CN117187143A - Lactobacillus plantarum ZF624 and application thereof - Google Patents
Lactobacillus plantarum ZF624 and application thereof Download PDFInfo
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Abstract
The application relates to the technical field of microorganisms, and particularly discloses lactobacillus plantarum ZF624 and application thereof. The application provides a lactobacillus plantarum ZF624, which has high pectin esterase activity, is used for preparing chopped peppers, can be used for carrying out demethylation on pectin in the peppers, converts high-methoxy pectin into low-methoxy pectin, and further reacts with Ca 2+ Generating pectate by using metal ions, and obviously improving brittleness of the chopped peppers; in addition, lactobacillus plantarum ZF624 can ferment chopped peppers and also can generate aroma substances of 3-methylthiophene and 3-)Methyl butyric acid increases the fragrance of the chopped peppers, so that the chopped peppers have strong fragrance, which is far higher than that of naturally fermented chopped peppers, and the flavor and quality of the chopped peppers are improved.
Description
Technical Field
The application belongs to the technical field of microorganisms, and particularly relates to lactobacillus plantarum ZF624 and application thereof.
Background
The chopped chilli is a chilli food which is prepared by taking chopped fresh chilli as a raw material, adding auxiliary materials such as salt and the like and pickling or fermenting in a closed jar, and can be directly eaten together with rice or bread due to unique flavor, fresh and spicy taste and crisp taste, and can be used for cooking vegetables and popular among people. In the current industrial production of chopped peppers, the production mode mainly comprises natural fermentation and salt embryo desalination fermentation. The natural fermentation period is long and the production is unstable; the salt embryo desalination and fermentation cost is lower, the processing technology is simple, but the loss of flavor, taste and nutrient substances in the longer storage period and the washing desalination process is serious.
In addition, the brittleness of the chopped peppers is reduced in shelf life, softening is easy to occur, and the reduction of commodity value is also a ubiquitous important problem for limiting the industrialized development of the chopped peppers. Aiming at softening of fermented fruits and vegetables, two methods of increasing pectate content in pickle tissues and shortening fermentation period by artificially inoculating lactobacillus are mainly adopted at present to improve the brittleness of pickle. The addition of exogenous pectin methylesterase or metal ions can increase pectate content, but can bring about poor flavor, while not meeting the needs of modern consumers for cleaning labels. The chopped peppers are fermented by artificially inoculating lactobacillus, and the quality of the product is relatively stable, but the flavor of the fermented product is common due to single fermentation bacteria. Studies have shown that proper high salt pre-curing and pre-heating treatments also increase the crispness of the cured vegetables, but tend to impair the flavor and mouthfeel of the vegetables. In the patent CN201510326039.7, fermentation broth prepared from lactobacillus, acetobacter, hydrocarbon-decomposing corynebacterium and saccharomycetes is poured into the blanched fresh red pepper, and the obtained chopped red pepper has unique flavor and is fresh red and crisp. However, the flavor of the blanched peppers is generally inferior to that of fresh peppers, and the fermentation mode involves more strains, has a complex process and is not beneficial to expanding production.
Disclosure of Invention
Based on the technical problems, the application mainly aims to overcome the defects of the background technology, and provides lactobacillus plantarum (Lactiplantibacillus plantarum) ZF624 which has high pectin esterase activity and is used for preparing chopped peppers, so that the fermentation period of the chopped peppers can be shortened, the brittleness of the shelf life of the chopped peppers can be obviously improved, and meanwhile, aroma substances 3-methylthiophene and 3-methylbutyric acid can be generated, the aroma of the chopped peppers can be increased, and the flavor and quality of the chopped peppers can be improved.
The inventors have conducted intensive studies to achieve the above object, and have completed obtaining the present application by repeating the study demonstration a plurality of times, specifically as follows:
in a first aspect, the application provides a lactobacillus plantarum ZF624, wherein the lactobacillus plantarum ZF624 has the preservation number of GDMCC No. 63685.
