CN105820967A - Lactobacillus brevis and application thereof - Google Patents
Lactobacillus brevis and application thereof Download PDFInfo
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- CN105820967A CN105820967A CN201510005242.4A CN201510005242A CN105820967A CN 105820967 A CN105820967 A CN 105820967A CN 201510005242 A CN201510005242 A CN 201510005242A CN 105820967 A CN105820967 A CN 105820967A
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/90—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in food processing or handling, e.g. food conservation
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Abstract
The invention relates to a strain of lactobacillus brevis, which is named as lactobacillus brevis AR-123. A preservation number of lactobacillus brevis AR-123 is CGMCC 9761. The lactobacillus brevis AR-123 can degrade nitrite with high efficiency, can be used as a vegetable starter for well keeping original flavor and nutrition of vegetables, and the nitrite content in the fermented vegetable after fermentation is complete is only 1.6 mg/L.
Description
Technical field
The present invention relates to microorganism, particularly to strain Lactobacillus brevis and an application thereof.
Technical background
Fermented vegetable refers to can be one of the Typical Representative of China's traditional zymotic food with the vegetable of long-term storage by fermentation, has the flavor characteristics of uniqueness.Fermented vegetable is mainly vegetables such as Chinese cabbage, Radix Raphani, Fructus Cucumidiss sativi under the effect of certain density Sal, water and flavoring agent, utilize the leavens such as the natural microorganism being attached to vegetable surface or artificial vaccination lactic acid bacteria, decompose the nutrient substance such as the fermentable saccharide in vegetable under anaerobic condition and produce the process of a large amount of lactic acid.During the fermentation, under substantial amounts of lactic acid and condition of salt stress, some putrefactive microorganisms in vegetable and the growth of other anaerobe can be produced the flavor substances such as ethanol, acetaldehyde, mannitol simultaneously by strong inhibition, thus form the fermented food of peculiar taste.The primary raw material of fermented vegetable is nutritious various fresh vegetabless, rich in nutrient and lactic acid such as vitamin, calcium, ferrum, phosphorus.Delicious tender crisp, appetite can not only be promoted, and there is certain health-care effect, such as prophylaxis of hypertension, cholesterol regulating content and the most aging.There are some researches show, fermented vegetable also has certain prophylactic activity to cancer.
Fermented vegetable is that China eats time-honored lactate fermentation vegetable product, has good local flavor and health care, but content of nitrite excessive problem limits the fast development of these type of cure foods all the time.In recent years, due to a large amount of uses of nitrogenous fertilizer, cause Nitrate in Vegetable to roll up, and vegetable is a kind of plant food being prone to be enriched with nitrate, in fermented vegetable curing process, the nitrate in vegetable can be become nitrite by the part bacterial reduction in Pickle.Research shows, content of nitrite in Pickle exceeds several times even tens times more than than fresh vegetables of the same race, and, due to Pickle in storage, to there may be preservation condition in transportation improper, the pollution microbes in product is caused to increase so that content of nitrite therein raises further.Therefore, the most strictly control to pickle the content of the nitrite in Pickles, it is ensured that the safety in utilization of this traditional food of Pickles so that it is commercially preferably circulation is urgent problem.
Summary of the invention
The purpose of the present invention, it is aiming in prior art lacking the deficiency of the lactic acid bacteria of safe and efficient degrading nitrite, Lactobacillus brevis and the application thereof of one strain energy effectively degrading nitrite are provided, the bacterial strain of present invention fermentation pickled vegetable under optimum conditions, can well keep the original local flavor of vegetable and nutrition, after fermentation ends, Nitrite in Pickles content is only 1.6mg/L.
In order to realize the purpose of the present invention, present invention employs techniques below scheme: a strain Lactobacillus brevis (Lactobacillusbrevis), named Lactobacillus brevis AR-123, this bacterial strain on October 13rd, 2014 in China Committee for Culture Collection of Microorganisms's common micro-organisms center (address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3) preservation, its deposit number is CGMCC9761, and its gene order is as shown in SEQIDNO.1.
The Lactobacillus brevis AR-123 bacterial strain screening of the present invention is from conventional Kimchi.Pickles sample is carried out separation screening, utilize gradient dilution method, sample liquid after dilution is coated MRS flat board, 37 DEG C of constant temperature culture, after line separates, the lactic acid bacteria separating acquisition is carried out morphologic observation, being defined as effectively degrading nitrite bacterial strain in conjunction with degrading nitrite fermenting experiment, its degradation efficiency is up to 99.5%.
Above-mentioned Lactobacillus brevis AR-123 is as the application of fermented vegetable agent.
Above-mentioned Lactobacillus brevis AR-123 is as follows as the application process of fermented vegetable agent:
(1) Lactobacillus brevis AR-123 is activated as inoculation liquid；
(2) in sterilized container, 300mL saline, condiment and cleaned, the vegetable of stripping and slicing are added；
(3) inoculation liquid is inoculated in the above-mentioned container being placed with vegetable by the inoculum concentration of 5-15% (v/v), 25 DEG C of standing for fermentation 7 days, obtains the fermented vegetable of low nitrite content.
