CN103571782A

CN103571782A - Weissella confuse and application thereof

Info

Publication number: CN103571782A
Application number: CN201310612545.3A
Authority: CN
Inventors: 李崎; 朱林江; 蒯辉; 王金晶; 田金凤; 李永仙
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2013-11-26
Filing date: 2013-11-26
Publication date: 2014-02-12
Anticipated expiration: 2033-11-26
Also published as: CN103571782B

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

One strain confusus and application thereof

Technical field

The present invention relates to strain confusus and an application thereof, belong to fermentation technical field.

Background technology

Milk-acid bacteria refers to that a group can fermentable carbohydrates, produces the bacterium general name of a large amount of lactic acid.This bacterioid extensively exists in nature and food, be the earliest by the mankind for one of microorganism of food-processing and food storage.Milk-acid bacteria can be given tart flavour and the fragrance that food is soft, improves quality and the nutrition of food, improves the keeping property of food, promotes the maturation of the leavened foods such as milk-product, fermented sausages, pickles, sweet sauce.

Bacterium lacticum is lactobacillus, makes one of conventional bacterium of yogurt, and acid resistance is strong, and it is positive that gramstaining becomes, and is human body probiotics, the effect of helpful digestion.Bacterium lacticum is acidproof, and optimum pH is 5.5～5.8, even lower.The milk-acid bacteria that comprises Bacterium lacticum is distributed widely in the animals and plants leavened prod that contains carbohydrate, in plant surface, milk-product, meat product, beer, grape wine, fruit juice, wort, bread dough, sewage and human and animal excreta, equal separable arriving.The food that China is traditional, if the making preservation technology of pickles, hot pickled mustard tube, salted vegetables and wine brewing etc. is exactly this effect that has utilized milk-acid bacteria.

Confusus Gram-positive, does not form gemma, and amphimicrobian or micro-aerobic, can not utilize rhamnosyl, can utilize most of carbon sources, utilizes arginine, does not produce ammonia, and catalase is negative, and decomposition glucose produces CO ₂.Confusus has been widely used in, in the production of multiple traditional fermented food and beverage, being usually used in the research of fermenting bean milk.

Broad bean paste is to take half of maintenance broad bean paste original shape that broad bean etc. forms as fermenting raw materials brew flow viscous body or semi-solid state seasonings.Broad bean paste has long history in China, nutritious, and delicious flavour has aid digestionly, improves a poor appetite, and is therefore deeply subject to consumers in general's welcome.The confusus the present invention relates to is screened from broad bean paste traditional zymotic sauce unstrained spirits, has obviously shortened the fermentation period of traditional zymotic bean cotyledon sauce.

Summary of the invention

The invention provides a strain Weissella confuse.Described Weissella confuse, on October 11st, 2013, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, 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.

W.confuse(confusus of the present invention) the sauce unstrained spirits of the separated traditional method brew of well-known sauce product from Anhui, this bacterial strain has the M13-PCR fingerprint of characteristic feature, i.e. the metastatic characteristic fingerprint of bacterial strain, as shown in Figure 1; Sequencing analysis, its 16S rDNA sequence gene type as shown in SEQ ID NO.1, blast search on NCBI website, with a strain confusus (serial ID: dbj|AB854267.1|) there is the highest matching degree.

Described W.confuse can suppress the growth of aerobic bacteria in broad bean paste traditional zymotic process, the fermentation period that shortens broad bean paste, tolerance 20%NaCl.

Described confusus (W.confuse) is for preferably screening gained the broad bean paste sauce unstrained spirits of traditional method brew from flavor taste, and plate count reaches 3 * 10 ⁴cFU/g.Its storage conditions is as follows: well-grown bacterium liquid that takes a morsel from MRS substratum anaerobism pipe moves in fresh MRS substratum, the 37 ℃ of standing cultivation of anaerobism 24h, get 0.75mL move into fill the aseptic glycerine 40%(v/v of 0.25mL) glycerine pipe in, put-70 ℃ of Refrigerator stores.

Described MRS substratum is: Tryptone10g L ^-1, Yeast Extract5g L ^-1, extractum carnis 10g L ^-1, glucose 20g L ^-1, sodium acetate 5g L ^-1, diammonium hydrogen citrate 2g L ^-1, sal epsom 0.58g L ^-1, manganous sulfate 0.25g L ^-1, tween-80 0.1g L ^-1, pH6.2～6.6.

Described confusus culture condition is: by the confusus access MRS liquid nutrient medium of glycerine pipe preservation, at the 37 ℃ of standing cultivation of anaerobism 24h to the logarithm middle and later periods, as seed liquor.With 2%(v/v) inoculum size is forwarded to seed liquor in fresh MRS substratum, and the 37 ℃ of standing cultivation of anaerobism 8h are to logarithmic phase.

The present invention also provides a kind of application of described confusus, by cultivating good confusus, in the mid-early stage of broad bean paste traditional zymotic process, is added into sauce unstrained spirits, suppresses the growth of the aerobic bacterias such as subtilis, shortens the fermentation period of broad bean paste.

The invention provides a strain confusus, can be applicable to high salt concentration traditional zymotic bean cotyledon sauce, suppress the growth of some aerobic bacteria, improve the local flavor of broad bean paste, shorten the fermentation period of broad bean paste.

Biomaterial preservation

Weissella confuse provided by the present invention, on October 11st, 2013, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number is CGMCC No.8321, preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica.

Accompanying drawing explanation

Fig. 1 Rep-PCR (M13-PCR) finger printing that increases, M:DS5000,1: sample.

Fig. 2 confusus salinity tolerance column diagram.

Embodiment

The screening of embodiment 1 confusus

From traditional zymotic bean cotyledon sauce, within 30 days to 50 days, sauce unstrained spirits, get 1g sample and join in 9mL sterilized water, suspension liquid is made in slight vibration, by suspension liquid gradient dilution, coats MRS flat board, and 32 ℃ of anaerobism are cultivated 60h.Picking oyster white list bacterium colony carries out plate streaking separation repeatedly, obtains in triplicate single bacterium colony of purifying, for next step, identifies.

Identify and mainly to pass through: 1) colony characteristics is observed: with the naked eye directly observe, choosing colony diameter between 1-1.5mm, circle, smooth surface, oyster whites etc. meet the bacterial strain of Bacterium lacticum colony characteristics.2) individual morphology is observed: with opticmicroscope gramstaining, observe.Select Gram-positive, nonspore-bearing bacterial strain.The bacterial strain that meets evaluation condition is chosen to the preservation of glycerine pipe.

With 2% inoculation, measure-70 ℃ of glycerine stock solutions and be inoculated in MRS substratum, the 37 ℃ of standing cultivation of anaerobism 24h.Get appropriate medium centrifugal 2min(4000rpm, 4 ℃), abandon most supernatant liquor, obtain appropriate thalline, with the bacterial genomes DNA extraction test kit (centrifugal column type) of day root, extract genomic dna.With bacterium universal primer to upstream primer 27F (sequence: 5 '-AGAGTTTGATCCTGGCTCAG-3 ') and downstream primer L1401R(5 '-GCGTGTGTACAAGACCC-3 ') amplifying genom DNA, reaction conditions is: after 94 ℃ of denaturation 3min, enter following circulation: 94 ℃ of sex change 40s, 56 ℃ of annealing 35s, 72 ℃ are extended 80s, 30 circulations; 72 ℃ are extended 10min.Good through 1% agarose gel electrophoresis qualification result.Amplified production is sent to Shanghai Hua Da gene sequencing, as shown in SEQ ID NO.1, on NCBI website, carry out BLAST sequence alignment, be defined as confusus.

The experiment of embodiment 2NaCl tolerance

The MRS substratum that the substratum that the experiment of confusus NaCl tolerance is used is different N aCl final concentration.MRS substratum is: Tryptone10g L ^-1, Yeast Extract5g L ^-1, extractum carnis 10g L ^-1, glucose 20g L ^-1, sodium acetate 5g L ^-1, diammonium hydrogen citrate 2g L ^-1, sal epsom 0.58g L ^-1, manganous sulfate 0.25g L ^-1, tween-80 0.1g L ^-1, pH6.2～6.6.NaCl concentration is respectively: 10%, 12%, 14%, 16%, 18%, 20%.Anaerobism pipe liquid amount is the every pipe of 15mL.Medium sterilization condition is 115 ℃, 15min.

With 2% inoculation, measure-70 ℃ of glycerine stock solutions and be inoculated in MRS substratum, the 37 ℃ of standing cultivation of anaerobism 24h.With 10% inoculum size, be forwarded in the MRS substratum of different N aCl final concentration, 37 ℃ of standing cultivations of anaerobism pipe 20 days, as shown in Figure 2, confusus is at NaCl final concentration 10%, 12%, 14%, 16%, 18%, 20%(%:g/100mL) all can grow under condition, wherein under NaCl final concentration 10% condition, the speed of growth is relatively very fast, and all the other condition growths are very slow.

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

CGMCC No.8321Weissella confuse bacterial strain anaerobism in MRS liquid nutrient medium is cultivated 2 days, as seed liquor.It is 14% sauce unstrained spirits that seed liquor is seeded to salinity, contrast is not for inoculating the sauce unstrained spirits that the salinity of seed liquor is 14%, and fermentation period has obvious variation, does not inoculate the sauce body of confusus after ripening in approximately 110 days, and the sauce body of inoculation confusus is after ripening in approximately 85 days, and aerobic miscellaneous bacteria is from 10 ⁸cFU/g(does not inoculate confusus) be down to 10 ⁷cFU/g(inoculates confusus).

Be understandable that, for those of ordinary skills, can be equal to replacement or change according to technical scheme of the present invention and inventive concept thereof, and all these changes or replacement all should belong to the protection domain of the appended claim of the present invention.

Claims

1. a strain Weissella confuse, on October 11st, 2013, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, 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. W.confuse claimed in claim 1, is characterized in that suppressing the growth of aerobic bacteria in broad bean paste traditional zymotic process.

3. W.confuse claimed in claim 1, is characterized in that shortening the fermentation period of broad bean paste.

4. W.confuse claimed in claim 1, is characterized in that tolerating 20%NaCl.

5. the cultural method of the arbitrary described W.confuse of claim 1-4, is by the W.confuse access MRS liquid nutrient medium of glycerine pipe preservation, at the 37 ℃ of standing cultivation of anaerobism 24h to the logarithm middle and later periods, as seed liquor.

6. method claimed in claim 5, is characterized in that, described MRS substratum is: Tryptone10g L ^-1, Yeast Extract5g L ^-1, extractum carnis 10g L ^-1, glucose 20g L ^-1, sodium acetate 5g L ^-1, diammonium hydrogen citrate 2g L ^-1, sal epsom 0.58g L ^-1, manganous sulfate 0.25g L ^-1, tween-80 0.1g L ^-1, pH6.2～6.6.

7. application rights requires the arbitrary described W.confuse of 1-4 to ferment in traditional broad bean paste, is that the mid-early stage in broad bean paste traditional zymotic process is added in sauce unstrained spirits by W.confuse seed liquor.