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CN111304108B - Leuconostoc lactis and application thereof - Google Patents

Leuconostoc lactis and application thereof Download PDF

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CN111304108B
CN111304108B CN201910645660.8A CN201910645660A CN111304108B CN 111304108 B CN111304108 B CN 111304108B CN 201910645660 A CN201910645660 A CN 201910645660A CN 111304108 B CN111304108 B CN 111304108B
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leuconostoc lactis
vegetable
fermented
vegetables
fermentation
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CN111304108A (en
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何艳平
卢伟
赵楠
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Sichuan Laotanzi Food Co ltd
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Abstract

The invention discloses leuconostoc lactis and application thereof. The Leuconostoc lactis is preserved in China Center for Type Culture Collection (CCTCC) M2019444. The leuconostoc lactis CCTCC M2019444 has acid resistance and salt resistance, can well grow in the environment of pH4.0 and 6 percent NaCl, and can produce n-nonanol at high yield; the strain has good growth characteristics in bittern and vegetable juice; the strain has good acid production property in bittern and vegetable juice, and the pH value after 18h fermentation is 5.58; the detection of the n-nonanol content and the determination of the volatile flavor compound types in the fermented vegetables and the fermented vegetable juice beverage prepared by the method show that the yield of the n-nonanol in the leuconostoc lactis is improved by 60.49-61.53%, the aroma quality of the product is improved, the sensory quality of the product is improved, and the method has a wide application prospect in the field of fermented vegetable products.

Description

Leuconostoc lactis and application thereof
Technical Field
The invention relates to the technical field of microorganisms, in particular to leuconostoc lactis and application thereof.
Background
The fermented food is a kind of food which is processed and manufactured by people by utilizing beneficial microorganisms, and has unique flavor, such as yoghurt, cheese, fermented glutinous rice, pickle, soy sauce, table vinegar, fermented soya beans, yellow rice wine, beer, wine and the like. Lactic acid bacteria are gram positive bacteria which can metabolize carbohydrates into lactic acid and have no spore structure, most of the lactic acid bacteria are beneficial bacteria, and because of the special metabolic characteristics, different strains of the genus lactic acid bacteria are widely used for food processing, and the contribution to the fermented food processing industry in China is not small.
While n-Nonanol (1-Nonols, C 9 H 20 O), one of the important flavor compounds in lactic acid fermented foods, generally imparts a rich floral, fruity, green, fresh greasy flavor to the food. Although only a trace amount of n-nonanol is contained in the fermented food, it can significantly improve and enrich the flavor quality of the fermented food, and when its concentration reaches 0.9 μg/kg, the characteristic flavor of the fermented food can be exhibited in flavor sense, so that the flavor of the fermented food is more gentle and rich. Normally, the yield of n-nonanol is lower in the food fermentation process, and in the production, the flavor of the fermented food can be improved by exogenously adding or increasing endogenous production for the purpose of flavoring.
The improvement of the n-nonanol content realized by the exogenous addition method often causes pollution due to impurities in exogenous additives, so that the safety of products is poor, the quality is unstable, the synthesis and extraction cost of the n-nonanol is high, and the natural flavor and sensory quality are difficult to be presented, so that the large-scale production and application of the exogenous addition type n-nonanol in the aspect of improving the flavor of fermented foods are limited. The extraction of n-nonanol from natural products has the disadvantage of expensive raw materials and high production costs, thus limiting the large-scale production applications.
Except for the addition of exogenous sources, endogenous production is also a mode of production of n-nonanol. The endogenous production is that the n-nonanol is directly synthesized by a series of biochemical reactions in the fermentation process by microorganisms, and the product is naturally released into the environment of fermented foods, so that the separation and purification steps are omitted, the method has the advantages of multiple microorganism types, quick growth, low raw materials, biodegradable produced substances, mellow and soft flavor and high acceptance, and is more in line with the consumption trend of the product which is not added in the nature of Chong, thus becoming a hot spot in the development field of the recent related technology.
In the process of food fermentation, the variety of lactic acid bacteria capable of producing n-nonanol is many, but at present, the variety of lactic acid bacteria capable of achieving the desired yield effect and the product flavoring effect by fermentation is not yet excavated.
Disclosure of Invention
The first object of the invention is to provide the leuconostoc lactis which is preserved in China Center for Type Culture Collection (CCTCC) M2019444. The leuconostoc lactis has higher capability of producing n-nonanol, and has larger industrial application value and application prospect.
The second object of the present invention is to provide a biologically pure culture of Leuconostoc lactis as described above.
The third object of the present invention is to provide a working starter comprising the above-mentioned Leuconostoc lactis.
A fourth object of the present invention is to provide a method for preparing the working starter.
A fifth object of the present invention is to provide a fermented food product comprising the above-mentioned leuconostoc lactis. The fermented food is fermented vegetable or fermented vegetable juice beverage.
A sixth object of the present invention is to provide a method for producing the above fermented vegetable.
The seventh object of the present invention is to provide a method for preparing the above fermented vegetable juice beverage.
The eighth object of the invention is to provide an application of the leuconostoc lactis in preparing a food fermentation inoculant or a fermented food.
The ninth object of the invention is to provide an application of the leuconostoc lactis in preparation of n-nonanol.
The tenth object of the present invention is to provide an application of the leuconostoc lactis to imparting any one or more of floral, fruit, green grass and fresh oil.
In order to achieve the above object of the present invention, the following technical solutions are adopted:
firstly, providing the Leuconostoc lactis which is preserved in the China Center for Type Culture Collection (CCTCC) M2019444. Leuconostoc lactis CCTCC M2019444 has acid resistance and salt resistance, and can well grow in the environment of pH4.0 and 6% NaCl; the strain has good growth characteristics in bittern and vegetable juice; the strain has good acid-producing property in bittern and vegetable juice, and the pH value is 5.58 after 18h fermentation.
Next, a biologically pure culture of Leuconostoc lactis as described above is provided. The biologically pure culture refers to the offspring obtained by culturing and propagating a cell or a group of the same cells.
The working starter is provided again with the leuconostoc lactis. The working starter is a starter obtained by culturing a mother starter or a seed starter in an expanded manner and used for the actual production of a direct production product. Preferably, the viable count in the working ferment is 10 9 CFU/mL。
And provides a preparation method of the working fermentation agent, which comprises the following steps:
s1, providing sterile skim milk;
weighing skim milk powder, glucose and glycerol with certain mass, adding water for dissolution, sterilizing and cooling to obtain sterile skim milk;
s2, activating the leuconostoc lactis strain;
the specific steps are that a ring of the leuconostoc lactis strains stored by a freeze drying tube dissolved by sterile water is taken by an inoculating ring, streaked on an MRS agar culture medium plate, and cultured in an incubator until single colony grows out, thus obtaining a plate activated strain; the MRS agar culture medium is a lactic acid bacteria culture medium, is not necessarily limited to the MRS agar culture medium, and can be used as all culture mediums capable of culturing strains.
S3, preparing a strain culture solution, adding sterile skim milk during centrifugal precipitation, and freeze-drying;
inoculating the plate activated strain obtained in the loop taking step into a triangular flask filled with broth liquid culture medium, and placing the triangular flask in an incubator for constant-temperature culture to obtain culture solution; and (3) centrifuging the obtained culture solution at a controlled rotating speed, cleaning a sediment obtained by centrifugation by using a buffer solution, adding a sterile skim milk solution into the cleaned sediment, carrying out vortex vibration to resuspension thalli, introducing the strain culture solution into a glass ampoule bottle under a sterile condition, putting the bottle stopper into a freezer for quick freezing, putting the glass ampoule bottle into a tray for containing, and putting the glass ampoule bottle into a freeze dryer for freeze drying. Thus obtaining the working starter containing leuconostoc lactis CCTCC M2019444. The buffer solution is preferably a sterile phosphate buffer solution.
