CN117965328A - Debaryomyces hansenii HRB1 with antioxidant function and application thereof - Google Patents
Debaryomyces hansenii HRB1 with antioxidant function and application thereof Download PDFInfo
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- CN117965328A CN117965328A CN202311844518.9A CN202311844518A CN117965328A CN 117965328 A CN117965328 A CN 117965328A CN 202311844518 A CN202311844518 A CN 202311844518A CN 117965328 A CN117965328 A CN 117965328A
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- hrb1
- debaryomyces hansenii
- starter
- hansenii
- debaryomyces
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- 241000235036 Debaryomyces hansenii Species 0.000 title claims abstract description 62
- 101100170514 Arabidopsis thaliana DI19-7 gene Proteins 0.000 title claims abstract description 47
- 101100339606 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) HRB1 gene Proteins 0.000 title claims abstract description 47
- APTZNLHMIGJTEW-UHFFFAOYSA-N pyraflufen-ethyl Chemical compound C1=C(Cl)C(OCC(=O)OCC)=CC(C=2C(=C(OC(F)F)N(C)N=2)Cl)=C1F APTZNLHMIGJTEW-UHFFFAOYSA-N 0.000 title claims abstract description 47
- 239000003963 antioxidant agent Substances 0.000 title abstract description 25
- 230000003078 antioxidant effect Effects 0.000 title abstract description 17
- 239000007858 starting material Substances 0.000 claims abstract description 30
- 235000013622 meat product Nutrition 0.000 claims abstract description 17
- 238000000855 fermentation Methods 0.000 claims description 39
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- 235000013580 sausages Nutrition 0.000 claims description 17
- 238000004321 preservation Methods 0.000 claims description 12
- 244000005700 microbiome Species 0.000 claims description 5
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- 235000015241 bacon Nutrition 0.000 claims description 2
- 238000009629 microbiological culture Methods 0.000 claims description 2
- 241000235035 Debaryomyces Species 0.000 claims 2
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- 238000012216 screening Methods 0.000 description 8
- MGJZITXUQXWAKY-UHFFFAOYSA-N diphenyl-(2,4,6-trinitrophenyl)iminoazanium Chemical compound [O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1N=[N+](C=1C=CC=CC=1)C1=CC=CC=C1 MGJZITXUQXWAKY-UHFFFAOYSA-N 0.000 description 7
- 238000000034 method Methods 0.000 description 7
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- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 3
- OCZVHBZNPVABKX-UHFFFAOYSA-N 1,1-diphenyl-2-(2,4,6-trinitrophenyl)hydrazine;ethanol Chemical compound CCO.[O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1NN(C=1C=CC=CC=1)C1=CC=CC=C1 OCZVHBZNPVABKX-UHFFFAOYSA-N 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
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- 102000004190 Enzymes Human genes 0.000 description 2
- 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 description 2
- 239000001888 Peptone Substances 0.000 description 2
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- 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 2
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- WQGWDDDVZFFDIG-UHFFFAOYSA-N pyrogallol Chemical compound OC1=CC=CC(O)=C1O WQGWDDDVZFFDIG-UHFFFAOYSA-N 0.000 description 2
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- 239000002994 raw material Substances 0.000 description 2
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- 239000012138 yeast extract Substances 0.000 description 2
- JIYLMPVCYFUNGG-UHFFFAOYSA-N 2,2,2-trichloroacetic acid;hydrochloride Chemical compound Cl.OC(=O)C(Cl)(Cl)Cl JIYLMPVCYFUNGG-UHFFFAOYSA-N 0.000 description 1
- HORQAOAYAYGIBM-UHFFFAOYSA-N 2,4-dinitrophenylhydrazine Chemical compound NNC1=CC=C([N+]([O-])=O)C=C1[N+]([O-])=O HORQAOAYAYGIBM-UHFFFAOYSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 241000436315 Candida metapsilosis Species 0.000 description 1
- 239000004278 EU approved seasoning Substances 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 206010016807 Fluid retention Diseases 0.000 description 1
- 240000006927 Foeniculum vulgare Species 0.000 description 1
- 235000004204 Foeniculum vulgare Nutrition 0.000 description 1
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 1
- WSMYVTOQOOLQHP-UHFFFAOYSA-N Malondialdehyde Chemical compound O=CCC=O WSMYVTOQOOLQHP-UHFFFAOYSA-N 0.000 description 1
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
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- 241001597632 Yarrowia divulgata Species 0.000 description 1
- 241001503229 Yarrowia galli Species 0.000 description 1
- 244000089698 Zanthoxylum simulans Species 0.000 description 1
- DGEZNRSVGBDHLK-UHFFFAOYSA-N [1,10]phenanthroline Chemical compound C1=CN=C2C3=NC=CC=C3C=CC2=C1 DGEZNRSVGBDHLK-UHFFFAOYSA-N 0.000 description 1
- 241000222295 [Candida] zeylanoides Species 0.000 description 1
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- SOIFLUNRINLCBN-UHFFFAOYSA-N ammonium thiocyanate Chemical compound [NH4+].[S-]C#N SOIFLUNRINLCBN-UHFFFAOYSA-N 0.000 description 1
