CN115960732A - Pichia anomala strain and microbial agent and application thereof - Google Patents
Pichia anomala strain and microbial agent and application thereof Download PDFInfo
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- CN115960732A CN115960732A CN202310082003.3A CN202310082003A CN115960732A CN 115960732 A CN115960732 A CN 115960732A CN 202310082003 A CN202310082003 A CN 202310082003A CN 115960732 A CN115960732 A CN 115960732A
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- lactobacillus plantarum
- dried powder
- pichia pastoris
- fermented
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Images
Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/90—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in food processing or handling, e.g. food conservation
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- Preparation Of Fruits And Vegetables (AREA)
Abstract
The invention relates to a pichia pastoris strain, a microbial agent and application thereof, and belongs to the technical field of microbial fermentation. The invention provides a Pichia anomala strain, which is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, and the preservation number is CGMCC No.25675; the pichia pastoris can produce alcohol flavor substances with flower and fruit fragrance such as ethanol, isobutanol, isoamylol and phenethyl alcohol, and can destroy polysaccharide ingredients in vegetables by producing various glycoside hydrolases, promote the release of linalool, terpenes and phenol bonding-state fragrance ingredients, produce flower fragrance and sweet fragrance, and enrich the flavor of fermented vegetables.
Description
Technical Field
The invention relates to a pichia pastoris strain, a microbial agent and application thereof, and belongs to the technical field of microbial fermentation.
Background
The fermented vegetable is a vegetable product obtained by fermenting fresh vegetables in an anaerobic environment through a microbial system. Various fresh vegetables including purple cabbage, chinese cabbage, carrot, beet, cucumber, celery, pepper, green bean, kidney bean, etc. can be used for the production of fermented vegetables. The fermented vegetable is not only a source of nutrient substances such as probiotics, vitamins, mineral substances and the like, but also has multiple functional characteristics.
At the present stage, the processing technology of fermented vegetables in China is mainly natural fermentation, and the natural fermentation has the problems of long fermentation period, overproof salt, high nitrite content and biogenic amine content, easy contamination by mixed bacteria and the like, so that the industrial development of green and healthy fermented vegetables is severely restricted, and the modern fermentation technology needs to be further developed.
Modern fermentation processes require stable fermentation under specific conditions using microbial agents consisting of specific strains of bacteria. At present, the microbial agent for vegetable fermentation is generally prepared by separating specific strains which are fast in acid production, rich in fragrance and stable in properties from naturally fermented vegetable products, then carrying out amplification culture on the specific strains, collecting thalli of the specific strains, mixing the thalli with a freeze-drying protective agent to prepare a high-concentration bacterial suspension, and finally carrying out vacuum freeze-drying.
The existing microbial agent for vegetable fermentation is usually prepared by adopting a single lactobacillus strain, which can accelerate the vegetable fermentation period to a certain extent, but because many original glucoside bonding state aroma substances in vegetables are stored in the plants in a glucoside form, and the lactobacillus cannot utilize polysaccharide substances in the plants, the glucoside bonding state flavor components are difficult to release, so that vegetable products obtained by fermenting the vegetables by using the microbial agent for vegetable fermentation prepared by the single lactobacillus strain have single taste and insufficient aroma, and are difficult to compare with natural fermentation products.
Disclosure of Invention
In order to solve the problems, the invention provides a Pichia pastoris (Pichia Galeiformis) F1705, which is characterized in that the Pichia pastoris is stored in the common microorganism center of China Committee for culture Collection of microorganisms, and the storage number is CGMCC No.25675.
The pichia kluyveri F1705 is separated from a fermented radish sample produced in the city of warfarin, nanping, fujian province, the 18S rDNA sequence of the strain is shown as SEQ ID No.1 through sequencing analysis, the sequence obtained through sequencing is compared with a nucleic acid sequence in a GeneBank, and the result shows that the strain is the pichia kluyveri F1705.
The invention also provides a microbial agent for fermenting vegetables, and the components of the microbial agent comprise the pichia pastoris and Lactobacillus plantarum.
In one embodiment of the invention, the lactobacillus plantarum is deposited in the general microbiological center of the China Committee for culture Collection of microorganisms with the deposit number of CGMCC1.3919.
In one embodiment of the invention, the components of the microbial agent comprise pichia pastoris freeze-dried powder and lactobacillus plantarum freeze-dried powder.
In one embodiment of the invention, the mass ratio of the pichia pastoris freeze-dried powder to the lactobacillus plantarum freeze-dried powder is 0.5-1.5: 1 to 3.
In one embodiment of the invention, the mass ratio of the pichia pastoris freeze-dried powder to the lactobacillus plantarum freeze-dried powder is 1:2.
in one embodiment of the invention, the preparation method of the pichia pastoris freeze-dried powder comprises the following steps: inoculating pichia pastoris into a liquid culture medium for culture to obtain a pichia pastoris bacterial liquid; centrifuging a pichia kluyveri bacterial liquid, and taking a pichia kluyveri thallus; mixing the Pichia pastoris with freeze-drying protective agent, and freeze-drying to obtain the final product with viable count of 1 × 10 9 ～1×10 10 CFU/g of pichia pastoris freeze-dried powder.
In an embodiment of the present invention, the lactobacillus plantarum freeze-dried powder preparation method comprises: inoculating lactobacillus plantarum into a liquid culture medium for culture to obtain lactobacillus plantarum liquid; centrifuging the lactobacillus plantarum bacterial liquid, and taking lactobacillus plantarum thalli; mixing Lactobacillus plantarum thallus with freeze-drying protective agent, and freeze-drying to obtain viable count of 1 × 10 9 ～1×10 10 CFU/g lactobacillus plantarum freeze-dried powder.