And provides a fermented food comprising the above Leuconostoc lactis. The fermented food is a kind of food processed and manufactured by people by utilizing beneficial microorganisms, and has unique flavor, such as yoghurt, cheese, fermented glutinous rice, pickle, soy sauce, table vinegar, fermented soya beans, yellow rice wine, beer, wine and the like.
The fermented food is preferably a fermented vegetable or a fermented vegetable juice beverage.
The preparation method of the fermented vegetable comprises the following steps:
s1 vegetable pretreatment
Removing impurities and inedible parts of vegetables, cutting the vegetables into blocks, cleaning for later use, and sterilizing the vegetables by using a sterilizing liquid; washing with clear water, and draining for later use; the disinfectant is preferably a disinfectant which can form hypochlorous acid or contains hypochlorous acid;
s2 preparing brine
Adding proper amount of flavoring into purified water, adding proper amount of salt and sugar according to water volume, heating, boiling, cooling, and cooling the boiled brine to room temperature to obtain brine; the flavoring is preferably star anise and pricklyash peel; the sugar is preferably glucose;
s3, preparing vegetable bittern water mixture
Adding the vegetables treated in the step S1 into the bittern water obtained in the step S2 to obtain a vegetable salt bittern mixture;
s4 fermentation culture
And (3) inoculating the working starter and the conventional starter containing the leuconostoc lactis into the vegetable bittern water mixture obtained in the step (S3) according to a certain proportion, and controlling the temperature to ferment to obtain the fermented vegetable containing the leuconostoc lactis. The working starter is the working starter containing leuconostoc lactis. The conventional starter is preferably any one or the combination of two of conventional lactobacillus plantarum and leuconostoc mesenteroides.
The preparation method of the fermented vegetable juice beverage comprises the following steps:
s1 vegetable pretreatment
Removing impurities and inedible parts of vegetables, cutting the vegetables into blocks, cleaning for later use, and sterilizing the vegetables by using a sterilizing liquid; washing with clear water, and draining for later use; the disinfectant is preferably a disinfectant which can form hypochlorous acid or contains hypochlorous acid;
s2 preparation of vegetable slurry
Taking a proper amount of vegetables and sugar, putting the vegetables and sugar into purified water, and putting the purified water into a beating machine for beating to obtain vegetable slurry; the sugar is preferably glucose; purified water is used to eliminate the influence of miscellaneous bacteria;
s3 preparation of fermentation base material
The working starter and the conventional starter containing the leuconostoc lactis are inoculated into the vegetable slurry obtained after the treatment of the step S2 according to a certain proportion, the temperature is controlled for fermentation, and the clear liquid obtained by filtration is refrigerated to obtain the fermentation base material containing the leuconostoc lactis; the conventional starter is preferably any one or the combination of two of conventional lactobacillus plantarum and leuconostoc mesenteroides;
s4 preparation of fermented vegetable juice
Adding purified water into sugar with certain mass, stirring at high speed for dissolving, sterilizing, and cooling to room temperature; adding a certain amount of fermentation base material into the solution, uniformly stirring, and regulating the acidity value by using a proper amount of acid; the acid is preferably citric acid; the sugar is preferably sucrose;
S5 homogenizing and sterilizing
Homogenizing the solution treated in the step S4, sterilizing, and cooling to obtain the fermented vegetable juice beverage containing the leuconostoc lactis.
And provides the application of the leuconostoc lactis in preparing a food fermentation inoculant or fermented food. The food fermenting microbial agent is a living microbial fermentation preparation prepared from beneficial microorganisms.
And provides the application of the leuconostoc lactis in preparing n-nonanol. The molecular formula of the n-nonanol is C 9 H 20 O. Colorless to yellow oily liquid. Boiling point 213-215 deg.C, dissolving in ethanol and oil, and acid value<1.0. Has strong fragrance of sweet and green rose wax and fruit wax. There is some orange-like, sweet orange smell. CAS number 143-08-8. The English name is 1-Nonols.
And provides the application of the leuconostoc lactis in imparting any one of floral, fruit, grass and fresh oil fragrance. Preferably, one or more of the above flavors are imparted to the fermented food product. The flower fragrance is preferably rose fragrance. The flavoring application of Leuconostoc lactate is based on the flavoring of n-nonanol for the harmonization preparation of flavor.
The beneficial effects of the invention are as follows:
the leuconostoc lactis CCTCC M2019444 has acid resistance and salt resistance, can well grow in the environment of pH4.0 and 6 percent NaCl, and can produce n-nonanol at high yield; the strain has good growth characteristics in bittern and vegetable juice; the strain has good acid production property in bittern and vegetable juice, and the pH value after 18h fermentation is 5.58; the detection of the n-nonanol content and the determination of the volatile flavor compound types in the fermented vegetables and the fermented vegetable juice beverage prepared by the method show that the yield of the n-nonanol in the leuconostoc lactis is improved by 60.49-61.53%, the fragrance quality of the product is improved, the sensory quality of the product is improved, and the method has a wide application prospect in the field of fermented vegetable products.
Preservation information
The invention provides a leuconostoc lactis (Leuconostoc lactis) strain (internal number: L7ZSAAS 01), the biological name of which is: leuconostoc lactis, deposited at China center for type culture Collection, with a deposit address: chinese, wuhan, university of Wuhan, post code: 430072, the strain has a deposit number of: cctccc M2019444.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram of a phylogenetic tree of Leuconostoc lactis CCTCC M2019444 according to example 2 of the present invention;
FIG. 2 is a cell morphology of Leuconostoc lactate CCTCC M2019444 (x 1600);
FIG. 3 is a graph showing the growth of Leuconostoc lactate CCTCC M2019444;
FIG. 4 is a graph of growth temperature of Leuconostoc lactate CCTCC M2019444;
FIG. 5 is a GC-MS total ion flow diagram of the fermented vegetable of Leuconostoc lactis CCTCC M2019444.
Detailed Description
The present invention will be described in further detail with reference to specific embodiments thereof in order to enable those skilled in the art to better understand the technical aspects of the invention.
The leuconostoc lactis and the application thereof according to the embodiment of the present invention are specifically described below.
The invention firstly screens out a lactobacillus L7ZSAAS01 from naturally fermented fruit and vegetable products, and utilizes the microbiological characteristics such as shape characteristics, culture characteristics, physiological and biochemical characteristics and the like and the genetic characteristics 16s rDNA thereof to identify the lactobacillus L7ZSAAS01 as Leuconostoc lacticum (Leuconostoc lactis), and the strain is preserved in China Center for Type Culture Collection (CCTCC) in the period of 6 and 10 of 2019, and the preservation number is CCTCC M2019444.
Biologically pure cultures of Leuconostoc lactis as described above. The biologically pure culture refers to the offspring obtained by culturing and propagating a cell or a group of the same cells.
A working starter comprising the above-mentioned Leuconostoc lactis. The working starter is a starter obtained by performing an expansion culture of a mother starter or a seed starter, and is used for actual production of a direct production product. Preferably, the viable count in the working ferment is 10 9 CFU/mL。
And provides a preparation method of the working fermentation agent, which comprises the following steps:
s1, providing sterile skim milk;
the method comprises the following specific steps: weighing 10-12% (m/v) of skim milk powder, 2-4% (m/v) of glucose and 1-2% of glycerol, adding purified water to fully dissolve the skim milk powder, sterilizing the skim milk powder at 95 ℃ for 20min, and cooling the skim milk powder to 37-40 ℃ to obtain the sterile skim milk.
