CN112646740A - Formate single-cell protein strain MA5 and application thereof - Google Patents
Formate single-cell protein strain MA5 and application thereof Download PDFInfo
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- CN112646740A CN112646740A CN202011356627.2A CN202011356627A CN112646740A CN 112646740 A CN112646740 A CN 112646740A CN 202011356627 A CN202011356627 A CN 202011356627A CN 112646740 A CN112646740 A CN 112646740A
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- 108010027322 single cell proteins Proteins 0.000 title claims abstract description 84
- BDAGIHXWWSANSR-UHFFFAOYSA-M Formate Chemical compound [O-]C=O BDAGIHXWWSANSR-UHFFFAOYSA-M 0.000 title claims abstract description 34
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims abstract description 122
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims abstract description 61
- 235000019253 formic acid Nutrition 0.000 claims abstract description 61
- 239000001963 growth medium Substances 0.000 claims abstract description 32
- 239000004280 Sodium formate Substances 0.000 claims abstract description 31
- HLBBKKJFGFRGMU-UHFFFAOYSA-M sodium formate Chemical compound [Na+].[O-]C=O HLBBKKJFGFRGMU-UHFFFAOYSA-M 0.000 claims abstract description 31
- 235000019254 sodium formate Nutrition 0.000 claims abstract description 31
- 238000004519 manufacturing process Methods 0.000 claims abstract description 26
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 24
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 23
- 229910052799 carbon Inorganic materials 0.000 claims abstract description 21
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 20
- 239000002028 Biomass Substances 0.000 claims abstract description 18
- 235000013305 food Nutrition 0.000 claims abstract description 11
- 238000012258 culturing Methods 0.000 claims abstract description 10
- 238000009629 microbiological culture Methods 0.000 claims abstract description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 48
- 229910052757 nitrogen Inorganic materials 0.000 claims description 24
- 239000002609 medium Substances 0.000 claims description 21
- 239000000843 powder Substances 0.000 claims description 21
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 19
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims description 12
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 12
- 241001057811 Paracoccus <mealybug> Species 0.000 claims description 11
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 10
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims description 6
- 235000019270 ammonium chloride Nutrition 0.000 claims description 6
- 125000001477 organic nitrogen group Chemical group 0.000 claims description 6
- 239000011780 sodium chloride Substances 0.000 claims description 6
- LLSDKQJKOVVTOJ-UHFFFAOYSA-L calcium chloride dihydrate Chemical compound O.O.[Cl-].[Cl-].[Ca+2] LLSDKQJKOVVTOJ-UHFFFAOYSA-L 0.000 claims description 5
- 229940052299 calcium chloride dihydrate Drugs 0.000 claims description 5
- 229940050906 magnesium chloride hexahydrate Drugs 0.000 claims description 5
- DHRRIBDTHFBPNG-UHFFFAOYSA-L magnesium dichloride hexahydrate Chemical compound O.O.O.O.O.O.[Mg+2].[Cl-].[Cl-] DHRRIBDTHFBPNG-UHFFFAOYSA-L 0.000 claims description 5
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 5
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 5
- 239000001103 potassium chloride Substances 0.000 claims description 5
- 235000011164 potassium chloride Nutrition 0.000 claims description 5
- 239000001888 Peptone Substances 0.000 claims description 4
- 108010080698 Peptones Proteins 0.000 claims description 4
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 4
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 4
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 4
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 4
- 235000015278 beef Nutrition 0.000 claims description 4
- 239000004202 carbamide Substances 0.000 claims description 4
- 235000013877 carbamide Nutrition 0.000 claims description 4
- 235000019319 peptone Nutrition 0.000 claims description 4
- 229910021529 ammonia Inorganic materials 0.000 claims description 3
- 238000003306 harvesting Methods 0.000 claims description 3
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 3
- 238000004321 preservation Methods 0.000 claims description 3
- 230000000813 microbial effect Effects 0.000 abstract description 6
- 241000535507 Paracoccus communis Species 0.000 abstract description 4
- 238000000855 fermentation Methods 0.000 description 21
- 230000004151 fermentation Effects 0.000 description 21
- 235000018102 proteins Nutrition 0.000 description 19
- 230000014616 translation Effects 0.000 description 17
- 230000000694 effects Effects 0.000 description 15
- 230000001580 bacterial effect Effects 0.000 description 12
- 230000012010 growth Effects 0.000 description 9
- 238000000034 method Methods 0.000 description 9
- 239000007788 liquid Substances 0.000 description 8
- CKUAXEQHGKSLHN-UHFFFAOYSA-N [C].[N] Chemical compound [C].[N] CKUAXEQHGKSLHN-UHFFFAOYSA-N 0.000 description 6
- 238000012216 screening Methods 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 241000894006 Bacteria Species 0.000 description 5