In one embodiment of the invention, the freeze-drying protective agent comprises, by mass, 1-2% of trehalose, 2-6% of sodium glutamate, 10-20% of sucrose, 10-20% of skim milk powder, 0.1-1.0% of calcium chloride, and the balance of water.
The invention also provides a method for preparing fermented vegetables, which comprises the following steps: and (3) fermenting the vegetables by using the microbial agent to obtain the fermented vegetables.
In one embodiment of the present invention, the method is: soaking vegetables below the liquid level of a salt solution to obtain a mixture; adding the microbial agent into the mixture to obtain a fermentation system; fermenting the fermentation system at 25-30 deg.c for 1-7 days to obtain fermented vegetable.
In one embodiment of the present invention, the method is: soaking vegetables below the liquid level of a salt solution to obtain a mixture; firstly, adding the pichia pastoris freeze-dried powder into the mixture, fermenting for 12-24 h at 25-30 ℃, then adding the lactobacillus plantarum freeze-dried powder into the mixture, and continuing to ferment for 1-3 d at 25-30 ℃ to obtain fermented vegetables; the mass ratio of the helmet-shaped pichia pastoris freeze-dried powder to the lactobacillus plantarum freeze-dried powder is 0.5-1.5: 1 to 3; the total mass of the helmet-shaped pichia pastoris freeze-dried powder and the lactobacillus plantarum freeze-dried powder accounts for 0.2 to 0.4 percent of the total mass of the vegetables.
In one embodiment of the invention, the mass ratio of the pichia pastoris freeze-dried powder to the lactobacillus plantarum freeze-dried powder is 1:2.
in one embodiment of the present invention, the salt solution is present in an amount of 2 to 5% by mass.
The invention also provides application of the pichia pastoris or the microbial agent or the method in preparation of fermented vegetables.
The technical scheme of the invention has the following advantages:
1. the invention provides a Pichia anomala (Pichia Galeiformis) strain, which is preserved in the common microorganism center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No.25675; the pichia pastoris can produce alcohol flavor substances with flower and fruit flavor, such as ethanol, isobutanol, isoamylol, phenethyl alcohol and the like, and can destroy polysaccharide ingredients in vegetables by producing various glycoside hydrolases, promote the release of linalool, terpenes and phenol bonding-state flavor ingredients, generate flower flavor and sweet flavor, and enrich the flavor of fermented vegetables.
2. The invention provides a microbial agent, wherein the components of the microbial agent comprise pichia pastoris F1705 and Lactobacillus plantarum (Lactobacillus plantarum), wherein the pichia pastoris can produce alcohol flavor substances with flower and fruit fragrance such as ethanol, isobutanol, isoamylol, phenethyl alcohol and the like, the pichia pastoris can destroy polysaccharide components in vegetables by producing various glycoside hydrolases, release of linalool, terpenes and phenolic bonding state fragrance components is promoted, flower fragrance and sweet fragrance are generated, the flavor of a vegetable product obtained by fermentation can be obviously improved when the pichia pastoris is used for vegetable fermentation, excessive growth of the pichia pastoris in a vegetable fermentation system can degrade a large amount of pectin in the vegetables, the texture of the fermented vegetables is softened, the brittleness is reduced, the product quality is influenced, the Lactobacillus plantarum is added into the fermentation system, the monosaccharide components can rapidly grow to produce acid by utilizing the monosaccharide components, the glycoside fragrance components are further modified to form the flavor of the fermented vegetables, the low pH environment formed by the Lactobacillus plantarum can inhibit excessive growth, the pichia pastoris is prevented from being degraded to cause the softened texture and the softened fragrance of the vegetables, the Lactobacillus plantarum itself can further enhance the flavor of the fermented vegetables, and the flavor of the fermented vegetable product, so that the pectin and the fermented vegetable product can be used for enhancing the flavor of the fermented vegetables.
Further, the microbial agent comprises a pichia pastoris freeze-dried powder and a lactobacillus plantarum freeze-dried powder, and in the microbial agent, the mass ratio of the pichia pastoris freeze-dried powder to the lactobacillus plantarum freeze-dried powder is 1:2. under the mass ratio, the flavor enhancement and brittleness keeping effects of the microbial agent are better.
3. The invention provides a method for preparing fermented vegetables, which comprises the step of fermenting the vegetables by using a microbial agent comprising pichia kluyveri F1705 and lactobacillus plantarum to obtain the fermented vegetables, wherein the pichia kluyveri can produce alcohol flavor substances with flower and fruit flavor such as ethanol, isobutanol, isoamylol, phenethyl alcohol and the like, and can destroy polysaccharide components in the vegetables by producing a plurality of glycoside hydrolases, promote the release of linalool, terpenes and phenol bonding-state flavor components to generate flower fragrance and sweet fragrance, the flavor of the vegetable products obtained by fermentation can be obviously improved when the pichia kluyveri is used for vegetable fermentation, but pectin in the vegetables can be greatly degraded when the pichia kluyveri grows excessively in a vegetable fermentation system, the fermented vegetable is softened in texture, the brittleness is reduced, the product quality is influenced, the lactobacillus plantarum is added into a fermentation system, monosaccharide components can be used for rapidly growing to produce acid, glucoside-state aroma components are further modified to form the special flavor of the fermented vegetable, the low pH environment formed by the lactobacillus plantarum can inhibit excessive growth of the pichia pastoris, the texture softening and the peculiar smell of the vegetable caused by the degradation of pectin in the vegetable are prevented, in addition, the lactobacillus plantarum can generate a large amount of organic acid, aromatic substances and flavor-developing amino acids, the vegetable is endowed with acid aroma and delicate flavor, the flavor of the fermented vegetable is further enhanced and improved, and therefore, the method disclosed by the invention is good in aroma-enhancing and crispness-keeping effects, can be used for vegetable fermentation to obviously improve the flavor of the fermented vegetable product, and has a great application prospect.