The lactobacillus plantarum ZF624 provided by the application is obtained by screening from thick broad-bean sauce, the form of the lactobacillus plantarum ZF624 on a solid MRS flat plate culture medium is 3-5 mm in colony diameter, the colony is milky white, the colony is convex and round, the surface is smooth, and the edge of the colony is neat. The strain was deposited at 25.2023, 7.and 25.with the cantonese collection of microorganism strains.
In a second aspect, the application also provides a zymophyte agent, which comprises the lactobacillus plantarum ZF624.
In a third aspect, the application also provides application of the lactobacillus plantarum ZF624 or the zymophyte agent in preparation of chopped peppers, in particular to application of improving the content of 3-methylthiophene and/or 3-methylbutyric acid in chopped peppers.
In a fourth aspect, the application also provides a preparation method of chopped peppers, which is a method for adding the lactobacillus plantarum ZF624 or a fermentation inoculant into peppers for fermentation.
Further, the addition amount of the lactobacillus plantarum ZF624 is 10 5 ～10 6 CFU/mL, preferably 10 6 CFU/mL。
Further, the fermentation temperature of the method is 28-32 ℃.
Further, the fermentation time of the method is 20-28h.
In a fifth aspect, the present application also provides chopped peppers prepared by the preparation method of the present application.
One or more technical solutions provided in the embodiments of the present application at least have the following technical effects or advantages:
1. the lactobacillus plantarum ZF624 provided by the application has high pectin esterase activity, can be used for preparing chopped peppers, can be used for carrying out demethylation and esterification on pectin in the peppers, and can be used for converting high-methoxy pectin into low-methoxy pectin so as to further react with Ca 2+ And pectate is generated by the plasma metal ions, so that the brittleness of the chopped peppers is obviously improved.
2. The lactobacillus plantarum ZF624 provided by the application can ferment chopped peppers to generate high-concentration 3-methylthiophene and 3-methylbutyric acid; 3-methylthiophene mainly has the flavor of orange, lemon, fruit, etc., and has a smell similar to that of fried foods, and is widely used as a food essence and flavor additive; 3-methylbutyric acid mainly has creaminess, fruity flavour and sourness, and is often used as a component of food additives and fragrances for enhancing the fruity flavour of foods; lactobacillus plantarum ZF624 can ferment chopped peppers to increase the content of 3-methylthiophene and 3-methylbutyric acid in chopped peppers, and the fermented chopped peppers have strong fragrance without using additives, which is far higher than that of naturally fermented chopped peppers.
3. The storage process of chopped peppers fermented by lactobacillus plantarum ZF624 provided by the application has a slow tendency of brittleness reduction, the content of volatile flavor substances is 3-methylthiophene, the variation range of the 3-methylthiophene in the shelf life is small, the better brittleness and fragrance can be kept in the shelf life, and the flavor and quality of chopped peppers are improved.
Drawings
FIG. 1 is a colony morphology of Lactobacillus plantarum ZF624 on MRS plates;
FIG. 2 is a morphological image of Lactobacillus plantarum ZF624 after gram staining by microscopy;
the lactobacillus plantarum (Lactiplantibacillus plantarum) ZF624 provided by the application is preserved in the Guangdong microbiological strain collection center at the address: building 5 of No. 59 of Mitsui 100 of Guangzhou City of Guangdong with the preservation number of GDMCC No. 63685; the strain was received from the collection at 25.7.2023 and registered, and was detected as a viable strain by the collection at 25.7.2023.
Detailed Description
The present application will be described more fully hereinafter in order to facilitate an understanding of the present application. This application may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
Unless otherwise indicated, the reagents, methods and apparatus employed in the present application are conventional food-grade reagents, methods and apparatus in the art.
The terminology used herein in the description of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.
EXAMPLE 1 screening of strains of interest
1. Raw material treatment
1g of soybean paste is taken into 9mL of sterile physiological saline, homogenized into uniform suspension, and diluted to 10 times by 10 times of sterile water -8 100 mu L of each concentration is respectively coated on MRS solid culture medium containing 0.012 per mill bromocresol purple solution, and the culture is carried out at the constant temperature of 37 ℃ for 48-72h. After the cultivation is finished, single colonies which turn yellow around the colonies are selected on a solid MRS plate, the MRS solid culture medium is inoculated, plate streaking separation is carried out, and the steps are repeated for 2 to 3 times until pure single colonies are obtained.
