Lactobacillus plantarum CMRC 1L and its applications

Abstract

The present invention belongs to the technical field of food fermentation, and provides a plant lactobacillus CMRC 1L and an application thereof. Lactobacillus plantarum ) CMRC 1L was isolated from a sour meat sample in Maojia Village, Lijiang City, Yunnan Province. It grows well on MRS culture medium, produces acid quickly, has biological characteristics such as salt resistance, frostbite resistance, and inhibition of the growth and reproduction of corrupt microorganisms. It has a good fermentation effect on a variety of plant proteins and is an excellent strain for making a starter for production. The present invention also provides a starter containing the above-mentioned strain and its application in plant protein meat products. Compared with the plant protein jerky to which no starter is added, the preparation method of the plant protein jerky product provided by the present invention has stable product quality, a sweet fermented aroma, no beany smell, and a good flavor and taste. The application of strain CMRC 1L in the production of plant protein meat products has great potential.

Description

Technical Field

[0001] The present invention belongs to the technical field of food fermentation, and specifically relates to a Lactobacillus plantarum CMRC 1L and its application. Background Art

[0002] Plant protein is a more environmentally friendly protein with a wider source than animal protein and is an indispensable part of people's daily diet. With the rapid development of human society, it is estimated that by 2050, the global total population will reach 9.8 billion, and the demand for food is also increasing continuously. In recent years, due to the increasing awareness of low-carbon environmental protection and animal welfare among people, the demand for animal protein substitutes has been increasing day by day. Therefore, the market position of plant protein has been continuously improved. Plant-based protein meat is mainly made from plant proteins such as soybean protein, corn protein, and wheat protein, added with auxiliary materials, and formed through synthesis and processing into a food with similar nutritional value, taste, and flavor to meat products. Its raw material sources are rich, and a variety of plant proteins provide more possibilities for the production of plant-based protein meat. For example, soybean protein, chickpea protein, and pea protein in legume proteins; corn protein in cereal proteins, and rapeseed protein in oilseed proteins can all be used to make plant-based protein meat. Among them, soybean protein has a low price, is easy to obtain, and has high nutritional value, showing excellent food processing characteristics such as gelation and water retention, and is recognized as a good choice for making plant-based protein meat. However, plant-based protein meat products related to soybean protein often have an obvious "soybean smell", which is difficult to be accepted by consumers, the product texture and flavor quality are poor, and the product form is single; in addition, due to the tight structure of legume proteins and the anti-nutritional factors present in legumes, the digestibility of legume proteins is weak, which also does not meet the pursuit of consumers for nutritious and healthy foods. In recent years, the problem of how to improve the flavor quality of plant-based protein meat products and enhance their nutritional value has received wide attention.

[0003] As a traditional food processing method, microbial fermentation is a mild and energy-saving processing technology. It does not require complex equipment and processes, and has the characteristics of simplicity, economy, and convenience. Fermented foods have certain advantages in flavor and nutritional value. China has a long history of fermenting legume proteins, such as fermented soybeans and fermented bean curd. Bacteria, molds, and yeasts can all be used as fermenting agent strains for legume proteins. Among them, bacteria and molds are the main microorganisms in naturally fermented fermented soybeans, Rhizopus and Mucor are used to make fermented bean curd, and salt-tolerant lactic acid bacteria and yeasts are in a dominant position in the production of soy sauce. At present, there are relatively few studies on fermenting agent strains with flavor improvement effects applicable to plant-based protein meat products. In particular, there are few reports on the application of Lactobacillus plantarum in the production of plant-based protein meat-related products in order to improve product flavor, texture and other characteristics. Summary of the Invention

[0004] The object of the present invention is to provide a Lactobacillus plantarum CMRC 1L and its applications.

[0005] To achieve the object of the present invention, in the first aspect, the present invention provides a Lactobacillus plantarum CMRC 1L isolated and purified from a self-made farmhouse sour meat sample, and classified and named as Lactobacillus plantarum Lactobacillus plantarum , which has now been deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, postal code 100101, deposit number CGMCC No. 32440, deposit date October 31, 2024.

[0006] In the second aspect, the present invention provides a microbial preparation containing the above-mentioned Lactobacillus plantarum.

[0007] Furthermore, the content of Lactobacillus plantarum in the microbial preparation is 10 9 ~10 11 CFU / g.

[0008] The preparation method of the microbial preparation includes: fermenting and culturing the Lactobacillus plantarum, centrifuging the obtained fermentation broth, collecting the bacterial cell precipitate, resuspending the bacterial cells with sterile physiological saline and then centrifuging to discard the supernatant, adding a small amount of physiological saline again and mixing evenly to obtain a concentrated bacterial solution, and adding an appropriate amount of freeze-drying protectant and then freeze-drying to obtain the product.

[0009] Furthermore, the mass ratio of the concentrated bacterial solution to the freeze-drying protectant is (0.1 - 10):1.

[0010] The freeze-drying protectant can be selected from at least one of skim milk, glycerol, sucrose, etc.

