CN110934224A - Yeast hydrolysate with high free nucleotide content and its prepn and application - Google Patents
Yeast hydrolysate with high free nucleotide content and its prepn and application Download PDFInfo
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- CN110934224A CN110934224A CN201811118416.8A CN201811118416A CN110934224A CN 110934224 A CN110934224 A CN 110934224A CN 201811118416 A CN201811118416 A CN 201811118416A CN 110934224 A CN110934224 A CN 110934224A
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- 240000004808 Saccharomyces cerevisiae Species 0.000 title claims abstract description 373
- 239000002773 nucleotide Substances 0.000 title claims abstract description 184
- 125000003729 nucleotide group Chemical group 0.000 title claims abstract description 182
- 239000000413 hydrolysate Substances 0.000 title claims abstract description 84
- 239000008267 milk Substances 0.000 claims abstract description 170
- 210000004080 milk Anatomy 0.000 claims abstract description 170
- 235000013336 milk Nutrition 0.000 claims abstract description 170
- 238000000855 fermentation Methods 0.000 claims abstract description 36
- 230000004151 fermentation Effects 0.000 claims abstract description 36
- 238000002360 preparation method Methods 0.000 claims abstract description 27
- 230000020477 pH reduction Effects 0.000 claims abstract description 22
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 16
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 16
- 108010009736 Protein Hydrolysates Proteins 0.000 claims abstract description 10
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 8
- 150000001413 amino acids Chemical class 0.000 claims abstract description 7
- 230000002787 reinforcement Effects 0.000 claims abstract description 6
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 claims description 370
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 96
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 54
- 229910052757 nitrogen Inorganic materials 0.000 claims description 48
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 claims description 42
- 229910052698 phosphorus Inorganic materials 0.000 claims description 42
- 239000011574 phosphorus Substances 0.000 claims description 42
- 229910052799 carbon Inorganic materials 0.000 claims description 33
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 32
- 101710163270 Nuclease Proteins 0.000 claims description 28
- 108090000526 Papain Proteins 0.000 claims description 25
- 239000004365 Protease Substances 0.000 claims description 25
- 229940055729 papain Drugs 0.000 claims description 25
- 235000019834 papain Nutrition 0.000 claims description 25
- 208000035404 Autolysis Diseases 0.000 claims description 24
- 206010057248 Cell death Diseases 0.000 claims description 24
- RQFCJASXJCIDSX-UUOKFMHZSA-N guanosine 5'-monophosphate Chemical compound C1=2NC(N)=NC(=O)C=2N=CN1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H]1O RQFCJASXJCIDSX-UUOKFMHZSA-N 0.000 claims description 24
- 235000013928 guanylic acid Nutrition 0.000 claims description 24
- 238000000034 method Methods 0.000 claims description 24
- 230000028043 self proteolysis Effects 0.000 claims description 24
- GRSZFWQUAKGDAV-UHFFFAOYSA-N Inosinic acid Natural products OC1C(O)C(COP(O)(O)=O)OC1N1C(NC=NC2=O)=C2N=C1 GRSZFWQUAKGDAV-UHFFFAOYSA-N 0.000 claims description 23
- 241001465754 Metazoa Species 0.000 claims description 23
- 239000004226 guanylic acid Substances 0.000 claims description 23
- 235000013902 inosinic acid Nutrition 0.000 claims description 23
- DJJCXFVJDGTHFX-XVFCMESISA-N uridine 5'-monophosphate Chemical compound O[C@@H]1[C@H](O)[C@@H](COP(O)(O)=O)O[C@H]1N1C(=O)NC(=O)C=C1 DJJCXFVJDGTHFX-XVFCMESISA-N 0.000 claims description 23
- AUHDWARTFSKSAC-HEIFUQTGSA-N (2S,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)-2-(6-oxo-1H-purin-9-yl)oxolane-2-carboxylic acid Chemical compound [C@]1([C@H](O)[C@H](O)[C@@H](CO)O1)(N1C=NC=2C(O)=NC=NC12)C(=O)O AUHDWARTFSKSAC-HEIFUQTGSA-N 0.000 claims description 22
- UDMBCSSLTHHNCD-UHFFFAOYSA-N Coenzym Q(11) Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(O)=O)C(O)C1O UDMBCSSLTHHNCD-UHFFFAOYSA-N 0.000 claims description 22
- 229930183912 Cytidylic acid Natural products 0.000 claims description 22
- DJJCXFVJDGTHFX-UHFFFAOYSA-N Uridinemonophosphate Natural products OC1C(O)C(COP(O)(O)=O)OC1N1C(=O)NC(=O)C=C1 DJJCXFVJDGTHFX-UHFFFAOYSA-N 0.000 claims description 22
- FOGRQMPFHUHIGU-UHFFFAOYSA-N Uridylic acid Natural products OC1C(OP(O)(O)=O)C(CO)OC1N1C(=O)NC(=O)C=C1 FOGRQMPFHUHIGU-UHFFFAOYSA-N 0.000 claims description 22
- UDMBCSSLTHHNCD-KQYNXXCUSA-N adenosine 5'-monophosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)[C@H]1O UDMBCSSLTHHNCD-KQYNXXCUSA-N 0.000 claims description 22
- 229950006790 adenosine phosphate Drugs 0.000 claims description 22
- IERHLVCPSMICTF-XVFCMESISA-N cytidine 5'-monophosphate Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(O)=O)O1 IERHLVCPSMICTF-XVFCMESISA-N 0.000 claims description 22
- IERHLVCPSMICTF-UHFFFAOYSA-N cytidine monophosphate Natural products O=C1N=C(N)C=CN1C1C(O)C(O)C(COP(O)(O)=O)O1 IERHLVCPSMICTF-UHFFFAOYSA-N 0.000 claims description 22
- 239000004245 inosinic acid Substances 0.000 claims description 22
- 229940028843 inosinic acid Drugs 0.000 claims description 22
- 235000013379 molasses Nutrition 0.000 claims description 18
- 238000004321 preservation Methods 0.000 claims description 16
- LFVGISIMTYGQHF-UHFFFAOYSA-N ammonium dihydrogen phosphate Chemical compound [NH4+].OP(O)([O-])=O LFVGISIMTYGQHF-UHFFFAOYSA-N 0.000 claims description 15
- 229910000387 ammonium dihydrogen phosphate Inorganic materials 0.000 claims description 15
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 15
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 15
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 15
- 235000019837 monoammonium phosphate Nutrition 0.000 claims description 15
- 238000002635 electroconvulsive therapy Methods 0.000 claims description 13
- 238000012258 culturing Methods 0.000 claims description 11
- 238000004519 manufacturing process Methods 0.000 claims description 11
- -1 disodium nucleotide salt Chemical class 0.000 claims description 10
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 7
- PAWQVTBBRAZDMG-UHFFFAOYSA-N 2-(3-bromo-2-fluorophenyl)acetic acid Chemical compound OC(=O)CC1=CC=CC(Br)=C1F PAWQVTBBRAZDMG-UHFFFAOYSA-N 0.000 claims description 6
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 6
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 6
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 5
- 230000008569 process Effects 0.000 claims description 3
- 230000007071 enzymatic hydrolysis Effects 0.000 claims 2
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 claims 2
- 235000014590 basal diet Nutrition 0.000 claims 1
- 235000019624 protein content Nutrition 0.000 description 31
- 239000000243 solution Substances 0.000 description 27
- 238000001035 drying Methods 0.000 description 25
- 238000002156 mixing Methods 0.000 description 20
- 239000000047 product Substances 0.000 description 16
- 230000000052 comparative effect Effects 0.000 description 15
- 239000007788 liquid Substances 0.000 description 14
- QXNVGIXVLWOKEQ-UHFFFAOYSA-N Disodium Chemical class [Na][Na] QXNVGIXVLWOKEQ-UHFFFAOYSA-N 0.000 description 13
- 108090000790 Enzymes Proteins 0.000 description 13
- 102000004190 Enzymes Human genes 0.000 description 13
- 230000002358 autolytic effect Effects 0.000 description 13
- 238000006243 chemical reaction Methods 0.000 description 13
- 229940088598 enzyme Drugs 0.000 description 13
- 238000010438 heat treatment Methods 0.000 description 13
- 150000003839 salts Chemical class 0.000 description 13
- 230000000415 inactivating effect Effects 0.000 description 12
- 230000035939 shock Effects 0.000 description 12
- 230000001954 sterilising effect Effects 0.000 description 12
- 238000001816 cooling Methods 0.000 description 10
- 150000007523 nucleic acids Chemical class 0.000 description 10
- 238000003756 stirring Methods 0.000 description 10
- 102000039446 nucleic acids Human genes 0.000 description 9
- 108020004707 nucleic acids Proteins 0.000 description 9
- 230000000694 effects Effects 0.000 description 6
- 230000000968 intestinal effect Effects 0.000 description 6
- 230000008901 benefit Effects 0.000 description 5
- 210000001035 gastrointestinal tract Anatomy 0.000 description 5
- 230000012010 growth Effects 0.000 description 5
- 239000002994 raw material Substances 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 4
- 230000037213 diet Effects 0.000 description 4
- 235000005911 diet Nutrition 0.000 description 4
- 235000019750 Crude protein Nutrition 0.000 description 3
- 241000186660 Lactobacillus Species 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 235000016709 nutrition Nutrition 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 239000013589 supplement Substances 0.000 description 3
- 210000005253 yeast cell Anatomy 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 241000186000 Bifidobacterium Species 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 2
- 239000004472 Lysine Substances 0.000 description 2
- 241000700159 Rattus Species 0.000 description 2
- 238000009395 breeding Methods 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
- 229940041514 candida albicans extract Drugs 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 150000004676 glycans Chemical class 0.000 description 2
- 210000003405 ileum Anatomy 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 210000004347 intestinal mucosa Anatomy 0.000 description 2
- 244000144972 livestock Species 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- 229920001282 polysaccharide Polymers 0.000 description 2