2. Bacterial strain primary screening
The selected colonies were amplified with the use of universal primers for sequencing analysis of the 16S rDNA sequences, after sequence acquisition, BLAST analysis was performed at NCBI to determine the colony species from which edible representative strains were selected for subsequent testing. 8 strains were screened together and were numbered FJ041-FJ048.
3. Bacterial strain re-screening
(1) Strain activation: sucking 0.1mL of preserved bacterial liquid by using a sterile pipetting gun, inoculating the preserved bacterial liquid into a 100mLMRS liquid culture medium for activation, controlling the temperature to 37 ℃, and standing and culturing for 24 hours; inoculating 1mL of activated bacterial liquid into a 100mLMRS liquid culture medium for activation, controlling the temperature to 37 ℃, and performing stationary culture for 24 hours; and then 1mL of secondary activated bacteria liquid is inoculated into a 100mLMRS liquid culture medium for expansion culture, the temperature is controlled to be 37 ℃, the static culture is carried out for 24 hours, after the culture is finished, the upper fermentation liquid is centrifugally removed, and 100mL of distilled water is added into the lower sediment to prepare the fermentation seed liquid for standby.
(2) And (3) fermentation of chopped peppers: cleaning the red pepper and the pricklyash peel with tap water, removing pedicel, and cutting into 0.5cm x 0.5cm blocks for standby; cutting peeled Bulbus Allii into Bulbus Allii powder; boiling the glass jar in boiling water for 10min, taking out and cooling for standby; adding 17% tap water, 3% salt and 2% white sugar into the sterilized glass jar in turn, stirring to dissolve the salt and white sugar, adding 48% red pepper, 12% pricklyash peel and 2% garlic in turn after the salt and white sugar are completely dissolved, stirring uniformly, inoculating 12% seed liquid, fermenting, controlling the fermentation temperature to 30 ℃, standing and fermenting for 24 hours, and adding 2% salt and 2% white wine after the fermentation is finished.
(3) And (3) physical and chemical index detection: after the fermentation, samples were taken from each jar to detect the crispness and pectin esterase activity of the chopped peppers and the content of calcium pectate, and the detection results are recorded in table 1.
TABLE 1 detection results of physicochemical indexes of chopped peppers fermented by the primary screening strain
| Numbering device | Friability F max (g) | Pectin esterase activity (U/g) | Calcium pectate content (ug/g) |
| FJ041 | 3942 | 14.05 | 159.18 |
| FJ042 | 2523 | 8.77 | 132.18 |
| FJ043 | 3324 | 13.25 | 155.72 |
| FJ044 | 3125 | 9.60 | 140.72 |
| FJ045 | 3721 | 13.72 | 158.86 |
| FJ046 | 3256 | 9.21 | 135.13 |
| FJ047 | 2689 | 8.95 | 133.25 |
| FJ048 | 2601 | 8.83 | 132.79 |
| Natural fermentation group | 2975 | 10.71 | 147.27 |
As shown in Table 1, the crispness of the chopped peppers fermented by FJ041, FJ045 and FJ043 is highest, namely 3942g, 3721g and 3324g, respectively, the pectin esterase activities are 14.05U/g, 13.72U/g and 13.25U/g, and the calcium pectate contents are 159.18ug/g, 158.86ug/g and 155.72ug/g, respectively.
(4) Detecting the content of volatile flavor substances: detecting the contents of aroma substances 3-methylthiophene and 3-methyl butyric acid in the chopped pepper sample by GC-MS, and screening the strain with the highest aroma substance content. The results of the chopped pepper volatile flavor content are shown in table 2.
TABLE 2 results of the content of volatile flavor substances of chopped peppers by fermentation of different strains
| Strain name | 3-methylthiophene (μg/kg) | 3-methylbutyric acid (μg/kg) |
| FJ041 | 197.85 | 134.21 |
| FJ042 | 160.42 | 101.25 |
| FJ043 | 101.76 | 82.39 |
| FJ044 | 154.47 | 125.78 |
| FJ045 | 146.53 | 109.98 |
| FJ046 | 85.64 | 98.23 |
| FJ047 | 96.54 | 95.12 |
| FJ048 | 77.56 | 67.12 |
| Non-inoculated control group | 46.24 | 37.34 |
As is clear from the results in Table 2, the FJ041 samples had the highest volatile flavor contents, and the 3-methylthiophene and 3-methylbutyric acid contents were 197.85. Mu.g/kg and 134.21. Mu.g/kg, respectively.