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- HHEAADYXPMHMCT-UHFFFAOYSA-N dpph Chemical compound [O-][N+](=O)C1=CC([N+](=O)[O-])=CC([N+]([O-])=O)=C1[N]N(C=1C=CC=CC=1)C1=CC=CC=C1 HHEAADYXPMHMCT-UHFFFAOYSA-N 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- LJQKCYFTNDAAPC-UHFFFAOYSA-N ethanol;ethyl acetate Chemical compound CCO.CCOC(C)=O LJQKCYFTNDAAPC-UHFFFAOYSA-N 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 229960002089 ferrous chloride Drugs 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 235000011194 food seasoning agent Nutrition 0.000 description 1
- 229960000789 guanidine hydrochloride Drugs 0.000 description 1
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 description 1
- 150000003278 haem Chemical class 0.000 description 1
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- 230000002401 inhibitory effect Effects 0.000 description 1
- NMCUIPGRVMDVDB-UHFFFAOYSA-L iron dichloride Chemical compound Cl[Fe]Cl NMCUIPGRVMDVDB-UHFFFAOYSA-L 0.000 description 1
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 235000020997 lean meat Nutrition 0.000 description 1
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- 230000000813 microbial effect Effects 0.000 description 1
- 230000002906 microbiologic effect Effects 0.000 description 1
- LPUQAYUQRXPFSQ-DFWYDOINSA-M monosodium L-glutamate Chemical compound [Na+].[O-]C(=O)[C@@H](N)CCC(O)=O LPUQAYUQRXPFSQ-DFWYDOINSA-M 0.000 description 1
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- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 239000007800 oxidant agent Substances 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 231100000683 possible toxicity Toxicity 0.000 description 1
- 229940079877 pyrogallol Drugs 0.000 description 1
- 238000012599 radical scavenging assay Methods 0.000 description 1
- 235000020995 raw meat Nutrition 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
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- 230000035807 sensation Effects 0.000 description 1
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- 238000002864 sequence alignment Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- FQENQNTWSFEDLI-UHFFFAOYSA-J sodium diphosphate Chemical compound [Na+].[Na+].[Na+].[Na+].[O-]P([O-])(=O)OP([O-])([O-])=O FQENQNTWSFEDLI-UHFFFAOYSA-J 0.000 description 1
- 229940048086 sodium pyrophosphate Drugs 0.000 description 1
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- 231100000167 toxic agent Toxicity 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- 239000007222 ypd medium Substances 0.000 description 1
Classifications
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- 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
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
- C12N1/165—Yeast isolates
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
- A23B4/00—General methods for preserving meat, sausages, fish or fish products
- A23B4/14—Preserving with chemicals not covered by groups A23B4/02 or A23B4/12
- A23B4/18—Preserving with chemicals not covered by groups A23B4/02 or A23B4/12 in the form of liquids or solids
- A23B4/20—Organic compounds; Microorganisms; Enzymes
- A23B4/22—Microorganisms; Enzymes; Antibiotics
-
- 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
- A23L13/00—Meat products; Meat meal; Preparation or treatment thereof
- A23L13/70—Tenderised or flavoured meat pieces; Macerating or marinating solutions specially adapted therefor
- A23L13/72—Tenderised or flavoured meat pieces; Macerating or marinating solutions specially adapted therefor using additives, e.g. by injection of solutions
- A23L13/74—Tenderised or flavoured meat pieces; Macerating or marinating solutions specially adapted therefor using additives, e.g. by injection of solutions using microorganisms or enzymes
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- 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
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; 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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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Mycology (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Polymers & Plastics (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Food Science & Technology (AREA)
- Botany (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- General Chemical & Material Sciences (AREA)
- Nutrition Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Debaryomyces hansenii HRB1 with antioxidant function and application thereof belong to the technical field of food processing. The invention provides debaryomyces hansenii HRB1 with an antioxidant function and a starter containing the strain, and the starter is applied to fermented meat products, aiming at solving the technical problems of low safety of artificially synthesized antioxidants and complex extraction process of plant extract antioxidants. The Debaryomyces hansenii HRB1 provided by the invention has better growth capability under H 2O2 stress, has higher scavenging capability and reducing capability for DPPH free radical, hydroxyl free radical and superoxide free radical, and proves that the oxidation resistance is stronger; the provided starter has the function of reducing the content of peroxide, thiobarbituric acid and carbonyl, and achieves the aims of reducing nutrition loss, poor flavor and spoilage caused by excessive oxidation of lipid and protein.
Description
Technical Field
The invention belongs to the technical field of food processing, and particularly relates to debaryomyces hansenii HRB1 with an antioxidant function and application thereof.
