CN116286438A

CN116286438A - Lactobacillus plantarum from bean curd acid pulp and application thereof

Info

Publication number: CN116286438A
Application number: CN202210964770.2A
Authority: CN
Inventors: 朱运平; 赵炳钰; 赵红玲; 司雪晨; 任斐
Original assignee: Beijing Technology and Business University
Current assignee: Beijing Technology and Business University
Priority date: 2022-08-11
Filing date: 2022-08-11
Publication date: 2023-06-23
Anticipated expiration: 2042-08-11
Also published as: CN116286438B

Abstract

The invention provides lactobacillus plantarum from bean curd acid pulp and application thereof, belonging to the technical field of microorganism and food processing, wherein the lactobacillus plantarum SJ-L-1 has the preservation number of: CGMCC No.25209. The lactobacillus plantarum SJ-L-1 applied in the invention has the advantages of high growth speed, large acid yield, strong freezing resistance and high transglycosylation activity. The lactobacillus plantarum SJ-L-1 applied in the invention is a safe strain for food, and can be used for preparing the acid pulp of the fermented bean curd without the coagulant and the acid pulp bean curd by directly inoculating the fermentation liquor or directly throwing the starter. The method for preparing the acid pulp by inoculating SJ-L-1 shortens the preparation period of the acid pulp and avoids the safety risks such as mixed bacteria or pathogenic bacteria pollution caused by natural fermentation. By comparing the quality of the fermented bean curd without coagulant of the brine bean curd, the sour slurry bean curd, the L-1-16 and the SJ-L-1, the SJ-L-1 fermented bean curd is more uniform in structure and higher in flavor substance content. The lactobacillus plantarum SJ-L-1 is fermented to enable the glycosidic isoflavone to be converted into aglycone isoflavone to the greatest extent, so that the nutritional value of the bean curd is improved.

Description

Lactobacillus plantarum from bean curd acid pulp and application thereof

Technical Field

The invention relates to lactobacillus plantarum from bean curd acid pulp and application thereof, and belongs to the technical field of microorganisms and food processing.

Background

Bean curd is taken as a traditional food with Chinese characteristics, and is deeply favored by people from ancient times to the past. Not only is delicious, but also contains various essential amino acids and non-essential amino acids required by human body, and biological active ingredients such as isoflavone, flavone and vitamin, etc., and has certain health care effect on human body. The bean curd is prepared from soybean by cleaning, soaking, pulping, boiling, solidifying, pressing, and shaping. Wherein coagulation is critical, the coagulant can affect the microstructure, degree of formation and uniformity of the tofu. Common bean curd coagulants are gypsum, bittern, glucono Delta Lactone (GDL), acid pulp, etc. The sour slurry tofu prepared by taking the sour slurry as a coagulant is a traditional characteristic food in China, and the sour slurry tofu with characteristics in Shandong places in China is listed as a characteristic food as 'non-matter cultural heritage'. The sour slurry bean curd has the characteristics of good water retention, fine texture, high nutritive value and low salt content. However, since the acid pulp is a product of natural fermentation, and is greatly influenced by environmental factors, bacterial groups in the acid pulp are unstable, the quality of the acid pulp bean curd is unstable, the quality guarantee period is short, and industrial production is not realized yet.

Although partial researches show that the dominant microorganism flora in the acid pulp is lactic acid bacteria and partial strains are also screened, the functional researches on the strains are not deep enough, the strains still stay in a laboratory stage, and no direct-injection type strain which can be directly applied to industrial production exists. Some researchers transplanted industrial strains in dairy fermentation to the preparation of the acid pulp, but the defects of poor strain adaptability, requirement for mixed use of multiple strains, long fermentation time, improper flavor, non-ideal obtained bean curd texture and the like exist. Therefore, the specific lactobacillus strain of the acid pulp, which is suitable for industrialized pure seed preparation, is selected from the acid pulp, so that the acid pulp is free from the existing manual workshop production, and the problems of standardized and industrialized production are solved at first. The acid pulp is prepared by industrial pure species, so that the preparation efficiency of the acid pulp can be improved, and the food safety problem caused by mixed bacteria or pathogenic bacteria pollution caused by natural fermentation of the acid pulp can be effectively avoided.

On the other hand, the lactobacillus is used as human intestinal probiotics, has high edible safety, and has various probiotics effects of promoting digestion and absorption, maintaining intestinal flora microecological balance, regulating immunity and the like. If lactobacillus can be directly inoculated to soybean milk for fermentation to prepare fermented soybean curd, the steps of preparing the sour milk can be reduced, the nutrition composition in a soybean milk system can be changed, and the quality of the soybean curd is further improved. For example, lactobacillus can utilize nutrient components such as sugar, mineral matters and the like to carry out growth metabolism, various bioactive substances are generated, the absorption and utilization rate of the nutrient substances in the fermented food are improved, and the fermented finished product has unique flavor and higher nutritive value; organic acid generated by lactic acid bacteria metabolism in the fermentation process can directly induce aggregation and solidification of soybean protein. The key factors influencing the quality of fermented bean curd in the process are still the characteristics of the strain, the growth characteristics and the metabolic characteristics of the strain not only influence the aggregation behavior of soybean protein and further influence the quality of bean curd, but also influence the bioconversion way of raw materials in a soybean milk system and further influence the nutrition composition, the flavor characteristics and the like of bean curd, so that the screening of special strains suitable for the preparation of fermented bean curd is particularly important. Although some researches have been made to try the process of preparing fermented bean curd without coagulant by lactobacillus fermentation, the research on the quality of bean curd by the strain is not deep enough, and it is not easy to screen the strain which meets the requirements of strong adaptability, fast fermentation and good flavor and texture of bean curd.

