CN117187141A - Lactobacillus plantarum for shallow fermentation of fruits and vegetables and preparation method of fermented fruits and vegetables - Google Patents

Lactobacillus plantarum for shallow fermentation of fruits and vegetables and preparation method of fermented fruits and vegetables Download PDF

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CN117187141A
CN117187141A CN202311300722.4A CN202311300722A CN117187141A CN 117187141 A CN117187141 A CN 117187141A CN 202311300722 A CN202311300722 A CN 202311300722A CN 117187141 A CN117187141 A CN 117187141A
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fermented
lactobacillus plantarum
microbial inoculum
vegetables
fermentation
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迟原龙
覃书漫
龙文玲
黄伦杰
刘振宇
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Sichuan University
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Sichuan University
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Abstract

The invention relates to the technical field of microorganisms, in particular to a preparation method of lactobacillus plantarum agent fermented fruits and vegetables for shallow fermentation of fruits and vegetables; the preservation number of the lactobacillus plantarum is CCTCC M20231830; the composite microbial inoculum also comprises lactobacillus plantarum and Weissella fusion; the lactobacillus plantarum provided by the invention is a strain screened from natural fermented pickle, and has high activity and stable fermentation performance; and preparing a weak post-acidification fermented fruit and vegetable product under specific fermentation conditions, so as to radically solve the problem of post-fermentation acidification; the composite microbial inoculum can obtain fermented fruit and vegetable products with richer tastes, and the fermented vegetables have higher acidity preference and obvious weak post-acidification effect.

Description

Lactobacillus plantarum for shallow fermentation of fruits and vegetables and preparation method of fermented fruits and vegetables
Technical Field
The invention relates to the technical field of fermentation, in particular to lactobacillus plantarum, a microbial inoculum, a composite microbial inoculum, application of the microbial inoculum and the composite microbial inoculum, fermented food and a preparation method thereof.
Background
The fermented fruit and vegetable products are popular with consumers because of unique taste and flavor and the advantage of being rich in probiotics, the fermented fruit and vegetable products are mainly fermented by lactic acid bacteria to endow food with unique sensory quality, and when the pH of pickle brine is lower than 4.0 and the total acid is higher than 0.3g/100g lactic acid, the pickle can be considered to be fermented and ripe. Lactobacillus plantarum, one of the typical species of lactobacillus fermentum, has been shown to be a strain with potential probiotic properties, with antioxidant, anti-inflammatory, immunostimulating and antibacterial activities; the method is involved in the fermentation process of various foods such as meat, dairy products and the like, and endows the products with unique color, flavor, texture and nutritional value, but due to the characteristics of high acid production rate and strong acid resistance, the fermentation and maturation can still utilize nutrient substances in the fermented foods to produce acid and metabolize, so that the quality of the products is reduced in the storage process, and the post-acidification is a representative main problem. Post acidification can cause the problems of reduced flavor, softened texture, deteriorated color, reduced activity and quantity of probiotics and the like of the fermented fruit and vegetable products, and is a difficult problem to be researched in the fermentation industry of the fermented fruit and vegetable at present.
Meanwhile, in view of the defect of single fermentation product and lacking flavor, the weak post-acidification fusion Weissella PCYLJY-1 is combined for compound fermentation. However, at present, a mode of regulating the shallow fermentation of lactobacillus plantarum through the environment is not researched and reported, and the acidogenic metabolic characteristics of lactobacillus under the combined conditions of temperature, acidity and salinity are not known yet.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention provides lactobacillus plantarum for shallow fermentation, a composite microbial inoculum, fermented fruits and vegetables and a preparation method.
The technical scheme adopted by the invention is as follows: a lactobacillus plantarum (lactobacillus plantarum), wherein the preservation number of the lactobacillus plantarum is CCTCC M20231830.
A microbial agent comprising the lactobacillus plantarum (lactobacillus plantarum) of claim 1.
A complex microbial agent comprising lactobacillus plantarum (lactobacillus plantarum) and fusogenic weissella (Weissella confusa) of claim 1; the number of the viable bacteria of the lactobacillus plantarum and the Weissella fusion in the composite microbial inoculum is more than or equal to 1 to 3 multiplied by 10 9 CFU/g; when in use, the lactobacillus plantarum and the Weissella fusion in the composite microbial inoculum are added into the fermented vegetables according to the mass ratio of the microbial powder of 3:7 and the total mass of the raw materials of 0.1-0.4 percent.
The application of a microbial inoculum in preparing fermented food.
The application of the composite microbial inoculum in preparing fermented food.
A method of preparing a fermented food product comprising the steps of:
inoculating a microbial inoculum into the fermented food to be fermented for fermentation; the microbial inoculum is one of the microbial inoculum of claim 2 and the composite microbial inoculum of claim 3.