- 239000005017 polysaccharide Substances 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 230000008439 repair process Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 210000000813 small intestine Anatomy 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 239000012138 yeast extract Substances 0.000 description 2
- 239000004475 Arginine Substances 0.000 description 1
- 108090000145 Bacillolysin Proteins 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 235000019733 Fish meal Nutrition 0.000 description 1
- 108010058643 Fungal Proteins Proteins 0.000 description 1
- 102100024023 Histone PARylation factor 1 Human genes 0.000 description 1
- 101001047783 Homo sapiens Histone PARylation factor 1 Proteins 0.000 description 1
- 150000008539 L-glutamic acids Chemical class 0.000 description 1
- 102000035092 Neutral proteases Human genes 0.000 description 1
- 108091005507 Neutral proteases Proteins 0.000 description 1
- 101710149004 Nuclease P1 Proteins 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 102000016679 alpha-Glucosidases Human genes 0.000 description 1
- 108010028144 alpha-Glucosidases Proteins 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 238000003975 animal breeding Methods 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 1
- 239000004327 boric acid Substances 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 229910000366 copper(II) sulfate Inorganic materials 0.000 description 1
- JZCCFEFSEZPSOG-UHFFFAOYSA-L copper(II) sulfate pentahydrate Chemical compound O.O.O.O.O.[Cu+2].[O-]S([O-])(=O)=O JZCCFEFSEZPSOG-UHFFFAOYSA-L 0.000 description 1
- 235000021051 daily weight gain Nutrition 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 230000023011 digestive tract development Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 210000001198 duodenum Anatomy 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000010812 external standard method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000004467 fishmeal Substances 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000031891 intestinal absorption Effects 0.000 description 1
- 230000003871 intestinal function Effects 0.000 description 1
- 229940039696 lactobacillus Drugs 0.000 description 1
- 238000002386 leaching Methods 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000012982 microporous membrane Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000004877 mucosa Anatomy 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 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
- OTYBMLCTZGSZBG-UHFFFAOYSA-L potassium sulfate Chemical compound [K+].[K+].[O-]S([O-])(=O)=O OTYBMLCTZGSZBG-UHFFFAOYSA-L 0.000 description 1
- 229910052939 potassium sulfate Inorganic materials 0.000 description 1
- 235000011151 potassium sulphates Nutrition 0.000 description 1
- 229920002477 rna polymer Polymers 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000009423 ventilation Methods 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
Classifications
-
- 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
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/153—Nucleic acids; Hydrolysis products or derivatives thereof
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/163—Sugars; Polysaccharides
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/20—Inorganic substances, e.g. oligoelements
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/20—Inorganic substances, e.g. oligoelements
- A23K20/26—Compounds containing phosphorus
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Polymers & Plastics (AREA)
- Engineering & Computer Science (AREA)
- Animal Husbandry (AREA)
- Zoology (AREA)
- Food Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Inorganic Chemistry (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Physiology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to the field of biological fermentation and feed, in particular to a yeast hydrolysate with high free nucleotide, a preparation method and application thereof. The invention provides a high-free nucleotide yeast hydrolysate which comprises 60-75% of protein, more than 35% of free nucleotide, amino acid and small peptide by mass of dry matter of the yeast hydrolysate, and is prepared by yeast milk enzymolysis, nucleotide acidification and nucleotide reinforcement.
Description
Technical Field
The invention relates to the field of biological fermentation and feed, in particular to a yeast hydrolysate with high free nucleotide, a preparation method and application thereof.
Background
The nucleotide has important significance for maintaining, improving and repairing gastrointestinal tract functions of animals, and the supplement of exogenous nucleotide can accelerate the differentiation, growth and repair of intestinal cells, promote the maturation of small intestines and promote the growth of bifidobacteria and lactobacilli in intestinal tracts. Hoffmann (Hoffmann. the use of nucleotides in animal feed. feed Mix,2008(15):14-16.) studies show that the ratio between E.coli and Lactobacillus flora changes in favor of beneficial microorganisms after addition of nucleotides to the pig ration. Sauer et al (Sauer, Bauer, Vahjen et al, Nucleotides modif grown of selected endogenous bacteria in vitro Livestock Science,2010,133(1): 161-. Di Giancamilo et al (Di Giancamilo, Domeneghini, Paratte, et al. Oral feeding with L-glutamic and nucleic acids: impact on some GALT (gut associated with lysine) parameters and cell promotion/death rates in genetic peptides of Animal Journal of Animal Science 2011,2(1S):364 once 366) mention that the addition of Nucleotides to the diet has a positive effect on ileal morphology, increasing the villus height and decreasing crypt depth in the ileum of piglets. Studies of Zyongqing and the like (Zyongqing, Wanglong, Chen Anguo. exogenous nucleotide has influence on the production performance and small intestine development of weaned piglets. China livestock raising journal, 2007,43 (21): 19-21.) find that the 21d test shows that the feed utilization rate of the weaned piglets is obviously improved compared with the control group by feeding the weaned piglets with daily ration added with 0.2% of nucleotide, and the feed conversion rate is obviously improved by 16.90% compared with the control group, in addition, the nucleotide has the tendency of improving the daily weight gain of the piglets, and can obviously improve the duodenum villus height of the piglets at 7 days after weaning and the ileum mucosa DNA content of the piglets at 14 days, and respectively improve 44.79% and 11.65% compared with the control group. Kehoe et al (Kehoe, Heinrichs, Baumrucker, et al. effects of nucleotide deletion in milk replacer on small intestinal absorption capacity in enzymes. journal of medicine Science,2008,91(7):2759 2770) mention that adding yeast source nucleotide to daily ration improves intestinal mucosa morphology and intestinal environment, and is beneficial to improving calf intestinal function. Uauy et al (Uauy, Stringel, Thomas, et. Effect of diabetes on growth and regulation of the degravinggut in the rate. J. Peditr Gastroenterol Nutr,1990,10(4):497-503) fed the purified diets separately from the diets added with 0.8% exogenous nucleotides for two weeks, found that the intestinal villus height, the intestinal proximal mucosal protein content and the maltase activity in the intestinal mucosa were all higher in the rats added with the group than in the purified diets without nucleotides. The results of adding nucleotide mixtures or nucleotides to the daily ration of rats lacking proteins show that the addition of exogenous nucleotide groups can significantly increase villus height, duct depth and intestinal wall thickness.
The nucleotide content in the feed is extremely low, and the requirement of stress young animals on the nucleotide cannot be met. The yeast cell contains abundant nucleic acid substances, the content of nucleic acid (nucleotide) is about 20 times of that in fish meal, and the yeast cell is a very good nucleic acid source. However, currently, about 300 yeast enterprises exist around the world, and yeast hydrolysates produced by yeast protein raw material production companies are all common enzymolysis processes, and contain low free nucleotides (less than 0.3%) and exist mainly in nucleic acid form (5-8%). Because of the low activity of the nuclease in the animal body, the absorption and utilization rate of nucleic acid in yeast hydrolysate by the animal is reduced, and the effect of supplementing nucleotide source cannot be fully realized. At present, a novel enzymolysis process is adopted, and the technology for producing the yeast hydrolysate with high nucleotide content is still blank.
The prior published patents mainly comprise beer yeast extract nucleotide, and no patent for producing high nucleotide yeast hydrolysate by using pure culture yeast is found, so the prior published patents have novelty.
The literature of the nucleotide reported at present mainly focuses on the necessity of the nucleotide as a young animal, the content research in feed and the improvement of the animal production performance after exogenous addition. Yeast is used as a main nucleotide source to produce high-nucleotide yeast hydrolysate, so that the culture benefit can be increased.
In chinese patent publication No. 103704543a (kokai No. 2014-04-09, application No. 201310677654.3) entitled "a high nucleotide yeast powder and a method for preparing the same", nucleic acid in yeast is further converted into nucleotide by adding a nucleic acid degradation promoter to active baker yeast or a mixture of the active baker yeast and beer yeast, and the nucleotide content of the prepared high nucleotide yeast powder can reach 8000 mg/kg.
