CN114958631A - Method for producing single-cell protein by using heavy-phase lactic acid - Google Patents
Method for producing single-cell protein by using heavy-phase lactic acid Download PDFInfo
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- CN114958631A CN114958631A CN202210526116.3A CN202210526116A CN114958631A CN 114958631 A CN114958631 A CN 114958631A CN 202210526116 A CN202210526116 A CN 202210526116A CN 114958631 A CN114958631 A CN 114958631A
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- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 title claims abstract description 138
- 239000004310 lactic acid Substances 0.000 title claims abstract description 68
- 235000014655 lactic acid Nutrition 0.000 title claims abstract description 68
- 108010027322 single cell proteins Proteins 0.000 title claims abstract description 49
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 15
- 238000000855 fermentation Methods 0.000 claims abstract description 182
- 241000235015 Yarrowia lipolytica Species 0.000 claims abstract description 54
- 238000000034 method Methods 0.000 claims abstract description 31
- 241000233671 Schizochytrium Species 0.000 claims abstract description 17
- 239000006227 byproduct Substances 0.000 claims abstract description 9
- 239000002994 raw material Substances 0.000 claims abstract description 7
- 230000004151 fermentation Effects 0.000 claims description 180
- 239000001963 growth medium Substances 0.000 claims description 71
- 239000002609 medium Substances 0.000 claims description 43
- 239000007788 liquid Substances 0.000 claims description 35
- 241000003595 Aurantiochytrium limacinum Species 0.000 claims description 32
- 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 claims description 28
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 claims description 28
- 239000000047 product Substances 0.000 claims description 24
- 238000011081 inoculation Methods 0.000 claims description 22
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 claims description 22
- 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 21
- 239000008103 glucose Substances 0.000 claims description 21
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 17
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 17
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 14
- 240000008042 Zea mays Species 0.000 claims description 11
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 11
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 11
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 11
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 11
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 11
- LLSDKQJKOVVTOJ-UHFFFAOYSA-L calcium chloride dihydrate Chemical compound O.O.[Cl-].[Cl-].[Ca+2] LLSDKQJKOVVTOJ-UHFFFAOYSA-L 0.000 claims description 11
- 229940052299 calcium chloride dihydrate Drugs 0.000 claims description 11
- 229940041514 candida albicans extract Drugs 0.000 claims description 11
- 235000005822 corn Nutrition 0.000 claims description 11
- 239000004323 potassium nitrate Substances 0.000 claims description 11
- 235000010333 potassium nitrate Nutrition 0.000 claims description 11
- 239000012137 tryptone Substances 0.000 claims description 11
- 239000012138 yeast extract Substances 0.000 claims description 11
- 238000007599 discharging Methods 0.000 claims description 10
- 238000001914 filtration Methods 0.000 claims description 9
- 238000003756 stirring Methods 0.000 claims description 8
- 229910052751 metal Inorganic materials 0.000 claims description 6
- 238000005273 aeration Methods 0.000 claims description 5
- 229910019142 PO4 Inorganic materials 0.000 claims description 4
- 239000002184 metal Substances 0.000 claims description 4
- 239000010452 phosphate Substances 0.000 claims description 4
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 4
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 3
- 235000001727 glucose Nutrition 0.000 claims description 3
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 3
- 229940093928 potassium nitrate Drugs 0.000 claims description 3
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 3
- 150000003839 salts Chemical class 0.000 claims description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 claims description 2
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 claims description 2
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 claims description 2
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims description 2
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 claims description 2
- PWHULOQIROXLJO-UHFFFAOYSA-N Manganese Chemical compound [Mn] PWHULOQIROXLJO-UHFFFAOYSA-N 0.000 claims description 2
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 claims description 2
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 claims description 2
- 239000002253 acid Substances 0.000 claims description 2
- 150000001868 cobalt Chemical class 0.000 claims description 2
- 229910017052 cobalt Inorganic materials 0.000 claims description 2
- 239000010941 cobalt Substances 0.000 claims description 2
- GUTLYIVDDKVIGB-UHFFFAOYSA-N cobalt atom Chemical compound [Co] GUTLYIVDDKVIGB-UHFFFAOYSA-N 0.000 claims description 2
- 229910052802 copper Inorganic materials 0.000 claims description 2
- 150000001879 copper Chemical class 0.000 claims description 2
- 239000010949 copper Substances 0.000 claims description 2
- 239000011777 magnesium Substances 0.000 claims description 2
- 229910052749 magnesium Inorganic materials 0.000 claims description 2
- 159000000003 magnesium salts Chemical class 0.000 claims description 2
- 150000002696 manganese Chemical class 0.000 claims description 2
- 239000011591 potassium Substances 0.000 claims description 2
- 229910052700 potassium Inorganic materials 0.000 claims description 2
- XAEFZNCEHLXOMS-UHFFFAOYSA-M potassium benzoate Chemical compound [K+].[O-]C(=O)C1=CC=CC=C1 XAEFZNCEHLXOMS-UHFFFAOYSA-M 0.000 claims description 2
- 239000011734 sodium Substances 0.000 claims description 2
- 229910052708 sodium Inorganic materials 0.000 claims description 2
- 159000000000 sodium salts Chemical class 0.000 claims description 2
- 229910052748 manganese Inorganic materials 0.000 claims 1
- 239000011572 manganese Substances 0.000 claims 1
- 108090000623 proteins and genes Proteins 0.000 abstract description 8
- 102000004169 proteins and genes Human genes 0.000 abstract description 8
- 239000000126 substance Substances 0.000 abstract description 5
- 235000020660 omega-3 fatty acid Nutrition 0.000 abstract description 4
- 239000004626 polylactic acid Substances 0.000 abstract description 4
- CYQFCXCEBYINGO-IAGOWNOFSA-N delta1-THC Chemical compound C1=C(C)CC[C@H]2C(C)(C)OC3=CC(CCCCC)=CC(O)=C3[C@@H]21 CYQFCXCEBYINGO-IAGOWNOFSA-N 0.000 abstract description 3
- 239000000178 monomer Substances 0.000 abstract description 3
- 235000015097 nutrients Nutrition 0.000 abstract description 3
- 229940012843 omega-3 fatty acid Drugs 0.000 abstract description 3
- 229920000747 poly(lactic acid) Polymers 0.000 abstract description 3
- 239000013589 supplement Substances 0.000 abstract description 3
- 230000007812 deficiency Effects 0.000 abstract 1
- 238000002360 preparation method Methods 0.000 description 24
- 230000001954 sterilising effect Effects 0.000 description 22
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 18
- 238000012544 monitoring process Methods 0.000 description 18
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 12
- 238000001816 cooling Methods 0.000 description 10
- 241001052560 Thallis Species 0.000 description 8