Further, the microbial agent comprises pichia kluyveri freeze-dried powder and lactobacillus plantarum freeze-dried powder, and in the microbial agent, the mass ratio of the pichia kluyveri freeze-dried powder to the lactobacillus plantarum freeze-dried powder is 1:2. under the quality ratio, the method has better effects of increasing aroma and keeping crisp.
Furthermore, the pichia kluyveri F1705 and the lactobacillus plantarum are directly inoculated into the fermented vegetables, the growth rate of the two strains and the growth competitive relationship between the two strains are difficult to monitor and control, the quality of products is easy to be unstable, and the excessive growth of the pichia kluyveri can cause the degradation of pectin in the vegetables, so that the brittleness of the vegetables is reduced, and the mouthfeel of the fermented vegetables is influenced. According to the method, firstly, pichia pastoris with a slow growth rate is put into fermented vegetables, and lactobacillus plantarum with a fast growth rate is put into the fermented vegetables after fermentation is carried out for 12-24 hours. The mixed strain two-stage inoculation fermentation can complement the advantages of the strains, maintain the good crispness of the vegetables and obviously improve the flavor of the fermented vegetable products.
Biological material preservation
A Pichia helmet (Pichia Galeiformis) F1705 is classified and named as Pichia Galeiformis, and is deposited in China general microbiological culture Collection center on 09.09.18 years, with the deposition number of CGMCC No.25675, and the deposition address of No.3, xilu No.1, north Cheng, of the Korean district, beijing.
Drawings
FIG. 1: colony and thallus morphology of Pichia anomala (Pichia Galeiformis) F1705.
FIG. 2 is a schematic diagram: and (3) performing gas quality detection ion flow diagram on volatile aroma components produced by the pichia pastoris.
FIG. 3: and (3) a gas detection ion flow diagram of the flavor substance in the fermented pepper prepared in the comparative example 5.
FIG. 4: and (3) performing gas detection ion-flow graph on the flavor substances in the fermented pepper prepared in the comparative example 4.
FIG. 5: and (3) performing gas detection ion flow diagram on the flavor substances in the fermented hot pepper prepared in the comparative example 3.
FIG. 6: example 3 gas detection ion-flow profile of flavor in fermented peppers made in accordance with the present disclosure.
FIG. 7: and (5) sensory evaluation results of the fermented peppers.
Detailed Description
The following examples are provided to further understand the present invention, not to limit the scope of the present invention, but to provide the best mode, not to limit the content and the protection scope of the present invention, and any product similar or similar to the present invention, which is obtained by combining the present invention with other prior art features, falls within the protection scope of the present invention.
The media and reagents involved in the following examples are as follows:
YPD liquid medium: 10g of yeast extract, 20g of peptone, 20g of glucose and 1000g of water.
YPD solid Medium: 10g of yeast extract, 20g of peptone, 20g of glucose, 15g of agar and 1000g of water.
MRS liquid culture medium: 10g of peptone, 0.2g of magnesium sulfate, 2g of dipotassium phosphate, 5g of sodium acetate, 0.054g of manganese sulfate, 20g of glucose, 2g of diammonium hydrogen citrate, 5g of yeast powder, 10g of beef extract, 80 g of tween-80 and 1000g of water.
Mineral salt culture medium: 50g of glucose, 1.7g of nutrient medium Y1251 (purchased from sigma company) without amino acid and ammonium sulfate, 0.19444g of ammonium sulfate and 1000g of water.
0.85% physiological saline: 0.85g of sodium chloride and 100g of water.
Freeze-drying protective agent: 1.22wt% of trehalose, 5.80wt% of sodium glutamate, 15.80wt% of sucrose, 16.50wt% of skim milk, 0.5wt% of calcium chloride and the balance of water.
The detection methods referred to in the following examples are as follows:
the detection method of viable count comprises the following steps: the method adopts the national standard GB 4789.35-2016 food safety national standard food microbiology lactobacillus detection and a methyl blue staining blood counting plate counting method.
The nitrite determination method comprises the following steps: GB5009.33-2016 determination of nitrite-determination of nitrate in vegetables and fruits by the third method, and determination of nitrite content in fermented food by ultraviolet spectrophotometry.
The detection method of the aroma components comprises the following steps: and detecting the content of the volatile flavor substances by a gas chromatography-mass spectrometer by adopting a solid phase microextraction method.
