CN112625946A - Bacillus subtilis, microecological preparation, feed and application of bacillus subtilis to improvement of production performance and eggshell quality of hens - Google Patents
Bacillus subtilis, microecological preparation, feed and application of bacillus subtilis to improvement of production performance and eggshell quality of hens Download PDFInfo
- Publication number
- CN112625946A CN112625946A CN202011476999.9A CN202011476999A CN112625946A CN 112625946 A CN112625946 A CN 112625946A CN 202011476999 A CN202011476999 A CN 202011476999A CN 112625946 A CN112625946 A CN 112625946A
- Authority
- CN
- China
- Prior art keywords
- liu
- bacillus subtilis
- eggshell
- group
- percent
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 210000003278 egg shell Anatomy 0.000 title claims abstract description 159
- 108010000912 Egg Proteins Proteins 0.000 title claims abstract description 135
- 102000002322 Egg Proteins Human genes 0.000 title claims abstract description 135
- 244000063299 Bacillus subtilis Species 0.000 title claims abstract description 95
- 235000014469 Bacillus subtilis Nutrition 0.000 title claims abstract description 95
- 238000002360 preparation method Methods 0.000 title claims abstract description 72
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 19
- 230000006872 improvement Effects 0.000 title description 2
- 238000009395 breeding Methods 0.000 claims abstract description 48
- 230000001488 breeding effect Effects 0.000 claims abstract description 48
- 238000000034 method Methods 0.000 claims abstract description 31
- 238000000855 fermentation Methods 0.000 claims description 64
- 230000004151 fermentation Effects 0.000 claims description 64
- 239000001963 growth medium Substances 0.000 claims description 35
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 20
- 241000186605 Lactobacillus paracasei Species 0.000 claims description 18
- 230000001580 bacterial effect Effects 0.000 claims description 16
- 239000006041 probiotic Substances 0.000 claims description 15
- 235000018291 probiotics Nutrition 0.000 claims description 15
- 239000000843 powder Substances 0.000 claims description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 14
- 229940041514 candida albicans extract Drugs 0.000 claims description 12
- 239000012138 yeast extract Substances 0.000 claims description 12
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 11
- 239000008103 glucose Substances 0.000 claims description 11
- 238000002156 mixing Methods 0.000 claims description 11
- 239000012137 tryptone Substances 0.000 claims description 11
- 239000000306 component Substances 0.000 claims description 10
- 238000004321 preservation Methods 0.000 claims description 10
- 239000011780 sodium chloride Substances 0.000 claims description 10
- 230000000529 probiotic effect Effects 0.000 claims description 8
- 238000009629 microbiological culture Methods 0.000 claims description 7
- 238000005507 spraying Methods 0.000 claims description 7
- 229920002774 Maltodextrin Polymers 0.000 claims description 6
- 239000005913 Maltodextrin Substances 0.000 claims description 6
- 229940035034 maltodextrin Drugs 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 6
- 238000007865 diluting Methods 0.000 claims description 5
- 238000009472 formulation Methods 0.000 claims description 3
- 244000005700 microbiome Species 0.000 claims description 3
- 239000012092 media component Substances 0.000 claims description 2
- 235000013601 eggs Nutrition 0.000 abstract description 110
- 239000011575 calcium Substances 0.000 abstract description 59
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 abstract description 58
- 229910052791 calcium Inorganic materials 0.000 abstract description 58
- 230000001965 increasing effect Effects 0.000 abstract description 57
- 108090000623 proteins and genes Proteins 0.000 abstract description 33
- 230000012447 hatching Effects 0.000 abstract description 27
- 230000014509 gene expression Effects 0.000 abstract description 21
- 230000002829 reductive effect Effects 0.000 abstract description 19
- 230000002308 calcification Effects 0.000 abstract description 18
- 210000004291 uterus Anatomy 0.000 abstract description 18
- 210000002303 tibia Anatomy 0.000 abstract description 15
- 210000002966 serum Anatomy 0.000 abstract description 13
- 230000008569 process Effects 0.000 abstract description 12
- 238000012546 transfer Methods 0.000 abstract description 5
- 235000013351 cheese Nutrition 0.000 abstract description 4
- 238000011160 research Methods 0.000 abstract description 3
- 238000012360 testing method Methods 0.000 description 35
- 239000002131 composite material Substances 0.000 description 30
- 239000000523 sample Substances 0.000 description 29
- 239000010410 layer Substances 0.000 description 26
- 230000008859 change Effects 0.000 description 23
- 239000000243 solution Substances 0.000 description 21
- 230000000694 effects Effects 0.000 description 20
- 241000894006 Bacteria Species 0.000 description 19
- 239000000835 fiber Substances 0.000 description 15
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 13
- 210000002969 egg yolk Anatomy 0.000 description 12
- 230000017448 oviposition Effects 0.000 description 12
- 229920001817 Agar Polymers 0.000 description 11
- 239000008272 agar Substances 0.000 description 11
- 150000001875 compounds Chemical group 0.000 description 11
- 238000012258 culturing Methods 0.000 description 11
- 235000018102 proteins Nutrition 0.000 description 10
- 102000004169 proteins and genes Human genes 0.000 description 10
- 108010058846 Ovalbumin Proteins 0.000 description 9
- 238000001514 detection method Methods 0.000 description 9
- 239000003085 diluting agent Substances 0.000 description 9
- 230000035611 feeding Effects 0.000 description 9
- 239000002609 medium Substances 0.000 description 9
- 229940092253 ovalbumin Drugs 0.000 description 9
- 238000012797 qualification Methods 0.000 description 9
- 108010026206 Conalbumin Proteins 0.000 description 8
- 238000004458 analytical method Methods 0.000 description 8
- 238000002474 experimental method Methods 0.000 description 8
- 239000002299 complementary DNA Substances 0.000 description 7
- 239000012153 distilled water Substances 0.000 description 7
- 244000144977 poultry Species 0.000 description 7
- 235000013594 poultry meat Nutrition 0.000 description 7
- 238000003753 real-time PCR Methods 0.000 description 7
- 239000006228 supernatant Substances 0.000 description 7
- 102000003846 Carbonic anhydrases Human genes 0.000 description 6
- 108090000209 Carbonic anhydrases Proteins 0.000 description 6
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- 108060003393 Granulin Proteins 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 229920002472 Starch Polymers 0.000 description 6
- DKGAVHZHDRPRBM-UHFFFAOYSA-N Tert-Butanol Chemical compound CC(C)(C)O DKGAVHZHDRPRBM-UHFFFAOYSA-N 0.000 description 6
- 239000003795 chemical substances by application Substances 0.000 description 6
- 230000002354 daily effect Effects 0.000 description 6
- 238000011081 inoculation Methods 0.000 description 6
- 238000001543 one-way ANOVA Methods 0.000 description 6
- 239000008107 starch Substances 0.000 description 6
- 235000019698 starch Nutrition 0.000 description 6
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 5
- 241000287828 Gallus gallus Species 0.000 description 5
- 230000003321 amplification Effects 0.000 description 5
- 230000015572 biosynthetic process Effects 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 5
- 235000013330 chicken meat Nutrition 0.000 description 5
- 238000000151 deposition Methods 0.000 description 5
- 230000008021 deposition Effects 0.000 description 5
- 238000009826 distribution Methods 0.000 description 5
- 239000003651 drinking water Substances 0.000 description 5
- 235000020188 drinking water Nutrition 0.000 description 5
- 238000009630 liquid culture Methods 0.000 description 5
- 210000001595 mastoid Anatomy 0.000 description 5
- 238000003199 nucleic acid amplification method Methods 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 238000010839 reverse transcription Methods 0.000 description 5
- 238000012216 screening Methods 0.000 description 5
- 238000002791 soaking Methods 0.000 description 5
- 239000004382 Amylase Substances 0.000 description 4
- 102000013142 Amylases Human genes 0.000 description 4
- 108010065511 Amylases Proteins 0.000 description 4
- 102100029751 Plasma membrane calcium-transporting ATPase 1 Human genes 0.000 description 4
- 108050001987 Plasma membrane calcium-transporting ATPase 1 Proteins 0.000 description 4
- 235000019418 amylase Nutrition 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 4
- 238000013461 design Methods 0.000 description 4
- 235000019441 ethanol Nutrition 0.000 description 4
- 230000001788 irregular Effects 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 239000011159 matrix material Substances 0.000 description 4
- 230000001954 sterilising effect Effects 0.000 description 4
- 238000005303 weighing Methods 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- BVKZGUZCCUSVTD-UHFFFAOYSA-M Bicarbonate Chemical compound OC([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-M 0.000 description 3
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 3
- 238000010009 beating Methods 0.000 description 3
- 238000010241 blood sampling Methods 0.000 description 3
- VTYYLEPIZMXCLO-UHFFFAOYSA-L calcium carbonate Substances [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 3
- 238000001816 cooling Methods 0.000 description 3
- 238000005336 cracking Methods 0.000 description 3
- 238000001035 drying Methods 0.000 description 3
- 235000013345 egg yolk Nutrition 0.000 description 3
- 230000027632 eggshell formation Effects 0.000 description 3
- 210000004696 endometrium Anatomy 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 235000015097 nutrients Nutrition 0.000 description 3
- 210000000993 outer shell membrane Anatomy 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 239000003223 protective agent Substances 0.000 description 3
- 239000010802 sludge Substances 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 239000007858 starting material Substances 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- GVJHHUAWPYXKBD-UHFFFAOYSA-N (±)-α-Tocopherol Chemical compound OC1=C(C)C(C)=C2OC(CCCC(C)CCCC(C)CCCC(C)C)(C)CCC2=C1C GVJHHUAWPYXKBD-UHFFFAOYSA-N 0.000 description 2
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 229910021532 Calcite Inorganic materials 0.000 description 2
- 102000014914 Carrier Proteins Human genes 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- 238000003794 Gram staining Methods 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 description 2
- 108091005804 Peptidases Proteins 0.000 description 2
- 102000002141 Plasma Membrane Calcium-Transporting ATPases Human genes 0.000 description 2
- 108010040945 Plasma Membrane Calcium-Transporting ATPases Proteins 0.000 description 2
- 239000004365 Protease Substances 0.000 description 2
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 2
- AUNGANRZJHBGPY-SCRDCRAPSA-N Riboflavin Chemical compound OC[C@@H](O)[C@@H](O)[C@@H](O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O AUNGANRZJHBGPY-SCRDCRAPSA-N 0.000 description 2
- 108090000787 Subtilisin Proteins 0.000 description 2
- 235000015278 beef Nutrition 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 108091008324 binding proteins Proteins 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 230000037396 body weight Effects 0.000 description 2
- 229910000019 calcium carbonate Inorganic materials 0.000 description 2
- 229910001424 calcium ion Inorganic materials 0.000 description 2
- 238000006555 catalytic reaction Methods 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000004140 cleaning Methods 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 238000005520 cutting process Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- XBDQKXXYIPTUBI-UHFFFAOYSA-N dimethylselenoniopropionate Natural products CCC(O)=O XBDQKXXYIPTUBI-UHFFFAOYSA-N 0.000 description 2
- 238000002224 dissection Methods 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 230000000977 initiatory effect Effects 0.000 description 2
- 210000000994 inner shell membrane Anatomy 0.000 description 2
- 238000007689 inspection Methods 0.000 description 2
- 239000011630 iodine Substances 0.000 description 2
- 229910052740 iodine Inorganic materials 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 230000000670 limiting effect Effects 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000011880 melting curve analysis Methods 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 2
- 230000000877 morphologic effect Effects 0.000 description 2
- 235000016709 nutrition Nutrition 0.000 description 2
- 238000005457 optimization Methods 0.000 description 2
- -1 pH 6.5 +/-0.2 Chemical compound 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 2
- 235000020183 skimmed milk Nutrition 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- FPIPGXGPPPQFEQ-UHFFFAOYSA-N 13-cis retinol Natural products OCC=C(C)C=CC=C(C)C=CC1=C(C)CCCC1(C)C FPIPGXGPPPQFEQ-UHFFFAOYSA-N 0.000 description 1
- 108020004465 16S ribosomal RNA Proteins 0.000 description 1
- 239000001763 2-hydroxyethyl(trimethyl)azanium Substances 0.000 description 1
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 description 1
- 108091006112 ATPases Proteins 0.000 description 1
- 102000057290 Adenosine Triphosphatases Human genes 0.000 description 1
- 235000001674 Agaricus brunnescens Nutrition 0.000 description 1
- 208000031648 Body Weight Changes Diseases 0.000 description 1
- 102000005701 Calcium-Binding Proteins Human genes 0.000 description 1
- 108010045403 Calcium-Binding Proteins Proteins 0.000 description 1
- 235000019743 Choline chloride Nutrition 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- AUNGANRZJHBGPY-UHFFFAOYSA-N D-Lyxoflavin Natural products OCC(O)C(O)C(O)CN1C=2C=C(C)C(C)=CC=2N=C2C1=NC(=O)NC2=O AUNGANRZJHBGPY-UHFFFAOYSA-N 0.000 description 1
- 102000007260 Deoxyribonuclease I Human genes 0.000 description 1