Disclosure of Invention

In order to solve the existing problems, the invention provides lactobacillus plantarum from bean curd acid pulp and application thereof, and the specific technical scheme is as follows:

the invention provides a bean curd acid pulp-derived bacterial strain SJ-L-1 which is preserved in China general microbiological culture Collection center (CGMCC) with a preservation registration number of 25209 and a preservation date of 2022, 6 and 29 days.

Individual morphological characteristics of the strain SJ-L-1 provided by the invention: rod-like or short rod-like, single, paired or chain-like, gram-positive. Colony morphology characterization: the front surface of the colony is round, the side surface is slightly raised, milky white or light yellow, opaque, neat in edge, smooth in surface, soft and elastic in texture. The optimal growth temperature is 37 ℃, and the optimal pH is 6.2-6.4.

Physiological and biochemical characteristics of the strain SJ-L-1 provided by the application: carbon sources such as ribose, galactose, glucose, fructose, mannose, rhamnose, mannitol and the like can be used, and carbon sources such as glycerol, sorbose, inositol, starch, hepatic glucose, xylitol and the like cannot be used. Tween 40 cannot be hydrolyzed and citric acid cannot be utilized. Glucuronic acid and glucuronamide can be used, and serine, alanine, arginine, aspartic acid, histidine, acetoacetic acid, propionic acid, acetic acid, formic acid, mucic acid, quinic acid, D-glucaric acid, and the like cannot be used. Sensitive to lincomycin and insensitive to vancomycin.

Gene characterization of Strain SJ-L-1 provided herein: dDH values for Lactobacillus pentosus (Lactobacilli), lactobacillus plantarum (Lactobacilli splantarum) and Lactobacillus Argentina (Lactiplantibacillus argentoratensis) were 24.20%, 93.9% and 63.40%, respectively; ANI values with the proximal strains Lactobacillus pentosus (Lactobacilli), lactobacillus plantarum (Lactiplantibacillus plantarum) and Lactobacillus argentina (Lactiplantibacillus argentoratensis) were 82.11%, 99.24% and 95.56%, respectively; the Lactobacillus plantarum strain has a dDH value greater than the threshold value of 70% of the bacterial species and an ANI value greater than the threshold value of 95-96% of the proposed demarcation species.

The strain SJ-L-1 accords with the characteristics of lactobacillus plantarum through morphological characteristics, physiological and biochemical characteristics and genetic characteristic analysis.

The application provides an application of lactobacillus plantarum SJ-L-1 in preparation of a direct vat set starter, which comprises the following steps:

after the strain is activated, the strain is subjected to expansion culture in a 50mL conical flask for 24 hours at 37 ℃ to prepare concentrated bacterial liquid, then a protective agent solution is added into the bacterial suspension, the bacterial suspension is subjected to vacuum freeze drying, and finally the freeze-dried direct vat starter is preserved.

In the above steps, preferably, in the process of preparing the bacterial liquid, the cultured bacterial liquid is placed in a centrifuge, centrifuged at 8000 Xg at 4℃for 10min, and the bacterial cells are washed twice with 0.85% sterile physiological saline.

In the above steps, preferably, the protectant solution is skim milk powder+5% sucrose, and the ratio (v/v) of the bacterial liquid to the protectant is 1:1.

optionally, the application of the lactobacillus plantarum SJ-L-1 in preparing the sour slurry bean curd comprises the following steps:

inoculating lactobacillus plantarum SJ-L-1 into sterilized yellow serofluid by directly adding 0.1-0.5g/L starter or 1-3% (volume percentage) strain culture solution. The pH of the acid pulp can be reduced to pH3.6-3.8 after fermentation for 10-15h, the pH range of the acid pulp is reduced to curd after the completion of squatting at 90 ℃, and the acid pulp bean curd finished product with excellent texture, unique flavor and high yield can be prepared.

The application also provides application of the lactobacillus plantarum SJ-L-1 in preparation of the coagulant-free fermented bean curd.

Optionally, the application of lactobacillus plantarum SJ-L-1 in preparing the coagulant-free fermented bean curd comprises the following steps:

(1) The preparation of the bean cheese comprises the following steps: inoculating lactobacillus plantarum into sterilized soybean milk according to the inoculation amount of 0.1-0.5g/L of direct-vat starter, and fermenting for 20-26 hours at the temperature of 30-35 ℃ to form soybean cheese;

(2) And (3) bean curd fermentation: inoculating the soybean cheese into sterilized soybean milk according to the inoculation amount of 1-3% (volume percentage) strain culture solution, and fermenting at 30-35 ℃ until the pH is 5.0-6.2 respectively.

(3) Determination of squatting brain temperature: when the soybean milk is fermented to pH5.0-6.2, immediately heating to 75-95deg.C for squatting brain.

In the step (1), lactobacillus plantarum is preferably inoculated in the form of a bacterial suspension with an inoculum size of 3%. The preparation method of the lactobacillus plantarum bacterial suspension specifically comprises the following steps: inoculating lactobacillus plantarum glycerol tube preserved at-18deg.C into sterilized MRS liquid culture medium under aseptic operation, culturing at 37deg.C for 24h, and passaging for 2 times to obtain lactobacillus plantarum bacterial suspension.