[0011] In the third aspect, the present invention provides the application of the above-mentioned Lactobacillus plantarum or the microbial preparation in food fermentation.

[0012] In the fourth aspect, the present invention provides a preparation method of a plant protein meat product, including the following steps:

[0013] 1) Soaking and shredding plant tissue protein: Soak the plant tissue protein in water for 1 - 2 h to fully rehydrate and then drain the water;

[0014] 2) Mixing and flavoring raw materials: Mix plant protein powder, plant tissue protein, seasonings and water evenly according to the ratio;

[0015] 3) Inoculating the fermentation agent: Add the above-mentioned microbial preparation according to the ratio;

[0016] 4) Fermentation: Put it into a constant temperature and humidity box for fermentation;

[0017] 5) Molding and drying: Extrude the fermented raw materials into corresponding shapes according to production needs;

[0018] 6) Baking: Place in an oven for baking;

[0019] 7) Cooling and packaging: Cut according to production needs, cool to room temperature and then package.

[0020] Furthermore, the plant tissue protein can be selected from soy tissue protein and / or pea tissue protein, etc.

[0021] Furthermore, the plant protein powder can be selected from at least one of soy protein isolate, soy protein powder, wheat protein powder, rice protein powder, pea protein powder, etc.;

[0022] Furthermore, the seasonings can be selected from at least one of plant-derived seasonings such as table salt, white sugar, brewed soy sauce, five-spice powder, yeast extract, etc.

[0023] Preferably, by weight, the ingredients in step 2) are as follows: 50 - 80 parts of soy tissue protein, 20 - 50 parts of soy protein isolate, 20 - 50 parts of vital wheat gluten, 2 - 20 parts of granulated sugar, 2 - 6 parts of salt, 2 - 10 parts of brewed soy sauce, 1 - 3 parts of spice powder or five-spice powder, 1 - 5 parts of yeast extract, and 100 - 150 parts of water.

[0024] Preferably, in step 3), the inoculation amount of Lactobacillus plantarum is 10 4 CFU / g of raw materials, and the microbial preparation is dissolved in advance with a 2% glucose solution, and the dosage of the glucose solution is 5 - 10% of the total weight of the raw materials.

[0025] Preferably, the fermentation conditions in step 4) are: temperature 20 - 25°C, for 12 - 36 hours;

[0026] Preferably, in step 5), the fermented raw material blank is extruded into a tray, in the shape of meat strips, and placed in a smoke oven to dry the moisture, and the drying conditions are: temperature 50 - 55°C, for 1 - 2 h;

[0027] Preferably, the baking conditions in step 6) are: temperature 120 - 150°C, for 10 - 20 min.

[0028] In the fifth aspect, the present invention provides a plant protein meat product prepared according to the said method.

[0029] By means of the above technical solutions, the present invention has at least the following advantages and beneficial effects:

[0030] (1) The Lactobacillus plantarum provided by the present invention ( Lactobacillus plantarum)CMRC 1L, a strain isolated from a sour meat sample in Maojiacun area, Lijiang City, Yunnan Province, grows well on MRS medium, has a fast acid production rate, and has biological characteristics such as salt tolerance, frost injury tolerance, and inhibition of the growth and reproduction of spoilage microorganisms. It is an excellent strain for making production starters.

[0031] (II)The Lactobacillus plantarum CMRC 1L provided by the present invention has good fermentation effects on various plant proteins such as soybean protein, peanut protein, and pea protein. It can grow and reproduce using plant protein as a substrate, produce specific fermentation flavors during the fermentation process, effectively improve the "soybean fishy smell" and other unpleasant odors in plant proteins such as soy protein isolate and wheat protein, improve the texture of plant protein meat products, promote the degradation of proteins and the formation of flavors, and improve the nutritional value of products.

[0032] The products produced according to the method for preparing artificial fermented plant protein meat jerky provided by the present invention have good elasticity and chewiness, significantly improved flavors, increased overall acceptance, and have good market prospects. Applying Lactobacillus plantarum CMRC 1L to the production of plant protein meat products has great potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 It is the colony morphology of Lactobacillus plantarum CMRC 1L in the specific embodiment of the present invention.

[0034] Figure 2 It is the strain morphology of Lactobacillus plantarum CMRC 1L in the specific embodiment of the present invention.

[0035] Figure 3 It is the growth curve of Lactobacillus plantarum CMRC 1L strain in the specific embodiment of the present invention.

[0036] Figure 4 It is the acid production curve of Lactobacillus plantarum CMRC 1L strain in the specific embodiment of the present invention.

[0037] Figure 5 It is the influence of different pH values on the growth of Lactobacillus plantarum CMRC 1L strain in the specific embodiment of the present invention.

[0038] Figure 6 It is the influence of different ethanol concentrations on the growth of CMRC 1L strain and CICC 21792 strain in the specific embodiment of the present invention.