(5) Sensory evaluation: a researcher 20 in the field of pepper fermentation research is selected to carry out sensory evaluation on chopped peppers, all samples are placed randomly, 4 indexes of color, aroma, taste and shape of each chopped peppers are evaluated and scored by an evaluator, the scoring interval is 1 to 100 points, and the average value of the scores of the samples is calculated. The evaluation criteria are detailed in Table 3, and the sensory scores are shown in Table 4.
TABLE 3 sensory evaluation criteria for chopped peppers
| Project | Color | Brittleness of the product | Sour feel | Fermented flavor |
| 20 to 25 minutes | Bright color and luster | Very fragile | Appropriately, is | Is rich in weight |
| 13 to 19 minutes | Slightly brown in color | Has brittleness of | Stronger or lighter | Is stronger |
| 6 to 12 minutes | Light yellow brown | Slightly fragile | Stronger or lighter | Lighter and lighter |
| 0 to 5 minutes | Dark brown | Soft rot | Very strong or very weak | Without any provision for |
TABLE 4 results of sensory scores of different strains fermented chopped peppers
| Strain name | Color scoring | Critical score | Acid feel score | Flavor scoring | Comprehensive score |
| FJ041 | 19.0 | 19.4 | 17.7 | 18.1 | 18.55 |
| FJ042 | 16.1 | 17.1 | 16.5 | 16.9 | 16.65 |
| FJ043 | 17.9 | 18.1 | 16.8 | 17.5 | 17.58 |
| FJ044 | 19.7 | 18.0 | 17.6 | 17.8 | 18.28 |
| FJ045 | 18 | 18.8 | 18.7 | 17.2 | 18.18 |
| FJ046 | 18.1 | 18.6 | 17.7 | 16.5 | 17.73 |
| FJ047 | 17.3 | 17.2 | 18.0 | 17.9 | 17.60 |
| FJ048 | 18.1 | 17.3 | 17.9 | 18.6 | 17.98 |
As is clear from the results in Table 4, the chopped pepper after FJ041 fermentation gave a maximum score of 18.55, and the strain had the highest content of 3-methylthiophene and 3-methylbutanoic acid, which are volatile flavors, and therefore FJ041 was the best choice.
The strain is lactobacillus plantarum, the morphological observation on a solid MRS plate culture medium is shown in figure 1, the colony diameter is 3mm-5mm, the colony is milky white, the colony is convex and circular, the surface is smooth, and the edge of the colony is neat. As shown in FIG. 2, the sample was designated as Lactobacillus plantarum ZF624, deposited at the Cantonese microorganism strain collection at 7.25.2023 under the accession number GDMCC No. 63685.
EXAMPLE 2 Lactobacillus plantarum ZF624 passage stability assay
Inoculating 0.2mL of lactobacillus plantarum ZF624 glycerol frozen stock into 100mL of MRS broth culture medium, standing and culturing for 24h at 37 ℃, adding 1% of the frozen stock into 100mL of MRS broth culture medium, standing and culturing for 24h at 37 ℃ to obtain first-generation fermentation liquor, culturing for 10 generations under the same culture conditions, and sampling for later use at 1 generation, 5 generation and 10 generation respectively.
Taking 1 generation, 5 generation and 10 generation fermentation liquor 1mL, centrifuging for 2 minutes at 12000rpm/min, washing with sterile physiological saline for 2-3 times, re-suspending, and diluting with proper multiple to obtain seed liquor. Inoculating seed solution according to the method of example 1 for chopped pepper fermentation, lactobacillus plantarum ZF624 with an inoculation ratio of 12% and a final concentration of 1×10 7 CFU/g, controlling the fermentation temperature to be 30 ℃, and standing and fermenting for 24 hours. After fermentation, the stability of the fermentation performance of lactobacillus plantarum ZF624 passage is determined by taking brittleness, pectin methylesterase activity, 3-methylthiophene and 3-methylbutyric acid content as indexes. The results are recorded in table 5, respectively.
