WO2023061098A1 - 一种酵母、乳酸菌联合菌、培育方法及其应用 - Google Patents

一种酵母、乳酸菌联合菌、培育方法及其应用 Download PDF

Info

Publication number
WO2023061098A1
WO2023061098A1 PCT/CN2022/116841 CN2022116841W WO2023061098A1 WO 2023061098 A1 WO2023061098 A1 WO 2023061098A1 CN 2022116841 W CN2022116841 W CN 2022116841W WO 2023061098 A1 WO2023061098 A1 WO 2023061098A1
Authority
WO
WIPO (PCT)
Prior art keywords
bacteria
lactobacillus
yeast
lactic acid
fermentation
Prior art date
Application number
PCT/CN2022/116841
Other languages
English (en)
French (fr)
Inventor
耿胜利
Original Assignee
命之源(杭州)生物科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 命之源(杭州)生物科技有限公司 filed Critical 命之源(杭州)生物科技有限公司
Publication of WO2023061098A1 publication Critical patent/WO2023061098A1/zh

Links

Images

Classifications

    • 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
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
    • A01N63/32—Yeast
    • 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
    • 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
    • A23B7/00—Preservation or chemical ripening of fruit or vegetables
    • A23B7/14—Preserving or ripening with chemicals not covered by groups A23B7/08 or A23B7/10
    • A23B7/153—Preserving or ripening with chemicals not covered by groups A23B7/08 or A23B7/10 in the form of liquids or solids
    • A23B7/154—Organic compounds; Microorganisms; Enzymes
    • A23B7/155—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
    • A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
    • A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
    • A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
    • A23L3/3463—Organic compounds; Microorganisms; Enzymes
    • A23L3/3571—Microorganisms; Enzymes
    • 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
    • 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
    • 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
    • C12N1/205—Bacterial isolates
    • 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
    • C12N1/36—Adaptation or attenuation of cells
    • 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
    • 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
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • 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
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/225—Lactobacillus
    • 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
    • C12R2001/72—Candida