In the step (1), lactobacillus plantarum is preferably inoculated in the form of a direct vat set, and the inoculation amount is 0.2g/L. The preparation method of the lactobacillus plantarum direct vat set starter specifically comprises the following steps: the protective agent solution is skimmed milk powder and 5% sucrose, and the ratio (v/v) of the bacterial liquid to the protective agent is 1:1, freeze drying to obtain the direct vat set starter.

In the step (1), the culture conditions are preferably as follows: fermenting at 37deg.C for 24 hr.

In the step (2), the inoculating amount of the soybean cheese is preferably 3%. The 3% fermentation amount can reach the required fermentation result in a shorter time relative to the fermentation amount lower than 3%; with respect to 3% fermentation, an inoculum size higher than 3% does not have a significant advantage in achieving the same fermentation result.

In the step (2), the culture conditions are preferably as follows: fermenting at 37deg.C until pH is 5.8, and making soybean milk in semi-coagulated state, wherein the obtained bean curd has high water retention and good texture; when the pH is lower than 5.6, the soybean milk is in a completely coagulated state, and the soybean milk is seriously dehydrated after being heated to squat the brain, so that the soybean milk is hard in texture; when the pH is higher than 6.0, the soybean milk is insufficiently coagulated, and the shaped bean curd cannot be obtained by heating the squatting pan.

In the step (3), the temperature of the squatting pan is preferably 90 ℃. Compared with the squatting temperature of 75-85 ℃ and 95 ℃, the bean curd has higher yield at the squatting temperature of 90 ℃. Namely, under the conditions that the quality and the dosage of the soybean milk are the same and the other process conditions are the same, the weight of the obtained bean curd is higher at the squat temperature of 90 ℃ relative to the squat temperature of 75-85 ℃ and 95 ℃; that is, the yield of the bean curd is higher.

Advantageous effects

1. The lactobacillus plantarum SJ-L-1 applied in the invention is a special strain which is screened from the many polyacid pulp lactobacillus and is suitable for preparing the acid pulp and preparing the bean curd without the coagulant by fermenting, and has the following excellent characteristics in various aspects, which cannot be achieved by the strain reported in the prior art.

2. The lactobacillus plantarum SJ-L-1 applied in the invention has the advantages of high growth speed, high acid production speed and high acid production capacity, the acid production amount is 17.38g/L in 24 hours, and the growth condition of the SJ-L-1 is optimized, so that the acid production amount can be further improved to 26.01g/L. SJ-L-1 can be used for preparing the sour slurry bean curd after fermenting in yellow slurry water for 10-15 hours, and the sour slurry is prepared by mixing and fermenting the yellow slurry water with multiple strains usually for 24 hours. Through SJ-L-1 pure fermentation, single-strain short-time fermentation is realized, the production period is shortened, and the defect of unstable multi-strain mixed fermentation process is avoided; the fermentation end point can be reached after the soybean milk is fermented for 4-6 hours, and the soybean milk is used for producing the fermented bean curd without the coagulant, compared with the production period of the sour slurry bean curd, the production period of the fermented bean curd without the coagulant is further shortened because the process of preparing the sour slurry alone is not needed, and the cost is saved.

3. The lactobacillus plantarum SJ-L-1 has strong freezing resistance, the survival rate of the strain is close to 90 percent by adding the conventional strain protectant into the bacterial liquid, and the viable count can reach 1.2 multiplied by 10 ¹² The CFU/g has high viable count level, can be prepared into a high-activity direct-vat starter, has the advantages of convenient operation, continuous production realization, good stability and the like, and provides guarantee for standardized and industrialized production of the acid pulp and the coagulant-free fermented bean curd.

4. Based on the whole genome analysis of the lactobacillus plantarum SJ-L-1, the carbohydrate metabolism genes of the strain have the largest proportion in the genome, and 13 genes related to beta-glucosidase are identified through CAZy database annotation. The beta-glucosidase can convert isoflavone from glycoside type to aglycone type with higher bioactivity, thereby improving the bioavailability of soybean isoflavone. Therefore, SJ-L-1 can effectively convert the glycoside type isoflavone into aglycone type with higher bioavailability in the fermentation process of yellow serofluid and soybean milk, and improve the nutritive value of the sour slurry tofu and the non-solidification fermented tofu.

5. The lactobacillus plantarum SJ-L-1 of the invention has high total isoflavone content of the fermented bean curd without the coagulant, which is more than 3 times of that of the brine bean curd, and is also higher than that of the L-1-16 fermented bean curd. The conversion rate of isoflavone from the combined glycoside to aglycone in the SJ-L-1 fermented bean curd is highest, so that the nutritional and functional value of the bean curd is improved.

6. The genome of the lactobacillus plantarum SJ-L-1 has a gene cluster for synthesizing antibacterial peptide, and antibacterial experiments show that the lactobacillus plantarum SJ-L-1 can generate antibacterial substances, and the performance is that the physalis prepared by the lactobacillus plantarum SJ-L-1 have natural antibacterial property, so that the shelf life of the physalis bean curd prepared by pure fermentation of the physalis can be longer than that of the physalis bean curd prepared by natural fermentation of the physalis.