Example 1: obtaining Pichia anomala
Taking fermented radish produced in the city of Wuyi mountain in Nanping city, fujian province as a sample, taking 0.5g of the sample, fully grinding the sample in a sterile mortar, adding 5mL of sterile physiological saline, and uniformly mixing to obtain a sample mixed solution; sucking 0.5mL of sample mixed solution, adding the sample mixed solution into 5mL of YPD liquid culture medium, and culturing at 30 ℃ and 200rpm for 24h for enrichment to obtain an enriched sample; sucking 0.5mL of the enriched sample and adding to 4.5mL of sterile physiological saline to obtain 10 -1 The diluted solution was then 0.5mL of 10 -1 The dilution was taken in 4.5mL of physiological saline to give 10 -2 The dilution was carried out in this order to give 10 -3 、10 -4 、10 -5 、10 -6 Diluting the solution; draw 100. Mu.L of the gradient diluent and spread on MRS solid medium, 10 -4 、10 -5 、10 -6 Culturing each gradient 1 plate at 30 deg.C and 200rpm for 48h to obtain bacterial colony; selecting a colony with typical characteristics of the helmet-shaped pichia pastoris on a YPD solid culture medium according to the shape, the size, the edge, the transparency and the like of the colony, picking the colony by using an inoculating loop, streaking the colony on the YPD solid culture medium, and culturing at 30 ℃ and 200rpm for 48h to obtain a purified single colony (the colony is white, the edge is irregular, and the specific figure is 1); picking purified single colony to inoculate respectivelyCulturing in 5mL YPD liquid culture medium at 30 deg.C and 200rpm for 24 hr to obtain bacterial liquid; numbering each strain corresponding to each bacterial liquid, performing gram staining, strain identification, physiological and biochemical experiments and genome identification and analysis according to the steps recorded in textbook "microbiology" (Shen Nu, chen Dong main eds.), and selecting a strain with the typical characteristics of the pichia pastoris to obtain a strain F1705;
wherein the strain identification process is as follows:
taking F1705 thallus, extracting the genome of F1705 by using a fungus genome extraction kit, and amplifying by using a primer pair with nucleotide sequences shown as SEQ ID NO.2 and SEQ ID NO.3 respectively and taking the extracted genome of F1705 as a template to obtain 18S rRNA of F1705 (the 16S rDNA sequence of F1705 is shown as SEQ ID NO. 1); the 18S rDNA of F1705 is subjected to nucleic acid sequence alignment in a Blastn program of NCBI, and the result shows that the sequence homology of the strain and the Pichia anomala is 99.78%, the strain is identified as the Pichia anomala and named as the Pichia anomala (Pichia Galeiformis) F1705.
Experimental example 1: detection of flavor substances produced by pichia capitata
Pichia anomala (Pichia Galeiformis) F1705 from example 1 was inoculated into mineral salt medium to the initial OD 600 After =0.500, the culture was carried out at 30 ℃ and 200rpm, and the OD of the culture solution was periodically monitored during the culture 600 Changing values until the growth state is stable, sampling the culture solution when culturing for 72h, and analyzing the volatile aroma components by the following steps:
extracting volatile aroma components in the culture solution by adopting a headspace solid phase microextraction method to obtain a sample; sucking 3mL of sample into a 10mL headspace bottle, adding 1g of NaCl, immediately sealing, placing in a magnetic stirrer after 1min of vortex, balancing for 15min at 40 ℃ at 500r/min, and adding 3-octanol (0.05 mu g/mL) as an internal standard (40 mu L) by using a gas-tight syringe; inserting the aged 50/30 μm DVB/CAR/PDMS extraction probe into the headspace of a sample bottle, adsorbing at 40 deg.C and 500r/min for 40min, inserting the extraction probe into a GC sample inlet, and desorbing at 250 deg.C for 5min; separating and detecting the volatile matter by using a DB-5MS chromatographic column (30 m multiplied by 0.32mm multiplied by 0.25 mu m); helium with the purity of more than 99.99 percent is taken as carrier gas, the flow is constant and is 2.0mL/min, and split-flow sample injection is not carried out; the initial temperature was first maintained at 45 ℃ for 5 minutes, then increased to 85 ℃ at a rate of 4 ℃/min, then increased to 250 ℃ at a rate of 5 ℃/min; the scanning range of the mass spectrum is 35-500 m/z, and the electron ionization mode is 70eV; the ion source and transfer line temperatures of the mass spectrometer were 230 ℃ and 280 ℃, respectively; the solvent delay time was 1.0min. The volatile content of each sample was determined in 4 replicates and the results are shown in FIG. 2.
Volatile aroma components were passed through a database of comparative NIST 2014 libraries, and under the same operating conditions as normal alkanes (C) 5 -C 25 ) For external reference, preliminary characterization of Retention Index (RI) and literature data determined experimentally; to each sample was added 40. Mu.L of 3-octanol (5X 10) -5 μ g/L) as an internal standard, and the calculation results are shown in table 1.
As can be seen from fig. 2 and table 1, pichia pastoris itself can produce alcoholic flavors having flower and fruit flavors such as ethanol, isobutanol, isoamyl alcohol, and phenethyl alcohol. The flavor substances have rich flower and fruit fragrance and are important flavor substances in fermented foods.