TABLE 5 influence of seed fermentation on chopped peppers by different passage times
As shown in Table 5, the chopped peppers fermented by the lactobacillus plantarum ZF624 of the 1 st generation, the 5 th generation and the 10 th generation have no obvious difference in the indexes such as brittleness, pectin methylesterase activity, calcium pectate content, 3-methylthiophene, 3-methylbutyric acid content and the like, and the fermentation characteristics of the lactobacillus plantarum ZF624 are not changed and are relatively stable during the continuous culture of the 10 th generation, so that the chopped peppers are safe strains capable of being used for a long time.
Example 3 Effect of Lactobacillus plantarum ZF624 inoculation concentration on fermentation of chopped Capsici fructus
Inoculating 0.2mL of lactobacillus plantarum ZF624 glycerol frozen stock into 100mL of MRS broth culture medium, standing and culturing for 24h at 37 ℃, inoculating 1% of the frozen stock into 100mL of MRS broth culture medium, standing and culturing for 24h at 37 ℃, taking 1mL of fermentation liquor, centrifuging at 12000rpm for 2 minutes, washing 2-3 times with sterile physiological saline, re-suspending, diluting by a proper multiple, and preparing seed liquid.
Inoculating seed solution according to the method described in example 1, and fermenting with chopped pepper, wherein the seed solution addition ratio is kept to be 12%, and the inoculation final concentration of Lactobacillus plantarum ZF624 is 1×10 respectively 4 CFU/g、1×10 5 CFU/g、1×10 6 CFU/g、1×10 7 CFU/g, controlling the fermentation temperature to be 30 ℃, and standing and fermenting for 24 hours. The effect of different inoculation concentrations on the fermentation of chopped peppers was determined using crispness, pectin methylesterase activity, 3-methylthiophene, 3-methylbutyric acid content as indicators, and the results are recorded in table 6.
TABLE 6 Effect of different concentration Strain fermentation on chopped Capsici fructus results
As is clear from Table 6, inoculating Lactobacillus plantarum ZF624 at a certain concentration to ferment chopped peppers can significantly increase the brittleness of chopped peppers, and in a certain range, the higher the inoculating concentration, the higher the brittleness of chopped peppers, the higher the pectin esterase activity, calcium pectate, 3-methylthiophene and 3-methylbutyric acid content thereof, and when Lactobacillus plantarum ZF624 inoculating concentration is 1×10 6 When CFU/g, the crispness of chopped peppers reaches to be 3942g at the maximum, and simultaneously, the activity of pectin methylesterase, the content of calcium pectate and the content of volatile flavor substances 3-methylthiophene and 3-methylbutyric acid also reach to be the maximum; however, when the inoculum size of Lactobacillus plantarum ZF624 is 1×10 7 At CFU/g, the crispness of chopped peppers, the activity of pectate methyl esterase, the content of pectate calcium, the content of volatile flavor substances 3-methylthiophene and 3-methylbutyric acid are all reduced.
Example 4 Lactobacillus plantarum ZF624 fermentation chopped Capsicum shelf life experiment
Cleaning the red bell peppers and the pricklyash peppers with tap water, and removing pedicel for later use; cutting peeled Bulbus Allii into Bulbus Allii powder; boiling the glass jar in boiling water for 10min, taking out and cooling for standby; adding 17% tap water, 3% salt and 2% white sugar into sterilized glass jar, stirring to dissolve salt and white sugar, adding 48% red pepper, 12% pricklyash peel and 2% Bulbus Allii, stirring, adding 12% lactobacillus plantarum ZF624 seed solution (final concentration of 1×10) 6 CFU/g), controlling the fermentation temperature to be 30 ℃, standing and fermenting for 24 hours, and adding 2% of salt and 2% of white spirit after the fermentation is finished. Meanwhile, a naturally fermented sample is used as a shelf life control group.