Further, the fermented food is a fermented vegetable or a fermented fruit and vegetable juice, the fermented vegetable includes but is not limited to a fermented radish, a fermented pepper, a fermented cucumber, and a fermented cabbage; the fermented fruit and vegetable juice includes, but is not limited to, fermented green melon juice, fermented apple juice, and fermented carrot juice.
A fermented vegetable is obtained by the above preparation method, and has a salt content of 1wt.% to 5wt.%, an initial pH of 3.6 to 4.4, and a storage temperature of 5 ℃ or 10 ℃.
Further, the fermentation process is as follows: the vegetable water ratio of the fermented vegetables is 1:2, and the salt content is 3wt.%; inoculating a microbial inoculum containing the fusion Weissella in fermented vegetables, fermenting for one day, inoculating a microbial inoculum containing the lactobacillus plantarum of claim 1, fermenting at normal temperature to pH4.0, and storing at 5 ℃.
A fermented fruit and vegetable juice is prepared by the above preparation method.
Further, the fermentation process is as follows: the fruit and vegetable juice is obtained by squeezing vegetables or fruits and water according to a ratio of 1:2; inoculating the microbial inoculum to fruit and vegetable juice, fermenting at normal temperature to pH4.0, and storing at 5deg.C.
The beneficial effects of the invention are as follows:
(1) The lactobacillus plantarum provided by the invention is a strain screened from natural fermented pickle, and has high activity and stable fermentation performance; and preparing a weak post-acidification fermented fruit and vegetable product under specific fermentation conditions, so as to radically solve the problem of post-fermentation acidification;
(2) The composite microbial inoculum provided by the invention can greatly shorten the fermentation time, the acidity is kept stable in the storage period, and the pH is kept stable at 3.7-3.8;
(3) The lactobacillus plantarum and the compound microbial inoculum provided by the invention have excellent fermentation quality, and can be used for fermenting vegetables, so that the original texture and color of the fermented vegetables can be maintained within a week, and the quantity of lactobacillus is abundant;
(3) The composite microbial inoculum formed by the lactobacillus plantarum and the Weissella fusion can obtain fermented fruit and vegetable products with richer tastes, and the fermented vegetables have higher acidity preference and obvious weak post-acidification effect.
Preservation of organisms
The lactobacillus plantarum (lactobacillus plantarum) provided by the invention is preserved in China Center for Type Culture Collection (CCTCC) in 9 months and 28 days in 2023, wherein the preservation number is CCTCC M20231830, and the preservation address is 211 room (abbreviated as CCTCC) of China center for type culture collection (China) in Wuhan university of Wuchang district in Wuhan City, hubei province.
Drawings
FIG. 1 is a graph showing the growth and acid production change of Lactobacillus plantarum (lactobacillus plantarum) abbreviated as SCSL-1 in example 1 of the present invention under different conditions.
FIG. 2 is a transmission electron microscope image of Lactobacillus plantarum (lactobacillus plantarum) abbreviated as SCSL-1 in example 1 of the present invention, wherein A is 5 ℃, B is 10 ℃, and C is 20 ℃ under different temperature conditions at pH 4.4.
FIG. 3 shows fermentation pH and TTA of Lactobacillus plantarum (lactobacillus plantarum) abbreviated as SCSL-1 in example 2 of the present invention.
FIG. 4 shows the changes of pH (a) and TTA (b) at different storage temperatures (20 ℃ C., 5 ℃ C.) after the composite microbial inoculum of example 3 of the present invention ferments kimchi with a salt content of 3wt.% to pH 4.0.
FIG. 5 shows the change of lactic acid bacteria at different storage temperatures (20 ℃ C., 5 ℃ C.) after the composite microbial inoculum of example 3 of the present invention ferments kimchi with a salt content of 3wt.% to pH 4.0.
FIG. 6 is a diagram showing the sensory evaluation at various storage temperatures (20 ℃ C., 5 ℃ C.) after the complex microbial agent of example 3 of the present invention ferments kimchi with a salt content of 3wt.% to pH 4.0.
Detailed Description
The invention will be further described with reference to the drawings and specific examples.
Lactobacillus plantarum (lactobacillus plantarum) with a preservation number of CCTCC M20231830, SCSL-1 for short, is screened from dominant strains in the later period of natural fermentation, and is preserved in 25% glycerol at-80 ℃.
The lactobacillus plantarum DNA sequence provided by the invention is shown as SEQ ID No. 1.