- 238000012258 culturing Methods 0.000 description 8
- 239000000203 mixture Substances 0.000 description 8
- 238000000199 molecular distillation Methods 0.000 description 8
- GFHNAMRJFCEERV-UHFFFAOYSA-L cobalt chloride hexahydrate Chemical compound O.O.O.O.O.O.[Cl-].[Cl-].[Co+2] GFHNAMRJFCEERV-UHFFFAOYSA-L 0.000 description 7
- 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 7
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 7
- MHWHABOIZXUXES-UHFFFAOYSA-L manganese(II) sulfate hexahydrate Chemical compound O.O.O.O.O.O.[Mn+2].[O-]S([O-])(=O)=O MHWHABOIZXUXES-UHFFFAOYSA-L 0.000 description 7
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 6
- 229910021529 ammonia Inorganic materials 0.000 description 6
- 235000011114 ammonium hydroxide Nutrition 0.000 description 6
- 239000000919 ceramic Substances 0.000 description 6
- 239000000498 cooling water Substances 0.000 description 6
- 238000011049 filling Methods 0.000 description 6
- 238000010438 heat treatment Methods 0.000 description 6
- 239000012528 membrane Substances 0.000 description 6
- 238000005070 sampling Methods 0.000 description 6
- 238000001694 spray drying Methods 0.000 description 6
- 230000000052 comparative effect Effects 0.000 description 5
- 244000005700 microbiome Species 0.000 description 5
- 238000004659 sterilization and disinfection Methods 0.000 description 5
- 239000012530 fluid Substances 0.000 description 4
- 241001138401 Kluyveromyces lactis Species 0.000 description 3
- 241000186604 Lactobacillus reuteri Species 0.000 description 3
- 239000006052 feed supplement Substances 0.000 description 3
- 229940001882 lactobacillus reuteri Drugs 0.000 description 3
- 238000009423 ventilation Methods 0.000 description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- 230000006978 adaptation Effects 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 239000002054 inoculum Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000000049 pigment Substances 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 239000003674 animal food additive Substances 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 229920000704 biodegradable plastic Polymers 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004042 decolorization Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 229910000397 disodium phosphate Inorganic materials 0.000 description 1
- 235000019800 disodium phosphate Nutrition 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 235000010755 mineral Nutrition 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 238000001728 nano-filtration Methods 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 239000005416 organic matter Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 239000006041 probiotic Substances 0.000 description 1
- 235000018291 probiotics Nutrition 0.000 description 1
- 239000010453 quartz Substances 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
- 239000002910 solid waste Substances 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000011426 transformation method Methods 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
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- 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
- C12N1/14—Fungi; Culture media therefor
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- C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
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Abstract
The invention provides a method for producing single-cell protein by using heavy-phase lactic acid. The invention takes the black lactic acid as the raw material, ferments the yarrowia lipolytica to produce the single-cell protein, and in addition, adds the schizochytrium to carry out the co-fermentation, supplements the lacked natural omega-3 fatty acid and other nutrient substances, promotes the reasonable application of the black lactic acid, and also effectively supplements the deficiency in the aspect of feed protein resources. The method provides a green, environment-friendly and efficient solution for solving the problem that a large amount of black lactic acid byproducts are difficult to treat in the production process of the polylactic acid monomer.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for producing single-cell protein by using heavy-phase lactic acid.
Background
Lactic acid, chemically known as alpha-hydroxypropionic acid, is an important raw material widely used in the fields of food, pharmacy, organic synthesis and materials. The demand for biodegradable plastics is greatly increasing due to the increasing environmental problems caused by "white pollution", of which polylactic acid (PLA) polymerized from lactic acid as a monomer is one of the most important and broadest market. Since lactic acid monomers are required to have extremely high optical purity for the production of polylactic acid, the fermentation method is the mainstream method for the production of lactic acid. However, the fermentation of the microorganism needs to add nutrients such as yeast powder and inorganic salts, and the metabolic products in the process are complex, and besides lactic acid, a large amount of proteins, pigments, heteroacids, fusel and the like exist, which affect the chemical purity of the lactic acid, and further affect the quality and application performance of the polylactic acid. In the lactic acid industry which is currently industrialized, most processes of plate-and-frame filtration, activated carbon decolorization, nanofiltration, scraper concentration and molecular distillation are adopted to obtain polymer-grade lactic acid, however, in the process of molecular distillation, a part of lactic acid and oligomers thereof, and impurities such as pigments, saccharides, proteins and the like in original fermentation liquor can remain in heavy components, and about 15% of viscous black byproducts (also called black lactic acid or red lactic acid) are generated. Generally, the black lactic acid can only be used as a feed additive, but with the increasing requirements on the quality of raw materials in the feed field at present, the application of the black lactic acid as a byproduct is more and more limited, so that a proper method needs to be developed to reasonably treat the black lactic acid, reduce solid waste pollution and promote reasonable utilization of resources.
Because the organic matter content in the black lactic acid is rich, the microbial transformation method can utilize the organic by-products in the black lactic acid to carry out thallus growth and amplification, and the obtained microbial thallus is called as single-cell protein. The protein content is high (50-80%), the amino acid content is rich, the protein contains microorganisms, mineral substances and other bioactive substances, the nutritive value is high, and the protein can be used as a good protein source in feed. The single cell protein is produced by using the black lactic acid as a fermentation raw material, so that the black lactic acid byproduct in the lactic acid preparation process can be well treated, the single cell protein product with high added value can be produced, and the increasing demand of feed protein is met.
Disclosure of Invention
The invention aims to provide a method for producing single-cell protein by using heavy-phase lactic acid, which can convert black lactic acid at a high value, and the produced single-cell protein can be used as an excellent feed protein source, thereby improving the commercial value of a lactic acid industrial chain.