S2, activating the leuconostoc lactis strain;
the method comprises the following specific steps: the culturing process includes inoculating Leuconostoc lactis CCTCC M2019444 strain with inoculating loop, freeze drying, and culturing in MRS agar culture medium for 24-48 hr at 37 deg.c to form single colony. The MRS agar culture medium is a lactic acid bacteria culture medium, is not necessarily limited to the MRS agar culture medium, and can be used as all culture mediums capable of culturing strains.
S3, preparing a strain culture solution, adding sterile skim milk during centrifugal precipitation, and freeze-drying;
the method comprises the following specific steps: inoculating the plate activated strain obtained in the step S2 into a 250mL triangular flask filled with 50mL MRS culture medium, and culturing at a constant temperature in an incubator at 37 ℃ for 12-18 hours to obtain a culture solution; centrifuging the culture solution at 4000-6000 r/min for 20-30 min, washing the precipitate with sterile phosphate buffer solution (50 mM, pH 6.8) for 2-3 times, adding 50mL sterile skim milk solution into the washed precipitate, vortex shaking at 4000-6000 rpm to resuspension thallus, introducing the strain culture solution into glass ampoule bottle under aseptic condition, covering bottle stopper, quick freezing in-30deg.C refrigerator, packaging glass ampoule bottle with tray, and freeze drying in freeze dryer.
And provides a fermented food comprising the above Leuconostoc lactis. The fermented food can be cereal fermented product, bean fermented product, milk fermented product, and vegetable fermented product. Preferably vegetable fermented product, and vegetable comprises various Chinese cabbage, beet, radish, cucumber, celery, green tomato, chili, green bean, kidney bean, onion, ginger, cowpea, etc. The vegetable fermentation product is more preferably a fermented vegetable or a fermented vegetable juice beverage.
The preparation method of the fermented vegetable comprises the following steps:
s1 vegetable pretreatment
The method comprises the following specific steps: removing impurities and inedible parts of vegetables, processing the vegetables into blocks or strips or other shapes, cleaning for standby, sterilizing the vegetables by using a sterilizing liquid capable of forming hypochlorous acid for 3-5 min, and controlling the microbial quantity of the vegetables to be less than or equal to 300CFU/g; washing with clear water for 3-5 min, and draining for later use;
s2 preparing brine
The method comprises the following specific steps: weighing 1-2% (m/v) star anise, 2-4% (m/v) pepper, 2-6% (m/v) salt and 3-6% glucose, adding purified water to fully dissolve the star anise, heating and boiling for 10-60 minutes, cooling, and cooling the boiled brine to room temperature to obtain pickle brine, thus obtaining salt brine;
S3, preparing vegetable bittern water mixture
The method comprises the following specific steps: mixing the pretreated vegetables treated in the step S1 with the bittern water obtained in the step S2 according to the proportion of 1:5-1:10 (m/v) to obtain a vegetable bittern water mixture;
s4 fermentation culture
The method comprises the following specific steps: the inoculation amount of the working starter and the conventional starter is 1-5% (m/v), the temperature is controlled at 25-30 ℃, and the fermentation is carried out for 3-7 days until the total acid is 0.6-2.4% of the vegetable, thus obtaining the fermented vegetable containing the leuconostoc lactis. The working starter is the working starter containing leuconostoc lactis. The conventional starter is preferably any one or the combination of two of conventional lactobacillus plantarum and leuconostoc mesenteroides.
The preparation method of the fermented vegetable juice beverage comprises the following steps:
s1 vegetable pretreatment
The method comprises the following specific steps: removing impurities and inedible parts of vegetables, cutting the vegetables into blocks, cleaning for standby, sterilizing the vegetables by using a sterilizing liquid capable of forming hypochlorous acid for 3-5 min, and controlling the microorganism quantity of the vegetables to be less than or equal to 300CFU/g; washing with clear water for 3-5 min, and draining for later use;
s2 preparation of vegetable slurry
The method comprises the following specific steps: weighing 10-20% (m/v) vegetables and 6-10% (m/v) glucose, adding purified water, and pulping in a pulping machine to obtain vegetable slurry;
S3 preparation of fermentation base material
The method comprises the following specific steps: inoculating a working starter and a conventional starter which contain Leuconostoc lactis into vegetable slurry according to the inoculum size of 1-5% (m/v), controlling the temperature to be 25-30 ℃, fermenting for 3-7 days until the total acid is 0.6-2.4%, and filtering the vegetable fermentation liquor by using filter cloth with the mesh number of 150-300 meshes to obtain a fermentation base material containing the Leuconostoc lactis; the conventional starter is preferably any one or the combination of two of conventional lactobacillus plantarum and leuconostoc mesenteroides;
s4 preparation of fermented vegetable juice
The method comprises the following specific steps: taking 10-15% (m/v) sucrose, treating with purified water at 70-80 ℃, stirring at high speed for dissolving for 20-30 min, sterilizing at 95 ℃ for 5-10 min, and cooling to 20-30 ℃; adding 20-30% of fermentation base material into the solution, stirring for 10-15 min, and regulating the pH value to 3.6-4.0 by using a proper amount of citrate;
s5 homogenizing and sterilizing
The method comprises the following specific steps: homogenizing at 20-30 deg.c and 20-30 MPa; sterilizing at 95 ℃ for 5-10 min, and refrigerating at 4 ℃ to obtain the fermented vegetable juice beverage containing the leuconostoc lactis.
And provides the application of the leuconostoc lactis in preparing a food fermentation inoculant or fermented food. The food fermenting microbial agent is a living microbial fermentation preparation prepared from beneficial microorganisms.
And provides the application of the leuconostoc lactis in preparing n-nonanol. The molecular formula of the n-nonanol is C 9 H 20 O. Colorless to yellow oily liquid. Boiling point 213-215 deg.C, dissolving in ethanol and oil, and acid value<1.0. Has strong fragrance of sweet and green rose wax and fruit wax. There is some orange-like, sweet orange smell. CAS number 143-08-8. The English name is 1-Nonols.
And provides the application of the leuconostoc lactis in imparting any one of floral, fruit, grass and fresh oil fragrance. Preferably, one or more of the above flavors are imparted to the fermented food product.
The morphological characteristics of the Leuconostoc lactis CCTCC M2019444 of the present invention are described in further detail below with reference to the examples
Colony characteristics: the strain is streaked and separated on an MRS plate, and is subjected to anaerobic culture for 48 hours at 37 ℃, so that the strain grows well. The colony has a diameter of about 1mm-1.5mm, is round, concave, smooth, moist, opaque and milky or yellowish.
Characteristics of the cells: the cells were spherical (FIG. 2), were arranged in a plurality of rows in a chain shape of different lengths, and were also arranged in a single dispersion, and the cell size was generally 0.5. Mu.m.times.1.5. Mu.m, and gram staining was positive.
The culturing characteristics of the leuconostoc lactis CCTCC M2019444 are as follows:
as shown in fig. 4, the minimum growth temperature of the leuconostoc lactis CCTCC M2019444 is 15 ℃, the maximum growth temperature is 40 ℃, and the growth temperature is optimal at 30-40 ℃; the highest and lowest initial growth pH values are 9.0 and 4.0, and the optimal initial growth pH value is 6.0; as shown in FIG. 3, the strain CCTCC M2019444 has a relatively short delay period, 6 hours enters the logarithmic growth phase, and 16 hours reaches the stationary phase.