7. The antibacterial property of the lactobacillus plantarum SJ-L-1 enables the shelf life of the SJ-L-1 non-coagulant fermented bean curd to be longer than 8 days, the shelf life of the L-1-16 fermented bean curd is about 6 days, and the shelf life of the brine bean curd and the sour slurry bean curd is about 2 days.

8. The lactobacillus plantarum SJ-L-1 non-coagulant fermented bean curd has the characteristic of moderate hardness, and the hardness of the lactobacillus plantarum SJ-L-1 non-coagulant fermented bean curd is close to that of fresh brine bean curd, lower than that of L-1-16 fermented bean curd, but higher than that of acid pulp bean curd; and compared with the L-1-16 fermented bean curd, the structure of the SJ-L-1 fermented bean curd is more uniform.

9. The lactobacillus plantarum SJ-L-1 non-coagulant fermented bean curd has high structural stability and is superior to brine tofu and sour slurry tofu. The hardness of the brine tofu and the sour slurry tofu is obviously reduced on the 4 th day, and the brine tofu and the sour slurry tofu are deteriorated. The hardness of the L-1-16 fermented bean curd is obviously reduced at the 8 th day, compared with the hardness of the lactobacillus plantarum fermented bean curd at the 8 th day, which is still not obviously different.

10. The lactobacillus plantarum SJ-L-1 of the invention has high total flavor substance content of the non-coagulant fermented bean curd.

11. Pure lactobacillus fermentation, no pathogenic bacteria pollution such as mixed bacteria, and the like, improves the production speed and ensures the edible safety of the bean curd.

Drawings

FIG. 1 shows growth curves of 7 strains of the acid pulp-derived lactic acid bacteria (SJ-L-1, SJ-L-2, SJ-L-3, SJ-L-4, SJ-L-5, SJ-L-6 and L-1-16) in the fermentation process;

FIG. 2 shows acid production curves of 7 strains of the lactic acid bacteria (SJ-L-1, SJ-L-2, SJ-L-3, SJ-L-4, SJ-L-5, SJ-L-6 and L-1-16) from the acid slurry in the fermentation process;

FIG. 3 shows colony morphology of Lactobacillus plantarum SJ-L-1 according to the present invention;

FIG. 4 shows the microscopic examination result of Lactobacillus plantarum SJ-L-1 according to the present invention;

FIG. 5 is a phylogenetic tree of Lactobacillus plantarum SJ-L-1 based on the 16S rRNA gene sequence of the present invention;

FIG. 6 shows the detection result of lactobacillus plantarum SJ-L-1 direct vat set starter in the present invention;

FIG. 7 is a scanning electron microscope image of lactobacillus plantarum SJ-L-1 fermented bean curd according to the present invention;

FIG. 8 is a scanning electron microscope image of the marinated bean curd of the present invention;

FIG. 9 is a scanning electron microscope image of the acid pulp tofu of the present invention;

FIG. 10 is a scanning electron microscope image of L-1-16 fermented bean curd according to the present invention.

Preservation information

Preservation unit: china general microbiological culture Collection center address: the date of preservation of the microbiological institute of national academy of sciences, no. 3, north chen west way 1, region of korea, beijing city: 2022, 6, 29

Preservation number: CGMCC No.25209

Classification naming: lactobacillus plantarum

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the embodiments of the present invention will be described in further detail with reference to the accompanying drawings.

EXAMPLE 1 preliminary screening of lactic acid bacteria

In order to screen the special lactobacillus suitable for the fermentation of the acid pulp and the processing of the bean curd without the coagulant, the invention collects more than 80 parts of acid pulp samples from different processing workshops such as Yunnan, shandong, guizhou and the like, obtains more than 200 acid-producing strains through separation and screening, and identifies 7 strains with stronger acid-producing capacity according to preliminary sorting of the colony morphology of a flat plate, the size of a calcium dissolving ring, the density of bacteria cultured in an MRS test tube, the microscopic morphology, the coagulation time of fermented soybean milk, the smell of fermentation broth and the like, namely SJ-L-1, SJ-L-2, SJ-L-3, SJ-L-4, SJ-L-5, SJ-L-6 and L-1-16. In order to further screen the special strain with excellent performance, the invention examines the growth performance, acid production performance, enzyme production performance, bacteriostasis performance, fermentation soybean milk performance and other aspects of the 7 strains to obtain the strain with better comprehensive evaluation of SJ-L-1 and L-1-16.

EXAMPLE 2 growth curve of lactic acid bacteria in MRS

7 strains of lactic acid bacteria (SJ-L-1, SJ-L-2, SJ-L-3, SJ-L-4, SJ-L-5, SJ-L-6 and L-1-16) from the physalis alkekengi are inoculated into 5mL of MRS liquid culture medium, and the bacteria liquid is obviously cloudy after shaking culture for 24 hours at 37 ℃ and 180r/min, namely the grown lactic acid bacteria are collected. The collected lactobacillus was diluted and spread on MRS solid medium by plate streaking method, and cultured in an incubator at 37℃for 24 hours. Single colonies were selected and inoculated again into MRS liquid medium, and shake cultured at 37℃and 180r/min for 48 hours. Determination of OD at 2h, 4h, 8h, 10h, 12h, 24h, 36h, 48h using an ultraviolet spectrophotometer ₆₀₀ The absorbance was plotted against time and the results are shown in figure 1. As can be seen from FIG. 1, the growth rate of Lactobacillus plantarum SJ-L-1 was significantly higher than that of the other 6 strains before 24 hours.

