subtilis LYS，該LYS菌株與枯草芽孢桿菌168菌株（Bacillus subtilis subsp. subtilis str. 168）之16S rRNA基因序列進行比對，168菌株為枯草芽孢桿菌之標準菌株，比對結果顯示本發明LYS菌株與168菌株之相似性為99.53%，證明本發明之LYS菌株為枯草芽孢桿菌之一種。The present invention is to isolate and purify a new Bacillus strain Bacillus subtilis LYS from nature. The LYS strain is compared with the 16S rRNA gene sequence of Bacillus subtilis subsp. subtilis str. 168. The 168 strain is The comparison result of the standard strain of Bacillus subtilis shows that the similarity between the LYS strain of the present invention and the 168 strain is 99.53%, proving that the LYS strain of the present invention is a type of Bacillus subtilis.
於是本發明提供一種具有顯著高量表面素之新穎枯草芽孢桿菌LYS菌株（Bacillus subtilis LYS），寄存編號為BCRC911131。Therefore, the present invention provides a novel Bacillus subtilis LYS strain (Bacillus subtilis LYS) with a significantly high amount of surface proteins, and the registration number is BCRC911131.
於本發明之一項具體實施態樣，所述之枯草芽孢桿菌LYS菌株以大豆粕發酵生產表面素發酵產物，其表面素產量為10.79-18.40 mg/g，經分析得知本發明表面素所含β羥基脂肪酸鏈種類有7種，其表面素組成比率為C 12：4.70-5.86%；C 13：21.01-27.53%；C 14：31.87-39.22%；C 15：31.73-33.06%；線型C 15：0.26-0.45%；C 16 ：1.66-3.20%；C 17：0.09-0.17%。此表面素種類與比率與現有菌株不同，其中線型C 15為表面素之胜肽鏈未形成環化之結構，為本菌株之特色之一。 In one specific embodiment of the present invention, the Bacillus subtilis LYS strain ferments soybean meal to produce a surfactin fermentation product, and its surfactin yield is 10.79-18.40 mg/g. After analysis, it is found that the surface toxin of the present invention is There are 7 types of β-hydroxy fatty acid chains, and their surface prime composition ratios are C 12 : 4.70-5.86%; C 13 : 21.01-27.53%; C 14 : 31.87-39.22%; C 15 : 31.73-33.06%; Linear C 15 : 0.26-0.45%; C 16 : 1.66-3.20%; C 17 : 0.09-0.17%. The types and ratios of this surface peptide are different from those of existing strains. Linear C 15 is a structure in which the peptide chain of the surface peptide does not form a cyclization, which is one of the characteristics of this strain.
本發明進一步提供一種飼料蛋白質原料，其特徵在於包含本發明之枯草芽孢桿菌LYS菌株。於本發明之較佳具體實施態樣，所述之枯草芽孢桿菌LYS發酵產物係用於改善動物腸道中的絨毛發育以促進動物生長，並可完全取代飼料中之優質魚粉。The present invention further provides a feed protein raw material, which is characterized by containing the Bacillus subtilis LYS strain of the present invention. In a preferred embodiment of the present invention, the Bacillus subtilis LYS fermentation product is used to improve villus development in the intestinal tract of animals to promote animal growth, and can completely replace high-quality fish meal in feeds.
本發明之上述目的與優點，不難從以下所選用實施例之詳細說明與附圖中獲得深入了解。The above objects and advantages of the present invention can be easily understood from the following detailed description of selected embodiments and the accompanying drawings.