Chinese patent publication No. 104855733A (published Japanese 2015-08-26, application No. 201510315328.7) is titled as "application of yeast containing active polysaccharide and nucleotide in cow feed", and is characterized by fermenting Saccharomyces cerevisiae, separating solid and liquid of the fermentation liquor, adding water into the solid part to prepare yeast liquor, adding neutral protease and nuclease P1 to enzymolyze mannoprotein and ribonucleic acid contained in the yeast, thereby obtaining the yeast containing active polysaccharide and nucleotide, wherein the components containing nucleotide are adenylic acid, cytidylic acid, guanylic acid and uridylic acid, and the total content of nucleotide is 1-20 wt% of the yeast.
Disclosure of Invention
Therefore, the technical problem solved by the invention is as follows: research shows that the nucleotide content in the feed raw materials is very low, the amount of the nucleotide required by the young animals is higher, and the requirement of the nucleotide of the young animals suffering from stress cannot be met only by taking the nutrient substances of the feed. Yeast is one of the accepted nucleotide sources in the industry, but the content of free nucleotides which can be rapidly absorbed in the products is extremely low at present.
The invention aims to provide a yeast hydrolysate with high free nucleotide content and a preparation method thereof, which are used for solving the problem of low free nucleotide content in yeast hydrolysate for commercial feed, meeting the nutritional requirements of young animals on nucleotide and maximizing the production benefit.
The invention provides a yeast hydrolysate with high free nucleotides, which comprises protein, free nucleotides, amino acids and small peptides, wherein the content of the protein is 60-75% by mass of dry matter of the yeast hydrolysate, the content of the free nucleotides is more than 35%, and the yeast hydrolysate is obtained by yeast milk enzymolysis, nucleotide acidification and nucleotide reinforcement.
The invention provides a preparation method of yeast hydrolysate with high free nucleotide, which comprises the following steps:
(1) fermentation culture: fermenting and culturing a saccharomyces cerevisiae strain to obtain first yeast milk;
(2) autolysis and enzymolysis: performing heat shock treatment on the yeast milk of the first yeast milk accounting for 60-80 wt%, preferably 65-70 wt% obtained in the step (1) to perform autolysis, and then adding papain for enzymolysis to obtain second yeast milk;
(3) and (3) nucleotide acidification: adding yeast RNA and nuclease into the second yeast milk obtained in the step (2) for enzymolysis to obtain third yeast milk, preferably adding the yeast RNA firstly, and then adding the nuclease for enzymolysis;
(4) nucleotide fortification: and (4) adding disodium nucleotide salt into the third yeast milk obtained in the step (3) to obtain the yeast hydrolysate with high free nucleotides.
The invention also provides the use of the yeast hydrolysate in feed, preferably in young animals.
Specifically, the present invention proposes the following technical solutions.
The invention provides a yeast hydrolysate with high free nucleotides, which comprises protein, free nucleotides, amino acids and small peptides, wherein the content of the protein is 60-75% by mass of dry matter of the yeast hydrolysate, the content of the free nucleotides is more than 35%, and the yeast hydrolysate is obtained by yeast milk enzymolysis, nucleotide acidification and nucleotide reinforcement.
Preferably, the yeast hydrolysate contains more than 40% of free nucleotides, preferably 40.9-41.38%; the content of the protein is 66.72-74.17%.
Preferably, in the yeast hydrolysate as described above, wherein the nucleotides comprise cytidylic acid, adenylic acid, guanylic acid, uridylic acid and inosinic acid, preferably, the content of cytidylic acid is 2.0-4.0% by mass, the content of the sum of adenylic acid and inosinic acid is 15.0-20.0% by mass, the content of guanylic acid is 15.0-19.0% by mass, the content of uridylic acid is 3.0-5.0% by mass, more preferably, the content of cytidylic acid is 2.82-3.01% by mass, the content of the sum of adenylic acid and inosinic acid is 17.28-18.48% by mass, the content of guanylic acid is 15.35-16.99% by mass, and the content of uridylic acid is 3.84-4.13% by mass.
Preferably, the yeast hydrolysate is prepared by fermenting saccharomyces cerevisiae FX-2, wherein the preservation number of the saccharomyces cerevisiae strain is CCTCC NO: m2016418.
The invention provides a preparation method of the yeast hydrolysate, which comprises the following steps:
(1) fermentation culture: fermenting and culturing a saccharomyces cerevisiae strain to obtain first yeast milk;
(2) autolysis and enzymolysis: performing hot shock treatment on the yeast milk of the first yeast milk accounting for 60-80 wt%, preferably 65-70 wt% in the step (1) to perform autolysis, and adding papain for enzymolysis to obtain second yeast milk;
(3) and (3) nucleotide acidification: adding yeast RNA and nuclease into the second yeast milk obtained in the step (2) for enzymolysis to obtain third yeast milk, preferably adding the yeast RNA firstly, and then adding the nuclease for enzymolysis;
(4) nucleotide fortification: and (4) adding disodium nucleotide salt into the third yeast milk obtained in the step (3) to obtain the yeast hydrolysate with high free nucleotides.
Preferably, in the above production method, wherein, in the step (2), the temperature of the heat shock treatment is 85 to 90 ℃.
Preferably, in the above production method, in the step (2), the heat shock treatment time is 50 to 60 seconds, preferably 55 to 60 seconds.
Preferably, in the preparation method described above, in the step (2), citric acid is added after the heat shock treatment for heat preservation, and preferably, the addition amount of citric acid is 2 to 4%, preferably 3 to 4%, by mass of yeast milk dry matter.
Preferably, in the preparation method, the heat preservation temperature of the heat preservation treatment is 40-55 ℃, preferably 40-50 ℃; the heat preservation time is 8-12h, preferably 8-10 h.
Preferably, in the preparation method, in the step (2), the addition amount of the papain is 1 to 5%, preferably 2 to 4%, and more preferably 3 to 3.4% by mass of the dried yeast milk.
Preferably, in the preparation method, the enzymolysis temperature of the papain is 50-65 ℃, preferably 55-59 ℃, the pH is 5-6.5, preferably 5-6, and the enzymolysis time is 5-10 hours, preferably 6-8 hours.
Preferably, in the preparation method described above, in the step (3), the yeast RNA is added in an amount of 8 to 15% by mass, preferably 10 to 12% by mass, based on the mass of the second yeast milk dry matter.
Preferably, in the preparation method described above, the yeast RNA is extracted from yeast milk that accounts for 20 to 40 wt%, preferably 30 to 35 wt%, of the first yeast milk in step (1).
Preferably, in the preparation method, the nuclease is added in an amount of 3 to 8%, preferably 4 to 5.4%, and more preferably 4.8 to 5.4% by mass of the second yeast milk dry matter.
Preferably, in the preparation method, the enzymolysis temperature of the nuclease is 50-60 ℃, the pH is 5-6.5, and the enzymolysis time is 10-20 hours, preferably 12-15 hours.
Preferably, in the preparation method, the disodium nucleotide salt is added in an amount of 20 to 30% by mass, preferably 24 to 25% by mass, based on the dry matter of the third yeast milk.
Preferably, in the above preparation method, in step (1), the fermentation culture is fed with a carbon source, a nitrogen source and a phosphorus source in a feeding manner;
the carbon source is 9000g of 5000-;
the carbon source is molasses, more preferably 25-35% molasses;
the nitrogen source is selected from one of ammonium sulfate or ammonium nitrate, preferably ammonium sulfate;
the phosphorus source is selected from one of ammonium dihydrogen phosphate or potassium dihydrogen phosphate, and ammonium dihydrogen phosphate is preferred.
The nitrogen content of the nitrogen source comprises the nitrogen content of the nitrogen source and the nitrogen content of the phosphorus source.
Preferably, in the above preparation method, in the step (1), the fermentation temperature of the fermentation culture is 29 to 35 ℃, the fermentation time is 10 to 18 hours, and the fermentation pH is 4.5 to 6.4.
The invention provides the use of said yeast hydrolysate in animal feed, preferably in young animal feed.
The invention provides a feed which comprises the yeast hydrolysate and basic ration, wherein the addition amount of the yeast hydrolysate is 0.2-1.0%, and preferably 0.3-0.5%.
The beneficial effects obtained by the invention are as follows:
1. the invention can obtain high-content free nucleotides, and the total amount of the free nucleotides accounts for more than 35% of the mass of the yeast hydrolysate.
2. The preparation method is simple, and the high nucleotide yeast hydrolysate produced by the method is a natural product, green and safe.
3. The high free nucleotide yeast hydrolysate obtained by the invention can relieve the shortage of the nucleotide source for feeding, increase the breeding benefit and has wide application prospect.
Information on the preservation of the strains
The strain used by the invention, namely the Saccharomyces cerevisiae FX-2(Saccharomyces cerevisiae FX-2), is preserved in China Center for Type Culture Collection (CCTCC) in 2016, 8, 1 and 8, with the preservation number of CCTCC NO: M2016418, and the preservation address is as follows: china, wuhan university, zip code: 430072; telephone: (027)68754052.
The saccharomyces cerevisiae FX-2 used in the invention is derived from fermented dough, the fermented dough contains various wild bacteria, the fermented dough is used as a sample to prepare a dough leaching solution, a pure strain is obtained by separation through a dilution coating flat plate separation method, and the strain is identified to belong to the saccharomyces cerevisiae.