In order to achieve the purpose of the invention, the invention adopts the following technical scheme:
a method for producing single-cell protein by using heavy-phase lactic acid, which takes the heavy-phase lactic acid as a raw material and prepares the single-cell protein by mixed fermentation of two strains of yarrowia lipolytica and schizochytrium; wherein the heavy phase lactic acid is a heavy component byproduct formed in the production process of lactic acid.
Aiming at the problem that the black lactic acid byproduct is difficult to treat, the black lactic acid is used as a raw material, the yarrowia lipolytica is fermented to produce single-cell protein, and in addition, schizochytrium and other probiotics are added for co-fermentation; the yarrowia lipolytica can metabolize and grow by using the black lactic acid, and simultaneously convert the black lactic acid into an organic component which can be directly absorbed by the schizochytrium, so that the schizochytrium can quickly grow, secrete natural omega-3 fatty acid, and supplement omega-3 and other nutrient substances which are lacked in the yarrowia lipolytica single-cell protein; and the schizochytrium can also consume byproducts generated by the metabolism of the yarrowia lipolytica, so that the purity of the product is improved.
In one embodiment, the method for producing single-cell protein comprises adding heavy-phase lactic acid to a single-cell protein culture medium, then adding yarrowia lipolytica and schizochytrium limacinum, fermenting to obtain a fermentation liquid rich in single-cell protein, and separating to obtain single-cell protein product.
In the present invention, the yarrowia lipolytica is cultured in the seed solution before the fermentation in the upper tank; preferably, the culture medium of the yarrowia lipolytica is one or more of glucose, ammonium sulfate, potassium dihydrogen phosphate, magnesium sulfate heptahydrate, tryptone; preferably, the culture medium of the yarrowia lipolytica is 3-20 g/L of glucose, 0.5-4 g/L of ammonium sulfate, 0.5-3 g/L of monopotassium phosphate, 0.1-1 g/L of magnesium sulfate heptahydrate and 5-20 g/L of tryptone.
In the invention, the culture temperature of the yarrowia lipolytica seed liquid is 25-40 ℃, and the culture time is 16-48 h.
In the invention, the schizochytrium limacinum is cultured with seed liquid before in-tank fermentation; preferably, the schizochytrium culture medium is one or more of glucose, yeast extract, corn steep liquor, potassium nitrate, magnesium sulfate heptahydrate and calcium chloride dihydrate; preferably, the schizochytrium culture medium is 10-50 g/L of glucose, 1-20 g/L of yeast extract, 0.1-3 g/L of corn steep liquor, 0.2-3 g/L of potassium nitrate, 0.2-2 g/L of magnesium sulfate heptahydrate and 0.05-0.2 g/L of calcium chloride dihydrate.
In the invention, the culture temperature of the schizochytrium limacinum seed liquid is 20-30 ℃, and the culture time is 24-72 h.
In the invention, the culture medium in the fermentation process contains one or more of potassium, sodium, magnesium, copper, cobalt and manganese metal elements; preferably, the metal element is in the form of one or more of phosphate, acid phosphate, chloride, sulfate, citrate; preferably, the addition amount of the metal element salt is 5-20 g/L of potassium salt, 10-20 g/L of sodium salt, 0.5-4 g/L of magnesium salt, 0.1-2 g/L of copper salt, 0.01-0.1 mg/L of cobalt salt and 0.001-0.02 mg/L of manganese salt.
In the invention, the method adds heavy phase lactic acid into a fermentation medium; preferably, the added heavy phase lactic acid accounts for 1-10 wt% of the total mass of the culture medium, and preferably 3-7 wt%.
In the present invention, the yarrowia lipolytica is inoculated in the fermentation medium in an amount of 5% to 20%, preferably 10% to 17%, by volume of the medium.
In the present invention, the culture temperature after inoculation of yarrowia lipolytica is 25 to 40 ℃, preferably 28 to 37 ℃.
In the invention, the inoculation amount of the schizochytrium limacinum in the fermentation medium is 10-20% of the volume of the medium, and preferably 13-18%.
In the invention, the culture temperature after the inoculation of the schizochytrium limacinum is 20-30 ℃, and the preferable temperature is 23-28 ℃.
In the invention, the pH value in the fermentation process is 6.0-8.0, preferably 6.5-7.4, the fermentation ventilation amount is 0.5-3.5 vvm, preferably 1.0-2.5 vvm, and the stirring speed is 20-500 rpm, preferably 100-300 rpm.
In the invention, in the fermentation process, the discharging is started when the OD value of the thallus of the fermentation liquor reaches 100-150, and preferably, the discharging is started when the OD value reaches 120-130.
In the invention, the fermentation liquor is discharged from the fermentation tank, the clear solution returns to the fermentation tank for continuous fermentation after filtration, and the concentrated solution is dried to obtain the single-cell protein product.
In the present invention, the continuous fermentation operation period is 60 to 90 days.
In the invention, the unit yield of the single-cell protein product is 2-4 g/L/h based on dry weight.
Another object of the present invention is to provide a single-cell protein.
A single-cell protein is prepared by the method for producing the single-cell protein by using the heavy-phase lactic acid.
Compared with the prior art, the invention has the advantages that:
(1) the invention can efficiently produce the single-cell protein product rich in natural omega-3 fatty acid, and the unit yield of the single-cell protein product can reach 4 g/L/h.
(2) The invention has simple process, simple and convenient and easily obtained materials and fully utilizes waste.
Detailed Description
In order to further illustrate the method for culturing single-cell protein by using black lactic acid provided by the present invention, the present invention is illustrated in detail by the following examples, but the present invention is not limited thereto.
