CN115820515A - Preparation of cheap lactobacillus PTG medium and application of cheap lactobacillus PTG medium in pickled vegetable fermentation - Google Patents
Preparation of cheap lactobacillus PTG medium and application of cheap lactobacillus PTG medium in pickled vegetable fermentation Download PDFInfo
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- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
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Abstract
The invention provides a cheap lactobacillus PTG culture medium and application thereof in pickled vegetable fermentation, belonging to the technical field of food fermentation. Lactic acid bacteria OD by single factor and Box-Behnken response surface optimization test 600 The value is an evaluation index, and the optimized lactobacillus PTG culture medium is as follows: 20% of potato extract, 21% -26% of tomato juice, 2% -2.5% of glucose and 6.0-6.2 of initial pH. Culturing lactobacillus seed liquid with the PTG culture medium, artificially inoculating fermented pickled Chinese cabbage at room temperatureThe finished pickled Chinese cabbage fermented by the optimized process has mellow taste, soft sour taste and nitrite content far lower than the national standard. Compared with the traditional method for artificially fermenting the pickled Chinese cabbage, the method disclosed by the invention fully utilizes the organic combination and potential effects of natural raw materials, replaces the traditional expensive raw materials such as peptone, lactalbumin and the like, has the advantages of good thallus proliferation effect, low cost, strong operability, short fermentation time and quick acid production, and is an effective method for fermenting the pickled Chinese cabbage.
Description
Technical Field
The invention belongs to the technical field of food microbial fermentation, and particularly relates to an application of a cheap culture medium PTG (Potatto Tomato Glucose) in culture of lactic acid bacteria and fermentation of pickled Chinese cabbage.
Background
Lactic Acid Bacteria (LAB) are a general term for a group of bacteria that can utilize fermentable carbohydrates to produce large amounts of lactic acid. The bacteria are widely distributed in nature, have abundant species diversity, at least comprise 18 genera, and more than 200. Except a few, many species of the lactic acid bacteria are human intestinal host bacteria, and substances such as organic acid, special enzyme systems, lactobacillin and the like generated by the fermentation of the lactic acid bacteria have important significance in the aspects of maintaining the balance of human intestinal flora, decomposing harmful substances in intestinal tracts, improving the immunity of organisms and the like. Lactic acid bacteria are not only ideal materials for research classification, biochemistry, heredity, molecular biology and genetic engineering, but also have extremely high application values in important fields closely related to human life, such as industry, agriculture and animal husbandry, food, medicine and the like.
The lactobacillus is a heterotrophic bacterium, needs to obtain a carbon source, a nitrogen source, growth factors and the like from a living environment to finish the metabolic growth and the propagation process, has strong dependence on vitamins, and is a growth factor necessary for the growth and the metabolism of part of the lactobacillus. The potato contains rich nutrient components such as plant polysaccharide, protein, vitamins and the like, and can provide a carbon source, a nitrogen source and vitamins for the growth of lactic acid bacteria; the tomato contains more vitamins, inorganic salts and saccharides, which can provide energy and nutrient substances for the growth and metabolism of lactic acid bacteria. Therefore, the selection of materials in the low-cost PTG culture medium designed by the invention can meet the growth and metabolism requirements of lactic acid bacteria.
Disclosure of Invention
The common lactobacillus culture medium is an MRS culture medium, the traditional MRS culture medium is composed of beef extract, peptone, yeast extract and the like, and the raw materials are expensive; the Tween 80 reagent in MRS is viscous, and the sampling accuracy in operation is low; the MRS culture medium has more component types, larger change range of the concentration of the required trace elements and difficult operation. Meanwhile, nitrogen sources selected for culturing lactic acid bacteria at present generally come from animal proteins such as beef extract, peptone, lactalbumin and the like, are expensive, and the preparation cost of downstream fermented pickled Chinese cabbage is increased. Aiming at the problems, the invention selects common cheap natural plant raw materials, makes full use of organic combination and potential efficacy of the natural raw materials, replaces the complex and expensive raw materials, is used for the seed culture medium of the lactic acid bacteria, and can culture a large amount of stable lactic acid bacteria by the PTG culture medium obtained by optimizing the components of the culture medium, thereby achieving good proliferation effect. The PTG culture medium is applied to the fermentation of the artificial pickled Chinese cabbage, compared with the current similar fermented pickled Chinese cabbage, the fermentation time is obviously shortened, the problem that the low-temperature long-time fermentation is needed for fermenting the pickled Chinese cabbage at present is solved, and the prepared pickled Chinese cabbage finished product is savoury and mellow in taste and soft in sourness.