Definitions

  • the invention relates to the technical field of microbial application, in particular to a yeast, a lactic acid bacteria combination, a cultivation method and an application thereof.
  • Joint flora also known as joint microbial colony or mixed flora usually refers to a microbial ecosystem composed of two or more artificially selected excellent strains.
  • the microorganisms in this system have nutritional mutual benefits, complementary advantages, and ecological balance. , strong survivability, and strong adaptability. By ingeniously combining different strains, it can perform better than a single strain.
  • Lactic acid bacteria refer to a group of bacteria that produce a large amount of lactic acid by fermenting sugars. They can be divided into cocci and bacilli in terms of morphology. And all are Gram-positive facultative anaerobic or anaerobic bacteria that grow well in an oxygen-deficient environment. It is widely distributed in nature, and widely exists in human, livestock and poultry intestines, many foods, materials and a few clinical samples. Except for a few, most of them are essential flora with important physiological functions in humans and animals. Yeast is a typical single-cell microorganism with a simple structure and belongs to the fungus class.
  • yeast Because yeast has the characteristics of large individual, high protein content, strong omnivorousness, easy separation, easy cultivation, many metabolites, and wide comprehensive utilization, it is not only used in wine making, but also used in glycerin, organic acids, and enzyme preparations in the industry. Wait for production. Both lactic acid bacteria and yeast are microorganisms that are beneficial to the human body and have similar growth conditions, that is, a symbiotic basis.
  • the present invention provides a yeast, lactic acid bacteria combined bacteria, cultivation method and application thereof, which are composed of stable symbiotic bacterial strains, can promote each other and naturally balance, and expand the scope of application for it; and through
  • the biological enzyme preparation prepared by the fermentation product of the combined strains can be used to prepare a natural biological antiseptic and fresh-keeping agent with good anti-oxidation and fresh-keeping functions.
  • a kind of yeast, lactic acid bacteria combination bacteria it comprises yeast and lactic acid bacteria, described yeast comprises Candida ethanolophilus B-JJ1, and described lactic acid bacteria comprises Lactobacillus Brucella B-JR1, Lactobacillus paracasei B-JR2, corn At least one of Lactobacillus B-JR4, Lactobacillus plantarum B-JR5, and Lactobacillus B-JR6;
  • the preservation number of the Candida ethanolica B-JJ1 is CCTCC NO: M2021136, and its classification is named as Candida ethanolica;
  • Lactobacillus buchneri B-JR1 The preservation number of the Lactobacillus buchneri B-JR1 is CCTCC NO: M2021132, and its classification is named Lactobacillus buchneri;
  • Lactobacillus casei B-JR2 The preservation number of said Lactobacillus casei B-JR2 is CCTCC NO: M2021133, and its classification is named as Lactobacillus paracasei;
  • Lactobacillus zeae B-JR4 The preservation number of the Lactobacillus zeae B-JR4 is CCTCC NO: M2021135, and its classification is named Lactobacillus zeae;
  • Lactobacillus plantarum B-JR5 The preservation number of said Lactobacillus plantarum B-JR5 is CCTCC NO: M2021501, and its classification is named Lactobacillus plantarum;
  • the preservation number of the Lactobacillus B-JR6 is CCTCC NO: M2021502, and its classification is named Lactobacillus chiayiensis.
  • yeast includes yeast and lactic acid bacteria
  • the yeast is Candida ethanolophilus B-JJ1
  • the lactic acid bacteria are composed of Lactobacillus brauchneri B-JR1, Lactobacillus paracasei B-JR2, Lactobacillus corn B-JR4, Composed of Lactobacillus plantarum B-JR5 and Lactobacillus B-JR6.
  • the present invention also provides a method for cultivating the above-mentioned yeast and lactic acid bacteria combined bacteria, which includes the following steps:
  • Seed bacteria extraction take the pit mud fermentation product of the liquor brewing pit, add sterile water into the pit mud fermentation product according to the weight ratio, mix it and leave it for later use;
  • Substratum preparation the substratum is prepared from mashed potato powder, wheat germ, and glutinous rice as the main ingredients; after taking the raw materials, add water according to the weight ratio, and stir evenly, and then treat the granules through steam distillation After expansion, it can be put into a sterile container and spread out to dissipate heat, and then set aside after drying to a certain temperature;
  • Bacterial strain inoculation the pit mud fermentation product of step (1) and the prepared medium of step (2) are mixed evenly according to the weight ratio, and then placed in a sterile environment for constant temperature and aerobic cultivation;
  • Sampling detection and identification of the obtained symbiotic colonies detection of the activity of the combined bacteria in step (6), when the average live bacteria of yeast is 6.4 ⁇ 10 4 cfu/ml or more; the average live bacteria of lactic acid bacteria is 4 ⁇ When the concentration is above 10 8 cfu/ml, the first-generation yeast and lactic acid combined bacteria are obtained;
  • Combined bacteria acclimatization Mix the culture medium in step (2) with the first-generation yeast and lactic acid bacteria combined bacteria in step (7), and spray sterile water or steamer water cooled to 37°C until it is wetted to It is wet and loose, and water drips out when it is held in the hand; then it is activated aerobically until white or milky white plaques are evenly distributed on the surface of the culture medium, and the surface culture medium feels wet and sticky and overflows with a yeast aroma; then the mixed culture The base material is placed in a closed environment and continues to ferment until the surrounding oxygen is gradually consumed for anaerobic fermentation; anaerobic fermentation until the joint colony of yeast and lactic acid bacteria grows vigorously, that is, the acclimation of the joint colony of yeast and lactic acid is completed.
  • a method for cultivating the above-mentioned yeast and lactic acid bacteria combined bacteria comprises the following steps:
  • Seed bacteria extraction take the pit mud fermented product from the liquor brewing cellar, add 35-38°C sterile water into the pit mud fermented product according to the weight ratio of 2:1, mix it and let it stand for later use;
  • the medium is prepared from mashed potato powder, wheat germ, and glutinous rice as main ingredients; take mashed potato powder, wheat germ, and glutinous rice, and add water according to a weight ratio of 1:1 , and stir evenly, and then steam distilled for about 40 minutes. After the granules expand, they can be spread out in a sterile container to dissipate heat, and aired to below 45 degrees Celsius for later use;
  • the above-mentioned medium also includes sorghum rice and buckwheat.
  • the ratio of each raw material of the medium is:
  • 5% of sorghum rice and buckwheat can be replaced with sugar.
  • Sampling detection and identification of the obtained symbiotic colonies detection of the activity of the combined bacteria in step (6), when the average live bacteria of yeast is 6.4 ⁇ 10 4 cfu/ml or more; the average live bacteria of lactic acid bacteria is 4 ⁇ When the concentration is above 10 8 cfu/ml, the first-generation yeast and lactic acid combined bacteria are obtained;
  • Combined bacteria acclimatization mix the medium in step (2) with the first-generation yeast and lactic acid bacteria combined bacteria in step (7) at a ratio of 1:1, and spray sterile water or cool to 37°C Steam the water in a steamer until it becomes wet and loose, and water drips out when you hold it in your hand; then place it at a sterile temperature above 26°C for aerobic activation for more than 48 hours, until white or milky white plaques appear on the surface of the culture medium and evenly distribute, remove The surface culture medium has a wet and sticky feeling and overflows with the aroma of yeast; then the mixed culture medium is placed in a closed environment to continue fermentation until the surrounding oxygen is gradually consumed for anaerobic fermentation; after 20-30 days of anaerobic fermentation, yeast, The combined colony of lactic acid bacteria grew vigorously, that is, the combined colony of yeast and lactic acid was domesticated.
  • step (9) Combined bacteria packaging: vacuum pack the culture medium of the yeast and lactic acid combined bacteria in step (8) at a rate of 5-10 kg/bag.
  • the average live bacteria of the yeast and lactic acid combined bacteria in step (9) is above 6.4 ⁇ 10 4 cfu/ml, and the average live bacteria of lactic acid bacteria is 4 ⁇ 10 8 cfu/ml above.
  • the present invention also provides a preparation method of a biological enzyme preparation, the biological enzyme preparation is made from the fermentation product of the above-mentioned yeast and lactic acid combined bacteria.
  • preparation method of the above-mentioned biological enzyme preparation comprises the following steps:
  • the culture medium is formulated with mashed potato powder, wheat germ, and glutinous rice as the main ingredients. After the culture medium is added with water and stirred evenly, it is steamed, and then aired in the aseptic environment until Below 45 degrees Celsius, the medium preparation is completed;
  • step (3) the humidity of the culture medium after step (2) inoculation is adjusted to ensure that the dough has water stains, and the culture medium as a whole remains moist and breathable;
  • step (3) Carry out aerobic fermentation to the substratum of step (3) until evenly visible white and milky white plaques appear in the substratum, transfer the substratum into the fermenter;
  • the fermented product of the culture medium continues to be cultured aerobically in the tank until the oxygen in the tank is exhausted and enters the state of anaerobic fermentation by itself;
  • step (6) Add the solid-state fermented product of step (5) into sterile water, and fully stir to dilute the metabolic substrate in the solid-state fermentation process, and carry out aerobic activation culture until the surface of the liquid fermented product is densely covered with white or milky white plaques, All the passages of the fermenter can be closed, and the fermented product gradually depletes oxygen and enters the anaerobic fermentation process;
  • the obtained fermentation product can be the prepared biological enzyme preparation.
  • step (8) After the fermentation substrate obtained in step (7) is heated and inactivated, the supernatant of the fermented product is extracted to obtain an inactivated biological enzyme preparation.
  • the present invention also provides the application of the above-mentioned biological enzyme preparation, which can be used for the preparation of biological preservatives.
  • the biological preservation agent can be applied to preservation and preservation of vegetables, fruits or fresh or dried meat.
  • the biological preservative can be used to prepare human or animal body specimens.
  • the present invention adopts processes such as inoculation, breeding, optimization of combined bacteria, and combined bacteria domestication to cultivate strong symbiotic ability and good stability of yeast and lactic acid bacteria combined bacteria.
  • the cultivated combined bacteria have higher activity and can be According to actual needs, after the combined bacteria are purified and separated, they are mixed and formulated into combined bacteria with different properties;
  • the combined bacteria of the present invention undergoes complex biochemical reactions through fermentation, and the generated fermentation product is rich in various amino acid residues, amides, coenzymes, lactic acid, linoleic acid and other active groups, which can be used to prepare nutritious Biological enzyme preparations, and the biological enzyme preparations prepared by the present invention can be used to prepare antiseptic preservatives;
  • the antiseptic preservative prepared by the present invention not only has nutritional components such as various amino acids, various enzymes, and enzyme precursors that can maintain the activity of animal and plant tissue cells and can repair defective cells, but also can The inhibition of enzyme activity delays the saccharification process of organisms, and can kill microorganisms such as Pythium mold, which has strong anti-oxidation, anti-corrosion, and fresh-keeping effects;
  • the antiseptic preservative prepared by the biological enzyme preparation of the present invention is natural, without any additives, safe and efficient. It can not only be used to keep fresh plants or meat fresh, but also keep animal and plant cells active for a long time and prolong their fresh-keeping time , and it can also preserve the freshness of dried meat and prevent oxidation and discoloration.