Activation described in step (1) refers to: be inoculated in MRS culture medium by Lactobacillus brevis AR-123,37 DEG C of quiescent culture 24h, secondary amplification culture is carried out under similarity condition, 4000rpm is centrifuged 8min and collects thalline, suspending thalline with normal saline, being modulated into Lactobacillus brevis AR-123 concentration is 10 again8The inoculation liquid of cfu/mL.
In saline described in step (2), the weight percent concentration of salt is 3-10%, and the amount adding vegetable is 75-200g.
The Lactobacillus brevis AR-123 that the present invention filters out can effectively degrading nitrite, under optimum conditions fermentation pickled vegetable, the original local flavor of vegetable and nutrition can well be kept, after fermentation ends, Nitrite in Pickles content is only 1.6mg/L.Utilizing this strain fermentation vegetable, have the local flavor of uniqueness, fresh perfume (or spice) is tender crisp, in good taste, and content of nitrite is extremely low.It is simple that the Lactobacillus brevis AR-123 of the present invention is used for preparing fermented vegetable process, it is possible to achieve heavy industrialization is applied.
Accompanying drawing explanation
Fig. 1 is the growth curve of Lactobacillus brevis AR-123.
Specific implementation method
The present invention is expanded on further below in conjunction with specific embodiment.For concrete grammar used in embodiment or material, those skilled in the art on the basis of the technology of the present invention thinking, can carry out the replacement of routine and select according to existing technology, are not limited solely to the concrete record of the embodiment of the present invention.
Experimental technique used in following embodiment if no special instructions, is conventional method；The material that used, reagent etc., if no special instructions, the most commercially obtain.
MRS solid medium: peptone 10g/L, Carnis Bovis seu Bubali cream 10g/L, yeast extract 5g/L, K2HPO42g/L, dibasic ammonium citrate 2g/L, sodium acetate 5g/L, glucose 20g/L, Tween 80 1mL, MgSO4·7H2O0.58g/L, MnSO4·4H2O0.25g/L, agar 20g/L, regulate pH to 6.4.
The screening of embodiment 1 Lactobacillus brevis AR-123 and qualification
1.1 sample diluting liquid preparation and strains separation
Gather from Sichuan farmers' homemade fermented vegetable sample, shred, add 15mL sterilized water, acutely shake 20min.Use Concentraton gradient dilution method (10-1-10-4), taking diluent 100uL and coat on MRS agar culture medium flat board, 37 DEG C of Anaerobic culturel 48h, choosing colony is big, grow fast bacterial strain, and line is isolated and purified further.
1.2 bacterial strain screening
By 37 DEG C of quiescent culture 24h in the inoculation in step 1.1 to MRS fluid medium, it is prepared as seed liquor, inoculum concentration according to 5% accesses in the MRS fluid medium containing 0.2mg/mL nitrite, 37 DEG C of quiescent culture 48h, after cultivation terminates, 4000rpm, 10min centrifugation medium, takes supernatant.How ethylenediamine method measures the NaNO of residual in culture fluid to use hydrochloric acid2Content.Experimental result is as shown in table 1.
Table 1 primary dcreening operation strains for degrading nitrite screens
1.3 identification of strains
By a named AR-123 of strain bacterium of isolated.Bacterium colony ellipse or similar round, neat in edge, bacterium colony size is generally 2～3mm, milky.By 16SrDNA sequence comparative analysis, result shows that this sequence reaches 99% with the homology of Lactobacillus brevis in ncbi database, can identify that this bacterium is Lactobacillus brevis (Lactobacillusbrevis), named Lactobacillus brevis AR-123.
1.4 strain growth characteristics
Accessing in MRS fluid medium by the Lactobacillus brevis AR-123 activated by 1% (v/v) inoculum concentration, 37 DEG C of quiescent culture 24h, the bacterium measuring culture fluid at 600nm every 2h is dense.Being plotted against time, so that bacterium is dense, the growth curve obtaining strains A R-123 in MRS, its result is as shown in Figure 1.Test result indicate that, Lactobacillus brevis AR-123 grows rapidly in MRS culture medium, enters logarithmic (log) phase at about 6h, and about 12h enters stable phase.
Embodiment 2 Lactobacillus brevis AR-123 prepares low nitrite fermented vegetable
(1) adding Sterile Saline and the condiment that 300mL concentration is 3% in sterilized container, take fresh Chinese cabbage, clean, drain excessive moisture, be cut into the fritter that length and width is 5*3cm and load in container under aseptic condition, putting into vegetable weight is 75g；
(2) accessing the activated Lactobacillus brevis AR-123 of 5% (v/v) in container, add water-stop and be placed at 25 DEG C, ferment 7d, obtains fermented vegetable.After testing, the content of nitrite of the present embodiment products obtained therefrom is 8.4mg/L.Result shows, the content of nitrite of gained fermented vegetable is relatively low, and product has peculiar flavour, and the fresh perfume (or spice) of mouthfeel is tender crisp.