本發明由自然界經分離且純化出一種枯草芽孢桿菌LYS（Bacillus subtilis LYS）（寄存編號：BCRC 911131），枯草芽孢桿菌LYS進行菌株16S rRNA 全長定序，再將所得之序列利用BLAST（Basic Local Alignment Search Tool ）與NCBI（National Center for Biotechnology Information ）資料庫中 16S rRNA 序列進行比對。結果顯示，本發明之枯草芽孢桿菌LYS菌株與標準菌株枯草芽孢桿菌168菌株（Bacillus subtilis subsp. subtilis str. 168）之16S rRNA基因序列相似性為99.53%。The present invention separates and purifies Bacillus subtilis LYS (registration number: BCRC 911131) from nature. Bacillus subtilis LYS performs full-length sequencing of strain 16S rRNA, and then uses BLAST (Basic Local Alignment) to obtain the sequence. Search Tool) to compare with the 16S rRNA sequence in the NCBI (National Center for Biotechnology Information) database. The results show that the 16S rRNA gene sequence similarity between the Bacillus subtilis LYS strain of the present invention and the standard strain Bacillus subtilis 168 strain (Bacillus subtilis subsp. subtilis str. 168) is 99.53%.
本發明使用枯草芽孢桿菌LYS（Bacillus subtilis LYS）以大豆粕作為固態發酵基質，將水分調整至55-70%（v/w），並進行高壓滅菌（121℃，1.2 kg/cm 2），待冷卻後分別接種5-10%（v/w）菌液（菌液濃度為8.74 Log CFU/mL 以上），於30-40℃發酵2天，待發酵終點後，以55℃烘乾進行乾燥及粉碎以製備發酵產物。 The present invention uses Bacillus subtilis LYS (Bacillus subtilis LYS) and soybean meal as a solid fermentation substrate, adjusts the moisture to 55-70% (v/w), and performs high-pressure sterilization (121°C, 1.2 kg/cm 2 ), and waits for After cooling, inoculate 5-10% (v/w) bacterial liquid (bacterial liquid concentration is 8.74 Log CFU/mL or above), ferment at 30-40℃ for 2 days, after the end of fermentation, dry at 55℃ for drying and Crush to prepare fermentation products.
將萃取樣品以高解析度軌道阱質譜儀（LC-ESI-HRMS）（Thermo Fisher Scientific, Germany）進行分析，LC-ESI-HRMS分析條件：以正電荷模式進行全質量掃描（full scan），分析管柱為Mightysil RP-18 GP（250 × 4.6 mm; 5 μm, Kanto Kagaku, Japan），流速1.0 mL/min，以0.1%甲酸（formic acid, Merk, Germany）水溶液和0.1%甲酸之乙腈（acetonitrile, Anaqua™ Chemicals Supply, USA）溶液為移動相（mobile phase），並將表面素標準品（surfactin, ≥ 98.0%, Sigma-Aldrich ®, USA）之質荷比（ m/z）作為定性依據，並以表面素標準品之檢量線進行定量，以連續稀釋方法，製備不同濃度之表面素，每濃度3重複，以此定量樣品之表面素濃度。 The extracted samples were analyzed with a high-resolution orbital trap mass spectrometer (LC-ESI-HRMS) (Thermo Fisher Scientific, Germany). LC-ESI-HRMS analysis conditions: full mass scan (full scan) in positive charge mode, analysis The column is Mightysil RP-18 GP (250 × 4.6 mm; 5 μm, Kanto Kagaku, Japan), with a flow rate of 1.0 mL/min, and 0.1% formic acid (Merk, Germany) aqueous solution and 0.1% formic acid in acetonitrile (acetonitrile). , Anaqua™ Chemicals Supply, USA) solution is the mobile phase, and the mass-to-charge ratio ( m/z ) of the surfactin standard (surfactin, ≥ 98.0%, Sigma-Aldrich ® , USA) is used as the qualitative basis. Quantification was carried out based on the calibration curve of the surfactin standard. Different concentrations of surfactin were prepared using the serial dilution method. Each concentration was repeated three times to quantify the surfactin concentration of the sample.