The strain identification method is that the sequence of the D1/D2 region of the 26S rDNA of the strain is subjected to homology analysis with the sequence in a GenBank nucleic acid database, and the result shows that the sequence homology is more than 99 percent, so that the strain obtained by the separation of the invention is determined to belong to Saccharomyces cerevisiae (Saccharomyces cerevisiae) which is biologically classified as Saccharomyces cerevisiae FX-2(Saccharomyces cerevisiae) and is described in the patent publication No. CN 201611141122.8.
Detailed Description
As described above, the present invention provides a yeast hydrolysate, which comprises protein, free nucleotides, amino acids and small peptides, wherein the content of the protein is 60-75% by mass of dry matter of the yeast hydrolysate, the content of the free nucleotides is more than 35%, and the yeast hydrolysate is obtained by yeast milk enzymolysis, nucleotide acidification and nucleotide reinforcement.
In a preferred embodiment of the present invention, the content of free nucleotides is above 40%, preferably 40.9-41.38%, and the content of proteins is 66.72-74.17%.
In a more preferred embodiment of the present invention, the free nucleotides comprise cytidylic acid, adenylic acid, guanylic acid, uridylic acid and inosinic acid, preferably, the content of cytidylic acid is 2.0-4.0% by mass, the content of the sum of adenylic acid and inosinic acid is 15.0-20.0% by mass, the content of guanylic acid is 15.0-19.0% by mass, the content of uridylic acid is 3.0-5.0% by mass, more preferably, the content of cytidylic acid is 2.82-3.01% by mass, the content of the sum of adenylic acid and inosinic acid is 17.28-18.48% by mass, the content of guanylic acid is 15.35-16.99% by mass, and the content of uridylic acid is 3.84-4.13% by mass.
In a preferred embodiment of the invention, the yeast milk is obtained by fermenting saccharomyces cerevisiae FX-2, and the preservation number of the saccharomyces cerevisiae strain is CCTCC NO: m2016418.
Preferably, the yeast hydrolysate is obtained by carrying out liquid ventilation fermentation culture, autolytic enzymolysis, nucleotide acidification, nucleotide enrichment, concentration and drying on saccharomyces cerevisiae.
The invention provides a preparation method of yeast hydrolysate, which comprises the following steps:
(1) fermentation culture: fermenting and culturing a saccharomyces cerevisiae strain to obtain first yeast milk;
(2) autolysis and enzymolysis: performing hot shock treatment on 60-80 wt%, preferably 65-70 wt% of the yeast milk of the first yeast milk in the step (1) to perform autolysis, and adding papain for enzymolysis to obtain second yeast milk;
(3) and (3) nucleotide acidification: adding yeast RNA and nuclease into the second yeast milk obtained in the step (2) for enzymolysis to obtain third yeast milk, preferably adding the yeast RNA firstly, and then adding the nuclease for enzymolysis;
(4) nucleotide fortification: and (4) adding disodium nucleotide salt into the third yeast milk obtained in the step (3) to obtain the yeast hydrolysate with high free nucleotides.
In a preferred embodiment of the present invention, wherein, in the step (2), the temperature of the heat shock treatment is 85 to 90 ℃, preferably, the time of the heat shock treatment is 50 to 60s, more preferably 55 to 60 s.
In a preferred embodiment of the invention, in the step (2), citric acid is added after the heat shock treatment for heat preservation, preferably, the addition amount of citric acid is 2-4 per thousand, preferably 3-4 per thousand based on the dry matter mass of the yeast milk.
In a more preferable embodiment of the present invention, the temperature of the heat preservation treatment is 40 to 55 ℃, preferably 40 to 50 ℃; the heat preservation time is 8-12h, preferably 8-10 h.
In a preferred embodiment of the present invention, in step (2), the addition amount of the papain is 1 to 5%, preferably, the addition amount of the papain is 2 to 4%, and more preferably, the addition amount of the papain is 3 to 3.4%, based on the mass of the dry matter of the yeast milk.
Preferably, the enzymolysis temperature of the papain is 50-65 ℃, preferably 55-59 ℃, the pH is 5-6.5, preferably 5-6, and the enzymolysis time is 5-10 hours, preferably 6-8 hours.
Wherein the papain (pH5-6.5) can hydrolyze carboxyl terminals of arginine and lysine in proteins and polypeptides, and can limitedly hydrolyze peptide bonds of amino acids having two carboxyl groups at the N-terminal of the peptide bonds or aromatic L-amino acids, thereby increasing the content of the polypeptides and amino acids in the yeast hydrolysate.
In a preferred embodiment of the present invention, in step (3), the yeast RNA is added in an amount of 8-15%, preferably 10-12% by mass of the dry matter of the second yeast milk.
Wherein, the yeast RNA is obtained by extracting yeast milk accounting for 20-40 wt% of the first yeast milk in the step (1), preferably 30-35 wt%. It is extracted by a high-temperature extraction method: 10% salt is used to break yeast cells at 90 plus or minus 2 deg.C, and RNA is extracted.
In a preferred embodiment of the invention, the nuclease is added in an amount of 3-8%, preferably 4-5.4%, and more preferably 4.8-5.4% by mass of the dry matter of the second yeast milk.
Wherein the enzymolysis temperature of the nuclease is 50-60 ℃, the pH value is 5-6.5, and the enzymolysis time is 10-20 hours, preferably 12-15 hours.
Wherein nuclease (pH5-6.5) acts on nucleic acid to form a phosphodiester bond between a hydroxyl group on the 3 '-carbon atom of the RNA single strand and a phosphate to obtain 5' -nucleotide, thereby increasing the content of free nucleotides in the yeast hydrolysate.
In a preferred embodiment of the present invention, the nucleotide disodium salt is added in an amount of 20-30%, preferably 24-25% by mass of the third yeast milk dry matter.
The disodium nucleotide salt contains high-content inosinic acid and guanylic acid, and has the effects of optimizing the nucleotide proportion and improving the delicate flavor of products by adding the disodium nucleotide salt.
In a preferred embodiment of the invention, in step (1), the fermentation culture is fed-batch with a carbon source, a nitrogen source and a phosphorus source;
the carbon source is 9000g of 5000-;
the carbon source is molasses, more preferably 25-35% molasses;
the nitrogen source is selected from one of ammonium sulfate or ammonium nitrate, preferably ammonium sulfate;
the phosphorus source is selected from one of ammonium dihydrogen phosphate or potassium dihydrogen phosphate, and is preferably ammonium dihydrogen phosphate; preferably, in the step (1), the fermentation temperature of the fermentation culture is 29-35 ℃, the fermentation time is 10-18 hours, and the fermentation pH is 4.5-6.4.
The invention provides the use of the yeast hydrolysate described above in animal feed, preferably in young animal feed; the yeast hydrolysate can supplement nucleotide nutrition for animals, and increase breeding benefit.
The invention provides a feed which comprises the yeast hydrolysate and basic ration, wherein the addition amount of the yeast hydrolysate is 0.2-1.0%, and preferably 0.3-0.5%.
The yeast hydrolysate can be added into feed for animal breeding.
The manufacturers of the raw materials and equipment used in the present example, and the equipment and analysis method used in the product analysis are described below, wherein the chemical substances are not indicated as being chemically pure grades of conventional reagents. Information on the raw materials used in the examples is shown in Table 1
Table 1 information on the raw materials used in the examples
EXAMPLE one preparation of Yeast hydrolysate
(1) Fermentation culture of yeast
The culture solution contains a carbon source, a nitrogen source and a phosphorus source, wherein the carbon source is 7500g of 35% molasses, the nitrogen source is 450g of ammonium sulfate, and the phosphorus source is 420g of ammonium dihydrogen phosphate. Sterilizing the culture solution at 121 deg.C for 10min, feeding into tank, inoculating yeast, fermenting at 29 deg.C for 14 hr to obtain pure culture liquid fermented first yeast milk (wherein nitrogen content is 144.9g, and phosphorus content is 113.4g), and culturing at pH 4.5.
(2) Autolytic enzymolysis of yeast
And (2) performing heat shock on the yeast milk accounting for 60 wt% of the first yeast milk in the step (1) at the temperature of 85 ℃ for 55s, controlling the temperature to be 45 ℃, adding citric acid accounting for 3 thousandth of the weight of the dry matter added in the yeast milk, and performing autolysis for 10 h.
Controlling the temperature to 55 ℃, adjusting the pH value to 5.0, adding 4 per mill of papain, and carrying out enzymolysis for 8 hours to obtain second yeast milk.
(3) Nucleotide acidification
Adding yeast RNA accounting for 10% of the dry matter weight of the second yeast milk, mixing uniformly, cooling to 50 ℃, adjusting the pH value to 5.0, adding 5 thousandths of nuclease, performing enzymolysis for 15 hours to obtain third yeast milk, wherein the yeast RNA is obtained by crushing the residual first yeast milk in the step (1) at the high temperature of 90 +/-2 ℃ by adopting 10% of salt, and extracting RNA.
(4) Nucleotide fortification
And (4) adding disodium nucleotide (I + G) accounting for 25% of the dry matter weight of the third yeast milk in the step (3), stirring for 10 minutes, and uniformly mixing.