- 108010008532 Deoxyribonuclease I Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000192125 Firmicutes Species 0.000 description 1
- 101000834253 Gallus gallus Actin, cytoplasmic 1 Proteins 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 239000007836 KH2PO4 Substances 0.000 description 1
- 241000238367 Mya arenaria Species 0.000 description 1
- 101100353526 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) pca-2 gene Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 238000010802 RNA extraction kit Methods 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 238000000944 Soxhlet extraction Methods 0.000 description 1
- 229910000831 Steel Inorganic materials 0.000 description 1
- FPIPGXGPPPQFEQ-BOOMUCAASA-N Vitamin A Natural products OC/C=C(/C)\C=C\C=C(\C)/C=C/C1=C(C)CCCC1(C)C FPIPGXGPPPQFEQ-BOOMUCAASA-N 0.000 description 1
- 229930003427 Vitamin E Natural products 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- 235000011054 acetic acid Nutrition 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- FPIPGXGPPPQFEQ-OVSJKPMPSA-N all-trans-retinol Chemical compound OC\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C FPIPGXGPPPQFEQ-OVSJKPMPSA-N 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 235000014590 basal diet Nutrition 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 230000023555 blood coagulation Effects 0.000 description 1
- 230000004579 body weight change Effects 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 238000010805 cDNA synthesis kit Methods 0.000 description 1
- FAPWYRCQGJNNSJ-UBKPKTQASA-L calcium D-pantothenic acid Chemical compound [Ca+2].OCC(C)(C)[C@@H](O)C(=O)NCCC([O-])=O.OCC(C)(C)[C@@H](O)C(=O)NCCC([O-])=O FAPWYRCQGJNNSJ-UBKPKTQASA-L 0.000 description 1
- 229960002079 calcium pantothenate Drugs 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 238000003763 carbonization Methods 0.000 description 1
- 239000005018 casein Substances 0.000 description 1
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 1
- 235000021240 caseins Nutrition 0.000 description 1
- 229960003178 choline chloride Drugs 0.000 description 1
- SGMZJAMFUVOLNK-UHFFFAOYSA-M choline chloride Chemical compound [Cl-].C[N+](C)(C)CCO SGMZJAMFUVOLNK-UHFFFAOYSA-M 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 230000002508 compound effect Effects 0.000 description 1
- 238000013329 compounding Methods 0.000 description 1
- 239000013068 control sample Substances 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 238000013211 curve analysis Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000005238 degreasing Methods 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 239000012470 diluted sample Substances 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 229940012466 egg shell membrane Drugs 0.000 description 1
- 210000000969 egg white Anatomy 0.000 description 1
- 235000014103 egg white Nutrition 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 210000002615 epidermis Anatomy 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 210000003746 feather Anatomy 0.000 description 1
- 235000021050 feed intake Nutrition 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- WIGCFUFOHFEKBI-UHFFFAOYSA-N gamma-tocopherol Natural products CC(C)CCCC(C)CCCC(C)CCCC1CCC2C(C)C(O)C(C)C(C)C2O1 WIGCFUFOHFEKBI-UHFFFAOYSA-N 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 238000006703 hydration reaction Methods 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 235000015141 kefir Nutrition 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 239000002932 luster Substances 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 description 1
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 description 1
- WPBNNNQJVZRUHP-UHFFFAOYSA-L manganese(2+);methyl n-[[2-(methoxycarbonylcarbamothioylamino)phenyl]carbamothioyl]carbamate;n-[2-(sulfidocarbothioylamino)ethyl]carbamodithioate Chemical compound [Mn+2].[S-]C(=S)NCCNC([S-])=S.COC(=O)NC(=S)NC1=CC=CC=C1NC(=S)NC(=O)OC WPBNNNQJVZRUHP-UHFFFAOYSA-L 0.000 description 1
- CDUFCUKTJFSWPL-UHFFFAOYSA-L manganese(II) sulfate tetrahydrate Chemical compound O.O.O.O.[Mn+2].[O-]S([O-])(=O)=O CDUFCUKTJFSWPL-UHFFFAOYSA-L 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 239000002068 microbial inoculum Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 239000006872 mrs medium Substances 0.000 description 1
- 210000003097 mucus Anatomy 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 229960003512 nicotinic acid Drugs 0.000 description 1
- 235000001968 nicotinic acid Nutrition 0.000 description 1
- 239000011664 nicotinic acid Substances 0.000 description 1
- 210000002445 nipple Anatomy 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 239000012044 organic layer Substances 0.000 description 1
- 210000003101 oviduct Anatomy 0.000 description 1
- 108010071539 ovocleidin 17 Proteins 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 239000003208 petroleum Substances 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 235000019260 propionic acid Nutrition 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- IUVKMZGDUIUOCP-BTNSXGMBSA-N quinbolone Chemical compound O([C@H]1CC[C@H]2[C@H]3[C@@H]([C@]4(C=CC(=O)C=C4CC3)C)CC[C@@]21C)C1=CCCC1 IUVKMZGDUIUOCP-BTNSXGMBSA-N 0.000 description 1
- 230000001850 reproductive effect Effects 0.000 description 1
- 229960002477 riboflavin Drugs 0.000 description 1
- 235000019192 riboflavin Nutrition 0.000 description 1
- 239000002151 riboflavin Substances 0.000 description 1
- 239000012898 sample dilution Substances 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 239000004576 sand Substances 0.000 description 1
- 238000013341 scale-up Methods 0.000 description 1
- 238000004062 sedimentation Methods 0.000 description 1
- 230000011218 segmentation Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 210000000998 shell membrane Anatomy 0.000 description 1
- 210000003491 skin Anatomy 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- 210000004872 soft tissue Anatomy 0.000 description 1
- 238000004544 sputter deposition Methods 0.000 description 1
- 239000010959 steel Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000002344 surface layer Substances 0.000 description 1
- 238000009966 trimming Methods 0.000 description 1
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 description 1
- 229940038773 trisodium citrate Drugs 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 238000009777 vacuum freeze-drying Methods 0.000 description 1
- 235000019155 vitamin A Nutrition 0.000 description 1
- 239000011719 vitamin A Substances 0.000 description 1
- 239000011715 vitamin B12 Substances 0.000 description 1
- 235000019165 vitamin E Nutrition 0.000 description 1
- 229940046009 vitamin E Drugs 0.000 description 1
- 239000011709 vitamin E Substances 0.000 description 1
- 229940045997 vitamin a Drugs 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/02—Breeding vertebrates
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K40/00—Shaping or working-up of animal feeding-stuffs
- A23K40/30—Shaping or working-up of animal feeding-stuffs by encapsulating; by coating
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/70—Feeding-stuffs specially adapted for particular animals for birds
- A23K50/75—Feeding-stuffs specially adapted for particular animals for birds for poultry
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/165—Paracasei
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Polymers & Plastics (AREA)
- Microbiology (AREA)
- Health & Medical Sciences (AREA)
- Animal Husbandry (AREA)
- Food Science & Technology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- Environmental Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Birds (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Tropical Medicine & Parasitology (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Physiology (AREA)
- Animal Behavior & Ethology (AREA)
- Biodiversity & Conservation Biology (AREA)
- Virology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides bacillus subtilis, a microecological preparation, a feed and application thereof in improving the production performance and the eggshell quality of breeding hens, and relates to the technical field of biology. The bacillus subtilis Liu-c1 provided by the invention is derived from traditional cheese, and the inventor adds the bacillus subtilis Liu-c1 as a microecological preparation into the daily ration of a breeding hen to research and find that the laying rate of the breeding hen is improved by 5.63%, the qualified rate of hatching eggs is improved by 9.15%, and the feed-egg ratio is reduced by 4.09%; the superfine structure of the eggshell is improved, the thickness of the eggshell is increased by 3.67 percent, the strength of the eggshell is increased by 17.21 percent, and the calcium content of the eggshell is increased by 2.34 percent; and the calcium content of the serum, the tibia and the uterus of the breeding hen are respectively increased by 11.62 percent, 5.06 percent and 19.63 percent, and the relative expression quantity of genes participating in calcium transfer and calcification in the egg shell forming process of the uterus is improved.
Description
Technical Field
The invention relates to the technical field of biology, in particular to bacillus subtilis, a microecological preparation, a feed and application thereof in improving the production performance and the eggshell quality of breeding hens.
Background
China is the largest egg producing country and the largest consuming country in the world at present, the total stock quantity of laying hens in China in 2019 is about 14.54 hundred million, the egg yield is more than 3000 ten thousand t, and the egg yield accounts for 46% of the global yield. The egg laying period of the laying hens is generally 17-72 weeks old, the laying hens enter the later egg laying period when the laying hens are 46 weeks old, due to long-term egg laying activities, the laying performance of the laying hens at the period is reduced, the reproductive cavities are gradually enlarged, egg contents are gradually increased, egg shells are gradually thinned, and the egg shell quality is also seriously affected. According to statistics, in commercial laying hen breeding production, the number of eggs which have the problem of eggshell quality (broken eggs, soft-shell eggs and deformed eggs) in the later stage of egg laying is increased by 20 percent compared with the peak period of egg laying, and the economic loss caused by the eggs is as high as 5 billion yuan.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The first purpose of the invention is to provide a bacillus subtilis Liu-c1 which is preserved in China general microbiological culture Collection center in 9-29 th of 2020 with the preservation number as follows: CGMCC No. 20840.
The second purpose of the invention is to provide a fermentation method of the bacillus subtilis Liu-c1, which is simple and high in efficiency.
The third purpose of the invention is to provide a microecological preparation, which comprises bacillus subtilis Liu-c 1.
The fourth purpose of the invention is to provide a using method of the microecological preparation.
The fifth purpose of the invention is to provide a feed which comprises the bacillus subtilis Liu-c1 or the microecological preparation.
The sixth purpose of the invention is to provide application of bacillus subtilis Liu-c1 and microbial inoculum or feed thereof in improving the production performance and eggshell quality of breeding hens at the later period of laying.
In a first aspect, the invention provides bacillus subtilis Liu-c1, wherein the bacillus subtilis Liu-c1 is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms with the preservation number: CGMCC No. 20840.
In a second aspect, the invention provides a fermentation method of bacillus subtilis Liu-c1, which comprises the following steps: the Bacillus subtilis Liu-c1 is inoculated into a culture medium for culture.
As a further technical scheme, the fermentation condition of the bacillus subtilis Liu-c1 at least meets one of the following conditions: the fermentation temperature is 37-45 ℃, the fermentation time is 12-16h, and the pH value is 6.5-7.5.
Preferably, the fermentation conditions of the bacillus subtilis Liu-c1 at least meet one of the following conditions: the fermentation temperature is 45 ℃, the fermentation time is 16h, and the pH value is 6.5.
As a further technical scheme, the components of the culture medium of the bacillus subtilis Liu-c1 comprise: 5.0-20.0g/L of tryptone, 2.5-20g/L of yeast extract powder, 1-10g/L of glucose and 0-5.0g/L of sodium chloride.
Preferably, the culture medium components of the bacillus subtilis Liu-c1 comprise: 10.0g/L of tryptone, 10g/L of yeast extract powder, 10g/L of glucose and 2.5g/L of sodium chloride.
In a third aspect, the invention provides a microecological formulation comprising Bacillus subtilis Liu-c 1.
As a further technical proposal, the lactobacillus paracasei KL1 is also included;
the lactobacillus paracasei KL1 is preserved in the general microorganism center of China general microbiological culture Collection Committee (CGMCC) at 10-22 months in 2015, and the preservation numbers are as follows: CGMCC No. 11533;
preferably, the bacterial quantity ratio of the bacillus subtilis Liu-c1 to the lactobacillus paracasei KL1 in the microecological preparation is (1-2) to (1-2), and preferably 1: 1.
As a further technical scheme, the dosage of the microecological preparation is 107～108CFU/day, preferably 6.0X 107～8.0×107CFU/day.
In a fourth aspect, the present invention provides a method for using a microecological preparation, comprising the steps of: diluting the microecological preparation with maltodextrin, mixing, dissolving in water, and spraying onto the surface of feed.
In a fifth aspect, the invention provides a feed comprising Bacillus subtilis Liu-c1 and/or a probiotic.
In a sixth aspect, the invention provides application of bacillus subtilis Liu-c1, a microecological preparation or a feed in improving the production performance of breeding hens at the later period of laying.