After fermentation, solid-liquid separation is carried out by using sterilized gauze, and the mass of fermentation liquid and solid is respectively weighed. The fermentation broth is placed in a closed container and heated at 70 ℃ for 20min. After the temperature of the fermentation broth was reduced to 60 ℃, the fermentation broth was added to the solids and heated at 60 ℃ for 10min. After cooling to room temperature, the total mass of fermentation broth and solids was weighed and the lost water was made up with sterile water.
The fermentation liquor and the solid matters are put into 250mL sauce bottles according to the proportion, 150g of the total of the fermentation liquor and the solid matters are put into each bottle, and the bottles are respectively placed into a normal temperature oven, a 40 ℃ oven and a 50 ℃ oven. Physicochemical indexes such as brittleness, 3-methylthiophene, 3-methylbutyric acid content, etc. were sampled and examined according to the sampling arrangement of Table 7, and the results are recorded in Table 8.
Table 7 shelf life sample times and amounts
| Temperature (temperature) | Second week | Fourth periphery | Eighth week | Twelfth week | Sixteenth week |
| Normal temperature | 1 bottle each | 1 bottle each | 1 bottle each | 1 bottle each | 1 bottle each |
| 40℃ | 1 bottle each | 1 bottle each | 1 bottle each | 1 bottle each | 1 bottle each |
| 50℃ | 1 bottle each | 1 bottle each | / | / | / |
Table 8 results of detection of physical and chemical indicators of shelf life of chopped peppers
As can be seen from table 8, the overall crispness of the chopped peppers tended to decrease during shelf life, and the overall crispness of the samples inoculated with ZF624 fermentation tended to decrease less than the naturally fermented samples. The sample after being stored for sixteenth week at 40 ℃ and inoculated with ZF624 for fermentation has the brittleness of 2978, which is reduced by 24.5 percent compared with the initial sample; the brittleness of the naturally fermented sample was 1411, which was reduced by 52.6% compared to the initial sample. The brittleness of the sample inoculated with ZF624 fermentation is 2864 when stored for the fourth week at 50 ℃, and is reduced by 27.3% compared with the initial sample; the brittleness of the naturally fermented sample is 1396, which is 53.1% lower than that of the original sample. Therefore, the inoculation of ZF624 has better crisp-keeping effect on chopped pepper products in the shelf life and smaller change of brittleness. The content of the volatile flavor substances is 3-methylthiophene, the variation range of the 3-methylthiophene in the shelf life is smaller, and the fragrance of the chopped pepper product inoculated with ZF624 for fermentation in the shelf life is far higher than that of the chopped pepper product subjected to natural fermentation.
The above examples illustrate only a few embodiments of the application, which are described in detail and are not to be construed as limiting the scope of the application. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the application, which are all within the scope of the application. Accordingly, the scope of protection of the present application is to be determined by the appended claims.
Claims (10)
1. A lactobacillus plantarum ZF624, wherein the lactobacillus plantarum ZF624 has a deposit number of GDMCC No. 63685.
2. A fermenting bacterial agent, comprising lactobacillus plantarum ZF624 according to claim 1.
3. Use of lactobacillus plantarum ZF624 according to claim 1 or the zymophyte agent according to claim 2 for the preparation of chopped peppers.
4. Use according to claim 3, characterized in that the use is to increase the content of 3-methylthiophene and/or 3-methylbutanoic acid in chopped peppers.
5. A method for preparing chopped peppers, which is characterized by adding lactobacillus plantarum ZF624 according to claim 1 or the fermentation inoculant according to claim 2 to peppers for fermentation.
6. The preparation method according to claim 5, wherein the lactobacillus plantarum ZF624 is added in an amount of 10 5 ～10 7 CFU/mL。
7. The preparation method according to claim 6, wherein the lactobacillus plantarum ZF624 is added in an amount of 10 6 CFU/mL。
8. The process according to any one of claims 5 to 7, wherein the fermentation temperature of the process is 28 to 32 ℃.
9. The method of claim 8, wherein the fermentation time of the method is 20 to 28 hours.
10. Chopped chilli, characterized in that it is prepared by the preparation method according to any one of claims 5-9.
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