(5) Concentrating and drying
After the enzymolysis reaction is finished, heating to 87 ℃, preserving the temperature for 2 hours, inactivating the enzyme, and carrying out active concentration and drying to obtain the yeast hydrolysate product with high free nucleotide.
The hydrolysate obtained above was subjected to protein content and nucleotide content measurement.
1. Determination of protein content in Yeast hydrolysates
The crude protein is determined according to the determination method of the crude protein in the feed GB/T6432-94, and the specific operation is as follows: taking 1g of sample, adding 12ml of concentrated sulfuric acid for digestion under the action of a mixed catalyst (0.4g of blue vitriol and 6g of potassium sulfate); then distilling, and absorbing the product ammonia by boric acid; and titrating with 0.1mol/l hydrochloric acid, reading data, calculating nitrogen content, selecting N (nitrogen content) × 6.25 as protein coefficient, and calculating to obtain crude protein content.
The content of protein in the yeast hydrolysate obtained by the above method was 66.72%.
2. Determination of nucleotide content in Yeast hydrolysates
The nucleotide is determined by adopting a method for determining the nucleotide in the GB/T23530-2009 yeast extract, and the specific operation method is as follows:
analysis was performed using a high performance liquid chromatograph equipped with a low pressure quaternary pump, uv detector and column oven system (Waters, E2695), column C18(250mm X4.6 mm). A1 g sample was taken and diluted to 100ml (dilution factor F). Filtering with 0.45 μm water phase microporous membrane, and sampling 20 μ L of sample, wherein the specific chromatographic conditions are as follows:
the chromatographic peaks of IMP/GMP/UMP/CMP in the sample were characterized according to the retention time of the standard, and the percentage of each component was calculated by external standard method according to the peak area of the sample.
After the determination by the above method, the total content of adenylic acid and inosinic acid in the yeast hydrolysate prepared in example one was 17.28%, the content of uridylic acid was 3.84%, the content of cytidylic acid was 2.82%, the content of guanylic acid was 16.99%, and the content of total free nucleotides was 40.93%.
Example two
(1) Fermentation culture of yeast
The culture solution contains a carbon source, a nitrogen source and a phosphorus source, wherein the carbon source is 8000g of 35% molasses, the nitrogen source is 400g of ammonium sulfate, and the phosphorus source is 450g of ammonium dihydrogen phosphate. Sterilizing the culture solution at 121 deg.C for 10min, feeding into tank, inoculating yeast, fermenting at 30 deg.C for 15 hr to obtain pure culture liquid fermented first yeast milk (wherein nitrogen content is 138g, and phosphorus content is 121.5g), and fermenting at pH 5.0.
(2) Autolytic enzymolysis of yeast
And (2) performing heat shock on the yeast milk of the first yeast milk accounting for 65 wt% of the yeast milk in the step (1) at 88 ℃ for 60s, controlling the temperature to be 50 ℃, adding citric acid accounting for 4 per mill of the added weight of the yeast milk dry matter, and performing autolysis for 8 h.
Controlling the temperature to be 58 ℃, adjusting the pH value to be 6.0, adding 2 per mill of papain, and carrying out enzymolysis for 6 hours to obtain second yeast milk.
(3) Nucleotide acidification
Adding yeast RNA accounting for 11% of the dry matter weight of the second yeast milk, mixing uniformly, cooling to 55 ℃, adjusting the pH value to 6.0, adding nuclease 4 per mill, performing enzymolysis for 12 hours to obtain third yeast milk, wherein the yeast RNA is obtained by crushing the residual first yeast milk in the step (1) at a high temperature of 90 +/-2 ℃ by using 10% of salt and extracting RNA.
(4) Nucleotide fortification
And (4) adding disodium nucleotide (I + G) accounting for 25% of the dry matter weight of the third yeast milk in the step (3), stirring for 20 minutes, and uniformly mixing.
(5) Concentrating and drying
After the enzymolysis reaction is finished, heating to 85 ℃, preserving the temperature for 2 hours, inactivating the enzyme, and carrying out active concentration and drying to obtain the yeast hydrolysate product with high free nucleotide.
The determination was performed using the method for determining the protein content and the nucleotide content in example one, wherein the protein content in example two was 71.35%, the sum of the contents of adenylic acid and inosinic acid in the yeast hydrolysate prepared in example two was 18.48%, the content of uridylic acid was 4.13%, the content of cytidylic acid was 2.94%, the content of guanylic acid was 15.35%, and the content of total free nucleotides was 40.90%.
EXAMPLE III
(1) Fermentation culture of yeast
The culture solution contains carbon source, nitrogen source and phosphorus source, wherein the carbon source is 7000g of 35% molasses, the nitrogen source is 380g of ammonium sulfate, and the phosphorus source is 420g of ammonium dihydrogen phosphate. Sterilizing the culture solution at 121 deg.C for 10min, feeding into tank, inoculating yeast, fermenting at 33 deg.C for 15 hr, and fermenting at pH 6.0 to obtain pure culture liquid fermented first yeast milk (wherein nitrogen content is 130.2g, and phosphorus content is 113.4 g).
(2) Autolytic enzymolysis of yeast
And (2) performing heat shock on the yeast milk of the first yeast milk accounting for 70 wt% of the yeast milk in the step (1) at the temperature of 89 ℃ for 55s, controlling the temperature to be 40 ℃, adding citric acid accounting for 3.5 thousandths of the added weight of the yeast milk dry matter, and performing autolysis for 10 h.
Controlling the temperature to 59 ℃, adjusting the pH value to 5.5, adding 3.4 per mill of papain, and carrying out enzymolysis for 7 hours to obtain second yeast milk.
(3) Nucleotide acidification
Adding yeast RNA accounting for 12% of the dry matter weight of the second yeast milk, mixing uniformly, cooling to 50 ℃, adjusting the pH value to 6.5, adding 5.4 thousandths of nuclease, and performing enzymolysis for 13 hours to obtain third yeast milk, wherein the yeast RNA is obtained by crushing the residual first yeast milk in the step (1) at the high temperature of 90 +/-2 ℃ by adopting 10% of salt and extracting RNA.
(4) Nucleotide fortification
And (4) adding nucleotide disodium (I + G) accounting for 24% of the dry matter weight of the third yeast milk in the step (3), stirring for 30 minutes, and uniformly mixing.
(5) Concentrating and drying
After the enzymolysis reaction is finished, heating to 90 ℃, preserving the temperature for 2 hours, inactivating the enzyme, and carrying out active concentration and drying to obtain the yeast hydrolysate product with high free nucleotide.
The determination was performed using the method for determining the protein content and the nucleotide content in example one, wherein the protein content in example three was 74.17%, the sum of the contents of adenylic acid and inosinic acid in the yeast hydrolysate prepared in example three was 18.27%, the content of uridylic acid was 3.94%, the content of cytidylic acid was 3.01%, the content of guanylic acid was 15.78%, and the content of total free nucleotides was 41.00%.
Example four
(1) Fermentation culture of yeast
The culture solution contains a carbon source, a nitrogen source and a phosphorus source, wherein the carbon source is 35% molasses 8700g, the nitrogen source is ammonium sulfate 540g, and the phosphorus source is ammonium dihydrogen phosphate 480 g. Sterilizing the culture solution at 121 deg.C for 10min, feeding into tank, inoculating yeast, fermenting at 33 deg.C for 14 hr to obtain pure culture liquid fermented first yeast milk (wherein the mass of nitrogen is 171g, and the amount of phosphorus is 129.6g), and culturing at pH 5.4.
(2) Autolytic enzymolysis of yeast
And (2) performing heat shock on the yeast milk of the first yeast milk accounting for 80 wt% of the yeast milk in the step (1) at the temperature of 90 ℃ for 60s, controlling the temperature to be 50 ℃, adding citric acid accounting for 3.6 per mill of the added weight of the yeast milk dry matter, and performing autolysis for 10 h.
Controlling the temperature to 65 ℃, adjusting the pH value to 6.5, adding 2.8 per mill of papain, and carrying out enzymolysis for 7 hours to obtain second yeast milk.
(3) Nucleotide acidification
Adding yeast RNA accounting for 11% of the dry matter weight of the second yeast milk, mixing uniformly, cooling to 55 ℃, adjusting the pH value to 6.5, adding nuclease 4.8 per mill, performing enzymolysis for 14 hours to obtain third yeast milk, wherein the yeast RNA is obtained by crushing the residual first yeast milk in the step (1) at the high temperature of 90 +/-2 ℃ by adopting 10% of salt and extracting RNA.
(4) Nucleotide fortification
And (4) adding nucleotide disodium (I + G) accounting for 26% of the dry matter weight of the third yeast milk in the step (3), stirring for 25 minutes, and uniformly mixing.
(5) Concentrating and drying
After the enzymolysis reaction is finished, heating to 95 ℃, preserving the temperature for 2 hours, inactivating the enzyme, and carrying out active concentration and drying to obtain the yeast hydrolysate product with high free nucleotide.