本發明之分析結果如第１圖所示，其中：A為表面素標準品；B為枯草芽孢桿菌LYS發酵產物；C為C 12表面素；D為C 13表面素；E為C 14表面素；F為C 15表面素；G為C 15表面素（線型）；H為C 16表面素；I，C 17表面素。而A-B為總離子層析圖；C-I為萃取離子層析圖。結果顯示，可於標準品及枯草芽孢桿菌LYS發酵產物檢測到C 12-17之表面素組成波峰，並且發現C 15表面素含有線型及環形結構。將其組成質荷比依大小分別為994.64、1008.66、1022.67、1036.69、1054.70、1050.71及1064.72。 The analysis results of the present invention are shown in Figure 1, in which: A is a surface tin standard; B is a Bacillus subtilis LYS fermentation product; C is a C 12 surface tin; D is a C 13 surface tin; E is a C 14 surface tin. ; F is C 15 surface prime; G is C 15 surface prime (linear); H is C 16 surface prime; I, C 17 surface prime. AB is the total ion chromatogram; CI is the extracted ion chromatogram. The results showed that the C 12-17 surface peptide composition peak could be detected in the standard product and the Bacillus subtilis LYS fermentation product, and it was found that the C 15 surface peptide contains linear and cyclic structures. The mass-to-charge ratios of their compositions are 994.64, 1008.66, 1022.67, 1036.69, 1054.70, 1050.71 and 1064.72 respectively.
枯草芽孢桿菌LYS發酵產物之表面素定量結果顯示，枯草芽孢桿菌LYS表面素產量為10.79-18.40 mg/g。而標準菌株枯草芽孢桿菌168菌株由於sfp基因突變無法生產表面素（Tsuge et al., 2001）。Zhu et al.（2013b）將大豆粉以液化澱粉芽孢桿菌XZ-173（Bacillus amyloliquefaciens XZ-173）配合反應曲面法生產表面素，其表面素產量高達15.03 mg/g。本發明枯草芽孢桿菌LYS以水分55-70%發酵，其表面素產量最高可達18.40 mg/g (18400 ppm)，超越目前文獻已知的最高表面素產量（15030 ppm）。本發明之表面素產量與其他文獻方法之產量差異如下表所示。 Quantitative results of surface tin of Bacillus subtilis LYS fermentation products showed that the surface tin production of Bacillus subtilis LYS was 10.79-18.40 mg/g. The standard strain Bacillus subtilis strain 168 is unable to produce surface proteins due to a mutation in the sfp gene (Tsuge et al., 2001). Zhu et al. (2013b) used soybean flour with Bacillus amyloliquefaciens XZ-173 and reaction surface method to produce surface peptides, and the surface peptide yield was as high as 15.03 mg/g. The Bacillus subtilis LYS of the present invention is fermented with 55-70% moisture, and its surface peptide yield can reach up to 18.40 mg/g (18400 ppm), exceeding the highest surface peptide yield (15030 ppm) known in the current literature. The difference between the yield of surfactin of the present invention and that of other literature methods is shown in the table below.
枯草芽孢桿菌LYS發酵產物之表面素組成比率如第２圖所示。其表面素組成比率分別如下：C 12：4.70-5.86%；C 13：21.01-27.53%；C 14：31.87-39.22%；C 15：31.73-33.06%；線型C 15：0.26-0.45%；C 16：1.66-3.20%；C 17：0.09-0.17%。表面素組成比率為枯草芽孢桿菌LYS生產表面素之特異性指標，與現有已知菌株均不相同，且較目前已知菌株多出C 12、C 16、C 17之表面素種類。本發明之表面素組成比率與其他文獻方法之組成比率差異如下表所示。 The surface peptide composition ratio of the Bacillus subtilis LYS fermentation product is shown in Figure 2. The surface prime composition ratios are as follows: C 12 : 4.70-5.86%; C 13 : 21.01-27.53%; C 14 : 31.87-39.22%; C 15 : 31.73-33.06%; Linear C 15 : 0.26-0.45%; C 16 : 1.66-3.20%; C 17 : 0.09-0.17%. The surface peptide composition ratio is a specific indicator of surface peptide production by Bacillus subtilis LYS. It is different from currently known strains and has more types of C 12 , C 16 , and C 17 surface peptides than currently known strains. The difference between the surface peptide composition ratio of the present invention and the composition ratio of other literature methods is shown in the table below.