Yarrowia lipolytica, accession number CICC 32520, purchased from China center for Industrial culture Collection of microorganisms;
schizochytrium limacinum, deposit number ATCC 20888, purchased from american type strain collection;
kluyveromyces lactis with the preservation number CICC 32413 purchased from China center for Collection of Industrial microorganism strains
Lactobacillus reuteri with the collection number CICC 6128, purchased from China center for culture Collection of Industrial microorganisms
Black lactic acid, wanhua chemistry;
glucose, ammonium sulfate, potassium dihydrogen phosphate, magnesium sulfate heptahydrate, potassium nitrate, magnesium sulfate heptahydrate, calcium chloride dihydrate, disodium hydrogen phosphate and copper sulfate pentahydrate by 1.8g/L, wherein cobalt chloride hexahydrate and manganese sulfate hexahydrate are analytically pure and purchased from Tianjin Kemiou chemical reagent Limited;
tryptone, yeast extract, corn steep liquor were chemically pure and purchased from Angel Yeast, Inc.
And (3) OD value detection adopts an ultraviolet spectrophotometry, the fermentation liquor is diluted to a proper multiple, the fermentation liquor is transferred to a quartz cuvette, and an OD value under 600nm is detected by using a spectrophotometer. The ultraviolet spectrophotometer is available from Shanghai spectral element instruments Inc., model number Alpha-1500.
Example 1
(1) Preparation and culture of seed liquid
In a shake flask, a seed solution suitable for yarrowia lipolytica was prepared, and the formulation of 1L of medium was: 17g/L glucose, 0.77g/L ammonium sulfate, 2g/L potassium dihydrogen phosphate, 0.1g/L magnesium sulfate heptahydrate and 8g/L tryptone, wherein after the preparation of the culture medium is finished, the culture medium is sterilized at the high temperature of 121 ℃ for 30min, and is kept stand and cooled to about 30 ℃. Inoculating yarrowia lipolytica into the culture medium under aseptic operation condition, and shaking culturing at 200rpm in a shaker at 30 ℃ for 36h to obtain yarrowia lipolytica seed liquid.
Preparing seed liquid suitable for schizochytrium in a shake flask, wherein the formula of a 2L culture medium is as follows: 25g/L of glucose, 10g/L of yeast extract, 1.9g/L of corn steep liquor, 1.3g/L of potassium nitrate, 0.2g/L of magnesium sulfate heptahydrate and 0.05g/L of calcium chloride dihydrate. Sterilizing at 121 deg.C for 30min, standing, and cooling to about 30 deg.C. Inoculating the schizochytrium limacinum to the culture medium under the aseptic operation condition, and performing shake culture for 72h in a shaking table at the temperature of 28 ℃ and the rpm of 200 to obtain the schizochytrium limacinum seed liquid.
(2) Preparation of fermentation medium
The formula of the 12L fermentation medium is as follows: 13g/L of potassium dihydrogen phosphate, 16g/L of disodium hydrogen phosphate, 3.7g/L of magnesium sulfate heptahydrate, 1.8g/L of copper sulfate pentahydrate, 0.01mg/L of cobalt chloride hexahydrate and 0.01mg/L of manganese sulfate hexahydrate. And 95.25g of molecular distillation heavy component black lactic acid was added to make the composition of black lactic acid in the total medium 8.7 wt%. The pH was adjusted to 6.8 with pH monitoring by addition of ammonia. And (3) filling 12L of the fermentation medium into a 15L fermentation tank, sterilizing in a jacket steam heating mode for 30min at 121 ℃, opening a jacket cooling water inlet valve to start automatic temperature control after sterilization is finished, and preparing for inoculation until the temperature of the medium in the fermentation tank is reduced to 40 ℃.
(3) Supplemented culture
Inoculating the yarrowia lipolytica seed solution cultured in the step (1) into a culture medium according to the inoculation amount of 5% (v/v), starting culture, starting automatic monitoring of the pH of a fermentation tank in the culture process, adjusting the pH to be stable at 6.8 by using ammonia water and dilute acetic acid, adjusting the fermentation aeration quantity to be 1.8vvm, stirring the fermentation speed to be 250rpm, the culture temperature to be 40 ℃, sampling at regular time to detect the OD value of fermentation liquor, inoculating the schizochytrium limacinum seed solution into the fermentation tank according to the inoculation amount of 10% (v/v) when the OD value reaches 60, adjusting the fermentation temperature to be 20 ℃, and starting mixed culture of two thalli. Monitoring the OD value of the fermentation liquor at any time in the culture process, starting a discharge valve on the fermentation tank when the OD value reaches 100, discharging the fermentation liquor at a discharge speed of 40mL/min, filtering the fermentation liquor by a 200nm sterile ceramic membrane, and returning the clear solution to the fermentation tank for continuous fermentation; collecting the concentrated solution, and spray drying (inlet temperature of 120 deg.C, feed flow of 200mL/min) to obtain dry thallus, i.e. single cell protein product. While the fermentation liquid is discharged from the fermentation tank, a supplementary culture medium (containing black lactic acid) is prepared according to the same formula as the fermentation culture medium and is sterilized at high temperature, and the supplementary culture medium is introduced into the fermentation tank through a supplementary valve on the fermentation tank, so that the liquid level in the fermentation tank is kept stable at 60%.
The continuous fermentation culture time is 90 days, and the unit yield of the single-cell protein product is 2.5g/L/h on a dry weight basis.
Example 2
(1) Preparation and culture of seed liquid
In a shake flask, a seed solution suitable for yarrowia lipolytica was prepared, and the 2L medium formulation was: 20g/L glucose, 1.67g/L ammonium sulfate, 1.5g/L potassium dihydrogen phosphate, 0.7g/L magnesium sulfate heptahydrate and 20g/L tryptone, and sterilizing at 121 ℃ for 30min after the preparation of the culture medium, standing and cooling to about 30 ℃. Inoculating yarrowia lipolytica into the culture medium under aseptic operation condition, and shaking culturing in a shaker at 25 deg.C and 200rpm for 16h to obtain yarrowia lipolytica seed solution.
Preparing a seed solution suitable for schizochytrium in a shake flask, wherein the formula of a 2L culture medium is as follows: 45g/L of glucose, 4g/L of yeast extract, 2.6g/L of corn steep liquor, 2g/L of potassium nitrate, 1.5g/L of magnesium sulfate heptahydrate and 0.08g/L of calcium chloride dihydrate. After the preparation of the culture medium is finished, sterilizing at the high temperature of 121 ℃ for 30min, standing and cooling to about 30 ℃. Inoculating the schizochytrium limacinum to the culture medium under the aseptic operation condition, and performing shake culture in a shaking table at 23 ℃ and 200rpm for 60 hours to obtain a schizochytrium limacinum seed solution.