Compared with the prior art, the invention has the following beneficial effects:
the bacillus subtilis Liu-c1 provided by the invention is derived from traditional cheese, and the inventor adds the bacillus subtilis Liu-c1 as a microecological preparation into daily ration of hens, so that on the premise that performance indexes such as the average weight, the average egg weight, the egg shape index, the egg yolk color and the Ha's unit of breeding hens are not influenced, the egg laying rate of the hens is improved by 5.63%, the egg qualification rate is improved by 9.15%, and the feed-egg ratio is reduced by 4.09%; the superfine structure of the eggshell is improved, the thickness of the eggshell is increased by 3.67 percent, the strength of the eggshell is increased by 17.21 percent, and the calcium content of the eggshell is increased by 2.34 percent; and the calcium content of the serum, the tibia and the uterus of the breeding hen are respectively increased by 11.62 percent, 5.06 percent and 19.63 percent, and the relative expression quantity of genes participating in calcium transfer and calcification in the egg shell forming process of the uterus is improved. Therefore, the bacillus subtilis Liu-c1 provided by the invention can be used for improving the production performance and the eggshell quality of the breeding hens at the later period of laying.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a scanning electron microscope (800X) of the ultrastructure on the outer surface of an eggshell, wherein A is a blank group, B is a KL1 group, C is a Liu-C1 group, and D is a composite group;
FIG. 2 is a scanning electron microscope (2000X) of the ultrastructure on the inner surface of the eggshell, wherein A is a blank group, B is a KL1 group, C is a Liu-C1 group, and D is a composite group;
FIG. 3 is a scanning electron microscope (500X) of the ultra-micro structure of the cross section of the eggshell, wherein A is a blank group, B is KL1 group, C is Liu-C1 group, and D is a composite group.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to embodiments and examples, but those skilled in the art will understand that the following embodiments and examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. Those who do not specify the conditions are performed according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
In a first aspect, the invention provides a bacillus subtilis Liu-c1, which is classified and named as: bacillus subtilis Liu-c1, latin literature name: bacillus subtilis Liu-c1, which is preserved in China general microbiological culture Collection center, and the preservation address is as follows: west road No.1 hospital No.3, north jing, chaoyang district, preservation date: 29/9/2020, accession number: CGMCC No. 20840.
The bacillus subtilis Liu-c1 is separated and screened from the conventional cheese. The bacillus subtilis Liu-c1 is a gram-positive bacterium, two ends of the bacterium are blunt, spores are located in the center and near the center of the bacterium, and the bacterium is peritrichogenous and non-capsulated; the colonies were characterized by dry, rough surface, irregular edges, wavy, matte, opaque, off-white.
In a second aspect, the invention provides a fermentation method of bacillus subtilis Liu-c1, which comprises the following steps: inoculating the bacillus subtilis Liu-c1 into a culture medium for culturing;
in some preferred embodiments, the fermentation conditions of the Bacillus subtilis Liu-c1 satisfy at least one of the following: the fermentation temperature is 37-45 ℃, the fermentation time is 12-16h, and the pH value is 6.5-7.5.
In the present invention, the temperature of fermentation of Bacillus subtilis Liu-c1 is typically, but not limited to, 37 deg.C, 39 deg.C, 41 deg.C, 43 deg.C or 45 deg.C; the fermentation time is typically, but not limited to, 12h, 13h, 14h, 15h or 16 h; the pH is typically, but not limited to, 6.5, 6.7, 6.9, 7.1, 7.3, or 7.5. The Bacillus subtilis Liu-c1 can be fermented better under the conditions.
Preferably, the fermentation conditions of the bacillus subtilis Liu-c1 at least meet one of the following conditions: the fermentation temperature is 45 ℃, the fermentation time is 16h, and the pH value is 6.5.
The optimal fermentation conditions of the bacillus subtilis Liu-c1 are obtained by further optimizing and adjusting the fermentation conditions, namely the fermentation temperature is 45 ℃, the fermentation time is 16h and the pH value is 6.5.
In some preferred embodiments, the media components of Bacillus subtilis Liu-c1 include: 5.0-20.0g/L of tryptone, 2.5-20g/L of yeast extract powder, 1-10g/L of glucose and 0-5.0g/L of sodium chloride.
In the present invention, the concentration of tryptone in the medium is typically, but not limited to, 5.0g/L, 10.0g/L, 15.0g/L or 20.0 g/L; the concentration of yeast extract is typically, but not limited to, 2.5g/L, 5g/L, 10g/L, 15g/L, or 20 g/L; the concentration of glucose is typically, but not limited to, 1g/L, 3g/L, 5g/L, 7g/L, 9g/L, or 10 g/L; the concentration of sodium chloride is typically, but not limited to, 0g/L, 1.0g/L, 2.0g/L, 3.0g/L, 4.0g/L, or 5.0 g/L.
Preferably, the culture medium components of the bacillus subtilis Liu-c1 comprise: 10.0g/L of tryptone, 10g/L of yeast extract powder, 10g/L of glucose and 2.5g/L of sodium chloride.
The optimal component distribution ratio of the culture medium is obtained by further optimizing and adjusting the components of the culture medium of the bacillus subtilis Liu-c 1.
In a third aspect, the present invention provides a probiotic, including but not limited to Bacillus subtilis Liu-c 1.
The microecological preparation provided by the invention comprises the bacillus subtilis Liu-c1, or the bacillus subtilis Liu-c1 and auxiliary materials or other bacteria, for example, the microecological preparation can be a microbial agent prepared by mixing the bacillus subtilis Liu-c1 and other bacteria, or a microbial agent prepared by mixing the bacillus subtilis Liu-c1 and auxiliary materials.
The inventor adds the bacillus subtilis Liu-c1 into the daily ration of the breeding hens as a microecological preparation, and finds that the egg laying rate of the breeding hens is improved by 5.63%, the egg qualification rate is improved by 9.15% and the feed-egg ratio is reduced by 4.09% on the premise that performance indexes such as the average weight, the average egg weight, the egg shape index, the egg yolk color and the Haugh unit of the breeding hens are not influenced; the superfine structure of the eggshell is improved, the thickness of the eggshell is increased by 3.67 percent, the strength of the eggshell is increased by 17.21 percent, and the calcium content of the eggshell is increased by 2.34 percent; and the calcium content of the serum, the tibia and the uterus of the breeding hen are respectively increased by 11.62 percent, 5.06 percent and 19.63 percent, and the relative expression quantity of genes participating in calcium transfer and calcification in the egg shell forming process of the uterus is improved.
In some preferred embodiments, lactobacillus paracasei KL 1;
the lactobacillus paracasei KL1 is separated and screened from Tibetan mushroom (also called kefir grains) in common families in Jilin and Hohehaote city, and the classification of the strain is named as: lactobacillus paracasei KL1, latin literature name: lactobacillus paracasei KL1, deposited in China general microbiological culture Collection center, with the deposition address: west road No.1 hospital No.3, north jing, chaoyang district, preservation date: 22/10/2015, accession number: CGMCC No. 11533.
The inventor researches and discovers that the egg yield of the breeding hens is improved by 8.84%, the egg qualification rate is improved by 14.43% and the feed-egg ratio is reduced by 5.45% on the premise that performance indexes such as the average weight, the average egg weight, the egg shape index, the egg yolk color and the Ha's unit of the breeding hens are not affected by adding the bacillus subtilis Liu-c1 and the lactobacillus paracasei KL1 serving as microecologics into the daily ration of the breeding hens; the superfine structure of the eggshell is improved, the thickness of the eggshell is increased by 8.33 percent, the strength of the eggshell is increased by 31.82 percent, and the calcium content of the eggshell is increased by 2.91 percent; and the calcium content of the serum, the tibia and the uterus of the breeding hen are respectively increased by 26.10%, 9.64% and 60.37%, and the relative expression quantity of genes participating in calcium transport and calcification in the egg shell forming process of the uterus is improved. Therefore, the bacillus subtilis Liu-c1 provided by the invention can be used for improving the production performance and the eggshell quality of the breeding hens at the later period of laying.
Preferably, the bacterial quantity ratio of the bacillus subtilis Liu-c1 to the lactobacillus paracasei KL1 in the microecological preparation is (1-2): (1-2), and can be, for example and without limitation, 1:1, 1:2 or 2:1, preferably 1: 1.
In some preferred embodiments, the probiotic is used at a dose of 107～108CFU/day, for example, but not limited to, 1.0X 107CFU/day 2.0X 107CFU/day, 4.0X 107CFU/day, 6.0X 107CFU/day, 8.0X 107CFU/day or 1.0X 108CFU/day, preferably 6.0X 107～8.0×107CFU/day.
In the invention, the production performance and the eggshell quality of the breeding hens at the later period of laying can be better improved by further optimizing and adjusting the using amount of the microecological preparation and the ratio of the two bacteria in the composite microecological preparation.
In a fourth aspect, the present invention provides a method for using a microecological preparation, comprising the steps of: diluting the microecological preparation with maltodextrin, mixing, dissolving in water, and spraying onto the surface of feed.
The application method of the invention is simple and easy to operate, can ensure that all hens can take in the microecological preparation simultaneously and quickly, can avoid the loss of the viable bacteria quantity of the microecological preparation in the using process, and ensures the effective intake of the microecological preparation.
For example, one possible implementation method is: will be described in the inventionDiluting the microecological preparation with maltodextrin, and mixing to obtain viable bacteria with viable count of 8.0 × 108The preparation method comprises the following steps of (1) accurately weighing the microecological preparation premix according to the dosage of 1kg of microecological preparation for feeding 1 ten thousand chickens, dissolving 1kg of microecological preparation in 35kg of poultry drinking water to obtain microecological preparation premix diluent, and finally uniformly spraying each group of microecological preparation premix diluent on the surface of the feed in a feed trough of a breeding hen, wherein the moist feed containing bacteria on the surface can be eaten by the breeding hen within 10 min.
In a fifth aspect, the invention provides a feed comprising Bacillus subtilis Liu-c1 and/or a probiotic.
The feed provided by the present invention comprises the bacillus subtilis Liu-c1 or the microecological agent of the present invention mixed with a nutrient, and may be, for example, a feed obtained by mixing the bacillus subtilis Liu-c1 of the present invention with a nutrient or a feed obtained by mixing the microecological agent of the present invention with a nutrient. The feed has all the beneficial effects of the bacillus subtilis Liu-c1 or the microecological preparation of the invention.
The term "and/or" means that the feed may include bacillus subtilis Liu-c1, a microecological preparation, or bacillus subtilis Liu-c1 and a microecological preparation.
In a sixth aspect, the invention provides application of bacillus subtilis Liu-c1, a microecological preparation or a feed in improving the production performance and the eggshell quality of breeding hens at the later stage of laying eggs.
The inventor tests show that the bacillus subtilis Liu-c1, the microecological preparation or the feed has the effects of improving the laying rate and the qualified rate of hatching eggs, reducing the feed-egg ratio, improving the ultrastructure of eggshells, increasing the calcium content of serum, shin bones and uterus, improving the relative expression quantity of genes participating in calcium transfer and calcification in the eggshell forming process of uterus, promoting the calcium deposition of the eggshells, increasing the eggshell thickness, enhancing the eggshell strength and the like on the premise of not influencing the performance indexes of the breeding hens such as the average weight, the average egg weight, the egg shape index, the yolk color and the Haugh unit, and the like, so that the bacillus subtilis Liu-c1, the microecological preparation or the feed can be used for improving the production performance and the eggshell quality of.
The invention is further illustrated by the following specific examples and comparative examples, but it should be understood that these examples are for purposes of illustration only and are not to be construed as limiting the invention in any way.
If not specifically stated, all the breeding hens used in the invention are 54-week-old 'Jinghong No. 1' parental breeding hens which have consistent breeding conditions and no significant difference in weight, and are selected from Huadu valley poultry industry, Limited liability company in Beijing City.
The basic ration was provided by Huadu valley poultry, llc, beijing, and the composition and nutritional levels are shown in table 1.
TABLE 1 composition and nutritional level of basal diets (air-dried basis)
1) The premix can be provided for each kilogram of daily ration: 65mg of iron, 10mg of copper, 101mg of zinc, 90mg of manganese, 9975IU of vitamin A, VD 34970 IU, 80IU of vitamin E, 10mg of riboflavin, 50.1mg of nicotinic acid, 18mg of calcium pantothenate and vitamin B120.03mg, biotin 0.4mg, choline chloride 450 mg.
2) Metabolic energy was calculated, and the rest were measured values.
The main reagents comprise: (1) PCA culture medium: 5g of tryptone, 2.5g of yeast extract powder, 1g of glucose, pH7.0 +/-0.2 and 1000mL of distilled water. The above formula is a liquid culture medium, and on the basis, 17g of agar powder is added to obtain a solid culture medium.
(2) Protein agar medium (for testing protein decomposing bacteria):
solution A: 50g of skim milk powder and 500mL of distilled water.