枯草芽孢桿菌LYS發酵產物之動物試驗，試驗分為3組，1. 對照組：餵飼一般飼料；2. 魚粉組：飼料中添加2.5%丹麥白魚粉 （粗蛋白質70%，TripleNine Group）；3. 枯草芽孢桿菌LYS發酵產物組：飼料中添加2.5%發酵產物（飼料表面素含量為270-460 mg/kg）。試驗為期35天。枯草芽孢桿菌LYS發酵產物對白肉雞體增重、生產效率因子之影響分別如第３圖及第４圖所示。結果顯示，枯草芽孢桿菌LYS發酵產物組對雞隻生長表現最佳，其可較對照組改善白肉雞之體增重9.55%、生產效率因子13.26%，並與魚粉組無差異。枯草芽孢桿菌LYS發酵產物對白肉雞腸道組織型態之影響分別如第５圖及第６圖所示。結果顯示，枯草芽孢桿菌LYS發酵產物組對雞隻腸道組織型態表現最佳，可較對照組分別改善十二指腸、空腸之絨毛高度21.76%、9.39%及十二指腸、空腸之絨毛高度/腺窩深度比90.22%、76.85%，並與魚粉組無差異。魚粉是一種易消化且富含營養的蛋白質飼料原料，其為家禽提供高量的粗蛋白質及必需胺基酸，如離胺酸和含硫胺基酸，可有效改善雞隻生長性狀（Frempong et al., 2019）。然而，由於魚粉成本高，導致其在飼料使用上受到限制（Premathilaka et al., 2020）。本發明所使用魚粉為業界公認最佳之丹麥白魚粉，而當雞隻整體獲益以對照組為100時，枯草芽孢桿菌LYS發酵產物組可分別較對照組與魚粉組之整體獲益提高8.70%及4.53%。Animal test of Bacillus subtilis LYS fermentation product, the test was divided into 3 groups, 1. Control group: fed with general feed; 2. Fish meal group: 2.5% Danish white fish meal (70% crude protein, TripleNine Group) was added to the feed; 3 . Bacillus subtilis LYS fermentation product group: 2.5% fermentation product is added to the feed (feed surface protein content is 270-460 mg/kg). The trial lasts for 35 days. The effects of Bacillus subtilis LYS fermentation products on body weight gain and production efficiency factors of white broiler chickens are shown in Figures 3 and 4 respectively. The results showed that the Bacillus subtilis LYS fermentation product group had the best performance on chicken growth. It could improve the body weight gain of white broiler chickens by 9.55% and the production efficiency factor by 13.26% compared with the control group, and was no different from the fish meal group. The effects of Bacillus subtilis LYS fermentation products on the intestinal tissue morphology of white broiler chickens are shown in Figures 5 and 6 respectively. The results showed that the Bacillus subtilis LYS fermentation product group performed best on the intestinal tissue type of chickens, and could improve the villus height of the duodenum and jejunum by 21.76% and 9.39% and the villus height/crypt depth of the duodenum and jejunum respectively compared with the control group. Ratios were 90.22% and 76.85%, and there was no difference between them and the fish meal group. Fishmeal is an easily digestible and nutritious protein feed ingredient that provides poultry with a high amount of crude protein and essential amino acids, such as lysine and sulfur-containing amino acids, and can effectively improve the growth traits of chickens (Frempong et al. al., 2019). However, the high cost of fishmeal has limited its use in feed (Premathilaka et al., 2020). The fishmeal used in the present invention is the Danish white fishmeal recognized as the best in the industry. When the overall benefit of the chickens is 100 in the control group, the Bacillus subtilis LYS fermentation product group can increase the overall benefit by 8.70% compared with the control group and the fishmeal group respectively. % and 4.53%.