- 241001465754 Metazoa Species 0.000 description 5
- 241001052560 Thallis Species 0.000 description 5
- 239000002689 soil Substances 0.000 description 5
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 4
- 150000001875 compounds Chemical class 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 240000002044 Rhizophora apiculata Species 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 235000021120 animal protein Nutrition 0.000 description 3
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 3
- 239000012153 distilled water Substances 0.000 description 3
- 238000011081 inoculation Methods 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 238000005457 optimization Methods 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 238000009210 therapy by ultrasound Methods 0.000 description 3
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 238000012300 Sequence Analysis Methods 0.000 description 2
- 230000010261 cell growth Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 235000021118 plant-derived protein Nutrition 0.000 description 2
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 2
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 239000013535 sea water Substances 0.000 description 2
- 238000011218 seed culture Methods 0.000 description 2
- 238000012807 shake-flask culturing Methods 0.000 description 2
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical compound [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 description 2
- 239000008223 sterile water Substances 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 108010064851 Plant Proteins Proteins 0.000 description 1
- 108010073771 Soybean Proteins Proteins 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 235000011114 ammonium hydroxide Nutrition 0.000 description 1
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- 235000013373 food additive Nutrition 0.000 description 1
- 239000002778 food additive Substances 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000005984 hydrogenation reaction Methods 0.000 description 1
- 230000008676 import Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
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- 230000004060 metabolic process Effects 0.000 description 1
- -1 monocarbon compound Chemical class 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 238000005192 partition Methods 0.000 description 1
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- 238000004153 renaturation Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000004317 sodium nitrate Substances 0.000 description 1
- 235000010344 sodium nitrate Nutrition 0.000 description 1
- 229940001941 soy protein Drugs 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 239000012137 tryptone Substances 0.000 description 1
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- 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
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/195—Proteins from microorganisms
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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Abstract
The invention discloses a formate single-cell protein strain MA5, which is classified and named as follows: paracoccus communis, deposited in China general microbiological culture Collection center with the following deposit numbers: CGMCC No. 21106. The invention also discloses a method for producing a single-cell protein, comprising: 1) culturing MA 5; 2) single-cell proteins were harvested from the culture medium in the form of biomass of strain MA5 grown in step 1). The invention also discloses the application of the single-cell protein product, the food or feed product and the strain MA5 in producing food or feed by using formic acid or sodium formate. The invention uses formic acid or sodium formate as the only carbon source to transform the formic acid efficiently to produce the microbial strain MA5 of the protein, produces the protein with high added value on the basis of using formic acid or sodium formate, and provides technical support for producing single-cell protein in green and low cost.
Description
Technical Field
The invention belongs to the technical field of microorganisms and fermentation, and relates to a formate-based single-cell protein strain MA5 and application thereof.
Background
In China, which is a large population, the demand for proteins is enormous. China, the largest protein importing country all over the world, costs up to 1500 billion yuan each year in protein import, and mainly comprises imported soy protein and animal protein. However, since plants and animals have a long growth cycle, it undoubtedly increases the production cost of animal and plant proteins greatly. The problem is avoided by the application of the single-cell protein, namely, the single-cell protein produces protein by utilizing the metabolism of microorganisms, and then the microbial thallus is prepared into dry powder to be added into feed or other edible substances. The single-cell protein has abundant amino acid types and abundant nutrition, and the microorganisms can convert inorganic nitrogen into organic nitrogen, so that the production cost is reduced and the environment is protected compared with plant and animal proteins.