Example 1 Leuconostoc lactate CCTCC M2019444 collection, separation and selection method
(1) Obtaining a proper dilution gradient and culturing
Sampling from natural fermented sauerkraut product collected from Sichuan, jiangsu, etc. The collected sample is put into an ice box for refrigeration, kept at a lower temperature and brought back to a laboratory and placed at-80 ℃ for refrigeration for standby. 1mL of the sample is weighed and added into 9mL of sterile water, then 1mL of bacterial liquid is sequentially taken and diluted into 9mL of sterile water, and the concentration of the sample is diluted to 10 in a gradient manner -4 Taking 4 dilutions of 10-10 respectively 4 Each 50 mu L of the bacterial suspension of (B) is respectively coated on an MRS agar plate, and is cultured for 36-48 hours under the condition of facultative anaerobism and the constant temperature of 37 ℃.
(2) Separation and purification
The single bacterial colony with different sizes, bulges, micro white color, wetting, regular edges and yellow bacterial colony back is selected by a plate streaking method, and the strain with excellent properties is obtained after repeated culture and selection operations.
(3) Gram staining and catalase experiments
Selecting single colony, performing gram staining and catalase experiment, purifying gram positive and hydrogen peroxide negative bacteria by four generations, selecting typical single colony by plate streaking method, repeating the culture selection operation to obtain strain with excellent properties, inoculating to MRS, culturing for three generations, centrifuging at 5000rpm for 5min, adding 30% glycerol as protective agent, and storing in 30% glycerol tube at-20deg.C.
(4) Determination of n-nonanol from different strains
The isolate obtained from the plate was inoculated into MRS liquid medium for 24 hours, then inoculated into MRS-containing liquid medium in an inoculum size of 2% (v/v), cultured at 37℃for 10 hours, and activated 2 times successively. Centrifuging the fermentation liquor at 4 ℃ for 20min at 5000r/min, washing the obtained precipitate with sterile phosphate buffer solution (50 mM, pH 6.8) for 3 times, adding 50mL of sterile skim milk into the washed precipitate, and carrying out vortex vibration at 5000rpm to resuspend the thallus for later use;
weighing 12% (m/v) skimmed milk powder, 6% (m/v) glucose and 2% glycerol, adding purified water to dissolve thoroughly, sterilizing at 95deg.C for 20min, and cooling to 37deg.C to obtain sterilized skimmed milk;
Inoculating the resuspension thallus and conventional starter into sterilized skimmed milk at 5% (m/v), mixing, fermenting at 25deg.C until titrating acidity is 70-80 deg.C, cooling to room temperature, and storing at 4deg.C.
7g of the above refrigerated fermented vegetables were weighed and placed in a headspace solid phase microextraction vial (15 mL). Placing the headspace bottle in a constant temperature water bath kettle with the temperature of 55 ℃, inserting an extraction head (50/30 mu m DVB/CAR/PDMS), adsorbing the headspace for 40min, transferring the extraction head to a GC-MS sample inlet for 5min after extraction is finished, and completing sample injection.
Wherein the fermentation time of the fermentation vegetable of the leuconostoc lactis is preferably 3 days.
Wherein, the measurement conditions of the headspace solid-phase microextraction are as follows: 7g of the fermented vegetables are placed in a 15mL headspace bottle, extracted for 40min in a constant temperature water bath at 55 ℃, and the extraction head is placed on GC-MS for 5min.
GC-MS is carried out at an initial temperature of 40 ℃ for 3min,5 ℃/min is heated to 230 ℃ and a speed of 10min is maintained for heating, so that the detection of the aroma compounds of the fermented vegetables is completed, and the n-nonanol content in the fermented vegetables is determined. The strain B-MS-PC-12 can produce n-nonanol, and the strain is selected and named as L7ZSAAS01.
EXAMPLE 2 identification of Leuconostoc lactate L7ZSAAS01
(1) Physiological and biochemical identification
Strain L7ZSAAS01 is a gram positive, peroxidase negative, motionless bacillus capable of growth at 15 ℃ and 40 ℃.
(2) PCR amplification 16S rDNA sequence analysis of Strain L7ZSAAS01
1) Sucking 1mL of the liquid culture medium after shaking and mixing, centrifuging, discarding the supernatant, blowing and cleaning 2 times with 1mL of sterile water, centrifuging, discarding the supernatant, and using the supernatant as a template for colony PCR. The PCR system was 50. Mu.L, wherein Mix was 25. Mu.L, 27F was 1. Mu.L, 1492R was 1. Mu.L, and ddH2O was 23. Mu.L.
The primer used was (16s27F:GAGAGTTTGATCCTGGCTCAG;16s 1492R:CGGCTACCTTGTTACGACTT). The nucleotide sequence of the amplified fragment is shown as SEQ ID No. 3, and the length is 429bp.
And (3) carrying out phylogenetic tree analysis on the sequence obtained by sequencing, wherein an experimental result is shown in a figure 1, and the strain obtained by the experiment is Leuconostoc lactis.
2) PCR conditions:
Lid：105℃mBY-16s V：20μL
the DNA double strand was denatured at 94℃for 10min, cooled at 50℃after 30s, rapidly warmed to 72℃for 80s and cycled 29 times for 30s, and finally maintained at 72℃for 7min.
3) Agar gel electrophoresis (80 mL)
0.8g agarose and 80mL 1 xTAE are added into a triangular flask, and heated by microwaves until the mixture is clear, and after the mixture is cooled slightly, 8 mu L of EB dye is added; adding an electrophoresis plate, cooling for half an hour, and condensing into solid colloid; 3-5 mu L of sample is injected into the small hole of the rubber plate by a liquid-transfering gun, and a Marker is added at the tail end of each row; inserting an electrode, regulating the voltage to 120V, and running for half an hour; taking out the rubber plate, exposing for 10s under UV, and storing the image of the electrophoresis strip; sample sequencing will result in a clear electrophoretic band.
4) 16S rRNA sequence analysis and identification
According to the sequencing report given by Beijing Liuhua Dairy Gene technologies Inc., the isolated lactobacillus 16S rRNA sequence was compared and identified with the corresponding sequence of the known strain in the (GenBank/EMBL/DDBJ) database by combining with BLAST analysis tool, and was identified as Leuconostoc lactis (Leuconostoc lactis) by analysis, and the Accession Number was MK855188. Leuconostoc lactis L7ZSAAS01 has the morphological characteristics of rod shape, white color, smooth and moist surface, regular edge and raised colony.
The 16s rDNA is identified as Leuconostoc lactis (Leuconostoc lactis) according to the microbiological characteristics such as morphological characteristics, physiological and biochemical characteristics and the like and the genetic characteristics of the Leuconostoc lactis L7ZSAAS01, and the strain is preserved in China Center for Type Culture Collection (CCTCC) for type 6 and 10 days in 2019, and the preservation number is CCTCC M2019444.
Example 3 growth and fermentation Properties of CCTCC M2019444 Strain
(1) Drawing of CCTCC M2019444 strain growth curve
Inoculating activated Leuconostoc lactis CCTCC M2019444 into MRS liquid culture medium according to an inoculum size of 2% (v/v), culturing at a constant temperature of 37 ℃ for 18 hours, measuring the OD value of the culture solution at 600nm every 1-2 hours, and plotting the OD value versus time to obtain a growth curve of a strain CCTCC M2019444 in MRS, wherein the result (figure 3) shows that: leuconostoc lactis CCTCC M2019444 grows rapidly in MRS culture medium, enters the logarithmic phase about 4 hours, and enters the stationary phase about 12 hours.