  • Fig. 1 is the bacterial strain morphological feature photo of Candida ethanolophilus B-JJ1 of the present invention; Wherein (a) is optical microscope microscopic examination photo (400X); (b) is front plate photo;
  • Fig. 2 is the bacterial strain morphological feature photo of Lactobacillus brucelli B-JR1 of the present invention; Wherein (a) is optical microscope microscopic examination photo (1000X); (b) is front plate photo;
  • Fig. 3 is the strain morphological feature photo of Lactobacillus casei B-JR2 of the present invention; Wherein (a) is optical microscope microscopic examination photo (1000X); (b) is front plate photo;
  • Fig. 4 is the strain morphological feature photo of Lactobacillus zea B-JR4 of the present invention; Wherein (a) is optical microscope microscopic examination photo (1000X); (b) is front plate photo;
  • Fig. 5 is the bacterial strain morphological feature photo of Lactobacillus plantarum B-JR5 of the present invention; Wherein (a) is optical microscope microscopic examination photo (1000X); (b) is front plate photo;
  • Fig. 6 is the bacterial strain morphological feature photo of Lactobacillus B-JR6 of the present invention; Wherein (a) is optical microscope microscopic examination photo (1000X); (b) is front plate photo;
  • Fig. 7 is the photograph that the biological enzyme preparation of embodiment 10 of the present invention is applied to the fresh-keeping experiment of garlic moss;
  • Fig. 8 is the photo that the present invention implements 11 biological enzyme preparations and is applied to pear fresh-keeping experiment;
  • Fig. 9 is the photograph that biological enzyme preparation of embodiment 12 of the present invention is applied to the pear fresh-keeping experiment through injury;
  • Fig. 10 is the photograph that the biological enzyme preparation of embodiment 13 of the present invention is applied to fresh pork preservation experiment
  • Fig. 11 is the photograph that the biological enzyme preparation of embodiment 14 of the present invention is applied to fresh pork preservation experiment
  • Fig. 12 is the photograph that the biological enzyme preparation of embodiment 15 of the present invention is applied to fresh pig's head fresh-keeping experiment;
  • Fig. 13 is the photograph that the biological enzyme preparation of embodiment 16 of the present invention is applied to fresh shrimp;
  • Fig. 14 is the photo of the anti-mold experiment of the embodiment of the present invention 17 biological enzyme preparations
  • Fig. 15 is a photo of the inhibition saccharification experiment of the biological enzyme preparation of Example 18 of the present invention.
  • Fig. 16 is the photomicrograph of the snowflake mud grouping molecule of the biological enzyme preparation of the embodiment of the present invention 16;
  • the preservation number of the Candida alcoholicus B-JJ1 of the present invention is CCTCC NO: M2021136; the preservation date is January 21, 2021; the preservation institution is the China Center for Typical Culture Collection; the preservation place is Wuhan University, China.
  • the preservation number of the Lactobacillus buchneri B-JR1 is CCTCC NO: M2021132; the preservation date is January 21, 2021; the preservation institution is the China Center for Typical Culture Collection; the preservation place is Wuhan University, China.
  • the preservation number of the Lactobacillus paracasei B-JR2 is CCTCC NO: M2021133; the preservation date is January 21, 2021; the preservation institution is the China Center for Typical Culture Collection; the preservation place is Wuhan University, China.
  • the preservation number of the Lactobacillus zea B-JR4 is CCTCC NO: M2021135; the preservation date is January 21, 2021; the preservation institution is the China Center for Typical Culture Collection; the preservation place is Wuhan University, China.
  • the preservation number of the Lactobacillus plantarum B-JR5 is CCTCC NO: M2021501; the preservation date is May 07, 2021; the preservation institution is the China Center for Typical Culture Collection; the preservation place is Wuhan University, China.
  • the preservation number of the Lactobacillus B-JR6 is CCTCC NO: M2021502; the preservation date is May 07, 2021; the preservation institution is the China Center for Typical Culture Collection; the preservation place is Wuhan University, China.
  • Embodiment 1 a kind of yeast, lactic acid bacteria combined bacteria, its cultivation method is that the steps are as follows:
  • Sampling detection and identification of the obtained symbiotic colonies detection of the activity of the combined bacteria in step (6), when the average live bacteria of yeast is 6.4 ⁇ 10 4 cfu/ml or more; the average live bacteria of lactic acid bacteria is 4 ⁇ When the concentration is above 10 8 cfu/ml, the first-generation yeast and lactic acid combined bacteria are obtained;
  • the highest value of live yeast bacteria is 3.1 ⁇ 10 5 cfu/ml
  • the highest live bacteria value of lactic acid bacteria is 1.2 ⁇ 10 9 cfu/ml.
  • step (2) due to the acquired joint colonies, after the complementation of large groups such as Aspergillus and Mucor, a new small group of symbiotic adaptation, adaptation to the medium, and aerobic and anaerobic environment are formed.
  • the adaptation of the change of living environment such as conversion therefore needs to domesticate combined bacterium;
  • the substratum in step (2) is mixed with the first generation yeast of step (7), lactic acid bacteria combined bacterium according to quantity quality 1:1, and spray Spray sterile water until it becomes wet and loose, and water drips out from the hand; then place it in a sterile environment above 26°C for aerobic activation for more than 48 hours, until white or milky white plaques appear on the surface of the culture medium and evenly distribute.
  • the culture medium on the open surface has a wet and sticky feeling and overflows with the aroma of yeast; then the mixed culture medium is placed in a closed environment to continue fermentation until the surrounding oxygen is gradually consumed for anaerobic fermentation; after 25 days of anaerobic fermentation, yeast, lactic acid bacteria The joint colonies grew vigorously, that is, the joint colonies of yeast and lactic acid were domesticated.
  • step (8) Vacuum-pack the culture medium of yeast and lactic acid combined bacteria in step (8) at 5-10 kg/bag, and isolate the air to prevent aerobic bacteria infection and completely kill the possible first-generation strains Residual fungal spores such as Aspergillus, Mucor, etc. can facilitate industrial transportation and application.
  • the yeast and lactic acid combined bacteria after subpackaging in step (9) are detected, and the average value of viable bacteria of yeast is 6.4 ⁇ 104 cfu/ml or more, and the average value of viable bacteria of lactic acid bacteria is 4 ⁇ 10 More than 8 cfu/ml, mold among harmful bacteria ⁇ 10cfu/ml; Escherichia coli ⁇ 30cfu/ml; Salmonella ND; Shigella ND; Staphylococcus aureus ND (ND means not detected).
  • Embodiment 2 a kind of yeast, lactic acid bacteria combined bacteria, its cultivation method is that the steps are as follows:
  • the brewing pool of this embodiment is a traditional liquor brewing pool in Yaowan Ancient Town, Subei.
  • Sampling detection and identification of the obtained symbiotic colonies detection of the activity of the combined bacteria in step (6), when the average live bacteria of yeast is 6.4 ⁇ 10 4 cfu/ml or more; the average live bacteria of lactic acid bacteria is 4 ⁇ When the concentration is above 10 8 cfu/ml, the first-generation yeast and lactic acid combined bacteria are obtained;
  • the highest value of live yeast bacteria is 3.1 ⁇ 10 5 cfu/ml
  • the highest live bacteria value of lactic acid bacteria is 1.2 ⁇ 10 9 cfu/ml.
  • step (2) due to the acquired joint colonies, after the complementation of large groups such as Aspergillus and Mucor, a new small group of symbiotic adaptation, adaptation to the medium, and aerobic and anaerobic environment are formed.
  • the adaptation of the change of living environment such as conversion therefore needs to domesticate combined bacterium;
  • the substratum in step (2) is mixed with the first generation yeast of step (7), lactic acid bacteria combined bacterium according to quantity quality 1:1, and spray Spray sterile water until it becomes wet and loose, and water drips out from the hand; then place it in a sterile environment above 26°C for aerobic activation for more than 48 hours, until white or milky white plaques appear on the surface of the culture medium and evenly distribute.
  • the culture medium on the open surface has a wet and sticky feeling and overflows with the aroma of yeast; then the mixed culture medium is placed in a closed environment to continue fermentation until the surrounding oxygen is gradually consumed for anaerobic fermentation; after 25 days of anaerobic fermentation, yeast, lactic acid bacteria The joint colonies grew vigorously, that is, the joint colonies of yeast and lactic acid were domesticated.
  • step (8) Vacuum-pack the culture medium of yeast and lactic acid combined bacteria in step (8) at 5-10 kg/bag, and isolate the air to prevent aerobic bacteria infection and completely kill the possible first-generation strains Residual fungal spores such as Aspergillus, Mucor, etc. can facilitate industrial transportation and application.
  • the yeast and lactic acid combined bacteria after subpackaging in step (9) are detected, and the average value of viable bacteria of yeast is 6.4 ⁇ 104 cfu/ml or more, and the average value of viable bacteria of lactic acid bacteria is 4 ⁇ 10 More than 8 cfu/ml, mold among harmful bacteria ⁇ 10cfu/ml; Escherichia coli ⁇ 30cfu/ml; Salmonella ND; Shigella ND; Staphylococcus aureus ND (ND means not detected).
  • Embodiment 3 a kind of yeast, lactic acid bacteria combined bacteria, its cultivation method is that the steps are as follows:
  • the brewing pool of this embodiment is a traditional liquor brewing pool in Yaowan Ancient Town, Subei.
  • Sampling detection and identification of the obtained symbiotic colonies detection of the activity of the combined bacteria in step (6), when the average live bacteria of yeast is 6.4 ⁇ 10 4 cfu/ml or more; the average live bacteria of lactic acid bacteria is 4 ⁇ When the concentration is above 10 8 cfu/ml, the first-generation yeast and lactic acid combined bacteria are obtained;
  • the highest value of live yeast bacteria is 3.1 ⁇ 10 5 cfu/ml
  • the highest live bacteria value of lactic acid bacteria is 1.2 ⁇ 10 9 cfu/ml.
  • step (2) due to the acquired joint colonies, after the complementation of large groups such as Aspergillus and Mucor, a new small group of symbiotic adaptation, adaptation to the medium, and aerobic and anaerobic environment are formed.
  • the adaptation of the change of living environment such as conversion therefore needs to domesticate combined bacterium;
  • the substratum in step (2) is mixed with the first generation yeast of step (7), lactic acid bacteria combined bacterium according to quantity quality 1:1, and spray Spray sterile water until it becomes wet and loose, and water drips out from the hand; then place it in a sterile environment above 26°C for aerobic activation for more than 48 hours, until white or milky white plaques appear on the surface of the culture medium and evenly distribute.
  • the culture medium on the open surface has a wet and sticky feeling and overflows with the aroma of yeast; then the mixed culture medium is placed in a closed environment to continue fermentation until the surrounding oxygen is gradually consumed for anaerobic fermentation; after 25 days of anaerobic fermentation, yeast, lactic acid bacteria The joint colonies grew vigorously, that is, the joint colonies of yeast and lactic acid were domesticated.
  • step (8) Vacuum-pack the culture medium after the yeast and lactic acid combined bacteria in step (8) are 5-10 kg/bag, and isolate the air to prevent aerobic bacteria infection and completely kill the first-generation strains Residual fungal spores such as Aspergillus, Mucor, etc. can facilitate industrial transportation and application.
  • the yeast and lactic acid combined bacteria after subpackaging in step (9) are detected, and the average value of viable bacteria of yeast is 6.4 ⁇ 104 cfu/ml or more, and the average value of viable bacteria of lactic acid bacteria is 4 ⁇ 10 More than 8 cfu/ml, mold among harmful bacteria ⁇ 10cfu/ml; Escherichia coli ⁇ 30cfu/ml; Salmonella ND; Shigella ND; Staphylococcus aureus ND (ND means not detected).
  • the combined bacteria in the step (9) of Example 1 to Example 3 were separated and purified to obtain 6 kinds of bacteria, which were respectively Candida alcoholophilus, Lactobacillus Brucella, Lactobacillus paracasei, and Lactobacillus bread , Lactobacillus zea, Lactobacillus plantarum, Lactobacillus chiayiensis, this application is respectively named as Candida alcoholophilus B-JJ1, Lactobacillus brauchneri B-JR1, Lactobacillus paracasei B-JR2, Lactobacillus zea B-JR4 , Lactobacillus plantarum B-JR5, and Lactobacillus B-JR6 were separated and purified for detection.