Background
Fermented meat products are a type of meat product that under natural or artificial control conditions, by means of the fermentation action of microorganisms, produces a product with a typical fermented flavor, color and texture, and a long shelf life. Traditional fermented meat products are produced by means of endogenous enzymes, spices in the raw meat or naturally occurring microorganisms or artificially inoculated starter substances to effect fermentation and maturation of the meat. However, since the muscle tissue of the fermented meat product contains high concentrations of unsaturated lipids, heme pigments, metal catalysts and various oxidizing agent components, lipid oxidation and protein oxidation are liable to occur during storage, processing, transportation and the like. Lipid oxidation in fermented meat products is mainly enzymatic oxidation and autooxidation, protein oxidation is performed by complex free radical chain reactions. Proper oxidation can promote the formation of quality and flavor of fermented meat products, but excessive oxidation can cause deterioration of product quality, reduction of nutritional value and generation of toxic compounds, which is unfavorable for the health of consumers. In addition, oxidation of proteins can also disrupt the muscle tissue structure, reduce the water retention of the product, and affect the final organoleptic sensation. Therefore, the degree of oxidation of lipids and proteins is an important factor affecting the safety and quality of fermented meat products, and the use of antioxidants is currently an effective means of solving oxidative deterioration during processing and storage of fermented meat products.
Antioxidants can be classified into artificial antioxidants and natural antioxidants according to sources, wherein:
The artificially synthesized antioxidant has potential toxicity, and the natural antioxidant has wide source, higher safety and better oxidation resistance, plays the role of inhibiting bacteria and preventing corrosion in the meat processing and storage process, and can improve the quality of meat products and the edible value.
The natural antioxidant is mainly derived from plant extracts of fruits, vegetables, spices, herbal medicines and the like and microorganisms, but the development and application of the plant extracts are limited due to the complex extraction process of the plant extracts, and the variety of the microorganisms is rich.
The art has long been desirous of developing a starter with antioxidant function for fermented meat products to solve the technical problem of excessive oxidation of fat and protein in the fermented meat products.
Disclosure of Invention
The invention provides debaryomyces hansenii HRB1 with an antioxidant function and application thereof, and aims to solve the technical problems of low safety of artificially synthesized antioxidants and complex extraction process of plant extract antioxidants.
The aim of the invention is achieved by the following technical scheme:
One of the purposes of the invention is to provide debaryomyces hansenii HRB1 with an antioxidant function, wherein the debaryomyces hansenii is named HRB1, belongs to Debaryomyces hansenii (debaryomyces hansenii) new species, is preserved in China general microbiological culture Collection center (China Committee for culture Collection), has a preservation address of North Star Xidelu No.1, the national academy of China academy of sciences No.3 in the Korean area of Beijing, has a preservation date of 2023, 11 months and 10 days, and has a preservation number of CGMCC No. 28944.
Another object of the present invention is to provide a starter comprising the above debaryomyces hansenii HRB1.
In a preferred embodiment of the invention, the effective concentration of Debaryomyces hansenii in the starter is 1X 10 6 CFU/mL.
The invention further aims to provide an application of the debaryomyces hansenii or the starter in fermented meat products.
In a preferred embodiment of the present invention, the fermented meat product comprises air-dried sausage, bacon or ham.
The invention has the beneficial effects that:
(1) The survival rate of the debaryomyces hansenii HRB1 provided by the invention after being cultured for 48 hours in a culture medium containing 2.0mM H 2O2 is up to 50.11%, and the death rate of colony numbers after being cultured for 1 hour in a culture medium containing 12.0mM H 2O2 is less than or equal to 1lg CFU mL -1, so that the debaryomyces hansenii HRB1 has better growth capacity under the high oxidation condition.
(2) The DPPH free radical clearance of the fermentation supernatant, the thalli cells and the intracellular extracts of the debaryomyces hansenii HRB1 provided by the invention are respectively 25.49%, 28.37% and 30.24%, the hydroxyl free radical clearance is respectively 59.40%, 47.91% and 48.75%, the clearance of the superoxide free radical is respectively 31.36%, 22.39% and 37.55%, and the reduction capacity is respectively 0.84, 0.75 and 0.25, so that the whole of the debaryomyces hansenii HRB1 has better antioxidant capacity.
(3) The Debaryomyces hansenii HRB1 starter provided by the invention has the capability of obviously reducing the contents of oxide, thiobarbituric acid and carbonyl in the air-dried sausage, proves that the starter has a certain inhibition effect on the oxidation of lipid and protein, and reduces the risks of nutrition loss, poor flavor, spoilage and the like caused by the excessive oxidation of lipid and protein.
(4) The invention provides a natural microbial antioxidant which is simple and convenient in preparation process, nontoxic and harmless and high in safety, and is suitable for processing large-scale industrial fermented meat products.
