CN115960732B - Pichia glabra strain, microbial agent and application thereof - Google Patents

Pichia glabra strain, microbial agent and application thereof Download PDF

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CN115960732B
CN115960732B CN202310082003.3A CN202310082003A CN115960732B CN 115960732 B CN115960732 B CN 115960732B CN 202310082003 A CN202310082003 A CN 202310082003A CN 115960732 B CN115960732 B CN 115960732B
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pichia
glabra
freeze
lactobacillus plantarum
dried powder
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CN115960732A (en
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张炜炜
易俊洁
刘志佳
孙钰薇
李红
肖玥
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Kunming University of Science and Technology
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Abstract

The invention relates to pichia glabra strain, a microbial agent and application thereof, and belongs to the technical field of microbial fermentation. The invention provides a strain of Pichia glabra which is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No.25675; the pichia helminth can produce alcohol flavor substances with flower and fruit flavor such as ethanol, isobutanol, isoamyl alcohol, phenethyl alcohol and the like, and can destroy polysaccharide components in vegetables by producing various glycoside hydrolase, promote the release of linalool, terpenes and phenolic bonding state aroma components, produce flower flavor and sweet flavor, enrich the flavor of fermented vegetables, and in addition, the pichia helminth and the lactobacillus plantarum are subjected to 2-stage inoculation fermentation to avoid the reduction of the brittleness of the fermented vegetables caused by overgrowth of the yeast, and simultaneously, endow the fermented vegetables with the due sour flavor, thereby having great application prospect.

Description

Pichia glabra strain, microbial agent and application thereof
Technical Field
The invention relates to pichia glabra strain, a microbial agent and application thereof, and belongs to the technical field of microbial fermentation.
Background
The fermented vegetable refers to a vegetable product obtained by fermenting fresh vegetables in an anaerobic environment through a microorganism system. Various fresh vegetables including purple cabbage, chinese cabbage, carrot, beet, cucumber, celery, capsicum, green bean, kidney bean, etc. can be used for the production of fermented vegetables. The fermented vegetables are not only sources of nutrients such as probiotics, vitamins, minerals and the like, but also have various functional characteristics.
At present, the processing technology of the fermented vegetables in China mainly comprises natural fermentation, the natural fermentation has the problems of long fermentation period, excessive salt, high nitrite content and biogenic amine content, easiness in pollution by mixed bacteria and the like, the industrialized development of the 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 composed of specific strains. At present, the microbial agent for vegetable fermentation is generally prepared by separating specific strains with quick acid production, more aroma production and stable characters from naturally fermented vegetable products, performing expansion culture on the specific strains, collecting thalli of the specific strains, mixing the thalli with a freeze-drying protective agent to prepare high-concentration bacterial suspension, and finally performing vacuum freeze-drying, and has the advantages of strong activity, short fermentation period, predictable fermentation process, stable quality of the fermented vegetable products and the like.
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 glycoside-bonded aroma substances in vegetables are stored in plants in the form of glycosides, and the lactobacillus cannot utilize polysaccharide substances in the plants to make the glycoside-bonded flavor components difficult to release, the vegetable product obtained by fermenting the vegetables by using the microbial agent for vegetable fermentation prepared by adopting the single lactobacillus strain has single taste, insufficient aroma and difficult comparison with natural fermentation products.
Disclosure of Invention
In order to solve the problems, the invention provides a Pichia glabra (Pichia Galeiformis) F1705, which is characterized in that the Pichia glabra is preserved in the China general microbiological culture collection center with the preservation number of CGMCC No.25675.
The Pichia glabra F1705 is separated from a fermented radish sample produced in Wuyi mountain city of Ping-Chen City of Fujian, the strain is subjected to sequencing analysis, the 18S rDNA sequence of the strain is shown as SEQ ID NO.1, the sequence obtained by sequencing is subjected to nucleic acid sequence comparison in GeneBank, and the result shows that the strain is Pichia glabra and is named Pichia glabra F1705.
The invention also provides a microbial agent for fermenting vegetables, and the microbial agent comprises the pichia delbrueckii and lactobacillus plantarum (Lactobacillus plantarum).
In one embodiment of the invention, the lactobacillus plantarum is preserved in China general microbiological culture Collection center with the preservation number of CGMCC1.3919.
In one embodiment of the invention, the microbial agent comprises pichia glabra freeze-dried powder and lactobacillus plantarum freeze-dried powder.
In one embodiment of the invention, the mass ratio of the pichia glabra 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 glabra 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 glabra freeze-dried powder comprises the following steps: inoculating pichia glabra into a liquid culture medium for culturing to obtain pichia glabra liquid; centrifuging the Pichia glabra bacterial liquid, and taking Pichia glabra bacterial bodies; mixing pichia glabra thallus with freeze-drying protecting agent, and freeze-drying to obtain live bacteria with number of 1×10 9 ～1×10 10 CFU/g Pichia glabra freeze-dried powder.