(2) Determination of optimum growth temperature of CCTCC M2019444 strain
The activated Leuconostoc lactis CCTCC M2019444 is inoculated into 10mL of MRS liquid culture medium according to the inoculation amount of 2% (V/V), and is respectively placed into the conditions of 15 ℃, 25 ℃, 32 ℃, 37 ℃, 40 ℃ and 45 ℃ for constant-temperature culture for 12 hours, the unvaccinated MRS liquid culture medium is used as a control, the OD value of the culture solution cultured at different temperatures is measured at 600nm, and the optimal growth temperature is determined according to the OD value. The results show that: (FIG. 4) Leuconostoc lactate CCTCC M2019444 has a wide growth temperature range, grows well at 30-40 ℃ from 15 ℃ to 45 ℃ and has an optimal growth temperature of 37 ℃.
(3) Change of pH and titrated acidity of Leuconostoc lactate CCTCC M2019444 in skim milk for 16h
Inoculating Leuconostoc lactis CCTCC M2019444 of the present invention preserved at-80deg.C into MRS liquid culture medium, culturing at 37deg.C for 24 hr, subculturing for 2-3 times, and collecting the culture medium 8 ～10 9 cfu/mL. Taking out the bacterial liquid activated in MRS, inoculating the bacterial liquid in skim milk according to the volume ratio of 2-4% to make the bacterial amount in the yoghourt reach 10 5 cfu/g. The inoculated samples are placed into an incubator at 42 ℃ for fermentation and sampling is carried out every 4 hours, the pH and the titrating acidity change in the fermentation process are detected, and the experimental results are shown in fig. 3 and 4. From FIGS. 3 and 4, the pH of Leuconostoc lactis CCTCC M2019444 was reduced by 0.98 when fermented for 16 hours.
EXAMPLE 4 determination of Leuconostoc lactate CCTCC M2019444 for n-nonanol production Capacity
(1) Preparation of sterile skim milk
Weighing 12% (m/v) skimmed milk powder, 6% (m/v) glucose and 2% glycerol, adding purified water to dissolve thoroughly, sterilizing at 95deg.C for 20min, and cooling to 37deg.C to obtain sterile skimmed milk;
(2) Strain activation
Taking a ring of Leuconostoc lactis CCTCC M2019444 strain preserved by a freeze drying tube dissolved by sterile water through an inoculating ring, scribing on an MRS agar culture medium plate, and growing single bacterial colony in an incubator at 37 ℃ for 24 hours to obtain a plate activated strain;
(3) Preparation of working starter
Inoculating the plate-activated strain obtained in the step (2) into a 250mL triangular flask containing 50mL of MRS broth liquid culture medium, and culturing at constant temperature in a 37 ℃ incubator for 18 hours to obtain a culture solution; centrifuging the culture solution at 5000r/min for 20min, washing the precipitate with sterile phosphate buffer solution (50 mM, pH 6.8) for 3 times, adding 50mL sterile skim milk into the washed precipitate, vortex shaking at 5000rpm to resuspension thallus, introducing strain culture solution into glass ampoule bottle under aseptic condition, rapidly freezing at-30deg.C, placing the glass ampoule bottle in tray, freeze drying in freeze dryer to obtain working ferment, and viable count preferably 10 9 cfu/mL or more;
(4) Vegetable pretreatment
Removing impurities and inedible parts of vegetables, processing the vegetables into blocks, strips or other shapes, cleaning for standby, sterilizing the vegetables by using a sterilizing liquid capable of forming hypochlorous acid for 5min, and controlling the microbial quantity of the vegetables to be less than or equal to 300CFU/g; washing with clear water for 5min, and draining for later use;
(5) Preparation of brine
Weighing 2% (m/v) of old ginger, 3% (m/v) of wild pepper, 4% (m/v) of salt and 4% of glucose, adding purified water to fully dissolve the materials, heating and boiling for 45 minutes, cooling, and cooling the boiled brine to room temperature to obtain the pickle brine.
(6) Preparation of vegetable bittern water mixture
And (3) mixing the pretreated vegetables obtained in the step (4) with the bittern water obtained in the step (5) according to the proportion of 1:10 (m/v) to obtain a vegetable bittern water mixture.
(7) Fermentation culture
The inoculum size of the working starter, the lactobacillus plantarum conventional starter and the leuconostoc mesenteroides conventional starter is 2% (m/v), the temperature is controlled at 30 ℃, and the fermentation is carried out for 3 days until the total acid is 1.5%, so that the fermented vegetable containing the leuconostoc lactis is obtained.
(8) GC-MS (gas chromatography-tandem mass spectrometry) for measuring n-nonanol content in vegetable fermentation process
The chromatographic conditions are as follows: the fermented vegetables obtained above were weighed 5g, 1.3g of NaCl was added thereto, and methyl heptanoate (1 mg/mL, loading amount 1. Mu.L) was quantitatively used as an internal standard and placed in a headspace solid phase microextraction vial. The detection of the fragrant gas compound of the fermented vegetables is completed, so that the content of n-nonanol in the fermented vegetables is determined, and the total ion flow diagram is shown in figure 5.DB-WAX-UI capillary column; column specification: 30m x 0.25 m, inlet temperature 240 ℃, column flow rate 35cm/s, carrier gas helium; programming temperature: the initial temperature is 40 ℃, and the temperature is kept for 10min; heating to 90 ℃ at 10 ℃/min, and keeping for 15 minutes; heating to 130 at 20 ℃/min, and keeping for 5min; heating to 250 ℃ per minute at 20 ℃ per minute, and keeping for 5 minutes.
The mass spectrum conditions are as follows: ionization mode EI, emission energy is 74eV, ion source temperature 230 ℃, interface temperature 280 ℃, four-level rod temperature: 150 ℃ and 15-500 mass-to-charge ratio. The qualitative analysis was performed by searching and comparing the NIST 2001 standard spectrum library with the standard substance, and the peak area of each component was calculated by a peak area normalization method, and expressed as μg/kg vegetable product.
(9) Variation of n-nonanol content during fermentation
Leuconostoc lactis CCTCC M2019444 has higher n-nonanol yield in each fermentation period, the n-nonanol yield is 1.30 mug/kg at 18 hours, the conventional starter lactobacillus plantarum is 0.11 mug/kg, the conventional starter leuconostoc mesenteroides is 0.32 mug/kg, and the screened L7ZSAAS01 strain can produce n-nonanol in an amount far exceeding that of the conventional starter lactobacillus plantarum.
Application example 1 preparation of working fermentation agent containing Leuconostoc lactis CCTCC M2019444
(1) Preparation of sterile skim milk
Weighing 10% (m/v) skimmed milk powder, 6% (m/v) glucose and 2% glycerol, adding purified water to dissolve thoroughly, sterilizing at 95deg.C for 20min, and cooling to 37deg.C to obtain sterile skimmed milk;
(2) Strain activation
Taking a ring of leuconostoc lactis CCTCC M2019444 strain preserved by a freeze drying tube dissolved by sterile water through an inoculating ring, streaking on an MRS agar culture medium plate, and culturing in a 37 ℃ incubator for 48 hours until single colonies grow out, thus obtaining a plate activated strain;
(3) Preparation of working starter
Culturing in a constant temperature incubator at 37 ℃ for 12 hours to obtain a culture solution; centrifuging the obtained culture solution at a rotation speed of 5000r/min for 20min, washing the precipitate obtained by centrifugation with sterile phosphate buffer solution (50 mM, pH 6.8) for 3 times, adding 50mL of sterile skim milk solution into the washed precipitate, shaking by vortex at 5000rpm to resuspension thallus, introducing strain culture solution into glass ampoule bottle under aseptic condition, rapidly freezing at-30deg.C after bottle stopper covering, placing the glass ampoule bottle in tray, freeze-drying in freeze dryer to obtain working fermentation agent containing Leuconostoc lacticum CCTCC M2019444, and the viable count in the working fermentation agent is 10 9 cfu/mL or more.