[0039] Figure 7 It is the influence of different sodium chloride concentrations on the growth of CMRC 1L strain and CICC 21792 strain in the specific embodiment of the present invention.

[0040] Figure 8This shows the effects of different glucose concentrations on the growth of CMRC 1L strain and CICC 21792 strain in specific embodiments of the present invention.

[0041] Figures 9 - 13 These are the sensory evaluation radar charts of fermented products of soy protein, rice protein, peanut protein, wheat protein, and pea protein in specific embodiments of the present invention in sequence. Detailed implementation manners

[0042] The present invention isolates and screens a Lactobacillus plantarum strain suitable for production operations and capable of effectively improving the quality such as the flavor of plant proteins such as soy protein. Based on this, a processing method for plant protein jerky is established, which is of great significance for developing nutritious and delicious plant protein meat products.

[0043] The present invention adopts the following technical solutions:

[0044] The inventors collected various traditional homemade sour meat samples from areas such as Maojia Village in Lijiang City, Yunnan Province, and Jialiang Town in Libo County, Guizhou Province. Through separation, screening, purification, and further biological characteristics and fermentation characteristics tests, a Lactobacillus plantarum strain suitable for use as a fermenting agent for plant protein meat products was obtained and submitted to the General Microbiology Center of the China Microbial Culture Collection Center. The strain preservation number is CGMCC No. 32440, and this strain was isolated from the Maojia sour meat sample.

[0045] The present invention also provides a method for isolating, screening, and identifying the Lactobacillus plantarum strain.

[0046] Enrich the culture of the collected various samples. After 10-fold serial dilution, pipette an appropriate concentration of the sample solution, inoculate it on MRS medium (containing 6% NaCl by mass fraction) by the pour plate method, mix well, solidify and then culture at 37 °C for 48 h. Select single colonies according to colony morphology, size, transparency, etc., and repeatedly streak and purify until a single strain is obtained. Pick a single colony on the plate and inoculate it into MRS broth, culture it at 37 °C and 200 r / min with shaking for 24 h. Drop the culture solution on a precision test paper with a pH of 5.4 - 7.0, select the strain corresponding to the test paper turning yellow (pH < 5.4) for Gram staining to observe the cell morphology, and preserve the strain.

[0047] The obtained Lactobacillus plantarum was initially screened for fermentation performance, including acid production characteristics experiment, glucose fermentation gas production experiment, mucus production experiment, H2S production experiment, and Oxford cup method antibacterial experiment (Staphylococcus aureus and Escherichia coli). Strains with strong acid production ability, no mucus production, no gas production during glucose fermentation, no H2S production, and obvious inhibitory effect on spoilage bacteria were selected for re-screening. The strains obtained from the initial screening were inoculated into a liquid medium supplemented with soy protein isolate and glucose, and after culturing at 37 °C for 24 h, the strains were screened by the smelling method. According to the results of the initial screening and re-screening, Lactobacillus plantarum CMRC 1L with good growth, strong fermentation ability, and the ability to ferment using soy protein as a substrate to produce characteristic flavors was comprehensively selected for subsequent identification and application research.

[0048] The colony morphological characteristics of the Lactobacillus plantarum provided by the present invention: It grows well on solid MRS medium, the colonies are white, round or oval, convex, with smooth edges and surfaces, and the colony diameter is about 1 - 2 mm. The cell morphological characteristics: Gram-positive, short rod-shaped, without spores and flagella. The colony morphology of Lactobacillus plantarum CMRC 1L is shown in Figure 1 , and the strain morphology is shown in Figure 2 .

[0049] Molecular biological identification of strain CMRC 1L: The bacterial genomic DNA was extracted using a bacterial genomic DNA extraction kit, and the quality and fragment size of the extracted DNA were detected by 0.8% agarose gel electrophoresis. Using universal primers 27F and 1492R, the 16S rRNA sequence was amplified with genomic DNA as a template. After the PCR products were analyzed by gel electrophoresis, they were sent to Invitrogen Trading Co., Ltd. (Beijing) for sequencing, and the sequencing results were compared with the sequences in the GenBank database by Blast homology. The results showed that the sequence of this strain was highly homologous to the gene sequence of strain Lactobacillus plantarum MLG - 17, and it was identified as Lactobacillus plantarum ( Lactobacillus plantarum ), and the 16S rRNA gene sequence of the strain is shown in SEQ ID NO:1.

[0050] The present invention also provides a plant protein meat product fermenting agent containing the above-mentioned Lactobacillus plantarum CMRC 1L.

[0051] After the strain preserved in a glycerol tube was transferred and activated 2 times, it was inoculated into MRS broth medium at an inoculation ratio of 2 - 4% and cultured on a shaker at 100 - 150 r / min at 37 °C for 18 - 24 hours to obtain a bacterial suspension. After centrifuging at 3000 - 5000 rpm for 10 - 15 min, the supernatant was discarded, the cells were resuspended with sterile normal saline and then centrifuged to discard the supernatant. After adding a small amount of normal saline and mixing evenly again, a concentrated bacterial solution was obtained. After adding a certain volume of freeze-drying protectant, it was freeze-dried to prepare the fermenting agent.