TABLE 1 Pichia kluyveri self-producible flavor substances
| Name of Chinese | CAS number | RI | Content (μ g/Kg) | Description of the smell |
| Ethanol | 64-17-5 | 431 | 108.55±39.56 | Taste of alcohol |
| Isobutanol | 78-83-1 | 630 | 74.50±10.50 | Wine flavor |
| Isoamyl alcohol | 123-51-3 | 738 | 436.31±23.54 | Oil, fruit, banana |
| 2-Ethyl hexanol | 104-76-7 | 1033 | 10.61±2.54 | Orange, flower fragrance, sweet fragrance |
| Phenylethanolic acid | 60-12-8 | 1117 | 114.41±19.70 | Rose fragrance |
| Alpha-terpineol | 98-55-5 | 1199 | 1.73±0.26 | Orange, woody and flowery odour |
| Geraniol | 106-24-1 | 1254 | 4.54±0.88 | Sweet, fruity, rose-scented, citrus |
Example 2: microbial agent for fermenting vegetables and preparation thereof
The embodiment provides a microbial agent for fermenting vegetables, which consists of freeze-dried powder of Pichia pastoris (Pichia Fermentans) and freeze-dried powder of Lactobacillus plantarum (Lactobacillus plantarum); in the microbial agent, the mass ratio of the helmet-shaped pichia pastoris freeze-dried powder to the lactobacillus plantarum freeze-dried powder is 1:2.
the preparation method of the pichia pastoris freeze-dried powder comprises the following steps: streaking and inoculating a bacterial liquid of Pichia pastoris (Pichia Galeiformis) F1705 in example 1 on a YPD solid culture medium, inversely placing the YPD solid culture medium in a constant temperature incubator, and culturing for 3 days at 30 ℃ to obtain a single colony; selecting a single colony, inoculating the single colony in 4.5mL YPD liquid culture medium, and performing activation culture at 30 ℃ and 200rpm for 3 days to obtain activated culture bacterial liquid; the activated culture broth was diluted to 1.0X 10 in YPD liquid medium 8 CFU/mL to obtain a diluent; inoculating the diluent into YPD liquid culture medium at 3% (v/v), and performing amplification culture at 30 deg.C and 200rpm for 5 days to obtain amplification culture bacterial liquid; putting the bacterial liquid obtained by the enlarged culture into a sterilized centrifugal tube, centrifuging for 20min at 4000rpm/min, collecting thalli, resuspending the thalli by using 0.85% sterile physiological saline, centrifuging for 20min at 4000rpm/min, washing repeatedly for three times, and collecting the thalli again; adding a freeze-drying protective agent with the volume 1.5 times that of the thalli to obtain a bacterial suspension; pre-freezing the bacterial suspension with liquid nitrogen, transferring into vacuum freezing desiccant, and freeze-drying at-60 deg.C for 2 days to obtain helmet-shaped Pichia pastoris lyophilized powder (viable count of 1 × 10) 9 CFU/g)。
The preparation method of the lactobacillus plantarum freeze-dried powder comprises the following steps: streaking a bacterial solution of lactobacillus plantarum CGMCC1.3919 (purchased from China general microbiological culture Collection center), inoculating the bacterial solution on an MRS solid culture medium, inverting the MRS solid culture medium in an anaerobic incubator, and culturing for 2 days at 37 ℃ to obtain a single bacterial colony; selecting a single colony, inoculating the single colony in 4.5mL of MRS liquid culture medium, placing the single colony in an anaerobic incubator, and culturing for 1 day at 37 ℃ to obtain activated culture bacterial liquid; using MRS liquid culture medium to dilute the activated culture bacterial liquid to the total number of bacterial colonies of 1.0 multiplied by 10 8 CFU/mL to obtain a diluent; inoculating the diluent into an MRS liquid culture medium in an inoculation amount of 2% (v/v), placing the MRS liquid culture medium in an anaerobic incubator, and carrying out amplification culture at 37 ℃ for 2 days to obtain an amplification culture bacterial liquid; placing the enlarged culture bacterial liquid into a sterilized centrifugal tube, centrifuging for 20min at 4000rpm/min, collecting thalli, resuspending the thalli by using 0.85% sterile physiological saline, centrifuging for 20min at 4000rpm/min, washing for three times, and collecting the thalli again; adding a freeze-drying protective agent with the volume 1.5 times that of the thalli to obtain a bacterial suspension; pre-freezing the bacterial suspension with liquid nitrogen, transferring into vacuum freezing desiccant, and freeze-drying at-60 deg.C for 2 days to obtain Lactobacillus plantarum lyophilized powder (viable count is 1 × 10) 9 CFU/g)。
Example 3: preparation method of fermented Capsici fructus
The present example provides a method for preparing fermented pepper, the method comprising the steps of:
the method comprises the following steps: cleaning fresh Capsici fructus, draining, and sterilizing with ultraviolet irradiation for 2 hr to obtain sterilized Capsici fructus; placing the sterilized hot pepper in a fermentation container, adding 4% (w/v) of salt solution to completely immerse the hot pepper below the liquid level of the salt solution to obtain a mixture; adding 1.5g of pichia pastoris freeze-dried powder into every 1kg of pepper, and fully dissolving the pichia pastoris freeze-dried powder of the microbial agent in the embodiment 2 into the mixture to obtain a fermentation system A;
step two: sealing the fermentation container filled with the fermentation system A, placing the sealed fermentation container in a constant temperature incubator, and fermenting at the temperature of 30 ℃; after fermenting for 24 hours, adding 3g of lactobacillus plantarum freeze-dried powder into each 1kg of pepper, and fully dissolving the lactobacillus plantarum freeze-dried powder of the microbial inoculant in the embodiment 2 into the fermentation system A to obtain a fermentation system B; and sealing the fermentation container filled with the fermentation system B, placing the sealed fermentation container in a constant-temperature incubator, and continuously fermenting for 48 hours at the temperature of 30 ℃ to obtain the fermented chili.