SEQ ID No.1
TATCTGTCACTTAGGCGGCTGGTTCCTAAAAGGTTACCCCACCGACTTTGGGTGTTACAAACTCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCGACTTCATGTAGGCGAGTTGCAGCCTACAATCCGAACTGAGAATGGCTTTAAGAGATTAGCTTACTCTCGCGAGTTCGCAACTCGTTGTACCATCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTCACCAGAGTGCCCAACTTAATGCTGGCAACTGATAATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTATCCATGTCCCCGAAGGGAACGTCTAATCTCTTAGATTTGCATAGTATGTCAAGACCTGGTAAGGTTCTTCGCGTAGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGCCTTGCGGCCGTACTCCCCAGGCGGAATGCTTAATGCGTTAGCTGCAGCACTGAAGGGCGGAAACCCTCCAACACTTAGCATTCATCGTTTACGGTATGGACTACCAGGGTATCTAATCCTGTTTGCTACCCATACTTTCGAGCCTCAGCGTCAGTTACAGACCAGACAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTTCACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTTTCCCAGTTTCCGATGCACTTCTTCGGTTGAGCCGAAGGCTTTCACATCAGACTTAAAAAACCGCCTGCGCTCGCTTTACGCCCAATAAATCCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGGCACGTAGTTAGCCGTGGCTTTCTGGGTTAAATACC
The fusion Weissella used was a strain which has been publicly preserved, and at 2021101832274 Lactobacillus plantarum and its use in fruit and vegetable juice fermentation have been disclosed.
The actual use in the following examples of the invention is:
0.01M sodium hydroxide, potassium dihydrogen phosphate, ammonium molybdate, concentrated hydrochloric acid, concentrated sulfuric acid, ascorbic acid, anhydrous tin chloride, bovine serum albumin, coomassie Brilliant blue G-250, 90% ethanol, 85% phosphoric acid, tris-HCl buffer (75 mmol/L, pH=7, containing 10mmol/L MgSO) 4 ) Toluene, tris-HCl buffer (50 mmol/L, pH=7.5, containing 10mmol/L MgSO) 4 ) Adenosine 5' -triphosphate disodium salt (ATP), 50% trichloroacetic acid, etc., all reagents were analytically pure.
The main instrument is:
the main instrument is as follows: PHS-3C acidimeter, V-1800PC ultraviolet spectrophotometer, JEM-1400FLASH transmission electron microscope, lumin fluorescence spectrophotometer, C-2030 high performance liquid chromatograph, 1300-TSQ 9000 gas chromatograph-mass spectrum, TA.XT Plus texture instrument, CM-5 color difference instrument, HC-3018R high speed refrigerated centrifuge, SW-CJ-1F biological clean bench, LRH-250F biochemical incubator, LDZF-50L-I autoclave, etc.
The evaluation methods employed for the strains were as follows:
(1) Strain activation: the strain was activated by inoculating Lactobacillus plantarum SCSL-1, an experimental strain deposited at-20deg.C, to fresh MRS broth medium at 37deg.C for 24h at 2% (v/v) and transferring to culture for 2 passages.
(2) Acid production characteristics: the activated bacteria solution was inoculated to fresh MRS broth at 2% (v/v) for different salt contents (1 wt.%, 3wt.%, 5 wt.%) and different pH conditions (pH 4.4, pH4.0 and pH 3.6), and then the culture solutions which had been inoculated with different pH values were respectively cultured at 20 ℃,10 ℃,5 ℃ until the acidity reached equilibrium, and the pH and TTA of the fermentation supernatant were measured every 1 day. The pH was determined using a pH meter and TTA was determined with reference to GB 12456-2021. Maximum acid production rate: acid production rate was analyzed using a modified Gompertz model:
L(t)＝n+a exp(-exp(-k(t-M)))
wherein L (t) is the total acid concentration (g 100 mL) corresponding to different fermentation days -1 Calculated as lactic acid), n is the initial TTA value, a is the increase in final total acid concentration (g 100mL -1 ) K is TTA maximum acid production rate (g 100 mL) -1 day -1 ) M is the time (d) required to reach the maximum acid production rate.
(3) Growth characteristics: according to the culture mode of (2), the OD of the fermentation liquid is measured at intervals 600 Viable count of lactic acid bacteria. Determination of bacterial suspension OD by ultraviolet spectrophotometer 600 And determining the viable count of the lactic acid bacteria by a pour culture method. Maximum growth rate: calculated according to the modified Gompertz model:
L(t)＝n+a exp(-exp(-k(t-M)))
where k is the maximum growth rate (Ka).