[0052] Optionally, the content of Lactobacillus plantarum strains in the starter is 10 9 ~10 11 CFU / g.

[0053] Optionally, the mass ratio of the concentrated bacterial liquid to the lyoprotectant is (0.1 - 10):1.

[0054] Optionally, the physiological saline added for the second time is 1 / 5 - 1 / 10 of the volume of the initial bacterial suspension, so that the bacterial concentration in the finally obtained concentrated bacterial liquid reaches 10 10 CFU / mL.

[0055] Optionally, the composition components of the lyoprotectant may include skim milk, glycerol and sucrose.

[0056] Optionally, the culture conditions for inoculation in MRS broth medium are pH 6.0 - 6.4 and the culture temperature is 30 - 37°C.

[0057] The present invention also provides the application of the Lactobacillus plantarum CMRC 1L or the microbial inoculant containing the Lactobacillus plantarum in the production of plant protein meat products.

[0058] The present invention further provides a preparation method of an artificially fermented plant protein meat jerky containing the Lactobacillus plantarum strain, comprising the following steps:

[0059] 1) Soaking and shredding of plant tissue protein: The plant tissue protein is pre-soaked in water for 1 - 2 h to fully rehydrate and then drained.

[0060] 2) Mixing and flavoring of raw materials: Mix the plant protein powder, plant tissue protein, seasonings and water evenly according to the ratio.

[0061] 3) Inoculating the starter: Add the starter strain according to the ratio.

[0062] 4) Fermentation: Place it in a constant temperature and humidity box for fermentation.

[0063] 5) Shaping and drying: Extrude the fermented raw materials into the corresponding shape according to the production needs.

[0064] 6) Baking: Place it in an oven for baking.

[0065] 7) Cooling and packaging: It can be cut according to the demand, cooled to room temperature and then packaged.

[0066] Optionally, the plant tissue protein in step 1) may be selected from one or more of soybean tissue protein, pea tissue protein, etc., and can be shredded according to the shape of the tissue protein.

[0067] Optionally, the plant protein powder described in step 2) can be selected from one or several of soy protein isolate, soy protein powder, wheat protein powder, rice protein powder, pea protein powder, etc. The seasonings can be selected from at least one of plant-derived seasonings such as table salt, white sugar, brewed soy sauce, five-spice powder, yeast extract, etc.

[0068] Preferably, the ingredients in step 2) are as follows: 50 - 80 parts of textured soy protein, 20 - 50 parts of soy protein isolate, 20 - 50 parts of vital wheat gluten, 2 - 20 parts of granulated sugar, 2 - 6 parts of salt, 2 - 10 parts of brewed soy sauce, 1 - 3 parts of spice powder (or five-spice powder), 1 - 5 parts of yeast extract, and 100 - 150 parts of water. The "parts" here are parts by weight.

[0069] Preferably, in step 3), the inoculation amount of Lactobacillus plantarum is 10 4 CFU / g of raw materials. The starter culture is dissolved in advance with a 2% glucose solution, and the amount of the glucose solution used is 5 - 10% of the total weight of the raw materials.

[0070] Preferably, in step 4), the fermentation conditions are a temperature of 20 - 25°C for 12 - 36 hours. To ensure the uniformity of the distribution of the starter culture in the raw materials, intermittent stirring can be carried out during fermentation.

[0071] Optionally, in step 5), the fermented raw material blank is extruded into a tray, in the shape of meat strips, and placed in a smokehouse to dry the moisture. The drying stage conditions are a temperature of 50 - 55°C for 1 - 2 h.

[0072] Preferably, in step 6), the baking conditions are a temperature of 120 - 150°C for 10 - 20 min.

[0073] The following examples are used to illustrate the present invention, but do not limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0074] The unit "parts" representing weight in the present invention can be grams, kilograms, kilograms, catties, taels, tons, etc., depending on the production scale.

[0075] Example 1 Screening by Sniffing Method

[0076] The obtained Lactobacillus plantarum was initially screened for fermentation performance, including acid production characteristics experiment, glucose fermentation gas production experiment, mucus production experiment, H2S production experiment, and Oxford cup method antibacterial experiment (Staphylococcus aureus and Escherichia coli). Strains with strong acid production ability, no mucus production, no gas production during glucose fermentation, no H2S production, and obvious inhibitory effect on spoilage bacteria were selected for re-screening. The strains obtained from the initial screening were inoculated into a liquid medium supplemented with soy protein isolate and glucose, and after culturing at 37 °C for 24 h, the fermentation effect of lactic acid bacteria on soy protein was analyzed by smelling the aroma. The results were expressed in terms of the strength of the fermentation aroma and the strength of the beany flavor. The flavor characteristics of the fermentation aroma included sweet and fresh aroma, fruity aroma, fatty aroma, and creamy aroma, and the intensity was expressed as "-" (no aroma), "+" (slight aroma), "++" (relatively fragrant), and "+++" (strong aroma); the intensity of the beany flavor was expressed as "-" (none), "+" (slight beany flavor), and "++" (strong beany flavor). The blank liquid medium without any inoculant was used as the negative control, and the fermentation liquid medium inoculated with Lactobacillus plantarum CICC 21792 was used as the positive control.