Example 4: preparation method of fermented chayote
The embodiment provides a method for preparing fermented chayote, which comprises the following steps:
the method comprises the following steps: cleaning fresh fructus Sechii edulis, draining, cutting into pieces with thickness of 2mm, and sterilizing with ultraviolet irradiation for 2 hr to obtain sterilized fructus Sechii edulis; placing the sterilized chayote into a fermentation container, adding 2% (w/v) of a salt solution, and completely soaking the chayote below the liquid level of the salt solution to obtain a mixture; adding 1g of pichia pastoris freeze-dried powder into each 1kg of chayote, and fully dissolving the pichia pastoris freeze-dried powder of the microbial agent in the embodiment 2 into the mixture to obtain a fermentation system A;
step two: sealing the fermentation container filled with the fermentation system A, placing the sealed fermentation container in a constant temperature incubator, and fermenting at the temperature of 30 ℃; after fermenting for 12 hours, adding 2g of lactobacillus plantarum freeze-dried powder into each 1kg of chayote, and fully dissolving the lactobacillus plantarum freeze-dried powder of the microbial inoculant in the embodiment 2 in the fermentation system A to obtain a fermentation system B; and sealing the fermentation container filled with the fermentation system B, placing the fermentation container in a constant-temperature incubator, and continuously fermenting for 48 hours at the temperature of 30 ℃ to obtain the fermented chayote.
Example 5: preparation of fermented radish
This example provides a method of preparing fermented radish, comprising the steps of:
the method comprises the following steps: cleaning fresh radish peel, draining, cutting into pieces with thickness of 3mm, and sterilizing for 2h by ultraviolet irradiation to obtain sterilized radish peel; placing sterilized radish skin into a fermentation container, adding 3% (w/v) salt solution, and soaking radish skin below the salt solution to obtain mixture; adding 2g of pichia kluyveri freeze-dried powder into every 1kg of radish peel, and fully dissolving the pichia kluyveri freeze-dried powder of the microbial agent in the embodiment 2 into the mixture to obtain a fermentation system A;
step two: sealing the fermentation container filled with the fermentation system A, placing the sealed fermentation container in a constant temperature incubator, and fermenting at the temperature of 30 ℃; after fermenting for 24 hours, adding 4g of lactobacillus plantarum freeze-dried powder into 1kg of radish skin, and fully dissolving the lactobacillus plantarum freeze-dried powder of the microbial inoculant in the embodiment 2 into the fermentation system A to obtain a fermentation system B; and sealing the fermentation container filled with the fermentation system B, placing the fermentation container in a constant-temperature incubator, and continuously fermenting for 72 hours at the temperature of 30 ℃ to obtain the fermented radish.
Comparative example 1: microbial agent for fermenting vegetables and preparation method thereof
The comparative example provides a microbial agent for fermenting vegetables, and the microbial agent consists of freeze-dried powder of Pichia pastoris (Pichia Fermentans).
The preparation method of the pichia pastoris freeze-dried powder comprises the following steps: the bacterial liquid of the Pichia pastoris (Pichia Galeiformis) F1705 in the example 1 is streaked and inoculated on a YPD solid culture medium, and is inverted in a constant temperature incubator and cultured for 3 days at the temperature of 30 ℃ to obtain a single colony; selecting a single colony, inoculating the single colony in 4.5mL YPD liquid culture medium, and performing activation culture at 30 ℃ and 200rpm for 3 days to obtain activated culture bacterial liquid; the activated culture broth was diluted to 1.0X 10 in YPD liquid medium 8 CFU/mL to obtain a diluent; inoculating the diluent into YPD liquid culture medium at an inoculation amount of 3% (v/v), and performing amplification culture at 30 deg.C and 200rpm for 5 days to obtain amplification culture bacterial liquid; putting the bacterial liquid obtained by the enlarged culture into a sterilized centrifugal tube, centrifuging for 20min at 4000rpm/min, collecting thalli, resuspending the thalli by using 0.85% sterile physiological saline, centrifuging for 20min at 4000rpm/min, washing for three times, and collecting the thalli again; adding a freeze-drying protective agent with the volume 1.5 times that of the thalli to obtain a bacterial suspension; pre-freezing the bacterial suspension with liquid nitrogen, transferring into vacuum freezing desiccant, and freeze-drying at-60 deg.C for 2 days to obtain helmet-shaped Pichia pastoris lyophilized powder (viable count of 1 × 10) 9 CFU/g)。
Comparative example 2: microbial agent for fermenting vegetables and preparation thereof
The comparative example provides a microbial agent for fermenting vegetables, which consists of Lactobacillus plantarum freeze-dried powder.
