Acetobacter sengaricum for one-step fermentation of fruit vinegar and application of acetobacter sengaricum

Abstract

The invention belongs to the field of microorganisms and fermentation industry, and particularly relates to acetobacter sengaricum for one-step fermentation of fruit vinegar and application of the acetobacter sengaricum. The preservation number of the acetobacter senecii is CGMCC (China General Microbiological Culture Collection Center) NO.33406, and the acetobacter senecii is preserved in China General Microbiological Culture Collection Center on January 14, 2025. According to the fermentation method provided by the invention, a mode of one-step fermentation and no addition of saccharomyces cerevisiae is adopted, a microbial strain acetobacter sesgaricus with special metabolic capability is directly utilized to be compounded with commercial acetic acid bacteria for fermentation, saccharides in the fruit raw materials are converted into acetic acid at one time, and only acetic acid fermentation conditions need to be controlled, so that the production cost is reduced, and the production efficiency is improved. According to the method, the operation links of strain propagation, inoculation, fermentation condition conversion and the like in the yeast alcohol fermentation stage are omitted, so that the whole production process is more compact and coherent, the time period from raw materials to finished products is greatly shortened, the production efficiency is improved, the market demand can be quickly responded, and the method is suitable for a large-scale industrial continuous production mode.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms and fermentation industry, and particularly relates to a strain of Acetobacter senegalensis used for one-step fermentation of fruit vinegar and application thereof. Background Art

[0002] In recent years, fruit vinegar, as a fermented product with both nutritional and health functions, has attracted widespread attention worldwide. With the increasing demand of consumers for natural and healthy food, the fruit vinegar market is showing a rapid growth trend.

[0003] Traditional fruit vinegar production requires the use of brewer's yeast for alcohol fermentation, and then the introduction of acetic acid bacteria to carry out acetic acid fermentation. The steps are cumbersome, and the two-step fermentation requires the control of the fermentation conditions of different strains of bacteria (such as temperature, pH, and dissolved oxygen content) respectively. The total time is as long as 15-30 days. Multi-stage operations are prone to the introduction of foreign bacterial contamination, and equipment conversion and repeated sterilization lead to energy waste. At the same time, some flavor substances and functional ingredients will also be lost during the lengthy fermentation process, reducing the comprehensive utilization value of the raw materials and increasing the cost of raw materials. Summary of the invention

[0004] In view of the shortcomings of the prior art, the present invention isolates and screens a strain of Acetobacter senegalensis from blackened jujube vinegar ( Acetobacter senegalensis ), the strain is combined with commercial acetic acid bacteria for fermentation. Unlike traditional fermentation, the strain can directly produce acid to obtain fruit vinegar through a one-step fermentation without the need to add brewer's yeast; the fruit vinegar obtained by fermentation has good flavor quality and greatly improved production efficiency.

[0005] Another object of the present invention is to provide the use of the above-mentioned Acetobacter Senegalensis in the preparation of fruit vinegar.

[0006] The technical solution adopted by the present invention to achieve the above-mentioned purpose is: The present invention provides a strain of Acetobacter senegalenicae ( Acetobacter senegalensis ), the Senegalese Acetobacter has a deposit number of CGMCC NO.33406, and was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on January 14, 2025.

[0007] The present invention also provides the use of the Acetobacter Senegalensis in the preparation of fruit vinegar by fermentation.

[0008] The specific fermentation process of the present invention using Acetobacter Senegal to ferment and prepare fruit vinegar is as follows: (1) Raw material screening: select red dates with full grains, no mold, no deterioration, and no insect pests; (2) Blackening of red dates: soaking the red dates in water; then performing a blackening treatment on the soaked red dates to obtain blackened red dates; (3) Jujube juice extraction: add water to the blackened red dates, beat them, add pectinase, extract, squeeze the juice and set aside to obtain blackened red dates juice; (4) Activation of bacterial strains: Under sterile conditions, aspirate the purified strain Acetobacter senegalensis and inoculate it into a sterilized and cooled liquid culture medium for overnight culture; (5) Fermentation: Inoculate acetic acid bacteria and Acetobacter senegalenica into the blackened red date juice and culture under constant temperature to obtain fruit vinegar.

[0009] Preferably, in step (2), the humidity of the red dates after soaking is 70-80%; the conditions for the blackening treatment are: blackening for 84-96 h at a constant temperature of 65-75°C and a constant humidity of 70%-80%RH.

[0010] Preferably, in step (3), the solid-liquid ratio of the blackened red dates to water is 1:5; the amount of pectinase added is 0.1%-0.3% of the date pulp mixture; and the extraction is performed at a temperature of 55-60° C. for 4-5 hours.