[0077] The results showed that there were obvious differences in the flavors of the fermentation products of different lactic acid bacteria strains. Some strains could not adapt well to the fermentation of soy protein, perhaps they did not convert the beany flavor compounds in soy protein into other flavor components, and as a result, only a weak fermentation aroma or no fermentation aroma was presented. The fermentation broth of Lactobacillus plantarum CMRC 1L had sweet and fresh aroma, fruity aroma, and creamy aroma, and the fermentation aroma was stronger, while the beany flavor was significantly weakened. As shown in Table 1, compared with the control without inoculant, Lactobacillus plantarum CMRC 1L effectively improved the overall flavor characteristics of the sample, indicating that this strain plays an important role in enhancing the sensory quality of plant protein meat products mainly made of soy protein. Lactobacillus plantarum CICC 21792 had a normal sweet and fresh fermentation aroma, but at the same time had a stimulating and sour smell, the sweet and fresh aroma was relatively weak, and the fresh aroma was more prominent.

[0078] Table 1

[0079]

[0080] Example 2 Biological characteristics of Lactobacillus plantarum ( Lactobacillus plantarum ) CGMCC No. 32440

[0081] 1. Growth curve determination

[0082] The glycerol stock-preserved Lactobacillus plantarum ( Lactobacillus plantarum ) CGMCC No. 32440 was inoculated into MRS nutrient broth for activation twice, and then transferred to fresh MRS broth medium at an inoculation amount of 2%, and cultured with shaking at 37 °C and 200 r / min for 48 h. Samples were taken every 2 h to measure the turbidity of the bacterial suspension (OD600), and three replicates were made for each time point.Figure 3 This is the growth curve of Lactobacillus plantarum CMRC 1L strain of the present invention.

[0083] 2. Determination of acid production curve

[0084] The strain was cultured and sampled at regular intervals as above. The pH value change of the bacterial suspension was measured with a pH meter, and 3 replicates were made at each time point. Figure 4 This is the acid production curve of Lactobacillus plantarum CMRC 1L strain of the present invention.

[0085] Example 3 A fermented plant protein meat product and its processing method

[0086] 1. Soaking and shredding of soy protein isolate: 500 g of soy protein isolate was presoaked in water for 1 h to fully rehydrate and then drained.

[0087] 2. Mixing and flavoring of raw materials: 600 g of soy protein isolate, 400 g of soy protein powder, 400 g of wheat gluten, 100 g of granulated sugar, 40 g of salt, 40 g of brewed soy sauce, 20 g of five-spice powder, 20 g of yeast extract and 1200 g of water were mixed evenly according to the ratio.

[0088] 3. Inoculation of starter culture: 0.005 g of starter culture dry powder was accurately weighed and dissolved in 200 g of 2% glucose solution, and then added to the raw materials and stirred evenly, so that the addition amount of Lactobacillus plantarum ( Lactobacillus plantarum ) CGMCC No. 32440 was 10 4 CFU / g of raw materials.

[0089] 4. Fermentation: It was placed in a constant temperature and humidity incubator for fermentation. The fermentation conditions were temperature 20 °C for 16 hours. To ensure the uniformity of the distribution of the starter culture in the raw materials, intermittent stirring was carried out during fermentation.

[0090] 5. Shaping and drying: The fermented raw materials were extruded into meat strip shapes. They were placed in a smokehouse to dry the moisture. The drying stage conditions were temperature 55 °C for 1 h.

[0091] 6. Baking: It was placed in an oven for baking. The baking conditions were temperature 130 °C for 10 min.

[0092] 7. Cooling and packaging: After cooling to room temperature, it was packaged.

[0093] Comparative Example 1 A fermented plant protein meat product and its processing method

[0094] Same as Example 3, the difference is that:

[0095] 3. Inoculation of starter: Accurately weigh 0.005g of starter powder, dissolve it in 200g of 2% glucose solution, and add it to the raw material and stir evenly, so that the amount of Lactobacillus plantarum CICC 21792 added to the raw material is 10 4 CFU / g raw material.

[0096] Comparative Example 2 A plant protein jerky product and its processing method

[0097] Same as Example 3, except that:

[0098] No starter culture was inoculated.

[0099] Experimental Example 1 Sensory Evaluation

[0100] A sensory evaluation panel consisting of 15 experienced professionals aged between 20 and 40 was selected to evaluate the color, smell, taste and mouthfeel of the plant protein jerky products through blind scoring. The total score for each item was 25 points. The acceptance was the sum of the scores of the above four items. The mouth was rinsed with water between the evaluations of each sample. The sensory evaluation standards are shown in Table 2, and the sensory evaluation results are shown in Table 3.