The determination was performed using the method for determining the protein content and the nucleotide content in example one, wherein the protein content in example four was 69.37%, the sum of the contents of adenylic acid and inosinic acid in the yeast hydrolysate prepared in example four was 17.63%, the content of uridylic acid was 3.86%, the content of cytidylic acid was 2.74%, the content of guanylic acid was 16.59%, and the content of total free nucleotides was 40.82%.
EXAMPLE five
(1) Fermentation culture of yeast
The culture solution contains carbon source, nitrogen source and phosphorus source, wherein the carbon source is 8300g of 35% molasses, the nitrogen source is 550g of ammonium sulfate, and the phosphorus source is 420g of ammonium dihydrogen phosphate. Sterilizing the culture solution at 121 deg.C for 10min, feeding into tank, inoculating yeast, fermenting at 33 deg.C for 14 hr to obtain pure culture liquid fermented first yeast milk (wherein nitrogen content is 165.9g, and phosphorus content is 113.4g), and culturing at pH 5.4.
(2) Autolytic enzymolysis of yeast
And (2) performing heat shock on the yeast milk of the first yeast milk accounting for 68 wt% of the yeast milk in the step (1) at the temperature of 85 ℃ for 60s, controlling the temperature to be 50 ℃, adding citric acid accounting for 4 per mill of the added weight of the yeast milk dry matter, and performing autolysis for 10 h.
Controlling the temperature to 59 ℃, adjusting the pH value to 5.0, adding 3 per mill of papain, and carrying out enzymolysis for 7.5 hours to obtain second yeast milk.
(3) Nucleotide acidification
Adding yeast RNA accounting for 12% of the dry matter weight of the second yeast milk, uniformly mixing, heating to 60 ℃, adjusting the pH value to 6.0, adding 5.0 thousandths of nuclease, and performing enzymolysis for 13 hours to obtain third yeast milk, wherein the yeast RNA is obtained by crushing the residual first yeast milk in the step (1) at the high temperature of 90 +/-2 ℃ by adopting 10% of salt and extracting RNA.
(4) Nucleotide fortification
And (4) adding disodium nucleotide (I + G) accounting for 25% of the dry matter weight of the third yeast milk in the step (3), stirring for 20 minutes, and uniformly mixing.
(5) Concentrating and drying
After the enzymolysis reaction is finished, heating to 95 ℃, preserving the temperature for 2 hours, inactivating the enzyme, and carrying out active concentration and drying to obtain the yeast hydrolysate product with high free nucleotide.
The method for measuring the protein content and the nucleotide content in example one was used, wherein the protein content in example five was 72.39%, the total content of adenylic acid and inosinic acid in the yeast hydrolysate prepared in example five was 18.37%, the content of uridylic acid was 4.03%, the content of cytidylic acid was 2.88%, the content of guanylic acid was 16.10%, and the content of total free nucleotides was 41.38%.
EXAMPLE six
(1) Fermentation culture of yeast
The culture solution contains 5000g of 35% molasses as carbon source, 400g of ammonium nitrate as nitrogen source and 500g of potassium dihydrogen phosphate as phosphorus source. Sterilizing the culture solution at 121 deg.C for 10min, feeding into tank, inoculating yeast, fermenting at 30 deg.C for 10 hr to obtain pure culture liquid fermented first yeast milk (wherein nitrogen content is 140g, and phosphorus content is 115 g).
(2) Autolytic enzymolysis of yeast
And (2) performing heat shock on the yeast milk accounting for 60 wt% of the first yeast milk in the step (1) at the temperature of 90 ℃ for 50s, controlling the temperature to be 55 ℃, adding citric acid accounting for 2 thousandth of the weight of the dry matter of the yeast milk, and performing autolysis for 12 h.
Controlling the temperature to be 50 ℃, adjusting the pH value to be 5.5, adding 1 per mill of papain, and carrying out enzymolysis for 5 hours to obtain second yeast milk.
(3) Nucleotide acidification
Adding yeast RNA accounting for 8% of the dry matter weight of the second yeast milk, mixing uniformly, continuously controlling the temperature to be 50 ℃, adjusting the pH value to be 5.0, adding 3.0 thousandth of nuclease, performing enzymolysis for 10 hours to obtain third yeast milk, wherein the yeast RNA is obtained by crushing the residual first yeast milk in the step (1) at the high temperature of 90 +/-2 ℃ by adopting 10% of salt, and extracting RNA.
(4) Nucleotide fortification
And (4) adding nucleotide disodium (I + G) accounting for 20% of the dry matter weight of the third yeast milk in the step (3), stirring for 10 minutes, and uniformly mixing.
(5) Concentrating and drying
After the enzymolysis reaction is finished, heating to 95 ℃, preserving the temperature for 2 hours, inactivating the enzyme, and carrying out active concentration and drying to obtain the yeast hydrolysate product with high free nucleotide.
The method for measuring the protein content and the nucleotide content in example one was used, wherein the protein content in example six was 61.35%, the total content of adenylic acid and inosinic acid in the yeast hydrolysate prepared in example six was 15.64%, the content of uridylic acid was 3.21%, the content of cytidylic acid was 2.18%, the content of guanylic acid was 15.22%, and the content of total free nucleotides was 36.25%.
EXAMPLE seven
(1) Fermentation culture of yeast
The culture solution contains a carbon source, a nitrogen source and a phosphorus source, wherein the carbon source is 6000g of 35% molasses, the nitrogen source is 500g of ammonium nitrate, and the phosphorus source is 450g of monopotassium phosphate. Sterilizing the culture solution at 121 deg.C for 10min, feeding into tank, inoculating yeast, fermenting at 35 deg.C for 18 hr to obtain pure culture liquid fermented first yeast milk (wherein nitrogen content is 175g, and phosphorus content is 103.5g), and fermenting at pH 6.0.
(2) Autolytic enzymolysis of yeast
And (2) performing heat shock on the yeast milk accounting for 80 wt% of the first yeast milk in the step (1) at 88 ℃ for 60s, controlling the temperature to be 50 ℃, adding citric acid accounting for 3 thousandth of the weight of the dry matter of the yeast milk, and performing autolysis for 8 h.
Controlling the temperature to be 60 ℃, adjusting the pH value to be 6.0, adding papain with the concentration of 5 per mill, and carrying out enzymolysis for 10 hours to obtain second yeast milk.
(3) Nucleotide acidification
Adding yeast RNA accounting for 15% of the dry matter weight of the second yeast milk, mixing uniformly, cooling to 55 ℃, adjusting the pH value to 5.5, adding 8.0 thousandths of nuclease, performing enzymolysis for 20 hours to obtain third yeast milk, wherein the yeast RNA is obtained by crushing the residual first yeast milk in the step (1) at the high temperature of 90 +/-2 ℃ by adopting 10% of salt, and extracting RNA.
(4) Nucleotide fortification
And (4) adding disodium nucleotide (I + G) accounting for 30% of the dry matter weight of the third yeast milk in the step (3), stirring for 15 minutes, and uniformly mixing.
(5) Concentrating and drying
After the enzymolysis reaction is finished, heating to 95 ℃, preserving the temperature for 2 hours, inactivating the enzyme, and carrying out active concentration and drying to obtain the yeast hydrolysate product with high free nucleotide.
The determination was performed using the method for determining the protein content and the nucleotide content in example one, wherein the protein content in example seven was 65.98%, the sum of the contents of adenylic acid and inosinic acid in the yeast hydrolysate prepared in example seven was 17.02%, the content of uridylic acid was 3.60%, the content of cytidylic acid was 2.65%, the content of guanylic acid was 15.34%, and the content of total free nucleotides was 38.61%.
Example eight
(1) Fermentation culture of yeast
The culture solution contains carbon source, nitrogen source and phosphorus source, wherein the carbon source is 9000g of 35% molasses, the nitrogen source is 450g of ammonium nitrate, and the phosphorus source is 500g of potassium dihydrogen phosphate. Sterilizing the culture solution at 121 deg.C for 10min, feeding into tank, inoculating yeast, fermenting at 33 deg.C for 12 hr, and fermenting at pH5 to obtain pure culture solution fermented first yeast milk (wherein nitrogen is 157.5g, and phosphorus is 115 g).
(2) Autolytic enzymolysis of yeast
And (2) performing heat shock on the yeast milk accounting for 70 wt% of the first yeast milk in the step (1) at the temperature of 85 ℃ for 55s, controlling the temperature to be 40 ℃, adding citric acid accounting for 4 per mill of the added weight of the yeast milk dry matter, and performing autolysis for 10 h.
Controlling the temperature to 65 ℃, adjusting the pH value to 6.5, adding 4 per mill of papain, and carrying out enzymolysis for 8 hours to obtain second yeast milk.
(3) Nucleotide acidification
Adding yeast RNA accounting for 12% of the dry matter weight of the second yeast milk, mixing uniformly, cooling to 60 ℃, adjusting the pH value to 6.0, adding nuclease 6.0 per mill, performing enzymolysis for 18 hours to obtain third yeast milk, wherein the yeast RNA is obtained by crushing the residual first yeast milk in the step (1) at the high temperature of 90 +/-2 ℃ by adopting 10% of salt and extracting RNA.