And B, liquid B: 10g of soluble starch and 5g of yeast extract powder.0g，KH2PO4 1.0g，MgSO4 0.2g。
17g of agar powder and 500mL of distilled water, and the pH value is 7.0-7.6.
Respectively preparing solution A and solution B, sterilizing at 0.07MPa for 20min, and mixing at a ratio of 1: 1.
(3) Starch agar medium (for testing starch hydrolyzing bacteria): 10g of peptone, 5g of beef extract, 5g of NaCl, 2g of soluble starch, 17g of agar, pH7.2, 1000mL of distilled water, and sterilizing at 0.1MPa for 15-20 min.
(4) Modified MRS medium (for culturing lactobacillus paracasei KL1 strain): 5.00g of tryptone, 5.00g of acid hydrolyzed casein, 10.00g of beef extract, 5.00g of yeast extract powder, 2.00g of monopotassium phosphate, 2.00g of trisodium citrate, 2.00g of sodium acetate, 20.00g of glucose, 0.58g of magnesium sulfate heptahydrate, 0.25g of manganese sulfate tetrahydrate, 801.00 mL of tween-801.00 mL of pH 6.5 +/-0.2, adding distilled water to 1000mL of the mixture, taking the formula as a liquid culture medium, and adding 17g of agar on the basis to obtain the solid culture medium.
(5) Modified PCA Medium (for culturing Bacillus subtilis Liu-c1 Strain): 10.00g of tryptone, 10.00g of yeast extract powder, 10.00g of glucose, 2.50g of sodium chloride, pH 6.5 +/-0.2, and distilled water to 1000mL, wherein the formula is a liquid culture medium, and 17g of agar is added on the basis to obtain a solid culture medium.
(6) The microecological preparation protective agent comprises: 5.5 percent (mass volume percent) of skim milk powder, 14.5 percent (mass volume percent) of maltodextrin and 0.07Mpa are sterilized for 20min and then are taken out of the sterilizing pot immediately to prevent the protective agent from browning to influence the protective effect.
EXAMPLE 1 preparation of Bacillus subtilis Liu-c1 Microecological preparation
(1) Screening of Bacillus subtilis
1.0g of a conventional cheese sample is aseptically weighed, put into a sterile homogeneous bag containing 99mL of physiological saline containing 0.1% Tween, mixed uniformly and hermetically sealed. Beating with a beating homogenizer (SCIENTZ-11, Ningbo Xin Zhi Biotech Co., Ltd.) at 6T/S for 20min to dissolve the sample sufficiently to obtain a sample dilution. And (3) gradually diluting 1mL of sample diluent by 10-fold gradient to obtain a plurality of concentration sample diluent liquid. Selecting 100 mu L of each of 3-4 sample diluents with different concentrations, inoculating the sample diluents on a PCA culture medium plate by a coating method, culturing the sample diluents at 37 ℃ for 24 hours, selecting single colonies which are characterized by dry and rough surface, irregular edge, ripple, dull luster, opaque and grey white on the plate, continuously coating the single colonies for three times and inoculating the single colonies on the PCA plate, and further separating and culturing a target strain. Selecting 1 ring of target strain from PCA plate, streaking and inoculating in PCA slant test tube culture medium, culturing at 37 deg.C for 24 hr, gram staining and microscopic examining strain purity, and storing pure target strain in 4 deg.C refrigerator.
(2) Screening of protease and amylase producing Bacillus subtilis
Inoculating the screened target strain slant culture in 100mL of PCA liquid culture medium, culturing at 37 ℃ for 18h, centrifuging at 6000rpm for 5min, taking supernatant, taking 10 mu L of supernatant by using a sterile 200 mu L pipette gun, respectively inoculating the supernatant on the surfaces of a protein agar culture medium plate and a starch agar culture medium plate, respectively measuring the diameters of a transparent ring and a bacterial colony around the bacterial colony after the protein agar culture medium plate is cultured at 45 ℃ for 2d, calculating the difference value between the diameter of the transparent ring of each strain and the diameter of the bacterial colony, and comparing the difference value of each strain; after 12h of starch agar medium flat plate at 45 ℃, open the plate lid, drip into a small amount of luger iodine solution on the culture medium surface, rotate the plate gently, make iodine solution evenly distributed whole flat plate, measure the diameter of transparent circle and bacterial colony around the bacterial colony respectively, calculate the difference of the transparent circle diameter of each bacterial strain and bacterial colony circle diameter, compare each bacterial strain difference size. The results are shown in Table 2.
TABLE 2 subtilisin and amylase test results
As can be seen from the results of the subtilisin and amylase tests in Table 2, the Liu-c1 strain has the greatest difference in diameter not only between the protease-decomposed clearing zone and the colony zone, but also between the amylase-decomposed clearing zone and the colony zone, as compared with the Liu-c2 and Liu-c6 strains. The results show that the yield of protease and amylase secreted by the Liu-c1 strain is higher, and the Liu-c1 strain can become an excellent strain for preparing a microecological preparation.
(3) Colony characteristics, morphological identification and molecular biological identification of Liu-c1 strain
And streaking the selected Liu-c1 excellent strain onto a PCA culture medium plate, culturing at 37 ℃ for 24h, observing the colony characteristics, picking out a single colony, carrying out gram staining, and observing the cell morphology under an optical microscope. The colonies were characterized by dry, rough surface, irregular edges, wavy, matte, opaque, off-white. Morphological observation shows that the Liu-c1 strain is gram-positive bacteria, the two ends of the bacteria are blunt circles, spores are located in the center and near the center of the bacteria, and the bacteria are periflagellated and non-capsulated.
Selecting a single colony on the Liu-c1 strain plate, inoculating the single colony into 10mL of PCA liquid culture medium, culturing at 37 ℃ for 18h at the rotating speed of 180rpm, centrifuging at 4 ℃ for 2min at 8000rpm, removing supernatant, taking bacterial sludge, extracting DNA of bacillus subtilis by adopting a bacterial genome DNA rapid extraction kit [ engineering biology (Shanghai) engineering Limited company ], and carrying out PCR amplification by using a bacterial 16S rDNA universal primer, wherein the primer sequence is as follows:
the upstream primer 27F: 5 '-AGAGTTTGATCM TGGCTCAG-3' (SEQ ID No. 1);
a downstream primer 1492R: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID No. 2).
The obtained PCR product was sent out and sequenced [ Probiotics (Shanghai) engineering Co., Ltd ], and the nucleotide sequence information was as shown in SEQ ID No. 3:
GGGTGTGGCGGCTGCTATACATGCAAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTTTGAACCGCATGGTTCAAACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTACCGTTCGAATAGGGCGGTACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCCTGTCCTGAAAGTTGGGTTAGTCCCCAACGAGCGAACCCTTGATTTTATTGCCGCATTCAGTTGGGCACCTAAGGTACTGCCGGTGAAACTTCTGTAACCTTCGGCGGCTGGCTCCTAAAAGGTTACCTCACCGACTTCGGGTGTTACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCAGCTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACTGAGAACAGATTTGTGGGATTGGCTTAACCTCGCGGTTTCGCTGCCCTTTGTTCTGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTGAATGCTGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACTCTGCCCCCGAAGGGGACGTCCTATCTCTAGGATTGTCAGAGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTAAGGGGCGGAAACCCCCTAACACTTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTTACAGACCAGAGAGTCGCCTTCGCCACTGGTGTTCCTCCACATCTCTACGCATTTCACCGCTACACGTGGAATTCCACTCTCCTCTTCTGCACTCAAGTTCCCCAGTTTCCAATGACCCTCCCCGGTTGAGCCGGGGGCTTTCACATCAGACTTAAGAAACCGCCTGCGAGCCCTTTACGCCCAATAATTCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCAAGGTACCGCCCTATTCGAACGGTACTTGTTCTTCCTAACACAGAGCTTTACGATCGAAAACCTTCATCCTCAGCGGCGTTGCTCGT。
BLAST homology sequence comparison analysis is carried out on the sequencing result in an NCBI database, and finally the Liu-c1 strain is determined to be Bacillus subtilis (Bacillus subtilis), and the identification result is shown in Table 3.
TABLE 3 identification results of excellent strains screened by Bacillus subtilis Liu-c1
(4) Liu-c1 strain activation and scale-up culture
Transferring a glycerol storage tube of the bacillus subtilis Liu-c1 at the temperature of-80 ℃ into 10mL of improved PCA culture medium at the inoculation amount of 2 percent by volume, culturing at 37 ℃ for 18h at the rotation speed of 180rpm, and carrying out passage twice to obtain an activated strain; the activated strain was inoculated into 100mL of the modified PCA medium at a volume percentage of 2% under the same conditions as above to obtain an expanded culture of the Liu-c1 strain.
(5) Optimization of fermentation conditions of bacillus subtilis Liu-c1 strain
Transferring 2 percent of inoculum size of 2 percent by volume into Liu-c1 strain expanded culture solution in a 500mL triangular flask containing 200mL of LPCA culture medium, setting the rotating speed to be 180rpm, and designing three-factor three levels (L) of fermentation temperature, fermentation time and initial pH of the culture medium9(33)]Orthogonal tests (see table 4) are carried out, each group is repeated for 3 times, the viable count in PCA fermentation liquor is taken as a test index, and the superior fermentation condition of the Liu-c1 strain is determined by the extreme difference analysis (R) and the K value analysis of the result.
TABLE 4 optimization of fermentation conditions and results of Bacillus subtilis Liu-c1 strain orthogonal test
The results of the Liu-c1 strain optimized fermentation conditions orthogonal experiments are shown in Table 4. As can be seen from Table 4, R was analyzed according to the orthogonality testA＞RC＞RBIt can be seen that the sequence of the influence of different fermentation conditions on the viable count of the strain Liu-c1 is as follows: the fermentation temperature is higher than the initial pH of the fermentation medium and is longer than the fermentation time; analysis of K from the orthogonal test K valueA3＞KA1＞KA2、KB3＞KB1＞KB2、KC1＞KC2＞KC3It is known that the optimum fermentation conditions for the strain Liu-c1 are: the temperature is 45 ℃, the time is 16h, and the initial pH of the culture medium is 6.5.
(6) Improved test of PCA culture medium of Bacillus subtilis Liu-c1 strain
In a 5L intelligent fermentation tank, the expanded culture solution of the Liu-c1 strain is transferred into 2.0L of improved PCA culture medium according to the inoculation amount of 2%, the results of the optimized fermentation conditions of the Liu-c1 strain are adopted to carry out high-density fermentation respectively according to the table 5, and the viable count in different PCA fermentation liquids is determined.
TABLE 5 viable cell count of Bacillus subtilis Liu-c1 strain in different PCA cultures
As can be seen from Table 5, the number of viable bacteria in the fermentation broth using the modified PCA-2 medium was the highest, 1.11X 10, as compared with the PCA medium9CFU/mL, therefore, the improved PCA culture formula of the Bacillus subtilis Liu-c1 strain is determined to be 10g/L of tryptone, 10g/L of yeast extract, 10.0g/L of glucose and 2.5g/L of sodium chloride.
(7) High-density fermentation of bacillus subtilis Liu-c1 strain
Transferring the Liu-c1 strain expanded culture solution into a 5L intelligent fermentation tank (5L/DM 9000A, Shanghai Biotechnology (Shanghai) Co., Ltd.) containing 2.0L of improved PCA culture medium according to the inoculation amount of 2%, controlling the fermentation temperature to be 45 ℃, controlling the pH of the fermentation solution to be 6.5 (adjusting the pH of the fermentation solution by automatically adding 2mol/L NaOH into the fermentation tank), setting the stirring speed of the fermentation tank to be 600-700 rpm or the associated dissolved oxygen to be 60-70%, and fermenting for 16h to obtain the Liu-c1 strain high-density fermentation solution.
(8) Collecting thallus
After fermentation, transferring the high-density fermentation broth into a centrifuge bottle under aseptic condition, centrifuging at 8000rpm for 20min at 4 deg.C by using high-speed vertical refrigerated centrifuge (TGL-21M, Shanghai Luxiang apparatus centrifuge Co., Ltd.), discarding supernatant, and collecting to obtain bacterial sludge. And re-dissolving the obtained bacterial sludge by using a sterilized microecological preparation protective agent with the volume of 1/10 fermentation liquor to obtain the concentrated fermentation agent.