[ Biological preservation information ]: debaryomyces hansenii is named HRB1, the preservation number is CGMCC NO 28944, the classification is named Debaryomyces hansenii (Debaryomyces hansenii), the Debaryomyces hansenii is preserved in China center for general microbiological preservation, the preservation time is 2023, 11 months and 10 days, and the preservation address is North Star Xiyang area of Beijing, national academy of sciences of China, no. 3.
Drawings
FIG. 1 is a graph showing the results of the test for the survival rate of the primary screening strain in example 1;
FIG. 2 is a graph showing the statistical result of the colony count of the re-screened strain in example 1;
FIG. 3 is a graph showing the measurement results of DPPH radical scavenging rate in example 2;
FIG. 4 is a graph showing the results of the hydroxyl radical scavenging assay in example 2;
FIG. 5 is a graph showing the results of the superoxide radical scavenging test in example 2;
FIG. 6 is a graph showing the results of the measurement of the reducing power in example 2;
FIG. 7 is a sequence alignment of 26S rRNA species in example 2;
FIG. 8 is a standard curve of Peroxide (POV) detection in example 4;
FIG. 9 is a graph showing the results of Peroxide (POV) detection in example 4;
FIG. 10 is a graph showing the results of thiobarbituric acid assay in example 4;
FIG. 11 is a graph showing the results of measurement of carbonyl group content in example 4.
Detailed Description
Those skilled in the art can, with the benefit of this disclosure, suitably modify the process parameters to achieve this. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included herein. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the methods and applications described herein can be modified or adapted and combined to implement and utilize the technology of this invention without departing from the spirit and scope of this invention.
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be further described in detail with reference to the following detailed description and the accompanying drawings. The experimental methods used in the examples below were conventional, and the materials, reagents, methods and apparatus used, unless otherwise indicated, were all conventional in the art and commercially available to those skilled in the art.
In the following examples, the Debaryomyces hansenii HRB1 obtained by the invention is abbreviated as HRB1;
YPD liquid medium: peptone (20.0 g/L), glucose (20.0 g/L), yeast extract (20.0 g/L);
YPD solid medium: peptone (10.0 g/L), glucose (20.0 g/L), yeast extract powder (5.0 g/L), agar (14.0 g/L).
Example 1: screening of antioxidant Debaryomyces hansenii HRB1
1. Screening of antioxidant strains
S1: adding H 2O2 solution into the sterile YPD liquid medium to obtain YPD liquid medium with H 2O2 concentration of 0mM, 0.5mM, 1.0mM, 1.5mM and 2.0mM respectively; the 33 strains (Table 1) separated from the traditional air-dried sausage in northeast China are respectively inoculated into YPD liquid culture medium, and are cultured and activated for 2 generations at 28 ℃ and 180r/min for 12 hours per generation, so that bacterial liquids of the strains are obtained.
S2: adding 33 strains to be tested with the concentration of 3.0X10 8 CFU/mL into YPD liquid culture medium obtained in S1 respectively according to the inoculation amount of 2%, performing shake culture at 28 ℃ for 48 hours at 180r/min, and measuring absorbance of the culture solution at 600nm by using an enzyme-labeled instrument, wherein the survival rate of H 2O2 tolerant yeast strains is as follows:
survival (%) = (a i/A0) ×100
Wherein A i is absorbance after 48 hours of culture in YPD liquid medium, i is H 2O2,H2O2 concentration of 0.5mM, 1.0mM, 1.5mM and 2.0mM of different addition concentration in YPD liquid medium; a 0 is absorbance after 48 hours of culture in YPD liquid medium, wherein 0 is the concentration of 0mM H 2O2 added to YPD liquid medium.
S3: selecting a strain with the survival rate of not less than 50% after culturing in YPD liquid culture medium with the H 2O2 concentration of 2.0mM for 48 hours as a primary screening strain bacterial liquid resistant to high-concentration H 2O2;
S4: adding H 2O2 solution into the sterile YPD liquid medium to obtain YPD liquid medium with H 2O2 concentration of 0mM, 2mM, 4mM, 6mM, 8mM, 10mM and 12mM respectively;
S5: adding the strain solution of the primary screening strain obtained in the step S3 into YPD liquid culture medium according to the inoculation amount of 2%, performing shake culture for 1H at 28 ℃ and 180r/min, then adopting YPD solid culture medium, pouring and inoculating by a flat plate, selecting dilution factors of 10 -5、10-6 and 10 -7, performing colony counting after culturing for 48H at 28 ℃, multiplying the total colony number by the corresponding dilution factor to obtain the number of saccharomycetes, and finally selecting the strain with the death rate less than or equal to 1lg CFU mL -1 after culturing in YPD liquid culture medium with the H 2O2 concentration of 12.0mM as the re-screened HRB1 strain with high-concentration H 2O2 resistance.