The working starter used in application examples 2 to 5 below is the working starter prepared in application example 1.
Application example 2 preparation of fermented vegetables containing Leuconostoc lactis CCTCC M2019444
(1) Vegetable pretreatment
Removing impurities and inedible parts of vegetables, processing the vegetables into blocks, strips or other shapes, cleaning for standby, sterilizing the vegetables by using a sterilizing liquid capable of forming hypochlorous acid for 3min, and controlling the microbial quantity of the vegetables to be less than or equal to 300CFU/g; washing with clear water for 3min, and draining for use;
(2) Preparation of brine
Weighing 1% (m/v) star anise, 2% (m/v) pepper, 3% (m/v) salt and 3% glucose, adding purified water to fully dissolve, heating and boiling for 60 minutes, cooling, and cooling the boiled brine to room temperature to obtain the brine of pickled vegetables.
(3) Preparation of vegetable bittern water mixture
And (3) mixing the pretreated vegetables obtained in the step (1) with the bittern water obtained in the step (2) according to the proportion of 1:10 (m/v) to obtain a vegetable bittern water mixture.
(4) Fermentation culture
The inoculation amount of the working starter containing the leuconostoc lactis CCTCC M2019444 is 2% (M/v), the temperature is controlled to be 30 ℃, and the fermentation is carried out for 3 days until the total acid is 1.5%, so that the fermented vegetable containing the leuconostoc lactis is obtained.
Application example 3 preparation of fermented vegetables containing Leuconostoc lactis CCTCC M2019444
(1) Vegetable pretreatment
Removing impurities and inedible parts of vegetables, processing the vegetables into blocks, strips or other shapes, cleaning for standby, sterilizing the vegetables by using a sterilizing liquid capable of forming hypochlorous acid for 5min, and controlling the microbial quantity of the vegetables to be less than or equal to 300CFU/g; washing with clear water for 5min, and draining for later use;
(2) Preparation of brine
Weighing 2% (m/v) star anise, 3% (m/v) pepper, 4% (m/v) salt and 4% glucose, adding pure water to fully dissolve the star anise, the 3% (m/v) pepper, the 4% (m/v) salt and the 4% glucose, heating and boiling for 45 minutes, and cooling the boiled salt water to room temperature to obtain the pickle brine, namely the salt brine.
(3) Preparation of vegetable bittern water mixture
And (3) mixing the pretreated vegetables obtained in the step (1) with the bittern water obtained in the step (2) according to the proportion of 1:10 (m/v) to obtain a vegetable bittern water mixture.
(4) Fermentation culture
The inoculation amount of the working starter containing the leuconostoc lactis CCTCC M2019444 is 4% (M/v), the temperature is controlled at 30 ℃, and the fermentation is carried out for 3 days until the total acid is 2.4%, so that the fermented vegetable containing the leuconostoc lactis is obtained.
Application example 4 preparation of fermented vegetable juice beverage containing Leuconostoc lactis CCTCC M2019444
(1) Vegetable pretreatment
Removing impurities and inedible parts of vegetables, cutting the vegetables into blocks, cleaning for standby, sterilizing the vegetables by using a sterilizing liquid capable of forming hypochlorous acid for 5min, and controlling the microorganism quantity of the vegetables to be less than or equal to 300CFU/g; washing with clear water for 5min, and draining for later use;
(2) Preparation of vegetable slurry
Weighing 10% (m/v) vegetables and 6% (m/v) glucose, adding purified water, pulping in a pulping machine to obtain vegetable slurry.
(3) Preparation of fermentation base
The working starter containing the leuconostoc lactis CCTCC M2019444 is inoculated into vegetable slurry according to the inoculum size of 2% (M/v), then the temperature is controlled at 30 ℃, and fermentation is carried out for 3 days until the total acid is 0.6%, thus obtaining the fermentation base material containing the leuconostoc lactis.
(4) Preparation of fermented vegetable juice
Taking 15% (m/v) sucrose, treating with 80 deg.C purified water, stirring at high speed for 30min, sterilizing at 95deg.C for 10min, and cooling to 30deg.C; adding 30% of fermentation base material into the above solution, stirring for 15min, and adjusting pH to 4.0 with appropriate amount of citrate.
(5) Homogenizing, sterilizing
Homogenizing at 30deg.C under 20 Mpa; sterilizing at 95deg.C for 10min, and refrigerating at 4deg.C to obtain fermented vegetable juice beverage containing Leuconostoc lactis.
Application example 5 preparation of fermented vegetable juice beverage containing Leuconostoc lactis CCTCC M2019444
(1) Vegetable pretreatment
Removing impurities and inedible parts of vegetables, cutting the vegetables into blocks, cleaning for standby, sterilizing the vegetables by using a sterilizing liquid capable of forming hypochlorous acid for 5min, and controlling the microorganism quantity of the vegetables to be less than or equal to 300CFU/g; washing with clear water for 5min, and draining for later use;
(2) Preparation of vegetable slurry
Weighing 20% (m/v) vegetables and 10% (m/v) glucose, adding purified water, pulping in a pulping machine to obtain vegetable slurry.
(3) Preparation of fermentation base
In the preparation of the fermentation base material: the working starter containing the leuconostoc lactis is inoculated into vegetable slurry according to the inoculation amount of 4% (m/v), then the temperature is controlled at 30 ℃, fermentation is carried out for 3 days until the total acid is 1.8%, filter cloth with 200 meshes is used for filtering vegetable fermentation liquor, and clear liquid is the fermentation base material containing the leuconostoc lactis.
(4) Preparation of fermented vegetable juice
And (3) preparing fermented vegetable juice: taking 12% (m/v) sucrose, treating with 80 deg.C purified water, stirring at high speed for 30min, sterilizing at 95deg.C for 10min, and cooling to 20deg.C; 25% of the fermentation base material is added into the solution, stirred for 15min, and the pH value is regulated to 3.6 by a proper amount of citrate.
(5) Homogenizing, sterilizing
Homogenizing and sterilizing: homogenizing at 30deg.C under 30 Mpa; sterilizing at 95deg.C for 10min, and refrigerating at 4deg.C to obtain fermented vegetable juice beverage containing Leuconostoc lactis.
Comparative example 1 preparation of control fermented vegetables
(1) Vegetable pretreatment
Removing impurities and inedible parts of vegetables, processing the vegetables into blocks, strips or other shapes, cleaning for standby, sterilizing the vegetables by using a sterilizing liquid capable of forming hypochlorous acid for 5min, and controlling the microbial quantity of the vegetables to be less than or equal to 300CFU/g; washing with clear water for 3min, and draining for use;
(2) Preparation of brine
Weighing 2% (m/v) star anise, 4% (m/v) pepper, 5% (m/v) salt and 5% glucose, adding pure water to fully dissolve the star anise, the 4% (m/v) salt and the 5% glucose, heating and boiling for 60 minutes, cooling, and cooling the boiled brine to room temperature to obtain the pickle brine, namely the salt brine.
(3) Preparation of vegetable bittern water mixture
The pretreated vegetables obtained in the step (1) and the bittern water obtained in the step (2) are mixed according to the proportion of 1:5 (m/v) to obtain the vegetable bittern water mixture.
(4) Fermentation culture
The inoculation amount of the lactobacillus plantarum conventional starter or the leuconostoc mesenteroides conventional starter is 2% (m/v), the temperature is controlled to be 30 ℃, and fermentation is carried out for 3 days until the total acid is 1.6%, so that the fermented vegetable is obtained.