EXAMPLE 3 acid production of lactic acid bacteria in MRS

7 strains of lactic acid bacteria (SJ-L-1, SJ-L-2, SJ-L-3, SJ-L-4, SJ-L-5, SJ-L-6 and L-1-16) from the physalis alkekengi are inoculated into 3mL of liquid MRS culture medium according to an inoculum size of 1%, and are cultured for 24 hours at 37 ℃ to be activated to the third generation. The fermentation broth was taken at 2h, 4h, 8h, 10h, 12h, 24h, 36h, 48h, respectively, diluted 25-fold with distilled water, 5 drops of phenolphthalein indicator were added dropwise, and titration was carried out with 0.1mol/L sodium hydroxide solution to the end point. The sterilized MRS liquid medium was used as a blank. Acid yield (X) of lactic acid bacteria in MRS was calculated according to the following formula:

wherein V is ₀ (mL) is the volume of the fermentation broth before dilution, V ₁ (mL) is the volume of sodium hydroxide solution consumed by the fermentation broth, V ₂ (mL) is the volume of sodium hydroxide solution consumed by the blank. C (C) _NaOH (0.1 mol/L) is forThe titrated sodium hydroxide concentration, M (g/mol), is the molar mass of lactic acid.

The results of the acid production of the lactic acid bacteria used in the embodiment of the invention within 48 hours are shown in figure 2, and the acid production rate of SJ-L-1 before 18 hours is higher than that of other strains and is 17.38g/L at 24 hours as shown in figure 2.

EXAMPLE 4 glycosidase production and bacteriostatic Properties of lactic acid bacteria

7 strains of lactic acid bacteria (SJ-L-1, SJ-L-2, SJ-L-3, SJ-L-4, SJ-L-5, SJ-L-6 and L-1-16) from the physalis alkekengi are inoculated into 3mL of liquid MRS culture medium according to an inoculum size of 1%, and are cultured for 24 hours at 37 ℃ to be activated to the third generation. As can be seen from Table 1, SJ-L-1 and L-1-16 have higher glycosidase activity and stronger antibacterial performance.

Table 17 strain lactobacillus glycosidase and bacteriostatic properties

Note that: "+++" means bacteriostatic the diameter of the ring exceeds 12mm, "++" refers to a zone of inhibition of 6-12mm in diameter, "+" refers to a zone of inhibition of 0.5-6mm in diameter, and "-" refers to no bacteriostatic activity.

Example 5 lactic acid bacteria fermented Soybean milk

And respectively inoculating 7 activated lactic acid bacteria into the sterilized soybean milk, wherein the inoculation amount is 1%. Placing in an incubator, and standing at 37 ℃. And observing the state of the sample every 30min, and recording the time required by solidification when the sample is completely solidified.

7 strains of lactobacillus ferment soybean milk, the solidification time is different, but the soybean milk is completely solidified within 10 hours. The soybean milk fermented by the strain SJ-L-1 is solidified fastest, and can be solidified for about 4 hours, which has a correlation with the growth speed of the strain SJ-L-1, the acid production speed and the acid production amount. Secondly, the strains L-1-16 and SJ-L-6 solidify for about 5 hours. The soybean milk fermented by the strains SJ-L-2, SJ-L-4 and SJ-L-5 is solidified for about 7 hours. The longest time consuming is the strain SJ-L-3, which takes about 9 hours to ferment the soymilk for coagulation.

10 students were invited to perform sensory evaluation on the bean cheese obtained by fermenting 7 strains of lactic acid bacteria for 24 hours, and the scoring criteria are as follows:

TABLE 2 sensory evaluation scoring criteria

The sensory evaluation results were as follows: from the aspect of appearance, the 7-strain fermented soybean cheese is milky white and has better color. From the aspect of smell, the soy cheese acid taste of the strains SJ-L-1 and L-1-16 is soft, the soy cheese acid taste of the strains SJ-L-6 is more stimulated, and the soy cheese acid taste of the other strains is acceptable. From the viewpoint of water retention, the soybean cheese fermented by the strains SJ-L-1 and L-1-16 is better, and the soybean cheese fermented by the strains SJ-L-2, SJ-L-4 and SJ-L-5 is worse. In general, the strains SJ-L-1 and L-1-16 have outstanding fermentation performance in soybean milk matrix.

EXAMPLE 6 identification and annotation of Lactobacillus plantarum

6.1 morphological characterization

The colony characteristics of strain SJ-L-1 are: the strain is in a form on a solid MRS culture medium as shown in figure 3, and the colony is white at about 24 hours, and has round edge, opaque, convex and smaller edge. As shown in FIG. 4, the cells were rod-shaped or short rod-shaped, and the gram-stained cells were purple, i.e., gram-positive cells.

6.2 physiological Biochemical characteristics

The detection method comprises the following steps: the physiological and biochemical characteristic detection method of the strain is carried out by referring to the ' Berger's bacteria identification manual ' and the ' common bacteria System identification manual '.

The test results are shown in Table 2.

TABLE 3 physiological and biochemical identification results of strain SJ-L-1

Note that: "-" means negative (not detected), "+" means positive (detected) "W" means no detection.