[0101] Table 2 Sensory evaluation standards

[0102]

[0103] Table 3 Sensory evaluation results

[0104]

[0105] As can be seen from Table 3, the products made in Example 3 and Comparative Example 1 are significantly higher than those in Comparative Example 2 in terms of smell, flavor, mouthfeel and overall acceptance, which are specifically manifested as having a sweet fermented aroma without a beany smell; the taste is palatable and the aftertaste is mellow. The flavor of Comparative Example 2 is relatively simple, the taste is average, and there is a significant beany smell. The plant protein jerky product made in Example 3 is superior to that in Comparative Example 1 in terms of smell and taste, and is manifested as a richer aroma without unpleasant odors such as irritation and acridity. There is no significant difference in color between Example 3 and Comparative Examples 1-2. It can be seen that the present invention uses Lactobacillus plantarum CMRC 1L to prepare fermented plant protein jerky, which can significantly improve the flavor characteristics of the product, enhance the taste and quality of plant protein meat products, and make it easier for consumers to accept.

[0106] Experimental Example 2: Environmental tolerance test of Lactobacillus plantarum

[0107] The pH tolerance, ethanol tolerance, glucose tolerance, and sodium chloride tolerance of the strains were measured separately: After the strains were transferred and activated twice, they were inoculated at an inoculation amount of 2% into MRS liquid media containing different ethanol volume fractions (0%, 3%, 6%, 9%, 12%, 15%), pH values (2, 3, 4, 5, 6, 7, 8, 9, 10), glucose mass concentrations (100, 200, 300, 400, 500, 600, 700 g / L), and sodium chloride mass concentrations (0, 20, 40, 60, 80, 100 g / L). Incubate at 36 °C and 120 r / min for 1 day, and use an enzyme-linked immunosorbent assay (ELISA) reader to measure the OD value at 600 nm ( Figure 5 , Figure 6 Figure 7 and Figure 8 ).

[0108] As Figure 5 shown, the OD values at different pHs showed a trend of first increasing and then decreasing. An overly acidic or alkaline environment would inhibit the growth of the strains. The OD values of Lactobacillus plantarum CMRC 1L were close to 0 when measured at pH 3 and pH 9, and the highest OD value was measured at pH 5. The OD values of strain CICC 21792 were close to 0 when measured at pH 4 and pH 9, and the highest OD value was measured at pH 6.

[0109] As Figure 6 shown, as the ethanol addition amount increased, the OD value gradually decreased, indicating that the growth and reproduction ability of the strains gradually decreased. The higher the ethanol concentration, the more obvious the inhibitory effect on its growth, and it could even cause its growth to stop. The maximum ethanol tolerance concentration of strain CMRC 1L was 12%, and that of strain CICC 21792 was 9%.

[0110] As Figure 7 shown, when the sodium chloride mass concentration was less than 40 g / L, it had basically no effect on the growth and reproduction of the two strains. When the sodium chloride mass concentration was greater than 40 g / L, the growth and reproduction ability of the strains gradually decreased. When the sodium chloride mass concentration increased to 80 g / L, both strains basically stopped growing.

[0111] As Figure 8 shown, as the initial glucose mass concentration increased, the growth ability of the strains gradually decreased; in a 600 g / L glucose solution, both strains could basically not grow; in a 500 g / L glucose solution, strain CMRC 1L could grow slowly, with an OD600nm of 0.994, while under this condition, the OD600nm of strain CICC 21792 was 0.688, indicating that strain CMRC 1L had good tolerance to glucose.

[0112] Experimental Example 3 Growth and Reproduction of Lactobacillus plantarum Utilizing Different Plant Proteins

[0113] (1)Preparation of plant protein medium: Add 15% soy protein powder and 2% glucose to 100 mL of water, shake and mix well, sterilize at 105 °C for 10 min, and reserve as soy protein liquid medium. Similarly, prepare rice protein, wheat protein, pea protein, and peanut protein liquid media.

[0114] (2)Plant protein liquid fermentation method: After activation, inoculate lactic acid bacteria into the above medium at an inoculation amount of 2%. At the same time, use the un-inoculated plant protein liquid medium as a control, and culture at 36 °C and 120 r / min for 24 - 48 h.

[0115] (3)Viable count of lactic acid bacteria in different plant protein fermentations

[0116] The viable count method for lactic acid bacteria is the plate count method. Take plant protein fermentations with different dilution gradients, coat them on MRS agar medium, and count after culturing at 36 °C for 48 h.

[0117] According to the plate count method, gradient-dilute and coat the fermentations of two lactic acid bacteria cultured in different plant protein media for 24 h on mRS medium. The results are shown in Table 4. The growth of the two lactic acid bacteria using rice protein is poor, and their utilization ability for the other plant proteins is good. By comparing the viable cell numbers in the fermentations, it is found that the fermentation effect of CMRC 1L strain on different plant proteins is better than that of CICC 21792 strain, that is, the viable cell number is higher.