Comparative example 2 preparation of control fermented vegetables
(1) Vegetable pretreatment
Removing impurities and inedible parts of vegetables, processing the vegetables into blocks, strips or other shapes, cleaning for standby, sterilizing the vegetables by using a sterilizing liquid capable of forming hypochlorous acid for 5min, and controlling the microbial quantity of the vegetables to be less than or equal to 300CFU/g; washing with clear water for 3min, and draining for use;
(2) Preparation of brine
Weighing 1% (m/v) star anise, 3% (m/v) pepper, 6% (m/v) salt and 4% glucose, adding pure water to fully dissolve the star anise, the 3% (m/v) salt and the 4% glucose, heating and boiling for 45 minutes, cooling, and cooling the boiled brine to room temperature to obtain the pickle brine, namely the salt brine.
(3) Preparation of vegetable bittern water mixture
The pretreated vegetables obtained in the step (1) and the bittern water obtained in the step (2) are mixed according to the proportion of 1:5 (m/v) to obtain the vegetable bittern water mixture.
(4) Fermentation culture
The inoculation amount of the lactobacillus plantarum conventional starter or the leuconostoc mesenteroides conventional starter is 4% (m/v), the temperature is controlled to be 30 ℃, and fermentation is carried out for 3 days until the total acid is 2.4%, so that the fermented vegetable is obtained.
Comparative example 3 preparation of control fermented vegetable juice beverage
(1) Vegetable pretreatment
Removing impurities and inedible parts of vegetables, cutting the vegetables into blocks, cleaning for standby, sterilizing the vegetables by using a sterilizing liquid capable of forming hypochlorous acid for 5min, and controlling the microorganism quantity of the vegetables to be less than or equal to 300CFU/g; washing with clear water for 5min, and draining for later use;
(2) Preparation of vegetable slurry
Weighing 15% (m/v) vegetables and 6% (m/v) glucose, adding purified water, and pulping in a pulping machine to obtain vegetable pulp.
(3) Preparation of fermentation base
In the preparation of the fermentation base material: inoculating lactobacillus plantarum conventional starter or leuconostoc mesenteroides conventional starter into vegetable slurry according to an inoculum size of 2% (m/v), controlling the temperature to 30 ℃, fermenting for 3 days until the total acid is 1.6%, and filtering the vegetable fermentation liquid by using filter cloth with the mesh number of 150 meshes, wherein clear liquid is a fermentation base material.
(4) Preparation of fermented vegetable juice
And (3) preparing fermented vegetable juice: taking 10% (m/v) sucrose, treating with 80 deg.C purified water, stirring at high speed for 30min, sterilizing at 95deg.C for 10min, and cooling to 20deg.C; adding 30% of fermentation base material into the above solution, stirring for 15min, and adjusting pH to 4.0 with appropriate amount of citrate.
(5) Homogenizing, sterilizing
Homogenizing and sterilizing: homogenizing at 30deg.C under 20 Mpa; sterilizing at 95deg.C for 10min, and refrigerating at 4deg.C to obtain fermented vegetable juice beverage.
Comparative example 4 preparation of control fermented vegetable juice beverage
(1) Vegetable pretreatment
Removing impurities and inedible parts of vegetables, cutting the vegetables into blocks, cleaning for standby, sterilizing the vegetables by using a sterilizing liquid capable of forming hypochlorous acid for 5min, and controlling the microorganism quantity of the vegetables to be less than or equal to 300CFU/g; washing with clear water for 5min, and draining for later use;
(2) Preparation of vegetable slurry
Weighing 18% (m/v) vegetables and 10% (m/v) glucose, adding purified water, pulping in a pulping machine to obtain vegetable slurry.
(3) Preparation of fermentation base
In the preparation of the fermentation base material: inoculating lactobacillus plantarum conventional starter or leuconostoc mesenteroides conventional starter into vegetable slurry according to an inoculum size of 4% (m/v), controlling the temperature to 30 ℃, fermenting for 3 days until the total acid is 2.0%, and filtering the vegetable fermentation liquid by using filter cloth with 300 meshes, wherein clear liquid is a fermentation base material.
(4) Preparation of fermented vegetable juice
And (3) preparing fermented vegetable juice: taking 15% (m/v) sucrose, treating with 70 deg.C purified water, stirring at high speed for dissolving for 20min, sterilizing at 95deg.C for 10min, and cooling to 20deg.C; adding 20% of fermentation base material into the above solution, stirring for 10min, and adjusting pH to 3.6 with appropriate amount of citrate.
(5) Homogenizing, sterilizing
Homogenizing and sterilizing: homogenizing at 25deg.C under 30 Mpa; sterilizing at 95deg.C for 10min, and refrigerating at 4deg.C to obtain fermented vegetable juice beverage.
Effect example 1 application of Leuconostoc lactate in fermented vegetables
This example compares the ability of Leuconostoc lactis CCTCC M2019444 to produce n-nonanol with conventional fermenters.
Fermented peppers were prepared by setting the preparation processes in application examples 2 and 3 and comparative examples 1 and 2, respectively, and n-nonanol content of the fermented vegetables in application examples 2 and 3 and comparative examples 1 and 2 was measured, respectively. Wherein comparative example 1 and comparative example 2 were each provided with an experimental group of two species of Lactobacillus plantarum, leuconostoc mesenteroides, respectively.
The results are shown in Table 1.
TABLE 1 fermentation results
From table 1, it can be seen that leuconostoc lactis cctccc M2019444 has higher n-nonanol production capability in fermented vegetables, can greatly improve the endogenous aroma of the fermented vegetables in the fermentation process, and has wide application prospects in the preparation of the fermented vegetables.
Effect example 2 application of Leuconostoc lactate in fermented vegetable juice beverage
The volatile flavor compound species in the fermented vegetable juice beverage containing leuconostoc lactis cctccc M2019444 is compared with the control fermented vegetable juice beverage.
Fermented radish juice beverages were prepared by setting the preparation processes in application examples 4, 5 and comparative examples 3, 4, respectively, and n-nonanol content of the fermented vegetable juice beverages in application examples 4, 5 and comparative examples 3, 4 was measured, respectively. Wherein comparative example 3 and comparative example 4 were each provided with an experimental group of two species of Lactobacillus plantarum, leuconostoc mesenteroides. The results are shown in Table 2.
TABLE 2 fermentation results
From Table 2, it can be seen that Leuconostoc lactis CCTCC M2019444 has higher n-nonanol production capability in the fermented vegetable juice beverage, can greatly improve the endogenous aroma of the fermented vegetables in the fermentation process, and has wide application prospects in the preparation of the fermented vegetable juice beverage.
The foregoing is merely a preferred embodiment of the present invention, and it should be noted that the above-mentioned preferred embodiment should not be construed as limiting the invention, and the scope of the invention should be defined by the appended claims. It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the invention.