The invention comprises the following steps:
1. the preparation of the PTG culture medium and the application operation steps in the fermentation of the pickled Chinese cabbage are as follows:
(1) Subpackaging PTG liquid culture medium into conical flask, and sterilizing at 121 deg.C for 20-25min in autoclave; inoculating activated lactobacillus into PTG culture medium, standing at 37 deg.C in constant temperature incubator for 18-24 hr, and measuring OD 600 A value;
(2) Immersing the pretreated Chinese cabbage in 1.5% (m/v) salt solution containing 2% lactobacillus seed solution and having initial pH of 5.5, and fermenting at room temperature for 9 days.
2. The PTG culture medium related to the invention is as follows: the concentration of the potato extract is 20 percent, the concentration of the tomato juice is 21 to 26 percent, the concentration of the glucose is 2 to 2.5 percent, the initial pH value is 6.0 to 6.2, and the lactobacillus is inoculated for culturing for 18 to 24 hours;
(1) The preparation method of the potato leachate comprises the steps of cleaning a certain amount of potatoes, peeling, cutting into small pieces, adding 1000ml of distilled water, boiling, filtering with gauze, slightly cooling, and then supplementing water to 1000ml for later use;
(2) The tomato juice is prepared by weighing a certain amount of tomatoes, crushing for 1min in a juicer, filtering the crushed liquid with three layers of gauze to obtain tomato filtrate, centrifuging at 4000r/min in a centrifuge for 15min, and collecting the supernatant.
3. The lactobacillus sauerkraut seed liquid is prepared by mixing lactobacillus paracasei and leuconostoc mesenteroides PTG seed liquid according to the proportion of 1:1, mixing in proportion;
(1) Lactobacillus paracasei seed liquid: inoculating lactobacillus paracasei into a PTG liquid culture medium, and culturing at 37 ℃ for 18-24h;
(2) Leuconostoc mesenteroides seed liquid: leuconostoc mesenteroides is inoculated into PTG liquid culture medium and cultured for 18-24h at 37 ℃.
4. The pickled Chinese cabbage fermented by the optimal process is fermented for 7-9 days, the pH value of the prepared pickled Chinese cabbage is stabilized at 3.3, the nitrite content is stabilized at 1.5mg/kg, and the pickled Chinese cabbage finished product is fragrant and tasty, has soft sour taste and is clear in vegetable juice.
5. The PTG culture medium of the invention can be applied to all lactic acid bacteria including leuconostoc mesenteroides, lactobacillus paracasei and pediococcus acidilactici: the invention also comprises the application of the cheap lactic acid bacteria PTG culture medium prepared by the method in any one of the 1-5 in the fermentation of the pickled Chinese cabbage.
Aiming at the prior art, the invention has the advantages that:
1. the lactobacillus culture medium is cheap, the raw materials are easy to obtain, the thallus multiplication effect is good, and the operability is strong;
2. the PTG culture medium is adopted to culture the lactobacillus seed liquid, the pickled Chinese cabbage is fermented at room temperature, the fermentation time is short, the acid production is quick, the finished pickled Chinese cabbage product is fragrant and tasty, the sour taste is soft, the color of the leaf is bright, and the juice is sour and fragrant and clear. The invention provides a feasible, low-cost, convenient and effective preparation method for industrially producing artificially fermented pickled Chinese cabbage, and provides effective parameters for annual production of pickled Chinese cabbage.