  • the colony and cell morphology of each strain were shown in Figure 1 to Figure 7, respectively, and the physical, chemical and biochemical identification of each strain was carried out. The results are shown in Table 1 to Table 12.
  • Table 8 Physiological and biochemical characteristics of Lactobacillus plantarum B-JR5 - enzyme activity, carbon source oxidation
  • the molecular biology method of 18S rRNA gene sequencing was used to identify the above-mentioned Candida alcoholophilus B-JJ1, whose gene sequence is shown in SEQ. JR1, Lactobacillus paracasei B-JR2, Lactobacillus zea B-JR4, Lactobacillus plantarum B-JR5, Lactobacillus B-JR6 were sequenced, and their gene sequences were SEQ.ID.NO.2, SEQ.ID.NO .3, shown in SEQ.ID.NO.4, SEQ.ID.NO.5, SEQ.ID.NO.6.
  • the present invention can purify and separate the combined bacteria according to actual needs, and then mix and prepare combined bacteria with different properties.
  • the yeast and lactic acid combined bacteria of the present invention can include Candida alcoholophilus B-JJ1, Lactobacillus Brucella B-JR1, Lactobacillus B-JR6; or Candida ethanophilia B-JJ1, Lactobacillus Brucella B-JR1, Lactobacillus paracasei B-JR2, Lactobacillus B-JR6; or Alcoholophilia Candida B-JJ1, Lactobacillus Brucella B-JR1, Lactobacillus paracasei B-JR2, Lactobacillus corn B-JR4, Lactobacillus B-JR6.
  • Example 1 to Example 3 Its specific operations can be prepared in the step (9) of Example 1 to Example 3, including Candida ethanolophilus B-JJ1, Lactobacillus Brucella B-JR1, Lactobacillus paracasei B-JR2, corn milk Bacillus B-JR4, Lactobacillus plantarum B-JR5, and Lactobacillus B-JR6 were separated and purified according to conventional methods in the field, and after obtaining 6 kinds of bacteria, they were expanded and cultivated according to requirements, and then mixed culture was carried out to cultivate different strains. Joint bacteria.
  • the combined bacteria of the present invention can also directly adopt the combined bacteria obtained in step (9) of embodiment 1 to embodiment 3.
  • Embodiment 4 a kind of biological enzyme preparation, it is made by the fermentation product of yeast, lactic acid bacteria combination bacteria, and its preparation process is:
  • preparation medium said medium is formulated from 30 parts of mashed potato powder, 30 parts of wheat germ, 20 parts of glutinous rice, 10 parts of sorghum rice, and 10 parts of buckwheat;
  • yeast and lactic acid combined bacteria include Candida ethanolophilus B-JJ1, Lactobacillus Brucella B-JR1, and Lactobacillus B-JR6;
  • step (3) the humidity of the culture medium after step (2) inoculation is adjusted to ensure that the dough has water stains, and the culture medium as a whole remains moist and breathable;
  • step (3) Carry out aerobic fermentation to the substratum of step (3) for more than 48 hours;
  • the substratum appears uniform white, milky white plaque visible to the naked eye, the substratum is transferred in the fermenter, and the solid-state fermented product occupies the part of the fermenter.
  • One third is enough, when the aerobic fermentation exhausts the oxygen in the fermenter, it enters the anaerobic fermentation;
  • step (4) When step (4) continues to ferment until the solid-state fermentation reaches saturation, add sterile water and stir to enter the liquid-state fermentation stage, and ensure that there is at least 1/5 of the space in the fermenter, and then carry out aerobic activation for more than 72 hours , until the surface of the liquid fermented product is densely covered with white or milky white plaques, when the aerobic fermentation exhausts the oxygen in the fermenter, enter anaerobic fermentation again;
  • the obtained fermentation product is the prepared biological enzyme preparation.
  • Embodiment 5 a kind of biological enzyme preparation, it is made by the fermentation product of yeast, lactic acid bacteria combination bacteria, and its preparation process is:
  • preparation medium said medium is formulated from 30 parts of mashed potato powder, 30 parts of wheat germ, 20 parts of glutinous rice, 10 parts of sorghum rice, and 10 parts of buckwheat;
  • yeast and lactic acid combined bacteria include Candida ethanolophilus B-JJ1, Lactobacillus Brucella B-JR1, Lactobacillus paracasei B-JR2 , Lactobacillus B-JR6;
  • step (3) the humidity of the culture medium after step (2) inoculation is adjusted to ensure that the dough has water stains, and the culture medium as a whole remains moist and breathable;
  • step (3) Carry out aerobic fermentation to the substratum of step (3) for more than 48 hours;
  • the substratum appears uniform white, milky white plaque visible to the naked eye, the substratum is transferred in the fermenter, and the solid-state fermented product occupies the part of the fermenter.
  • One third is enough, when the aerobic fermentation exhausts the oxygen in the fermenter, it enters the anaerobic fermentation;
  • step (4) When step (4) continues to ferment until the solid-state fermentation reaches saturation, add sterile water and stir to enter the liquid-state fermentation stage, and ensure that there is at least 1/5 of the space in the fermenter, and then carry out aerobic activation for more than 72 hours , until the surface of the liquid fermented product is densely covered with white or milky white plaques, when the aerobic fermentation exhausts the oxygen in the fermenter, enter anaerobic fermentation again;
  • the obtained fermentation product is the prepared biological enzyme preparation.
  • Embodiment 6 a kind of biological enzyme preparation, it is made by the fermentation product of yeast, lactic acid bacteria combination bacteria, and its preparation process is:
  • preparation medium said medium is formulated from 30 parts of mashed potato powder, 30 parts of wheat germ, 20 parts of glutinous rice, 10 parts of sorghum rice, and 10 parts of buckwheat;
  • yeast and lactic acid combined bacteria are inoculated on the medium for fermentation;
  • the yeast and lactic acid combined bacteria include Candida ethanolophilus B-JJ1, and lactic acid bacteria include Lactobacillus Brucella B-JR1 and Lactobacillus paracasei B-JR2, Lactobacillus zea B-JR4, Lactobacillus plantarum B-JR5, Lactobacillus B-JR6;
  • step (3) the humidity of the culture medium after step (2) inoculation is adjusted to ensure that the dough has water stains, and the culture medium as a whole remains moist and breathable;
  • step (3) Carry out aerobic fermentation to the substratum of step (3) for more than 48 hours;
  • the substratum appears uniform white, milky white plaque visible to the naked eye, the substratum is transferred in the fermenter, and the solid-state fermented product occupies the part of the fermenter.
  • One third is enough, when the aerobic fermentation exhausts the oxygen in the fermenter, it enters the anaerobic fermentation;
  • step (4) When step (4) continues to ferment until the solid-state fermentation reaches saturation, add sterile water and stir to enter the liquid-state fermentation stage, and ensure that there is at least 1/5 of the space in the fermenter, and then carry out aerobic activation for more than 72 hours , until the surface of the liquid fermented product is densely covered with white or milky white plaques, when the aerobic fermentation exhausts the oxygen in the fermenter, enter anaerobic fermentation again;
  • the obtained fermentation product is the prepared biological enzyme preparation.
  • Embodiment 7 a kind of biological preservative preservation agent, it is prepared by the biological enzyme preparation of embodiment 4 to embodiment 6.
  • Example 8 The biological enzyme preparations prepared in Examples 4 to 7 are directly used as biological preservatives.
  • Example 9 After the biological enzyme preparation prepared in Example 6 was heat-inactivated, the supernatant of the fermented product was extracted to obtain a biological enzyme preparation, which was directly used as a biological preservative.
  • Embodiment 10 when the biological enzyme preparation that embodiment 8 obtains is used as a biological antiseptic preservative, its method of use is:
  • the closed warehouse can be thoroughly sterilized, and then the fresh-keeping items can be uniformly Spread out on the shelf with clear layers and do not stack and squeeze.
  • the active enzyme preparation After the storage is completed, take the active enzyme preparation and cover it with a one-time fine spray, until the humidity in the warehouse is above 90%, and the temperature is kept below room temperature and stored in a closed range above zero degrees Celsius to avoid infection with miscellaneous bacteria.
  • Example 10 In this example, the antiseptic preservative prepared in Example 9 is used to keep garlic moss fresh:
  • Figure 7(a) shows the state of garlic moss before the experiment.
  • One group of garlic moss after grouping is sprayed with fermentation substrate on its surface and placed in a fresh-keeping bag.
  • the other group was directly put into the fresh-keeping bags without any treatment, and then the two groups of fresh-keeping bags were placed at room temperature for two months.
  • the photograph of garlic moss, Fig. 7 (c) is the photo of untreated garlic moss, compares the state of Fig. 7 (b) and Fig.
  • Example 11 In this example, the antiseptic preservative prepared in Example 9 is used to keep pears fresh:
  • Embodiment 12 In this embodiment, the antiseptic preservative prepared in Embodiment 9 is used to preserve the damaged pears:
  • FIG. 9(a) shows the pears with obvious depressions after collision before the experiment, and then spray the antiseptic preservative of Example 7 on the surface of the sunken pears, put it into a fresh-keeping bag, and store it at room temperature for 9 months
  • Figure 9(b) the state of the pear is shown in Figure 9(b). Comparing the state of the same pear in Figure 9(a) and Figure 9(b) after 9 months, it can be clearly seen that the pear is in its There is self-recovery at the collision damage, and the depression is not obvious, so it can be seen that the antiseptic and fresh-keeping agent of the present invention not only has excellent anti-corrosion and fresh-keeping effect, but also has a strong repair effect on damaged cell tissue.
  • Embodiment 13 This embodiment adopts the antiseptic antistaling agent prepared in Example 9 to preserve the freshness of the fresh meat: take the fresh pork on the market and see Figure 10 (a), after the antiseptic antistaling agent is evenly sprayed on the surface of the fresh pork, Place it at room temperature and store it for 1 month, see Figure 10 (b), compare the photos before and after the fresh pork of this embodiment, it can be seen that the meat is dehydrated and dry except for part of the surface (this is normal, because it has not been processed). Airtight storage, water evaporation is a normal phenomenon), other colors are normal, and there is no peculiar smell.
  • Example 14 In this example, the antiseptic preservative prepared in Example 9 is used to preserve the freshness of fresh meat: take fresh pork on the market, spray the antiseptic antistaling agent evenly on the surface of the pork, and then place it at room temperature for air-drying. After completely air-dried, cut the surface and observe its internal conditions, see Figure 11(a), it can be clearly seen from Figure 11(a) that although the surface is dried and whitish, the meat inside is still fresh in color, but It is also obvious that its water has evaporated, showing a pan-drying phenomenon.
  • Example 15 In this example, the antiseptic and antistaling agent prepared in Example 9 is used to preserve the freshness of the pig's head. Take the fresh pig's head and soak it in the antistaling agent for 24 hours, remove it and place it on a ventilated shelf to drain the water such as the enzyme preparation until the pig's head The surface is dry, the cross-section is dry, and there is no residual moisture such as preservatives in the mouth, trachea, brainstem, and under the tongue, and then sealed for 4 years. See Figure 12 after 4 years.
  • Example 16 In this example, the antiseptic preservative prepared in Example 9 is used to treat fresh shrimps, and the fresh freshwater shrimps are placed in a tank filled with antiseptic preservation solution, and then sealed for 3 months, as shown in Figure 13. It can be seen from Figure 13 that even after 4 months, the color of the shrimp has not changed, such as the redness of the rot, and the color is not much different from that of the fresh shrimp.
  • Embodiment 17 This embodiment adopts the anti-mold experiment of the antiseptic preservative prepared in Example 9, and the specific experiments are as follows:
  • Example 18 In this example, the non-inactivated biological enzyme preparation in Example 6 was used to inhibit saccharification experiment on potato snowflake puree