6.3 Gene characterization

The strain SJ-L-1 of the present application was DNA sequenced by Megaku biosystems. The genome sequence data of the lactobacillus plantarum SJ-L-1 shows that the whole genome sequence is 320,994,0bp, and the G+C content in DNA is 44.64%. The gene sequences of the strain YP1 of the present application were aligned by gene bank (Genebank) sequences, and phylogenetic tree was established using MEGA7.0, and the results are shown in FIG. 5. By phylogenetic analysis of the 16S rDNA sequences, ANI and dDH values analysis, strain SJ-L-1 was identified as Lactobacillus plantarum.

6.4 Gene annotation

The coding gene of the lactobacillus plantarum SJ-L-1 is functionally annotated through a COG (http:// eggnog.embl.de /) and KEGG (http:// www.genome.jp/KEGG /) database, and the carbohydrate-active enzyme is analyzed through a CAZy database (http:// www.cazy.org /). The results of the COG database annotation showed that the most abundant in Lactobacillus plantarum SJ-L-1 are carbohydrate transport and metabolism (278 genes) and transcribed (242 genes) amino acid transport and metabolism (203 genes). The results noted in the KEGG database showed that carbohydrate metabolism and amino acid metabolism were dominant in Lactobacillus plantarum SJ-L-1, consistent with the COG noted results. The results show that the genes involved in SJ-L-1 carbohydrate have a large ratio, and the SJ-L-1 is presumed to have good glycoside hydrolysis capability.

The CAZy annotation results of Lactobacillus plantarum SJ-L-1 of the present application indicate that the glycoside hydrolase is the largest (46.73%) in the carbohydrate-active enzyme. 13 beta-glucosidase genes were identified in the glycoside hydrolase class and beta-glucosidase can convert isoflavone from sugar ketone to aglycone form, so that SJ-L-1 is presumed to have great potential in isoflavone conversion.

EXAMPLE 7 preparation of Lactobacillus plantarum direct vat set starter

Lactobacillus plantarum SJ-L-1 of the third generation was inoculated into 50mL MRS flasks at an inoculum size of 2% for expansion culture, and cultured at 37℃for 24 hours. Placing the cultured bacterial liquid into a centrifuge, centrifuging for 10min at 8000 Xg and 4 ℃, discarding supernatant, washing the bacterial body twice with 0.85% sterile physiological saline, and centrifuging under the same conditions to obtain bacterial body precipitate.

The method is characterized in that skim milk powder and 5% sucrose are taken as freeze-drying protective agent solution, and bacterial suspension is prepared according to the ratio (v/v) of bacterial liquid (volume before centrifugation) to protective agent of 1:1. Packaging the bacterial liquid, pre-freezing at-80deg.C, freeze-drying in vacuum freeze drier for 24 hr, and storing in-20deg.C refrigerator. SJ-L-1 has good freezing resistance, and the viable bacteria rate after freeze drying is approximately 90% before freeze drying. The lactobacillus plantarum SJ-L-1 direct-vat-set starter is sent to Shannon Ka Biotechnology (Suzhou) for detection, and the result is shown in figure 6, wherein the number of viable bacteria of the direct-vat-set starter is up to 1.2 multiplied by 10 ¹² CFU/g。

Example 8 preparation of Lactobacillus plantarum acid syrup

Inoculating lactobacillus plantarum SJ-L-1 into sterilized yellow serofluid by directly adding 0.1-0.5g/L starter or 1-3% (volume percentage) of strain fermentation liquor. The pH of the acid pulp can be reduced to pH3.6-3.8 after fermentation for 10-15 h. The pH range of the sour slurry is that the sour slurry is squatted to curd at 90 ℃, and the sour slurry is pressed and molded after the squatting is finished, so that the sour slurry bean curd finished product with excellent texture, unique flavor and high finished product yield can be prepared.

The method comprises the steps of preparing the sour slurry bean curd by natural fermentation under the same processing conditions, preserving the natural fermentation sour slurry bean curd and pure fermentation sour slurry bean curd at room temperature, observing the form of the sour slurry bean curd by sampling every 1 day, and measuring the total number of bacterial colonies, wherein the phenomenon that the sour slurry bean curd prepared by natural fermentation is turbid in slurry water is found in the next day, and the total number of bacterial colonies is also out of standard. The puree fermented sour slurry bean curd does not start to have slurry turbidity until the 3 rd day.

Example 9 application of Lactobacillus plantarum in the preparation of coagulant-free fermented Bean curd

Inoculating lactobacillus plantarum SJ-L-1 into sterilized soybean milk according to an inoculum size of 3%, and fermenting at 37 ℃ for 24 hours to obtain soybean cheese; inoculating bean cheese into sterilized soybean milk according to an inoculum size of 3%, culturing at 37 ℃, taking out when the soybean milk is in a semi-coagulated state, and putting into a water bath kettle at 90 ℃ for squatting; and after the squatting brain is finished, pressing and forming to obtain the SJ-L-1 fermented bean curd.

Naturally fermenting the bean curd yellow serofluid without the coagulant for 15 hours, wherein the fermentation liquid is the sour slurry coagulant; squatting the brain at 90deg.C to curd; and (5) after the squatting brain is finished, pressing and forming to obtain the acid pulp bean curd.

L-1-16 fermented bean curd was prepared by the method of example 4 using Lactobacillus rhamnosus L-1-16 as an inoculating strain.

With MgCl ₂ The bittern is prepared by performing squatting to curd at 90 ℃, and pressing and forming after squatting is finished.