Formic acid is an important monocarbon compound, which can be obtained by fixing carbon dioxide through a physicochemical method, and belongs to an inexpensive carbon source, and the route thereof includes electrochemical reduction and hydrogenation. In addition, formic acid can be used in food production as a food additive.
At present, no microbial strain which takes formic acid as a carbon source to produce protein is seen.
Disclosure of Invention
An object of the present invention is to solve at least the above problems and/or disadvantages and to provide at least the advantages described hereinafter.
It is a further object of the present invention to provide the formate-based single cell protein strain MA 5.
It is another object of the present invention to provide a method for producing a single-cell protein.
It is another object of the invention to provide a single-cell protein product.
It is a further object of the invention to provide a food or feed product.
The invention also aims to provide the application of the formate-based single-cell protein strain MA5 in producing food or feed by using formic acid or sodium formate.
Therefore, the technical scheme provided by the invention is as follows:
formate-based single cell protein strain MA5, comprising: the formate-based single cell protein strain MA5, which is classified and named: paracoccus comunii, Paracoccus comunii MA5, was deposited in the China general microbiological culture Collection center with the following numbers: CGMCC No.21106, the preservation time is as follows: the address of the depository unit is as follows, at 11/05 of 2020: xilu No. 1, Beijing, Chaoyang, Beijing, and institute for microbiology, China academy of sciences. The invention relates to a formate single-cell protein strain MA5 and application thereof. The MA5 strain has short rod-shaped thallus, mostly appears in pairs, belongs to gram-negative bacteria, is facultative anaerobic, and has a light yellow colony form after being cultured on a formic acid culture medium for 24 hours, a smooth and moist surface and a radial periphery.
A method for producing a single-cell protein, comprising the steps of:
1) inoculating the formate-based single-cell protein strain MA5 into a culture medium for culture;
2) harvesting the single-cell protein from the culture medium in the form of biomass of paracoccus grown in step 1).
Preferably, in the method for producing a single-cell protein, the culture is performed at a temperature of 26 ℃ to 40 ℃ and a pH of 6.5 to 8.5 for 6 to 168 hours. More preferably, in the method for producing a single-cell protein, the culture is performed at 37 ℃ and at 7 for 24 hours.
Preferably, in the method for producing a single-cell protein, the medium used in the culture may be formic acid or sodium formate alone as a sole carbon source, or other formate-based compounds.
Preferably, in the method for producing a single-cell protein, the ratio of the carbon source to the nitrogen source in the medium during the culture is 6.8-10.6: 1.
Preferably, in the method for producing a single-cell protein, the culture medium used in the culture has an organic nitrogen source including, but not limited to, beef extract, peptone or yeast powder, or various combinations thereof, or an inorganic nitrogen source including, but not limited to, ammonia, ammonium sulfate, ammonium chloride, urea, and the like, and combinations thereof, as a nitrogen source.
Preferably, in the method for producing a single-cell protein, the culture medium comprises: 5g/L of sodium formate or formic acid, 8g/L of yeast powder, 0.5g/L of sodium chloride, 0.5g/L of magnesium chloride hexahydrate, 0.1g/L of calcium chloride dihydrate, 0.5g/L of potassium chloride and 0.2g/L of potassium dihydrogen phosphate.
A single cell protein product comprising protein from said formate-based single cell protein strain MA 5.
A food or feed product comprising protein from said formate-based single cell protein strain MA 5.
The application of the formate-based single-cell protein strain MA5 in producing food or feed by using formic acid or sodium formate.
The invention at least comprises the following beneficial effects:
in the invention, a 5L fermentation system is adopted to ferment the formate-based single-cell protein strain MA5, and the result shows that the biomass of the strain MA5 reaches 14.1g/L, the protein content reaches 49.1 percent, and the consumption rate of formic acid reaches 0.84g/L/h when the highest consumption rate of formic acid exists in the presence of yeast powder. The formate-based single-cell protein strain MA5 can efficiently utilize formate-based protein, and can be used as a candidate strain for providing a theoretical basis for producing animal feed by using formic acid or formate-based compounds.