(9) Prefreezing and lyophilizing
Pre-freezing the concentrated starter culture in a refrigerator at-45 deg.C for 10h to obtain pre-frozen starter culture, and freeze-drying the pre-frozen starter culture with a vacuum freeze-drying machine (6LLABCONCO, LABCONCO Co., USA) at-55 deg.C under a vacuum degree of 0.16mBar for 48h to obtain Bacillus subtilis Liu-c1 microecological preparation, and storing in a refrigerator at-20 deg.C for use. Through detection, the viable count of the bacillus subtilis Liu-c1 microecological preparation is 2.2 multiplied by 1010CFU/g。
(10) Preparation of Lactobacillus paracasei KL1 microecological preparation
Transferring a glycerol storage tube of the Lactobacillus paracasei KL1 strain into 10mL of improved MRS culture medium according to the inoculation amount of 3 percent of volume percentage, standing and culturing for 14h at 37 ℃, and carrying out passage twice to obtain an activated strain; the activated strain is transferred into 100mL of improved MRS culture medium by the inoculation amount of 3 percent of volume percentage, and is statically cultured for 14h at 37 ℃ to obtain KL1 strain expansion culture solution. Transferring the KL1 strain expanded culture solution into a 5L intelligent fermentation tank containing 2.5L of improved MRS culture medium according to the inoculation amount of 3 percent of volume percentage, controlling the fermentation temperature to be 37 ℃ and the pH of fermentation liquor to be 6.5 (adjusting the pH of the fermentation liquor by automatically feeding 2mol/L NaOH into the fermentation tank), setting the stirring speed of the fermentation tank to be 90rpm and the fermentation time to be 14h, and obtaining the KL1 strain high-density fermentation liquor. And (4) preparing the lactobacillus paracasei KL1 microecological preparation through the steps (8) and (9). Through detection, the viable count of the lactobacillus paracasei KL1 microecological preparation is 2.5 multiplied by 1011CFU/g。
Example 2 method for Using Microecological preparation and measurement of production Performance and quality of hatching eggs of hen
(1) Animal test grouping design
384 healthy 54-week-old 'Jinghong No. 1' parent breeder hens which are close in weight under the same feeding condition are selected and randomly divided into 4 groups, 96 hens in each group (6 in each group and 16 in each group) are subjected to animal tests with the period of 8 weeks (54-61 weeks old), and the grouping and the addition amount of the microecologics are shown in table 6. The feeding conditions are strictly operated according to the feeding management regulation of Beijing Huadu valley poultry industry Limited company: three-layer A-shaped stepped cage culture is carried out, 4 feathers are planted in each cage, the illumination intensity is 10-15 lx, the duration is constant for 15.5h, the temperature is 18-25 ℃, and the relative humidity is 40-70%. Feeding three times every day, feeding and drinking water freely for chickens, cooling the chickens in the henhouse by adopting a wet curtain, ventilating by a fan, cleaning excrement regularly, and sterilizing the environment and tools.
TABLE 6 test grouping of Jinghong No.1 parent breeder hens
(2) Use method of microecological preparation
The number of viable bacteria to be prepared is 2.5 × 1011KL1 microecological formulation of CFU/g and 2.2X 1010Diluting CFU/g Liu-c1 microecological preparation with maltodextrin respectively, and mixing to obtain viable bacteria with viable count of 8.0 × 108The CFU/g microecological preparation premix is stored in a refrigerator at the temperature of 20 ℃ below zero for later use. Accurately weighing the microecological preparation premix of each group according to the dosage of 1kg of microecological preparation for feeding 1 ten thousand chickens at 14:00 (the feed intake of the breeding hens is in the peak period in one day) each day when the premix is temporarily used, and then respectively dissolving the microecological preparation premix of each group in the drinking water of the poultry according to the proportion that 1kg of microecological preparation is dissolved in 35kg of the drinking water of the poultry to obtain microecological preparation premix diluent; uniformly spraying the micro-ecological preparation premix diluent on the feed surface in the trough of the group of the breeding hens (spraying poultry drinking water without the micro-ecological preparation in the trough of the blank group), wherein the moist feed containing the bacteria on the surface can be eaten by the breeding hens within 10 min.
(3) Hen breeding productivity determination
During the test period, the weight is weighed once every two weeks, the total weight of eggs of each group, the total number of eggs, the number of qualified eggs (non-shell eggs, soft eggs, sand shell eggs, malformed eggs and preserved steel eggs, and the weight of the eggs is between 53 and 72 g) and the daily feed consumption are counted and recorded, the average egg weight, the laying rate, the egg qualification rate and the feed-egg ratio are calculated, the growth state and the excrement color of the breeding hens are observed, and whether the breeding hens are ill or not is recorded.
Average egg weight (g) is total egg weight/total egg count;
laying rate (%) — total number of eggs/number of chickens × 100%;
the percent of qualified hatching eggs/the total number of eggs laid is multiplied by 100%.
(4) Determination of egg quality
Every two weeks, 4 egg samples with intact appearance and approximate average egg weight (62g) were selected from each repeat group for egg quality index detection. The indexes of egg weight, Ha's unit and yolk color are measured by an egg full-function quality tester (EA-01, ORKA, Israel); the eggshell thickness index was measured using an ultrasonic thickness gauge (ETG-1061A, Karl deutsch, Germany); the eggshell strength index was measured using a texture analyzer (ta.xt plus, Stable Micro systems, uk); the egg shape index was measured using an egg shape index meter (FHK, Robotmation, Japan).
(5) Data processing
Experimental data One-way analysis of variance (One-way ANOVA), Duncan's multiple comparison test, was performed using IBM SPSS Statistics 22.0 statistical software. Experimental data are presented as mean ± standard deviation, P <0.05 for significant differences and P <0.01 for very significant differences.
The results and analysis of the production performance and the quality of the hatching eggs of the breeding hens are as follows:
1) weight change of breeding hens
Table 7 shows the body weight change of the hens of each group during the test. As can be seen from Table 7, the average body weight of the various groups of hens increased with the day-old during the test period, but there was no significant difference (P > 0.05), indicating that the addition of three different probiotics had no effect on the body weight of the individual hens.
TABLE 7 weight change (g) of various groups of hens tested for 2-8 weeks
Note: the shoulder marks on the same row have no letters or the same letters represent no significant difference (P is more than 0.05), different lower case letters represent significant difference (P is less than 0.05), and different upper case letters represent significant difference (P is less than 0.01).
2) Average egg weight variation
Table 8 shows the average egg weight change of each group of hens during the test. As can be seen from Table 8, there was no significant difference in the average egg weight of each group (P > 0.05) during the test period, indicating that the addition of three different probiotics had no effect on the average egg weight of the breeder hens.
TABLE 8 average egg weight change (g) for various groups of hens tested for 1-8 weeks
Note: the shoulder marks on the same row have no letters or the same letters represent no significant difference (P is more than 0.05), different lower case letters represent significant difference (P is less than 0.05), and different upper case letters represent significant difference (P is less than 0.01).
3) Change in laying rate
Table 9 shows the change in laying rate for each group of hens during the test. As can be seen from Table 9, compared with the blank group, the KL1 group, the Liu-c1 group and the composite group were not significantly different at the initial stage of the test (P > 0.05); the KL1 group is remarkably increased by 3.85 percent and 4.60 percent (P is less than 0.05) in 1-2 weeks and 3-4 weeks respectively, and is remarkably increased by 5.79 percent and 5.77 percent (P is less than 0.01) in 5-6 weeks and 7-8 weeks respectively; the laying rate of the Liu-c1 group is remarkably increased by 4.48 percent and 4.82 percent respectively at 1-2 and 3-4 weeks (P is less than 0.05), and is remarkably increased by 5.63 percent and 5.37 percent respectively at 5-6 and 7-8 weeks (P is less than 0.01). The compound groups are remarkably increased by 3.87 percent and 3.96 percent (P is less than 0.05) in 1-2 and 3-4 weeks respectively, and are remarkably increased by 8.16 percent and 8.84 percent (P is less than 0.01) in 5-6 and 7-8 weeks respectively; the results show that after the three different microecologics are added, the laying rate of the breeding hens in the later period of laying is remarkably improved at 1-4 weeks, the laying rate is remarkably improved at 5-8 weeks, and the effect of the composite group is optimal, namely the KL1 group and the Liu-c1 group.
TABLE 9 change in laying rate (%)
Note: the shoulder marks on the same row have no letters or the same letters represent no significant difference (P is more than 0.05), different lower case letters represent significant difference (P is less than 0.05), and different upper case letters represent significant difference (P is less than 0.01).
4) Change of egg yield
Table 10 shows the change in egg yield for each group of hens during the test. As can be seen from Table 10, there was no significant difference in the percent of pass of the initial groups of hatching eggs (P > 0.05); with the increase of the day age, the blank hatching egg qualification rate is reduced from initial 89.16% to 79.65% of the 4 th week, and is kept at about 80% from the 4 th week to the 8 th week; and the qualification rate of the KL1 group, the Liu-c1 group and the compound group hatching eggs is always kept between 87 percent and 93 percent. At week 8, compared with the blank group, the qualification rates of the hatching eggs of the KL1 group, the Liu-c1 group and the compound group are respectively and remarkably improved by 12.65 percent, 9.15 percent and 14.43 percent (P is less than 0.01). The three different microecologics can increase the hatching egg qualification rate of the parent generation hens of 'Jinghong No. 1' parent generation in different degrees, wherein the composite group is the best, and the KL1 group and the Liu-c1 group are the second time.
TABLE 10 change in the percent of pass of hatching eggs from 1-8 weeks for various groups of hens (%)
Note: the shoulder marks on the same row have no letters or the same letters represent no significant difference (P is more than 0.05), different lower case letters represent significant difference (P is less than 0.05), and different upper case letters represent significant difference (P is less than 0.01).
5) Change of ratio of feed to egg
Table 11 shows the change in feed-to-egg ratio of the hens of each group during the test. As can be seen from Table 11, the feed-egg ratios of the KL1, Liu-c1 and the compound groups were significantly decreased (P < 0.05) at weeks 1-4 compared with the blank group; in 5-6 weeks, the KL1 group and the Liu-c1 group are respectively and remarkably reduced by 2.25 percent and 2.70 percent (P is less than 0.05), and the composite group is remarkably reduced by 4.50 percent (P is less than 0.01); in 7-8 weeks, KL1 group was significantly reduced by 2.27% (P < 0.05), and Liu-c1 group and the complex group were significantly reduced by 4.09% and 5.45% (P < 0.01). The three different microecologics can reduce the feed-egg ratio of 'Jinghong No. 1' parent-generation hens to different degrees, wherein the effect of the composite group is the best, and the KL1 group and the Liu-c1 group are the second time.
TABLE 11 feed-egg ratio variation for groups of hens tested for 1-8 weeks
Note: the shoulder marks on the same row have no letters or the same letters represent no significant difference (P is more than 0.05), different lower case letters represent significant difference (P is less than 0.05), and different upper case letters represent significant difference (P is less than 0.01).
6) Variation in eggshell thickness
The eggshell thickness changes of the various groups of hatching eggs during the test are shown in Table 12, and compared with the blank group, the KL1 group, the Liu-c1 group and the compound group have no significant difference (P is more than 0.05) at the 2 nd week; at week 4, KL1 and Liu-c1 increased by 2.30% and 1.97%, respectively (P > 0.05), and the composite group increased by 3.61% (P < 0.05); at week 6, KL1 and Liu-c1 increased by 2.97% and 2.31%, respectively (P > 0.05), and the composite group increased by 4.95% (P < 0.05); at week 8, the Liu-c1 group increased 3.67% (P > 0.05), and the KL1 group and the composite group increased significantly by 7.00% and 8.33%, respectively (P < 0.05). Shows that the eggshell thickness of the hatching egg is increased after three different microecologics are added, wherein the effect of the composite group is the best, and the KL1 group and the Liu-c1 group are the second time.
TABLE 12 Eggshell thickness variation (mm) for each group of eggs tested for 2-8 weeks
Note: the shoulder marks on the same row have no letters or the same letters represent no significant difference (P is more than 0.05), different lower case letters represent significant difference (P is less than 0.05), and different upper case letters represent significant difference (P is less than 0.01).
7) Variation in eggshell strength
The eggshell strength changes of the various groups of hatching eggs during the test are shown in Table 13, and compared with the blank group, the eggshell strength of the KL1 group, the Liu-c1 group and the compound group at the 2 nd week is not significantly different (P is more than 0.05); at week 4, the KL1 group and the Liu-c1 group were increased by 13.65% and 9.84%, respectively (P > 0.05), and the composite group was significantly increased by 21.59% (P < 0.05); at week 6, KL1 and Liu-c1 increased by 6.97% and 7.27%, respectively (P > 0.05), and the composite group increased by 14.24% (P < 0.05); at week 8, the Liu-c1 group was significantly increased by 17.21% (P < 0.05), and the KL1 group and the composite group were significantly increased by 20.45% and 31.82% (P < 0.01), respectively. Shows that the eggshell strength of the hatching eggs is increased after the three different microecologics are added, wherein the composite group has the best effect, and the KL1 group and the Liu-c1 group are the second time.