As shown in FIG. 1, the survival rate of the primary screening strain is shown in FIG. 1, in this example, 33 strains to be tested are cultivated in a culture medium containing H 2O2, and are found to be viable, the survival rate gradually decreases along with the increase of the concentration of H 2O2 in the culture medium, when the concentration of H 2O2 is increased to 1.5mM, the survival rate of 19 strains in the 33 strains to be tested is higher than 50%, when the concentration of H 2O2 is increased to 2.0mM, the survival rate of only 11 strains exceeds 50%, which are D.hansenii SH4、D.hansenii HRB1、D.hansenii HRB3、C.zeylanoides SH10、T.asahii HRB8、Y.divulgata SH1、R.mucilaginosa SH8、R.mucilaginosa SH9、Y.galli SH11、C.metapsilosis MDJ7 and T.coremiiforme MDJ8 respectively; the result shows that 11 strains obtained by the preliminary screening show better survival ability under the stress of lower concentration of H 2O2, and have certain antioxidation capability.
In this example, 11 primary strains were cultured in a medium containing H 2O2 at a high concentration for 1 hour, and then the colony numbers were counted, and as a result, as shown in FIG. 2, the colony numbers of the respective strains gradually decreased with an increase in the concentration of H 2O2 in the medium, and when the concentration of H 2O2 was 12.0mM, only 7 strains of CFU mL -1 having a mortality rate of 1lg, C.metasilosis MDJ7, D.hansenii SH4, D.hansenii HRB1, D.hansenii HRB3, Y.gali SH11, R.mucilargineasa SH8 and R.muciliarosa SH9, respectively, were obtained. In summary, 7 strains obtained by the screening show better survival ability and better oxidation resistance under the condition of higher concentration stress of H 2O2, wherein the survival rate of D.hansenii HRB1 after being cultured for 48 hours in a culture medium containing 2.0mM H 2O2 is 50.11%, the colony number after being cultured for 1 hour in a culture medium containing 12.0mM H 2O2 is reduced from 8.5168 g CFU mL -1 to 7.7954 g CFU mL -1, and the death rate is not higher than 1 g CFU mL -1.
TABLE 1 names of strains to be tested
2. Determination of in vitro antioxidant capacity of strain
(1) Preparation of strain fermentation supernatant, somatic cells and intracellular extracts
S1: fully activating 7 re-screened strains obtained in the embodiment, and then placing activated bacterial liquid at 4 ℃ and centrifuging at 6000r/min for 10min to obtain bacterial strain fermentation supernatant, wherein the obtained precipitate is bacterial cells;
S2: then, the cells obtained in S1 were washed 3 times with a phosphate buffer (PBS: 0.2m, ph=7.4) and resuspended in PBS to obtain a cell suspension, and the final concentration of the cell suspension was about 3.0x10 8 CFU/mL to obtain a cell suspension;
s3: the cell suspension is crushed by ultrasonic in ice bath for 15min (ultrasonic for 2s, intermittent for 2s, 600W), and then centrifuged at 6000r/min for 10min at 4 ℃ to remove cell debris sediment, and the obtained supernatant is the intracellular extract.
(2) Detection of DPPH radical scavenging Rate
Taking 1.0mL of each of the strain fermentation supernatant, the thalli cells and the intracellular extracts of the 7 re-screened strains obtained in the example, adding 2.0mL of a 0.05mM freshly prepared DPPH-ethanol solution, immediately mixing, standing the mixed solution at a dark place at room temperature for 30min, centrifuging for 10min under 10000g to obtain a supernatant, measuring the absorbance A i of the supernatant at a wavelength of 517nm, and removing DPPH free radical percentage:
Clearance (%) = [1- (a i-A)/A0 ] ×100)
Wherein A is absorbance after the reaction of the equal volume of absolute ethyl alcohol instead of DPPH-ethanol solution, A i is sample absorbance, i is strain fermentation supernatant of 7 re-screened strains, thallus cells and intracellular extract samples, and A 0 is absorbance after the reaction of the equal volume of PBS instead of sample solution.
The result of the DPPH radical scavenging rate test in this example is shown in fig. 3, and the fermentation supernatant, cell and intracellular extract of 7 strains of yeast obtained in this example all have certain DPPH radical scavenging ability, and DPPH radical scavenging rates of different strains have certain differences, wherein the cell DPPH radical scavenging rate of d.hansenii HRB1 is the highest (P < 0.05). The results of the clearance rate of the three parts of the fermentation supernatant, the somatic cells and the intracellular extract of 7 strains of saccharomycetes are comprehensively shown that the strain D.hansenii HRB1 has the strongest capacity of scavenging DPPH free radicals on the whole.