The preparation method of the lactobacillus plantarum freeze-dried powder comprises the following steps: streaking a bacterial solution of lactobacillus plantarum CGMCC1.3919 (purchased from China general microbiological culture Collection center), inoculating the bacterial solution on an MRS solid culture medium, inverting the MRS solid culture medium in an anaerobic incubator, and culturing for 2 days at 37 ℃ to obtain a single bacterial colony; selecting a single colony, inoculating the single colony in 4.5mL of MRS liquid culture medium, placing the single colony in an anaerobic incubator, and culturing for 1 day at 37 ℃ to obtain activated culture bacterial liquid; using MRS liquid culture medium to dilute the activated culture bacterial liquid to the total number of bacterial colonies of 1.0 multiplied by 10 8 CFU/mL to obtain a diluent; inoculating the diluent into an MRS liquid culture medium in an inoculation amount of 2% (v/v), placing the MRS liquid culture medium in an anaerobic incubator, and carrying out amplification culture at 37 ℃ for 2 days to obtain an amplification culture bacterial liquid; putting the expanded bacterial liquid into a sterilized centrifugal tube, centrifuging for 20min at 4000rpm/min, collecting thalli, resuspending the thalli by using 0.85% sterile physiological saline, centrifuging for 20min at 4000rpm/min, washing repeatedly for three times, and collecting the thalli again; adding a freeze-drying protective agent with the volume 1.5 times that of the thalli to obtain a bacterial suspension; pre-freezing the bacterial suspension with liquid nitrogen, transferring into vacuum freezing desiccant, and freeze-drying at-60 deg.C for 2 days to obtain Lactobacillus plantarum lyophilized powder (viable count is 1 × 10) 9 CFU/g)。
Comparative example 3: preparation method of fermented Capsici fructus
The present comparative example provides a method of preparing fermented peppers, the method including the steps of:
the method comprises the following steps: cleaning fresh Capsici fructus, draining, and sterilizing with ultraviolet irradiation for 2 hr to obtain sterilized Capsici fructus; placing the sterilized pepper in a fermentation container, adding 4% (w/v) of salt solution, and soaking the pepper below the liquid level of the salt solution to obtain a mixture; adding 3g of lactobacillus plantarum freeze-dried powder into each 1kg of pepper, and fully dissolving the lactobacillus plantarum freeze-dried powder of the microbial inoculant in the comparative example 2 into the mixture to obtain a fermentation system;
step two: and sealing the fermentation container filled with the fermentation system, placing the fermentation container in a constant-temperature incubator, and fermenting for 72 hours at the temperature of 30 ℃ to obtain the fermented pepper.
Comparative example 4: preparation method of fermented Capsici fructus
The present comparative example provides a method for preparing fermented peppers, the method comprising the steps of:
the method comprises the following steps: cleaning fresh Capsici fructus, draining, and sterilizing with ultraviolet irradiation for 2 hr to obtain sterilized Capsici fructus; placing the sterilized pepper in a fermentation container, adding 4% (w/v) of salt solution, and soaking the pepper below the liquid level of the salt solution to obtain a mixture; adding 1.5g of pichia pastoris freeze-dried powder into every 1kg of hot pepper, and fully dissolving the pichia pastoris freeze-dried powder of the microbial agent in the comparative example 1 into the mixture to obtain a fermentation system;
step two: and sealing the fermentation container filled with the fermentation system, placing the sealed fermentation container in a constant-temperature incubator, and fermenting for 72 hours at the temperature of 30 ℃ to obtain the fermented chili.
Comparative example 5: preparation method of fermented Capsici fructus
The present comparative example provides a method for preparing fermented peppers, the method comprising the steps of:
the method comprises the following steps: cleaning fresh Capsici fructus, draining, and sterilizing with ultraviolet irradiation for 2 hr to obtain sterilized Capsici fructus; placing the sterilized hot peppers into a fermentation container, adding 4% (w/v) of a salt solution, and completely soaking the hot peppers below the liquid level of the salt solution to obtain a fermentation system;
step two: and sealing the fermentation container filled with the fermentation system, placing the sealed fermentation container in a constant-temperature incubator, and fermenting for 72 hours at the temperature of 30 ℃ to obtain the fermented chili.
Experimental example 1: performance testing of fermented vegetables
1. Nitrite content
The nitrite contents of the fermented vegetables obtained in examples 3 to 5 and those of the fermented vegetables obtained in comparative examples 3 to 5 were measured, and the results are shown in table 2. As can be seen from Table 2, the nitrite content in the fermented vegetables was below the 20mg/kg limit.
TABLE 2 nitrite content of fermented vegetables
| Sample(s) | Content (mg/kg) |
| Example 4 | 5.30±0.84 |
| Example 5 | 8.56±1.55 |
| Example 6 | 14.38±1.42 |
| Comparative example 3 | 9.25±0.66 |
| Comparative example 4 | 12.71±2.01 |
| Comparative example 5 | 16.57±1.63 |
2. pH value
Changes in pH can affect metabolites and metabolic pathways of the strains, and thus affect the flavor and quality of the fermented vegetables. The pH values of the fermented vegetables obtained in examples 3 to 5 and the fermented vegetables obtained in comparative examples 3 to 5 were measured, and the results of the measurements are shown in table 3. As can be seen from Table 3, the pH of the vegetables fermented with the added lactic acid bacteria was low, while the pH of the samples inoculated with yeast and the samples not inoculated with the bacterial strain varied slightly.
TABLE 3 pH of fermented vegetables
3. Microbiological indicator
This experiment examined the microbial indicators of the fermented vegetables obtained in examples 3 to 5 on the 15 th day of storage, and the results of the examination are shown in table 4. As can be seen from Table 4, the fermented vegetables prepared in examples 3 to 5 still meet the national standards when stored for 15 days, and have a long shelf life.
TABLE 4 microbial indicators of fermented vegetables
| Detecting items | National standard | Example 3 | Example 4 | Example 5 |
| Total number of colonies, CFU/g | ≤100 | 25 | 32 | 44 |
| Escherichia coli, MPN/100g | ≤30 | 8 | 10 | 14 |
| Mold, CFU/g | ≤20 | 5 | 9 | 10 |
| Pathogenic bacteria | Cannot be detected | Is free of | Is composed of | Is free of |
4. Flavor component detection
The fermented peppers obtained in example 3 and the fermented peppers obtained in comparative examples 3 to 5 were examined for their flavor components, and the results of the examination are shown in fig. 3 to 6 and table 5. As can be seen from fig. 3 to 6 and table 5, compared with the non-inoculated fermented pepper, the lactic acid bacteria inoculation fermentation mainly produces lactic acid, creates a lower pH environment, is beneficial to pepper preservation, but contributes less to pepper flavor components. The yeast inoculated fermented pepper has great change on flavor components, and promotes the release of bonding state flavor substances such as linalool, terpenes, phenols, phenyl derivatives and the like in the pepper. The pepper inoculated and fermented in the two-section mode by mixing the yeast and the lactic acid bacteria has rich flavor substances and can also promote the release of bonding state air substances in the pepper.