Drawings
FIG. 1 is a flow chart of the production process of the present invention
FIG. 2 concentration of potato extract versus three lactic acid bacteria OD 600 Influence of the value
FIG. 3 concentration of tomato juice versus three lactic acid bacteria OD 600 Influence of the value
FIG. 4 glucose concentration versus OD of three lactic acid bacteria 600 Influence of the value
FIG. 5 initial pH of the medium versus OD of the three lactic acid bacteria 600 Influence of the value
FIG. 6 growth curves of three lactic acid bacteria in MRS and PTG media
FIG. 7 influence of different salt concentrations on pickled Chinese cabbage quality
FIG. 8 influence of different inoculum sizes on pickled Chinese cabbage quality
FIG. 9 Effect of different initial pH on pickled vegetable quality
FIG. 10 shows the pH and nitrite content trend of pickled Chinese cabbage fermentation for nine days
FIG. 11 sensory evaluation of pickled Chinese cabbage products
Detailed Description
Example 1: PTG medium preparation and strain activation
(1) PTG Medium preparation
Mixing potato leachate and tomato juice, adding 2 vol% glucose, adjusting pH to 6.2, packaging into conical flask, and sterilizing at 121 deg.C for 20-25 min.
1) Preparing a potato leaching solution: cleaning a certain amount of potatoes, peeling, cutting into small pieces, adding 1000ml of distilled water, boiling, filtering with gauze, slightly cooling, and supplementing water to 1000ml for later use.
2) Preparing tomato juice: weighing a certain amount of tomatoes, crushing for 1min in a juicer, filtering the crushed liquid through three layers of gauze to obtain tomato filtrate, centrifuging for 15min at 4000r/min in a centrifuge, and taking supernatant for later use.
(2) Activation of bacterial cells
Inoculating lactobacillus preserved in a glycerol tube into a sterilized MRS liquid culture medium according to the inoculation amount of 1%, standing and culturing at 37 ℃ for 48-72h for activation, and then transferring for two generations for later use.
Example 2: single factor test
(1) Selection of optimum concentration of potato extract
Respectively taking 800ml of 10%,15%,20%,25%,30% potato extract, 200ml of tomato juice, and 2% glucose, inoculating lactobacillus into the culture medium at 3%, standing at 37 deg.C for 20 hr, and measuring OD 600 As can be seen from the results of FIG. 2, OD was obtained in the range of 10% to 20% in the potato extract concentration as the concentration of the potato extract increased 600 Gradually increase when the concentration exceedsAt 20%, the absorbance of the three strains tended to decrease significantly. Therefore, the optimum adding amount of the potato extract is determined to be 20%.
(2) Selection of optimum concentration of tomato juice
The concentration of the potato extract is added according to the optimum value determined by the test, the tomato juice is added according to the proportion of 10 percent, 15 percent, 20 percent, 25 percent and 30 percent (namely the volume percentage of the tomato juice in 1000mL culture medium), the concentration of glucose is 2 percent, and the shortage of water is supplemented to 1000mL. Inoculating lactobacillus into the culture medium at 3%, standing at 37 deg.C for 20 hr, and as shown in FIG. 3, the absorbance increases significantly with the increase of tomato juice concentration, and when the tomato juice concentration is 20%, pediococcus acidilactici and Leuconostoc mesenteroides OD 600 The maximum value, lactobacillus paracasei OD, when the tomato juice concentration is 25% 600 The value is maximum.
(3) Selection of optimal concentration of glucose
Adding potato extract and tomato juice at the optimum concentration determined by the above test, adding glucose at 1%,1.5%,2%,2.5%, and 3% by volume, inoculating lactobacillus at 3% into the above culture medium, standing at 37 deg.C for 20 hr, and measuring OD 600 The value is obtained. As can be seen from the results in FIG. 4, the OD of Leuconostoc mesenteroides and Pediococcus acidilactici reached 2% when the glucose concentration reached 600 The value was the maximum, and the cell concentration was the highest. When the glucose concentration reached 2.5%, lactobacillus paracasei OD 600 The value is maximum.