The invention uses formic acid or formate-based compounds as the only carbon source, and the microbial strain MA5 for efficiently converting formic acid to produce protein can produce protein with high added value on the basis of utilizing formic acid, thereby providing technical support for green and low-cost production of single-cell protein.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.
Drawings
FIG. 1 is a diagram of the MA 516 SrDNA phylogenetic tree according to the present invention;
FIG. 2 is a graph showing the effect of pH on MA5 single cell protein production in the present invention;
FIG. 3 is a graph showing the effect of temperature on MA5 single cell protein production in the present invention;
FIG. 4 is a graph showing the effect of different carbon source concentrations on MA5 single cell biomass and protein production in accordance with the present invention;
FIG. 5 is a graph showing the effect of different nitrogen sources on MA5 single cell biomass and protein production in accordance with the present invention;
FIG. 6 is a graph showing the effect of different carbon-nitrogen ratios on MA5 single cell protein production in the present invention.
Detailed Description
The present invention is further described in detail below with reference to the attached drawings so that those skilled in the art can implement the invention by referring to the description text.
It will be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.
The invention provides a formate single-cell protein strain MA5, and the formate single-cell protein strain MA5 is classified and named as follows: paracoccus comunii, Paracoccus comunii MA5, was deposited in the China general microbiological culture Collection center with the following numbers: CGMCC No.21106, the preservation time is as follows: the address of the depository unit is as follows, at 11/05 of 2020: xilu No. 1, Beijing, Chaoyang, Beijing, and institute for microbiology, China academy of sciences. The MA5 strain has short rod-shaped thallus, mostly appears in pairs, belongs to gram-negative bacteria, is facultative anaerobic, and has a light yellow colony form after being cultured on a formic acid culture medium for 24 hours, a smooth and moist surface and a radial periphery.
The present invention also provides a method for producing a single-cell protein, comprising the steps of:
1) inoculating the formate-based single-cell protein strain MA5 into a culture medium for culture;
2) harvesting the single-cell protein from the culture medium in the form of biomass of paracoccus grown in step 1).
In some embodiments of the invention, preferably, the method for producing single-cell protein is performed by culturing for 6-144 h at 26-40 ℃ and pH 6.5-8.5. In the above scheme, preferably, the method for producing a single-cell protein comprises culturing at 37 ℃ and pH7 for 24 h.
In some embodiments of the present invention, preferably, the method for producing a single-cell protein is performed by using the culture medium only containing formic acid or sodium formate as the sole carbon source, or other formate-based compounds, wherein the concentration of the sodium formate or formic acid is 0.5g/L to 25 g/L. More preferably, the concentration of sodium formate or formic acid is 2g/L to 14 g/L. Most preferably the concentration of sodium formate or formic acid is 5 g/L.
In some embodiments of the invention, the ratio of the carbon source to the nitrogen source in the culture medium is 6.8-10.6: 1. More preferably, the carbon-nitrogen ratio in the culture medium is 7 to 7.9. Most preferably, the carbon to nitrogen ratio in the medium is 7.20: 1.
In some embodiments of the present invention, it is preferable that the culture medium is an organic nitrogen source or an inorganic nitrogen source, wherein the organic nitrogen source includes, but is not limited to, beef extract, peptone or yeast powder or various combinations thereof, and the inorganic nitrogen source includes, but is not limited to, ammonia, ammonium sulfate, ammonium chloride, urea, and the like, and combinations thereof.
In some embodiments of the invention, preferably, the method for producing a single-cell protein comprises culturing in a medium comprising: 5g/L of sodium formate or formic acid, 8g/L of yeast powder, 0.5g/L of sodium chloride, 0.5g/L of magnesium chloride hexahydrate, 0.1g/L of calcium chloride dihydrate, 0.5g/L of potassium chloride and 0.2g/L of potassium dihydrogen phosphate.
A single cell protein product comprising protein from said formate-based single cell protein strain MA 5.
A food or feed product comprising protein from said formate-based single cell protein strain MA 5.
The invention also provides application of the formate-based single-cell protein strain MA5 in producing food or feed by using formic acid or sodium formate.