TABLE 13 Eggshell Strength Change (kg/cm) for groups of eggs tested for 2-8 weeks2)
Note: the shoulder marks on the same row have no letters or the same letters represent no significant difference (P is more than 0.05), different lower case letters represent significant difference (P is less than 0.05), and different upper case letters represent significant difference (P is less than 0.01).
8) Change in egg shape index
The change in egg shape index of each group of eggs during the experiment is shown in table 14. As can be seen from the data in Table 14, there was no significant difference in egg shape index between the groups tested (P > 0.05), indicating that the addition of three different probiotics had no effect on the egg shape index of the hatching eggs.
TABLE 14 egg shape index changes for groups of eggs tested at 2-8 weeks
Note: the shoulder marks on the same row have no letters or the same letters represent no significant difference (P is more than 0.05), different lower case letters represent significant difference (P is less than 0.05), and different upper case letters represent significant difference (P is less than 0.01).
9) Change in Ha's unit
The change in hards units of the various groups of hatching eggs during the experiment is shown in table 15. As can be seen from the data in Table 15, there was no significant difference in Ha's units among the groups tested (P > 0.05), indicating that the addition of three different probiotics had no effect on the Ha's units of the hatching eggs.
TABLE 15 variation in Ha's units for each group of hatching eggs tested at 2-8 weeks
Note: the shoulder marks on the same row have no letters or the same letters represent no significant difference (P is more than 0.05), different lower case letters represent significant difference (P is less than 0.05), and different upper case letters represent significant difference (P is less than 0.01).
10) Color change of yolk
The change in yolk color of each group of eggs during the test is shown in table 16. As can be seen from Table 16, there was no significant difference in the yolk color between the groups tested (P > 0.05), indicating that the addition of three different probiotics had no effect on the yolk color of the eggs.
TABLE 16 yolk color change of various groups of eggs tested for 2-8 weeks
Note: the shoulder marks on the same row have no letters or the same letters represent no significant difference (P is more than 0.05), different lower case letters represent significant difference (P is less than 0.05), and different upper case letters represent significant difference (P is less than 0.01).
Discussion and conclusion: the growth of the laying hens is gradually stopped after the laying peak period, the weight growth is basically completed at the age of 36-38 weeks, and the average egg weight also reaches a stable high peak value. The thickness and strength of the eggshell play an important role in the quality, transportation, preservation and hatching of the hatching eggs. The egg shape index is also one of important factors influencing hatching of hatching eggs, eggs cannot be over-round or over-long, and are generally in the best elliptical shape, and the generation of deformed eggs can be reduced by the egg shape index. The Ha's unit is one of important indexes for measuring the freshness of eggs, reflects the freshness and the preservation time of the eggs, and the lower the Ha's unit is, the worse the viscosity of protein is, the lower the freshness of the eggs is. The color of the yolk reflects the deposition of the fat-soluble pigment. The three microecologics added in the test obviously increase the laying rate, the egg qualification rate, the eggshell thickness and the strength of the breeding hens in the later period of laying, and obviously reduce the feed-egg ratio, and indexes such as average weight, average egg weight, egg shape index, Ha's unit, yolk color and the like have no obvious change, which shows that the three different microecologics can not generate adverse effects on the breeding hens and the eggs while improving the production performance and the egg quality of the breeding hens in the later period of laying.
EXAMPLE 3 Observation of Eggshell ultrastructures
(1) Sampling of eggshells
At the end of 8 weeks of the experiment, 6 fresh hatching eggs (1 egg in each of 6 replicates in each group) were selected for each group, the same parts of 6 eggshells in each group were collected, eggshell membranes were removed, impurities such as egg white attached to the eggshells were cleaned with PBS and trimmed to 0.5cm2Left and right. The samples were fixed by soaking in 2.5% glutaraldehyde solution in PBS and stored in a refrigerator at 4 ℃.
(2) Pretreatment of eggshells
And (3) cleaning the fixed eggshell sample again by using a PBS solution, and sequentially soaking the fixed eggshell sample by using 30%, 50%, 70%, 80%, 90%, 95% and 100% ethanol, ethanol with different gradients for about 15min, soaking the eggshell sample by using 100% ethanol for 3 times, and replacing water in the eggshell cell tissue, wherein the process cannot be carried out overnight. Soaking the dehydrated eggshell sample in tert-butyl alcohol for 3 times, each time for 30min, replacing ethanol in eggshell cell tissue, adding about 0.2mL in the first two times, adding tert-butyl alcohol for the third time to ensure that the eggshell sample just submerges, facilitating the drying of the sample, placing the eggshell sample in a refrigerator at-20 ℃ for 10min, and then drying in a freeze dryer for 30 min.
(3) Ultrastructural observation of eggshells
Trimming the dried eggshells to about 0.2cm2The outer surface, the inner surface and the cross section of the eggshell are respectively stuck on a sample table, and the eggshell is placed in a scanning electron microscope cavity for observation and photographing after an ion sputtering instrument carries out gold spraying treatment.
The observation and analysis of the eggshell ultrastructure are as follows:
1) observation of ultrastructure on outer surface of eggshell
The observation result of the scanning electron microscope for the ultrastructure of the outer surface of the eggshell is shown in figure 1, the outer surface of the eggshell of the blank group is obviously chapped, and the crack of the crack is wider; the cracked area of the outer surface of the Liu-c1 eggshell is reduced, and the crack of the crack is narrower; the cracking area of the outer surface of the KL1 group eggshell is obviously reduced, cracks are reduced, and no crack exists; the cracks on the outer surface of the composite eggshell are obviously reduced, and no crack is generated. The three different microecologics are shown to improve the outer surface of the eggshell of the breeding hens at the later period of laying, wherein the composite group has the best effect, and the KL1 group and the Liu-c1 group are the second group.
2) Ultrastructural observation of inner surface of eggshell
The observation result of the scanning electron microscope for the ultrastructure of the inner surface of the eggshell is shown in fig. 2, the fiber structure of the inner surface of the eggshell in the blank group is disordered, the gaps among the fibers are large, the fibers are not uniformly distributed, and the diameters of the fibers are small; KL1 and Liu-c1 groups of eggshells are relatively uniform in inner surface layer distribution, and gaps among fibers are relatively small; the composite eggshell has thick inner surface fiber, compact and clear structure arrangement and uniform layer distribution. The results show that the three different microecologics can improve the inner surface structure of the eggshell of the breeding hens at the later period of laying, wherein the effect of the composite group is the best, and the KL1 group and the Liu-c1 group are the second group.
3) Observation of ultrastructure of cross section of eggshell
The observation result of the scanning electron microscope for the ultrastructure of the cross section of the eggshell is shown in figure 3, the fence layer structure of the cross section of the eggshell in the blank group is loose, the number of air holes is large, and the nipple gaps of the papilla layer are uneven and large; compared with the blank group, the KL1 group and the Liu-c1 group have compact structure of the cross-section fence layer of the eggshell, obviously reduce air holes, and have uniform mastoid layer and mastoid distribution and regular arrangement; the cross section of the composite eggshell has a dense fence layer structure, basically has no air holes, the papilla layer has small and wide papilla quantity, and the papilla gaps are dense. The three microecologics are shown to improve the sparse structure of the eggshell palisade layer of the breeding hens at the later period of laying, increase the mastoid width and reduce the mastoid gap, thereby improving the eggshell quality, wherein the composite group has the best effect, and the KL1 group and the Liu-c1 group are the second group.
Discussion and conclusion: the eggshell is a complex biological material, and the eggshell structure comprises 6 layers as observed by a scanning electron microscope: inner and outer shell membranes, papillary layers, palisade layers, calcite crystalline layers, and epidermal layers. The innermost 2 layers are inner and outer shell membranes in a non-calcified state, the thinner inner membrane wraps the content to form the innermost barrier of the egg, and the thicker outer shell membrane is formed by criss-cross protein fibers into a net shape and is arranged in multiple layers. The surface of the protein fiber layer is distributed with a plurality of granular protrusions and nodules, and the nodules play a role in connecting and fixing the protein fibers and maintain the structural stability of the protein fibers. The calcified irregular papillary layer, the palisade layer and the vertical crystalline layer together form a calcified layer of the eggshell. The outermost skin layer is composed of an organic layer deposited on the surface of the eggshell. The study on the relationship between the quality of the eggshell and the ultrastructure shows that the number of cracks on the outer surface of the eggshell and the strength of the eggshell are in positive correlation, the width of the cracks on the outer surface and the strength of the eggshell are in negative correlation, and the small and dense chaps on the outer surface can help to resist internal stress, thereby preventing the eggshell from cracking. In the experiment, after the microecologics are added into the daily ration of the laying hens for 8 weeks, the cracking degree of the outer surface of the eggshell of the KL1 group, the Liu-c1 group and the composite group is obviously improved, the crack width of cracks is reduced, the fiber gap of the inner surface is reduced, and the fiber diameter is increased; in addition, a plurality of small nodules are visible to be attached to the fiber mesh structure, the nodules in the composite group are distributed most, the diameters of the nodules are also largest, the fence layer structure on the cross section is compact, and air holes are obviously reduced. The results show that the single or compound addition of the lactobacillus paracasei KL1 and the bacillus subtilis Liu-c1 microecological preparation can improve the eggshell ultrastructure of the Jinghong No.1 hen, and the compound effect of the two is the best.
EXAMPLE 4 detection of serum calcium, tibial calcium, eggshell calcium and uterine calcium levels in hens
(1) Serum calcium
After the test is finished, selecting 6 breeding hens (1 hen is randomly extracted from each of 6 repetitions of each group) per group for conducting infrawing venous blood sampling, adopting a sterile injector to conduct infrawing venous blood sampling on the breeding hens which are fasting for 12 hours, wherein the infrawing venous blood sampling is about 5mL, and carrying out blood coagulation for 2 hours at room temperature to obtain a blood sample; centrifuging the blood sample at 4 deg.C and 10000rpm for 10min, respectively taking supernatant in centrifuge tube, and storing in refrigerator at 4 deg.C for inspection. The calcium content in serum was determined using a fully automatic biochemical analyzer.
(2) Tibia calcium
Dissecting 6 hens selected from each group, taking shin bones, removing residual muscles and soft tissues, soaking in absolute ethyl alcohol for 48h for dehydration, taking out, degreasing with petroleum ether by a Soxhlet extraction method, drying in an oven at 105 ℃ for 6h, cooling in a dryer, and measuring the weight of the shin bones. Crushing the dried shinbone, putting the crushed shinbone into a crucible, putting the crucible into a muffle furnace for carbonization at 550 ℃, then burning the shinbone for 24h, cooling the shinbone by using a dryer, and weighing the ash weight of the shinbone, wherein the detection method of calcium in the ash is determined according to the determination method of calcium in food of national standard GB 5009.90-2016.
(3) Egg shell calcium
At the end of 8 weeks of the test, 6 fresh hatching eggs are selected from each group (1 egg is randomly extracted from each of 6 repeats of each group), eggshells of the 6 hatching eggs in each group are respectively collected, mucus and dirt on the eggshells are washed clean by running water, the eggshells are dried for 24 hours at 70 ℃ and then crushed, and the eggshells are entrusted to a feed titer and safety supervision and inspection test center (Beijing) in rural agriculture to be detected according to a method for measuring calcium in food of national standard GB 5009.90-2016.
(4) Uterus calcium
Cutting endometrium after dissection, accurately weighing 2g endometrium, cutting into pieces, grinding, uniformly mixing with 18mL physiological saline at 4 ℃, beating in a sterile homogenizing bag for 15-20min to obtain uterus homogenate, centrifuging the homogenate at 4 ℃ and 10000rpm for 10min, taking supernatant into a centrifuge tube, storing at 4 ℃, and entrusting to Beijing and the Lanbo clinical laboratory Co., Ltd to determine according to the operation of a kit which is provided by Nanjing institute of bioengineering.
(5) Data processing
Experimental data One-way analysis of variance (One-way ANOVA), Duncan's multiple comparison test, was performed using IBM SPSS Statistics 22.0 statistical software. Experimental data are presented as mean ± standard deviation, with P <0.05 indicating significant differences.
The detection results and analysis of the contents of serum calcium, tibial calcium, eggshell calcium and uterine calcium are as follows:
the results for serum calcium, tibial calcium, eggshell calcium, and uterine calcium levels are shown in table 17. As can be seen from Table 17, the serum calcium content of the KL1 group, the Liu-c1 group and the compound group was significantly increased by 12.57%, 11.62% and 26.10%, respectively (P < 0.01), compared with the blank group; the calcium content of the tibia is respectively and remarkably increased by 6.99 percent, 5.06 percent and 9.64 percent (P is less than 0.01); the content of eggshell calcium is respectively and remarkably increased by 2.57 percent, 2.34 percent and 2.91 percent (P is less than 0.01); the uterus calcium content is respectively and obviously increased by 43.33 percent (P is less than 0.01), 19.63 percent (P is more than 0.05) and 60.37 percent (P is less than 0.01). The three microecological preparations can increase the contents of eggshell calcium, serum calcium, tibia calcium and uterus calcium of the egg-laying hens at the later period of egg laying to different degrees, wherein the compound group has the best effect, and the KL1 group and the Liu-c1 group are the second group.