[0011] Preferably, in step (4), the amount of Acetobacter senegalensis added is 1%-5% of the volume of the liquid culture medium; and the culture temperature is 30-32°C.

[0012] Preferably, in step (5), the acetic acid bacteria is Shanghai Niang 1.01; the inoculation amount of the acetic acid bacteria is 2%-4%; and the inoculation amount of the Senegalese Acetobacter is 1%-5%.

[0013] Preferably, in step (5), the culturing is carried out at a temperature of 30-32° C. for 8 days.

[0014] The fermentation method provided by the present invention adopts a one-step fermentation method without adding brewer's yeast, directly utilizes the microbial strain Senegal Acetobacter with special metabolic ability, and performs composite fermentation with commercial acetic acid bacteria, so as to convert the sugars in the fruit raw material into acetic acid at one time, and only needs to control the acetic acid fermentation conditions, thereby eliminating the operation links such as strain expansion, inoculation and fermentation condition conversion in the yeast alcohol fermentation stage, making the whole production process more compact and coherent, greatly shortening the time cycle from raw materials to finished products, improving production efficiency, being able to quickly respond to market demand, and being suitable for large-scale industrial continuous production mode.

[0015] The beneficial effects of the present invention are: (1) The present invention isolates and screens a strain of Acetobacter senegalensis TLT07.78, and the screening method is simple and efficient; (2) The Acetobacter Senegalensis obtained by the present invention has the advantages of high acid production, good vinegar flavor and acid resistance; and the one-step fermentation method without adding brewer's yeast can improve the vinegar production efficiency, save production costs, produce high-quality fruit vinegar, and significantly improve the economic benefits of the enterprise.

[0016] Collection Information Storage time: January 14, 2025; Depository: General Microbiology Center, China Microbiological Culture Collection Administration; Deposit number: CGMCC NO.33406; Address of the depository: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; Postal code: 100101; Classification name: Acetobacter senegalensis Acetobacter senegalensis . BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the cell morphology of Shannong TLT07.78 under a microscope; Figure 2 This is the colony morphology of Shannong TLT07.78; Figure 3 This is the result diagram of the one-step fermentation acid production capacity of Shannong TLT07.78. DETAILED DESCRIPTION

[0018] The present invention is described in detail below in conjunction with specific drawings and embodiments. The examples described below are only preferred embodiments of the present invention. It should be noted that the following description is only for the purpose of explaining the present invention and does not limit the present invention in any form. Any simple modification, equivalent changes and modifications made to the embodiments according to the technical essence of the present invention are within the scope of the technical solution of the present invention.

[0019] In the following examples, the materials, reagents, etc. used were obtained from commercial sources unless otherwise specified.

[0020] The present invention utilizes Acetobacter senegalensis to brew fruit vinegar, which specifically comprises the following steps: (1) Raw material screening: Select red dates with full grains, no mold, no deterioration, and no insect pests.

[0021] (2) Blackening of red dates: Weigh 500.0 g of red dates and place them in a sealed bag. Add 300 mL of water and soak for 1 h until the relative humidity of the red date sample reaches 80%. Then place the sealed bag containing the sample in an incubator and blacken it for 96 h under constant temperature (70°C) and constant humidity (80%RH).

[0022] (3) Jujube juice extraction: extract jujube juice at a solid-liquid ratio of 1:5, add pectinase at a ratio of 0.2 g / L, extract at 55°C for 4 hours, and squeeze the juice for later use.

[0023] (4) Activation of bacterial strains: Under sterile conditions, take 3% of the volume of the liquid culture medium and inoculate it into sterilized and cooled liquid culture medium (20 g / L anhydrous glucose, 5 g / L peptone, 5 g / L yeast powder, 1.15 g / L citric acid, 4 g / L disodium hydrogen phosphate, pH 6.0, sterilize at 121°C for 20 min, cool to 50°C and then add 20 mL / L ethanol), and culture at 30°C overnight.

[0024] (5) Fermentation: Inoculate 3% Shanghai Brewing 1.01 and 1.5% Acetobacter senegalensis (Shannong TLT07.78) and culture in an incubator at 30°C for 8 days.

[0025] Example 1 Screening of Acetobacter Senegalensis Take 10 mL of black jujube vinegar and put it into a 500 mL Erlenmeyer flask mixed with glass beads and 90 mL of enrichment medium, and incubate at 30°C and 200 r / min overnight. The bacterial suspension was diluted 10 times with sterile saline (10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 ), spread 200 μL of appropriate gradient dilution on the separation medium, culture at 30℃ for 3-5 days, pick a single colony with a transparent circle for streak culture to obtain pure bacteria, purify 3-4 times, pick a single colony in liquid culture medium and culture at 30℃, 180 r / min for 2 days, further study and identify the strain that can produce gel-like substances, and preserve the pure strain at -80℃ with a final volume fraction of 20% glycerol and name it Shannong TLT07.78.