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Mycology (AREA)
  • Virology (AREA)
  • Food Science & Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Polymers & Plastics (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Biomedical Technology (AREA)
  • Environmental Sciences (AREA)
  • Dentistry (AREA)
  • Botany (AREA)
  • Agronomy & Crop Science (AREA)
  • Pest Control & Pesticides (AREA)
  • Plant Pathology (AREA)
  • Nutrition Science (AREA)
  • Cell Biology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

提供了一种酵母、乳酸菌联合菌，其包括酵母菌和乳酸菌，所述酵母菌包括嗜酒假丝酵母B-JJ1，所述乳酸菌包括布氏乳杆菌B-JR1、类干酪乳杆菌B-JR2、玉米乳杆菌B-JR4、植物乳杆菌B-JR5、乳杆菌B-JR6的至少一种，其经过种菌提取、菌株接种、优化联合菌、联合菌驯化等过程得到，提供的酵母、乳酸菌联合菌的发酵物可用来制备生物酶制剂，而所制备得到的生物酶制剂又可制备天然、绿色、无添加剂的具有强抗氧化性且能保持动植物细胞活性的防腐保鲜剂。

Description

一种酵母、乳酸菌联合菌、培育方法及其应用 技术领域
本发明涉及微生物应用技术领域，尤其涉及一种酵母、乳酸菌联合菌、培育方法及其应用。
背景技术
联合菌群(亦称联合微生物群体或混合菌群)通常是指两种或两种以上人工选育的优良菌种组成的微生物生态系统，该系统中的微生物具有营养互利、优势互补、生态平衡、存活力强、适应性强等特点。通过将不同的菌种进行巧妙地组合，能够使得其发挥高于单菌种的性能。
而乳酸菌是指一群通过发酵糖类，产生大量乳酸的细菌总称，从形态上可分为球菌和杆菌。并且均为革兰氏染色阳性、在缺少氧气的环境中生长良好的兼性厌氧性或厌氧性细菌。其在自然界分布极广，广泛存在于人畜禽肠道、许多食品、物料及少数临床样品中。除少数外，绝大部分是人和动物体内必不可少的具有重要生理功能的菌群。酵母菌是一类典型的单细胞微生物,其结构简单，属于真菌类。由于酵母具有个体大、蛋白质含量高、杂食性强、易分离、易培养、代谢产物多、综合利用广等特点，在现在工业中除了用于酿酒外，还用于甘油、有机酸、酶制剂等生产。乳酸菌和酵母菌均是对人体有益的微生物，具有相似的生长条件，即共生基础。
发明内容
因此，基于以上背景，本发明提供一种酵母、乳酸菌联合菌、培育方法及其应用，其为具有稳定的共生性的菌株构成的，能够相互促进、自然平衡，为其扩大应用范围；且通过联合菌株发酵的产物制备的生物酶制剂则可用于制备天然的具有良好的抗氧化和保鲜功能的生物防腐保鲜剂。
本发明提供的技术方案为：
一种酵母、乳酸菌联合菌，其包括酵母菌和乳酸菌，所述酵母菌包括嗜酒假丝酵母B-JJ1，所述乳酸菌包括布氏乳杆菌B-JR1、类干酪乳杆菌B-JR2、玉米乳杆菌B-JR4、植物乳杆菌B-JR5、乳杆菌B-JR6的至少一种；
所述嗜酒假丝酵母B-JJ1的保藏编号为CCTCC NO:M2021136，其分类命名为Candida ethanolica；
所述布氏乳杆菌B-JR1的保藏编号为CCTCC NO:M2021132，其分类命名为Lactobacillus buchneri；
所述类干酪乳杆菌B-JR2的保藏编号为CCTCC NO:M2021133，其分类命名为Lactobacillus paracasei；
所述玉米乳杆菌B-JR4的保藏编号为CCTCC NO:M2021135，其分类命名为Lactobacillus zeae；
所述植物乳杆菌B-JR5的保藏编号为CCTCC NO:M2021501，其分类命名为Lactobacillus plantarum；
所述乳杆菌B-JR6的保藏编号为CCTCC NO:M2021502，其分类命名为Lactobacillus chiayiensis。
进一步地，其包括酵母菌和乳酸菌，所述酵母菌为嗜酒假丝酵母B-JJ1，所述乳酸菌由布氏乳杆菌B-JR1、类干酪乳杆菌B-JR2、玉米乳杆菌B-JR4、植物乳杆菌B-JR5、乳杆菌B-JR6组成。
本发明还提供了一种上述的酵母、乳酸菌联合菌的培育方法，其包括如下步骤：
(1)种菌提取：取白酒酿造窖池的窖泥发酵物，按照重量比将无菌水加入窖泥发酵物中，拌开后静置备用；
(2)培养基配制：所述培养基由土豆泥雪花粉、小麦胚芽、糯米为主料进行配制而成；取原料后按照重量比加入水后，并搅拌均匀，然后经蒸汽蒸馏待粒状物膨胀即可放入无菌容器中摊开散热，晾至一定温度后备用；
(3)菌株接种：将步骤(1)的窖泥发酵物和步骤(2)的配制的培养基按照重量比进行混合均匀，然后放置在无菌环境中进行恒温有氧培植；
(4)无氧选育：当步骤(3)的培养基出现白色或乳白色菌斑均匀分布时，拨开料面有湿粘感并有淡淡的酵香味时，将培养基分袋包装，并将分袋包装后的培养基继续恒温发酵；
(5)获取兼氧性酵母、乳酸联合菌：当袋中的残余氧气被消耗尽以后，袋内培养基呈真空板结块，并有白色、乳白色菌斑，菌膜覆盖；
(6)优化兼氧性酵母、乳酸菌联合菌：将步骤(5)的袋中的培养基继续进行无氧发酵，以让兼氧菌继续增殖，进一步排除好氧菌，直至白色、乳白色菌膜完全覆盖培养基，完成占位并饱和发酵；
(7)取样检测鉴定所获共生菌落：对步骤(6)的联合菌活性进行检测，当酵母菌的活菌平均值为6.4×10 4cfu/ml以上；乳酸菌的活菌平均值为4×10 8cfu/ml以上时，即得到一代酵母、乳酸联合菌；
(8)联合菌驯化：将步骤(2)中的培养基与步骤(7)的一代酵母、乳酸菌联合菌进行混合，并喷淋无菌水或冷却至37℃的蒸锅水，直至濡湿至湿散，手攥握有水滴出；然后将其有氧活化，直至培养基表面出现白色或乳白色菌斑均匀分布，拨开表面培养基有湿黏感并伴有酵香味溢出；然后将混合培养基料置入密闭环境中进行继续发酵，直至逐渐耗周围氧气进行入无氧发酵；无氧发酵至酵母、乳酸菌联合菌落生长旺盛，即酵母、乳酸联合菌落驯化完成。
进一步地，一种上述的酵母、乳酸菌联合菌的培育方法，其包括如下步骤：
(1)种菌提取：取白酒酿造窖池的窖泥发酵物，按照2:1的重量比将35-38℃无菌水加入窖泥发酵物中，拌开后静置备用；
(2)培养基配制：所述培养基由土豆泥雪花粉、小麦胚芽、糯米为主料进行配制而成；取土豆泥雪花粉、小麦胚芽、糯米，按照1:1的重量比加入水后，并搅拌均匀，然后经蒸汽蒸馏约四十分钟，待粒状物膨胀即可放入无菌容器中摊开散热，晾至摄氏45度以下备用；
进一步地，上述的培养基还包括高粱米、荞麦。
更进一步地，所述培养基的各原料配比为：
高粱米4份，土豆泥雪花粉3份，小麦胚芽1份，荞麦1份，糯米1份。
更进一步地，5％的高粱米和荞麦可用糖来替代。
(3)菌株接种：将步骤(1)的窖泥发酵物和步骤(2)的配制的培养基按照重量比1:2比例进行混合均匀，然后放置在26℃以上的无菌环境中进行恒温有氧培植45小时以上，在此过程中需注意培养基变化；
(4)无氧选育：当步骤(3)的培养基出现白色或乳白色菌斑均匀分布时，拨开料面有湿粘感并有淡淡的酵香味时，将培养基按照1-5公斤分袋包装，并将分袋包装后的培养基放置在37℃中继续恒温发酵；
(5)获取兼氧性酵母、乳酸联合菌：当袋中的残余氧气被消耗尽以后，袋内培养基呈真空板结块，并有白色、乳白色菌斑，菌膜覆盖时，此时多种曲霉、毛霉类好氧菌即进入凋亡，能够生存并缓慢增殖的主要为兼氧性酵母菌、乳酸联合菌；
(6)优化兼氧性酵母、乳酸菌联合菌：将上述袋中的培养基继续进行无氧发酵，以让兼氧菌继续增殖，进一步排除好氧菌，直至白色、乳白色菌膜完全覆盖培养基，完成占位并饱和发酵；
(7)取样检测鉴定所获共生菌落：对步骤(6)的联合菌活性进行检测，当酵母菌的活菌平均值为6.4×10 4cfu/ml以上；乳酸菌的活菌平均值为4×10 8cfu/ml以上时，即得到一代酵母、乳酸联合菌；
(8)联合菌驯化：将步骤(2)中的培养基与步骤(7)的一代酵母、乳酸菌联合菌按量质量1:1的进行混合，并喷淋无菌水或冷却至37℃的蒸锅水，直至濡湿至湿散，手攥握有水滴出；然后将其放置于无菌26℃以上进行有氧活化48小时以上，直至培养基表面出现白色或乳白色菌斑均匀分布，拨开表面培养基有湿黏感并伴有酵香味溢出；然后将混合培养基料置入密闭环境中进行继续发酵，直至逐渐耗周围氧气进行入无氧发酵；无氧发酵20-30天后，酵母、乳酸菌联合菌落生长旺盛，即酵母、乳酸联合菌落驯化完成。
进一步地，其还包括以下步骤：
(9)联合菌包装：将步骤(8)的酵母、乳酸联合菌驯化后的培养基按照5–10公斤/袋进行真空包装。
进一步地，步骤(9)中分装后的酵母、乳酸联合菌中的酵母菌的活菌平均值为6.4×10 4cfu/ml以上，乳酸菌的活菌平均值为4×10 8cfu/ml以上。
本发明还提供了一种生物酶制剂的制备方法，所述生物酶制剂由上述的酵母、乳酸联合菌作的发酵产物制成。
进一步地，上述的生物酶制剂的制备方法，包括如下步骤：
(1)选用酵母、乳酸菌联合菌作菌种；
(2)培养基的制备和接种：所述培养基以土豆泥雪花粉、小麦胚芽、糯米为主料配制而成，将培养基加水后搅拌均匀后，蒸熟，然后无菌环境下晾至摄氏45度以下即培养基制备完成；
将制备的培养基与菌种进行混合均匀；
(3)对步骤(2)接种后的培养基的湿度进行调节，保证握团有水渍出，培养基整体保持湿润透气；
(4)将步骤(3)的培养基进行有氧发酵直至培养基出现均匀肉眼可见的白色、乳白色菌斑时，将培养基转入发酵罐中；封闭发酵罐的所有通道，以使得入罐的培养基发酵物在罐体中继续有氧培养，直至耗尽罐体中的氧气，自行进入无氧发酵状态；
(5)当发酵罐内压力无变化时，固态发酵物进入饱和发酵，开罐有浓郁的酵香味溢出，肉眼观察有丰茂的菌丝体覆盖在固态培养基表面，取发酵产物进行检测，酵母菌活菌总数6.4×10 4-5.9×10 5cfu/ml；乳酸菌总数4×10 8-5.1×10 9cfu/ml，此时固态发酵过程已经达到饱和，可以进入液态发酵阶段；
(6)将步骤(5)的固态发酵物加入无菌水，并充分搅拌，以稀释固态发酵过程中的代谢底物，进行有氧活化培养直至液态发酵物表面密布白色或乳白色菌斑时，即可封闭发酵罐所有通道，再次让发酵物逐渐耗尽氧气进入无氧发酵过程；
(7)当步骤(6)的液态发酵物表面菌斑折叠态，中上层清液呈现金黄色，清澈透明，底层固态发酵物和中层液体层次分明不粘连时，即固态发酵–液态发酵过程全部完成，所得发酵产物即可为制得的生物酶制剂。
进一步地，其还包括以下步骤：
(8)将步骤(7)所得发酵底物进行加热灭活后，提取发酵物的上清液，即制得灭活生物酶制剂。
本发明还提供了上述的生物酶制剂的应用，其可用于制备生物防腐保鲜剂。
进一步地，所述生物防腐保鲜剂可应用于对蔬菜、水果或新鲜肉或干肉进行防腐保鲜。
进一步地，所述生物防腐剂可用于制备人体或动物体标本。
采取上述技术方案，具有的有益效果如下：
①本发明采用接种、选育、优化联合菌、联合菌驯化等过程，培育出了共生能力强，稳定性好的酵母、乳酸菌联合菌，所培育的联合菌具有较高的活性，并且其可根据实际的需要，将联合菌进行纯化分离后，再进行混合配制成具有不同的性能的联合菌；
②并且本发明的联合菌通过发酵，经过复杂的生化反应，所生成的发酵产物中富含各种氨基酸残基，酰胺，辅酶和乳酸，亚油酸等活性基团，可用于制备营养丰富的生物酶制剂，而通过本发明制备的生物酶制剂则可用于制备防腐保鲜剂；
③本发明制备的防腐保鲜剂不仅具有能够保持动植物组织细胞活性并可对可对缺损细胞进行修复 的各种氨基酸、各种酶、酶前体等营养组分，并且可通过对α-淀粉酶活性的抑制，延缓生物体的糖化过程，并可杀死致腐霉菌等微生物，具有较强的抗氧化、防腐、保鲜效果；
④本发明的生物酶制剂制备得到的防腐保鲜剂天然、无任何添加剂，安全、高效，其不仅可用于对新鲜的植物或肉类进行保鲜，使得动植物细胞长时间保持活性，延长其保鲜时间，并且其还可对干肉等进行保鲜，防止氧化变色。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为本发明嗜酒假丝酵母B-JJ1的菌株形态特征照片；其中(a)为光学显微镜镜检照片(400X)；(b)为正面平板照片；
图2为本发明布氏乳杆菌B-JR1的菌株形态特征照片；其中(a)为光学显微镜镜检照片(1000X)；(b)为正面平板照片；
图3为本发明类干酪乳杆菌B-JR2的菌株形态特征照片；其中(a)为光学显微镜镜检照片(1000X)；(b)为正面平板照片；
图4为本发明玉米乳杆菌B-JR4的菌株形态特征照片；其中(a)为光学显微镜镜检照片(1000X)；(b)为正面平板照片；
图5为本发明植物乳杆菌B-JR5的菌株形态特征照片；其中(a)为光学显微镜镜检照片(1000X)；(b)为正面平板照片；
图6为本发明乳杆菌B-JR6的菌株形态特征照片；其中(a)为光学显微镜镜检照片(1000X)；(b)为正面平板照片；