In one embodiment of the invention, 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 bacterial liquid; centrifuging lactobacillus plantarum bacterial liquid, and taking lactobacillus plantarum bacterial bodies; 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 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 in percentage by mass.
The invention also provides a method for preparing the fermented vegetables, which comprises the following steps: the microbial agent is used for fermenting vegetables to obtain fermented vegetables.
In one embodiment of the invention, the method is: immersing vegetables below the level of the 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 d to obtain fermented vegetable.
In one embodiment of the invention, the method is: immersing vegetables below the level of the salt solution to obtain a mixture; firstly adding the pichia glabra freeze-dried powder into the mixture, fermenting for 12-24 hours at 25-30 ℃, then adding the lactobacillus plantarum freeze-dried powder into the mixture, and continuing fermenting for 1-3 d at 25-30 ℃ to obtain fermented vegetables; the mass ratio of the pichia glabra freeze-dried powder to the lactobacillus plantarum freeze-dried powder is 0.5-1.5: 1 to 3; the total mass of the pichia glabra freeze-dried powder and the lactobacillus plantarum freeze-dried powder accounts for 0.2-0.4% of the total mass of the vegetables.
In one embodiment of the invention, the mass ratio of the pichia glabra freeze-dried powder to the lactobacillus plantarum freeze-dried powder is 1:2.
in one embodiment of the present invention, the mass fraction of the common salt solution is 2 to 5%.
The invention also provides application of the pichia glabra or the microbial agent or the method in preparing fermented vegetables.
The technical scheme of the invention has the following advantages:
1. the invention provides a strain of Pichia glabra (Pichia Galeiformis), which is preserved in the China general microbiological culture Collection center (CGMCC) with the preservation number of 25675; the pichia glabra can produce alcohol flavor substances with flower and fruit flavor such as ethanol, isobutanol, isoamyl alcohol, phenethyl alcohol and the like, and can destroy polysaccharide components in vegetables by producing various glycoside hydrolase, promote the release of linalool, terpenes and phenolic bonding state aroma components, produce flower flavor and sweet flavor, enrich the flavor of fermented vegetables, so that the pichia glabra has good flavoring effect, can obviously promote the flavor of the vegetable products obtained by fermentation when used for vegetable fermentation, and has great application prospect.
2. The invention provides a microbial agent, which comprises Pichia glabra F1705 and lactobacillus plantarum (Lactobacillus plantarum), wherein the Pichia glabra can produce alcohol flavor substances with flower and fruit flavors such as ethanol, isobutanol, isoamyl alcohol and phenethyl alcohol, and the Pichia glabra can destroy polysaccharide components in vegetables by producing various glycoside hydrolase enzymes, promote release of linalool, terpenes and phenolic bonding state aroma components, produce flower flavor and sweet flavor, and can obviously promote the flavor of vegetable products obtained by fermentation of vegetables, but overgrowth of the Pichia glabra in a vegetable fermentation system can degrade pectin in the vegetables in a large amount, so that the texture of the fermented vegetables becomes soft and crisp, the quality of the products is reduced, after the lactobacillus plantarum is added into the fermentation system, the monosaccharide components can be utilized to rapidly grow to produce acid, and further modify the glycoside aroma components to form special flavor of the fermented vegetables, and the low-pH environment formed by the lactobacillus plantarum can inhibit overgrowth of the Pichia glabra, so as to prevent the degradation of pectin in the vegetables from causing the soft texture and the production of the vegetables, and the flavor of the vegetables can be greatly degraded, and the flavor of the obtained vegetable products can be greatly improved, and the flavor of the vegetable products can be further improved, and the obtained by the lactobacillus plantarum has the flavor has the advantage of the flavor of the vegetables.
Further, the microbial agent comprises pichia glabra freeze-dried powder and lactobacillus plantarum freeze-dried powder, and the mass ratio of the pichia glabra freeze-dried powder to the lactobacillus plantarum freeze-dried powder in the microbial agent is 1:2. compared with the quality, the microbial agent has better aroma enhancing and brittleness keeping effects.
3. The invention provides a method for preparing fermented vegetables, which comprises fermenting vegetables by using microbial agents comprising Pichia pastoris F1705 and lactobacillus plantarum, so as to obtain fermented vegetables, wherein the Pichia pastoris can produce alcohol flavor substances with flower and fruit flavors such as ethanol, isobutanol, isoamyl alcohol, phenethyl alcohol and the like, the Pichia pastoris can destroy polysaccharide components in the vegetables by producing various glycoside hydrolase, and release of linalool, terpenes and phenolic bonding state aroma components is promoted, flower flavor and sweet flavor are produced, the flavor of the fermented vegetables can be obviously improved by using the Pichia pastoris in vegetable fermentation, but the pectin in the vegetables can be degraded in a large amount by overgrowth in a vegetable fermentation system, so that the texture of the fermented vegetables becomes soft, the brittleness is reduced, the quality of the products is influenced, after the lactobacillus plantarum is added into the fermentation system, the monosaccharide components can be utilized to grow rapidly to produce acid, the special flavor of the fermented vegetables is further modified, the low pH environment formed by the lactobacillus plantarum can inhibit overgrowth of the Pichia pastoris, the pectin in the vegetables is prevented from being degraded, and the flavor of the vegetables is improved, and the flavor of the obtained vegetables is improved, and the flavor of the vegetables is improved.