(4) Nucleotide fortification
And (4) adding nucleotide disodium (I + G) accounting for 28% of the dry matter weight of the third yeast milk in the step (3), stirring for 15 minutes, and uniformly mixing.
(5) Concentrating and drying
After the enzymolysis reaction is finished, heating to 95 ℃, preserving the temperature for 2 hours, inactivating the enzyme, and carrying out active concentration and drying to obtain the yeast hydrolysate product with high free nucleotide.
The determination was performed using the method for determining the protein content and the nucleotide content in example one, wherein the protein content in example eight was 63.37%, the sum of the contents of adenylic acid and inosinic acid in the yeast hydrolysate prepared in example eight was 16.91%, the content of uridylic acid was 3.39%, the content of cytidylic acid was 2.47%, the content of guanylic acid was 15.09%, and the content of total free nucleotides was 37.86%.
Example nine
(1) Fermentation culture of yeast
The culture solution contains a carbon source, a nitrogen source and a phosphorus source, wherein the carbon source is 7500g of 35% molasses, the nitrogen source is 450g of ammonium sulfate, and the phosphorus source is 420g of ammonium dihydrogen phosphate. Sterilizing the culture solution at 121 deg.C for 10min, feeding into tank, inoculating yeast, fermenting at 29 deg.C for 14 hr to obtain pure culture liquid fermented first yeast milk (wherein nitrogen content is 144.9g, and phosphorus content is 113.4g), and culturing at pH 4.5.
(2) Autolytic enzymolysis of yeast
And (2) performing heat shock on the yeast milk accounting for 70 wt% of the first yeast milk in the step (1) at the temperature of 85 ℃ for 55s, controlling the temperature to be 45 ℃, adding citric acid accounting for 3 thousandth of the weight of the dry matter added in the yeast milk, and performing autolysis for 10 h.
Controlling the temperature to 55 ℃, adjusting the pH value to 5.0, adding 3.2 per mill of papain, and carrying out enzymolysis for 8 hours to obtain second yeast milk.
(3) Nucleotide acidification
Adding yeast RNA accounting for 10% of the dry matter weight of the second yeast milk, mixing uniformly, cooling to 50 ℃, adjusting the pH value to 5.0, adding nuclease 4.8 per mill, performing enzymolysis for 15 hours to obtain third yeast milk, wherein the yeast RNA is obtained by crushing the residual first yeast milk in the step (1) at the high temperature of 90 +/-2 ℃ by adopting 10% of salt and extracting RNA.
(5) Nucleotide fortification
And (4) adding disodium nucleotide (I + G) accounting for 25% of the dry matter weight of the third yeast milk in the step (3), stirring for 10 minutes, and uniformly mixing.
(5) Concentrating and drying
After the enzymolysis reaction is finished, heating to 87 ℃, preserving the temperature for 2 hours, inactivating the enzyme, and carrying out active concentration and drying to obtain the yeast hydrolysate product with high free nucleotide.
The determination was performed using the method for determining the protein content and the nucleotide content in example one, wherein the protein content in example eight was 71.42%, the sum of the contents of adenylic acid and inosinic acid in the yeast hydrolysate prepared in example eight was 18.17%, the content of uridylic acid was 3.97%, the content of cytidylic acid was 2.96%, the content of guanylic acid was 16.04%, and the content of total free nucleotides was 41.14%.
Comparative example 1
(1) Fermentation culture of yeast
The culture solution contains a carbon source, a nitrogen source and a phosphorus source, wherein the carbon source is 7500g of 35% molasses, the nitrogen source is 450g of ammonium sulfate, and the phosphorus source is 420g of ammonium dihydrogen phosphate. Sterilizing the culture solution at 121 deg.C for 10min, feeding into tank, inoculating yeast, fermenting at 29 deg.C for 14 hr to obtain pure culture liquid fermented first yeast milk (wherein nitrogen content is 144.9g, and phosphorus content is 113.4g), and culturing at pH 4.5.
(2) Autolytic enzymolysis of yeast
And (2) performing heat shock on all the first yeast milk in the step (1) at the temperature of 85 ℃ for 55s, controlling the temperature to be 45 ℃, adding citric acid which accounts for 3 per mill of the added weight of the yeast milk dry matter, and performing autolysis for 10 h.
Controlling the temperature to 55 ℃, adjusting the pH value to 5.0, adding 4 per mill of papain, and carrying out enzymolysis for 8 hours to obtain second yeast milk.
(3) Nucleotide acidification
And (3) cooling the second yeast milk obtained in the step (2) to 50 ℃, adjusting the pH value to 5.0, adding 5 per mill of nuclease, and performing enzymolysis for 15 hours to obtain third yeast milk, wherein the yeast RNA is obtained by crushing the remaining first yeast milk obtained in the step (1) at a high temperature of 90 +/-2 ℃ by using 10% of salt and extracting RNA.
(4) Concentrating and drying
After the enzymolysis reaction is finished, heating to 87 ℃, preserving the temperature for 2 hours, inactivating the enzyme, and carrying out active concentration and drying to obtain the yeast hydrolysate product with high free nucleotide.
The determination was performed using the method for determining the protein content and the nucleotide content in example one, wherein the protein content in comparative example one was 50.62%, the sum of the contents of adenylic acid and inosinic acid in the yeast hydrolysate prepared in comparative example one was 0.81%, the content of uridylic acid was 0.51%, the content of cytidylic acid was 0.36%, the content of guanylic acid was 0.68%, and the content of total free nucleotides was 2.36%.
Comparative example No. two
(1) Fermentation culture of yeast
The culture solution contains a carbon source, a nitrogen source and a phosphorus source, wherein the carbon source is 7500g of 35% molasses, the nitrogen source is 450g of ammonium sulfate, and the phosphorus source is 420g of ammonium dihydrogen phosphate. Sterilizing the culture solution at 121 deg.C for 10min, feeding into tank, inoculating yeast, fermenting at 29 deg.C for 14 hr to obtain pure culture liquid fermented first yeast milk (wherein nitrogen content is 144.9g, and phosphorus content is 113.4g), and culturing at pH 4.5.
(2) Autolytic enzymolysis of yeast
And (2) performing heat shock on the yeast milk accounting for 60 wt% of the first yeast milk in the step (1) at the temperature of 85 ℃ for 55s, controlling the temperature to be 45 ℃, adding citric acid accounting for 3 thousandth of the weight of the dry matter of the yeast milk, and performing autolysis for 10 h.
Controlling the temperature to 55 ℃, adjusting the pH value to 5.0, adding 4 per mill of papain, and carrying out enzymolysis for 8 hours to obtain second yeast milk.
(3) Nucleotide acidification
Adding yeast RNA accounting for 10% of the dry matter weight of the second yeast milk, mixing uniformly, cooling to 50 ℃, adjusting the pH value to 5.0, adding 5 thousandths of nuclease, performing enzymolysis for 15 hours to obtain third yeast milk, wherein the yeast RNA is obtained by crushing the residual first yeast milk in the step (1) at the high temperature of 90 +/-2 ℃ by adopting 10% of salt, and extracting RNA.
(4) Concentrating and drying
After the enzymolysis reaction is finished, heating to 87 ℃, preserving the temperature for 2 hours, inactivating the enzyme, and carrying out active concentration and drying to obtain the yeast hydrolysate product with high free nucleotide.
The determination of the protein content and the nucleotide content in example one was used, wherein the protein content in comparative example two was 56.63%, the sum of the contents of adenylic acid and inosinic acid in the yeast hydrolysate prepared in comparative example two was 6.43%, the content of uridylic acid was 3.71%, the content of cytidylic acid was 2.89%, the content of guanylic acid was 3.52%, and the content of total free nucleotides was 16.55%.
Comparative example No. three
(1) Fermentation culture of yeast
The culture solution contains a carbon source, a nitrogen source and a phosphorus source, wherein the carbon source is 7500g of 35% molasses, the nitrogen source is 450g of ammonium sulfate, and the phosphorus source is 420g of ammonium dihydrogen phosphate. Sterilizing the culture solution at 121 deg.C for 10min, feeding into tank, inoculating yeast, fermenting at 29 deg.C for 14 hr to obtain pure culture liquid fermented first yeast milk (wherein nitrogen content is 144.9g, and phosphorus content is 113.4g), and culturing at pH 4.5.
(2) Autolytic enzymolysis of yeast
And (2) performing heat shock on all the first yeast milk in the step (1) at the temperature of 85 ℃ for 55s, controlling the temperature to be 45 ℃, adding citric acid which accounts for 3 per mill of the added weight of the yeast milk dry matter, and performing autolysis for 10 h.
Controlling the temperature to 55 ℃, adjusting the pH value to 5.0, adding 4 per mill of papain, and carrying out enzymolysis for 8 hours to obtain second yeast milk.
(3) Nucleotide acidification
And (3) cooling the second yeast milk obtained in the step (2) to 50 ℃, adjusting the pH value to 5.0, adding 5 per mill of nuclease, and performing enzymolysis for 15 hours to obtain third yeast milk, wherein the yeast RNA is obtained by crushing the remaining first yeast milk in the step (1) at a high temperature of 90 +/-2 ℃ by using 10% of salt and extracting RNA.