(2) Preparation of fermentation medium
Preparing a fermentation medium of yarrowia lipolytica, wherein the formula is as follows: 20g/L of monopotassium phosphate, 20g/L of disodium phosphate, 3g/L of magnesium sulfate heptahydrate, 0.4g/L of copper sulfate pentahydrate, 0.05mg/L of cobalt chloride hexahydrate and 0.001mg/L of manganese sulfate hexahydrate. And 24.59g of molecular distillation heavy component black lactic acid was added to make the composition of black lactic acid in the whole medium 2.4 wt%. The pH was adjusted to 6.5 with pH monitoring by addition of ammonia. And (3) filling 12L of the fermentation medium into a 15L fermentation tank, sterilizing in a jacket steam heating mode for 30min at 121 ℃, opening a jacket cooling water inlet valve to start automatic temperature control after sterilization is finished, and preparing for inoculation until the temperature of the medium in the fermentation tank is reduced to about 25 ℃.
(3) Supplemented culture
Inoculating the yarrowia lipolytica seed solution cultured in the step (1) into a culture medium according to the inoculum size of 16% (v/v), starting culture, starting automatic monitoring of the pH of a fermentation tank in the culture process, adjusting the pH to be stabilized at about 6.5 by using ammonia water or dilute acetic acid, adjusting the fermentation ventilation volume to be 2vvm, stirring at the rotating speed of 300rpm, the culture temperature to be 25 ℃, sampling at regular time and detecting the OD value of the fermentation liquid, inoculating the schizochytrium limacinum seed solution into the fermentation tank according to the inoculum size of 14% (v/v) when the OD value reaches 50, adjusting the fermentation temperature to be 25 ℃, and starting mixed culture of two thalli. Monitoring the OD value of the fermentation liquor at any time in the culture process, opening a discharge valve on the fermentation tank when the OD value reaches 120, discharging the fermentation liquor at a discharge speed of 60mL/min, filtering the fermentation liquor by a 200nm sterile ceramic membrane, and returning the clear solution to the fermentation tank for continuous fermentation; collecting the concentrated solution, and obtaining dry thalli by a spray drying mode (the inlet temperature is 120 ℃, and the feed flow is 200mL/min), namely a single-cell protein product. While the fermentation liquid is discharged from the fermentation tank, a supplementary culture medium (containing black lactic acid) is prepared according to the same formula as the fermentation culture medium and is sterilized at high temperature, and the supplementary culture medium is introduced into the fermentation tank through a supplementary valve on the fermentation tank, so that the liquid level in the fermentation tank is kept stable at about 60 percent.
The continuous fermentation culture time is 60 days, and the unit yield of the single-cell protein product is 3g/L/h on a dry weight basis.
Example 3
(1) Preparation and culture of seed liquid
Preparing a seed solution suitable for yarrowia lipolytica in a shake flask, wherein the formula of a 2L culture medium is as follows: 3g/L glucose, 4g/L ammonium sulfate, 0.5g/L potassium dihydrogen phosphate, 0.5g/L magnesium sulfate heptahydrate, 12g/L tryptone, and sterilizing at 121 ℃ for 30min after the preparation of the culture medium, standing and cooling to about 30 ℃. Inoculating yarrowia lipolytica to the culture medium under aseptic operation condition, and shaking culturing at 200rpm in a shaker at 35 deg.C for 48h to obtain yarrowia lipolytica seed solution.
Preparing a seed solution suitable for schizochytrium in a shake flask, wherein the formula of a 3L culture medium is as follows: 30g/L of glucose, 20g/L of yeast extract, 0.1g/L of corn steep liquor, 3g/L of potassium nitrate, 1g/L of magnesium sulfate heptahydrate and 0.14g/L of calcium chloride dihydrate. After the preparation of the culture medium is finished, sterilizing at the high temperature of 121 ℃ for 30min, standing and cooling to about 30 ℃. Inoculating the schizochytrium limacinum to the culture medium under the aseptic operation condition, and performing shake culture in a shaker at 30 ℃ and 200rpm for 24h to obtain a schizochytrium limacinum seed solution.
(2) Preparation of fermentation medium
Preparing a fermentation medium of yarrowia lipolytica, wherein the formula is as follows: 8g/L potassium dihydrogen phosphate, 12g/L disodium hydrogen phosphate, 4g/L magnesium sulfate heptahydrate, 0.1g/L copper sulfate pentahydrate, 0.08mg/L cobalt chloride hexahydrate and 0.02mg/L manganese sulfate hexahydrate. And 71.81g of molecular distillation heavy component black lactic acid was added so that the composition of the black lactic acid in the whole medium was 6.7 wt%. The pH was adjusted to 7.4 with pH monitoring by addition of ammonia. Filling 12L of the fermentation medium into a 15L fermentation tank, sterilizing by jacket steam heating at 121 deg.C for 30min, opening a jacket cooling water inlet valve to automatically control temperature until the temperature of the medium in the fermentation tank is reduced to about 27 deg.C, and preparing for inoculation.
(3) Supplemented culture
Inoculating the yarrowia lipolytica seed solution cultured in the step (1) into a culture medium according to the inoculation amount of 9% (v/v), starting culture, starting automatic monitoring of the pH of a fermentation tank in the culture process, adjusting the pH to be stabilized at about 7.4 by using ammonia water or dilute acetic acid, adjusting the fermentation aeration quantity to be 2.5vvm, stirring the fermentation speed to be 200rpm, the culture temperature to be 27 ℃, sampling at regular time to detect the OD value of the fermentation liquid, inoculating the schizochytrium limacinum seed solution into the fermentation tank according to the inoculation amount of 18% (v/v) when the OD value reaches 70, adjusting the fermentation temperature to be 30 ℃, and starting mixed culture of two thalli. Monitoring the OD value of the fermentation liquor at any time in the culture process, opening a discharge valve on the fermentation tank when the OD value reaches 130, discharging the fermentation liquor at a discharge rate of 25mL/min, filtering the fermentation liquor by a 200nm sterile ceramic membrane, and returning the clear solution to the fermentation tank for continuous fermentation; collecting the concentrated solution, and spray drying (inlet temperature of 120 deg.C, feed flow of 200mL/min) to obtain dry thallus, i.e. single cell protein product. And (3) preparing a feed supplement culture medium (containing black lactic acid) according to the same formula as the fermentation culture medium while discharging the fermentation liquid from the fermentation tank, sterilizing at high temperature, introducing the feed supplement culture medium into the fermentation tank through a feed supplement valve on the fermentation tank, and keeping the liquid level in the fermentation tank stable at about 60 percent.