The invention relates to a bacterial strain capable of efficiently utilizing formic acid, which is obtained by screening sodium formate as a carbon source from a soil sample of a natural protection area of mangrove in Hongkong of east village. The strain was identified as Paracoccus (Paracoccus communis MA5) using 16SrDNA sequence analysis. The result of adopting a 5L fermentation system to ferment the MA5 shows that the biomass of the strain MA5 reaches 14.1g/L, the protein content reaches 49.1 percent, and the highest consumption rate of formic acid reaches 0.84 g/L/h. MA5 can efficiently utilize formic acid protein, and can be used as candidate strain to provide theoretical basis for producing animal feed by using formic acid.
In order to make the technical solution of the present invention better understood by those skilled in the art, the following examples are now provided for illustration:
the invention screens microbial strains for efficiently converting formic acid to produce protein by taking formic acid or sodium formate as a unique carbon source, optimizes the fermentation conditions of the microbial strains, produces protein with high added value on the basis of utilizing formic acid or sodium formate, and provides technical support for green and low-cost production of single-cell protein.
1 materials and methods
1.1 sample sources
The soil sample is from natural conservation area of Hongshan Hongshu Hao of south east China and institute of Industrial biotechnology, Tianjin institute of science and technology.
1.2 culture Medium
Screening a culture medium: 5g/L of sodium formate or formic acid, 0.5g/L of sodium chloride, 0.5g/L of magnesium chloride hexahydrate, 0.1g/L of calcium chloride dihydrate, 0.5g/L of potassium chloride, 0.2g/L of monopotassium phosphate, 0.2g/L of ammonium chloride and natural pH.
Seed culture medium
LB culture medium: 5g/L of yeast powder, 10g/L of tryptone and 10g/L of sodium chloride.
Fermentation medium
Formic acid fermentation medium: 5g/L of sodium formate or formic acid, 0.5g/L of sodium chloride, 0.5g/L of magnesium chloride hexahydrate, 0.1g/L of calcium chloride dihydrate, 0.5g/L of potassium chloride, 0.2g/L of monopotassium phosphate and 2g/L of yeast powder.
The formic acid solid culture medium is added in a formic acid fermentation culture medium: agar 18 g/L.
1.3 Primary screening of formic acid-resistant strains
Respectively weighing 1g of soil sample, 2 leaves and 1mL of seawater, adding the soil sample, 2 leaves and 1mL of seawater into a formic acid screening culture medium, performing shake culture for 48 hours, inoculating 50 mu L of bacterial liquid into a solid screening culture medium, and culturing at 37 ℃ until a single colony grows out.
1.4 resistant formic acid bacteria rescreening
And continuously transferring the primarily screened strains to a formic acid solid culture medium for three times, inoculating the finally obtained bacterial colonies to a liquid formic acid fermentation culture medium, performing shake flask culture for 48 hours, and then preserving the strains.
1.5 Medium optimization
1.5.1 Effect of pH on MA5 Single cell protein production
All liquid-liquid transfer operations are carried out according to the inoculation amount of 1%, MA5 is transferred from a seed culture medium to a formic acid fermentation culture medium, the initial pH values are set to be 6.5, 7, 7.5, 8 and 8.5, and the concentration of the bacterial liquid is measured at 600nm after the bacterial liquid is cultured for 24 hours in a shaking table.
1.5.2 Effect of temperature on MA5 Single cell protein production
Transferring MA5 into formic acid fermentation medium with pH of 7, designing five gradients at 26 deg.C, 30 deg.C, 35 deg.C, 37 deg.C, 40 deg.C, shake culturing for 24 hr, and measuring bacterial liquid concentration at 600 nm.
1.5.3 Effect of different carbon Source concentrations on MA5 Single cell protein production
MA5 was inoculated into a formic acid fermentation medium having a pH of 7, the culture temperature was set at 37 ℃ and the carbon source concentration was set at 5 gradients of 2g/L, 5g/L, 8g/L, 11g/L and 14g/L, respectively, after 24 hours of shake culture, the bacterial concentration was measured at 600nm and the cells were collected by centrifugation, and the protein content was measured at 595nm by the Coomassie Brilliant blue method.