图7为本发明实施例10生物酶制剂应用于蒜苔保鲜实验的照片；
图8为本发明实施11生物酶制剂应用于梨子保鲜实验的照片；
图9为本发明实施例12生物酶制剂应用于经伤害的梨子保鲜实验的照片；
图10为本发明实施例13生物酶制剂应用于鲜猪肉保鲜实验的照片；
图11为本发明实施例14生物酶制剂应用于鲜猪肉保鲜实验的照片；
图12为本发明实施例15生物酶制剂应用于新鲜猪头保鲜实验的照片；
图13为本发明实施例16生物酶制剂应用于新鲜虾的照片；
图14为本发明实施例17生物酶制剂的防霉菌实验的照片；
图15为本发明实施例18生物酶制剂的抑制糖化实验的照片；
图16为本发明实施例16生物酶制剂的对雪花泥分团分子的显微照片；
本发明的所述嗜酒假丝酵母B-JJ1的保藏编号为CCTCC NO:M2021136；保藏日期为2021年01月21日；保藏机构为中国典型培养物保藏中心；保藏地点为中国武汉大学。
所述布氏乳杆菌B-JR1的保藏编号为CCTCC NO:M2021132；保藏日期为2021年01月21日；保藏机构为中国典型培养物保藏中心；保藏地点为中国武汉大学。
所述类干酪乳杆菌B-JR2的保藏编号为CCTCC NO:M2021133；保藏日期为2021年01月21日；保藏机构为中国典型培养物保藏中心；保藏地点为中国武汉大学。
所述玉米乳杆菌B-JR4的保藏编号为CCTCC NO:M2021135；保藏日期为2021年01月21日；保藏机构为中国典型培养物保藏中心；保藏地点为中国武汉大学。
所述植物乳杆菌B-JR5的保藏编号为CCTCC NO:M2021501；保藏日期为2021年05月07日；保藏机构为中国典型培养物保藏中心；保藏地点为中国武汉大学。
所述乳杆菌B-JR6的保藏编号为CCTCC NO:M2021502；保藏日期为2021年05月07日；保藏机构为中国典型培养物保藏中心；保藏地点为中国武汉大学。
具体实施方式；
为使本发明实施例的目的、技术方案和优点更加清楚，下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
下面结合附图对本发明做进一步说明。
实施例1：一种酵母、乳酸菌联合菌，其培育方法为步骤如下：
(1)种菌提取：取白酒酿造窖池不同方位的窖泥发酵物，所说的不同方位指的东南西北，窖池壁和窖池底部，按照2:1的重量比将35-38℃无菌水加入窖泥发酵物中，拌开后静置备用；本实施例的酿造池为苏北窑湾古镇的某白酒传统酿造池。
(2)培养基配制：取高粱米4份，土豆泥雪花粉3份，小麦胚芽1份，荞麦1份，糯米1份后进行混合，按照1:1的重量比加入水后，并搅拌均匀，然后经蒸汽蒸馏约四十分钟，待粒状物膨胀即可放入无菌容器中摊开散热，晾至摄氏45度以下备用；
(3)菌株接种：将步骤(1)的窖泥发酵物和步骤(2)的配制的培养基按照重量比1:2比例进行混合均匀，然后放置在26℃以上的无菌环境中进行恒温有氧培植45小时以上，在此过程中需注意培养基变化；
(4)无氧选育：当步骤(3)的培养基出现白色或乳白色菌斑均匀分布时，拨开料面有湿粘感并有淡淡的酵香味时，将培养基按照1-5公斤分袋包装，并将分袋包装后的培养基放置在37℃中继续恒温发酵；
在此步骤中，由于之前的培养物伴有大量的好氧菌落，封袋后会有气体产出，应此需要注意适时放气减压。
(5)获取兼氧性酵母、乳酸联合菌：当袋中的残余氧气被消耗尽以后，袋内培养基呈真空板结块，并有白色、乳白色菌斑，菌膜覆盖时，此时多种曲霉、毛霉类好氧菌即进入凋亡，能够生存并缓慢增殖的主要为兼氧性酵母菌、乳酸联合菌；
(6)优化兼氧性酵母、乳酸菌联合菌：将上述袋中的培养基继续进行无氧发酵，以让兼氧菌继续增殖，进一步排除好氧菌，直至白色、乳白色菌膜完全覆盖培养基，完成占位并饱和发酵；此时好氧菌已经几乎没有生存的条件(可能会有极少数真菌孢子等存在但也会因为群落优势的压制及后续的工序中将其彻底杀灭)；同时兼氧菌也逐渐进入休眠状态，并等待复苏；
(7)取样检测鉴定所获共生菌落：对步骤(6)的联合菌活性进行检测，当酵母菌的活菌平均值为6.4×10 4cfu/ml以上；乳酸菌的活菌平均值为4×10 8cfu/ml以上时，即得到一代酵母、乳酸联合菌；
本实施例中，酵母菌的活菌的最高值为3.1×10 5cfu/ml，乳酸菌的活菌最高值为1.2×10 9cfu/ml。
(8)联合菌驯化：由于获取的联合菌落，脱离了曲霉，毛霉等等大群体互补后，形成新的小群体之间的共生性适应、对培养基的适应及有氧无氧环境的转换等生存环境的改变的适应，因此需要对联合菌进行驯化；将步骤(2)中的培养基与步骤(7)的一代酵母、乳酸菌联合菌按量质量1:1的进行混合，并喷淋无菌水，直至濡湿至湿散，手攥握有水滴出；然后将其放置于无菌26℃以上进行有氧活化48小时以上，直至培养基表面出现白色或乳白色菌斑均匀分布，拨开表面培养基有湿黏感并伴有酵香味溢出；然后将混合培养基料置入密闭环境中进行继续发酵，直至逐渐耗周围氧气进行入无氧发酵；无氧发酵25天后，酵母、乳酸菌联合菌落生长旺盛，即酵母、乳酸联合菌落驯化完成。
(9)联合菌包装：将步骤(8)的酵母、乳酸联合菌驯化后的培养基按照5–10公斤/袋进行真空包装，隔绝空气以杜绝好氧菌感染和彻底杀灭可能由一代株残留的曲霉、毛霉等真菌孢子，以此可便于工业化运输和应用。
本实施例在对步骤(9)中分装后的酵母、乳酸联合菌进行检测，其酵母菌的活菌平均值为6.4×10 4cfu/ml以上，乳酸菌的活菌平均值为4×10 8cfu/ml以上，有害菌中的霉菌<10cfu/ml；大肠杆菌<30cfu/ml；沙门氏菌ND；志贺氏菌ND；金黄色葡萄球菌ND(ND即未检测出)。
实施例2：一种酵母、乳酸菌联合菌，其培育方法为步骤如下：
(1)种菌提取：取白酒酿造窖池不同方位的窖泥发酵物，所说的不同方位指的东南西北，窖池壁和窖池底部，按照2:1的重量比将35-38℃无菌水加入窖泥发酵物中，拌开后静置备用；
本实施例的酿造池为苏北窑湾古镇的某白酒传统酿造池。
(2)培养基配制：取高粱米4份、土豆泥雪花粉3份，小麦胚芽1份，糯米1份后进行混合，按照1:1的重量比加入水后，并搅拌均匀，然后经蒸汽蒸馏约四十分钟，待粒状物膨胀即可放入无菌容器 中摊开散热，晾至摄氏45度以下备用；
(3)菌株接种：将步骤(1)的窖泥发酵物和步骤(2)的配制的培养基按照重量比1:2比例进行混合均匀，然后放置在26℃以上的无菌环境中进行恒温有氧培植45小时以上，在此过程中需注意培养基变化；
(4)无氧选育：当步骤(3)的培养基出现白色或乳白色菌斑均匀分布时，拨开料面有湿粘感并有淡淡的酵香味时，将培养基按照1-5公斤分袋包装，并将分袋包装后的培养基放置在37℃中继续恒温发酵；
在此步骤中，由于之前的培养物伴有大量的好氧菌落，封袋后会有气体产出，应此需要注意适时放气减压。
(5)获取兼氧性酵母、乳酸联合菌：当袋中的残余氧气被消耗尽以后，袋内培养基呈真空板结块，并有白色、乳白色菌斑，菌膜覆盖时，此时多种曲霉、毛霉类好氧菌即进入凋亡，能够生存并缓慢增殖的主要为兼氧性酵母菌、乳酸联合菌；
(6)优化兼氧性酵母、乳酸菌联合菌：将上述袋中的培养基继续进行无氧发酵，以让兼氧菌继续增殖，进一步排除好氧菌，直至白色、乳白色菌膜完全覆盖培养基，完成占位并饱和发酵；此时好氧菌已经几乎没有生存的条件(可能会有极少数真菌孢子等存在但也会因为群落优势的压制及后续的工序中将其彻底杀灭)；同时兼氧菌也逐渐进入休眠状态，并等待复苏；
(7)取样检测鉴定所获共生菌落：对步骤(6)的联合菌活性进行检测，当酵母菌的活菌平均值为6.4×10 4cfu/ml以上；乳酸菌的活菌平均值为4×10 8cfu/ml以上时，即得到一代酵母、乳酸联合菌；
本实施例中，酵母菌的活菌的最高值为3.1×10 5cfu/ml，乳酸菌的活菌最高值为1.2×10 9cfu/ml。
(8)联合菌驯化：由于获取的联合菌落，脱离了曲霉，毛霉等等大群体互补后，形成新的小群体之间的共生性适应、对培养基的适应及有氧无氧环境的转换等生存环境的改变的适应，因此需要对联合菌进行驯化；将步骤(2)中的培养基与步骤(7)的一代酵母、乳酸菌联合菌按量质量1:1的进行混合，并喷淋无菌水，直至濡湿至湿散，手攥握有水滴出；然后将其放置于无菌26℃以上进行有氧活化48小时以上，直至培养基表面出现白色或乳白色菌斑均匀分布，拨开表面培养基有湿黏感并伴有酵香味溢出；然后将混合培养基料置入密闭环境中进行继续发酵，直至逐渐耗周围氧气进行入无氧发酵；无氧发酵25天后，酵母、乳酸菌联合菌落生长旺盛，即酵母、乳酸联合菌落驯化完成。
(9)联合菌包装：将步骤(8)的酵母、乳酸联合菌驯化后的培养基按照5–10公斤/袋进行真空包装，隔绝空气以杜绝好氧菌感染和彻底杀灭可能由一代株残留的曲霉、毛霉等真菌孢子，以此可便于工业化运输和应用。
本实施例在对步骤(9)中分装后的酵母、乳酸联合菌进行检测，其酵母菌的活菌平均值为6.4×10 4cfu/ml以上，乳酸菌的活菌平均值为4×10 8cfu/ml以上，有害菌中的霉菌<10cfu/ml；大肠杆菌 <30cfu/ml；沙门氏菌ND；志贺氏菌ND；金黄色葡萄球菌ND(ND即未检测出)。
实施例3：一种酵母、乳酸菌联合菌，其培育方法为步骤如下：
(1)种菌提取：取白酒酿造窖池不同方位的窖泥发酵物，所说的不同方位指的东南西北，窖池壁和窖池底部，按照2:1的重量比将35-38℃无菌水加入窖泥发酵物中，拌开后静置备用；
本实施例的酿造池为苏北窑湾古镇的某白酒传统酿造池。
(2)培养基配制：取高粱米3.8份，土豆泥雪花粉3份，小麦胚芽0.95份，荞麦1份，糯米1份、0.25份果葡糖浆后进行混合，按照1:1的重量比加入水后，并搅拌均匀，然后经蒸汽蒸馏约四十分钟，待粒状物膨胀即可放入无菌容器中摊开散热，晾至摄氏45度以下备用；
(3)菌株接种：将步骤(1)的窖泥发酵物和步骤(2)的配制的培养基按照重量比1:2比例进行混合均匀，然后放置在26℃以上的无菌环境中进行恒温有氧培植45小时以上，在此过程中需注意培养基变化；
(4)无氧选育：当步骤(3)的培养基出现白色或乳白色菌斑均匀分布时，拨开料面有湿粘感并有淡淡的酵香味时，将培养基按照1-5公斤分袋包装，并将分袋包装后的培养基放置在37℃中继续恒温发酵；
在此步骤中，由于之前的培养物伴有大量的好氧菌落，封袋后会有气体产出，应此需要注意适时放气减压。
(5)获取兼氧性酵母、乳酸联合菌：当袋中的残余氧气被消耗尽以后，袋内培养基呈真空板结块，并有白色、乳白色菌斑，菌膜覆盖时，此时多种曲霉、毛霉类好氧菌即进入凋亡，能够生存并缓慢增殖的主要为兼氧性酵母菌、乳酸联合菌；
(6)优化兼氧性酵母、乳酸菌联合菌：将上述袋中的培养基继续进行无氧发酵，以让兼氧菌继续增殖，进一步排除好氧菌，直至白色、乳白色菌膜完全覆盖培养基，完成占位并饱和发酵；此时好氧菌已经几乎没有生存的条件(可能会有极少数真菌孢子等存在但也会因为群落优势的压制及后续的工序中将其彻底杀灭)；同时兼氧菌也逐渐进入休眠状态，并等待复苏；
(7)取样检测鉴定所获共生菌落：对步骤(6)的联合菌活性进行检测，当酵母菌的活菌平均值为6.4×10 4cfu/ml以上；乳酸菌的活菌平均值为4×10 8cfu/ml以上时，即得到一代酵母、乳酸联合菌；
本实施例中，酵母菌的活菌的最高值为3.1×10 5cfu/ml，乳酸菌的活菌最高值为1.2×10 9cfu/ml。
(8)联合菌驯化：由于获取的联合菌落，脱离了曲霉，毛霉等等大群体互补后，形成新的小群体之间的共生性适应、对培养基的适应及有氧无氧环境的转换等生存环境的改变的适应，因此需要对联合菌进行驯化；将步骤(2)中的培养基与步骤(7)的一代酵母、乳酸菌联合菌按量质量1:1的进行混合，并喷淋无菌水，直至濡湿至湿散，手攥握有水滴出；然后将其放置于无菌26℃以上进行有氧活化48小时以上，直至培养基表面出现白色或乳白色菌斑均匀分布，拨开表面培养基有湿黏感并伴有酵香味溢出；然 后将混合培养基料置入密闭环境中进行继续发酵，直至逐渐耗周围氧气进行入无氧发酵；无氧发酵25天后，酵母、乳酸菌联合菌落生长旺盛，即酵母、乳酸联合菌落驯化完成。
(9)联合菌包装：将步骤(8)的酵母、乳酸联合菌驯化后的培养基按照5–10公斤/袋进行真空包装，隔绝空气以杜绝好氧菌感染和彻底杀灭可能由一代株残留的曲霉、毛霉等真菌孢子，以此可便于工业化运输和应用。
本实施例在对步骤(9)中分装后的酵母、乳酸联合菌进行检测，其酵母菌的活菌平均值为6.4×10 4cfu/ml以上，乳酸菌的活菌平均值为4×10 8cfu/ml以上，有害菌中的霉菌<10cfu/ml；大肠杆菌<30cfu/ml；沙门氏菌ND；志贺氏菌ND；金黄色葡萄球菌ND(ND即未检测出)。
按照常规方法将实施例1至实施例3的步骤(9)的联合菌进行分离提纯，得到6种菌，分别为嗜酒假丝酵母菌、布氏乳杆菌、类干酪乳杆菌、面包乳杆菌、玉米乳杆菌、植物乳杆菌、乳杆菌Lactobacillus chiayiensis，本申请分别命名为嗜酒假丝酵母B-JJ1、布氏乳杆菌B-JR1、类干酪乳杆菌B-JR2、玉米乳杆菌B-JR4、植物乳杆菌B-JR5、乳杆菌B-JR6，分别分离提纯各发酵菌株进行检测，各菌株的菌落、细胞形态分别见图1至图7，并且对各菌株进行理化生化鉴定，结果分别见表1至表12。
表1：嗜酒假丝酵母B-JJ1生理生化特征
Figure PCTCN2022116841-appb-000001
Figure PCTCN2022116841-appb-000002