(4) Acid-producing related substances: the fresh MRS broth medium was inoculated with salt content (1 wt.%, 3wt.%, 5 wt.%) and different pH conditions (pH 4.4, pH4.0, pH 3.6), and then the culture solutions that had been inoculated with different pH values were incubated at 20℃and 10℃and 5℃respectively for 24 hours. And (3) measuring the content of organic acid and glucose in the fermentation liquor by adopting a High Performance Liquid Chromatography (HPLC). The measuring method comprises the following steps: accurately sucking 1mL of fermentation supernatant, adding 9mL of sterile water for dilution by 10 times, uniformly mixing, carrying out ultrasonic treatment, filtering and measuring. Measurement conditions: chromatographic separation column: sepax ME-H (NP) (7.8 mm. Times.300 mm); mobile phase: 2.5Mm H 2 SO 4 The method comprises the steps of carrying out a first treatment on the surface of the Column temperature: 55 ℃; sample injection amount: 10uL; flow rate: 0.5mL/min; a detector: an ultraviolet detector; detection wavelength: 210nm. Measuring the content of acetoin: to the sample, 0.1mL of 1M HCl and 2mL of 0.19% 2.4-Dinitrophenylhydrazine (DNPH) were added, then water was added to 5mL of the mixture to the mixture, water was put in a water bath at 30℃for 1 hour, and then 5mL of a 5.0 g/L sodium acetate solution was added, and after uniform mixing, the mixture was measured through a 0.45 μm filter membrane. C18 column (4.6 250mm,5 μm, sepax) with acetonitrile-water (55:45, v/v) as mobile phase, maintained at 30deg.C,the flow rate was 1.0mL/min and the detection wavelength was 363nm.
(5) Lactic dehydrogenase and H, key enzymes for acid production + -ATPase activity: lactate dehydrogenase: (a) permeabilizing the cell: after 24h incubation as in (2), centrifugation at 8000rpm for 10min, the pellet was resuspended in 50. Mu.L containing 10mM MgSO 4 In 75mM Tris-HCl buffer (pH 7.0). Then 50. Mu.L toluene was added, mixed vigorously at 37℃for 5min, frozen (-80℃for 2 h) and thawed (37℃for 20 min) 2 times successively to obtain permeable cells, and the cells were resuspended in a medium containing MgSO 4 (10 mM) Tris-HCl buffer (75 mM, pH 7.0) was stored at-80 ℃. (b) Lactate dehydrogenase (U g prot) -1 ) Activity measurement: a lactate dehydrogenase assay kit (NJCBIO, china) was used. The cellular protein content was determined using Bradford protein assay kit (NJJCBIO, china).
H + ATPase: (a) establishing a phosphorus content standard curve: molybdenum blue colorimetric method; (b) establishing a protein standard curve: bradford protein concentration assay kit; (c) Extraction of H + ATPase: extracting H by repeated freeze thawing at-80deg.C + -ATPase; (d) Determination of H + ATPase viability: 25 mu L H is taken + 1mL Tris-HCl (pH=7.5) buffer and 10. Mu.L 0.3mol/L ATP were added to the ATPase extract, and 20uL CCl was added immediately after 5min reaction at 37 ℃ 3 The reaction was terminated with COOH, and the supernatant was centrifuged to determine the phosphorus content and protein content. H + ATPase Activity catalyzes the release of ATP to one PO per unit time (min), per unit mass (mg) of enzyme protein 4 3- Amount (. Mu. Mol) as H + One viability unit U (. Mu. Mol/mg/min), H of ATPase + -ATPase activity calculation formula:
(6) Cell membrane fluidity: cells were collected by centrifugation at 8000rpm at 4℃for 10min, washed twice with cold brine (0.85% sodium chloride w/v), then mixed with 1ml of sodium hydroxide in distilled aqueous methanol (3:10:10 w/v/v), heated at 100℃for 30min, added with 2mL of 6M HCl-methanol (13:11, v/v) and rapidly cooled in 80℃environmentAnd 10min. FAMEs were extracted with 1.25mL of methyl tert-butyl ether hexane (1:1, v/v) for 10min and washed with 3mL of 0.33M NaOH. The upper organic phase (0.8 mL) was evaporated by transfer under a stream of nitrogen and then resuspended to 10mL with n-hexane. A1. Mu.L sample of the concentrated extract was taken and subjected to VF-WAX-MS capillary column (30.0m 0.25mm 0.25m;Agilent,USA) using a Trace1300-TSQ 9000GC mass spectrometer (Thermo Scientific, USA). During gas chromatography measurement, the carrier gas is helium, and the flow rate is 1mL min -1 The temperature of the sample inlet and the detector is 230 ℃, the temperature program is isothermal for 1min at 100 ℃, and then the temperature is increased by 4 ℃/min -1 And (5) heating to 230 ℃ and then keeping the temperature for 5min. Identification was performed based on mass spectra from a standard library of spectra. The relative content of FAMEs was calculated from the peak area.