(4) Selection of optimum starting pH
The potato extract, tomato juice and glucose concentrations were added at the optimum values determined in the previous experiment and the initial pH of the medium was adjusted to 5.8,6.0,6.2,6.4,6.6, respectively. Inoculating three kinds of lactobacillus into the above culture medium at 3%, standing at 37 deg.C for 20 hr, and measuring OD 600 As can be seen from FIG. 5, the absorbance was maximized at the initial pH of 6.2 for Lactobacillus paracasei and Pediococcus acidilactici, and at the initial pH of 6.0 for Leuconostoc mesenteroides.
Example 3: response surface test
Selecting the concentration (A) of the potato extract and the concentration (A) of the potato extract according to the factors and the ranges determined by the single-factor testThe concentration of tomato juice (B) and the concentration of glucose (C) are used as independent variables, and three strains OD are used 600 Value (R) 1 )(R 2 )(R 3 ) And (5) carrying out Box-Behnken response surface optimization test design for response values.
TABLE 1 Pediococcus acidilactici and Leuconostoc mesenteroides Box-Behnken test design factor level coding table
TABLE 2 Lactobacillus paracasei Box-Behnken test design factor level coding Table
Response surface test design and results
TABLE 3 Pediococcus acidilactici response surface test design and results
TABLE 4 Pediococcus acidilactici OD 600 Analysis of variance of value regression modeling equation
(1) Pediococcus acidilactici OD according to Design-Expert13 software 600 Optimizing the test result to carry out multiple regression fitting analysis, and obtaining a fitting equation as follows: r 1 ＝+0.5532-0.001875*A+0.006375*B-0.00075*C-0.004*A*B+0.00475*A*C-0.00425*B*C-0.04385*A 2 -0.02335*B 2 -0.0201*C 2 . In the response surface model, the coefficient R is determined by analysis of variance 2 =0.9838, integral dieType reaches a remarkable level (P)<0.0001 No outliers were significant (P = 0.7935)>0.05 Correction decision coefficient R) ("correction decision coefficient R"), 2 Adj =0.9630 and a coefficient of variation (c.v.) of 1.11%, the model better describes the test results.
TABLE 5 Leuconostoc mesenteroides response surface test design and results
TABLE 6 Leuconostoc mesenteroides OD 600 Analysis of variance of value regression modeling equation
(2) Performing multiple regression fitting analysis on the results of the optimization test of the table 6 by using Design-Expert13 software to obtain the Leuconostoc mesenteroides OD 600 The fitted equation with values as response values is: r 2 ＝+0.5502+0.001125*A+0.0044375*B+0.00125*C-0.00675*A*B+0.002*A*C-0.005*B*C-0.038975*A 2 -0.017475*B 2 -0.020225*C 2 . In the response surface model, the coefficient R is determined 2 =0.9613, significant levels were achieved for the global model (P =0.0004 < 0.05), insignificant terms for the outliers (P = 0.9652)>0.05 Correction decision coefficient R) ("correction decision coefficient R"), 2 Adj =0.9115, the coefficient of variation (c.v.) is 1.53%, and the model can better describe the test results.
TABLE 7 Lactobacillus paracasei response surface test design and results
TABLE 8 Lactobacillus paracasei OD 600 Analysis of variance of value regression modeling equation
(3) Performing multiple regression fitting analysis on the optimization test result of the lactobacillus paracasei 8 by using Design-Expert13 software to obtain lactobacillus paracasei OD 600 The fitted equation with values as response values is: r 3 ＝+0.6526+0.004875*A+0.005*B+0.000625*C-0.00975*A*B+0.0045*A*C-0.00125*B*C-0.0453*A 2 -0.01105*B 2 -0.0233*C 2 . In the response surface model, the coefficient R is determined 2 =0.9903, significant overall model level (P < 0.0001), insignificant mis-simulation term (P = 0.4891), and correction decision factor R 2 Adj =0.9778, the coefficient of variation (c.v.) is 0.7005%, and the model can better describe the experimental results.