1.5.4 Effect of different Nitrogen sources on the growth and protein production of MA5
MA5 was inoculated into a fermentation medium of pH7 and sodium formate concentration of 5g/L, the culture temperature was set at 37 ℃ and the target strain was cultured with ammonium chloride, sodium nitrate, ammonium sulfate, urea, beef extract, peptone and yeast powder as nitrogen sources, respectively, after 24h of shake cultivation, the concentration of the bacterial solution was measured at 600nm and the protein content was measured at 595 nm.
1.5.5 Effect of different carbon-nitrogen ratios on MA5 Single cell protein production
MA5 was inoculated into a fermentation medium having pH of 7, sodium formate concentration of 5g/L, and yeast powder as a nitrogen source, the culture temperature was set at 37 ℃, and the ratio of yeast powder to carbon and nitrogen (molar ratio) was 10.53:1, 7.86: 1. adding the mixture according to the ratio of 7.20:1, 6.98:1 and 6.79:1 respectively, after culturing for 24h in a shake flask, measuring the concentration of the bacterial liquid at 600nm, and measuring the protein content at 595 nm.
1.65 liter System fermentation
On the basis of a fermentation medium, formic acid is manually fed in before inoculation so that the medium contains 5g/L of formic acid, then ammonia water is fed in to adjust the pH to 7, the temperature is kept at 37 ℃, the initial rotation speed is set to be 200rpm, the fermentation medium is 3L, the addition amount of the formic acid is automatically adjusted, and yeast powder is fed in every 24h so that the medium contains 8g/L of yeast powder.
1.716 SrDNA extraction and strain identification
16SrDNA is extracted by using a colony PCR method, and the operation method comprises the following steps: a single colony was picked in 10. mu.L of sterile water, and then a 25. mu.L PCR system was prepared, which included 1. mu.L of the forward primer, 1. mu.L of the reverse primer, 12. mu.L of the mix, and 11. mu.L of sterile water + sample.
PCR procedure: pre-denaturation at 95 ℃ for 10 min; 35 cycles, each cycle having the parameters: denaturation at 95 ℃ for 30s, renaturation at 54 ℃ for 30s, and extension at 72 ℃ for 90 s; finally, extension is carried out for 5min at 72 ℃.
The primer uses a 16SrDNA universal primer, and the sequence of the primer is as follows:
27F:AGAGTTTGATCCTGGCTCAG(SEQ ID NO:1)
1492R：GGTTACCTTGTTACGACTT(SEQ ID NO:2)
the PCR products were sequenced from the wara gene, the results of the sequencing were compared at NCBI for homology with the 16SrDNA sequence published in the Genebank database, and phylogenetic tree analysis was performed using MEGA-X software.
1.8 analytical detection method
1.8.1 measurement of formic acid content in Medium
The invention utilizes high performance liquid chromatography to determine the content of formic acid in the solution. The experimental method is as follows:
mobile phase: 5mmoL/L sulfuric acid
Column: organic acid column
Flow rate: 0.5mL/min
Time: 20min
Sample pretreatment: diluting the sample with 10mmoL/L sulfuric acid to make the pH of the sample acidic, pH 1-2
1.8.2 bioassay
After the fermentation of the thalli is finished, centrifuging the bacterial liquid at 8000rpm for 10min to collect thalli, washing the collected thalli with physiological saline for three times, washing the thalli with distilled water for one time, then placing the thalli into a freeze dryer for processing for 24h, then weighing the bacterial powder, and recording data.
1.8.3 Coomassie brilliant blue method for determining single cell protein content
After a plurality of tests, the operation method is finally determined as follows: the bacteria were washed three times with physiological saline and once with distilled water (in order to completely wash the fermentation broth), 10mL of distilled water was added to the washed cell pellet, which was then disrupted on an ultrasonic disrupter, disruption protocol: the crushing power is 270W, the ultrasonic treatment is carried out for 3s, the ultrasonic treatment is stopped for 7s, and the ultrasonic treatment is carried out for 10 min; after obtaining a clear and transparent solution, 1mL of sample and 5mL of 0.1moL/L sodium hydroxide are uniformly mixed and boiled in boiling water for 10min to obtain a reaction solution, and finally, Coomassie brilliant blue dyeing reaction is carried out. The reaction time was strictly controlled to be 3min and then the protein content was determined at 595 nm.