The continuous fermentation culture time is 80 days, and the unit yield of the single-cell protein product is 2g/L/h on a dry weight basis.
Example 4
(1) Preparation and culture of seed liquid
Preparing a seed solution suitable for yarrowia lipolytica in a shake flask, wherein the formula of a 3L culture medium is as follows: 9g/L glucose, 3.5g/L ammonium sulfate, 2.4g/L potassium dihydrogen phosphate, 1g/L magnesium sulfate heptahydrate and 17g/L tryptone, after the preparation of the culture medium is finished, sterilizing at the high temperature of 121 ℃ for 30min, standing and cooling to about 30 ℃. Inoculating yarrowia lipolytica into the culture medium under aseptic operation condition, and shaking culturing in a shaker at 40 deg.C and 200rpm for 42h to obtain yarrowia lipolytica seed solution.
Preparing a seed solution suitable for schizochytrium in a shake flask, wherein the formula of a 2L culture medium is as follows: 10g/L of glucose, 8g/L of yeast extract, 2.5g/L of corn steep liquor, 0.2g/L of potassium nitrate, 2g/L of magnesium sulfate heptahydrate and 0.09g/L of calcium chloride dihydrate. After the preparation of the culture medium is finished, sterilizing at the high temperature of 121 ℃ for 30min, standing and cooling to about 30 ℃. Inoculating the schizochytrium limacinum to the culture medium under the aseptic operation condition, and performing shake culture for 45h in a shaking table at 20 ℃ and 200rpm to obtain a schizochytrium limacinum seed solution.
(2) Preparation of fermentation medium
Preparing a fermentation medium of yarrowia lipolytica, wherein the formula is as follows: 16g/L potassium dihydrogen phosphate, 10g/L disodium hydrogen phosphate, 2.4g/L magnesium sulfate heptahydrate, 1.5g/L copper sulfate pentahydrate, 0.1mg/L cobalt chloride hexahydrate and 0.0015mg/L manganese sulfate hexahydrate. And 52.63g of molecular distillation heavy component black lactic acid was added so that the composition of the black lactic acid in the whole medium was 5 wt%. The pH was adjusted to 7.0 with pH monitoring by addition of ammonia. And (3) filling 12L of the fermentation medium into a 15L fermentation tank, sterilizing in a jacket steam heating mode for 30min at 121 ℃, opening a jacket cooling water inlet valve to start automatic temperature control after sterilization is finished, and preparing for inoculation until the temperature of the medium in the fermentation tank is reduced to about 37 ℃.
(3) Supplemented culture
Inoculating the yarrowia lipolytica seed solution cultured in the step (1) into a culture medium according to the inoculation amount of 20% (v/v), starting culture, starting automatic monitoring of the pH of a fermentation tank in the culture process, adjusting the pH to be stabilized at about 7.0 by using ammonia water or dilute acetic acid, adjusting the fermentation aeration quantity to be 1.5vvm, stirring at 150rpm, culturing at 37 ℃, sampling at regular time and detecting the OD value of the fermentation liquid, inoculating the schizochytrium limacinum seed solution into the fermentation tank according to the inoculation amount of 10% (v/v) when the OD value reaches 65, adjusting the fermentation temperature to be 27 ℃, and starting mixed culture of two thalli. Monitoring the OD value of the fermentation liquor at any time in the culture process, opening a discharge valve on the fermentation tank when the OD value reaches 150, discharging the fermentation liquor at a discharge rate of 45mL/min, filtering the fermentation liquor by a 200nm sterile ceramic membrane, and returning the clear solution to the fermentation tank for continuous fermentation; collecting the concentrated solution, and obtaining dry thalli by a spray drying mode (the inlet temperature is 120 ℃, and the feed flow is 200mL/min), namely a single-cell protein product. While the fermentation liquid is discharged from the fermentation tank, a supplementary culture medium (containing black lactic acid) is prepared according to the same formula as the fermentation culture medium and is sterilized at high temperature, and the supplementary culture medium is introduced into the fermentation tank through a supplementary valve on the fermentation tank, so that the liquid level in the fermentation tank is kept stable at about 60 percent.
The continuous fermentation culture time is 75 days, and the unit yield of the single-cell protein product is 3.5g/L/h on a dry weight basis.
Example 5
(1) Preparation and culture of seed liquid
Preparing a seed solution suitable for yarrowia lipolytica in a shake flask, wherein the formula of a 2L culture medium is as follows: 12g/L glucose, 0.5g/L ammonium sulfate, 0.75g/L potassium dihydrogen phosphate, 0.36g/L magnesium sulfate heptahydrate, 5g/L tryptone, and the preparation of the culture medium, sterilizing at 121 ℃ for 30min, standing and cooling to about 30 ℃. Inoculating yarrowia lipolytica into the culture medium under aseptic operation condition, and shaking culturing at 200rpm in a shaker at 32 ℃ for 24h to obtain yarrowia lipolytica seed liquid.
Preparing a seed solution suitable for schizochytrium in a shake flask, wherein the formula of a 3L culture medium is as follows: 50g/L of glucose, 1g/L of yeast extract, 3g/L of corn steep liquor, 1.5g/L of potassium nitrate, 0.8g/L of magnesium sulfate heptahydrate and 0.18g/L of calcium chloride dihydrate. After the preparation of the culture medium is finished, sterilizing at the high temperature of 121 ℃ for 30min, standing and cooling to about 30 ℃. Inoculating the schizochytrium limacinum to the culture medium under the aseptic operation condition, and performing shake culture for 36h in a shaker at 25 ℃ and 200rpm to obtain a schizochytrium limacinum seed solution.