注：“+”为阳性，“-”为阴性。
表2：布氏乳杆菌B-JR1生理生化特性-酶活
Figure PCTCN2022116841-appb-000003
表3：布氏乳杆菌B-JR1生理生化特性-利用碳源产酸
Figure PCTCN2022116841-appb-000004
Figure PCTCN2022116841-appb-000005
注：“+”为阳性，“-”为阴性，“W”为弱阳性反应。
表4：类干酪乳杆菌B-JR2生理生化特性-酶活、碳源氧化
Figure PCTCN2022116841-appb-000006
表5：类干酪乳杆菌B-JR2生理生化特性-利用碳源产酸
Figure PCTCN2022116841-appb-000007
Figure PCTCN2022116841-appb-000008
表6：玉米乳杆菌B-JR4的生理生化特性–酶活
Figure PCTCN2022116841-appb-000009
表7：玉米乳杆菌B-JR4的生理生化特性–利用碳源产酸
Figure PCTCN2022116841-appb-000010
表8：植物乳杆菌B-JR5的生理生化特性–酶活、碳源氧化
Figure PCTCN2022116841-appb-000011
Figure PCTCN2022116841-appb-000012
表9：植物乳杆菌B-JR5的生理生化特性–利用碳源产酸
Figure PCTCN2022116841-appb-000013
表10：植物乳杆菌B-JR5的生理生化特性–利用碳源产酸
Figure PCTCN2022116841-appb-000014
Figure PCTCN2022116841-appb-000015
表11：乳杆菌B-JR6的生理生化特性–酶活
Figure PCTCN2022116841-appb-000016
表12：乳杆菌B-JR6的生理生化特性–利用碳源产酸
Figure PCTCN2022116841-appb-000017
Figure PCTCN2022116841-appb-000018
采用18S rRNA基因测序的分子生物学方法对上述的嗜酒假丝酵母菌B-JJ1进行鉴定，其基因序列见SEQ.ID.NO.1，采用16s rRNA基因测序方法对布氏乳杆菌B-JR1、类干酪乳杆菌B-JR2、玉米乳杆菌B-JR4、植物乳杆菌B-JR5、乳杆菌B-JR6进行测序，其基因序列分别为SEQ.ID.NO.2、SEQ.ID.NO.3、SEQ.ID.NO.4、SEQ.ID.NO.5、SEQ.ID.NO.6所示。
本发明可根据实际的需要，将联合菌进行纯化分离后，再进行混合配制成具有不同的性能的联合菌，例如本发明的酵母、乳酸均联合菌可包括嗜酒假丝酵母B-JJ1、布氏乳杆菌B-JR1、乳杆菌B-JR6；或嗜酒假丝酵母菌B-JJ1、布氏乳杆菌B-JR1、类干酪乳杆菌B-JR2、乳杆菌B-JR6；或嗜酒假丝酵母菌B-JJ1、乳酸菌由布氏乳杆菌B-JR1、类干酪乳杆菌B-JR2、玉米乳杆菌B-JR4、乳杆菌B-JR6。
其具体操作可以在实施例1至实施例3的步骤(9)所制备得到的包括有嗜酒假丝酵母B-JJ1、布氏乳杆菌B-JR1、类干酪乳杆菌B-JR2、玉米乳杆菌B-JR4、植物乳杆菌B-JR5、乳杆菌B-JR6按照本领域常规方法进行分离提纯后，得到6种菌后，再根据需求分别扩大培养后，再进行混合培养，培育出不同的联合菌。
本发明的联合菌也可以直接采用实施例1至实施例3步骤(9)所得到的联合菌。
实施例4：一种生物酶制剂，其由酵母、乳酸菌联合菌的发酵产物制成，其制备过程为：
(1)配制培养基；所述培养基由土豆泥雪花粉30份、小麦胚芽30份、糯米20份、高粱米10份、荞麦10份配制而成；
(2)将酵母、乳酸联合菌接种至培养基上，进行发酵；所述酵母、乳酸联合菌包括嗜酒假丝酵母菌B-JJ1、布氏乳杆菌B-JR1、乳杆菌B-JR6；
(3)对步骤(2)接种后的培养基的湿度进行调节，保证握团有水渍出，培养基整体保持湿润透气；
(4)将步骤(3)的培养基进行有氧发酵48小时以上；当培养基出现均匀肉眼可见的白色、乳白色菌斑时，将培养基转入发酵罐中，固态发酵物占据发酵罐的三分之一即可，当有氧发酵耗尽发酵罐中的氧气后，进入无氧发酵；
(5)当步骤(4)继续发酵直至固态发酵达到饱和后，加入无菌水搅拌后进入液态发酵阶段，并确保发酵罐中至少有1/5的空间，然后进行有氧活化培养72小时以上，直至液态发酵物表面密布白色或乳白色菌斑时，当有氧发酵耗尽发酵罐中的氧气后，再次进入无氧发酵；
(6)将步骤(5)液态发酵物表面菌斑折叠态，中上层清液呈现金黄色，清澈透明，底层固态发酵物和中层液体层次分明不粘连时，即固态发酵–液态发酵过程全部完成，所得到的发酵产物即为制得的生物酶制剂。
实施例5：一种生物酶制剂，其由酵母、乳酸菌联合菌的发酵产物制成，其制备过程为：
(1)配制培养基；所述培养基由土豆泥雪花粉30份、小麦胚芽30份、糯米20份、高粱米10份、荞麦10份配制而成；
(2)将酵母、乳酸联合菌接种至培养基上，进行发酵；所述酵母、乳酸联合菌包括嗜酒假丝酵母B-JJ1、布氏乳杆菌B-JR1、类干酪乳杆菌B-JR2、乳杆菌B-JR6；
(3)对步骤(2)接种后的培养基的湿度进行调节，保证握团有水渍出，培养基整体保持湿润透气；
(4)将步骤(3)的培养基进行有氧发酵48小时以上；当培养基出现均匀肉眼可见的白色、乳白色菌斑时，将培养基转入发酵罐中，固态发酵物占据发酵罐的三分之一即可，当有氧发酵耗尽发酵罐中的氧气后，进入无氧发酵；
(5)当步骤(4)继续发酵直至固态发酵达到饱和后，加入无菌水搅拌后进入液态发酵阶段，并确保发酵罐中至少有1/5的空间，然后进行有氧活化培养72小时以上，直至液态发酵物表面密布白色或乳白色菌斑时，当有氧发酵耗尽发酵罐中的氧气后，再次进入无氧发酵；
(6)将步骤(5)液态发酵物表面菌斑折叠态，中上层清液呈现金黄色，清澈透明，底层固态发酵物和中层液体层次分明不粘连时，即固态发酵–液态发酵过程全部完成，所得到的发酵产物即为制得的生物酶制剂。
实施例6：一种生物酶制剂，其由酵母、乳酸菌联合菌的发酵产物制成，其制备过程为：
(1)配制培养基；所述培养基由土豆泥雪花粉30份、小麦胚芽30份、糯米20份、高粱米10份、荞麦10份配制而成；
(2)将酵母、乳酸联合菌接种至培养基上，进行发酵；所述酵母、乳酸联合菌包括嗜酒假丝酵母菌B-JJ1，乳酸菌包括布氏乳杆菌B-JR1、类干酪乳杆菌B-JR2、玉米乳杆菌B-JR4、植物乳杆菌B-JR5、乳杆菌B-JR6；
(3)对步骤(2)接种后的培养基的湿度进行调节，保证握团有水渍出，培养基整体保持湿润透气；
(4)将步骤(3)的培养基进行有氧发酵48小时以上；当培养基出现均匀肉眼可见的白色、乳白色菌斑时，将培养基转入发酵罐中，固态发酵物占据发酵罐的三分之一即可，当有氧发酵耗尽发酵罐中的氧气后，进入无氧发酵；
(5)当步骤(4)继续发酵直至固态发酵达到饱和后，加入无菌水搅拌后进入液态发酵阶段，并确保发酵罐中至少有1/5的空间，然后进行有氧活化培养72小时以上，直至液态发酵物表面密布白色或乳白色菌斑时，当有氧发酵耗尽发酵罐中的氧气后，再次进入无氧发酵；
(6)将步骤(5)液态发酵物表面菌斑折叠态，中上层清液呈现金黄色，清澈透明，底层固态发酵物和中层液体层次分明不粘连时，即固态发酵–液态发酵过程全部完成，所得到的发酵产物即为制得的 生物酶制剂。
实施例7：一种生物防腐保鲜剂，其由实施例4至实施例6的生物酶制剂制备。
实施例8：实施例4至实施例7所制备的生物酶制剂直接作为生物防腐保鲜剂进行使用。
实施例9：实施例6所制备的生物酶制剂进行加热灭活后，提取发酵物的上清液，即制得生物酶制剂，此生物酶制剂直接作为生物防腐保鲜剂进行使用。
实施例10：实施例8得到的生物酶制剂在作为生物防腐保鲜剂使用时，其使用方法为：
营造菌落优势小气候环境，将需保鲜物质放在环境中即可，此方法适应大面积、多质量物质的防腐保鲜抗氧化：例如可以将密闭的库房作彻底灭菌处理，然后将被保鲜物品均匀摊放在货架上，层次分明，不要堆垛挤压。入库完毕后取活性酶制剂一次性细密喷雾覆盖，至库房内湿度90％以上，温度保持在室温以下零摄氏度以上范围内密闭保存，避免感染杂菌。
或包裹、浸泡、涂抹等，此适合人体、动物体的标本制作；
或一过性浸浴，喷雾处理，此适合于果蔬的防腐保鲜等.
实施例10：本实施例将实施例9所制备的防腐保鲜剂用于对蒜苔进行保鲜：
具体操作为：将一批购买的蒜苔分为两组，图7(a)为蒜苔在实验前的状态，将分组后的其中一组蒜苔采用将发酵底物喷洒在其表面后放置在保鲜袋中，另一组则不经过任何处理后直接放入保鲜袋中，然后将两组保鲜袋置于室温下进行放置两个月，两个月后，图7(b)经过防腐保鲜剂处理后的蒜苔的照片，图7(c)为未处理的蒜苔的照片，对图7(b)和图7(c)蒜苔的状态进行比较，可以很明显看出，经过本发明的防腐保鲜剂处理过的蒜苔色泽新鲜、水分充足、形态饱满，几乎未出现任何干煸或发黄的地方，而未处理的失水严重，出现大面积的发黄，干枯等形态。
实施例11：本实施例采用实施例9所制备的防腐保鲜剂对梨子进行保鲜：
选取同一批的两个梨子，实验前状态见图8(a)，其中一个梨在其表面喷洒防腐保鲜剂后放置在保鲜袋中，另外一个不经过任何处理后直接放置在保鲜袋中，然后将两组保鲜袋置于室温下进行放置保存，半年后的两个梨子的状态见图8(b)，一年后的两个梨子的状态见图8(c)，其中图8(b)和图8(c)左边为经过处理后的梨子，右边为未经过处理后的梨子，从图8(b)和图8(c)可以明显看出本发明对水果的防腐保鲜的效果，左边经过处理在一年后还可保持色泽新鲜，水分充盈且饱满，而未经过处理的，则已经腐败成水。
实施例12：本实施例采用实施例9所制备的防腐保鲜剂对经伤害的梨子进行保鲜：
图9(a)为实验前经过碰撞有明显凹陷的梨子，然后将实施例7的防腐保鲜剂喷洒在有凹陷的梨子表面，将其放入保鲜袋后，置于室温下保存，9个月后，梨子的状态见图9(b)，将图9(a)和图9(b)的同一梨子时隔开9个月后的状态进行比较，明显可以看出9个月后梨子在其碰撞伤害处有进行自行恢复，凹陷处不明显，因此可以看出，本发明的防腐保鲜剂不仅具有优异的防腐保鲜效果，还可通过对 破损的细胞组织有很强的修复功效。
实施例13：本实施例采用实施例9所制备的防腐保鲜剂对经鲜肉进行保鲜：取市场上是猪鲜肉见图10(a)，将猪鲜肉表面均匀喷洒防腐保鲜剂后，置于室温下进行保存1个月，见图10(b)，本实施例的猪肉保鲜前后的照片对比，可以看出肉除了部分表面表现有些失水发干外(此为正常，因未进行密闭保存，水分蒸发为正常现象)，其他的色泽正常，且无任何异味。
实施例14：本实施例采用实施例9所制备的防腐保鲜剂对鲜肉进行保鲜：取市场上猪鲜肉，将猪肉表面均匀喷洒防腐保鲜剂后，置于室温下进行风干，待其表面完全风干后，切开表面对其内部情况进行观察，见图11(a)，从图11(a)中可以明显看出，虽然表面处于风干发白，但内部依旧的肉依旧色泽新鲜，但也很明显看出其水分有所蒸发，呈现泛干现象，将风干肉完全浸泡于水中后常温下两个小时左右即可恢复鲜肉状态，见图11(b)，从图11(b)可以看出猪肉的细胞依旧抱有活性，可再次吸收水分，以此猪肉的某些部位出现饱满状态。
实施例15：本实施例采用实施例9所制备的防腐保鲜剂对猪头进行保鲜，取新鲜猪头在所述保鲜剂中浸泡24小时，捞出放置透风架子上沥干酶制剂等水分，至猪头表面干燥，截面干燥，口腔、气管、脑干处、舌根下等部位无残留保鲜剂等水分，然后密闭保存4年，4年后见图12。
实施例16：本实施例采用实施例9所制备的防腐保鲜剂对新鲜虾进行处理，取新鲜青虾置于盛有防腐保鲜液中的罐中，然后密闭保存3个月，见图13，从图13可以看出即使在4个月后，虾的色泽未出现变化，例如腐烂所呈现的泛红等变化，且色泽与新鲜虾区别不大。
实施例17：本实施例采用实施例9所制备的防腐保鲜剂的防霉菌实验，具体实验如下：
取土豆雪花粉，用加入防腐保鲜剂的水浸润捏成团；然后采用未加入防腐保鲜剂的水将土豆雪花粉浸润后，捏成团作对比，将两个土豆雪花泥捏成的团置于常温下3天后，见图14，图14左边的未加入防腐保鲜剂的，右边为加入有防腐保鲜剂的，两个进行对比，可以很明显地看出本发明具有良好的防霉菌的效果。
实施例18：本实施例采用实施例6未灭活的生物酶制剂对土豆雪花泥团进行抑制糖化实验
取土豆雪花粉，用实施例6的生物酶制剂溶液浸润捏成团，另用未加入防腐保鲜剂的水将土豆雪花粉浸润后，捏成团作对比，将制作好的两组土豆泥雪花粉团在相同条件下，于室温中放置72小时，结果见图15，图15左上角为未采用生物酶制剂处理的土豆雪花粉团，右下角为采用生物酶制剂处理的土豆雪花粉团，可以明显看出应用了保鲜剂的雪花泥粉团形状、颜色、味型等未见明显变化，显微镜像的细胞结构完整(见图16a)，表面因脱水出现轻微干皮；而对照组则滩软黏出水无形，呈酸馊味，显微镜像显示分子结构被分解破碎，细胞呈空壳状(见图16b)，由此可见，本发明所制备的生物酶制剂具有抑制糖化的作用，分析通过对α-淀粉酶活性的抑制，延缓生物体的糖化过程。
以上对本发明及其实施方式进行了描述，这种描述没有限制性，附图中所示的也只是本发明的实 施方式之一，实际的结构并不局限于此。总而言之如果本领域的普通技术人员受其启示，在不脱离本发明创造宗旨的情况下，不经创造性的设计出与该技术方案相似的结构方式及实施例，均应属于本发明的保护范围。