The rotational diffusion of fatty acyl chains inside the membrane was studied using fluorescence anisotropy measurement methods. DPH (1, 6-diphenyl-1, 3, 5-hexatriene) probes are used to monitor changes in membrane dynamics. The steady state fluorescence anisotropy was measured at 37℃with a fluorescence spectrometer (Lumina, thermo Scientific, USA), excitation wavelength 360nm and emission wavelength 430nm. The degree of fluorescence polarization (p) was calculated as follows:
wherein I is V For corrected fluorescence, subscripts V and H are values obtained for the exciter and analyzer polarizers in either the vertical or horizontal directions, respectively. The film anisotropy (r) of the Plantarum SC-1 was calculated as:
(7) Transmission electron microscope: bacterial samples (8000 g, centrifuged at 4 ℃ C. For 20 min) were collected and washed 2 times with 3% glutaraldehyde. Then cells were doubly fixed (3% glutaraldehyde, 1% OsO) 4 ) Dewatering (acetone fractionation), infiltration and embedding (Epon 812 embedding agent). And preparing ultrathin slices of about 60-90 nm by using an ultrathin slicing machine and paving the ultrathin slices. Then the copper mesh is recovered, and is dyed (uranium acetate and lead citrate) in turn, and then observed by a transmission electron microscopeAnd (3) cells.
Example 1
Strains were cultured according to the above-described evaluation method, and the growth characteristics and acid-producing characteristics of the strains under the conditions of a predetermined salt content (3 wt.%) and an initial pH (4.4, 4.0, 3.6) and a low temperature (20 ℃,10 ℃,5 ℃) were analyzed. Meanwhile, the weak acidification effect, the acidogenesis metabolism (metabolic pathway, acidogenesis key enzyme activity) and the physiological condition (cell membrane morphology, cell membrane fatty acid composition and fluidity) are analyzed.
Growth thereof (OD) 600 ) And acid production (pH, TTA) results are shown in FIG. 1, and the corresponding acid production and growth characteristics are shown in Table 1.
In FIG. 1, panel A shows the pH change curve (delta: 5 ℃ C.) at different temperatures with a salt content of 3wt.%, an initial pH of 4.4; O10 ℃ and ∈20deg.C);
panel B shows the pH change profile (delta: 5 ℃ C.; O: 10 ℃ C.; 20 ℃ C.) at various temperatures with a salt content of 3wt.%, and an initial pH of 4.0;
panel C shows the pH change curve (delta: 5 ℃ C.; O: 10 ℃ C.; 20 ℃ C.) at various temperatures with a salt content of 3wt.%, and an initial pH of 3.6;
panel D shows TTA curves of variation (delta: 5 ℃ C.; O: 10 ℃ C.; 20 ℃ C.) at different temperatures, with a salt content of 3wt.%, and an initial pH of 4.4;
panel E shows the TTA change curve (delta: 5 ℃ C.; O: 10 ℃ C.; 20 ℃ C.) at a salt content of 3wt.%, an initial pH of 4.0, and at various temperatures;
panel F shows TTA curves with a salt content of 3wt.%, an initial pH of 3.6 (delta: 5 ℃ C.; O: 10 ℃ C.; O: 20 ℃ C.) at different temperatures;
panel G shows the OD change curve (delta: 5 ℃ C.; O: 10 ℃ C.; O: 20 ℃ C.) at different temperatures with a salt content of 3wt.%, and an initial pH of 4.4.
Panel H shows the OD change curve (delta: 5 ℃ C.; O: 10 ℃ C.; O: 20 ℃ C.) at different temperatures with a salt content of 3wt.%, and an initial pH of 4.0.
FIG. I shows the OD change curve (delta: 5 ℃ C.; O: 10 ℃ C.; 20 ℃ C.) at various temperatures with a salt content of 3wt.%, and an initial pH of 3.6.
TABLE 1 Lactobacillus plantarum SCSL-1 growth and acid production Properties at 3wt.% salt content, acid and Low temperature
Ka in the Table: maximum acid production rate (g 100 mL) -1 day -1 ) The method comprises the steps of carrying out a first treatment on the surface of the S: stabilizing the pH; m: time (days) corresponding to maximum acid production rate; kq maximum growth Rate (Log CFU mL -1 day -1 ) The method comprises the steps of carrying out a first treatment on the surface of the V: number of viable bacteria in stationary phase (Log CFU mL) -1 ). TD on the upper right of the two item names indicates that there is an interaction between low temperature and initial pH (P<0.05). ND: uncertainty.