9.1 texture determination

The tofu was cut into 1.5 cm. Times.1.5 cm squares, and analyzed by TMS-PRO texture analyzer. The experimental parameters are as follows: the trigger force is 0.5N, the pilot test speed is 30mm/s, the test speed is 20mm/s, and the post test speed is 20mm/s. Each tofu sample was assayed in triplicate.

As a result, as shown in Table 4, the hardness of the SJ-L-1 fermented bean curd was 290.96g, which is most similar to that of a marinated bean curd. The elastic, viscous and chewing change tendencies are similar, the L-1-16 fermented bean curd is the highest, and the sour slurry bean curd is the lowest. The above results indicate that the processing method and the fermentation strain affect the texture of tofu. Lactobacillus plantarum SJ-L-1 ferments to produce acid to gel the protein, and further completely gels by heating, thus forming a compact and uniform three-dimensional gel network structure, and further producing greater hardness, viscosity and chewiness.

The texture test hardness (g) was carried out using a texture analyzer with the results shown in Table 5, taking samples of the marinated tofu and the acid slurry tofu placed in 0d, 2d, 4d, 6d, 8d, L-1-16 fermented tofu, SJ-L-1 fermented tofu, respectively. The quality guarantee period of the brine bean curd and the acid pulp bean curd can be estimated to be about 2 days according to the texture change of the bean curd, the quality guarantee period of the L-1-16 fermented bean curd is about 6 days, and the quality guarantee period of the SJ-L-1 fermented bean curd is more than 8 days.

Table 4 texture characteristics of four tofu

Texture parameters	Marinated bean curd	Sour slurry bean curd	L-1-16 fermented bean curd	SJ-L-1 fermented bean curd
					Hardness (g)	285.26±45.62	181.34±41.38	395.93±42.30	290.96±22.36
Elasticity (mm)	2.66±0.34	2.04±0.64	3.01±0.20	2.93±0.21
					Cohesive property	0.60±0.00	0.55±0.58	0.63±0.05	0.65±0.58
Viscosity (g)	177.15±32.99	99.72±27.58	248.94±37.71	193.04±22.67
					Masticatory (mj)	4.7±1.50	2.15±1.34	7.63±1.21	5.58±1.05

TABLE 5 time-varying characteristics of hardness of four tofu

	0d	2d	4d	6d	8d
						Marinated bean curd	285.26	265.31	183.67	153.06
Sour slurry bean curd	181.34	142.86	91.84	81.63
						L-1-16 fermented bean curd	395.93	379.13	419.95	463.86	309.24
SJ-L-1 fermented bean curd	290.96	285.71	306.12	326.53	306.12

9.2 microstructural determination

Bean curd samples were cut into small pieces (5 mm. Times.2 mm), soaked in 2.5% glutaraldehyde at 4℃for 24 hours, and then rinsed 2 times with 0.1M phosphate buffer (pH=7.2-7.4). Before measurement, 30%, 50%, 70% and 90% ethanol was used for eluting for 1 hour, and finally 100% ethanol was used for eluting 3 times for 1 hour each. The samples were then lyophilized and surface blasted. The microstructure of the curd sample curd was observed under a Sigma300 scanning electron microscope.

As shown in FIG. 7, the SJ-L-1 fermented bean curd has uniform texture, compact structure and high hardness. SJ-L-1 fermentation is a slow acid production process, so that the obtained tofu gel has a uniform texture. As shown in fig. 8, magnesium chloride is a fast-acting coagulant, and the curding step is difficult to control, and the local fast curding causes the gel structure of the brine tofu to be uneven. As shown in fig. 9, the structure of the sour slurry bean curd is loose; as shown in FIG. 10, the microstructure of the L-1-16 fermented bean curd is dense, but has larger pores.

9.3 isoflavone content determination

1m soymilk was mixed with 4mL 80% (v/v) methanol and extracted by ultrasound for 60min. The extract was centrifuged at 10000rpm at 4℃for 10min and the supernatant was collected. And freeze-drying the bean curd sample, adding 10mL of 80% ethanol into each gram of freeze-dried product for redissolving, and performing ultrasonic extraction at room temperature for 6 hours with ultrasonic power of 70w. The extract was centrifuged at 10000rpm at 4℃for 10min. Ethanol in the ethanol extract supernatant was evaporated to dryness using a rotary evaporator and the remaining liquid was re-sized to 10mL with 80% methanol. The supernatant was filtered through a 0.22 μm filter membrane and assayed three times in parallel by HPLC analysis of the isoflavone content.

Chromatographic conditions: the chromatographic column is a Shimadzu AQ-C18 (4.6X105 mm,5 μm) column; mobile phase is 0.5% (v/v) glacial acetic acid aqueous solution (A) and 100% acetonitrile (B); the flow rate is 0.6mL/min; the column temperature is 30 ℃; the detection wavelength is 260nm; the sample injection amount is 15 mu L; the gradient elution conditions are shown in Table 6.