(2) Preparation of fermentation medium
Preparing a fermentation medium of yarrowia lipolytica, wherein the formula is as follows: 5g/L of potassium dihydrogen phosphate, 18g/L of disodium hydrogen phosphate, 0.9g/L of magnesium sulfate heptahydrate, 2g/L of copper sulfate pentahydrate, 0.07mg/L of cobalt chloride hexahydrate and 0.005mg/L of manganese sulfate hexahydrate. And 111.11g of molecular distillation heavy component black lactic acid was added to make the composition of black lactic acid in the total medium 10 wt%. The pH was adjusted to 7.2 with pH monitoring by addition of ammonia. And (3) filling 12L of the fermentation medium into a 15L fermentation tank, sterilizing in a jacket steam heating mode for 30min at 121 ℃, opening a jacket cooling water inlet valve to start automatic temperature control after sterilization is finished, and preparing for inoculation until the temperature of the medium in the fermentation tank is reduced to about 34 ℃.
(3) Supplemented culture
Inoculating the yarrowia lipolytica seed solution cultured in the step (1) into a culture medium according to the inoculation amount of 12% (v/v), starting culture, starting automatic monitoring of the pH of a fermentation tank in the culture process, adjusting the pH to be stabilized at about 7.2 by using ammonia water or dilute acetic acid, adjusting the fermentation aeration quantity to be 1.2vvm, stirring the fermentation speed to be 300rpm, the culture temperature to be 34 ℃, sampling at regular time to detect the OD value of the fermentation liquid, inoculating the schizochytrium limacinum seed solution into the fermentation tank according to the inoculation amount of 18% (v/v) when the OD value reaches 40, adjusting the fermentation temperature to be 22 ℃, and starting mixed culture of two thalli. Monitoring the OD value of the fermentation liquor at any time in the culture process, opening a discharge valve on the fermentation tank when the OD value reaches 120, discharging the fermentation liquor at a discharge rate of 10mL/min, filtering the fermentation liquor by a 200nm sterile ceramic membrane, and returning the clear solution to the fermentation tank for continuous fermentation; collecting the concentrated solution, and spray drying (inlet temperature of 120 deg.C, feed flow of 200mL/min) to obtain dry thallus, i.e. single cell protein product. While the fermentation liquid is discharged from the fermentation tank, a supplementary culture medium (containing black lactic acid) is prepared according to the same formula as the fermentation culture medium and is sterilized at high temperature, and the supplementary culture medium is introduced into the fermentation tank through a supplementary valve on the fermentation tank, so that the liquid level in the fermentation tank is kept stable at about 60 percent.
The continuous fermentation culture time is 68 days, and the unit yield of the single-cell protein product is 4g/L/h on a dry weight basis.
Example 6
(1) Preparation and culture of seed liquid
Preparing a seed solution suitable for yarrowia lipolytica in a shake flask, wherein the formula of a 1L culture medium is as follows: 10g/L glucose, 2.4g/L ammonium sulfate, 3g/L potassium dihydrogen phosphate, 0.73g/L magnesium sulfate heptahydrate and 10g/L tryptone, wherein after the preparation of the culture medium is finished, the culture medium is sterilized at the high temperature of 121 ℃ for 30min, and is kept stand and cooled to about 30 ℃. Inoculating yarrowia lipolytica to the culture medium under aseptic operation condition, and shaking culturing at 37 deg.C in shaker at 200rpm for 30h to obtain yarrowia lipolytica seed solution.
Preparing seed liquid suitable for schizochytrium in a shake flask, wherein the formula of a 2L culture medium is as follows: 40g/L of glucose, 16g/L of yeast extract, 1.8g/L of corn steep liquor, 2.4g/L of potassium nitrate, 0.7g/L of magnesium sulfate heptahydrate and 2g/L of calcium chloride dihydrate. After the preparation of the culture medium is finished, sterilizing at the high temperature of 121 ℃ for 30min, standing and cooling to about 30 ℃. Inoculating the schizochytrium limacinum to the culture medium under the aseptic operation condition, and performing shake culture in a shaking table at the temperature of 22 ℃ and the rpm of 200 for 48 hours to obtain the schizochytrium limacinum seed liquid.
(2) Preparation of fermentation medium
Preparing a fermentation medium of yarrowia lipolytica, wherein the formula is as follows: 10g/L of potassium dihydrogen phosphate, 14g/L of disodium hydrogen phosphate, 0.5g/L of magnesium sulfate heptahydrate, 1g/L of copper sulfate pentahydrate, 0.06mg/L of cobalt chloride hexahydrate and 0.017mg/L of manganese sulfate hexahydrate. And 10.10g of molecular distillation heavy component black lactic acid was added so that the composition of the black lactic acid in the whole medium was 1 wt%. Ammonia was added to adjust the pH to 7.0 with pH monitoring. And (3) filling 12L of the fermentation medium into a 15L fermentation tank, sterilizing in a jacket steam heating mode for 30min at 121 ℃, opening a jacket cooling water inlet valve to start automatic temperature control after sterilization is finished, and preparing for inoculation until the temperature of the medium in the fermentation tank is reduced to about 30 ℃.
(3) Supplemented culture
Inoculating the yarrowia lipolytica seed solution cultured in the step (1) into a culture medium according to the inoculation amount of 8% (v/v), starting culture, starting automatic monitoring of the pH of a fermentation tank in the culture process, adjusting the pH to be stabilized at about 7.0 by using ammonia water or dilute acetic acid, adjusting the fermentation ventilation amount to be 1.0vvm, stirring at the rotating speed of 100rpm, at the culture temperature of 30 ℃, sampling at regular time and detecting the OD value of the fermentation liquid, inoculating the schizochytrium limacinum seed solution into the fermentation tank according to the inoculation amount of 15% (v/v) when the OD value reaches 30, adjusting the fermentation temperature to be 28 ℃, and starting mixed culture of two thalli. Monitoring the OD value of the fermentation liquor at any time in the culture process, starting a discharge valve on the fermentation tank when the OD value reaches 100, discharging the fermentation liquor at a discharge speed of 50mL/min, filtering the fermentation liquor by a 200nm sterile ceramic membrane, and returning the clear solution to the fermentation tank for continuous fermentation; collecting the concentrated solution, and spray drying (inlet temperature of 120 deg.C, feed flow of 200mL/min) to obtain dry thallus, i.e. single cell protein product. While the fermentation liquid is discharged from the fermentation tank, a supplementary culture medium (containing black lactic acid) is prepared according to the same formula as the fermentation culture medium and is sterilized at high temperature, and the supplementary culture medium is introduced into the fermentation tank through a supplementary valve on the fermentation tank, so that the liquid level in the fermentation tank is kept stable at about 60 percent.