TABLE 5 fermented Pepper air test
5. Sensory evaluation
Taking out the fermented peppers prepared in the example 3 and the fermented peppers prepared in the comparative examples 3-5, cleaning the peppers by using clear water, observing the color and the shape of the fermented peppers, establishing a sensory evaluation group consisting of 25 persons, and carrying out sensory evaluation on the fermented peppers, wherein 3 samples are arranged in parallel. The evaluation indexes are the taste, appearance state, smell and color of the fermented pepper, the evaluation standards are shown in Table 6, and the sensory evaluation results are shown in FIG. 7.
The results in fig. 7 show that in each example, the sensory evaluation score of example 3 was 98 points at the maximum, and the sensory evaluation scores were all good in terms of taste, smell, appearance and color. In addition, the fermented vegetable products prepared by the microbial inoculum provided by the comparative example 3 have the special sour and hot taste and flavor of the fermented peppers, complete appearance, good brittleness and poor color; the product provided by the comparative example 4 has rich liquor fragrance, but lacks the acid fragrance flavor of fermented pepper, and has poor brittleness; comparative example 5 the taste of the fermented pepper without the bacteria was relatively flat. In contrast, the fermented pepper produced in example 3 has rich flavor components and high sensory evaluation, and is suitable for the production of fermented peppers.
TABLE 6 sensory evaluation criteria for fermented vegetables
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. This need not be, nor should it be exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
Claims (10)
1. A Pichia anomala (Pichia Galeiformis) is characterized in that the Pichia anomala is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No.25675.
2. A microbial inoculant for the fermentation of vegetables, wherein the ingredients of the microbial inoculant comprise pichia kluyveri and Lactobacillus plantarum (Lactobacillus plantarum) as defined in claim 1.
3. The microbial agent according to claim 2, wherein the lactobacillus plantarum is deposited in the China general microbiological culture Collection center (CGMCC), with the collection number of CGMCC1.3919.
4. The microbial agent according to claim 2 or 3, wherein the components of the microbial agent comprise freeze-dried powder of Pichia pastoris and freeze-dried powder of Lactobacillus plantarum.
5. The microbial agent according to claim 4, wherein the mass ratio of the Pichia pastoris freeze-dried powder to the Lactobacillus plantarum freeze-dried powder is 0.5-1.5: 1 to 3.
6. The microbial agent according to claim 4 or 5, wherein the preparation method of the pichia pastoris freeze-dried powder comprises the following steps: inoculating pichia pastoris into a liquid culture medium for culture to obtain a pichia pastoris bacterial liquid; centrifuging a pichia pastoris bacterial solution, and taking a pichia pastoris thallus; mixing the Pichia pastoris with a freeze-drying protective agent, and freeze-drying to obtain a mixture with a viable count of 1 × 10 9 ～1×10 10 CFU/g of helmet-shaped pichia pastoris freeze-dried powder;
the preparation method of the lactobacillus plantarum freeze-dried powder comprises the following steps: inoculating lactobacillus plantarum into a liquid culture medium for culture to obtain lactobacillus plantarum liquid; centrifuging the lactobacillus plantarum bacterial liquid, and taking lactobacillus plantarum thalli; mixing Lactobacillus plantarum thallus with a freeze-drying protective agent, and freeze-drying to obtain a viable count of 1×10 9 ～1×10 10 CFU/g lactobacillus plantarum freeze-dried powder.
7. A method of preparing a fermented vegetable, the method comprising: fermenting a vegetable with the microbial inoculant according to any one of claims 2 to 6 to obtain a fermented vegetable.
8. The method of preparing fermented vegetables according to claim 7, wherein the method comprises: soaking vegetables below the liquid level of a salt solution to obtain a mixture; adding the microbial agent as defined in any one of claims 2 to 6 to the mixture to obtain a fermentation system; fermenting the fermentation system at 25-30 deg.c for 1-7 days to obtain fermented vegetable.
9. The method of claim 7 or 8, wherein the method comprises: soaking vegetables below the liquid level of a salt solution to obtain a mixture; firstly, adding the pichia pastoris freeze-dried powder into the mixture, fermenting for 12-24 h at 25-30 ℃, then adding the lactobacillus plantarum freeze-dried powder into the mixture, and continuing to ferment for 1-3 d at 25-30 ℃ to obtain fermented vegetables; the mass ratio of the helmet-shaped pichia pastoris freeze-dried powder to the lactobacillus plantarum freeze-dried powder is 0.5-1.5: 1 to 3; the total mass of the pichia pastoris freeze-dried powder and the lactobacillus plantarum freeze-dried powder accounts for 0.2 to 0.4 percent of the total mass of the vegetables.
10. Use of pichia pastoris according to claim 1 or a microbial inoculant according to any one of claims 2 to 6 or a method according to any one of claims 7 to 9 for the preparation of fermented vegetables.
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