The acid production capacity of the strain is characterized by the maximum acid production rate of the strain under the co-action of acid and low temperature and the time corresponding to the pH value of the stable period and the maximum acid production rate, and the growth characteristics of the strain are measured by the maximum growth rate and the viable count of the stable period. As can be seen from fig. 1 and table 1, at 3wt.% salt content, the acid production change of the strain is consistent with the growth change, and the strain shows the advantage of slowing down acid production at low temperature at different initial pH, and the bacterial growth and acid production rate are significantly reduced (P < 0.05) with decreasing temperature; at the same pH, it is common that the temperature decrease is more pronounced for both the strain in terms of acidogenesis and growth, and at the same temperature, a lower initial pH slows down both strain growth and acidogenesis. There was a clear difference in acid production (P < 0.05) between the strains at pH4.4 and pH4.0 at 20 ℃. The time (M) corresponding to the maximum acid production rate can be used for estimating the time period with the maximum change of the acidification rate during storage approximately, the time range with the fastest change of the acid production of different acidity at 20 ℃ is 1 day after storage, compared with the time of 10 ℃ which truly prolongs the occurrence of the maximum acid production rate, and the maximum acid production rate is approximately generated on the 8 th to 9 th days of storage. Therefore, at a salt content of 3wt.%, a pH of 4.4 or below is a temperature of choice that can be used for short-term storage, any acidity of 5 ℃ will not grow and acid over a week, and will maintain the initial viable count.
The interaction between the pH (4.4, 4.0, 3.6) and the temperature (20 ℃,10 ℃,5 ℃) of the set parameters and the reduction of the growth capacity and the acid production capacity of the strain are the results of the joint addition of two environmental factors to the strain.
The major metabolites and key enzymes of the carbohydrate metabolism of Lactobacillus plantarum SCSL-1 are shown in Table 2.
TABLE 2 Lactobacillus plantarum SCSL-1 sugar metabolism major metabolites and key enzymes
As can be seen from Table 2, the metabolism of glucose and the production of lactic acid at 10℃and 5℃are lower than those at 20℃and the metabolism of sugar at low temperature also produces acetoin, which reduces the acid production of lactic acid, is particularly remarkable at 5℃ (P<0.05). Further by exploring the metabolic key enzyme activity, it was found that the lactate dehydrogenase and H had a salt content of 3wt.% at any acidity of 10, 5 ℃C + ATPase is very low, and a decrease in temperature affects not only the decrease in glucose metabolism and lactate production of the strain by metabolism, but also the decrease in mass exchange and metabolism by changing the cell membrane morphology and membrane fluidity of the strain.
The fatty acid composition changes and membrane fluidity changes of Lactobacillus plantarum SCSL-1 cell membranes at low temperature are shown in Table 3.
TABLE 3 fatty acid composition and fluorescence anisotropy of Lactobacillus plantarum SCSL-1 cell membranes
In the table: UFA = unsaturated fatty acid (CFA is not included in UFA), UFA/SFA = ratio of unsaturated fatty acid to saturated fatty acid, ND: uncertainty of
The low temperature can reduce the variety and content of fatty acid, which is very obvious at 5 ℃ and 10 ℃, the unsaturated fatty acid content is about 0 compared with the saturated fatty acid content, and the fluorescence anisotropy value is larger, which shows that the cell membrane fluidity of the strain is very low, which indicates that the low temperature leads to the reduction of the membrane fluidity of the strain, reduces the exchange of intracellular and extracellular substances, further influences the main metabolic process of bacteria, and leads to the reduction of acid production.
The cell membrane morphology of SCSL-1 at different temperatures is shown in FIG. 2, and the decrease in temperature does not affect the survival and integrity of the cells, but significantly alters the thickness of the cell membrane. Therefore, 3% salt content can be selected while selecting a low temperature of 10℃and a cold storage temperature of 5℃as a short-term and long-term storage temperature. The environmental factors act on the physiological metabolism of the strain to enable the strain to generate acid, reduce the key enzyme activity of acid generation, thicken cell membranes and reduce the fluidity of the membranes, so that the aim of delaying acid generation is achieved while the living bacteria are effectively maintained.
Further testing at 1wt.% salt content, at 5 ℃ and ph4.0 and below, gives the same results, which can be used as a long term storage parameter. And when the salt content is 5wt.%, it is possible to use as a short-term storage parameter at 10 ℃ and at ph4.4 and below.
Example 2
Genetic stability and strain identification of Lactobacillus plantarum
The Lactobacillus plantarum obtained in example 1 was subcultured with MRS broth for 10 passages, and the culture was performed at intervals of 8 hours with an inoculum size of 2% (v/v), and after sampling during the passage, the pH and TTA were measured after culturing under the same conditions, and the results are shown in FIG. 3. From the figure, it can be seen that the pH is kept substantially consistent, and the strain has good genetic stability in fermentation and acid production.
Bacterial genome DNA extraction kit is adopted to extract No. 12 strain DNA and carry out PCR amplification, after PCR amplification, the product is cut into gel, purified and recovered, and then the gel is sent to a biological engineering (Shanghai) limited company for sequencing, and the nucleotide sequence is shown as SEQ ID NO. 1. Sequencing results were aligned on-line by National Center for Biotechnology Information (https:// blast. Ncbi. Lm. Nih. Gov/blast. Cgi): the homology of the 16SrRNA of the strain with Lactobacillus plantarum strain is 99%, the strain has higher homology, but partial sequences have certain difference, and the strain belongs to a new strain and is named Lactobacillus plantarum SCSL-1.