TABLE 9 Pediococcus acidilactici response surface optimization results
TABLE 10 Leuconostoc mesenteroides response surface optimization results
TABLE 11 Lactobacillus paracasei response surface optimization results
And (3) determining the final culture medium components by referring to the best fitting extraction conditions and combining the actual conditions of a laboratory: the concentration of the potato extract is 20%, the concentration of the tomato juice is 21% -26%, the concentration of the glucose is 2% -2.5%, the initial pH value is 6.0-6.2, and the lactobacillus is inoculated for culture for 18-24h.
Example 3: growth Curve determination
Respectively inoculating lactobacillus into MRS liquid culture medium and optimized PTG liquid culture medium, culturing at 37 ℃ for 48h, sampling every 3h, diluting and coating a flat plate, calculating the number of viable bacteria, and drawing a growth curve.
The growth characteristics of the lactic acid bacteria are shown in FIG. 6.
Example 4: multi-strain compound pickled Chinese cabbage fermentation
(1) Preparation of lactic acid bacteria seed liquid
Lactobacillus paracasei and Leuconostoc mesenteroides were inoculated in the above-mentioned PTG liquid medium and cultured at 37 ℃ for 20 hours. When in use, two lactobacillus seed solutions are mixed according to the proportion of 1:1, mixing uniformly.
(2) Fermentation of pickled Chinese cabbage
1) Chinese cabbage pretreatment
Shredding fresh Chinese cabbage leaf, placing in a triangular flask, and microwave sterilizing for 30s each time for 2 min.
2) Influence of different salt concentrations on pickled vegetable quality
Cooling the Chinese cabbage leaves pretreated by microwave, respectively adding saline solution with the mass fractions of 1%,1.5%,2%,2.5%,3% and pH6, filling and sealing, inoculating lactic acid bacteria seed liquid with the mass fraction of 3% of the total mass of the Chinese cabbage in the saline solution, fastening with a sealing film, fermenting at room temperature, pulping the pickled Chinese cabbage sample at the 9 th day of fermentation, and measuring the pH and nitrite content of the pulp. As can be seen from fig. 7, the pH and nitrite content of the sample with a common salt content of 1% at the 9 th day of fermentation were not significantly different from those of the sample with a common salt content of 1.5%, and the flavor of sauerkraut was affected when the common salt content was low, and therefore, 1.5% was selected as the optimum common salt concentration.
3) Influence of different inoculation amounts on the quality of pickled Chinese cabbage
Cooling the Chinese cabbage leaves pretreated by microwave, adding saline solution with the mass fraction of 2% and the pH value of 6, filling and sealing, respectively inoculating lactic acid bacteria seed solutions with the total mass of 1%, 2%, 3%, 4% and 5% of the Chinese cabbage in the saline solution, fastening by using a sealing film, fermenting at room temperature, pulping the pickled Chinese cabbage sample at the 9 th day of fermentation, and measuring the pH value and the nitrite content of the pulp. As can be seen in FIG. 8, the pH of the 2% germ content pickled cabbage was lower and the nitrite content was the lowest on day 9 of fermentation. Therefore, 2% bacteria content was selected as the optimum bacteria content.
4) Effect of different initial pH on pickled vegetable quality
Cooling the cabbage leaves pretreated by microwave, adding salt water with pH of 5, 5.5, 6, 6.5, 7 and mass fraction of 2% to fill and seal, respectively inoculating lactobacillus seed liquid with 3% of total mass of cabbage in the salt water, fastening with a sealing film, fermenting at room temperature, pulping the pickled cabbage sample at the 9 th day of fermentation, and measuring the pH and nitrite content of the pulp. As can be seen from fig. 9, the initial pH of the pickled Chinese cabbage was lowest at 5.5, and the nitrite content was gradually increased as the initial pH was increased, so that the initial pH of 5.5 was selected as the optimum.