The continuous fermentation culture time is 74 days, and the unit yield of the single-cell protein product is 3.8g/L/h on a dry weight basis.
Comparative example 1
As in example 1, except that only yarrowia lipolytica seed fluid was inoculated, the yield of single-cell protein product was 1.2 g/L/h.
Comparative example 2
The difference from the example 1 is that only the schizochytrium limacinum seed solution is inoculated, the growth condition of the thallus is poor, the OD value is only 14 when the thallus is put into a tank, and no single-cell protein product is produced.
Comparative example 3
The same as example 1, except that the yarrowia lipolytica seed solution was replaced with the same volume of Kluyveromyces lactis seed solution, and the yield of single-cell protein was 0.3g/L/h by normal inoculation of the seed solution of Schizochytrium limacinum.
Comparative example 4
As in example 1, yarrowia lipolytica seed fluid was inoculated normally, except that the Schizochytrium limacinum seed fluid was replaced with the same volume of Lactobacillus reuteri seed fluid, and the single-cell protein yield was 1.6 g/L/h.
Comparative example 5
The same as example 1, except that the yarrowia lipolytica seed solution was replaced with the same volume of Kluyveromyces lactis seed solution, and the Schizochytrium limacinum seed solution was replaced with the same volume of Lactobacillus reuteri seed solution, the single-cell protein yield was 0.7 g/L/h.
It will be appreciated by those skilled in the art that modifications and adaptations to the invention may be made in light of the teachings of the present disclosure. Such modifications or adaptations are intended to be within the scope of the present invention as defined in the claims.
Claims (9)
1. A method for producing single-cell protein by using heavy-phase lactic acid is characterized in that the method takes the heavy-phase lactic acid as a raw material, and prepares the single-cell protein by mixed fermentation of yarrowia lipolytica and schizochytrium limacinum;
wherein the heavy phase lactic acid is a heavy component byproduct formed in the production process of lactic acid.
2. The method of claim 1, wherein yarrowia lipolytica cultures the seed solution prior to in-tank fermentation;
preferably, the culture medium of yarrowia lipolytica is one or more of glucose, ammonium sulfate, potassium dihydrogen phosphate, magnesium sulfate heptahydrate, tryptone;
preferably, the culture medium of the yarrowia lipolytica is 3-20 g/L of glucose, 0.5-4 g/L of ammonium sulfate, 0.5-3 g/L of monopotassium phosphate, 0.1-1 g/L of magnesium sulfate heptahydrate and 5-20 g/L of tryptone;
and/or the culture temperature of the yarrowia lipolytica seed solution is 25-40 ℃, and the culture time is 16-48 h.
3. The method of claim 1 or 2, wherein the schizochytrium limacinum cultures seed solution prior to tank fermentation;
preferably, the schizochytrium culture medium is one or more of glucose, yeast extract, corn steep liquor, potassium nitrate, magnesium sulfate heptahydrate and calcium chloride dihydrate;
preferably, the schizochytrium culture medium is 10-50 g/L of glucose, 1-20 g/L of yeast extract, 0.1-3 g/L of corn steep liquor, 0.2-3 g/L of potassium nitrate, 0.2-2 g/L of magnesium sulfate heptahydrate and 0.05-0.2 g/L of calcium chloride dihydrate;
and/or the culture temperature of the schizochytrium limacinum seed liquid is 20-30 ℃, and the culture time is 24-72 h.
4. The method according to any one of claims 1 to 3, wherein the medium of the fermentation process contains one or more of the metallic elements potassium, sodium, magnesium, copper, cobalt, manganese;
preferably, the metal element is in the form of one or more of phosphate, acid phosphate, chloride, sulfate and citrate;
preferably, the addition amount of the metal element salt is 5-20 g/L of potassium salt, 10-20 g/L of sodium salt, 0.5-4 g/L of magnesium salt, 0.1-2 g/L of copper salt, 0.01-0.1 mg/L of cobalt salt and 0.001-0.02 mg/L of manganese salt.
5. The method of any one of claims 1 to 4, wherein the method comprises adding heavy phase lactic acid to the fermentation medium;
preferably, the added heavy phase lactic acid accounts for 1-10 wt% of the total mass of the culture medium, and preferably 3-7 wt%.
6. The method of any one of claims 1 to 5, wherein the yarrowia lipolytica is inoculated in the fermentation medium in an amount of 5% to 20%, preferably 10% to 17%, by volume of the medium;
and/or the culture temperature after inoculation of the yarrowia lipolytica is 25-40 ℃, preferably 28-37 ℃;
and/or the inoculation amount of the schizochytrium in the fermentation medium is 10-20% of the volume of the medium, preferably 13-18%;
and/or the culture temperature after the schizochytrium limacinum is inoculated is 20-30 ℃, and preferably 23-28 ℃.
7. The method according to any one of claims 1 to 6, wherein the pH value during the fermentation is 6.0 to 8.0, preferably 6.5 to 7.4, the fermentation aeration is 0.5 to 3.5vvm, preferably 1.0 to 2.5vvm, and the stirring speed is 20 to 500rpm, preferably 100 to 300 rpm.
8. The method according to any one of claims 1 to 7, wherein in the fermentation process, the discharging is started when the OD value of the thallus of the fermentation liquor reaches 100-150, preferably the OD value reaches 120-130;
and/or, the fermentation liquor is discharged from the fermentation tank, after filtration, clear liquid returns to the fermentation tank for continuous fermentation, and the concentrated solution is dried to obtain a single-cell protein product;
and/or the continuous fermentation operation cycle is 60-90 days;
and/or, the unit yield of the single-cell protein product is 2-4 g/L/h based on dry weight.
9. A single-cell protein produced by the method for producing a single-cell protein using heavy-phase lactic acid according to any one of claims 1 to 8.
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