The experiments referred to in the present invention all designed three replicates and the data of the present invention are expressed as mean and standard deviation of the three replicates.
2 results
2.1 morphology of MA5 and analysis of 16SrDNA identification results
Through enrichment, plate partition streaking and transfer culture methods, the strain MA5 with the strongest formic acid utilization capacity and the fastest growth speed is screened out from the environment of the natural protected area of the mangrove forest in Hongkong of east village and is identified as Paracoccus communis. Therefore, the MA5 is selected as the target strain for the process research of producing the single-cell protein by transforming formic acid. The invention also made a phylogenetic tree for the MA5 strain, the results are shown in FIG. 1.
The MA5 strain was observed under a 1000-fold microscope, and it was found that the cells were short rod-shaped and appeared in pairs in many cases. The bacillus is a gram-negative bacterium and facultative anaerobic type, and the colony form is light yellow after being cultured on a formic acid culture medium for 24 hours, the surface is smooth and moist, and the periphery is radial.
2.2 Medium optimization
2.2.1 Effect of initial pH on MA5 Single cell protein production
As a result of measuring the growth of MA5 strain under different pH conditions, as shown in FIG. 2, it can be seen that the difference in cell growth rate was small in the case of MA5 strain at pH6.5 to 7, the biomass of cells increased with increasing pH, and the biomass reached a maximum value when the pH was increased to 7, and the pH continued to increase, and the biomass of cells began to decrease, so that pH7 was the optimum pH for growth of cells.
2.2.2 Effect of culture temperature on MA5 Single cell protein production
The strain was inoculated in formic acid medium at pH7, and growth of the strain was measured under different temperature culture conditions, and the results are shown in fig. 3. It can be seen from the figure that the biomass of the cells increased with the increase of the temperature in the temperature range of 26 ℃ to 37 ℃, the biomass of the cells reached the maximum value at the culture temperature of 37 ℃, and the biomass of the cells began to decrease as the temperature continued to increase. Therefore, 37 ℃ is the optimum growth temperature of the cells.
2.2.3 Effect of sodium formate concentration on MA5 Single cell protein production
The MA5 strain was tested for growth and protein production at different sodium formate concentrations and the results are shown in FIG. 4. As can be seen from the figure, when the concentration of sodium formate is less than 5g/L, the biomass of the thallus and the protein yield increase in a positive correlation relationship with the increase of the concentration of sodium formate, and when the concentration of sodium formate is increased to 5g/L, the single-cell protein yield reaches the highest value
1.99×10-3g/L, the nitrogen source is 2g/L ammonium chloride, but when the sodium formate concentration is increased, the biomass begins to decrease, and the eggs are hatchedThe white yield also decreased.
2.2.4 Effect of Nitrogen Source on MA5 Single cell protein production
The measured single-cell protein yields of MA5 strain after 24h of cultivation in medium containing different nitrogen sources are shown in FIG. 5. The result shows that the organic nitrogen source can obviously promote the yield of the MA5 strain single-cell protein compared with the inorganic nitrogen source, and the yield of the strain single-cell protein is the highest when the nitrogen source is yeast powder.
2.2.5 Effect of C/N ratio on MA5 Single cell protein production
Yeast powder is selected as a nitrogen source in the experiment, the total carbon content in the yeast powder is measured to be 63.60 percent and the total nitrogen content is measured to be 12 percent before the experiment, and the carbon-nitrogen ratio in the culture medium is calculated according to the data. Single cell protein production was measured after 24h shake flask culture and the results are shown in FIG. 6. It can be seen from the figure that the protein content of the MA5 strain is up to 60.70% at a carbon-nitrogen ratio of 7.20:1, and the yield of the single-cell protein is relatively high to 1.31g/L under the condition of the carbon-nitrogen ratio.