(3) Detection of hydroxyl radical scavenging rate
1.0ML of phenanthroline (0.1%, w/v) and 1.0mL of 2.5mM FeSO 4 were added to a 10mL reaction tube containing 0.1mL of PBS (0.2M, pH=7.4), then 0.5mL of each of the strain fermentation supernatant of the 7 re-screened species obtained in this example, the bacterial cells and the intracellular extract were added, and 1.0mL of H 2O2 (0.01%, v/v) were mixed, reacted at room temperature for 1.5 hours, and then centrifuged at 10000g for 10 minutes to obtain a supernatant, and the absorbance A i of the supernatant was measured at 536nm to remove hydroxyl radicals as follows:
Clearance (%) = (a i-A0)×100/(a-a0)
Wherein A i is the absorbance of the sample, wherein i is the strain fermentation supernatant of 7 re-screened species, the cell and the intracellular extract sample, A 0 is the absorbance of the control group containing no relative sample, and A is the absorbance of the blank group containing no H 2O2.
In this example, as shown in fig. 4, the results of the detection of the hydroxyl radical scavenging rate show that the fermentation supernatant, the somatic cells and the intracellular extracts of the 7 strains of yeasts obtained in this example all have a certain hydroxyl radical scavenging ability, and in the fermentation supernatants, the somatic cells and the intracellular extracts of different strains, the hydroxyl radical scavenging rate of the fermentation supernatant is found to be significantly higher than that of the somatic cells and the intracellular extracts (P < 0.05), wherein the hydroxyl radical scavenging rates of the fermentation supernatants, the somatic cells and the intracellular extracts of d.hansenii SH4 and d.hansenii HRB1 are higher, and the hydroxyl radical scavenging ability is better.
(4) Detection of superoxide radical scavenging
Taking 0.1mL of each of the strain fermentation supernatant, the thallus cells and the intracellular extracts of the 7 re-screened strains obtained in the example, mixing with 2.8mL of Tris-HCl solution with pH=8.2, reacting for 30min at room temperature, then adding 0.1mL of 0.05M pyrogallol, reacting the mixture at room temperature for 4min, finally adding 1.0mL of 8M HCl to terminate the reaction, measuring absorbance at 325nm, and removing the superoxide radical percentage to be:
clearance (%) = [ (a 0-Ai)/A0 ] ×100)
Wherein A i is the absorbance of the sample, wherein i is the sample of strain fermentation supernatant, somatic cells and intracellular extracts of 7 re-screened strains, and A 0 is the absorbance of the reaction of the sample replaced by an equal volume of deionized water.
As shown in FIG. 5, the results of the detection of superoxide radical scavenging rate in this example show that the fermentation supernatant, somatic cells and intracellular extracts of 7 strains of yeast obtained in this example all have a certain superoxide radical scavenging capacity, and in the fermentation supernatant, somatic cells and intracellular extracts of different strains, the superoxide radical scavenging rate of the somatic cells is found to be significantly lower than that of the fermentation supernatant and intracellular extracts (P < 0.05), wherein the superoxide radical scavenging rates of D.hansenii SH4 and D.hansenii HRB1 fermentation supernatant, somatic cells and intracellular extracts are higher, and all have a good superoxide radical scavenging capacity.
(5) Detection of reducing Capacity
The 7 re-screened strain fermentation supernatant, the cell and the intracellular extract obtained in this example were placed in a 10mL reaction tube with 0.5mL PBS (0.2M, pH 6.6), 0.5mL potassium ferricyanide (1.0%, w/v) solution was added to the tube, the mixture was stirred uniformly, then the mixture was subjected to water bath at 50℃for 20 minutes, the mixture was rapidly cooled after being taken out, then 0.5mL 10% trichloroacetic acid solution was added, the mixture was centrifuged at 6000g/min for 10 minutes after being stirred uniformly, the supernatant was obtained, 3.0mL of the supernatant was mixed with 3.0.1% ferric trichloride solution, the mixture was allowed to stand at room temperature for 10 minutes after being stirred uniformly, and the absorbance of the mixture was measured at 700nm wavelength.
The reduction capacity detection results of this example are shown in fig. 6, wherein the fermentation supernatant, the bacterial cells and the intracellular extracts of the 7 yeasts obtained in this example all have a certain reduction capacity, and the sequence from large to small in the fermentation supernatant, the bacterial cells and the intracellular extracts of different strains is found to be the fermentation supernatant > the bacterial cells > the intracellular extracts (P < 0.05), wherein R.mularginosa SH8, C.metaplasirosis MDJ7, D.hansenii SH4 and D.hansenii HRB1 fermentation supernatant, the bacterial cells and the intracellular extracts all have a good reduction capacity.
In summary, the DPPH radical scavenging rates of the fermentation supernatants, the somatic cells and the intracellular extracts of the D.hansenii HRB1 strain are 25.49%, 28.37% and 30.24%, the hydroxyl radical scavenging rates are 59.40%, 47.91% and 48.75%, the superoxide radical scavenging rates are 31.36%, 22.39% and 37.55% and the reducing power are 0.84, 0.75 and 0.25, respectively, and the results prove that the D.hansenii HRB1 strain has stronger antioxidant capacity.