TABLE 17 Effect of the Microecological Agents on the serum, shin, egg shell and uterine calcium content of the hens grown at the later stage of laying
Note: the shoulder marks on the same row have no letters or the same letters represent no significant difference (P is more than 0.05), different lower case letters represent significant difference (P is less than 0.05), and different upper case letters represent significant difference (P is less than 0.01).
Discussion and conclusion: the time for forming eggshells in egg-laying hens is mainly at night when the egg-laying hens stop feeding, during which the calcification process of eggshells in the uterine parts enters a linear sedimentation stage, and about 50% of calcium in marrow bones of the egg-laying hens is transported to the uterine parts to participate in the formation of eggshells. Research shows that the microecological preparation can metabolize a large amount of organic acids (lactic acid, acetic acid and propionic acid) in the laying hens, dissociate calcium element into an ionic state and promote the deposition of eggshell calcium. The main component of egg shell is CaCO3Therefore, the deposition amount of the eggshell calcium directly determines the superfine structure and the compactness of the eggshell,the calcium content is increased, and the eggshell strength is increased. The test result shows that the uterine calcium and eggshell contents of the KL1 group, the Liu-c1 group and the composite group are all obviously increased, the effect of the composite microecological preparation is superior to that of a single microecological preparation, and the reason is that the eggshell strength of the hatching eggs is improved and the eggshell ultrastructure is improved after the microecological preparation is added.
Example 5 detection of expression level of Gene involved in uterine segment and Eggshell formation
(1) Sample collection
At the end of the experiment, 6 hens (1 in each of 6 replicates in each group) were selected for dissection, endometrium was carefully cut into a centrifuge tube, frozen in liquid nitrogen and stored at-80 ℃, genes related to eggshell Calcium transport, Calcium-binding protein-d28k, capp-d 28k, Plasma membrane Calcium ATPase 1(Plasma membrane Calcium ATPase1, PMCA1) and Carbonic Anhydrase (CA), as well as genes related to eggshell calcification, eggshell specific matrix protein-17 (Ovocleidin17, OC17), Ovotransferrin, OVO) and Ovalbumin (Ovalbumin, OVA), were examined.
(2) Primer design
Sequences of the Cabp-d28k, PMCA1 target gene primer and the internal reference gene primer are shown in Table 18, and the internal reference gene is chicken beta-actin gene.
Primer sequences for genes of interest and reference genes of Table 18
(3) Extraction of total RNA from samples
Total RNA was extracted from the tissue sample using Total RNA Extraction Kit (DNase I) (Cat # GPQ1801, GenePool, China). The experimental procedures were carried out strictly according to the procedures of the product instructions.
(4) RNA electrophoresis
mu.L of the total RNA extracted above was subjected to electrophoresis on a 1% agarose gel to examine the integrity of the RNA.
(5) Reverse transcription
The reverse transcription is carried out by adopting an mRNA cDNA Synthesis Kit (Cat # GPQ1803, GenePool company, China), the experimental operation is strictly carried out according to the product instruction, and the steps are as follows: dissolving all components in the kit and placing the components on ice for later use; the components were added to the reaction tube in a total volume of 20. mu.L according to the reverse transcription system of Table 19, and mixed well. Incubation was carried out at 42 ℃ for 50min and at 85 ℃ for 5 min. Centrifuging for a short time after the reaction is finished, and collecting the solution on the tube wall to the tube bottom; the cDNA obtained by reverse transcription was stored at-20 ℃.
TABLE 19 reverse transcription System
(6)Real Time PCR
The Real Time PCR amplification is carried out by using a BIOER Line Gene 9600Plus type fluorescent quantitative PCR instrument, and the operation process is as follows:
1) reaction system: the mRNA qPCR Kit (Cat # GPQ1808, GenePool, China) was used for amplification, and the experimental procedures were performed according to the product instructions. The amplification procedure was: 30s at 95 ℃ (5 s at 95 ℃, 30s at 60 ℃) multiplied by 45 cycles, while melting curve analysis is performed at 60-95 ℃.
The Real Time PCR reaction system is as follows:
sterile distilled water was added to 20. mu.L.
2) Primer screening
After compounding cDNA of each sample, carrying out 5-fold gradient dilution by taking the cDNA as a template, taking 2 mu L of each diluted sample as the template, carrying out amplification by using a target gene primer (see table 20), simultaneously carrying out melting curve analysis at 60-95 ℃, and carrying out primer screening according to the principle of high amplification efficiency and single peak of the melting curve.
TABLE 20 primer screening Standard Curve Real Time PCR design
3) Sample Real Time PCR detection
After 10-fold dilution of each sample cDNA, 2. mu.L of each sample cDNA was used as a template and amplified with the target gene primer and the reference gene primer, respectively (see Table 21). At the same time, the dissolution curve analysis is carried out at 60-95 ℃.
Table 21 sample Real Time PCR detection design
| Form panel | Sample cDNA | Sample cDNA |
| Repeatedly detecting the number of channels | 3 | 3 |
| Primer and method for producing the same | Target gene primer | Internal reference gene primer |
(7) Data processing
By use of 2-△△CTThe relative quantitative method calculates the relative expression amount of the sample, and calculates 2 of each group-△△CTValues, set to 1 for 1 sample in the blank group, the remainderThe 23 samples were compared and a relative quantitative calculation was performed. The formula is as follows: 2-△△CT＝2- [ (destination sample CT-internal reference CT) - (control sample CT-internal reference CT)]. Experimental data One-way analysis of variance (One-way ANOVA), Duncan's multiple comparison test, was performed using IBM SPSS Statistics 22.0 statistical software. The data are expressed as mean. + -. standard deviation, P<0.05 means significant difference, P<0.01 indicates that the difference is extremely significant.
The results and analysis of the relative expression of the genes related to the formation of egg shells in uterus are as follows:
the results of changes in gene expression levels in the uterine regions associated with calcium transport and calcification are shown in Table 22. Compared with the blank group, the expression levels of the KL1 group and the calcium transport binding protein (Cabp-d28k) of the compound group are respectively and remarkably increased by 58.42 percent and 130.97 percent (P is less than 0.01), and the expression level of the Liu-c1 group Cabp-d28k is remarkably increased by 41.20 percent (P is less than 0.05); the expression levels of KL1, Liu-c1 and composite plasma membrane calcium ATPase 1(PMCA1) are increased by 23.81%, 22.62% and 50.00% (P > 0.05), and the expression levels of Carbonic Anhydrase (CA) are increased by 250.91%, 123.03% and 270.30% (P < 0.05) respectively; the expression levels of the eggshell-specific matrix protein-17 (OC17) of the KL1 group and the Liu-c1 group are respectively increased by 23.71 percent and 20.62 percent (P is more than 0.05), the expression levels of the ovotransferrin (OVO) are respectively increased by 20.59 percent and 17.65 percent (P is more than 0.05), and the expression levels of the eggshell-specific matrix protein-17 (OC17) and OVO of the compound group are respectively and remarkably increased by 89.69 percent and 64.71 percent (P is less than 0.01); the expression level of Ovalbumin (OVA) of each group has no significant difference. The results show that the three microecological preparations can increase the calcium transport and the expression level of calcification genes of the breeding hens at the later period of laying eggs to different degrees, wherein the effect of a composite group is the best, and the KL1 group and the Liu-c1 group are the second group.
TABLE 22 influence of probiotics on the amount of gene expression in uterus related to egg shell formation
Note: the shoulder marks on the same row have no letters or the same letters represent no significant difference (P is more than 0.05), different lower case letters represent significant difference (P is less than 0.05), and different upper case letters represent significant difference (P is less than 0.01).
Discussion and conclusion: the eggshell is formed by combining various inorganic substances and organic matrix proteins secreted by the uterine part of the oviduct, the essence of the eggshell is the process that calcium carbonate is orderly deposited on an eggshell membrane under the regulation and control action of various matrix proteins, related genes in a calcium transfer channel and the change rule of various organic matrix proteins in the uterus at the early stage of the calcification of the eggshell play a vital role in the initiation of the calcification, and the final formation of the eggshell and the quality of the eggshell are influenced. The process is that calcium transport binding protein (Cabp-d28k) and plasma membrane calcium ion ATPase 1(PMCA1) convert Ca2+Transport from extracellular to egg hen uterus fluid, catalysis of CO by Carbonic Anhydrase (CA)2Production of HCO by hydration3 -：H2O+CO2→H+HCO3 -Then HCO3 -Conversion to CO3 2-：HCO3 -→CO3 2-+ H, then Ca without enzyme catalysis2+And CO3 2-Reaction to CaCO in egg shell3。
In the early stage of eggshell calcification, the matrix proteins involved in the initiation of eggshell calcification are mainly eggshell-specific matrix protein-17 (OC17), ovotransferrin (OVO) and Ovalbumin (OVA). OC17 is a specific matrix protein in eggshells, present in glyco (based) form in the papillary, palisade and cuticle layers of eggshells, plays a fundamental role in the eggshell formation process and affects the structural and physical properties of eggshells. OVO is an eggshell matrix protein, which participates in the calcification of eggshells at the early stage of the calcification of eggshells, and can change the size of calcite crystals during the formation of eggshells, thereby affecting the compactness of eggshell structures. OVA, the first major protein found in eggshells, is present only in the papillary layer of eggshells and is particularly abundant during the initial stages of calcification of eggshells, a finding of which suggests that ovalbumin is involved in the formation of the centrum nucleus of the papillary layer of eggshells.