[0118] Table 4 Viable count of different plant proteins

[0119]

[0120] Experimental Example 4 Sensory Evaluation of the Fermentation Effect of Lactobacillus plantarum on Different Plant Proteins

[0121] Sensory evaluation by smelling method: The sensory evaluation panel consists of 20 experienced tasters aged 24 - 40 years old. The fermentation samples are randomly presented in the sensory test room. Determine the aroma characteristic descriptors of plant proteins: The members of the sensory evaluation panel develop and determine the aroma characteristic descriptors by directly smelling the control and plant protein fermentations. Finally, the determined descriptors are: nutty flavor, astringent flavor, greasy flavor, sour flavor, sweet and fresh fragrance, creamy fragrance, wine fragrance, and overall acceptance. Score them, and the flavor is divided into ten grades from weak to strong (Table 5).

[0122] Table 5 Sensory intensity scoring standard for fermentations

[0123]

[0124] The sensory evaluation of different protein fermentates and the control was carried out by the smelling method, and the results Figures 9 - 13 are shown (the soy protein fermentate corresponds to Figure 9 , the rice protein fermentate corresponds to Figure 10 , the peanut protein fermentate corresponds to Figure 11 , the wheat protein fermentate corresponds to Figure 12 , the pea protein fermentate corresponds to Figure 13 ). In the rice protein fermentation experiment, the flavor profiles of the lactic acid bacteria fermentation group and the control group were similar, with no significant differences. In other plant protein fermentation experiments, the astringent taste of the beans in the unfermented control group was more obvious, and at the same time, the nutty taste and oily taste were more prominent, and the overall flavor was weaker. After lactic acid bacteria fermentation, the astringent taste, nutty taste and oily taste were all weakened to varying degrees or even disappeared. Compared with the control, the sour taste characteristics in the fermentation group were more easily detected by people, and the sweet and fresh fragrance and creamy fragrance of the fermentation increased, and there was a little wine fragrance in some fermentates. The acceptability of different plant proteins all increased. Among them, the highest acceptability was peanut protein, followed by soy protein, and the improvement effect on soy protein was the best.

[0125] In summary, lactic acid bacteria fermentation can greatly improve the flavor of soy protein, wheat protein, pea protein and peanut protein, and the pleasant flavor characteristics such as sweet and fresh fragrance and creamy fragrance produced by the fermentation of CMRC 1L strain are more obvious, and the overall acceptability is better than that of the fermentation group of CICC 21792 strain.

[0126] Experimental Example 5 Comparison of Volatile Flavor Substances of Soy Protein Fermented by Lactobacillus plantarum

[0127] It was preliminarily judged by the smelling method that the improvement effect of lactic acid bacteria fermentation on the flavor quality of soy protein was the most significant, and the volatile flavor substances of the soy protein fermentate were further analyzed. Accurately weigh 5 mL of the sample to be tested and place it in a headspace sample bottle, add 5 μL of 2-methyl-3-heptanone with a concentration of 0.0816 μg / μL, tighten the bottle cap, insert a 50 / 30 μm DVB / CAR / PDMS extraction head, and place it in a 50 °C water bath to equilibrate for 10 min, then push out the fiber head to start extraction for 30 min. Then insert the extraction head into the GC-MS injection port for desorption for 10 min, and at the same time start the instrument to collect data. Each sample was repeated 3 times, and the average value was taken. The medium without inoculating lactic acid bacteria was used as the control.

[0128] GC conditions: TG-WaxmS polar column (30 m × 0.25 mm, 0.25 µm), splitless mode; carrier gas is high-purity helium; flow rate is maintained at 1.2 mL / min; inlet temperature is 230 °C. Temperature programming: column temperature is maintained at 40 °C for 3 min; heated to 200 °C at a rate of 5 °C / min; then heated to 230 °C at a rate of 10 °C / min and maintained for 5 min.

[0129] MS conditions: Electron ionization source (EI): ionization energy 70 eV, ion source temperature 300 °C; transfer line temperature 250 °C; full scan mode is adopted, mass scan range 40 - 400 m / z, start scanning time 3 min, scan for 48 min.

[0130] Qualitative analysis: Compare the compounds in the mass spectrum with databases such as NIST and Wiley, delete high-boiling compounds with retention time greater than 45 min and no fragrance, and determine the compounds based on positive and reverse similarity indices greater than 800.

[0131] Quantitative analysis: Calculate the content of each flavor substance relative to the internal standard through the peak area and content of the internal standard 2-methyl-3-heptanone. The calculation formula is as follows:

[0132]

[0133] Where: C is the content of the volatile substance to be measured / (μg / kg); Ax is the peak area of the volatile substance to be measured / (AU·min); C0 is the concentration of the internal standard (0.0816 μg / μL); A0 is the peak area of the internal standard / (AU·min); V is the injection volume of the internal standard / μL; m is the mass of the sample to be measured / g.