[0026] Enrichment medium: yeast powder 5 g / L, peptone 3 g / L, D-mannitol 25 g / L, sterilized at 121℃ for 20 min.

[0027] Isolation medium: 20 g / L anhydrous glucose, 8 g / L peptone, 5 g / L yeast powder, 12 g / L agar powder, pH 6.8, sterilize at 121°C for 20 min, cool to 50°C and add 5 mL / L ethanol.

[0028] Liquid culture medium: 20 g / L anhydrous glucose, 5 g / L peptone, 5 g / L yeast powder, 1.15 g / L citric acid, 4 g / L disodium hydrogen phosphate, pH 6.0, sterilize at 121°C for 20 min, cool to 50°C and add 20 mL / L ethanol.

[0029] Example 2 Morphological observation and physiological and biochemical characteristics of strain Shannong TLT07.78 The colony characteristics of Shannong TLT07.78, such as morphology, size, color, texture, transparency, and edge smoothness, were observed under a microscope, and the shape and size of the bacteria were recorded. Figure 1 and Figure 2 The culture temperature for the physiological and biochemical characterization tests of strain TLT07.78 was set at 30°C.

[0030] The biological characteristics of strain Shannong TLT07.78 are: usually rod-shaped, short or long, no spores, single cell size is (0.5 μm ~ 1.0 μm) × (1.0 μm ~ 3.0 μm). When cultured on solid at 30℃, the colonies are round, protruding, light white, opaque, with a moist surface, easy to pick, and neat edges.

[0031] The physiological and biochemical characteristics of strain Shannong TLT07.78 are: Gram staining negative, aerobic, optimal growth temperature is 30℃, and a more obvious sour taste is produced during the fermentation process.

[0032] The strain Shannong TLT07.78 screened in Example 1 was sent to Shanghai Paisonno Biotechnology Co., Ltd. for sequencing, and the genome sequence was compared with the Nucleotide Sequence Database (NT) to obtain the species information of the genome, as shown in Table 1.

[0033] Table 1 Through the GTDB database, the detailed genome information of Shannong TLT07.78 strain was obtained. The genome size is 4.1Mb, the GC content is 55.43%, and the species classification is bacterial domain ( Bacteria ), Proteobacteria ( Proteobacteria ), α-Proteobacteria ( Alphaproteobacteria ), Rhodospirillales ( Rhodospirillales ), Acetobacteraceae ( Acetobacteraceae ), Acetobacter spp. ( Acetobacter ), Acetobacter senegalensis ( Acetobacter senegalensis ).

[0034] Example 3 Analysis of acid resistance of strain Shannong TLT07.78 Acid tolerance test of strain Shannong TLT07.78: The bacterial solution of Shannong TLT07.78 (viable bacteria count 1.87×10 6CFU / mL) were added to a 250 mL conical flask containing 50 mL of liquid culture medium (pH 5.0, 3.0, 2.5, and 2.0, respectively) and cultured in a shaker at 30°C and 180 r / min. After culturing for 24 h and 72 h, the bacterial culture was taken to measure the growth rate of the strain OD 600nm Values ​​and viable counts of bacterial species.

[0035] OD 600nm Detection of the value: Take 3 mL of bacterial solution and select a wavelength of 600 nm for measurement under an ultraviolet spectrophotometer. If the fermentation solution is too turbid (the bacterial cell density is high), dilute the bacterial solution 10 times with sterile water before measurement.

[0036] Detection of viable bacterial count: Take 0.1 mL of bacterial solution into a sterile centrifuge tube, add 0.9 mL of sterile water, and shake to obtain 10 -1 Take another 0.1 mL of this bacterial solution and add it to a 2 mL centrifuge tube containing 0.9 mL of sterile water to obtain 10 -2 Dilute the bacterial solution. Similarly, take 0.1 mL of the bacterial solution with the appropriate dilution multiple and use the coating method to count the viable bacteria. Take three dilution multiples for each sample and perform three parallel tests for each dilution multiple.

[0037] The optimum growth pH of fermentation industrial bacteria Huniang 1.01 and AS1.41 is between 3.5 and 6.5, while the Acetobacter Shannong TLT07.78 of the present invention can tolerate high acid conditions below pH 3.0, the number of live bacteria still increases at pH 2.5, and can still survive at pH 2.0, as shown in Table 2.

[0038] Table 2 Example 4 Application of strain Shannong TLT07.78 in one-step fermentation of fruit vinegar (1) Bacterial strain selection: Shannong TLT07.78, Huniang 1.01 (purchased from Jiuwei Food Technology (Shanghai) Co., Ltd.); (2) Activation of bacterial strains: Under sterile conditions, take 3% of the volume of the frozen strain and inoculate it into sterilized and cooled liquid culture medium (20 g / L anhydrous glucose, 5 g / L peptone, 5 g / L yeast powder, 1.15 g / L citric acid, 4 g / L disodium hydrogen phosphate, pH 6.0, sterilize at 121°C for 20 minutes, cool to 50°C and then add 20 mL / L ethanol), and culture at 30°C overnight.

[0039] (3) Raw material screening: Select red dates with full grains, no mold, no deterioration, and no insect pests.

[0040] (4) Blackening of red dates: Weigh 500.0 g of red dates and place them in a sealed bag. Add 300 mL of water and soak for 1 h until the relative humidity of the red date sample reaches 80%. Then place the sealed bag containing the sample in an incubator and blacken it for 96 h under constant temperature (70°C) and constant humidity (80%RH).

[0041] (5) Jujube juice extraction: extract jujube juice at a solid-liquid ratio of 1:5, add pectinase at a ratio of 0.2 g / L, extract at 55°C for 4 hours, squeeze the juice to obtain jujube juice; (6) Fermentation culture: 3% Shanghai Niang 1.01 and 1.5% Shannong TLT07.78 were inoculated into a conical flask (500 mL) containing 100 mL of sterilized jujube juice; Shanghai Niang 1.01 was used instead of Shannong TLT07.78 as the control group and inoculated into a conical flask (500 mL) containing 100 mL of sterilized jujube juice and cultured at 30°C and 150 rpm.

[0042] (7) Comparison of acid production capacity: After 8 days of cultivation, the acidity of jujube vinegar was measured every two days. The results showed that ( Figure 3 ), without the addition of brewer's yeast, the acidity of the Shannong TLT07.78+Shanghai Brewing 1.01 group was significantly increased compared with the control group, and the highest total acid content reached 3.6 g / 100 mL, while the control group with only Shanghai Brewing 1.01 added did not undergo acetic acid fermentation.

Claims

1. A strain of Acetobacter senegalensis ( Acetobacter senegalensis ), characterized in that The deposit number of the Senegalese Acetobacter is CGMCC NO.33406, and it was deposited in the General Microbiology Center of the China Culture Collection Administration on January 14, 2025.

2. Use of Acetobacter senegalensis as claimed in claim 1 in the fermentation preparation of fruit vinegar.

3. The use according to claim 2, characterized in that: The specific fermentation process is: (1) Raw material screening: select red dates with full grains, no mold, no deterioration, and no insect pests; (2) Blackening of red dates: soaking the red dates in water; then performing a blackening treatment on the soaked red dates to obtain blackened red dates; (3) Jujube juice extraction: add water to the blackened red dates, beat them, add pectinase, extract, squeeze the juice and set aside to obtain blackened red dates juice; (4) Activation of bacterial strains: Under sterile conditions, aspirate the purified strain Acetobacter senegalensis and inoculate it into a sterilized and cooled liquid culture medium for overnight culture; (5) Fermentation: Inoculate acetic acid bacteria and Acetobacter senegalenica into the blackened red date juice and culture under constant temperature to obtain fruit vinegar.

4. The use according to claim 3, characterized in that: In step (2), the humidity of the red dates after soaking is 70-80%; the conditions for the blackening treatment are: blackening for 84-96 hours at a constant temperature of 65-75°C and a constant humidity of 70%-80%RH.

5. The use according to claim 3, characterized in that: In step (3), the solid-liquid ratio of the blackened red dates to water is 1:5; the amount of pectinase added is 0.1%-0.3% of the date pulp mixture; and the extraction is performed at a temperature of 55-60° C. for 4-5 hours.

6. The use according to any one of claims 3 to 5, characterized in that: In step (4), the amount of Acetobacter senegalensis added is 1%-5% of the volume of the liquid culture medium; and the culture temperature is 30-32°C.

7. The use according to claim 3 or 6, characterized in that: In step (5), the acetic acid bacteria is Shanghai Niang 1.01; the inoculation amount of the acetic acid bacteria is 2%-4%; and the inoculation amount of Senegalese Acetobacter is 1%-5%.

8. The use according to claim 7, characterized in that: In step (5), the culture is carried out at a temperature of 30-32°C for 8 days.

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