Example 3
The composite microbial inoculum was inoculated and cultured as described above, and the results were tested.
Adding a composite microbial inoculum according to 0.3% of the total mass of the raw materials into pickle containing 3wt.% of salt, fermenting at 20 ℃ for 1 day until reaching mature pH4.0, respectively storing the fermented pickle at 20 ℃ and 5 ℃, sampling and measuring the pH, TTA, viable count, texture, color and luster, sensory evaluation and volatile flavor substances of the pickle at 0d, 1d, 2d, 3d, 5d, 7d and 10d of the pickle storage, taking the storage at 20 ℃ as a control group, and observing the storage effect of the pickle at 5 ℃.
The pH and TTA change curves of the composite microbial inoculum fermented pickle respectively during storage at 20 ℃ and 5 ℃ are shown in figure 4. As can be seen from the figure, the pH and TTA of the kimchi slowly change during storage at 5 deg.C, and finally stabilize at about 3.74, and the TTA stabilizes at about 0.39g lactic acid/100 g kimchi. The pH at the storage stability period at 20℃was as low as 3.05 after 10 days of storage, and TTA was continuously increased to 0.84g of lactic acid per 100g of kimchi during the storage period.
The acidity and the acidity preference of the fermented kimchi were subjected to sensory evaluation, and the results are shown in fig. 6. The acidity score of the pickle stored at 20 ℃ is 4.5, and the acidity preference of the pickle stored at 2.45,5 ℃ which is significantly higher than that of the pickle stored at 5 ℃ is 3.82 minutes and higher than that of the pickle stored at 20 ℃ by 2.82 minutes. Lactobacillus plantarum SCSL-1 ferments pickle to pH4.0, and has obvious weak post-acidification effect when stored at 5 ℃, so that the quality guarantee period of the pickle can be prolonged.
After the composite microbial inoculum ferments kimchi with 3wt.% salt content to pH4.0, the change of lactic acid bacteria at different storage temperatures (20 ℃ C., 5 ℃ C.) is shown in FIG. 5. From the graph, the strain grows rapidly in the pickle fermentation system, and the number of viable bacteria reaches 8.54Log CFU mL on the 1 st day of fermenting pickle by inoculating the microbial inoculum -1 The number of viable bacteria is maintained at 10 at 20 ℃ and 5 DEG C 8 Log CFU mL -1 The number of viable bacteria is slightly reduced after the strain is stored for 10 days at 20 ℃, which indicates that the strain has high fermentation speed and strong viability and has more advantages on the number of viable bacteria when being stored at 5 ℃.
The texture and color of the fermented kimchi at 20 and 5 ℃ are shown in tables 4 and 5.
TABLE 4 texture variation of fermented kimchi during storage at 5 ℃ and 20 DEG C
TABLE 5 color Change of fermented kimchi during storage at 5.20 ℃ and 5 DEGC
As can be seen from tables 4 and 5, the fermented kimchi stored at 20deg.C has higher hardness, elasticity, chewiness, L at 5deg.C * And b * The value shows that the fermented pickle has better texture and color when stored at 5 ℃, has weak post-acidification effect, and effectively improves the problems of softening texture, yellowing color and browning of the fermented fruits and vegetables.
Table 6 shows the volatile flavor substances and contents of the kimchi by the single fermentation of Lactobacillus plantarum and the fermentation of the composite microbial inoculum.
TABLE 6 main volatile flavor component and relative content of SCSL-1 Mono-fungus compared with composite microbial agent fermented kimchi
In the table: "-" means not detected
As can be seen from table 6, the mixed fermented kimchi contains abundant and higher content of volatile flavor substances, among which sulfides and esters are the main volatile flavor substances, compared to the single SCSL-1 fermented kimchi. Sensory evaluation in fig. 6, five-point preference scale method is adopted to perform sensory evaluation on the kimchi, the average scores of the color, the crispness and the taste of the fermented kimchi are respectively 4.35, 4.20 and 4.15 when the fermented kimchi is stored at 5 ℃, and the average scores of the color, the crispness and the taste of the fermented kimchi are respectively 3.64, 3.25 and 3.58,5 ℃ when the fermented kimchi is stored at 20 ℃, so that the fermented kimchi has better color, texture and crispness, and the results show that the composite microbial inoculum has excellent fermentation effect, high fermentation speed, high fermentation food acceptability, obvious acidification effect after being combined with weak storage at 5 ℃, small acidity, texture and color change of the kimchi, and basically maintained viable count of the fruit and vegetable after weak fermentation.