[0134] Table 6 results show that in the fermented soy protein inoculated with lactic acid bacteria, the types and total amounts of flavor substances are significantly increased, specifically manifested in the obvious increase in the contents of acids, alcohols, aldehydes, and esters. Among them, in the fermentation groups of CMRC 1L strain and CICC 21792 strain, the acid contents are 711.40 and 652.90 μg / kg respectively, which are extremely significantly higher than 86.14 μg / kg in the control group. At the same time, small-molecule aldehydes and esters often have lower thresholds and play an important role in the formation of the overall flavor of the sample.

[0135] Comparative analysis of the two fermentation groups found that in the fermentation group of CMRC 1L strain, the total amount of volatile flavor substances was 1864.68 μg / kg, which was significantly higher than 1593.56 μg / kg in the fermentation group of CICC 21792 strain. It was mainly manifested as more alcohols, aldehydes and other substances. For example, the content of 2-n-amyl furan with the smell of green beans and butter and trans-2-octenal with the smell of nuts in the CMRC 1L group was significantly higher than that in the CICC 21792 group; for example, hexanoic acid with a sweet smell and hexanal with the smell of beef tallow were only detected in the CMRC 1L group. Combining with the sensory evaluation, it can be known that the CMRC 1L strain can improve the flavor of plant protein products mainly made of soy protein, and it has great potential to be applied to the production of plant protein meat products.

[0136] Table 6 Comparison of Volatile Flavor Substances in Fermented Soybean Protein by Lactobacillus plantarum

[0137]

[0138] Although the present invention has been described in detail with general descriptions and specific embodiments above, on the basis of the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection required by the present invention.

Claims

1. Lactobacillus plantarum Lactobacillus plantarum CMRC 1L, the deposit number is CGMCC No. 32440.

2. A microbial preparation containing the plant lactobacillus according to claim 1; The content of plant lactobacillus in the microbial preparation is 10 9 -10 11 CFU / g.

3. The microbial preparation according to claim 2, characterized in that The preparation method of the microbial preparation comprises: fermenting and culturing the plant lactobacillus, centrifuging the obtained fermentation liquid, collecting bacterial precipitates, resuspending the bacterial bodies with sterile physiological saline, centrifuging and discarding the supernatant, adding a small amount of physiological saline again to mix well to obtain concentrated bacterial liquid, adding an appropriate amount of freeze-drying protective agent and freeze-drying to obtain the concentrated bacterial liquid.

4. The microbial preparation according to claim 3, characterized in that The mass ratio of the concentrated bacterial solution to the freeze-drying protective agent is (0.1-10):1; The lyoprotectant is selected from at least one of skim milk, glycerol and sucrose.

5. Use of the plant lactobacillus according to claim 1 or the microbial preparation according to any one of claims 2 to 4 in food fermentation.

6. A method for preparing a vegetable protein meat product, characterized in that: The following steps are involved: 1) Soaking and untying plant texture protein: Soak the plant texture protein in water for 1-2 hours to fully rehydrate it and then drain the water; 2) Raw material mixing and flavoring: Mix the vegetable protein powder, vegetable textured protein, seasoning and water evenly according to the proportion; 3) Inoculating the starter: adding the microbial preparation according to any one of claims 2 to 4 according to the ratio; 4) Fermentation: Place in a constant temperature and humidity chamber for fermentation; 5) Molding and drying: according to production needs, the fermented raw materials are squeezed into corresponding shapes; 6) Baking: put it in the oven for baking; 7) Cooling and packaging: Cut according to production needs and package after cooling to room temperature.

7. The method according to claim 6, characterized in that The plant textured protein is selected from soybean textured protein and / or pea textured protein; The plant protein powder is selected from at least one of soy protein isolate powder, soy protein powder, wheat protein powder, rice protein powder and pea protein powder; The seasoning is selected from at least one of salt, sugar, brewed soy sauce, five-spice powder and yeast extract.

8. The method according to claim 7, characterized in that In terms of weight, the ingredients of step 2) are as follows: 50-80 parts of soy textured protein, 20-50 parts of soy protein isolate powder, 20-50 parts of gluten powder, 2-20 parts of white sugar, 2-6 parts of salt, 2-10 parts of brewed soy sauce, 1-3 parts of spice powder or five-spice powder, 1-5 parts of yeast extract and 100-150 parts of water; In step 3), the inoculum amount of Lactobacillus plantarum is 10 4 CFU / g raw material, the microbial preparation is dissolved in 2% glucose solution in advance, wherein the amount of glucose solution is 5-10% of the total weight of the raw material.

9. The method according to claim 8, characterized in that Step 4) The fermentation conditions are: temperature 20-25°C, 12-36 hours; Step 5) squeeze the fermented raw material into a tray in the shape of meat strips, and put it into a smoker to dry out the moisture. The drying conditions are: temperature 50-55°C, 1-2h; The baking conditions in step 6) are: temperature 120-150° C., 10-20 min.

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