(4) Nucleotide fortification
And (4) adding disodium nucleotide (I + G) accounting for 25% of the dry matter weight of the third yeast milk in the step (3), stirring for 30 minutes, and uniformly mixing.
(5) Concentrating and drying
After the enzymolysis reaction is finished, heating to 87 ℃, preserving the temperature for 2 hours, inactivating the enzyme, and carrying out active concentration and drying to obtain the yeast hydrolysate product with high free nucleotide.
The determination was performed using the protein content and nucleotide content measurements in example one, wherein the protein content in comparative example three was 63.29%, the sum of the contents of adenylic acid and inosinic acid in the yeast hydrolysate prepared in comparative example three was 12.13%, the content of uridylic acid was 0.41%, the content of cytidylic acid was 0.26%, the content of guanylic acid was 12.58%, and the content of total free nucleotides was 25.38%.
Comparing example one with comparative example one, comparative example two and comparative example three, it can be seen that when the yeast milk obtained in step (1) is totally used for autolysis (i.e. comparative example one), and nucleotide fortification is performed without adding disodium nucleotide, the protein content in the obtained fermented hydrolysate is relatively low, while the content of free nucleotides is lower, only 2.36%; when a part of the yeast milk in the step (1) is used for autolysis enzymolysis, and a part of the yeast milk is used for extracting yeast RNA, but nucleotide fortification is carried out without adding nucleotide disodium (namely, a comparative example II), the protein content of the yeast hydrolysate is relatively low, and the content of free nucleotides is very low, namely 16.55%; when the yeast milk in the step (1) is completely used for autolysis (namely, the comparative example III), the protein content in the yeast hydrolysate is relatively low, and the content of the free nucleotides is also relatively low and is only 25.38%, which indicates that the yeast milk in the step (1) is divided into two parts, one part is used for autolysis, the other part is used for extracting yeast RNA, and nucleotide reinforcement is performed by using nucleotide disodium, so that the protein content in the obtained yeast hydrolysate is 60-75%, and the content of the free nucleotides is more than 35%.
Examples two to eight the obtained yeast hydrolysates also had protein contents ranging from 60 to 75% and free nucleotides of over 35%.
In summary, the yeast hydrolysate with high free nucleotides prepared by the invention has the protein content accounting for 60-75% of the dry matter mass of the yeast hydrolysate, and the total free nucleotides accounting for more than 35% of the dry matter mass of the yeast hydrolysate. The high nucleotide yeast hydrolysate can meet the requirement of animals on free nucleotide and has wide application prospect.
The nucleotide has important significance for maintaining, improving and repairing gastrointestinal tract functions of animals, and the supplement of exogenous nucleotide can accelerate the differentiation, growth and repair of intestinal cells and promote the growth of bifidobacteria and lactobacilli in intestinal tracts. The yeast hydrolysate obtained by the invention can solve the problem of low free nucleotide content in the yeast hydrolysate for feeding on the market, can meet the nutritional requirements of young animals on nucleotide, and achieves the maximization of production benefits.
The foregoing is considered as illustrative and not restrictive in character, and that various modifications, equivalents, and improvements made within the spirit and principles of the invention are intended to be included within the scope of the invention.
Claims (20)
1. The yeast hydrolysate with high free nucleotides is characterized by comprising proteins, free nucleotides, amino acids and small peptides, wherein the content of the proteins is 60-75% by mass of dry matter of the yeast hydrolysate, the content of the free nucleotides is more than 35%, and the yeast hydrolysate is obtained by yeast milk enzymolysis, nucleotide acidification and nucleotide reinforcement.
2. The yeast hydrolysate according to claim 1, wherein the content of free nucleotides is above 40%, preferably 40.9-41.38%; the content of the protein is 66.72-74.17%.
3. The yeast hydrolysate according to claim 1 or 2, wherein the nucleotides comprise cytidylic acid, adenylic acid, guanylic acid, uridylic acid and inosinic acid, preferably, the cytidylic acid is present in an amount of 2.0 to 4.0% by mass, the sum of adenylic acid and inosinic acid is present in an amount of 15.0 to 20.0% by mass, the guanylic acid is present in an amount of 15.0 to 19.0% by mass, the uridylic acid is present in an amount of 3.0 to 5.0% by mass, more preferably, the cytidylic acid is present in an amount of 2.82 to 3.01% by mass, the sum of adenylic acid and inosinic acid is present in an amount of 17.28 to 18.48% by mass, the guanylic acid is present in an amount of 15.35 to 16.99% by mass, and the uridylic acid is present in an amount of 3.84.
4. The yeast hydrolysate according to any one of claims 1 to 3, wherein the yeast milk is obtained by fermentation of Saccharomyces cerevisiae FX-2 with a accession number CCTCC NO: m2016418.
5. A process for the preparation of a yeast hydrolysate according to any one of the claims 1 to 4 comprising the steps of:
(1) fermentation culture: fermenting and culturing a saccharomyces cerevisiae strain to obtain first yeast milk;
(2) autolysis and enzymolysis: performing hot shock treatment on the yeast milk of the first yeast milk accounting for 60-80 wt%, preferably 65-70 wt% in the step (1) to perform autolysis, and adding papain for enzymolysis to obtain second yeast milk;
(3) and (3) nucleotide acidification: adding yeast RNA and nuclease into the second yeast milk obtained in the step (2) for enzymolysis to obtain third yeast milk, preferably adding the yeast RNA firstly, and then adding the nuclease for enzymolysis;
(4) nucleotide fortification: and (4) adding disodium nucleotide salt into the third yeast milk obtained in the step (3) to obtain the yeast hydrolysate with high free nucleotides.
6. The production method according to claim 5, wherein, in the step (2), the temperature of the heat shock treatment is 85 to 90 ℃.
7. The production method according to claim 5 or 6, wherein, in the step (2), the time of the heat shock treatment is 50 to 60s, preferably 55 to 60 s.
8. The preparation method according to any one of claims 5 to 7, wherein in the step (2), citric acid is added after the heat shock treatment for heat preservation, preferably, the addition amount of the citric acid is 2 to 4 per thousand, preferably 3 to 4 per thousand, based on the mass of yeast milk dry matter.
9. The preparation method according to claim 8, wherein the heat preservation temperature of the heat preservation treatment is 40-55 ℃, preferably 40-50 ℃; the heat preservation time is 8-12h, preferably 8-10 h.
10. The preparation method according to any one of claims 5 to 9, wherein in the step (2), the addition amount of the papain is 1 to 5%, preferably 2 to 4%, and more preferably 3 to 3.4%, based on the mass of the yeast milk dry matter.
11. The preparation method according to any one of claims 5 to 10, wherein the papain has an enzymolysis temperature of 50 to 65 ℃, preferably 55 to 59 ℃, a pH of 5 to 6.5, preferably 5 to 6, and an enzymolysis time of 5 to 10 hours, preferably 6 to 8 hours.
12. The method according to any one of claims 5 to 11, wherein in step (3), the yeast RNA is added in an amount of 8 to 15%, preferably 10 to 12%, by mass of the dry matter of the second yeast milk.
13. The method according to any one of claims 5 to 12, wherein the yeast RNA is extracted from the yeast milk of step (1) which comprises 20 to 40 wt%, preferably 30 to 35 wt%, of the first yeast milk.
14. The preparation method according to any one of claims 5 to 13, wherein the nuclease is added in an amount of 3 to 8%, preferably 4 to 5.4%, more preferably 4.8 to 5.4%, by mass of the dry matter of the second yeast milk.
15. The method according to any one of claims 5 to 14, wherein the nuclease has an enzymatic hydrolysis temperature of 50 to 60 ℃, a pH of 5 to 6.5, and an enzymatic hydrolysis time of 10 to 20 hours, preferably 12 to 15 hours.
16. The preparation method according to any one of claims 5 to 15, wherein the disodium nucleotide salt is added in an amount of 20 to 30%, preferably 24 to 25%, by mass of the third yeast milk dry matter.
17. The production process according to any one of claims 5 to 16, wherein, in the step (1), the fermentation culture is fed with a carbon source, a nitrogen source and a phosphorus source in a fed-batch manner;
the carbon source is 9000g of 5000-;
the carbon source is molasses, more preferably 25-35% molasses;
the nitrogen source is selected from one of ammonium sulfate or ammonium nitrate, preferably ammonium sulfate;
the phosphorus source is selected from one of ammonium dihydrogen phosphate or potassium dihydrogen phosphate, and ammonium dihydrogen phosphate is preferred.
18. The preparation method according to claim 17, wherein, in the step (1), the fermentation temperature of the fermentation culture is 29-35 ℃, the fermentation time is 10-18 hours, and the fermentation pH is 4.5-6.4.
19. Use of a yeast hydrolysate according to any one of claims 1 to 4 or prepared by a method according to any one of claims 5 to 18 in an animal feed, preferably a young animal feed.
20. A feed comprising the yeast hydrolysate of any one of claims 1 to 4 or the yeast hydrolysate produced by the production method of any one of claims 5 to 18 and a basal diet, wherein the yeast hydrolysate is added in an amount of 0.2 to 1.0%, preferably 0.3 to 0.5%.
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