3. Isolation and identification of Debaryomyces hansenii HRB1
Homogenizing 10g of air-dried intestinal sample with 90mL of sterile normal saline, continuously performing 10-fold gradient dilution in the sterile normal saline, selecting 10 -5、10-6 and 10 -7 dilution multiples, respectively coating 200 mu L of dilution on YPD solid culture medium, selecting single colony conforming to the typical form of microzyme, repeatedly streaking and purifying to obtain a purified strain, sending the purified strain to biological medicine company in Jiangbei new area of Nanjing, jiangsu province for 26S rRNA sequence determination, wherein the sequence determination result is shown as SEQ ID NO.1, and performing BLAST (https;// BLAST. NCBI. Nlm. Gov/Blast. Cgi) comparison on the DNA sequence generated by sequencing through strain 26S rRNA of NCBI of a public database, and determining that the strain is Debaryomyces hansenii through homology detection and comparison, and the structure is shown in figure 7.
Example 2: preparation of Debaryomyces hansenii HRB1 starter
Debaryomyces hansenii HRB1 was passaged twice in YPD medium and cultured at 28℃for 12h; and centrifuging at 4deg.C and 10000g for 5min, removing supernatant, adding equal volume of sterile deionized water, and repeating for 2 times to obtain Debaryomyces hansenii HRB1 starter.
Example 3: application of debaryomyces hansenii HRB1 starter in air-dried sausage
S1: the raw material meat is lean meat (pig buttock meat) and fat meat (pig backfat) with the mass ratio of 9:1, and connective tissues such as lymph, tendon and blood vessel are removed;
S2: mincing with a meat mincer with a sieve plate with the aperture of 1.5cm, and adding the following seasonings according to the proportion: 2.5% of salt, 1% of Yuquan large yeast, 1% of soft white sugar, 0.3% of monosodium glutamate, 5% of water and 0.8% of mixed spice (comprising Chinese prickly ash, orange peel, fructus amomi, fennel, clove and the like) and 0.01% of nitrite (calculated by lean mass);
S3: inoculating the starter obtained in the example 2, adding 10 6 CFU/g of starter, taking a treatment group without starter as a control group, fully mixing the raw materials of each treatment group, filling the mixture into a small pig sausage casing, airing the pig sausage casing to a length of 15cm and a diameter of 2.5cm, airing the pig sausage casing for 24 hours in an environment with a temperature of 25+/-2 ℃ and a relative humidity of 30% -50%, and then transferring the pig sausage casing into a constant temperature and constant humidity fermentation box for fermentation for 8d at the temperature of 25+/-2 ℃ and the relative humidity of 65% -70%, thereby obtaining the treated air-dried sausage.
Example 4: effect of Debaryomyces hansenii HRB1 on the oxidative properties of air-dried intestines
This example is a test of the air-dried sausage obtained in example 3.
(1) Detection of Peroxide (POV)
Weighing 2.0g of the sheared air-dried sausage sample, putting the air-dried sausage sample into a 50mL centrifuge tube, adding 15mL of chloroform-methanol (2:1, V/V) solution stored at 4 ℃, and carrying out high-speed homogenizing centrifugation for 30s under 11000 r/min; adding 3mL of NaCl solution with the volume fraction of 0.5%, and centrifuging at 4 ℃ and 4000g for 5min; taking out 5mL of solution from the lower liquid phase of the centrifuged sample, transferring the solution into a clean test tube, adding 5mL of chloroform-methanol (2:1, V/V) solution stored at 4 ℃, and uniformly mixing by vortex for 3s; then adding 25 mu L of ferrous chloride solution and 25 mu L of ammonium thiocyanate (30%) solution, mixing uniformly by vortex for 3s, and standing for 5min at room temperature; the supernatant was taken and absorbance was measured at a wavelength of 500nm while the absorbance of reduced iron powder was used as a standard curve, and the POV results were expressed as mmol/kg, as shown in FIG. 8.
This example compares the air-dried intestinal peroxide values at days 0, 3, 6 and 9 of fermentation for air-dried intestines with and without debaryomyces hansenii HRB1 starter, and the results are shown in fig. 9, with no significant difference (P > 0.05) in air-dried intestinal peroxide values at initial fermentation (0 d) with and without debaryomyces hansenii HRB1 starter; the peroxide values of the air-dried intestines at the 3rd, 6 th and 9 th days of fermentation are respectively reduced from 0.65, 0.72 and 0.58 to 0.59, 0.66 and 0.50, which shows that the Debaryomyces hansenii HRB1 starter has a certain inhibition effect on lipid oxidation.