The test result shows that the calcium transport amount and the eggshell calcification gene expression amount of the Jinghong No.1 parent hen can be increased to different degrees when the two microecologics are used independently and compositely, and the composite use effect is better. In combination with the superfine structure of the eggshell, the calcium transport and the expression of calcification genes are improved, the corresponding shell membrane surface fiber is thicker, the structural arrangement is compact and clear, the hierarchical distribution is uniform, and the external surface chap area is reduced; the eggshell fiber inner membrane layer and the mastoid layer are more compact in structure and more orderly arranged, so that the eggshell thickness and the eggshell strength are improved.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
SEQUENCE LISTING
<110> Beijing college of agriculture
<120> bacillus subtilis, microecological preparation, feed and application thereof in improving production performance and eggshell of breeding hens
Application in quality
<160> 17
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> DNA
<213> Artificial sequence
<400> 1
agagtttgat cmtggctcag 20
<210> 2
<211> 19
<212> DNA
<213> Artificial sequence
<400> 2
ggttaccttg ttacgactt 19
<210> 3
<211> 2222
<212> DNA
<213> Bacillus subtilis
<400> 3
gggtgtggcg gctgctatac atgcaagtcg agcggacaga tgggagcttg ctccctgatg 60
ttagcggcgg acgggtgagt aacacgtggg taacctgcct gtaagactgg gataactccg 120
ggaaaccggg gctaataccg gatggttgtt tgaaccgcat ggttcaaaca taaaaggtgg 180
cttcggctac cacttacaga tggacccgcg gcgcattagc tagttggtga ggtaacggct 240
caccaaggca acgatgcgta gccgacctga gagggtgatc ggccacactg ggactgagac 300
acggcccaga ctcctacggg aggcagcagt agggaatctt ccgcaatgga cgaaagtctg 360
acggagcaac gccgcgtgag tgatgaaggt tttcggatcg taaagctctg ttgttaggga 420
agaacaagta ccgttcgaat agggcggtac cttgacggta cctaaccaga aagccacggc 480
taactacgtg ccagcagccg cggtaatacg taggtggcaa gcgttgtccg gaattattgg 540
gcgtaaaggg ctcgcaggcg gtttcttaag tctgatgtga aagcccccgg ctcaaccggg 600
gagggtcatt ggaaactggg gaacttgagt gcagaagagg agagtggaat tccacgtgta 660
gcggtgaaat gcgtagagat gtggaggaac accagtggcg aaggcgactc tctggtctgt 720
aactgacgct gaggagcgaa agcgtgggga gcgaacagga ttagataccc tggtagtcca 780
cgccgtaaac gatgagtgct aagtgttagg gggtttccgc cccttagtgc tgcagctaac 840
gcattaagca ctccgcctgg ggagtacggt cgcaagactg aaactcaaag gaattgacgg 900
gggcccgcac aagcggtgga gcatgtggtt taattcgaag caacgcgaag aaccttacca 960
ggtcttgaca tcctctgaca atcctagaga taggacgtcc ccttcggggg cagagtgaca 1020
ggtggtgcat ggttgtcgtc agctcctgtc ctgaaagttg ggttagtccc caacgagcga 1080
acccttgatt ttattgccgc attcagttgg gcacctaagg tactgccggt gaaacttctg 1140
taaccttcgg cggctggctc ctaaaaggtt acctcaccga cttcgggtgt tacaaactct 1200
cgtggtgtga cgggcggtgt gtacaaggcc cgggaacgta ttcaccgcgg catgctgatc 1260
cgcgattact agcgattcca gcttcacgca gtcgagttgc agactgcgat ccgaactgag 1320
aacagatttg tgggattggc ttaacctcgc ggtttcgctg ccctttgttc tgtccattgt 1380
agcacgtgtg tagcccaggt cataaggggc atgatgattt gacgtcatcc ccaccttcct 1440
ccggtttgtc accggcagtc accttagagt gcccaactga atgctggcaa ctaagatcaa 1500
gggttgcgct cgttgcggga cttaacccaa catctcacga cacgagctga cgacaaccat 1560
gcaccacctg tcactctgcc cccgaagggg acgtcctatc tctaggattg tcagaggatg 1620
tcaagacctg gtaaggttct tcgcgttgct tcgaattaaa ccacatgctc caccgcttgt 1680
gcgggccccc gtcaattcct ttgagtttca gtcttgcgac cgtactcccc aggcggagtg 1740
cttaatgcgt tagctgcagc actaaggggc ggaaaccccc taacacttag cactcatcgt 1800
ttacggcgtg gactaccagg gtatctaatc ctgttcgctc cccacgcttt cgctcctcag 1860
cgtcagttac agaccagaga gtcgccttcg ccactggtgt tcctccacat ctctacgcat 1920
ttcaccgcta cacgtggaat tccactctcc tcttctgcac tcaagttccc cagtttccaa 1980
tgaccctccc cggttgagcc gggggctttc acatcagact taagaaaccg cctgcgagcc 2040
ctttacgccc aataattccg gacaacgctt gccacctacg tattaccgcg gctgctggca 2100
cgtagttagc cgtggctttc tggttaggta ccgtcaaggt accgccctat tcgaacggta 2160
cttgttcttc ctaacacaga gctttacgat cgaaaacctt catcctcagc ggcgttgctc 2220
gt 2222
<210> 4
<211> 25
<212> DNA
<213> Artificial sequence
<400> 4
agacattaac aaccttgcga catac 25
<210> 5
<211> 22
<212> DNA
<213> Artificial sequence
<400> 5
gcacagagaa tgagagccag tt 22
<210> 6
<211> 24
<212> DNA
<213> Artificial sequence
<400> 6
gagccgtatt gaacaagaac agaa 24
<210> 7
<211> 22
<212> DNA
<213> Artificial sequence
<400> 7
ctaccatcct tccagagcct tc 22
<210> 8
<211> 19
<212> DNA
<213> Artificial sequence
<400> 8
ccatcgccat cagcaccaa 19
<210> 9
<211> 22
<212> DNA
<213> Artificial sequence
<400> 9
caccaacctg tagactccat cc 22
<210> 10
<211> 19
<212> DNA
<213> Artificial sequence
<400> 10
ttcgcttcgt ggcaccgaa 19
<210> 11
<211> 19
<212> DNA
<213> Artificial sequence
<400> 11
cgaaggcatt gcgctctgt 19
<210> 12
<211> 19
<212> DNA
<213> Artificial sequence
<400> 12
gcatgtcagc agcgttcac 19
<210> 13
<211> 24
<212> DNA
<213> Artificial sequence
<400> 13
tctttgtcgt catctttgga gttc 24
<210> 14
<211> 20
<212> DNA
<213> Artificial sequence
<400> 14
tcgttcagcc ttgccagtag 20
<210> 15
<211> 24
<212> DNA
<213> Artificial sequence
<400> 15
cctcctctat acagttcctt caca 24
<210> 16
<211> 21
<212> DNA
<213> Artificial sequence
<400> 16
gagaaattgt gcgtgacatc a 21
<210> 17
<211> 19
<212> DNA
<213> Artificial sequence
<400> 17
cctgaacctc tcattgcca 19
Claims (10)
1. The bacillus subtilis Liu-c1 is characterized in that the bacillus subtilis Liu-c1 is preserved in the China general microbiological culture Collection center of the China Committee for culture Collection of microorganisms with the preservation number as follows: CGMCC No. 20840.
2. The method for fermenting Bacillus subtilis Liu-c1 of claim 1, comprising the steps of: the Bacillus subtilis Liu-c1 is inoculated into a culture medium for culture.
3. The method for fermenting bacillus subtilis Liu-c1 according to claim 2, wherein the fermentation conditions of the bacillus subtilis Liu-c1 at least satisfy one of the following conditions: the fermentation temperature is 37-45 ℃, the fermentation time is 12-16h, and the pH value is 6.5-7.5;
preferably, the fermentation conditions of the bacillus subtilis Liu-c1 at least meet one of the following conditions: the fermentation temperature is 45 ℃, the fermentation time is 16h, and the pH value is 6.5.
4. The method for fermenting bacillus subtilis Liu-c1 of claim 2, wherein the media components of the bacillus subtilis Liu-c1 comprise: 5.0-20.0g/L of tryptone, 2.5-20g/L of yeast extract powder, 1-10g/L of glucose and 0-5.0g/L of sodium chloride;
preferably, the culture medium components of the bacillus subtilis Liu-c1 comprise: 10.0g/L of tryptone, 10g/L of yeast extract powder, 10g/L of glucose and 2.5g/L of sodium chloride.
5. A microecological preparation comprising the Bacillus subtilis Liu-c1 of claim 1.
6. The microecological formulation according to claim 5, further comprising Lactobacillus paracasei KL 1;
the lactobacillus paracasei KL1 is preserved in the China general microbiological culture Collection center of the culture Collection of microorganisms with the preservation number as follows: CGMCC No. 11533;
preferably, the bacterial quantity ratio of the bacillus subtilis Liu-c1 to the lactobacillus paracasei KL1 in the microecological preparation is (1-2) to (1-2), and preferably 1: 1.
7. The probiotic preparation according to claim 5 or 6, characterized in that it is used in a dose of 107～108CFU/day, preferably 6.0X 107～8.0×107CFU/day.
8. The method of using the probiotic of any of claims 5 to 7, characterized in that it comprises the following steps: diluting the microecological preparation with maltodextrin, mixing, dissolving in water, and spraying onto the surface of feed.
9. A feed comprising the Bacillus subtilis Liu-c1 of claim 1 and/or the probiotic of claim 5 or 7.
10. Use of the bacillus subtilis Liu-c1 of claim 1, the probiotic of claim 5 or 7 or the feed of claim 9 for improving the production performance and eggshell quality of breeding hens at the later stage of laying.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011476999.9A CN112625946B (en) | 2020-12-15 | 2020-12-15 | Bacillus subtilis, microecological preparation, feed and application of bacillus subtilis to improvement of production performance and eggshell quality of hens |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011476999.9A CN112625946B (en) | 2020-12-15 | 2020-12-15 | Bacillus subtilis, microecological preparation, feed and application of bacillus subtilis to improvement of production performance and eggshell quality of hens |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112625946A true CN112625946A (en) | 2021-04-09 |
| CN112625946B CN112625946B (en) | 2022-07-19 |
Family
ID=75313072
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011476999.9A Active CN112625946B (en) | 2020-12-15 | 2020-12-15 | Bacillus subtilis, microecological preparation, feed and application of bacillus subtilis to improvement of production performance and eggshell quality of hens |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112625946B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114507619A (en) * | 2022-01-28 | 2022-05-17 | 河北一然生物科技股份有限公司 | Liquid composite probiotic preparation, preparation method thereof and application thereof in aspects of improving diarrhea and protecting liver |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106635886A (en) * | 2016-11-18 | 2017-05-10 | 北京农学院 | Method for reducing egg cholesterol by utilizing lactobacillus paracasei of bile salt producing hydrolase |
| CN111642617A (en) * | 2020-06-19 | 2020-09-11 | 福建省大丰山禽业发展有限公司 | Special microecological preparation for laying hens and preparation method thereof |
-
2020
- 2020-12-15 CN CN202011476999.9A patent/CN112625946B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106635886A (en) * | 2016-11-18 | 2017-05-10 | 北京农学院 | Method for reducing egg cholesterol by utilizing lactobacillus paracasei of bile salt producing hydrolase |
| CN111642617A (en) * | 2020-06-19 | 2020-09-11 | 福建省大丰山禽业发展有限公司 | Special microecological preparation for laying hens and preparation method thereof |
Non-Patent Citations (5)
| Title |
|---|
| TAREKEGN: ""A Review on Effects of Probiotic Supplementation in Poultry Performance and Cholesterol Levels of Egg and Meat"", 《JOURNAL OF WORLD"S POULTRY RESEARCH》 * |
| 崔闯飞: ""枯草芽孢杆菌对产蛋后期蛋鸡生产性能及蛋壳品质的影响"", 《中国优秀硕士学位论文全文数据库（电子期刊）农业科技辑》 * |
| 潘宝海 等: ""芽孢杆菌对蛋鸡生产性能及蛋壳品质的影响"", 《饲料研究》 * |
| 聂彦芬 等: ""藏灵菇源副干酪乳杆菌KL1对蛋鸡脂质过氧化的研究"", 《食品科学》 * |
| 陈猜猜 等: ""微生态制剂对产蛋后期种母鸡生产性能、肠道菌群和消化吸收的影响"", 《动物营养学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114507619A (en) * | 2022-01-28 | 2022-05-17 | 河北一然生物科技股份有限公司 | Liquid composite probiotic preparation, preparation method thereof and application thereof in aspects of improving diarrhea and protecting liver |
| CN114507619B (en) * | 2022-01-28 | 2024-01-12 | 河北一然生物科技股份有限公司 | Liquid composite probiotic preparation, preparation method thereof and application thereof in aspects of improving diarrhea and protecting liver |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112625946B (en) | 2022-07-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110144304B (en) | Lactobacillus casei strain and application thereof | |
| CN110343642B (en) | Lactobacillus fermentum and preparation method of freeze-dried powder thereof | |
| CN109161509B (en) | Bacterial strain capable of preventing and treating bovine and sheep diarrhea | |
| CN114634901B (en) | Lactobacillus casei LC16 for promoting bone health and culture method and application thereof | |
| CN114231473B (en) | Probiotic lactobacillus plantarum and application thereof in preparation of low-salt fermented meat food | |
| CN110564638A (en) | Lactobacillus reuteri with probiotic characteristics and application thereof | |
| CN116083325B (en) | Lactobacillus rhamnosus for improving helicobacter pylori related gastrointestinal diseases and application thereof | |
| WO2017012571A1 (en) | Use of bacillus coagulans strain in increasing egg production in laying hen | |
| CN112574924A (en) | Bacillus subtilis strain, microecological preparation and application thereof | |
| CN112625946B (en) | Bacillus subtilis, microecological preparation, feed and application of bacillus subtilis to improvement of production performance and eggshell quality of hens | |
| CN109486732B (en) | Bifidobacterium longum and application thereof | |
| CN108373983B (en) | Pediococcus pentosaceus CCFM1012, fermented food thereof and application of pediococcus pentosaceus CCFM1012 in preparation of medicines for antagonizing campylobacter jejuni infection | |
| CN109645490A (en) | Application of the lactobacillus plantarum CQPC02 in the food or drug of preparation prevention diabetes | |
| CN109576165B (en) | Saccharomyces bayanus and application thereof | |
| CN116970512A (en) | Lactobacillus plantarum, and culture method and application thereof | |
| WO2019174002A1 (en) | Pediococcus pentosaceus ccfm1012, food fermented thereby and use thereof in preparation of medicine for antagonizing campylobacter jejuni infection | |
| CN113512559B (en) | Mycoplasma bovis Mbov _0701 mutant gene and mutant strain and application thereof | |
| CN113215057B (en) | Rana-sourced lactococcus lactis subspecies lactis LCLL1 and application thereof | |
| CN113717887B (en) | Goose-source lactobacillus plantarum and application thereof | |
| JP6261664B2 (en) | Lyophilized product containing Lactobacillus plantarum LLP5193, which is excellent in acid resistance, bile resistance and cell adhesion ability, as an active ingredient | |
| CN108102976B (en) | Lactobacillus reuteri SS23-27 and application thereof in preparation of pure probiotic yogurt | |
| CN114657093B (en) | Pigeon-derived lactobacillus crispatus BL4014 and application thereof | |
| CN109609409A (en) | Lactobacillus fermenti CQPC06 and its application in the food or drug that preparation improves ulcerative colitis | |
| CN116144525B (en) | Seawater fish probiotics lactococcus lactis strain and application thereof | |
| CN113430153B (en) | Lactobacillus reuteri ZJuuds 09 for reducing blood pressure and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |