Lactobacillus clavuliformis Z5-1 as well as fungicide and application of lactobacillus clavuliformis Z5-1

Abstract

The invention provides lactobacillus coryniformis Z5-1 as well as a culture method and application thereof, and belongs to the technical field of food fermentation. The preservation number of the lactobacillus clavuliformis Z5-1 is CGMCC (China General Microbiological Culture Collection Center) No.31547, the lactobacillus clavuliformis Z5-1 is preserved in the China General Microbiological Culture Collection Center (CGMCC) on August 5, 2024, and the lactobacillus clavuliformis Z5-1 has good degradation and conversion effects on ethanol. The lactobacillus clavuliformis Z5-1 is inoculated in a high-alcohol-degree or other fermentation environment, so that ethanol in the environment can be effectively degraded, or ethanol generated in the fermentation process is converted into various flavor substances, and the quality of fermented food is improved; and the strain culture mode is simple and convenient to operate, low in cost and easy to popularize and apply in laboratories and industrial production.

Description

Technical Field

[0001] The invention belongs to the technical field of food fermentation, and in particular relates to a rod-shaped putrefactive lactobacillus Z5-1 and a bacterial agent and application thereof. Background Art

[0002] Many traditional plant-based fermented foods, such as wine, fruit vinegar, kimchi, sauerkraut, soy milk, miso, fermented bean curd, etc., undergo natural fermentation, due to the metabolic effects of different microorganisms, and multiple complex biochemical reactions occur at the same time, producing a variety of metabolites, especially flavor substances represented by ketones, aldehydes, alcohols, acids, and esters. These substances give fermented foods a unique aroma and nutrition, and are deeply loved by people.

[0003] With the development of the fermentation industry, artificial inoculation to produce fermented foods has become the mainstream, but the single inoculation method leads to insufficient flavor of fermented foods, which cannot meet people's subjective impression and psychological needs for traditional fermented foods. Studies have found that lactic acid bacteria play an important role in traditional fermented foods and can affect the enrichment of flavor substances by producing diacetyl. However, the special existence conditions of ethanol will have many adverse effects on the quality of fermented foods. On the one hand, the white wine added before fermentation or the high ethanol environment produced during the fermentation process inhibits the growth of lactic acid bacteria, resulting in the obstruction of the production of flavor substances in the later stage. On the other hand, some plant-based fermented foods will also be affected by the yeast they carry in the early stage of fermentation, accumulating excessive ethanol and interfering with the normal fermentation process. In addition, the infection of undesirable microorganisms, rich nitrates and nitrites, low fermentation efficiency, long fermentation cycle and unstable post-production quality have seriously restricted the industrial development of traditional fermented foods.

[0004] As a type of microorganism that is widely used in fermented foods and is highly safe, lactic acid bacteria have unique advantages in improving the quality of fermented foods. At present, some studies have isolated lactic acid bacteria with the ability to degrade ethanol from traditional fermented foods and used them to prepare health foods for hangover and liver protection. However, most of the lactic acid bacteria traditionally used in plant-based food fermentation do not have the function of specifically degrading ethanol, or their ability to degrade ethanol is very limited, which cannot meet the actual needs of effectively regulating the ethanol content in the industrial production process of fermented foods.

[0005] Therefore, there is an urgent need to develop a new lactic acid bacteria resource that can tolerate ethanol while efficiently, moderately and safely degrading excess ethanol produced in the fermentation of plant-based foods, so as to ensure the quality and stability of fermented foods and promote the healthy development of the food industry. Summary of the invention

[0006] In view of this, the object of the present invention is to provide a rod-shaped putrefactive lactobacillus Z5-1, which has the ability to significantly degrade ethanol and can further convert the ethanol produced during the fermentation process into a variety of flavor substances, thereby improving the quality of fermented foods.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] A coryneform lactobacillus (Loigolactobacillus coryniformis) Z5-1, wherein the deposit number of the coryneform lactobacillus Z5-1 is CGMCC No.31547.

[0009] The present invention also provides a bacterial agent containing the coryneform Lactobacillus putrefactive Z5-1. Preferably, the viable count of the coryneform Lactobacillus putrefactive Z5-1 is ≥ 2×10 7 CFU / mL.

[0010] The present invention also provides a method for preparing the bacterial agent, comprising the following steps: the rod-shaped putrefactive lactobacillus Z5-1 is inoculated into an MRS solid culture medium for activation culture, and the activated colonies are inoculated into an MRS liquid culture medium to prepare a bacterial suspension, namely a liquid bacterial agent.

[0011] Preferably, the method further comprises the following steps: inoculating the bacterial suspension into an MRS broth medium to prepare a primary bacterial strain; adding a nutritional supplement to the MRS broth medium for continued fermentation and culture to prepare a secondary bacterial strain; and adding a thickener to prepare a paste bacterial agent. More preferably, the nutritional supplement comprises germinated brown rice paste, peanut paste, trace elements and growth factors.

[0012] The present invention also provides the use of the coryneform putrefactive lactobacillus Z5-1 or bacterial agent in degrading ethanol.

[0013] The present invention also provides the use of the coryneform putrefactive lactobacillus Z5-1 or the bacterial agent in the preparation of fermented food.

[0014] Preferably, the fermented food includes fruit wine, sauerkraut, fruit vinegar and wine-made fruit. More preferably, the fruit wine, sauerkraut or wine-made fruit is fermented by inoculating the bacterial agent on the basis of the fermentation raw material; and the fruit vinegar is fermented by inoculating acetic acid bacteria on the basis of fruit wine as the raw material.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The rod-shaped putrefactive lactobacillus Z5-1 of the present invention has excellent degradation and conversion capabilities for ethanol, and can effectively reduce the ethanol content in high alcohol content or other fermentation environments, reducing its adverse effects on the quality of fermented foods. At the same time, the strain can convert ethanol into a variety of flavor substances, significantly improve the aroma and taste of fermented foods, enrich their flavor levels, and meet consumers' demand for flavor diversity and high quality of fermented foods.

[0017] Rod-shaped Lactobacillus putrefaciens Z5-1 has a wide range of adaptability to ethanol, pH and temperature, and has high alcohol dehydrogenase and acetaldehyde dehydrogenase activities. It can grow and metabolize stably under different fermentation conditions, ensuring the smooth progress of the fermentation process and reducing the risk of fermentation failure caused by changes in environmental factors. It provides more reliable guarantees for industrial production and ensures the quality and safety of fermented foods.

[0018] The application effect of rod-shaped putrefactive Lactobacillus Z5-1 in the preparation of various fermented foods such as fruit wine, fruit vinegar, sauerkraut, and wine-made fruit is remarkable. In the fermentation of fruit wine, it can increase the alcohol content, reduce the pH value, reduce the content of organic acids, and increase the content of various flavor substances, making the fruit wine more fragrant, softer in taste, and more stable in quality; in the production of fruit vinegar, it can improve the activity of acetic acid bacteria, increase the content of acetic acid, optimize the content of phenols, amino acids and antioxidant capacity, and enhance the flavor and nutritional value of fruit vinegar; when used in sauerkraut fermentation, it can effectively degrade ethanol, increase the lactic acid content, optimize the flavor components, inhibit the growth of harmful microorganisms, and ensure the quality and safety of sauerkraut; in the production of wine dates, it can retain the nutrients of jujube, increase the content of soluble solids, vitamin C, flavonoids, etc., improve the antioxidant capacity, amino acid content and types of volatile substances, and give the wine dates a unique flavor and better preservation, which fully reflects the great potential and wide applicability of this strain in improving the quality of various fermented foods. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a microscopic photograph of the strain Lactobacillus putrefaciens Z5-1;

[0020] Figure 2 is the phylogenetic tree of Lactobacillus putrefaciens Z5-1;

[0021] Figure 3 is the growth curve of coryneform Lactobacillus putrefaciens Z5-1;

[0022] Figure 4 is the ethanol degradation ability of different strains.

[0023] Biological Deposit Description

[0024] The rod-shaped spoilage lactobacillus Z5-1 of the present invention is classified and named as rod-shaped spoilage lactobacillus (Loigolactobacillus coryniformis), the preservation unit is the General Microbiology Center (CGMCC) of the China Microbiological Culture Collection Administration, the preservation unit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the preservation number is CGMCC No. 31547, and the preservation date is August 5, 2024. DETAILED DESCRIPTION

[0025] The present invention provides a rod-shaped putrefactive lactobacillus (Loigolactobacillus coryniformis) Z5-1, the deposit number of which is CGMCC No.31547. The bacterium is derived from the bacterial flora of aged pit mud. 60 The strain was obtained by Co-γ-ray irradiation. The bacterial competition in the aged cellar mud environment is fierce. The strain can exist in such an environment, indicating that it has strong adaptability, which provides the possibility for its application in various fermentation industrial scenarios.

[0026] The present invention also provides a bacterial agent containing coryneform lactobacillus Z5-1; preferably, the number of viable bacteria of coryneform lactobacillus Z5-1 in the bacterial agent is ≥ 2×10 7 CFU / mL; preferably, the number of viable bacteria is ≥2×10 10 CFU / mL.

[0027] The present invention further provides a method for preparing the above-mentioned bacterial agent, comprising the following steps: inoculating the rod-shaped putrefactive lactobacillus Z5-1 into an MRS solid culture medium for activation culture, and inoculating the activated colonies into an MRS liquid culture medium to prepare a bacterial suspension, namely a liquid bacterial agent.

[0028] In the present invention, the preferred culture temperature is 32-38°C, within which the rod-shaped spoilage Lactobacillus Z5-1 grows well, and the preferred culture temperature is 35-37°C. The number of viable rod-shaped spoilage Lactobacillus Z5-1 in the bacterial suspension is preferably ≥ 2×10 7 CFU / mL.

[0029] As an implementable embodiment, the MRS solid culture medium formula of the present invention is: peptone 10g, beef extract powder 5g / L, yeast extract powder 4g / L, glucose 20g / L, Tween 80 1g / L, dipotassium hydrogen phosphate 2g / L, sodium acetate 5g / L, diammonium hydrogen citrate 2g / L, magnesium sulfate 0.2g / L, manganese sulfate 0.05g / L, agar powder 15g / L; MRS liquid culture medium formula is: yeast extract 5g / L, beef extract 10g / L, glucose 20g / L, magnesium sulfate 0.58g / L, anhydrous sodium acetate 3g / L, peptone 10g / L, diammonium hydrogen citrate 2g / L, manganese sulfate 0.25g / L.

[0030] In the present invention, it is preferred that the following steps are further included to further prepare the above-mentioned liquid bacterial agent into a paste bacterial agent: inoculating the above-mentioned bacterial suspension / liquid bacterial agent into an MRS broth culture medium to prepare a primary bacterial strain; adding a nutritional supplement to the MRS broth culture medium to continue fermentation and culture to prepare a secondary bacterial strain, and adding a thickener to prepare a paste bacterial agent.

[0031] In the present invention, the primary culture time is preferably 3-5 days, and the culture temperature is further preferably 32-38°C, and the viable cell count is ≥3×10 8 In the present invention, the secondary culture time is preferably 7 days, and the culture temperature is further preferably 30-37°C, and the viable count is ≥ 2×10 10 CFU / mL. As an embodiment, the MRS broth medium of the present invention is formulated as follows: peptone 10g / L, beef extract powder 8g / L, yeast extract powder 4g / L, glucose 20g / L, dipotassium hydrogen phosphate 2g / L, triammonium citrate 2g / L, sodium acetate trihydrate 5g / L, magnesium sulfate heptahydrate 0.2g / L, manganese sulfate tetrahydrate 0.05g / L, Tween 80 1g / L, pH 6.2±0.2.

[0032] In the present invention, preferably the nutritional supplement comprises germinated brown rice paste, peanut paste, trace elements and growth factors; further preferably the germinated brown rice paste is added at 10-20g / L, the peanut paste is added at 5-10g / L, and the addition amount is progressively reduced as the fermentation progresses, more preferably the germinated brown rice paste is added at 10-20g / L (the 1st day), 8g / L (the 3rd day), 6g / L (the 5th day), 4g / L (the 7th day), and the peanut paste is added at 5-10g / L (the 1st day), 8g / L (the 3rd day), 6g / L (the 5th day), 4g / L (the 7th day). The germinated brown rice paste is a semi-finished product formed by grinding brown rice after extrusion puffing, and 80°C hot water is added for gelatinization to a gelatinization degree of 70%-85%, without any other ingredients, and the original nutritional structure of brown rice is maintained, without chemical substances such as food additives and preservatives, and is conducive to the stability of the microbial growth environment, and germination can increase the enzyme activity and some nutrients in the brown rice. Peanut paste (protein content 20%-25%) is made by soaking peanuts for 8 hours until the peanut skin is easy to peel off, and the peeled peanuts are thoroughly rinsed with clean water. A soymilk machine is used to make peanut paste, and the peanuts are mixed with water in a ratio of 1:8 and crushed, and then filtered to obtain the peanut paste. No sugar, salt or other seasonings are added during the production process to ensure that it is pure and natural and without additives. The peanut paste with a high protein content can provide the necessary nitrogen source for microorganisms.

[0033] In the present invention, the preferred trace elements include 0.5 g / L of ferrous sulfate, 0.5 g / L of manganese sulfate, and 3 g / L of vitamin C, and the preferred growth factor is 100 g / L of potato juice. All of the above ingredients need to be sterilized before addition, using high-pressure steam sterilization at 121°C for 15 minutes. Ferrous sulfate is essential for the metabolism of microorganisms. It participates in processes such as the electron transport chain and redox reactions, and promotes the growth and reproduction of bacteria. Manganese sulfate contributes to a variety of biochemical reactions in cells, including DNA synthesis, protein synthesis, etc., and it can improve the tolerance and stress resistance of certain strains. Vitamin C is a powerful antioxidant that can protect cells from free radical damage, promote iron absorption, enhance immune system function, and support some metabolic processes in cells. Potato juice is rich in carbohydrates, vitamins and minerals, which can provide an additional source of nutrition for microorganisms, especially for those strains that require complex carbon sources, it can significantly promote their growth.

[0034] In the present invention, the thickener is preferably selected from one or more of carrageenan, xanthan gum, guar gum, and sodium carboxymethyl cellulose, and the addition amount is 1-2wt%; further preferably, 1-2wt% lecithin is added. Lecithin is a naturally occurring phospholipid with emulsifying properties, which helps prevent oil-water separation. In addition, lecithin can also provide phospholipid substances for microorganisms, which is very important for the construction and maintenance of cell membranes.

[0035] The present invention also provides the use of coryneform putrefactive Lactobacillus Z5-1 or a bacterial agent in degrading ethanol and preparing fermented food. Preferably, the fermented food includes fruit wine, sauerkraut, fruit vinegar and wine-made fruit; more preferably, the fruit wine, sauerkraut or wine-made fruit is fermented by inoculating the coryneform putrefactive Lactobacillus Z5-1 bacterial agent on the basis of the fermentation raw material; and the fruit vinegar is fermented by inoculating acetic acid bacteria with fruit wine as the raw material.

[0036] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0037] Example 1

[0038] Mutagenesis screening of strain Z5-1

[0039] (1) Source of bacterial flora

[0040] Use Liangshan high-quality strong-flavor aged cellar mud and vacuum pack it in 100g quantities for later use.

[0041] (2) Mutagenesis Methods

[0042] use 60The Co-γ ray static irradiation method has set irradiation doses of 0, 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 kGy, respectively. During the irradiation, it needs to be flipped 360° twice to ensure uniform dose.

[0043] (3) Preparation of bacterial solution

[0044] Add 50 mL of sterile water to the irradiated pit mud, stir evenly, culture at 37°C for 12-24 hours, filter, and repeatedly rinse the precipitate with the filtrate for 3-5 times to obtain a mixed bacterial solution induced by radiation.

[0045] (4) Screening of dominant bacterial flora

[0046] The mixed bacterial solution was inoculated into MRS broth at a ratio of 10% (V / V), and then cultured at 37°C for 48h after being shaken evenly. 10% vol anhydrous ethanol was added, and then cultured at 37°C for 48h after being shaken evenly. The operation was repeated for 5 times, and the number of colonies was determined, and the lethality was calculated. As shown in Table 1, the lethality of the bacterial solution after 0.8 kGy irradiation was 70%-80%, which was considered to have a high positive mutation rate, an absolute mutagenic advantage, and the optimal irradiation mutagenic dose.

[0047] Table 1 Effects of different irradiation doses on total colony count and mortality rate

[0048] Irradiation dose kGy Total colony count CFU / mL Fatality rate% 0 <![CDATA[205.92×10 6 ]]> 0 0.2 <![CDATA[121.10×10 6 ]]> 41.19 0.4 <![CDATA[103.13×10 6 ]]> 49.92 0.6 <![CDATA[69.38×10 6 ]]> 66.31 0.8 <![CDATA[45.90×10 6 ]]> 77.71 1.0 <![CDATA[23.18×10 6 ]]> 88.70 1.2 <![CDATA[8.45×10 6 ]]> 95.90

[0049] (5) Isolation and screening of single bacteria

[0050] The 0.8 kGy irradiated cellar mud bacterial solution was diluted to 10 -4 , 10 -5 , 10 -6 Gradient, 0.1 mL of each dilution was spread on MRS agar medium (containing 50% vol ethanol) plates, and inverted at 37°C for 48 h. Strains with different morphology, size and color were picked out, and streaked three times in succession according to the three-zone streak method. Then they were transferred to MRS broth medium (containing 50% vol ethanol) and cultured at 37°C in a shaking incubator for 3 days. The alcohol content in each bacterial solution was compared, and the dominant strain that degraded ethanol was screened out and named Z5-1. It was cultured as a pure strain and preserved with glycerol.

[0051] (6) Identification of strain Z5-1

[0052] The colony morphology of Z5-1, including size, color, edge, and protrusion, was observed and sent to Shanghai Paisonno Biotechnology Co., Ltd. for 16s rDNA identification to determine its species.

[0053] Streak strain Z5-1 on MRS agar medium and culture at 37℃ for 2 days. Observe the colony morphology, which is regular round, off-white, flat, translucent, with a smooth and shiny surface. Microscopic examination results show that the colony is rod-shaped, such as Figure 1 shown.

[0054] The 16s rDNA identification results showed that strain Z5-1 was Loigolactobacillus coryniformis. The phylogenetic tree of the screened strains was drawn with the help of MEGA7.0 software after alignment using the basic local alignment search tool (BLAST) module in NCBI. The results are shown in Figure 2 .

[0055] Example 2

[0056] Characteristics of coryneform Lactobacillus putrefaciens Z5-1

[0057] (1) Growth curve

[0058] Preparation of MRS liquid culture medium: 5g yeast extract, 10g beef extract, 20g glucose, 0.58g magnesium sulfate, 3g anhydrous sodium acetate, 10g peptone, 2g diammonium citrate, 0.25g manganese sulfate, 1g Tween 80, dissolved in distilled water, adjust the volume to 1L, sterilize at 121℃ for 20min; adjust the pH value to 6.2-6.5, cool and set aside. Inoculate the preserved rod-shaped putrefactive Lactobacillus Z5-1 into fresh MRS liquid culture medium and culture under appropriate conditions for 24-48h to ensure that the strain is in a good physiological state. Take out an appropriate amount of bacterial liquid from the activated culture and inoculate it into a test tube containing an appropriate volume of MRS culture medium so that the final inoculation volume is about 1% (v / v). The temperature is set to 37℃, and the culture time depends on the growth rate of the specific strain, generally observed for 32h. During the culture process, samples are taken every 4h and OD 600 The growth curve of Lactobacillus putrefaciens Z5-1 was determined by turbidimetry (optical density at 600 nm). Figure 3 It can be seen that the rod-shaped putrefactive Lactobacillus Z5-1 began to enter the logarithmic growth phase after 4 hours and entered the stable phase after 20 hours, and the number of microorganisms reached the maximum value and remained relatively stable.

[0059] (2) Ethanol resistance verification

[0060] The bacterial suspension was prepared with distilled water and inoculated into MRS broth culture medium at a ratio of 1:9. The initial ethanol concentrations in the broth were 10% vol, 20% vol, 30% vol, 40% vol, and 50% vol, respectively. The culture was shaken at 37°C for 24 hours, and the number of viable bacteria was determined.

[0061] As shown in Table 2, the viable count of Lactobacillus putrefaciens Z5-1 was high in the ethanol concentration range of 20%vol-30%vol. Even when the ethanol concentration exceeded 30%vol, the viable count remained at a certain level. At a high ethanol concentration of 40%vol-50%vol, the viable count dropped to 0.08×10 7 Around 10% vol, showing good ethanol tolerance. The viable counts of Lactobacillus plantarum CGMCC No.18390, Lactobacillus acidophilus CGMCC No.30824 and Lactobacillus acidophilus CGMCC No.14059 were relatively high in the ethanol concentration range of 10% vol-20% vol. As the ethanol concentration increased to above 30% vol, the viable counts decreased significantly. At high ethanol concentrations of 40% vol-50% vol, the viable counts were very low, at 0.05×10 7 The following. Lactobacillus CICC 6002 and Lactobacillus acidophilus CICC 6005 had a high number of viable bacteria in the low ethanol concentration range of 0-10%vol. However, once the ethanol concentration exceeded 10%vol, especially when it reached 40%vol-50%vol, the number of viable bacteria was almost zero, indicating that they had poor tolerance to ethanol.

[0062] Table 2 The number of viable bacteria of the strains at different ethanol concentrations

[0063]

[0064]

[0065] (3) Verification of ethanol degradation

[0066] The rod-shaped putrefactive lactobacillus Z5-1 was isolated and purified. At the same time, 5 strains of bacteria were used as controls. The 5 strains of bacteria were inoculated into ethanol culture medium (10% vol ethanol was added on the basis of MRS broth culture medium) at 5%, and the ethanol content was determined by potassium dichromate-sulfuric acid method, and the ethanol degradation rate was calculated. The calculation formula of ethanol degradation rate is as follows:

[0067]

[0068] Where: C0 is the volume fraction of ethanol in the blank control, %; C is the volume fraction of ethanol in the test group, %.

[0069] The results are as follows Figure 4 As shown, different strains have different effects on ethanol degradation. By comparison, it can be seen that the rod-shaped putrefactive Lactobacillus Z5-1 has the best ethanol degradation effect, with an average degradation rate of 15±2%, followed by Lactobacillus plantarum CGMCCNo.18390, and Lactobacillus CICC 6002 has the lowest ethanol degradation rate.

[0070] (4) Alcohol dehydrogenase activity assay

[0071] The alcohol dehydrogenase (ADH) activity detection kit was used to measure the alcohol dehydrogenase activity in the bacterial culture.

[0072] The results are shown in Table 3. The alcohol dehydrogenase activity of coryneform Lactobacillus Z5-1 was the highest, 76.3 U / mg, while the activity of Lactobacillus acidophilus CICC 6005 was the lowest, 53.5 U / mg. The activities of other strains were relatively close, concentrated between 60-63 U / mg. Due to its high alcohol dehydrogenase activity, coryneform Lactobacillus Z5-1 can be used as the preferred strain for metabolizing ethanol, and is more suitable for alcohol fermentation, alcohol degradation or the development of functional foods.

[0073] Table 3 Alcohol dehydrogenase activity of different strains

[0074] strain Alcohol dehydrogenase activity (U / mg) Z5-1 76.3 CGMCC No.18390 62.7 CGMCC No.30824 59.4 CGMCC No.14059 61.1 CICC6002 62.8 CICC6005 53.5

[0075] (5) Assay of acetaldehyde dehydrogenase activity

[0076] The acetaldehyde dehydrogenase (ALDH) activity detection kit was used to measure the acetaldehyde dehydrogenase activity in the bacterial culture.

[0077] As shown in Table 4, the acetaldehyde dehydrogenase activity of Lactobacillus putrefaciens Z5-1 was significantly higher than that of other strains, indicating that it was more efficient in metabolizing acetaldehyde. This helps to reduce acetaldehyde accumulation, thereby improving the overall acetaldehyde degradation efficiency. The activity of Lactobacillus plantarum CGMCC No.18390 and Lactobacillus acidophilus CGMCC No.14059 was between high-activity and low-activity strains, indicating that they have a certain ability to metabolize acetaldehyde; the acetaldehyde dehydrogenase activity of the three strains of Lactobacillus CGMCC No.30824, Lactobacillus CICC 6002 and Lactobacillus acidophilus CICC 6005 was low, which may be insufficient in metabolizing acetaldehyde.

[0078] Table 4 Acetaldehyde dehydrogenase activity of different strains

[0079] strain Acetaldehyde dehydrogenase activity (U / mg) Z5-1 37.6 CGMCC No.18390 25.2 CGMCC No.30824 14.2 CGMCC No.14059 20.0 CICC6002 14.9 CICC6005 14.6

[0080] (6) Fermentation adaptability verification

[0081] Prepare MRS solid medium and liquid medium, and adjust the pH value of MRS liquid medium to 2.0, 3.0, 4.0, 5.0, and 6.0. Autoclave at 121°C for 20 min. The solid medium is used for subsequent colony counting. Control the fermentation temperature at 29°C, 32°C, 35°C, 38°C, and 41°C. Inoculate the strains with the same inoculum of 5% into liquid medium under different pH and temperature conditions. Cultivate for 48 hours. Take 100 μL of bacterial solution from each culture and dilute 10 -3 , 10 -4 , 10 -5 Then spread it on a solid plate. Place the coated plate at 37°C and incubate for 48 hours. Count the number of colonies on each plate using the plate count method. Record the CFU / mL value under each condition, which represents the number of viable bacteria per milliliter of sample.

[0082] Table 5 pH tolerance of different strains

[0083] pH 2.0 3.0 4.0 5.0 6.0 Z5-1 <![CDATA[40.29×10 6 ]]> <![CDATA[108.20×10 6 ]]> <![CDATA[167.36×10 6 ]]> <![CDATA[175.46×10 6 ]]> <![CDATA[168.44×10 6 ]]> CGMCC No.18390 <![CDATA[37.95×10 6 ]]> <![CDATA[76.84×10 6 ]]> <![CDATA[156.01×10 6 ]]> <![CDATA[153.67×10 6 ]]> <![CDATA[154.74×10 6 ]]> CGMCC No.30824 <![CDATA[37.60×10 6 ]]> <![CDATA[76.23×10 6 ]]> <![CDATA[137.66×10 6 ]]> <![CDATA[154.85×10 6 ]]> <![CDATA[149.37×10 6 ]]> CGMCC No.14059 <![CDATA[28.93×10 6 ]]> <![CDATA[67.25×10 6 ]]> <![CDATA[139.08×10 6 ]]> <![CDATA[142.77×10 6 ]]> <![CDATA[138.65×10 6 ]]> CICC6002 <![CDATA[34.76×10 6 ]]> <![CDATA[68.30×10 6 ]]> <![CDATA[101.83×10 6 ]]> <![CDATA[109.33×10 6 ]]> <![CDATA[108.50×10 6 ]]> CICC6005 <![CDATA[38.63×10 6 ]]> <![CDATA[73.57×10 6 ]]> <![CDATA[125.71×10 6 ]]> <![CDATA[125.94×10 6 ]]> <![CDATA[127.00×10 6 ]]>

[0084] Table 6 Temperature tolerance of different strains

[0085] Temperature(℃) 29 32 35 38 41 Z5-1 <![CDATA[95.68×10 6 ]]> <![CDATA[156.80×10 6 ]]> <![CDATA[175.46×10 6 ]]> <![CDATA[168.44×10 6 ]]> <![CDATA[88.33×10 6 ]]> CGMCC No.18390 <![CDATA[84.32×10 6 ]]> <![CDATA[135.22×10 6 ]]> <![CDATA[153.67×10 6 ]]> <![CDATA[154.74×10 6 ]]> <![CDATA[72.02×10 6 ]]> CGMCC No.30824 <![CDATA[79.84×10 6 ]]> <![CDATA[147.61×10 6 ]]> <![CDATA[154.85×10 6 ]]> <![CDATA[149.37×10 6 ]]> <![CDATA[69.27×10 6 ]]> CGMCC No.14059 <![CDATA[94.36×10 6 ]]> <![CDATA[134.34×10 6 ]]> <![CDATA[142.77×10 6 ]]> <![CDATA[138.65×10 6 ]]> <![CDATA[87.56×10 6 ]]> CICC6002 <![CDATA[88.45×10 6 ]]> <![CDATA[100.06×10 6 ]]> <![CDATA[109.33×10 6 ]]> <![CDATA[108.50×10 6 ]]> <![CDATA[79.40×10 6 ]]> CICC6005 <![CDATA[94.57×10 6 ]]> <![CDATA[117.38×10 6 ]]> <![CDATA[125.94×10 6 ]]> <![CDATA[127.00×10 6 ]]> <![CDATA[80.29×10 6 ]]>

[0086] As shown in Tables 5 and 6, the pH tolerance and temperature tolerance of rod-shaped putrefactive Lactobacillus Z5-1 are better than those of other strains. When the pH is less than 3, the number of viable bacteria of other strains drops sharply; the fermentation tolerance temperature of rod-shaped putrefactive Lactobacillus Z5-1 is 32-38°C. Within this temperature range, the strain grows well and the number of viable bacteria is high.

[0087] Example 3

[0088] Preparation of coryneform Lactobacillus Z5-1 bacterial agent

[0089] Preparation process of microbial agent:

[0090] 1. Single bacteria activation: thaw the frozen strain stored in -80℃ refrigerator at room temperature, streak the bacterial liquid on MRS solid medium, and place it in a 37℃ constant temperature incubator for 24 hours of activation culture. Take the colony on the plate and inoculate it into MRS liquid medium and culture it at 37℃ for 24 hours.

[0091] 2. Preparation of bacterial suspension: Wash the slant repeatedly with sterile water and culture at 37℃ for 24h. The bacterial count reaches 2.89×10 7 CFU / mL.

[0092] 3.Liquid primary strains:

[0093] The bacterial suspension was inoculated into the culture medium, based on MRS broth, and cultured for 3-5 days until the bacterial count reached 3.18×108 CFU / mL.

[0094] 4. Industrial secondary strains:

[0095] Preparation of germinated brown rice paste: The germinated brown rice was purchased from Benxi Zhaixiang Ecological Agriculture Co., Ltd. and gelatinized by adding 80°C hot water to a gelatinization degree of 70%-85%. Before adding, ensure that the germinated brown rice paste has been sterilized.

[0096] Prepare sprouted peanut paste: Select sprouted peanuts and clean them. Soak the peanuts for 8 hours until the peanut skins are easy to peel off. Rinse the peeled peanuts thoroughly with clean water. Use a soymilk machine to make peanut paste. Mix the peanuts with water in a ratio of 1:8, crush them, and then filter to get peanut paste (protein content 20%-25%). Similarly, the peanut paste also needs to be sterilized.

[0097] Using MRS broth as the basic culture medium, the initial addition amount of germinated brown rice paste is 10-20g per liter of basic culture medium, and then added every 2 days, and the amount added each time is gradually reduced to 8g, 6g, and 4g; the initial addition amount of germinated peanut paste is 5-10g per liter of basic culture medium, and then added every 2 days, and gradually reduced to 4g, 3g, and 2g. In addition, 0.5g of trace elements ferrous sulfate, 0.5g of manganese sulfate, 3g of vitamin C, and 100g of growth factor potato juice are added to each liter of basic culture medium. The above ingredients also need to be sterilized in advance. Set the culture temperature to 30-37℃, culture for 7 days, and mix well when adding new nutritional supplements. The number of viable bacteria reaches 2.75×10 10 CFU / mL, 1%-2% of thickener carrageenan and 1%-2% of lecithin were added to make the final product paste-like.

[0098] The finished product 10-15mL is packaged into aluminum foil bags using heat-sealing technology to prevent leakage and external contamination and maintain product stability.

[0099] 5. Preparation before use:

[0100] Before use, add a certain amount of the finished product into warm water at 35°C and leave it to activate for 30 minutes. The purpose is to activate the dormant microorganisms and enable them to quickly enter an active metabolic state.

[0101] Example 4

[0102] Application of coryneform putrefactive Lactobacillus Z5-1 inoculant in fruit wine

[0103] 1. Fruit selection: Choose fresh and ripe fruits that are free of pests and diseases and are not damaged. Remove impurities such as branches, leaves, stems, and rotten fruits from the fruits to ensure that only high-quality parts are used.

[0104] 2. Wash the fruit: Gently wash the fruit with clean water or food-grade detergent to remove the dirt and pesticide residue on the surface. Be careful not to damage the fruit skin. After washing, use a clean towel or paper towel to wipe the moisture on the surface of the fruit, or let it dry naturally.

[0105] 3. Cut and core / seed: Cut the fruit appropriately according to its size and shape. Use grapes, blueberries, and hawthorns whole, and cut Nanguo pears into small pieces. Remove the core of hawthorns to avoid affecting the taste and fermentation process.

[0106] 4. Blanching: Blanching can inactivate enzymes in the fruit, preventing them from causing adverse changes during the fermentation process, and also helps release the juice. Put the cut fruit into boiling water and quickly blanch for about 1-2 minutes, then immediately remove and quickly cool (you can soak it in ice water). This step should be completed quickly to avoid overcooking and affecting the texture of the fruit.

[0107] 5. Put into sterilized wine jar: Choose clean, well-sealed glass or ceramic wine jars, and thoroughly clean and sterilize them with boiling water before use. Put the blanched fruits evenly into the wine jar, do not fill it too full, and leave some space for the fermentation gas to be discharged.

[0108] 6. Add sugar solution: Prepare sugar solution at a sugar content of 15% (i.e., add 15g sugar to every 100mL water). You can use white sugar, crystal sugar or other suitable sweeteners. Slowly pour the sugar solution into the wine jar containing the fruit, and gently stir to fully mix the sugar solution and the fruit. Be careful not to damage the integrity of the fruit.

[0109] 7. Add yeast: Activate the purchased brewer's yeast with warm water (about 30°C) for 15-30 minutes until the yeast starts to become active and foam. Sprinkle the activated yeast solution evenly on the surface of the fruit and sugar solution. Stir gently to ensure that the yeast is fully in contact with the material.

[0110] 8. Initial fermentation (15-20 days): Seal the jar and place it in a cool, dark place for initial fermentation. The temperature should be kept between 20-25℃, which is the most suitable temperature range for yeast activity. Check once a day. If a lot of gas is produced during the fermentation process, loosen the lid to deflate it slightly, and then seal it again.

[0111] 9. Add Z5-1 bacterial solution: After the first fermentation is completed, open the wine jar, stir gently, add the activated rod-shaped putrefactive Lactobacillus Z5-1 bacterial solution, seal again for secondary fermentation. Ferment for 30 days.

[0112] 10. Sterilization and filtration: Take out the fermentation liquid from the fermentation tank and filter it with 5 layers of gauze to remove the remaining pulp and impurities to make the juice clearer. Sterilize it in a constant temperature water bath at 60-85℃ for 30 minutes.

[0113] 11. Decanting and aging: If you want to further enhance the flavor, you can transfer the sterilized and filtered juice to a clean container and age it at 8°C for 20 days. During this period, decan the juice every 36 hours to separate the supernatant from the lees.

[0114] 12. Clarification treatment: Use the natural clarification method, place the fruit wine in a sealed container and leave it at 15-20℃ for 10 days. After the suspended matter in the fruit wine settles, the supernatant is the fruit wine.

[0115] The fruit wine prepared as above was compared with the fruit wine fermented naturally (without inoculation of Lactobacillus putrefaciens Z5-1), and the alcohol content, pH, organic acid, higher alcohols and various flavor substances were measured. The results are shown in Table 7.

[0116] Table 7 Effects of different fermentation methods on the quality and flavor of fruit wine

[0117]

[0118]

[0119] According to Table 7, the alcohol content of the Z5-1 fermentation group is generally higher than that of the natural fermentation group, which indicates that the rod-shaped spoilage lactobacillus Z5-1 can more effectively utilize sugar for fermentation. The pH value of the Z5-1 fermentation group is generally low, which helps to inhibit the growth of harmful microorganisms and improve the stability of fruit wine. The organic acid content of the Z5-1 fermentation group is generally low, which helps to improve the taste of the fruit wine and make it softer. The content of higher alcohols, volatile fatty acids, volatile phenols, terpenes, esters, aldehydes, ketones and isoprenoids in the Z5-1 fermentation group is generally higher than that in the natural fermentation group, which indicates that the rod-shaped spoilage lactobacillus Z5-1 can better produce and accumulate these compounds that are essential to the flavor and aroma of fruit wine.

[0120] The rod-shaped spoilage Lactobacillus Z5-1 has a higher fermentation efficiency and can more effectively convert sugar into lactic acid and synthesize other flavor compounds. The fruit wine of the Z5-1 fermentation group contains more flavor compounds such as higher alcohols, esters, and terpenes, which give the fruit wine a more complex aroma and taste, and enhance the consumer experience. The moderate organic acid content and low pH value make the acidity of the fruit wine softer and the taste more balanced. However, during the natural fermentation process, the generation of flavor compounds is unstable, resulting in a relatively single flavor of the fruit wine and a lack of layering; the high organic acid content and high pH value lead to excessive or low acidity of the fruit wine, affecting the taste. The fermentation group of the rod-shaped spoilage Lactobacillus Z5-1 strain shows obvious advantages in many aspects, and can produce fruit wines with higher quality, more complex flavors, and better stability, while the natural fermentation group has more uncertainties and risks.

[0121] Example 5

[0122] Application of coryneform putrefactive Lactobacillus Z5-1 inoculation in fruit vinegar

[0123] 1. Use the fruit wine in Example 4: Use fruit wine that has completed primary fermentation and clarified as raw material. The alcohol content of the fruit wine should be between 5% and 10%. Too high or too low alcohol content may affect the activity of acetic acid bacteria. Before inoculating acetic acid bacteria, filter the fruit wine to remove residual pulp, yeast and other impurities to prevent these substances from interfering with the fermentation process of acetic acid bacteria.

[0124] 2. Prepare acetic acid bacteria: Purchase acetic acid bacteria suitable for fruit vinegar brewing, make sure the product comes with detailed instructions, and perform activation treatment according to the instructions. Mix the acetic acid bacteria with an appropriate amount of sterile water or diluted fruit wine, and let it stand for a period of time at a temperature of about 30°C until obvious signs of activity (bubbles are generated) are observed.

[0125] 3. Add acetic acid bacteria: Add the activated acetic acid bacteria solution evenly to the prepared fruit wine. Add 1g of acetic acid bacteria to 250g of fruit wine, and gently stir the fruit wine to ensure that the acetic acid bacteria and the fruit wine are fully mixed. Be careful not to stir too much to avoid introducing too much air. Ferment for 1-2 weeks.

[0126] 4. Choice of fermentation container: Acetic acid bacteria need oxygen for fermentation, so ceramic jars with good air permeability should be selected. Avoid using completely sealed containers to prevent excessive pressure caused by accumulation of carbon dioxide. Make sure all tools and containers are thoroughly cleaned and disinfected before use to avoid contamination by bacteria.

[0127] 5. Fermentation conditions: Place the container containing the fruit wine and acetic acid bacteria in an environment with a temperature of 30°C for fermentation. Use a constant temperature box or fermentation room to maintain a stable temperature. Too high or too low a temperature will affect the activity of acetic acid bacteria. Avoid direct sunlight during the fermentation process. Choose a cool, dark place for fermentation.

[0128] 6. Stop fermentation: After fermentation, filter the fermented vinegar through multiple layers of gauze or filter paper to remove acetic acid bacteria membrane and other impurities. Put the filtered vinegar into a clean glass bottle, seal it well and store it in the refrigerator to extend the shelf life and maintain the flavor.

[0129] 7. Aging: If you want to further enhance the flavor of the vinegar, you can transfer the filtered vinegar to a clean container and age it at low temperature for a period of time. Aging can make the flavor of the vinegar more complex and soft. The vinegar can be drunk directly or diluted.

[0130] The fruit vinegar prepared as above was compared with the fruit vinegar prepared from the fruit wine by natural fermentation (without inoculation of Lactobacillus putrefaciens Z5-1), and the contents of acetic acid, organic acid, phenols, amino acids and antioxidant capacity in the fruit vinegar were determined. The results are shown in Table 8.

[0131] Table 8 Effects of different fermentation methods on the quality and flavor of fruit vinegar

[0132]

[0133] As shown in Table 8, the acetic acid content of the Z5-1 fermentation group was generally higher than that of the natural fermentation group, indicating that the acetic acid bacteria in the Z5-1 fermentation group were more active and the fermentation efficiency was better. The organic acid content of the Z5-1 fermentation group was generally lower than that of the natural fermentation group, because the organic acids in the fruit vinegar were further converted into acetic acid or other compounds. The phenolic content of the Z5-1 fermentation group was generally higher than that of the natural fermentation group, indicating that the Z5-1 fermentation group was better able to extract and retain phenolic substances, which made important contributions to the flavor and health benefits of the fruit vinegar. The antioxidant capacity of the Z5-1 fermentation group was generally higher than that of the natural fermentation group, indicating that the fruit vinegar in the Z5-1 fermentation group had stronger antioxidant activity and was beneficial to health. The amino acid content of the Z5-1 fermentation group was generally higher than that of the natural fermentation group, indicating that the Z5-1 fermentation group was better able to retain and produce amino acids, which made important contributions to the flavor and nutritional value of the fruit vinegar.

[0134] In general, the fruit vinegar of the Z5-1 fermentation group was superior to that of the natural fermentation group in terms of acetic acid content, phenolic content, antioxidant capacity and amino acid content. This indicates that the preparation of fruit vinegar from fruit wine fermented by Lactobacillus putrefaciens Z5-1 can significantly improve the quality and nutritional value of fruit vinegar.

[0135] Example 6

[0136] Application of coryneform lactobacillus Z5-1 in fermentation of sauerkraut

[0137] 1. Raw material preparation: First remove the old leaves, rotten leaves and other unnecessary parts on the outside of the cabbage, and keep the fresh and good texture parts.

[0138] 2. Cleaning: Wash the cabbage thoroughly with clean water to remove surface dirt and other impurities.

[0139] 3. Cut: Cut the cabbage into strips.

[0140] 4. Weighing: Determine the required weight of cabbage based on the size of the container used, and weigh 5kg.

[0141] 5. Blanching: Put the processed cabbage into boiling water and blanch for 30 seconds. This step can help kill surface bacteria and make the cabbage easier to absorb salt. The time should not be too long to avoid losing crispness.

[0142] 6. Cooling: Put the blanched cabbage into cold water quickly to cool down, or let it cool naturally to room temperature.

[0143] 7. Put into the tank: Choose a clean and oil-free fermentation tank as the fermentation container, put the cooled cabbage neatly into it, compact it, expel the air, and add 0.6±0.05kg of pure water.

[0144] 8. Add liquor: a liquor brand (loose liquor on the market, sorghum liquor), with an alcohol content of 40% vol, mix it evenly with an equal proportion of room temperature pure water, and pour it into the jar until it covers the cabbage.

[0145] 9. Inoculation: Inoculate 6% of rod-shaped putrefactive Lactobacillus Z5-1 (activated bacterial liquid) according to the mass ratio.

[0146] At the same time, natural fermentation and inoculation of ethanol-resistant Pediococcus CGMCC No.29506, Lactobacillus acidophilus CGMCCNo.31284, Lactobacillus CICC 6002, and Leuconostoc citrinum CICC 6056 were used as controls.

[0147] 10. Seal: Use a lid or other method to tightly seal the mouth of the container to prevent outside air from entering and affecting the fermentation quality. But at the same time, it is also necessary to ensure that the internal gas can be discharged to avoid damage to the container due to excessive pressure.

[0148] 11. Fermentation: Place the sealed container in a cool and ventilated place to allow it to ferment naturally. During this period, it is necessary to regularly check and remove the generated gas. Fermentation time is 30 days away from light.

[0149] 12. Finished product: You can take it out and eat it or transfer it to the refrigerator for storage.

[0150] The prepared sauerkraut was tested to determine the main flavor components and amino acid content, pH, ethanol degradation rate and lactic acid content in the finished sauerkraut.

[0151] Table 9 Effects of different fermentation methods on the main flavor components and other indicators of sauerkraut

[0152]

[0153]

[0154] Alcohols, aldehydes, acids, esters, ketones, sulfur compounds and nitrogen compounds in sauerkraut have a crucial impact on its flavor. Each compound can contribute different sensory properties, which together constitute the unique flavor, aroma and taste of sauerkraut. As shown in Table 8, esters and sulfur compounds in sauerkraut fermented by coryneformis putrefaction Lactobacillus Z5-1 were higher than those in other control groups. Esters are usually associated with pleasant fruity and floral aromas. Although their content in sauerkraut may not be high, even trace amounts can significantly improve the aroma quality of the product, making the flavor of sauerkraut richer and rounder. Sulfur compounds play an important role in the unique smell of sauerkraut. Amino acids and peptides are not only important components of flavor substances, but can also generate complex aroma compounds during heating through the Maillard reaction. Sauerkraut fermented by coryneformis putrefaction Lactobacillus Z5-1 has the highest lactic acid content, which can effectively inhibit the growth of most harmful microorganisms while maintaining appropriate sourness and crispness. If the lactic acid content is too low, the sauerkraut will lack sufficient sourness, resulting in a bland flavor that cannot meet the expectations of consumers who like sour taste. In addition, a lower lactic acid content means a higher pH value, which provides a living environment for some pathogens and increases food safety risks. Insufficient lactic acid content can also cause sauerkraut to deteriorate more easily during storage, resulting in a poor texture and affecting the eating experience. Based on ethanol degradation efficiency and pH, rod-shaped spoilage Lactobacillus Z5-1 is the best choice, with the highest ethanol degradation rate of 16.1%, which is suitable for situations where the ethanol content needs to be significantly reduced. The ethanol degradation rate of ethanol-resistant Pediococcus CGMCC No.29506 is 13.2%, second only to that of rod-shaped spoilage Lactobacillus Z5-1; the ethanol degradation rate of Lactobacillus acidophilus CGMCC No.31284 is 11.2%, which is better than other control groups. Ethanol-resistant Pediococcus CGMCC No.29506 caused the pH value to drop the most, reaching 3.1, while rod-shaped spoilage Lactobacillus Z5-1 caused the pH to drop to 3.5, indicating that the above strains produced acidic substances during metabolism, which may be organic acids such as lactic acid. The fermentation effects of the inoculated strains were better than those of the natural fermentation. The effects of other strains were not as good as that of Lactobacillus putrefaciens Z5-1, probably because they were inhibited by alcohol.

[0155] Example 7

[0156] Application of coryneform lactobacillus Z5-1 in wine-making of jujube

[0157] 1. Material selection: Select Chaoyang jujubes that are 90% mature, with uniform color change, no pests and diseases, no mechanical damage, and good hardness. Such jujubes not only ensure the quality of the raw materials, but also ensure the taste and flavor of the final product.

[0158] 2. Pretreatment: Wash the selected jujubes carefully with clean water to remove dust and impurities on the surface. Then blanch them. This step can not only further clean the jujubes, but also slightly soften the peel, which is conducive to the subsequent penetration of alcohol. After blanching, use clean air to dry the moisture on the surface of the jujubes to prevent the introduction of too much moisture during the canning process, which will affect the fermentation effect.

[0159] 3. Canning and soaking: Put the dried jujubes into pre-sterilized glass jars, 2kg per jar. Then, add 53%vol Fenjiu (a famous Chinese fragrant liquor) to completely immerse the jujubes in the liquor. Seal the glass jars and place them in a dark environment to soak for 1-2 days. During this time, the jujubes will gradually absorb the liquor, soften, and release their own sugars and other ingredients, preparing for the next fermentation process.

[0160] 4. Inoculation and fermentation: When the jujube has fully absorbed moisture and softened, inoculate the activated rod-shaped putrefactive Lactobacillus Z5-1 bacterial liquid at an inoculation rate of 6%-8%. Place the inoculated glass jar in a constant temperature environment at 37°C for closed culture for 3-5 days. This temperature range is the optimal growth temperature for most lactic acid bacteria, which can promote rapid reproduction of bacteria and start the fermentation process. After the initial fermentation is completed, move the glass jar to a cool and dark place to continue fermentation. Gently shake the bottle every two days to mix the internal substances evenly, promote gas exchange, and avoid local overheating or uneven fermentation. The entire fermentation cycle takes about 15-20 days. During this period, with the action of lactic acid bacteria, the sugar in the jujube will gradually be converted into lactic acid and other metabolites, giving the product a richer flavor.

[0161] In addition, the control group was inoculated with ethanol-resistant Pediococcus CGMCC No.29506, Lactobacillus acidophilus CGMCCNo.31284, Lactobacillus CICC 6002, and Leuconostoc citrinum CICC 6056.

[0162] 5. Finished product storage: After fermentation is completed, you can decide whether to extend the storage time to enhance the flavor according to personal preference. The finished product should be stored in a low temperature and dark environment to slow down the chemical reaction and maintain stable product quality.

[0163] The prepared wine-made dates were tested to determine their quality, antioxidant capacity, lactic acid content, ethanol degradation rate and types of volatile substances.

[0164] Table 10 Effects of different fermentation methods on the quality and function of wine jujube

[0165]

[0166] As shown in Table 10, after fermentation, the color of the wine dates turned purple or red, and the hardness decreased. The soluble solid content of the wine dates fermented by the rod-shaped spoilage Lactobacillus Z5-1 was 25%, and the sugar in the wine dates was better released, increasing the overall solid content. The titratable acid content of the wine dates fermented by the rod-shaped spoilage Lactobacillus Z5-1 was 0.6%, and the acid reduction ability of the rod-shaped spoilage Lactobacillus Z5-1 was significantly stronger than that of natural fermentation.

[0167] The vitamin C content of the wine dates fermented by rod-shaped Lactobacillus Z5-1Z5-1 is 48mg / 100g. It can be seen that it minimizes the loss of vitamin C in red dates and promotes the transformation of certain precursors. The flavonoid content of the wine dates fermented by rod-shaped Lactobacillus Z5-1 is 447mg / 100g, indicating that during the fermentation process, rod-shaped Lactobacillus Z5-1 can activate the enzyme activity in red dates, promote the release and transformation of flavonoids, and improve their bioavailability. The antioxidant capacity of the wine dates fermented by rod-shaped Lactobacillus Z5-1 is 4857umol / 100g, which is much higher than that of natural fermentation, indicating that the polyphenols, flavonoids and other antioxidants produced during the fermentation process work together to significantly enhance the antioxidant properties of the wine dates. The amino acid content of the wine dates fermented by coryneform Lactobacillus Z5-1 was 2.7 mg / 100 g, which was higher than that of the products fermented by natural fermentation and other strains, indicating that during the fermentation process, proteins were broken down into smaller peptides and free amino acids, especially the content of essential amino acids such as glutamic acid, aspartic acid, and leucine increased. The lactic acid content of the wine dates fermented by coryneform Lactobacillus Z5-1 was 0.8%, and the lactic acid content of other strains after fermentation was 0.5%-0.7%, both higher than that of natural fermentation, indicating that the production of lactic acid was effectively increased by introducing lactic acid bacteria for fermentation, giving the wine dates a unique sweet and sour flavor and better preservation. The volatile substances in the wine dates fermented by coryneform Lactobacillus Z5-1 were rich in variety, with a total content of 475 mg / L, which was higher than that of the products fermented by natural fermentation and other strains, indicating that the volatile compounds such as esters, aldehydes, ketones, and alcohols produced during the fermentation process gave the wine dates a unique aroma and flavor, forming a complex sensory experience.

[0168] In summary, fermenting red dates with inoculation of rod-shaped putrefactive Lactobacillus Z5-1 not only retains the original nutrients of red dates, but also produces a variety of beneficial metabolites during the fermentation process. Red dates show significant advantages in soluble solids, titratable acid, vitamin C, flavonoids, antioxidant capacity, amino acid content, lactic acid content, and types and contents of volatile substances. Red dates fermented with rod-shaped putrefactive Lactobacillus Z5-1 are rich in nutrients and are superior to fermentation with other strains. Red dates made by the above steps not only retain the original nutrients of red dates, but also add the fragrance of Fenjiu and the unique flavor of lactic acid bacteria fermentation, and are a very distinctive healthy food.

[0169] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A rod-shaped putrefactive lactobacillus (Loigolactobacillus coryniformis) Z5-1, characterized in that The deposit number of the coryneform putrefactive lactobacillus Z5-1 is CGMCC No.31547.

2. A bacterial agent containing the coryneform putrefactive lactobacillus Z5-1 according to claim 1.

3. The bacterial agent according to claim 2, characterized in that The viable count of the coryneform Lactobacillus putrefaciens Z5-1 is ≥ 2×10 7 CFU / mL.

4. A method for preparing the bacterial agent according to claim 2 or 3, characterized in that: The following steps are involved: The coryneform putrefactive lactobacillus Z5-1 is inoculated into an MRS solid culture medium for activation culture, and the activated colonies are inoculated into an MRS liquid culture medium to prepare a bacterial suspension, namely a liquid bacterial agent.

5. The preparation method according to claim 4, characterized in that: The following steps are also included: The bacterial suspension is inoculated into an MRS broth medium to prepare a primary bacterial strain; a nutrient supplement is added to the MRS broth medium to continue fermentation and culture to prepare a secondary bacterial strain, and a thickener is added to prepare a paste bacterial agent.

6. The preparation method according to claim 5, characterized in that: The nutritional supplement comprises germinated brown rice paste, peanut paste, trace elements and growth factors.

7. Use of the coryneform putrefactive lactobacillus Z5-1 according to claim 1 or the bacterial agent according to claim 2 or 3 in degrading ethanol.

8. Use of the coryneform putrefactive lactobacillus Z5-1 according to claim 1 or the bacterial agent according to claim 2 or 3 in the preparation of fermented food.

9. The use according to claim 8, characterized in that: The fermented food includes fruit wine, sauerkraut, fruit vinegar and wine-made fruit.

10. The use according to claim 9, characterized in that: The fruit wine, sauerkraut or wine-made fruit is produced by inoculating the bacterial agent described in claim 2 or 3 on the basis of fermented raw materials; the fruit vinegar is produced by inoculating acetic acid bacteria on the fruit wine as raw material and fermenting.

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