The isoflavone contents of the soybean milk and the four bean curd samples are shown in Table 7. Compared with soybean milk, marinated bean curd, sour milk bean curd and L-1-16 fermented bean curd, the SJ-L-1 fermented bean curd has the highest total isoflavone content. The isoflavone in soybean milk is mainly in the form of combined glycoside, the content of glycoside in bean curd is reduced, and the content of free aglycone is increased. The isoflavone conversion rate of the SJ-L-1 fermented bean curd is highest, and compared with soybean milk, the aglycone content in the SJ-L-1 fermented bean curd is improved by 60 times; compared with brine tofu, the aglycone content in the SJ-L-1 fermented tofu is improved to nearly four times. This is related to the fact that SJ-L-1 contains a large amount of beta-glucosidase, which is capable of hydrolyzing the glycosidic structure of soybean isoflavone in soybean milk and converting it into aglycone form with higher bioactivity, thereby improving the bioavailability of soybean isoflavone. The sour slurry of the SJ-L-1 fermented bean curd is used as a coagulant to be applied to the production of the sour slurry bean curd, the high-content aglycone isoflavone is reserved along with the fact that the sour slurry coagulant is added into the bean curd production process again, and finally the sour slurry bean curd rich in aglycone isoflavone is obtained, so that the nutritional value of the bean curd is greatly improved.

The SJ-L-1 fermented bean curd contains a large amount of soyabean glycoside and genistein, which respectively account for 52.50% and 44.21% of the total isoflavone content. The soybean flavone and genistein have stronger DPPH and hydroxyl radical scavenging capability, can protect cells from being damaged by free radicals, and have good antioxidant activity. In addition, daidzin and genistein also dominate the anticancer activity of soybean. Therefore, the lactobacillus plantarum SJ-L-1 serving as a functional lactobacillus has great potential in the development and application of fermented foods.

TABLE 6 gradient elution conditions for chromatographic columns

Retention time (min)	A:0.5% (v/v) aqueous glacial acetic acid solution	B:100% acetonitrile
			5	85	15
28	65	35
			42	55	45
47	10	90
			59	85	15

Table 7 soymilk and four Bean curd isoflavone contents

Note that: "-" means undetectable.

9.4 flavour determination

5g of fresh bean curd sample is crushed and placed in a headspace bottle of 20ml, and the sample bottle is incubated for 10min at 60 ℃ with shaking. The headspace was extracted at 50℃for 30min, followed by desorption at 240℃for 2min. The determination of volatile flavour substances was analysed by gas chromatography-mass spectrometry.

Chromatographic conditions: the chromatographic column is Agilent DB-WAX (60 m×0.25 μm×0.25 μm) capillary column; the temperature of the sample inlet is 240 ℃, sample injection is not split, and the flow rate of the column is 1.0mL/min; column temperature: the initial temperature was 35℃for 3min, and then increased to 220℃at a rate of 3℃per min for 10min. Mass spectrometry conditions: EI, ion source temperature 230 ℃, quadrupole temperature 150 ℃.

The results of gas chromatography-mass spectrometry detection were compared with the NIST mass spectrometry library to identify volatile compounds in bean curd samples, and the results are shown in table 8. In the SJ-L-1 fermented bean curd, 22 volatile compounds including 9 alcohols, 5 aldehydes, 2 ketones, 2 acids, 2 esters and 2 furans are identified, and the total content of the volatile compounds of the SJ-L-1 fermented bean curd is highest.

Table 8 volatile compound content of four tofu

Note that: "-" means undetectable.

9.5 sensory testing

Inviting 15 professional quality sensory evaluation staff to perform quality sensory evaluation on the fermented bean curd prepared in the example, wherein the scoring standard is shown in table 9; the evaluation results are shown in Table 10.

TABLE 9 sensory evaluation criteria

Table 10 four bean curd sensory evaluation scores

	Color	Flavor of	Hardness of
				Marinated bean curd	7.2	6.7	6.9
Sour slurry bean curd	6.9	6.5	5.7
				L-1-16 fermented bean curd	7.1	6	6
SJ-L-1 fermented bean curd	7.3	6.3	7.3

Claims

1. The lactobacillus plantarum from the bean curd acid pulp is characterized in that the strain is named as SJ-L-1, is classified and named as lactobacillus plantarum Lactobacillus plantarum, and is preserved in China general microbiological culture Collection center (CGMCC) No.25209.

2. A lactobacillus plantarum direct vat set starter, characterized in that it is made of the lactobacillus plantarum of claim 1.

3. Use of lactobacillus plantarum according to claim 1 or lactobacillus plantarum direct vat set starter according to claim 2 in the preparation of tofu pudding.

4. The use according to claim 3, characterized by the following steps: inoculating lactobacillus into sterilized yellow serofluid according to the inoculation amount of 0.1-0.5g/L of direct-vat starter or 1-3% (volume percentage) strain culture solution, fermenting for 10-15h, and reducing pH of the acid pulp to pH3.6-3.8, wherein the acid pulp with pH range can be used for preparing the acid pulp bean curd finished product with excellent texture, unique flavor and high yield.

5. The use according to claim 4, wherein the yellow serofluid is selected from yellow serofluid during pressing of fermented bean curd without coagulant.

6. Use of lactobacillus plantarum according to claim 1 or lactobacillus plantarum direct vat set starter according to claim 2 in the production of fermented tofu without coagulants.

7. The use according to claim 6, characterized by the steps of:

(1) The preparation of the bean cheese comprises the following steps: inoculating lactobacillus plantarum into sterilized soybean milk according to the inoculation amount of 0.1-0.5g/L of direct-vat starter or 1-3% (volume percentage) of strain culture solution, and fermenting for 20-26 hours at 30-35 ℃ to form soybean cheese;

(2) And (3) bean curd fermentation: inoculating 1-3% (by volume) of soybean cheese into sterilized soybean milk, fermenting at 30-35deg.C until pH is 5.8+ -0.2, and performing squatting brain at 90deg.C to obtain soybean curd with high water retention rate and good texture.