Further, the microbial agent comprises pichia glabra freeze-dried powder and lactobacillus plantarum freeze-dried powder, and the mass ratio of the pichia glabra freeze-dried powder to the lactobacillus plantarum freeze-dried powder in the microbial agent is 1:2. the method has better aroma-enhancing and crisp-keeping effects.
Further, pichia glabra F1705 and lactobacillus plantarum are directly inoculated into fermented vegetables, the growth rate of the two strains and the growth competition relationship between the two strains are difficult to monitor and regulate, unstable quality of products is easily caused, and excessive growth of the pichia glabra can lead to degradation of pectin in the vegetables, so that vegetable crispness is reduced, and the taste of the fermented vegetables is affected. According to the method, pichia glabra yeast with a slower growth rate is firstly put into fermented vegetables, and lactobacillus plantarum with a faster growth rate is put into the fermented vegetables after fermentation for 12-24 hours. The mixed strain two-stage inoculation fermentation can make the advantages among strains complementary, keep good brittleness of vegetables and obviously improve the flavor of vegetable products obtained by fermentation.
Preservation of biological materials
Pichia glabra (Pichia Galeiformis) F1705, taxonomic name Pichia Galeiformis, was deposited in China general microbiological culture Collection center of China Committee for culture Collection of microorganisms, with the preservation number of CGMCC No.25675 and the preservation address of North Chen West Lu No.1 of the Chaoyang area of Beijing city, at the year 09 of 2022.
Drawings
Fig. 1: colony and fungus morphology map of pichia glabra (Pichia Galeiformis) F1705.
Fig. 2: and (3) detecting an ion flow chart of the gas quality of the volatile aroma components produced by pichia glabra.
Fig. 3: comparative example 5 gas detection ion flow diagram of flavor substances in the prepared fermented peppers.
Fig. 4: comparative example 4 gas detection ion flow diagram of flavor substances in the fermented peppers prepared in example 4.
Fig. 5: ion flow diagram for detecting the gas quality of flavor substances in the fermented peppers prepared in comparative example 3.
Fig. 6: ion flow diagram for detecting the gas quality of flavor substances in the fermented peppers prepared in example 3.
Fig. 7: and (5) sensory evaluation results of the fermented peppers.
Detailed Description
The following examples are provided for a better understanding of the present invention and are not limited to the preferred embodiments described herein, but are not intended to limit the scope of the invention, any product which is the same or similar to the present invention, whether in light of the present teachings or in combination with other prior art features, falls within the scope of the present invention.
The media and reagents involved in the following examples were as follows:
YPD liquid medium: yeast extract 10g, peptone 20g, glucose 20g and water 1000g.
YPD solid medium: 10g of yeast extract, 20g of peptone, 20g of glucose, 15g of agar and 1000g of water.
MRS liquid medium: 10g of peptone, 0.2g of magnesium sulfate, 2g of dipotassium hydrogen 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, 1g of tween-80 and 1000g of water.
Mineral salt medium: glucose 50g, nutrient medium Y1251 (from sigma) free of amino acids and ammonium sulfate 1.7g, ammonium sulfate 0.19444g, water 1000g.
0.85% physiological saline: sodium chloride 0.85g and water 100g.
Lyoprotectant: trehalose 1.22wt%, sodium glutamate 5.80wt%, sucrose 15.80wt%, skim milk 16.50wt%, calcium chloride 0.5wt% and water for the rest.
The detection method involved in the following examples is as follows:
the method for detecting the number of living bacteria comprises the following steps: the national standard GB 4789.35-2016 food safety national standard food microbiology detection lactobacillus detection and the method of counting by using a methylene blue staining blood cell counting plate are adopted.
The nitrite determination method comprises the following steps: determination of nitrite in GB 5009.33-2016-determination of nitrate in vegetables and fruits according to the third method, and determination of nitrite content in fermented foods by ultraviolet spectrophotometry.
The detection method of the aroma components comprises the following steps: the content of the volatile flavor substances is detected by a gas chromatograph and mass spectrometer by adopting a solid-phase microextraction method.
Example 1: pichia glabra acquisition
Taking fermented radishes from Wuyi mountain city of south Ping City of Fujian as samples, taking 0.5g samples, fully grinding the samples in a sterile mortar, adding 5mL of sterile physiological saline, and uniformly mixing to obtain sample mixed liquid; 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 24 hours to enrich the sample, thereby obtaining an enriched sample; the 0.5mL of the enriched sample was pipetted into 4.5mL of sterile physiological saline to obtain 10 -1 Diluent and then aspirate 0.5mL 10 -1 The diluted solution was dissolved in 4.5mL of physiological saline to give 10 -2 The dilution was then operated to give 10 in sequence -3 、10 -4 、10 -5 、10 -6 A dilution liquid; suction is carried out100 mu L of gradient dilution is coated on MRS solid culture medium, 10 -4 、10 -5 、10 -6 1 plate per gradient, culturing at 30 ℃ and 200rpm for 48 hours to obtain colonies; selecting bacterial colony with typical characteristics of Pichia glabra on YPD solid culture medium according to bacterial colony shape, size, edge, transparency, etc., picking bacterial colony with inoculating loop, streaking on YPD solid culture medium, culturing at 30deg.C and 200rpm for 48 hr to obtain purified single bacterial colony (bacterial colony is white, edge is irregular, see figure 1 specifically); the purified single colony is selected and inoculated into 5mL YPD liquid culture medium respectively, and is cultured for 24 hours at 30 ℃ and 200rpm to obtain bacterial liquid; numbering each strain corresponding to each bacterial liquid, performing gram staining, strain identification, physiological and biochemical experiments and genome identification analysis by referring to the steps described in textbook (microbiology) (Shen Ping, chen Xiangdong main code), and selecting a strain with typical characteristics of Pichia glabra to obtain a strain F1705;
wherein, the strain identification process is as follows:
extracting genome of F1705 from F1705 thallus with fungus genome extracting kit, and amplifying with the extracted genome of F1705 as template to obtain 18S rRNA of F1705 (16S rDNA sequence of F1705 is shown as SEQ ID NO. 1) with nucleotide sequence shown as SEQ ID NO.2 and SEQ ID NO. 3; the nucleic acid sequence of the 18S rDNA of F1705 was aligned in the Blastn program of NCBI, and the result shows that the sequence homology of the strain and Pichia glabrata is 99.78%, and the strain is identified as Pichia glabrata, and is named as Pichia glabrata (Pichia Galeiformis) F1705.
Experimental example 1: detection of flavor substances produced by pichia glabra
Pichia glabra (Pichia Galeiformis) F1705 of example 1 was inoculated into mineral salt medium to an initial OD 600 After =0.500, culture was performed at 30 ℃ and 200rpm, and during the culture period, the OD of the culture broth was periodically monitored 600 And (3) the value is changed until the growth state is stable, sampling the culture solution when culturing for 72 hours, and analyzing the volatile aroma components, wherein the analysis process is as follows:
extracting volatile aroma components in the culture solution by adopting a headspace solid-phase microextraction method to obtain a sample; 3mL of the sample was sucked into a 10mL headspace bottle, 1g of NaCl was added, immediately sealed, vortexed for 1min, then placed in a magnetic stirrer, equilibrated at 40℃for 15min at 500r/min, and 3-octanol (0.05. Mu.g/mL) was added as an internal standard (40. Mu.L) using an airtight syringe; inserting an aged 50/30 mu m DVB/CAR/PDMS extraction probe into the headspace of a sample bottle, adsorbing at 40 ℃ for 40min at 500r/min, inserting the extraction probe into a GC sample inlet, and desorbing at 250 ℃ for 5min; the volatiles were separated and detected using a DB-5MS column (30 m. Times.0.32 mm. Times.0.25 μm); helium with purity of more than 99.99% is used as carrier gas, the flow is constant at 2.0mL/min, and sample injection is not carried out in a split way; the initial temperature was maintained at 45 ℃ for 5 minutes, then increased to 85 ℃ at a rate of 4 ℃/min, and 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 transmission line temperatures of the mass spectrum are 230 ℃ and 280 ℃, respectively; the solvent delay time was 1.0min. The volatile content of each sample was measured 4 times repeatedly, and the measurement results are shown in fig. 2.
Volatile aroma was obtained by comparing database of NIST 2014 library with normal alkane (C 5 -C 25 ) Preliminary identification of Retention Index (RI) as experimentally determined with literature data for external reference; mu.L of 3-octanol (5X 10) was added to each sample -5 μg/L) was used as an internal standard to calculate the relative amounts of each volatile aroma component, and the calculated results are shown in Table 1.
From fig. 2 and table 1, it can be seen that pichia pastoris itself can produce alcohol flavor substances having flower and fruit flavor such as ethanol, isobutanol, isoamyl alcohol, phenethyl alcohol, etc. The flavor substances have rich flower and fruit fragrance, and are important flavor substances in fermented foods.
TABLE 1 Pichia glabra itself may produce flavoring substances
Chinese name CAS number RI Content (μg/Kg) Odor description
Ethanol 64-17-5 431 108.55±39.56 Alcohol taste
Isobutanol 78-83-1 630 74.50±10.50 Grape wine flavor
Isoamyl alcohol 123-51-3 738 436.31±23.54 Oily, fruity, banana flavor
2-ethylhexanol 104-76-7 1033 10.61±2.54 Orange, flower and sweet
Phenethyl alcohol 60-12-8 1117 114.41±19.70 Rose fragrance
Alpha-terpineol 98-55-5 1199 1.73±0.26 Citrus, woody and flower fragrance
Geraniol 106-24-1 1254 4.54±0.88 Sweet, fruity, rose, citrus
Example 2: microbial agent for fermenting vegetables and preparation method thereof
The embodiment provides a microbial agent for fermenting vegetables, which consists of pichia helmet (Pichia Fermentans) freeze-dried powder and lactobacillus plantarum (Lactobacillus plantarum) freeze-dried powder; in the microbial agent, the mass ratio of pichia helmet-shaped freeze-dried powder to lactobacillus plantarum freeze-dried powder is 1:2.
the preparation method of the pichia glabra freeze-dried powder comprises the following steps: inoculating the bacterial liquid of Pichia glabra (Pichia Galeiformis) F1705 in the embodiment 1 on YPD solid culture medium in a streaking way, inverting the bacterial liquid in a constant temperature incubator, and culturing the bacterial liquid for 3 days at the temperature of 30 ℃ to obtain single bacterial colony; picking single colony, inoculating in 4.5mL YPD liquid culture medium, and performing activation culture at 30 ℃ and 200rpm for 3 days to obtain activated culture bacterial liquid; diluting the activated culture broth with YPD liquid medium to a total colony count of 1.0X10 8 CFU/mL, obtaining a diluent; inoculating the diluted solution into YPD liquid culture medium at 3% (v/v) and performing amplification culture at 30deg.C and 200rpm for 5 days to obtain amplified culture bacterial solutionThe method comprises the steps of carrying out a first treatment on the surface of the Loading the bacterial liquid obtained by the expansion culture into a sterilized centrifuge tube, centrifuging at 4000rpm/min for 20min, collecting bacterial cells, re-suspending bacterial cells with 0.85% sterile physiological saline, centrifuging at 4000rpm/min for 20min, washing, repeating for three times, and collecting bacterial cells again; adding a freeze-drying protective agent with the volume 1.5 times of that of the bacteria to obtain bacterial suspension; pre-freezing the bacterial suspension with liquid nitrogen, transferring into vacuum freeze drying agent, and freeze drying at-60deg.C for 2 days to obtain Pichia helmet-like lyophilized powder (viable count of 1×10) 9 CFU/g)。
The preparation method of the lactobacillus plantarum freeze-dried powder comprises the following steps: inoculating lactobacillus plantarum CGMCC1.3919 (purchased from China general microbiological culture Collection center) bacterial liquid streak on MRS solid culture medium, inverting the culture medium in an anaerobic incubator, and culturing for 2 days at 37 ℃ to obtain single colony; picking single colony, inoculating in 4.5mL MRS liquid culture medium, placing in an anaerobic incubator, and culturing at 37deg.C for 1 day to obtain activated culture bacterial liquid; diluting the bacterial liquid of the activation culture to the total colony count of 1.0X10 by using MRS liquid culture medium 8 CFU/mL, obtaining a diluent; inoculating the diluted solution into MRS liquid culture medium with an inoculum size of 2% (v/v), placing into an anaerobic incubator, and performing expansion culture at 37 ℃ for 2 days to obtain expanded culture bacterial solution; loading the bacterial liquid subjected to the expansion culture into a sterilized centrifuge tube, centrifuging at 4000rpm/min for 20min, collecting bacterial cells, re-suspending bacterial cells with 0.85% sterile physiological saline, centrifuging at 4000rpm/min for 20min, washing, repeating for three times, and collecting bacterial cells again; adding a freeze-drying protective agent with the volume 1.5 times of that of the bacteria to obtain bacterial suspension; pre-freezing the bacterial suspension with liquid nitrogen, transferring into vacuum freeze drying agent, and freeze drying at-60deg.C for 2 days to obtain Lactobacillus plantarum lyophilized powder (viable count is 1×10) 9 CFU/g)。
Example 3: preparation of fermented Capsici fructus
The embodiment provides a method for preparing fermented peppers, which comprises the following steps:
step one: cleaning fresh capsicum, draining, and sterilizing with ultraviolet irradiation for 2 hr to obtain sterilized capsicum; placing sterilized pepper into a fermentation container, adding 4% (w/v) salt solution, and soaking pepper below the salt solution to obtain a mixture; adding 1.5g of pichia glabra freeze-dried powder into each 1kg of chilli, and fully dissolving the pichia glabra freeze-dried powder of the microbial agent in the embodiment 2 into the mixture to obtain a fermentation system A;
step two: sealing a fermentation container filled with a fermentation system A, and then placing the fermentation container in a constant temperature incubator to ferment at 30 ℃; after 24h fermentation, adding 3g of lactobacillus plantarum freeze-dried powder into each 1kg of chilli, and fully dissolving the lactobacillus plantarum freeze-dried powder of the microbial agent in the embodiment 2 in the fermentation system A to obtain a fermentation system B; sealing the fermentation container filled with the fermentation system B, and then placing the fermentation container in a constant temperature incubator, and continuously fermenting for 48 hours at the temperature of 30 ℃ to obtain the fermented chilli.
Example 4: preparation of fermented chayote
The embodiment provides a method for preparing fermented chayote, which comprises the following steps:
step one: cleaning fresh chayote, draining, cutting into slices with the thickness of 2mm, and sterilizing for 2h by ultraviolet irradiation to obtain sterilized chayote; placing the sterilized chayote in a fermentation container, adding 2% (w/v) salt solution, and soaking the chayote below the salt solution to obtain a mixture; adding 1g of pichia glabra freeze-dried powder per 1kg of chayote, and fully dissolving the pichia glabra freeze-dried powder of the microbial agent in the embodiment 2 in the mixture to obtain a fermentation system A;
step two: sealing a fermentation container filled with a fermentation system A, and then placing the fermentation container in a constant temperature incubator to ferment at 30 ℃; after fermentation for 12 hours, adding 2g of lactobacillus plantarum freeze-dried powder into every 1kg of chayote, and fully dissolving the lactobacillus plantarum freeze-dried powder of the microbial agent in the embodiment 2 in a fermentation system A to obtain a fermentation system B; sealing the fermentation container filled with the fermentation system B, and then placing the fermentation container in a constant temperature incubator, and continuing to ferment for 48 hours at the temperature of 30 ℃ to obtain the fermented chayote.
Example 5: preparation of fermented radish
The present embodiment provides a method for preparing fermented radish, comprising the steps of:
step one: cleaning fresh radish skin, draining, cutting into slices with the thickness of 3mm, and sterilizing for 2 hours by ultraviolet irradiation to obtain sterilized radish skin; placing sterilized radish skin in a fermentation container, adding 3% (w/v) salt solution, and soaking radish skin part below the salt solution to obtain a mixture; adding 2g of pichia glabra freeze-dried powder per 1kg of radish skin, and fully dissolving the pichia glabra freeze-dried powder of the microbial agent in the embodiment 2 in the mixture to obtain a fermentation system A;
step two: sealing a fermentation container filled with a fermentation system A, and then placing the fermentation container in a constant temperature incubator to ferment at 30 ℃; after 24h fermentation, adding 4g of lactobacillus plantarum freeze-dried powder into each 1kg of radish skin, and fully dissolving the lactobacillus plantarum freeze-dried powder of the microbial agent in the embodiment 2 in a fermentation system A to obtain a fermentation system B; sealing the fermentation container filled with the fermentation system B, and then 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, which consists of pichia glabra (Pichia Fermentans) freeze-dried powder.
The preparation method of the pichia glabra freeze-dried powder comprises the following steps: inoculating the bacterial liquid of Pichia glabra (Pichia Galeiformis) F1705 in the embodiment 1 on YPD solid culture medium in a streaking way, inverting the bacterial liquid in a constant temperature incubator, and culturing the bacterial liquid for 3 days at the temperature of 30 ℃ to obtain single bacterial colony; picking single colony, inoculating in 4.5mL YPD liquid culture medium, and performing activation culture at 30 ℃ and 200rpm for 3 days to obtain activated culture bacterial liquid; diluting the activated culture broth with YPD liquid medium to a total colony count of 1.0X10 8 CFU/mL, obtaining a diluent; inoculating the diluted solution into YPD liquid culture medium with 3% (v/v) inoculum size, and performing expansion culture at 30deg.C and 200rpm for 5 days to obtain expanded culture bacterial solution; loading the bacterial liquid obtained by expansion culture into sterilized centrifuge tube, centrifuging at 4000rpm/min for 20min, collecting bacterial cells, and adding 0.85% sterile physiological salineRe-suspending the thalli, centrifuging at 4000rpm/min for 20min, washing, repeating for three times, and collecting the thalli again; adding a freeze-drying protective agent with the volume 1.5 times of that of the bacteria to obtain bacterial suspension; pre-freezing the bacterial suspension with liquid nitrogen, transferring into vacuum freeze drying agent, and freeze drying at-60deg.C for 2 days to obtain Pichia helmet-like lyophilized powder (viable count of 1×10) 9 CFU/g)。
Comparative example 2: microbial agent for fermenting vegetables and preparation method thereof
The comparative example provides a microbial agent for fermenting vegetables, which consists of lactobacillus plantarum (Lactobacillus plantarum) freeze-dried powder.
The preparation method of the lactobacillus plantarum freeze-dried powder comprises the following steps: inoculating lactobacillus plantarum CGMCC1.3919 (purchased from China general microbiological culture Collection center) bacterial liquid streak on MRS solid culture medium, inverting the culture medium in an anaerobic incubator, and culturing for 2 days at 37 ℃ to obtain single colony; picking single colony, inoculating in 4.5mL MRS liquid culture medium, placing in an anaerobic incubator, and culturing at 37deg.C for 1 day to obtain activated culture bacterial liquid; diluting the bacterial liquid of the activation culture to the total colony count of 1.0X10 by using MRS liquid culture medium 8 CFU/mL, obtaining a diluent; inoculating the diluted solution into MRS liquid culture medium with an inoculum size of 2% (v/v), placing into an anaerobic incubator, and performing expansion culture at 37 ℃ for 2 days to obtain expanded culture bacterial solution; loading the bacterial liquid subjected to the expansion culture into a sterilized centrifuge tube, centrifuging at 4000rpm/min for 20min, collecting bacterial cells, re-suspending bacterial cells with 0.85% sterile physiological saline, centrifuging at 4000rpm/min for 20min, washing, repeating for three times, and collecting bacterial cells again; adding a freeze-drying protective agent with the volume 1.5 times of that of the bacteria to obtain bacterial suspension; pre-freezing the bacterial suspension with liquid nitrogen, transferring into vacuum freeze drying agent, and freeze drying at-60deg.C for 2 days to obtain Lactobacillus plantarum lyophilized powder (viable count is 1×10) 9 CFU/g)。
Comparative example 3: preparation of fermented Capsici fructus
The present comparative example provides a method for preparing fermented capsicum, comprising the steps of:
step one: cleaning fresh capsicum, draining, and sterilizing with ultraviolet irradiation for 2 hr to obtain sterilized capsicum; placing sterilized pepper into a fermentation container, adding 4% (w/v) salt solution, and soaking pepper below the salt solution to obtain a mixture; adding 3g of lactobacillus plantarum freeze-dried powder into each 1kg of chilli, and fully dissolving the lactobacillus plantarum freeze-dried powder of the microbial agent in the comparative example 2 into the mixture to obtain a fermentation system;
step two: sealing the fermentation container filled with the fermentation system, and placing the fermentation container in a constant temperature incubator to ferment for 72 hours at 30 ℃ to obtain the fermented chilli.
Comparative example 4: preparation of fermented Capsici fructus
The present comparative example provides a method for preparing fermented capsicum, comprising the steps of:
step one: cleaning fresh capsicum, draining, and sterilizing with ultraviolet irradiation for 2 hr to obtain sterilized capsicum; placing sterilized pepper into a fermentation container, adding 4% (w/v) salt solution, and soaking pepper below the salt solution to obtain a mixture; 1.5g of pichia glabra freeze-dried powder is added into each 1kg of chilli, and the pichia glabra freeze-dried powder of the microbial agent in the comparative example 1 is fully dissolved in the mixture to obtain a fermentation system;
step two: sealing the fermentation container filled with the fermentation system, and placing the fermentation container in a constant temperature incubator to ferment for 72 hours at 30 ℃ to obtain the fermented chilli.
Comparative example 5: preparation of fermented Capsici fructus
The present comparative example provides a method for preparing fermented capsicum, comprising the steps of:
step one: cleaning fresh capsicum, draining, and sterilizing with ultraviolet irradiation for 2 hr to obtain sterilized capsicum; placing sterilized pepper into a fermentation container, adding 4% (w/v) salt solution, and soaking pepper below the salt solution to obtain a fermentation system;
step two: sealing the fermentation container filled with the fermentation system, and placing the fermentation container in a constant temperature incubator to ferment for 72 hours at 30 ℃ to obtain the fermented chilli.
Experimental example 1: performance detection of fermented vegetables
1. Nitrite content
The nitrite contents of the fermented vegetables prepared in examples 3 to 5 and the fermented vegetables prepared in comparative examples 3 to 5 were examined, and the examination results are shown in Table 2. As can be seen from Table 2, the nitrite content in the fermented vegetables was less than the 20mg/kg limit.
TABLE 2 nitrite content of fermented vegetables
Sample of Content (mg/kg)
Example 3 5.30±0.84
Example 4 8.56±1.55
Example 5 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 of
The change in pH can affect the metabolites and metabolic pathways of the strain, and thus the flavor and quality of the fermented vegetables. The pH values of the fermented vegetables prepared in examples 3 to 5 and the fermented vegetables prepared in comparative examples 3 to 5 were measured, and the measurement results are shown in Table 3. As is clear from Table 3, the pH of vegetables fermented with lactic acid bacteria was lower, while the pH of the samples inoculated with yeast and not inoculated with the strain was less changed.
TABLE 3 pH of fermented vegetables
3. Microorganism index
The microbial indicators of the fermented vegetables prepared in examples 3 to 5 were examined at 15 days of the storage, and the detection results 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 standard when stored for 15 days, and have a long shelf life.
TABLE 4 microbial indicator of fermented vegetables
Detecting items National standard Example 3 Example 4 Example 5
Colony count, CFU/g ≤100 25 32 44
Coli, MPN/100g ≤30 8 10 14
Mould, CFU/g ≤20 5 9 10
Pathogenic bacteria Cannot be detected Without any means for Without any means for Without any means for
4. Flavor component detection
The flavor components of the fermented peppers prepared in example 3 and those prepared in comparative examples 3 to 5 were examined, and the examination results are shown in fig. 3 to 6 and table 5. As can be seen from fig. 3 to 6 and table 5, lactic acid bacteria inoculated fermentation mainly produced lactic acid, created a lower pH environment, which facilitates the preservation of capsicum, but contributed less to the flavor components of capsicum, compared to non-inoculated fermentation of capsicum. The yeast inoculation fermentation chilli has great change on the flavor components, and promotes the release of bonding state flavor substances such as linalool, terpenes, phenols, phenyl derivatives and the like in the chilli. The flavor substances of the capsicum fermented by mixing the yeast and the lactobacillus and two-stage inoculation are rich, and the release of the bonded wind substances in the capsicum can be promoted.
TABLE 5 detection of fermented capsicum wind matter
5. Sensory evaluation
The fermented peppers prepared in example 3 and the fermented peppers prepared in comparative examples 3 to 5 were taken out, washed with clear water, and observed for color and morphology of the fermented peppers, while a sensory panel consisting of 25 persons was established for sensory evaluation of the fermented peppers, and 3 samples were set in parallel. The evaluation indexes are taste, appearance state, smell and color of the fermented peppers, 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 the sensory evaluation score of example 3 was 98 minutes at the highest in each example, and that the taste, smell, appearance shape and color index were all good. In addition, the fermented vegetable products prepared by the microbial agent provided in the comparative example 3 have the special sour and spicy taste and flavor of the fermented peppers, complete appearance, good crispness and poor color; the product provided in comparative example 4 has rich wine-like flavor, but lacks the acid flavor of the fermented peppers, and has poor crispness; comparative example 5 the taste of the capsicum without germ fermentation was lighter. In contrast, the fermented capsicum produced by the method of the example 3 has rich flavor components and higher sensory evaluation, and is suitable for the production of fermented capsicum.
TABLE 6 sensory evaluation criteria for fermented vegetables
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope of the invention.

Claims (10)

1. Pichia glabra strainPichia Galeiformis) The method is characterized in that the pichia glabra is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.25675.
2. A microbial agent for fermenting vegetables, wherein the microbial agent comprises the pichia glabra and lactobacillus plantarum (Lactobacillus plantarum) of claim 1.
3. The microbial agent according to claim 2, wherein the lactobacillus plantarum is deposited with the China general microbiological culture Collection center with a accession number of CGMCC1.3919.
4. The microbial agent of claim 2 or 3, wherein the microbial agent comprises pichia glabra freeze-dried powder and lactobacillus plantarum freeze-dried powder.
5. The microbial agent according to claim 4, wherein the mass ratio of the pichia glabra freeze-dried powder to the lactobacillus plantarum freeze-dried powder is 0.5-1.5: 1 to 3.
6. The microbial agent of claim 4 or 5, wherein the preparation method of the pichia glabra freeze-dried powder comprises the following steps: inoculating pichia glabra into a liquid culture medium for culturing to obtain pichia glabra liquid; centrifuging Pichia glabra strain liquid, and collecting Pichia glabra strainRed yeast cells; mixing pichia glabra thallus with freeze-drying protecting agent, and freeze-drying to obtain live bacteria with number of 1×10 9 ～1×10 10 CFU/g Pichia glabra 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 bacterial liquid; centrifuging lactobacillus plantarum bacterial liquid, and taking lactobacillus plantarum bacterial bodies; 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.
7. A method of preparing a fermented vegetable, the method comprising: fermenting vegetables with the microbial agent according to any one of claims 2 to 6 to obtain fermented vegetables.
8. A method of preparing a fermented vegetable according to claim 7, wherein the method is: immersing vegetables below the level of the salt solution to obtain a mixture; adding the microbial agent according to any one of claims 2 to 6 into the mixture to obtain a fermentation system; fermenting the fermentation system at 25-30 deg.c for 1-7 d to obtain fermented vegetable.
9. A method of preparing a fermented vegetable according to claim 7 or 8, wherein the method is: immersing vegetables below the level of the salt solution to obtain a mixture; firstly adding the pichia glabra freeze-dried powder into the mixture, fermenting for 12-24 hours at 25-30 ℃, then adding the lactobacillus plantarum freeze-dried powder into the mixture, and continuing fermenting for 1-3 d at 25-30 ℃ to obtain fermented vegetables; the mass ratio of the pichia glabra freeze-dried powder to the lactobacillus plantarum freeze-dried powder is 0.5-1.5: 1 to 3; the total mass of the pichia glabra freeze-dried powder and the lactobacillus plantarum freeze-dried powder accounts for 0.2-0.4% of the total mass of the vegetables.
10. Use of pichia glabra of claim 1 or a microbial agent of any one of claims 2 to 6 or a method of any one of claims 7 to 9 in the preparation of a fermented vegetable.
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