CN103571782B - Weissella confuse and application thereof - Google Patents

Abstract

The invention discloses weissella confuse and an application thereof and belongs to the technical field of fermentation. The weissella confuse has already been preserved in the CGMCC (China General Microbiological Culture Collection Center) since October 11th, 2013, the preservation number is CGMCC No. 8321, and the preservation address is Institute of Microbiology, Chinese Academy of Sciences, No.3, No.1 Precint, Beichen West Road, Chaoyang District, Beijing City. The lactobacillus has the higher osmotic-pressure-resistant capability, is used in the traditional fermentation production process for broad bean paste and has the characteristics of high salinity resistance and capability of shortening the fermentation period of the broad bean paste.

Description

A Fusion Lactobacillus and Its Application

technical field

The invention relates to a fusion lactobacillus strain and application thereof, belonging to the technical field of fermentation.

Background technique

Lactic acid bacteria refers to a group of bacteria that can ferment carbohydrates and produce large amounts of lactic acid. This type of bacteria widely exists in nature and food, and is one of the earliest microorganisms used by humans for food processing and food storage. Lactic acid bacteria can endow food with soft sour taste and aroma, improve food quality and nutrition, improve food preservation performance, and promote the maturity of fermented foods such as dairy products, fermented sausages, pickles, and sweet sauces.

Lactobacillus is Lactobacillus, one of the commonly used bacteria in making yogurt. It has strong acid resistance and is Gram-positive. It is a beneficial bacterium for the human body and has the effect of helping digestion. Lactobacillus is acid-resistant, and the optimum pH value is 5.5-5.8, or even lower. Lactic acid bacteria, including Lactobacillus, are widely distributed in animal and plant fermented products containing carbohydrates. They are found on the surface of plants, dairy products, meat products, beer, wine, fruit juice, wort, fermented dough, sewage, and human and animal manure. separable to. Traditional Chinese food, such as pickles, mustard mustard, pickled vegetables and wine making and preservation techniques, utilizes this effect of lactic acid bacteria.

Fusion Lactobacillus Gram-positive, non-spore forming, facultative anaerobic or microaerophilic, unable to utilize rhamnose, can utilize most carbon sources, utilize arginine, do not produce ammonia, negative catalase, decompose glucose Produces CO ₂ . Fusion Lactobacillus has been widely used in the production of a variety of traditional fermented foods and beverages, and is often used in the research of fermented soybean milk.

Broad bean paste is a semi-fluid viscous or semi-solid condiment that maintains the original shape of broad bean paste, which is fermented and brewed from broad beans. Broad bean paste has a long history in my country. It is rich in nutrition, delicious in taste, helpful in digestion, and increases appetite, so it is very popular among consumers. The fusion lactobacilli involved in the invention are selected from traditional fermented bean paste fermented grains of broad bean paste, which obviously shortens the fermentation cycle of traditional fermented bean paste.

Contents of the invention

The invention provides a strain of Weissella confuse. The Weissella confuse was deposited on October 11, 2013 in the General Microbiology Center of the China Microbiological Culture Collection Management Committee, the preservation number is CGMCC No.8321, and the preservation address is No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing. Institute of Microbiology, Chinese Academy of Sciences.

The W.confuse (fused Lactobacillus) described in the present invention is isolated from the sauce fermented grains brewed by a traditional method of a well-known sauce product in Anhui. The strain has a typical M13-PCR fingerprint, that is, the characteristic fingerprint of strain transfer, as shown in the figure 1; sequence analysis, its 16S rDNA sequence genotype is shown in SEQ ID NO.1, searched by BLAST on the NCBI website, and has the highest matching degree with a fusion Lactobacillus (sequence ID: dbj|AB854267.1| ) .

The W.confuse can inhibit the growth of aerobic bacteria in the traditional fermentation process of bean paste, shorten the fermentation period of bean paste, and tolerate 20% NaCl.

The fused Lactobacillus (W. confuse) is screened from bean paste fermented grains brewed by the traditional method with better flavor and taste, and the plate colony count reaches 3×10 ⁴ CFU/g. The storage conditions are as follows: Take a small amount of well-grown bacterial liquid from the anaerobic tube of the MRS medium and transfer it to a fresh MRS medium, and culture it anaerobically at 37°C for 24 hours. (v/v) in a glycerol tube and stored in a -70°C refrigerator.

The MRS medium is: Tryptone 10g L ^-1 , Yeast Extract 5g L ^-1 , beef extract 10g L ^-1 , glucose 20g L ^-1 , sodium acetate 5g L ^-1 , diammonium hydrogen citrate 2g L ^-1 , magnesium sulfate 0.58g L ^-1 , manganese sulfate 0.25g L ^-1 , Tween-80 0.1g L ^-1 , pH6.2～6.6.

The culture condition of the fusion lactobacillus is as follows: the fusion lactobacillus preserved in the glycerol tube is inserted into the MRS liquid medium, and the anaerobic static culture is carried out at 37° C. for 24 hours to the middle and late logarithm, as the seed solution. The seed liquid was transferred to fresh MRS medium with 2% (v/v) inoculum amount, and anaerobically cultured at 37°C for 8 hours to logarithmic phase.

The present invention also provides an application of the fused lactobacillus. The well-cultured fused lactobacillus is added into the fermented grains of soybean paste in the middle and early stages of the traditional fermentation process of bean paste, so as to inhibit the growth of aerobic bacteria such as Bacillus subtilis and shorten the time of fermented bean paste. fermentation cycle.

The invention provides a strain of fused lactobacillus, which can be applied to traditional fermented bean paste with high salt concentration, inhibits the growth of some aerobic bacteria, improves the flavor of the bean paste, and shortens the fermentation period of the bean paste.

biological material deposit

The Weissella confuse provided by the present invention has been preserved in the General Microbiology Center of China Microbiological Culture Collection Management Committee on October 11, 2013. The preservation number is CGMCC No.8321, and the preservation address is No. 1, Beichen West Road, Chaoyang District, Beijing No. 3, Institute of Microbiology, Chinese Academy of Sciences.

Description of drawings

Figure 1 Rep-PCR (M13-PCR) amplification fingerprint, M: DS5000, 1: sample.

Fig. 2 Histogram of salinity tolerance of fusion Lactobacillus.

Detailed ways

The screening of embodiment 1 fusion lactobacillus

Take 1g sample from traditional fermented bean paste 30 days to 50 days old fermented bean paste, add it to 9mL sterile water, shake slightly to make a suspension, dilute the suspension gradiently, spread it on the MRS plate, and incubate anaerobically at 32°C for 60h . Pick the milky white single colony and separate it by streaking on the plate repeatedly, and repeat three times to obtain a purified single colony for the next step of identification.

Identification is mainly through: 1) Observation of colony characteristics: observe directly with the naked eye, and select strains with a diameter of 1-1.5 mm, a round shape, a smooth surface, milky white, etc. that meet the characteristics of Lactobacillus colonies. 2) Observation of individual morphology: Observation by Gram staining with an optical microscope. Pick a Gram-positive, spore-free strain. Strains meeting the identification conditions were picked out of glycerol tubes for preservation.

The -70°C glycerol stock solution was inoculated in MRS medium with 2% inoculum amount, and anaerobically cultured at 37°C for 24 hours. Take an appropriate amount of culture solution and centrifuge for 2 minutes (4000rpm, 4°C), discard the supernatant to obtain an appropriate amount of bacterial cells, and use Tiangen’s Bacterial Genomic DNA Extraction Kit (spin column type) to extract genomic DNA. Use bacterial universal primers to amplify genomic DNA with the upstream primer 27F (sequence: 5'-AGAGTTTGATCCTGGCTCAG-3') and the downstream primer L1401R (5'-GCGTGTGTACAAGACCC-3'). The reaction conditions are: 94°C pre-denaturation for 3 minutes and then enter the following cycle : Denaturation at 94°C for 40s, annealing at 56°C for 35s, extension at 72°C for 80s, 30 cycles; extension at 72°C for 10min. The result of 1% agarose gel electrophoresis was good. The amplified product was sent to Shanghai Huada Gene for sequencing. As shown in SEQ ID NO.1, the BLAST sequence comparison was performed on the NCBI website, and it was determined to be a fusion lactobacillus.

Embodiment 2NaCl tolerance test

The medium used in the NaCl tolerance experiment of fusion Lactobacillus was MRS medium with different NaCl final concentrations. MRS medium: Tryptone 10g L ^-1 , Yeast Extract 5g L ^-1 , beef extract 10g L ^-1 , glucose 20g L ^-1 , sodium acetate 5g L ^-1 , diammonium hydrogen citrate 2g L ^-1 , magnesium sulfate 0.58g L ^-1 , manganese sulfate 0.25g L ^-1 , Tween-800.1g L ^-1 , pH6.2～6.6. NaCl concentrations were: 10%, 12%, 14%, 16%, 18%, 20%. The liquid volume of anaerobic tubes is 15mL per tube. The medium sterilization condition is 115°C for 15 minutes.

The -70°C glycerol stock solution was inoculated in MRS medium with 2% inoculum amount, and anaerobically cultured at 37°C for 24 hours. Transfer 10% of the inoculum to MRS medium with different NaCl final concentrations, and culture in an anaerobic tube at 37°C for 20 days. , 16%, 18%, 20% (%: g/100mL) conditions can grow, and the growth rate is relatively fast under the condition of NaCl final concentration of 10%, and the other conditions grow very slowly.

Embodiment 3 is applied to high salt concentration traditional fermented broad bean paste

CGMCC No.8321 Weissella confuse strain was anaerobically cultured in MRS liquid medium for 2 days as seed solution. The seed liquid was inoculated into the sauce unstrained spirits with a salinity of 14%, and the control was the uninoculated sauce unstrained spirits with a salinity of 14%. The sauce body inoculated with fused Lactobacillus matured after about 85 days, and the aerobic bacteria decreased from 10 ⁸ CFU/g (not inoculated with fused Lactobacillus) to 10 ⁷ CFU/g (inoculated with fused Lactobacillus).

It can be understood that those skilled in the art can make equivalent replacements or changes according to the technical solutions and inventive concepts of the present invention, and all these changes or replacements should belong to the protection scope of the appended claims of the present invention.

Claims (7)

1. a strain confusus ( weissella confuse), be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on October 11st, 2013, deposit number is CGMCC No.8321, and preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica.

2. confusus according to claim 1, is characterized in that the growth suppressing aerobic bacteria in thick broad-bean sauce traditional zymotic process.

3. confusus according to claim 1, is characterized in that the fermentation period shortening thick broad-bean sauce.

4. confusus according to claim 1, is characterized in that tolerating 20%NaCl.

5. the cultural method of the arbitrary described confusus of claim 1-4 is by the confusus of glycerine pipe preservation access MRS fluid nutrient medium, at 37 DEG C of anaerobism quiescent culture 24h to the logarithm middle and later periods, as seed liquor.

6. method according to claim 5, is characterized in that, described MRS culture medium is: Tryptone 10g L ^-1, Yeast Extract 5g L ^-1, beef extract 10g L ^-1, glucose 20g L ^-1, sodium acetate 5g L ^-1, diammonium hydrogen citrate 2g L ^-1, magnesium sulfate 0.58g L ^-1, manganese sulfate 0.25g L ^-1, Tween-80 0.1g L ^-1, pH 6.2 ~ 6.6.

7. application rights requires the method that the arbitrary described confusus of 1-4 is fermented in traditional thick broad-bean sauce, is be added in sauce unstrained spirits the mid-early stage of confusus seed liquor in thick broad-bean sauce traditional zymotic process.