2.35 liter System fermentation
Yeast powder is used as a nitrogen source, 300mL of seed solution is inoculated according to the inoculation amount of 1%, and finally 14.1g/L of biomass and 49.1% of protein yield are obtained.
The number of modules and the processing scale described herein are intended to simplify the description of the invention. Modifications and variations of the formate-based single cell protein strain of the present invention and its uses will be apparent to those skilled in the art.
The invention relates to a bacterial strain capable of efficiently utilizing formic acid, which is obtained by screening sodium formate as a carbon source from a soil sample of a natural protection area of mangrove in Hongkong of east village. The strain was identified as Paracoccus (Paracoccus communis MA5) using 16SrDNA sequence analysis. The growth conditions and the protein yield of the strain are optimized by adopting a single-factor method, and the culture medium optimization result shows that the optimal growth temperature of the strain is 37 ℃, the optimal initial pH value is 7, the optimal carbon source sodium formate concentration is 5g/L, and the optimal nitrogen source yeast powder concentration is 8 g/L. On the basis, a 5L fermentation system is adopted to optimize the fermentation process of MA5, and the result shows that the biomass of the strain MA5 reaches 14.1g/L, the protein content reaches 49.1 percent, and the highest consumption rate of formic acid reaches 0.84 g/L/h. MA5 can efficiently utilize formic acid protein, and can be used as candidate strain to provide theoretical basis for producing animal feed by using formic acid.
While embodiments of the invention have been described above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable in various fields of endeavor to which the invention pertains, and further modifications may readily be made by those skilled in the art, it being understood that the invention is not limited to the details shown and described herein without departing from the general concept defined by the appended claims and their equivalents.
Sequence listing
<110> institute of biotechnology for Tianjin industry of Chinese academy of sciences
<120> formate-based single-cell protein strain MA5 and application thereof
<130> 2019
<160> 2
<170> PatentIn version 3.5
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<211> 20
<212> DNA
<213> Artificial sequence
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Claims (10)
1. Formate-based single-cell protein strain MA5, said formate-based single-cell protein strain MA5, assigned by classification: paracoccus comunii, Paracoccus comunii MA5, was deposited in the China general microbiological culture Collection center with the following numbers: CGMCC No.21106, the preservation time is as follows: the address of the depository unit is as follows, at 11/05 of 2020: xilu No. 1, Beijing, Chaoyang, Beijing, and institute for microbiology, China academy of sciences.
2. A method for producing a single-cell protein, comprising the steps of:
1) inoculating the formate-based single-cell protein strain MA5 into a culture medium for culture;
2) harvesting the single-cell protein from the culture medium in the form of biomass of paracoccus grown in step 1).
3. The method for producing a single-cell protein according to claim 2, wherein the culturing is carried out at a temperature of 26 ℃ to 40 ℃ and a pH of 6.5 to 8.5 for 6 to 168 hours.
4. The method for producing a single-cell protein according to claim 2, wherein said culture medium used in said culturing uses only formic acid or sodium formate as a sole carbon source.
5. The method for producing a single-cell protein according to claim 2, wherein the ratio of the carbon source to the nitrogen source in the medium during the culture is 6.8-10.6: 1.
6. The method for producing a single-cell protein according to claim 2, wherein the culture medium used in the cultivation is an organic nitrogen source including but not limited to beef extract, peptone or yeast powder or various combinations thereof or an inorganic nitrogen source including but not limited to ammonia, ammonium sulfate, ammonium chloride, urea and combinations thereof.
7. The method for producing a single-cell protein according to claim 2, wherein in said culturing, said medium comprises: 5g/L of sodium formate or formic acid, 8g/L of yeast powder, 0.5g/L of sodium chloride, 0.5g/L of magnesium chloride hexahydrate, 0.1g/L of calcium chloride dihydrate, 0.5g/L of potassium chloride and 0.2g/L of potassium dihydrogen phosphate.
8. A single cell protein product comprising protein from the formate-based single cell protein strain MA5 of claim 1.
9. A food or feed product comprising protein from the formate-based single cell protein strain MA5 of claim 1.
10. Use of the formate-based single cell protein strain MA5 according to claim 1 in the production of food or feed from formic acid or sodium formate.
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