綜合以上結果，枯草芽孢桿菌LYS發酵產物可改善白肉雞腸道健康以促進對營養分之吸收而促進雞隻生長表現，並且於飼料中可完全取代優質魚粉之使用，可以降低肉雞生產成本。Based on the above results, the fermentation product of Bacillus subtilis LYS can improve the intestinal health of white broiler chickens to promote the absorption of nutrients and promote the growth performance of the chickens. It can completely replace the use of high-quality fish meal in feed and can reduce the production cost of broiler chickens.
惟以上實施例之揭示僅用以說明本發明，並非用以限制本發明，舉凡等效元件之置換仍應隸屬本發明之範疇。However, the disclosure of the above embodiments is only for illustrating the present invention and is not intended to limit the present invention. Substitution of equivalent components shall still fall within the scope of the present invention.
綜上所述，可使熟知本領域技術者明瞭本發明確可達成前述目的，實已符合專利法之規定，爰依法提出申請。To sum up, it can be understood by those skilled in the art that the present invention can clearly achieve the aforementioned objectives and is in compliance with the provisions of the Patent Law. The application can be filed in accordance with the law.
(無)(without)
第１圖為本發明枯草芽孢桿菌LYS發酵產物之表面素定性質譜層析圖； 第２圖為本發明枯草芽孢桿菌LYS發酵產物之表面素組成比率圖； 第３圖為本發明枯草芽孢桿菌LYS發酵產物對白肉雞體增重之比對示意圖； 第４圖為本發明枯草芽孢桿菌LYS發酵產物對白肉雞生產效率因子之比對示意圖； 第５圖為本發明枯草芽孢桿菌LYS發酵產物對白肉雞腸道絨毛高度之比對示意圖； 第６圖為本發明枯草芽孢桿菌LYS發酵產物對白肉雞腸道絨毛高度/腺窩深度比之比對示意圖。 Figure 1 is a qualitative mass spectrometry chromatogram of surface proteins of the Bacillus subtilis LYS fermentation product of the present invention; Figure 2 is a surface peptide composition ratio diagram of the Bacillus subtilis LYS fermentation product of the present invention; Figure 3 is a schematic diagram showing the comparison of the weight gain of white broiler chickens by the Bacillus subtilis LYS fermentation product of the present invention; Figure 4 is a schematic diagram comparing the production efficiency factors of white broiler chickens with the Bacillus subtilis LYS fermentation product of the present invention; Figure 5 is a schematic diagram showing the comparison of intestinal villus height of white broiler chickens with Bacillus subtilis LYS fermentation products of the present invention; Figure 6 is a schematic diagram showing the comparison of the intestinal villus height/crypt depth ratio of white broiler chickens with the Bacillus subtilis LYS fermentation product of the present invention.
枯草芽孢桿菌LYS（Bacillus subtilis LYS），寄存編號為BCRC 911131，寄存日為2022年5月25日，寄存地點於台灣新竹食品工業發展研究所。Bacillus subtilis LYS (Bacillus subtilis LYS), the deposit number is BCRC 911131, the deposit date is May 25, 2022, and the deposit location is the Food Industry Development Research Institute in Hsinchu, Taiwan.