Claims (4)

  1. 一种酵母、乳酸菌联合菌，其特征在于，
    其由嗜酒假丝酵母B-JJ1、布氏乳杆菌B-JR1、类干酪乳杆菌B-JR2、玉米乳杆菌B-JR4、植物乳杆菌B-JR5、乳杆菌B-JR6组成；
    所述嗜酒假丝酵母(Candida ethanolica)B-JJ1的保藏编号为：CCTCC NO:M2021136；
    所述布氏乳杆菌(Lactobacillus buchneri)B-JR1的保藏编号为：CCTCC NO:M2021132；
    所述类干酪乳杆菌(Lactobacillus paracasei)B-JR2的保藏编号为：CCTCC NO:M2021133；
    所述玉米乳杆菌(Lactobacillus zeae)B-JR4的保藏编号为：CCTCC NO:M2021135；
    所述植物乳杆菌(Lactobacillus plantarum)B-JR5的保藏编号为：CCTCC NO:M2021501；
    所述乳杆菌(Lactobacillus chiayiensis)B-JR6的保藏编号为：CCTCC NO:M2021502。
  2. 一种生物酶制剂的制备方法，其特征在于，其包括如下步骤：
    (1)选用权利要求1所述的酵母、乳酸菌联合菌作菌种；
    (2)培养基的制备和接种：所述培养基以土豆泥雪花粉、小麦胚芽、糯米为主料配制而成，将培养基加水后搅拌均匀后，蒸熟，然后无菌环境下晾至摄氏45度以下即培养基制备完成；
    将制备的培养基与菌种进行混合均匀；
    (3)对步骤(2)接种后的培养基的湿度进行调节，保证握团有水渍出，培养基整体保持湿润透气；
    (4)将步骤(3)的培养基进行有氧发酵直至培养基出现均匀肉眼可见的白色、乳白色菌斑时，将培养基转入发酵罐中；封闭发酵罐的所有通道，以使得入罐的培养基发酵物在罐体中继续有氧培养，直至耗尽罐体中的氧气，自行进入无氧发酵状态；
    (5)当发酵罐内压力无变化时，固态发酵物进入饱和发酵，开罐有浓郁的酵香味溢出，肉眼观察有丰茂的菌丝体覆盖在固态培养基表面，取发酵产物进行检测，酵母菌活菌总数6.4×10 4-5.9×10 5cfu/ml；乳酸菌总数4×10 8-5.1×10 9cfu/ml，此时固态发酵过程已经达到饱和，然后进入液态发酵阶段；
    (6)将步骤(5)的固态发酵物加入无菌水，并充分搅拌，以稀释固态发酵过程中的代谢底物，进行有氧活化培养直至液态发酵物表面密布白色或乳白色菌斑时，即可封闭发酵罐所有通道，再次让发酵物逐渐耗尽氧气进入无氧发酵过程；
    (7)当步骤(6)的液态发酵物表面菌斑折叠态，中上层清液呈现金黄色，清澈透明，底层固态发酵物和中层液体层次分明不粘连时，即固态发酵–液态发酵过程全部完成，所得到发酵产物即为制得生物酶制剂。
  3. 根据权利要求2所述的一种生物酶制剂的制备方法，其特征在于，其还包括如下步骤：
    (8)将步骤(7)所得发酵底物进行加热后，提取发酵物的上清液，即制得灭活生物酶制剂。
  4. 权利要求2或3所述的生物酶制剂的制备方法制备的生物酶制剂的应用，其特征在于，其可用于制备生物防腐保鲜剂。
PCT/CN2022/116841 2021-10-14 2022-09-02 一种酵母、乳酸菌联合菌、培育方法及其应用 WO2023061098A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202111198135.XA CN114456967B (zh) 2021-10-14 2021-10-14 一种酵母、乳酸菌联合菌、培育方法及其应用
CN202111198135.X 2021-10-14

Publications (1)

Publication Number Publication Date
WO2023061098A1 true WO2023061098A1 (zh) 2023-04-20

Family

ID=81405111

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/116841 WO2023061098A1 (zh) 2021-10-14 2022-09-02 一种酵母、乳酸菌联合菌、培育方法及其应用

Country Status (5)

Country Link
US (1) US20230120822A1 (zh)
EP (1) EP4166648A1 (zh)
CN (1) CN114456967B (zh)
WO (1) WO2023061098A1 (zh)
ZA (1) ZA202211197B (zh)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114456967B (zh) * 2021-10-14 2022-10-04 命之源(杭州)生物科技有限公司 一种酵母、乳酸菌联合菌、培育方法及其应用

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160338361A1 (en) * 2014-02-04 2016-11-24 Micro-Nature Llc Systems, methods, and compositions for promoting pathogen control and food preservation
RU2646109C1 (ru) * 2017-03-17 2018-03-01 Общество с ограниченной ответственностью "Зеленые линии" Способ производства консерванта продуктов питания
CN108522943A (zh) * 2017-03-06 2018-09-14 耿胜利 一种采用生物工程技术培植的鱼饵保鲜剂的制备方法
CN108651604A (zh) * 2018-05-21 2018-10-16 陕西科技大学 一种乳酸菌保鲜剂的制备方法及其在肉制品中的应用
CN114456967A (zh) * 2021-10-14 2022-05-10 耿胜利 一种酵母、乳酸菌联合菌、培育方法及其应用

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103468615B (zh) * 2013-09-23 2015-04-15 内蒙古农业大学 一种乳酸菌和酵母菌共生发酵的微生物制剂的生产方法
CA3059453A1 (en) * 2017-04-11 2018-10-18 Servatus Ltd Methods for the treatment of inflammation and inflammatory conditions
CN112167331A (zh) * 2020-09-27 2021-01-05 基因赛奥(大连)生物科技发展有限公司 一种生物果蔬保鲜剂及其制备方法

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160338361A1 (en) * 2014-02-04 2016-11-24 Micro-Nature Llc Systems, methods, and compositions for promoting pathogen control and food preservation
CN108522943A (zh) * 2017-03-06 2018-09-14 耿胜利 一种采用生物工程技术培植的鱼饵保鲜剂的制备方法
RU2646109C1 (ru) * 2017-03-17 2018-03-01 Общество с ограниченной ответственностью "Зеленые линии" Способ производства консерванта продуктов питания
CN108651604A (zh) * 2018-05-21 2018-10-16 陕西科技大学 一种乳酸菌保鲜剂的制备方法及其在肉制品中的应用
CN114456967A (zh) * 2021-10-14 2022-05-10 耿胜利 一种酵母、乳酸菌联合菌、培育方法及其应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BI WEN－HUI, YAO JIAN; LIU XUE-JUN; CHEN QING-MIN; CHEN YU-HANG; CHENG YUAN-YUAN; YUE FENG-LI: "Application and research progress of microorganism in fruit and vegetable storage", SCIENCE AND TECHNOLOGY OF FOOD INDUSTRY, GAI KAN BIANJIBU , BEIJING, CN, vol. 38, no. 20, 15 October 2017 (2017-10-15), CN , pages 347 - 351, XP093059056, ISSN: 1002-0306, DOI: 10.13386/j.issn1002-0306.2017.20.063 *
KWOFIE MABEL KYEI, BUKARI NAFISATU, ADEBOYE OLUWASEYI: "Probiotics Potential of Yeast and Lactic Acid Bacteria Fermented Foods and the Impact of Processing: A Review of Indigenous and Continental Food Products", ADVANCES IN MICROBIOLOGY, SCIENTIFIC RESEARCH PUBLISHING, INC., US, vol. 10, no. 09, 1 January 2020 (2020-01-01), US , pages 492 - 507, XP093059059, ISSN: 2165-3402, DOI: 10.4236/aim.2020.109037 *

Also Published As

Publication number Publication date
ZA202211197B (en) 2023-02-22
US20230120822A1 (en) 2023-04-20
CN114456967A (zh) 2022-05-10
EP4166648A1 (en) 2023-04-19
CN114456967B (zh) 2022-10-04

Similar Documents

Publication Publication Date Title
CN103725633B (zh) 一种泡菜发酵菌菌剂及制备方法及应用
CN108935972B (zh) 一种饲用大豆与甜高粱混贮的方法
CA1336749C (en) Obtaining edible material from fungus-digested medium
CN106103697B (zh) 速效乳杆菌菌株及其在改善青贮饲料的有氧稳定性方面的用途
CN109234204B (zh) 一种泡菜发酵剂及其制备和应用方法
CN113273639A (zh) 一种含益生菌的保鲜昆虫的制备方法
CN1961737A (zh) 一种即食含活益生菌的泡菜及其制备方法
TW201221065A (en) Method for producing Antrodia cinnamomea tea leaves
WO2023061098A1 (zh) 一种酵母、乳酸菌联合菌、培育方法及其应用
CN111286477B (zh) 一株丁香假单胞菌及其在防治苹果轮纹病中的应用
CN107889935A (zh) 一种半湿状益生菌制品及其制备方法与应用
CN114437994A (zh) 一种生物防治菌Bacillus siamensis HT1及其在制备生防菌剂中的应用
CN110584032A (zh) 一种臭氧联合隔氧微生物发酵的贝肉生物脱腥方法
CN117070427B (zh) 一株布氏乳杆菌及其青贮饲料发酵剂
CN109666597A (zh) 一种发酵青贮黄贮饲料的复合微生物处理剂及其制备方法
CN110591932B (zh) 一株控制果蔬采后病害的酵母菌ma及其使用方法
Sanni et al. Microbiological evaluation of Ghanaian maize dough co-fermented with cowpea
CN114854629A (zh) 一株植物乳杆菌c-37及其在发酵玉米胚芽粕中的应用
CN112063554B (zh) 一种生物防治菌Pantoea jilinensis D25及其应用
CN108522647A (zh) 植物乳杆菌mg-1在葡萄采后保鲜中的应用
CN108522943A (zh) 一种采用生物工程技术培植的鱼饵保鲜剂的制备方法
CN110042075B (zh) 一株荸荠内生枯草芽孢杆菌及其应用
CN108865919A (zh) 一种戊糖乳杆菌cicc6294及其在辣椒发酵中的应用
CN113773981A (zh) 一株高效拮抗青霉菌和灰霉菌的生防菌及其在猕猴桃贮藏中的应用
CN106615609A (zh) 一种可提高饲料青贮品质的布氏乳杆菌及其应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22880042

Country of ref document: EP

Kind code of ref document: A1