5) Determination of pickled Chinese cabbage parameters under optimal fermentation process
And (3) performing pickled vegetable fermentation by using the optimal salt concentration, the inoculation amount and the initial pH, fermenting for 9 days at room temperature, and measuring the pH and the nitrite content of the pickled vegetable for 9 continuous days. And performing sensory evaluation analysis on the pickled Chinese cabbage fermented on the 9 th day.
TABLE 12 sensory evaluation criteria
After the cabbage is subjected to microwave interval sterilization treatment, 1.5% of salt is added, 2% of composite lactobacillus suspension is inoculated, the initial pH value of fermentation is adjusted to be 5.5, the pH value and the nitrite content of the pickled cabbage are measured in different fermentation time, the pH value and the nitrite content are continuously measured for 9 days, and the test results are shown in figures 10-11. The taste, mouthfeel and color of the pickled Chinese cabbage finished product after fermentation are obviously improved compared with the pickled Chinese cabbage finished product before optimization, the taste is fragrant and mellow, the sour taste is soft, the vegetable juice is clear, and the color is bright.
Claims (6)
1. The preparation method of a cheap lactic acid bacteria PTG (lactic acid bacteria PTG) culture medium and the application of the PTG culture medium in pickled vegetable fermentation are characterized by comprising the following steps:
(1) Subpackaging PTG liquid culture medium into conical flask, sterilizing at 121 deg.C for 20-25min, inoculating activated lactobacillus into PTG culture medium, standing at 37 deg.C in constant temperature incubator for 18-24 hr, and measuring OD 600 A value;
(2) Immersing the pretreated Chinese cabbage in 1.5% (m/v) salt solution containing 2% lactobacillus seed solution and having initial pH of 5.5, and fermenting at room temperature for 9 days.
2. The method for producing a medium according to claim 1, wherein the PTG medium is: the concentration of the potato extract is 20 percent, the concentration of the tomato juice is 21 to 26 percent, the concentration of the glucose is 2 to 2.5 percent, the initial pH value is 6.0 to 6.2, and the lactobacillus is inoculated for culturing for 18 to 24 hours;
(1) The preparation method of the potato leachate comprises the steps of cleaning a certain amount of potatoes, peeling, cutting into small pieces, adding 1000ml of distilled water, boiling, filtering with gauze, slightly cooling, and then supplementing water to 1000ml for later use;
(2) The tomato juice is prepared by weighing a certain amount of tomato, crushing in a juicer for 1min, filtering the crushed liquid with three layers of gauze, centrifuging the obtained tomato filtrate in a centrifuge at 4000r/min for 15min, and collecting the supernatant for use.
3. The lactic acid bacteria seed solution according to claim 1, wherein the lactic acid bacteria seed solution is prepared by mixing lactobacillus paracasei and leuconostoc mesenteroides PTG seed solution in a ratio of 1:1, mixing in proportion;
(1) Lactobacillus paracasei seed liquid: inoculating lactobacillus paracasei into a PTG liquid culture medium, and culturing at 37 ℃ for 18-24h;
(2) Leuconostoc mesenteroides seed liquid: leuconostoc mesenteroides is inoculated into PTG liquid culture medium and cultured for 18-24h at 37 ℃.
4. The preparation method of claim 1, wherein the pH of the prepared sauerkraut is stabilized at 3.3 and the nitrite content in the sauerkraut is stabilized at 1.5mg/kg after 7-9 days of fermentation; the finished pickled Chinese cabbage product is fragrant and tasty, the sour taste is soft, and the vegetable juice is clear.
5. The method according to claim 1, wherein the PTG medium of the present invention is used in a range including all lactic acid bacteria including Leuconostoc mesenteroides, lactobacillus paracasei, and Pediococcus acidilactici.
6. Use of the cheap lactic acid bacteria PTG medium prepared according to the method of any one of claims 1 to 5 for fermenting sauerkraut.
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