(無)(without)
Claims (4)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| TW111132429A TWI824684B (en) | 2022-08-29 | 2022-08-29 | Bacillus subtilis LYS that can improve poultry intestinal health and promote growth |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| TW111132429A TWI824684B (en) | 2022-08-29 | 2022-08-29 | Bacillus subtilis LYS that can improve poultry intestinal health and promote growth |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| TWI824684B true TWI824684B (en) | 2023-12-01 |
| TW202409267A TW202409267A (en) | 2024-03-01 |
Family
ID=90052939
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| TW111132429A TWI824684B (en) | 2022-08-29 | 2022-08-29 | Bacillus subtilis LYS that can improve poultry intestinal health and promote growth |
Country Status (1)
| Country | Link |
|---|---|
| TW (1) | TWI824684B (en) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TWI659696B (en) * | 2014-12-12 | 2019-05-21 | 南京莎菲特生物科技有限公司 | Method for preparing feed additive containing high-concentration surface hormone |
-
2022
- 2022-08-29 TW TW111132429A patent/TWI824684B/en active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TWI659696B (en) * | 2014-12-12 | 2019-05-21 | 南京莎菲特生物科技有限公司 | Method for preparing feed additive containing high-concentration surface hormone |
Non-Patent Citations (3)
| Title |
|---|
| ;網路文獻 康雅雯， 篩選芽孢桿菌生產發酵或水解大豆粕產物對白肉雞之影響， 國立嘉義大學動物科學系研究所碩士論文， 2019年9月 * |
| 網路文獻 康雅雯， 篩選芽孢桿菌生產發酵或水解大豆粕產物對白肉雞之影響， 國立嘉義大學動物科學系研究所碩士論文， 2019年9月。 |
| 網路文獻 蔡一豪 篩選芽孢桿菌以製備發酵產物並探討其對於肉雞與仔豬飼養效果， 國立嘉義大學動物科學系研究所碩士論文， 2019 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108587957B (en) | Screening and application of probiotic bacillus licheniformis capable of highly producing complex enzyme | |
| KR101280811B1 (en) | Microorganism having zearalenone decomposing activity, method for degrading zearalenone using thereof and feed additives comprising the same | |
| US20100240577A1 (en) | Bacteriocins and Novel Bacterial Strains | |
| DK2379705T3 (en) | Bile resistant Bacillus composition | |
| Zhang et al. | Isolation and characterization of a novel gossypol-degrading bacteria Bacillus subtilis strain Rumen Bacillus Subtilis | |
| WO2023087499A1 (en) | Bacillus velezensis capable of producing complex enzyme at high yield and efficiently degrading mycotoxin and application thereof | |
| El-Baz et al. | Semi-industrial scale production of a new yeast with probiotic traits, Cryptococcus sp. YMHS, isolated from the Red Sea | |
| AU2005329462B2 (en) | Bacteriocins and novel bacterial strains | |
| CN1165547C (en) | Novel lantibiotic related to actagardine, and process for preparation and use thereof | |
| TWI824684B (en) | Bacillus subtilis LYS that can improve poultry intestinal health and promote growth | |
| CN111713607B (en) | Antibacterial peptide-containing feed prepared based on bacillus solid state fermentation, preparation process thereof and fermentation strain | |
| CN110607251A (en) | Salt-tolerant marinobacter capable of degrading vomitoxin and application thereof | |
| CN111996139B (en) | Composite microecological preparation for improving production benefit of meat calves and application thereof | |
| CN110628669B (en) | Bacillus subtilis LY-5, microbial inoculum and application thereof and product applying same | |
| CN112812159A (en) | Yeast polypeptide derived from saccharomyces boulardii as well as preparation method and application thereof | |
| TW202409267A (en) | Bacillus subtilis LYS that can improve poultry intestinal health and promote growth | |
| Josic et al. | Application of proteomics in biotechnology–microbial proteomics | |
| KR102327307B1 (en) | Lactobacillus paracasei strain with secretion activation of β-glucuronidase and Fermentative Scutellaria baicalensis and Feed supplement using the same | |
| CN111676155A (en) | Bacillus licheniformis and application thereof in aquaculture | |
| CN115093998A (en) | Bacillus belgii for fermenting soybean meal | |
| CN113072627B (en) | MccJ25 mutant and preparation method and application thereof | |
| CN114916610A (en) | Method for preparing selenium-rich feed additive by fermenting ginkgo leaf residues with bacillus subtilis and application of selenium-rich feed additive | |
| TW202239332A (en) | Feeding stuff for poultry with high surfactin content, fermentation product with high surfactin content, and its manufacturing method and use accelerating the growth of the poultry and lowering the manufacturing cost | |
| CN106883289B (en) | Antibacterial peptide BV21 | |
| CN112011484A (en) | Screening and application of probiotic bacillus |