(2) Detection of thiobarbituric acid (TBARS)
Weighing 2.0g of the sheared air-dried intestinal sample, putting the air-dried intestinal sample into a 50mL centrifuge tube, adding 3mL of thiobarbituric acid solution (1% TBA) and 17mL of trichloroacetic acid-hydrochloric acid solution (2.5% TCA), uniformly mixing, heating in a boiling water bath for 30min, taking out, rapidly cooling to room temperature, sucking 4mL of the supernatant after reaction, adding 4mL of chloroform, and centrifuging for 10min at 3000 r; the absorbance of the supernatant was then measured at a wavelength of 532nm, TBARs at a mass of malondialdehyde per kg lipid oxidation sample:
TBARS value (mg/kg) = (A 532nm/. Omega.) times.9.48
Wherein A 532nm is the absorbance of the sample; omega is the sample mass (g); 9.48 is a constant.
This example compares the TBARS values of the air-dried intestines at days 0, 3, 6 and 9 of fermentation with and without debaryomyces hansenii HRB1 starter, and the results are shown in fig. 10, in which there is no significant difference (P > 0.05) in the TBARS values of the air-dried intestines at initial fermentation (0 d) with and without debaryomyces hansenii HRB1 starter; the TBARS values of the air-dried sausage are respectively reduced from 0.40, 0.48 and 0.59 to 0.34, 0.37 and 0.51 at the 3 rd day, the 6 th day and the 9 th day of fermentation, which shows that the Debaryomyces hansenii HRB1 starter has a certain inhibition effect on lipid oxidation, and the risks of bad flavor, spoilage and the like caused by lipid over-oxidation are reduced.
(3) Detection of carbonyl content
1G of an air-dried intestinal sample is weighed, 10mL of sodium pyrophosphate buffer solution is added for homogenate, 2mL of homogenate is added with 2mL of 20% (mass fraction, the same applies below) trichloroacetic acid, and the mixture is centrifuged for 5min under the condition of 12000 r/min; discarding the supernatant, adding 2mL of 2, 4-dinitrophenylhydrazine, adding 2mL of 2mol/L HCl into a blank group without using a starter, and reacting for 30min at room temperature in a dark place; adding 2mL of 20% trichloroacetic acid, mixing, centrifuging at 12000r/min for 5min, and discarding supernatant; adding 5mL ethanol-ethyl acetate mixed solution (1:1, V/V) to wash the precipitate 3 times (12000 r/min, 5 min); 2mL of 6mol/L guanidine hydrochloride is added, and the mixture is placed in a water bath at 37 ℃ for heat preservation for 30min; the protein mass concentration rho/(mg/mL) is measured by a Coomassie brilliant blue method, a blank control group is used for zeroing, the absorbance A 370 nm of a read sample at the wavelength of 370nm is measured, the molar absorbance coefficient is 22000L/(mol cm), and the protein carbonyl content is:
Carbonyl content (n mol/mg) = (a 370 nm×107)/(0.625×22000×ρ)
Wherein A 370 nm is absorbance of the sample at 370nm, 22000 is molar absorptivity, ρ is mass concentration of protein measured by Coomassie Brilliant blue method, and 10. 10 7 and 0.625 are constants.
This example compares the carbonyl content of air-dried sausage at days 0, 3, 6 and 9 of fermentation with and without debaryomyces hansenii HRB1 starter, and shows that there is no significant difference (P > 0.05) in the carbonyl content of air-dried sausage at initial stage of fermentation (0 d) with and without debaryomyces hansenii HRB1 starter; the carbonyl content of the air-dried sausage is respectively reduced from 4.20, 5.80 and 9.20 to 4.01, 5.60 and 8.87 in the 3 rd, 6 th and 9 th days of fermentation, which shows that the Debaryomyces hansenii HRB1 starter has a certain inhibition effect on the oxidation of protein, and the risks of nutrition loss, poor flavor, spoilage and the like caused by the excessive oxidation of the protein are reduced.
The details of the present invention which are not described in detail in the present specification are known to those skilled in the art. While the invention has been described in terms of preferred embodiments, it is not intended to be limited thereto, but rather to enable any person skilled in the art to make various changes and modifications without departing from the spirit and scope of the present invention, which is therefore to be limited only by the appended claims.
Claims (5)
1. The Hansenula debaryomyces HRB1 with the antioxidation function is characterized in that the Hansenula debaryomyces has a preservation number of CGMCC NO 28944, is classified and named Debaryomyces hansenii, is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) and has a preservation address of China center for China, national institute of sciences of China, no. 3, north Star West Hirudo 1, and a preservation date of 2023, 11 months and 10 days.
2. A starter culture comprising the Debaryomyces hansenii HRB1 of claim 1.
3. The starter according to claim 2, wherein the effective concentration of debaryomyces hansenii in the starter is 1 x 10 6 CFU/mL.
4. Use of debaryomyces hansenii according to claim 1 or the starter according to claim 2 in the fermentation of meat products.
5. The use according to claim 4, wherein the fermented meat product comprises air-dried sausage, bacon or ham.
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