CN113151030A - Pediococcus pentosaceus, and method and application thereof for preparing natural strain old fertilizer - Google Patents
Pediococcus pentosaceus, and method and application thereof for preparing natural strain old fertilizer Download PDFInfo
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Abstract
The invention relates to Pediococcus pentosaceus (GBL-mf 1), which is preserved in China center for type culture Collection with the preservation number of CCTCC M20191128, and the preparation method comprises the steps of uniformly mixing fruits/grains, sugar and water to obtain fermentation liquor; adding the fermentation liquor into flour, and fermenting at normal temperature to obtain a first generation fermentation strain; taking the first generation fermentation strain, adding flour, fruit stock solution and water, and fermenting at normal temperature to obtain the second generation fermentation strain; placing the second generation fermentation strain into sterile water to obtain natural strain liquid; adding flour, water and soft sugar into the cultured natural strain bacterial liquid, and fermenting to obtain the natural strain old fertilizer. The invention not only improves the market competitiveness of related products, but also can create new vitality of traditional foods and comprehensively improve the industrial upgrading of the traditional Chinese wheaten foods.
Description
Technical Field
The invention belongs to the technical field of food fermentation, and particularly relates to pediococcus pentosaceus, a method for preparing natural strain old fertilizer by using the pediococcus pentosaceus and application of the pediococcus pentosaceus.
Background
The Chinese-style fermented flour food is a main part in the daily diet of Chinese people, such as steamed bread, steamed stuffed bun, bean paste bun and the like, has huge consumption, more and more consumers select industrial production of the fermented flour food, and the flour food processing industry is developing towards industrialization and presents the development trends of industrialization, mechanization and modernization. At present, the existing research at home and abroad shows that the yeast and the lactobacillus are suitable for the fermentation of the wheaten food, and the taste and the nutrient content are improved to a certain extent. However, a whole set of technical system for separating and screening natural strains from old fertilizers and fruits and applying the natural strains to Chinese traditional fermented wheaten food in cooperation with probiotic fermentation does not exist at home and abroad, and the application of the whole technical system in industrialization is not reported. For example, chinese patent — a pasta wrapper prepared by microbial fermentation and its preparation process (CN105918385A) disclose the following formulation of pasta wrappers prepared by microbial fermentation: high gluten flour: 20-30 parts of a solvent; buckwheat starch: 30-45 parts of a solvent; mustard flour: 4-6 parts; barley seedling powder: 0.5-0.8 part; powder baking: 0.25-0.5 part; yeast: 0.5-1 part; and (3) skim milk powder: 3.5-5 parts; salt: 2-3 parts of a solvent; food additive: 0.1 to 0.2 portion. However, pasta prepared by conventional yeast fermentation is deficient in both taste and nutritional ingredients.
Therefore, based on the problems, the pediococcus pentosaceus which not only improves the market competitiveness of related products, but also enables traditional foods to create new vitality and comprehensively improves the industrial upgrading of the traditional Chinese flour foods, the method for preparing the natural strain old fertilizer and the application thereof are provided, and the method has important practical significance.
Disclosure of Invention
The invention aims to solve the problems in the prior art and provides pediococcus pentosaceus which can not only improve the market competitiveness of related products, but also create new vitality of traditional foods and comprehensively improve the industry upgrading of the traditional Chinese flour foods, and a method and application for preparing natural strain old fertilizers thereof.
The technical scheme adopted by the invention to solve the problem is as follows:
the Pediococcus pentosaceus is classified as Pediococcus pentosaceus (Pediococcus pentosaceus) GBL-mf1, and is preserved in China center for type culture Collection (CCTCC NO): m20191128, preservation date of 2019, 12 months and 30 days.
The second purpose provided by the invention is as follows: the method for preparing the natural strain old fertilizer by pediococcus pentosaceus comprises the following steps:
the method comprises the following steps: uniformly mixing 90-110g of fruits/grains, 14-16g of sugar and 250g of water, and fermenting at normal temperature to obtain fermented fermentation liquor;
step two: adding 140 and 160g of fermented fermentation liquor into 150g of flour, fermenting at normal temperature for 24h, and refrigerating in a refrigerator at 4 ℃ for 10h to obtain a first-generation fermentation strain;
step three: taking 90-110g of the first generation fermentation strain, adding 100g of flour, 14-16g of fruit stock solution and 40g of water, fermenting at normal temperature for 24h, and transferring into a refrigerator at 4 ℃ for refrigeration to obtain the second generation fermentation strain;
step four: taking 9-11g of second-generation fermentation strain, putting into 90ml of sterile water, and stirring for 30min to obtain natural strain liquid, namely pediococcus pentosaceus liquid;
step five: culturing the obtained natural strain liquid until the viable count of the strain is 108CFU/mL, adding into flour, wherein 100 parts of flour, 9-11 parts of natural strain liquid, 51-61 parts of water and 0.9-1.1 parts of soft sugar, and fermenting at 25 ℃ and 40% RH for 24 hours to obtain the natural strain old fertilizer.
Preferably, the fruit is any one of plum and grape.
Preferably, the grain is wheat bran.
Preferably, the method also comprises a pediococcus pentosaceus purification identification experiment.
More preferably, 100 μ l of the natural strain liquid in step four is sucked for coating experiment, cultured at 28 ℃ for 48h, then a typical colony is picked for streaking separation, repeated for 2-3 times to obtain a single colony, the cell morphology is observed, and the pediococcus pentosaceus is obtained by screening.
Preferably, 10% of the natural strain old fertilizer in the fifth step is inoculated into 1kg of flour, 56% of water and 1% of soft sugar are added, and the mixture is fermented for 12 hours at the temperature of 25 ℃ and the relative humidity of 40%, so that the propagated natural strain old fertilizer is obtained.
Further preferably, the flour is wheat flour.
The third purpose provided by the invention is as follows: application of natural strain old fertilizer in preparation of flour products.
The pediococcus pentosaceus of the present invention has a taxonomic name of: pediococcus pentosaceus (GBL-mf 1 for short) has been deposited in China Center for Type Culture Collection (CCTCC) and Wuhan university Collection, and has a preservation number of CCTCC M20191128 and a preservation date of 2019, 12 months and 30 days, and the addresses of the preservation collections are as follows: in the Wuhan university school of eight routes 299 # Wuhan in the Wuchang area of Wuhan city, Hubei province (the first attached small opposite side of Wuhan university), the postal code is: 430061.
the invention has the advantages and positive effects that:
1. the invention screens natural strains from the traditional old fertilizer and fruits/grains, and prepares the product by multi-strain fermentation after strain purification and identification.
2. The quality of the Chinese fermented flour food is improved in the aspects of eating quality, nutritional characteristics and the like, the development of the key technology not only improves the market competitiveness of related products, but also enables the traditional food to create new vitality and comprehensively improves the industrial upgrading of the Chinese traditional flour food.
Drawings
The technical solutions of the present invention will be described in further detail below with reference to the accompanying drawings and examples, but it should be understood that these drawings are designed for illustrative purposes only and thus do not limit the scope of the present invention. Furthermore, unless otherwise indicated, the drawings are intended to be illustrative of the structural configurations described herein and are not necessarily drawn to scale.
FIG. 1 is a diagram illustrating the detection of the pH of a bolus made of a dough using a pH meter according to the present invention. Wherein, 1-4 in the abscissa represents 10%, 15%, 20% and 25% of the natural strain old fertilizer input amount in sequence, and the ordinate represents the PH value.
The pediococcus pentosaceus of the present invention has a taxonomic name of: pediococcus pentosaceus (GBL-mf 1), which is abbreviated as "GBL-mf 1", has been deposited in China Center for Type Culture Collection (CCTCC), Wuhan university Collection, with the Collection number of CCTCC M20191128, and the Collection date of 2019, 12 months and 30 days, and the address of the Collection is: in the Wuhan university school of eight routes 299 # Wuhan in the Wuchang area of Wuhan city, Hubei province (the first attached small opposite side of Wuhan university), the postal code is: 430061.
Detailed Description
First, it should be noted that the specific structures, features, advantages, etc. of the present invention will be specifically described below by way of example, but all the descriptions are for illustrative purposes only and should not be construed as limiting the present invention in any way. Furthermore, any single feature described or implicit in any embodiment or any single feature shown or implicit in any drawing may still be combined or subtracted between any of the features (or equivalents thereof) to obtain still further embodiments of the invention that may not be directly mentioned herein. In addition, for the sake of simplicity, the same or similar features may be indicated in only one place in the same drawing. The present invention will be specifically described with reference to fig. 1.
Example 1
Method for preparing natural strain old fertilizer
The natural wild strains in the grains, fruits or various old fertilizers and other microbial floras which exist together form a leaven, the leaven is mixed with flour and water to be fermented into sour dough, edible alkali is added to the sour dough to be made into Chinese wheaten food, the dough formed after the sour dough is mixed with the alkali can be used as the leaven for the next pattern of the noodles and can be recycled, and the old fertilizers are called as the old fertilizers.
The method comprises the following steps: uniformly mixing 90-110g of fruits/grains, 14-16g of sugar and 250g of water, and fermenting at normal temperature to obtain fermented fermentation liquor; preferably, 100g of wheat bran, 15g of sugar and 250g of water are uniformly mixed and fermented at normal temperature to obtain fermented fermentation liquor;
step two: adding 140 and 160g of fermented fermentation liquor into 150g of flour, fermenting at normal temperature for 24h, and refrigerating in a refrigerator at 4 ℃ for 10h to obtain a first-generation fermentation strain; preferably, 150g of fermented fermentation liquor is added with 150g of wheat flour, the mixture is fermented at normal temperature for 24h and then is refrigerated in a refrigerator at 4 ℃ for 10h to obtain a first-generation fermentation strain;
step three: taking 90-110g of the first generation fermentation strain, adding 100g of flour, 14-16g of fruit stock solution and 40g of water, fermenting at normal temperature for 24h, and transferring into a refrigerator at 4 ℃ for refrigeration to obtain the second generation fermentation strain; preferably, 100g of the first generation fermentation strain is taken, 100g of wheat flour, 15g of fruit stock solution and 40g of water are added, and after fermentation is carried out for 24 hours at normal temperature, the mixture is transferred to a refrigerator at 4 ℃ for refrigeration to obtain the second generation fermentation strain;
step four: taking 9-11g of second-generation fermentation strain, putting into 90ml of sterile water, and stirring for 30min to obtain natural strain liquid, namely pediococcus pentosaceus liquid; preferably, 10g of the second generation fermentation strain is put into 90ml of sterile water and stirred for 30min to obtain natural strain liquid;
step five: culturing the obtained natural strain liquid until the viable count of the strain is 108CFU/mL, adding into flour, wherein 100 parts of flour, 9-11 parts of natural strain liquid, 51-61 parts of water and 0.9-1.1 parts of soft sugar, fermenting at 25 ℃ and 40% RH for 24 hours to obtain natural strain old fertilizer; preferably, the wheat flour is 100 parts, the natural strain liquid is 10 parts, the water is 56 parts, and the soft sugar is 1 part.
Secondly, purifying and identifying strains in natural strain old fertilizer
The method comprises the following steps: screening
And (3) putting 10g of the obtained second-generation fermentation strain into 90ml of sterile water, stirring for 30min to obtain natural strain liquid, sucking 100 microliters of the liquid, performing a coating experiment, culturing for 48h at 28 ℃, selecting a typical bacterial colony, performing streak separation, repeating for 2-3 times to obtain a single bacterial colony, observing cell morphology, and screening to obtain the natural strain.
Culture medium: MRS broth culture medium (Beijing land bridge technology, Inc.), YPD broth culture medium (Biotechnology engineering (Shanghai), Inc.).
The pediococcus pentosaceus CCTCC M20191128 has the morphological characteristics that:
pediococcus pentosaceus, round colonies, opalescent, smooth, convex, neat-edged, opaque, gram-positive, cocci. Pediococcus pentosaceus (p. pentosaceus), spherical in cell shape, with a diameter of 0.8-1.0 μm. The optimal growth temperature is 35 ℃, and the maximum growth temperature is 42-45 ℃.
Step two: identification
The natural strain is sent to Beijing Liuhe Hua Dagen science and technology limited company for identification, the natural strain is judged to be Pediococcus pentosaceus (Pediococcus pentosaceus) GBL-mf1 according to the comprehensive analysis of the cell morphology, biochemical characteristics, 16S rRNA gene sequence and the like of the strain and the comparison in an NCBI database.
The identification experiment process comprises the following steps:
1. genomic DNA extraction
DNA electrophoresis detection
PCR amplification
16s primer sequence:
27f：AGAGTTTGATCMTGGCTCAG
1492R：TACGGYTACCTTGTTACGACTT
reaction system:
reaction conditions are as follows:
4. electrophoretic PCR product identification
5. Sequencing on the machine and outputting a peak diagram
The experimental results are as follows: the NCBI database comparison shows that the natural strain has the closest homology with the Pediococcus pentosaceus, is determined to belong to the Pediococcus pentosaceus and is named as Pediococcus pentosaceus (Pediococcus pentosaceus) GBL-mf 1.
Table 1: NCBI database alignment of the first 3 results
Third, natural strain old fertilizer sensory evaluation
The obtained natural strain old fertilizer, fruits, grains and industrial dry yeast are respectively inoculated into a 5% wheat flour solution, the pH value is measured after the natural strain old fertilizer, the fruits, the grains and the industrial dry yeast are cultured for 24 hours at normal temperature, and sensory evaluation is carried out on the flavor (shown in table 2).
Table 2: pH, odor evaluation and scoring
| Bacterial code | pH value | Type of producing | Scoring |
| Fruit | |||
| 1 | 5.91 | Sour, wheat-like smell, no |
3 |
| |
6.03 | Sour, special sweet and no peculiar smell | 3.5 |
| |
6.38 | Wheat fragrance without |
2 |
| |
6.47 | Slight sour taste, wheat flavor, no off- |
1 |
| Natural strain |
5.28 | Obvious sour taste, wheat fragrance and no peculiar smell | 5 |
| Natural strain |
5.46 | Obvious sour taste, wheat fragrance and no peculiar smell | 4.5 |
| Industrial dried yeast | 5.88 | Sour, wheat-like smell, no peculiar smell | 3.5 |
| Wheat blanc flour solution | 6.58 | Wheat fragrance without peculiar smell | 0 |
As can be seen from the table above, after the natural strain old fertilizer is added, the pH value of the flour solution is reduced, the pH value is smaller, the sourness is more obvious, and the score is higher.
Four, propagation of natural bacterial old fertilizer
10 parts of natural bacterial old fat dough/industrial dry yeast/fruit/grain was inoculated into 100 parts of wheat flour, 56 parts of water and 1 part of soft sugar were added, and fermentation was carried out at 25 ℃ and 40% RH for 12 hours to obtain dough, which was subjected to fermentation experiments (shown in Table 3).
Table 3: experimental results of dough fermentation
As can be seen from the above table, compared with industrial dry yeast, the dough yield is increased by adding the natural strain old fertilizer 1 and the natural strain old fertilizer 2, and the fermentation time is reduced.
Example 2
Application of natural strain old fertilizer in preparation of flour products
Adding the natural strain old fertilizer obtained in the example 2 into dry noodles according to the proportion of 1 part of wheat flour, 55-60% of water, 0.1-0.5% of edible alkali, 1% of soft sugar and 5-40% of the natural strain old fertilizer, and kneading to obtain the natural strain old fertilizer flour product.
Example 3
Application of natural strain old fertilizer in preparation of steamed bread
The obtained natural strain old fertilizer is added into dry noodles according to the following formula (shown in table 4) to knead the noodles. The ambient temperature is 25 ℃ and the humidity is 40%.
Table 4: formula for different old fertilizer input amounts
(ii) evaluation of quality
The quality analysis was performed on products made from different proportions of old fat dough (as shown in table 5).
Table 5: sensory evaluation results of products with different old fertilizer input amounts
| Formulation of | 25% old fertilizer | 30% old fertilizer | 35% old fertilizer | 40% old fertilizer |
| Specific volume | 2.36 | 2.53 | 2.52 | 2.48 |
| Height to diameter ratio | 0.618 | 0.656 | 0.647 | 0.633 |
| Compression relaxation property | 0.298 | 0.342 | 0.327 | 0.303 |
| PH | 5.19 | 5.11 | 5.07 | 4.98 |
| Sensory Scoring/100 | 85.9 | 87.5 | 87.8 | 85.2 |
The product with large specific volume and high height-diameter ratio has better appearance; the compression relaxation value is more than 0.3, and the product is soft and has good elasticity; the product has optimal flavor at pH 4.9-5.0. The quality of products with different old fertilizer input amounts is comprehensively evaluated, and the effect of adding 35% of natural strain old fertilizer is best.
Example 4
Application of natural strain old fertilizer in preparation of steamed stuffed buns
The obtained natural strain old fertilizer is added into dry noodles according to the following formula (shown in table 6) to be kneaded. The ambient temperature is 25 ℃ and the humidity is 40%.
Table 6: formula for different old fertilizer input amounts
Secondly, measuring the pH value of the agent prepared from the dough by using a pH meter, as shown in figure 1, the pH value of the dough is reduced after the natural strain old fertilizer is added, and the effect of reducing the pH value is more obvious when the old fertilizer is added. The obtained preparation was used for a preparation experiment, and the optimum pH during the use of the preparation was measured to be about 6.7.
(iii) quality evaluation
The quality analysis is carried out on products made of dough with different proportions of old fat, and because the products made of dough with 10% of old fat are poor in forming, the products are abandoned, and the products made of dough with 15%, 20%, 25% and 30% of old fat are selected.
Table 7: sensory evaluation results of products with different old fertilizer input amounts
| Formulation of | 15% old fertilizer | 20% old fertilizer | 25% old fertilizer | 30% old fertilizer |
| Specific volume | 1.28 | 1.32 | 1.29 | 1.23 |
| Height to diameter ratio | 0.551 | 0.602 | 0.582 | 0.558 |
| pH | 6.11 | 6.02 | 5.97 | 5.89 |
| Sensory Scoring/100 | 86.9 | 89.4 | 87.6 | 84.9 |
The product with large specific volume and large height-diameter ratio has better appearance, the lower the pH value, the richer the flavor substances, the pH value is between 4.9 and 5.0, the flavor is good, but the taste is not good, the most suitable pH value is between 5.8 and 6.2 from the aspects of overall taste and fermentation degree, the quality of the product with different old fertilizer input amounts is comprehensively evaluated, and the effect of adding 20 percent of the old fertilizer is the best from the table above.
Example 5
(Storage)
Coating a layer of wheat flour on the surface of the dough left every day, wrapping a preservative film, and storing in a sealed and cool place.
In conclusion, the pediococcus pentosaceus and the method for preparing the natural strain old fertilizer and the application thereof provided by the invention can not only improve the market competitiveness of related products, but also create new vitality of traditional foods and comprehensively improve the industrial upgrading of the traditional Chinese flour foods.
The present invention has been described in detail with reference to the above examples, but the description is only for the preferred examples of the present invention and should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made within the scope of the present invention shall fall within the scope of the present invention.
Sequence listing
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tacggytacc ttgttacgac tt 22
Claims (9)
1. The Pediococcus pentosaceus is classified as Pediococcus pentosaceus (Pediococcus pentosaceus) GBL-mf1, and is preserved in China center for type culture Collection (CCTCC M20191128) with the preservation number of CCTCC M20191128 and the preservation date of 30 months and 12 months in 2019.
2. The method for preparing natural strain old fertilizer according to claim 1, characterized in that: the method comprises the following steps:
the method comprises the following steps: uniformly mixing 90-110g of fruits/grains, 14-16g of sugar and 250g of water, and fermenting at normal temperature to obtain fermented fermentation liquor;
step two: adding 140 and 160g of fermented fermentation liquor into 150g of flour, fermenting at normal temperature for 24h, and refrigerating in a refrigerator at 4 ℃ for 10h to obtain a first-generation fermentation strain;
step three: taking 90-110g of the first generation fermentation strain, adding 100g of flour, 14-16g of fruit stock solution and 40g of water, fermenting at normal temperature for 24h, and transferring into a refrigerator at 4 ℃ for refrigeration to obtain the second generation fermentation strain;
step four: taking 9-11g of second-generation fermentation strain, putting into 90ml of sterile water, and stirring for 30min to obtain natural strain liquid, namely pediococcus pentosaceus liquid;
step five: culturing the obtained natural strain liquid until the viable count of the strain is 108CFU/mL, adding into flour, wherein 100 parts of flour, 9-11 parts of natural strain liquid, 51-61 parts of water and 0.9-1.1 parts of soft sugar, and fermenting at 25 ℃ and 40% RH for 24 hours to obtain the natural strain old fertilizer.
3. The method for preparing natural strain old fertilizer according to claim 2, characterized in that: the fruit is selected from one of fructus Pruni Salicinae and fructus Vitis Viniferae.
4. The method for preparing natural strain old fertilizer according to claim 2, characterized in that: the cereal is testa Tritici.
5. The method for preparing natural strain old fertilizer according to claim 2, characterized in that: and further comprises a pediococcus pentosaceus purification and identification experiment.
6. The method for preparing natural strain old fertilizer according to claim 5, characterized in that: and (3) sucking 100 mu l of the natural strain liquid in the step four for a coating experiment, culturing at 28 ℃ for 48h, selecting typical colonies, carrying out streaking separation, repeating for 2-3 times to obtain single colonies, observing cell morphology, and screening to obtain pediococcus pentosaceus.
7. The method for preparing natural strain old fertilizer according to claim 2, characterized in that: and (3) inoculating 10% of the natural strain old fertilizer in the fifth step into 1kg of flour, adding 56% of water and 1% of soft sugar, and fermenting at the temperature of 25 ℃ and the relative humidity of 40% for 12 hours to obtain the propagated natural strain old fertilizer.
8. The method for preparing natural strain old fertilizer according to claim 7, characterized in that: the flour is wheat flour.
9. Use of a natural bacterial manure according to any one of claims 2 to 8 in the preparation of a pasta product.
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