The composite microbial inoculum has the advantages of high fermentation rate and stable fermentation quality. Fermenting at 20deg.C for 1 day until the pickle is ripe, storing at 5deg.C, wherein pH of the fermented vegetable is 3.7-3.8, TTA is 0.38g lactic acid/100 g fermented vegetable-0.39 g lactic acid/100 g fermented vegetable, and the viable count is maintained at 10 days 8 CFU/g has obvious weak post-acidification effect, more flavor substances are added by mixed fermentation, and the mixed fermentation contains abundant viable bacteria, so that the shelf life of the fermented vegetables can be prolonged, and meanwhile, the prepared fermented vegetables have better color and texture.
In the fermentation process, the temperature is a main measure for solving the problem of post acidification maturation, and the temperature can cause the change of the fluidity of the membrane and reduce the activity of enzyme, thereby influencing the transportation and metabolism of nutrient substances inside and outside the cell. It is notable that acid treatment is an unavoidable obstacle to the survival of lactic acid bacteria during fermentation, and that lower pH is a major factor in preventing lactic acid formation. In addition, the salt content added during the actual fermentation process also affects the metabolic acidogenesis of the strain by affecting the microbial growth. Therefore, the temperature, the initial pH and the salinity are key factors influencing the acidogenesis and the metabolism of the lactic acid bacteria, and provide key control points for regulating the shallow fermentation of the strain in the storage process of the fermented vegetables. The invention is used for preparing the weak post-acidification fermented fruit and vegetable products by adjusting the concentration of fermentation salt, the initial pH of storage and low temperature, and solves the post-fermentation acidification problem from the point of causing continuous acidification of the fermented products, namely the lactobacillus plantarum with strong acid production.
The composite microbial inoculum disclosed by the invention is combined with the Weissella PCYLJY-1 to ferment fruits and vegetables in the early stage, more flavor substances are added, the fermented vegetables have higher acidity preference, and obvious weak post-acidification effect, in addition, the lactobacillus plantarum SCSL-1 has excellent fermentation quality, the fermented vegetables can still keep original texture and color within one week, and the quantity of lactobacillus is abundant, so that the composite microbial inoculum has great application potential in solving the post-acidification problem of fermented fruits and vegetables.
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Claims (11)

1. Lactobacillus plantarum strainlactobacillus plantarum) The method is characterized in that the preservation number of the lactobacillus plantarum is CCTCC M20231830.
2. A microbial inoculum comprising the Lactobacillus plantarum strain according to claim 1lactobacillus plantarum）。
3. A composite microbial inoculum, characterized in that the composite microbial inoculum comprises the lactobacillus plantarum of claim 1lactobacillus plantarum) And fusion of WeissellaWeissella confusa) The method comprises the steps of carrying out a first treatment on the surface of the The number of the viable bacteria of the lactobacillus plantarum and the Weissella fusion in the composite microbial inoculum is more than or equal to 1 to 3 multiplied by 10 9 CFU/g。
4. The use of a microbial agent according to claim 2, wherein the microbial agent is used in the preparation of a fermented food product.
5. The use of a composite microbial agent according to claim 3, wherein the microbial agent is used in the preparation of a fermented food product.
6. A method of preparing a fermented food product comprising the steps of:
inoculating a microbial inoculum into the fermented food to be fermented for fermentation; the microbial inoculum is one of the microbial inoculum of claim 2 and the composite microbial inoculum of claim 3.
7. The method for producing a fermented food according to claim 6, wherein the fermented food is a fermented vegetable or a fermented fruit and vegetable juice.
8. A fermented vegetable obtained by the method of claim 6, having a salt content of 1wt to 5 wt%, an initial pH of 3.6 to 4.4, and a storage temperature of 5 ℃ or 10 ℃.
9. A fermented vegetable according to claim 8, characterized in that the fermentation process is as follows: the vegetable water ratio of the fermented vegetables is 1:2, and the salt content is 3wt percent; inoculating a microbial inoculum containing the fusion Weissella in fermented vegetables, fermenting for one day, inoculating a microbial inoculum containing the lactobacillus plantarum of claim 1, fermenting at normal temperature to pH4.0, and storing at 5 ℃.
10. A fermented juice obtained by the method of claim 6.
11. The fermented fruit and vegetable juice according to claim 10, wherein the fermentation process is as follows: the fruit and vegetable juice is obtained by squeezing vegetables or fruits and water according to a ratio of 1:2; inoculating the microbial inoculum to fruit and vegetable juice, fermenting at normal temperature to pH4.0, and storing at 5deg.C.
CN202311300722.4A 2023-10-07 2023-10-07 Lactobacillus plantarum for shallow fermentation of fruits and vegetables and preparation method of fermented fruits and vegetables Pending CN117187141A (en)

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