SEQ ID No. 3 of the sequence Listing
Leuconostoc lactis L7ZSAAS01 16s RNA
GGGACTTGGGGGGCGTGCTATACATGCAAGTCGAACGCGCAGCGAAA GGTGCTTGCACCTTTCAAGCGAGTGGCGAACGGGTGAGTAACACGTGGATAACCTGCCTCAAGGCTGGGGATAACATTTGGAAACAGATGCTAATACCGA ATAAAACTTAGTATCGCATGATACAAAGTTGAAAGGCGCTACGGCGTCACCTAGAGATGGGTCCGCGGTGCATTAGTTAGTTGGTGGGGTAAAGGCCTACC AAGACAATGATGCATAGCCGAGTTGAGAGACTGATCGGCCACATTGGGAC TGAGACACGGCCCAAACTCCTACGGGAGGCTGCAGTAGGGAATCTTCCACAATGGGCGAAAGCCTGATGGAGCAACGCCGCGTGTGTGATGAAGGCTTTA GGGTCGTAAAGCACTGTTGTATGGGAAGAAATGCTAGAATAGGGAATGATTCTAGTTCGACGGTACCATACCAGAAAGGGACGGCTAAATACGTGCCAGC AGCCGCGGTAATACGTATGTCCCGAGCGTTATCCGGATTTATTGGGCGTAA AGCGAGCGCAGACGGTTGATTAAGTCTGATGTGAAAGCCCGGAGCTCAACTCCGGAATGGCATTGGAAACTGGTTAACTTGAGTGTTGTAGAGGTAAGTGG AACTCCATGTGTAGCGGTGGAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTTACTGGACAACAACTGACGTTGAGGCTCGAAAGTGTGG GTAGCAAACAGGATTAGATACCCTGGTAGTCCACACCGTAAACGATGAAT ACTAGGTGTTAGGAGGTTTCCGCCTCTTAGTGCCGAAGCTAACGCATTAAGTATTCAGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTTGA CGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGGAAAATCTTACCAGGTCTTGGACATTCTTTGAAGCTTTTAAGAGATATAAG TGTTC
Sequence listing
<110> Sichuan old jar food Co., ltd
<120> Leuconostoc lactate and use thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1007
<212> DNA
<213> Leuconostoc lactate (Leuconostoc lactis)
<400> 1
gggacttggg gggcgtgcta tacatgcaag tcgaacgcgc agcgaaaggt gcttgcacct 60
ttcaagcgag tggcgaacgg gtgagtaaca cgtggataac ctgcctcaag gctggggata 120
acatttggaa acagatgcta ataccgaata aaacttagta tcgcatgata caaagttgaa 180
aggcgctacg gcgtcaccta gagatgggtc cgcggtgcat tagttagttg gtggggtaaa 240
ggcctaccaa gacaatgatg catagccgag ttgagagact gatcggccac attgggactg 300
agacacggcc caaactccta cgggaggctg cagtagggaa tcttccacaa tgggcgaaag 360
cctgatggag caacgccgcg tgtgtgatga aggctttagg gtcgtaaagc actgttgtat 420
gggaagaaat gctagaatag ggaatgattc tagttcgacg gtaccatacc agaaagggac 480
ggctaaatac gtgccagcag ccgcggtaat acgtatgtcc cgagcgttat ccggatttat 540
tgggcgtaaa gcgagcgcag acggttgatt aagtctgatg tgaaagcccg gagctcaact 600
ccggaatggc attggaaact ggttaacttg agtgttgtag aggtaagtgg aactccatgt 660
gtagcggtgg aatgcgtaga tatatggaag aacaccagtg gcgaaggcgg cttactggac 720
aacaactgac gttgaggctc gaaagtgtgg gtagcaaaca ggattagata ccctggtagt 780
ccacaccgta aacgatgaat actaggtgtt aggaggtttc cgcctcttag tgccgaagct 840
aacgcattaa gtattcagcc tggggagtac gaccgcaagg ttgaaactca aaggaatttg 900
acggggaccc gcacaagcgg tggagcatgt ggtttaattc gaagcaacgc ggaaaatctt 960
accaggtctt ggacattctt tgaagctttt aagagatata agtgttc 1007

Claims (11)

1. The Leuconostoc lactis is characterized in that the Leuconostoc lactis is preserved in China center for type culture collection, and the preservation number is CCTCC M2019444.
2. A biologically pure culture comprising the leuconostoc lactis of claim 1.
3. A working starter comprising the leuconostoc lactis of claim 1.
4. A process for preparing a working starter culture according to claim 3, comprising the steps of:
s1, providing sterile skim milk;
s2, activating the leuconostoc lactis strain;
s3, preparing a strain culture solution, adding sterile skim milk during centrifugal precipitation, and freeze-drying.
5. A fermented food comprising the leuconostoc lactis of claim 1.
6. The fermented food product according to claim 5, characterized in that: the fermented food is fermented vegetable or fermented vegetable juice beverage.
7. A method of preparing the fermented vegetable of claim 6, characterized by: the method comprises the following steps:
S1 vegetable pretreatment
Removing impurities and inedible parts of vegetables, cutting the vegetables into blocks, cleaning for later use, and sterilizing the vegetables by using a sterilizing liquid; washing with clear water, and draining for later use;
s2 preparing brine
Adding proper amount of flavoring into purified water, adding proper amount of salt and sugar according to water volume, heating, boiling, cooling, and cooling the boiled brine to room temperature to obtain brine;
s3, preparing vegetable bittern water mixture
Adding the vegetables treated in the step S1 into the bittern water obtained in the step S2 to obtain a vegetable bittern water mixture;
s4 fermentation culture
And (3) inoculating the working starter and the conventional starter containing the leuconostoc lactis into the vegetable bittern water mixture obtained in the step (S3) according to a certain proportion, and controlling the temperature to ferment to obtain the fermented vegetable containing the leuconostoc lactis.
8. A method of preparing the fermented vegetable juice beverage of claim 6, wherein: the method comprises the following steps:
s1 vegetable pretreatment
Removing impurities and inedible parts of vegetables, cutting the vegetables into blocks, cleaning for later use, and sterilizing the vegetables by using a sterilizing liquid; washing with clear water, and draining for later use;
S2 preparation of vegetable slurry
Taking a proper amount of vegetables and sugar, putting the vegetables and sugar into purified water, and putting the purified water into a beating machine for beating to obtain vegetable slurry;
s3 preparation of fermentation base material
The working starter and the conventional starter containing the leuconostoc lactis are inoculated into the vegetable slurry obtained after the treatment of the step S2 according to a certain proportion, the temperature is controlled for fermentation, and the clear liquid obtained by filtration is refrigerated to obtain the fermentation base material containing the leuconostoc lactis;
s4 preparation of fermented vegetable juice
Adding purified water into sugar with certain mass, stirring at high speed for dissolving, sterilizing, and cooling to room temperature; adding a certain amount of fermentation base material into the solution, stirring uniformly, and regulating the acidity value by using a proper amount of acid;
s5 homogenizing and sterilizing
Homogenizing the solution treated in the step S4, sterilizing, and cooling to obtain the fermented vegetable juice beverage containing the leuconostoc lactis.
9. Use of leuconostoc lactis according to claim 1 for the preparation of a food fermentation inoculant or a fermented food.
10. Use of leuconostoc lactis according to claim 1 for the preparation of n-nonanol.
11. Use of leuconostoc lactis according to claim 1 for imparting any one or more of floral, fruit, green, fresh greasy notes.
CN201910645660.8A 2019-07-17 2019-07-17 Leuconostoc lactis and application thereof Active CN111304108B (en)

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Citations (1)

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CN109234204A (en) * 2018-11-07 2019-01-18 福建省农业科学院农业工程技术研究所 A kind of pickle starter and its methods for making and using same

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109234204A (en) * 2018-11-07 2019-01-18 福建省农业科学院农业工程技术研究所 A kind of pickle starter and its methods for making and using same

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* Cited by examiner, † Cited by third party
Title
AIR FLOW ASSISTED IONIZATION FOR REMOTE SAMPLING OF AMBIENT MASS SPECTROMETRY AND ITS APPLICATION;HE等;《RAPID COMMUNICATIONS IN MASS SPECTROMETRY》;第25卷(第7期);全文 *
牙签电喷雾电离质谱分析脐橙果汁成分;钟涛等;《质谱学报》;第37卷(第1期);全文 *
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