Embodiment 3 Lactobacillus brevis AR-123 prepares low nitrite fermented vegetable
(1) adding Sterile Saline and the condiment that 300mL concentration is 7% in sterilized container, take fresh Chinese cabbage, clean, drain excessive moisture, be cut into the fritter that length and width is 5*3cm and load in container under aseptic condition, putting into vegetable weight is 150g；
(2) accessing the activated Lactobacillus brevis AR-123 of 10% (v/v) in container, add water-stop and be placed at 25 DEG C, ferment 7d, obtains fermented vegetable.After testing, the content of nitrite of the present embodiment products obtained therefrom is 1.6mg/L.Result shows, the content of nitrite of gained fermented vegetable is relatively low, and product has peculiar flavour, and the fresh perfume (or spice) of mouthfeel is tender crisp.
Embodiment 4 Lactobacillus brevis AR-123 prepares low nitrite fermented vegetable
(1) adding Sterile Saline and the condiment that 300mL concentration is 7% in sterilized container, take fresh Chinese cabbage, clean, drain excessive moisture, be cut into the fritter that length and width is 5*3cm and load in container under aseptic condition, putting into vegetable weight is 200g；
(2) accessing the activated Lactobacillus brevis AR-123 of 15% (v/v) in container, add water-stop and be placed at 25 DEG C, ferment 7d, obtains fermented vegetable.After testing, the content of nitrite of the present embodiment products obtained therefrom is 1.8mg/L.Result shows, the content of nitrite of gained fermented vegetable is relatively low, and product has peculiar flavour, and the fresh perfume (or spice) of mouthfeel is tender crisp.
Embodiment 5 Lactobacillus brevis AR-123 prepares low nitrite fermented vegetable
(1) adding Sterile Saline and the condiment that 300mL concentration is 10% in sterilized container, take fresh Chinese cabbage, clean, drain excessive moisture, be cut into the fritter that length and width is 5*3cm and load in container under aseptic condition, putting into vegetable weight is 200g；
(2) accessing the activated Lactobacillus brevis AR-123 of 15% (v/v) in container, add water-stop and be placed at 25 DEG C, ferment 7d, obtains fermented vegetable.After testing, the content of nitrite of the present embodiment products obtained therefrom is 2.6mg/L.Result shows, the content of nitrite of gained fermented vegetable is relatively low, and product has peculiar flavour, and the fresh perfume (or spice) of mouthfeel is tender crisp.
Claims (5)
1. a strain Lactobacillus brevis (Lactobacillusbrevis), named Lactobacillus brevis AR-123, its deposit number is CGMCC9761, and its gene order is as shown in SEQIDNO.1.
2. Lactobacillus brevis AR-123 is as the application of fermented vegetable agent.
Lactobacillus brevis AR-123 the most according to claim 2 is as the application of fermented vegetable agent, it is characterised in that:
Application process is as follows:
(1) Lactobacillus brevis AR-123 is activated as inoculation liquid；
(2) in sterilized container, 300mL saline, condiment and cleaned, the vegetable of stripping and slicing are added；
(3) inoculation liquid is inoculated in the above-mentioned container being placed with vegetable by the inoculum concentration of 5-15% (v/v), 25 DEG C of standing for fermentation 7 days, obtains the fermented vegetable of low nitrite content.
Lactobacillus brevis AR-123 the most according to claim 3 is as the application of fermented vegetable agent, it is characterized in that: the activation described in step (1) refers to: Lactobacillus brevis AR-123 is inoculated in MRS culture medium, 37 DEG C of quiescent culture 24h, secondary amplification culture is carried out under similarity condition, 4000rpm is centrifuged 8min and collects thalline, suspending thalline with normal saline, being modulated into Lactobacillus brevis AR-123 concentration is 10 again8The inoculation liquid of cfu/mL.
Lactobacillus brevis AR-123 the most according to claim 3 is as the application of fermented vegetable agent, it is characterised in that: in the saline described in step (2), the weight percent concentration of salt is 3-10%, and the amount adding vegetable is 75-200g.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106962741A (en) * | 2017-03-16 | 2017-07-21 | 江南大学 | The method of microbial compound preparation efficient degradation salted fish nitrite |
| CN109645403A (en) * | 2018-11-30 | 2019-04-19 | 四川东坡中国泡菜产业技术研究院 | The Lactobacillus brevis of low temperature resistant antiphagin and its application in pickles |
| CN113416668A (en) * | 2021-06-08 | 2021-09-21 | 大连工业大学 | Lactobacillus brevis for reducing nitrite content and method and application thereof |
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| CN101326972A (en) * | 2008-07-28 | 2008-12-24 | 东北农业大学 | Directly thrown leaven for effectively degrading nitrite in pickled vegetables and preparation thereof |
| CN102018154A (en) * | 2010-10-26 | 2011-04-20 | 浙江大学 | Method for inoculating lactobacillus to rapidly reduce nitrite content in pickles |
| CN104187519A (en) * | 2014-09-05 | 2014-12-10 | 浙江正味食品有限公司 | Method for effectively inhibiting browning |
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| CN101326972A (en) * | 2008-07-28 | 2008-12-24 | 东北农业大学 | Directly thrown leaven for effectively degrading nitrite in pickled vegetables and preparation thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106962741A (en) * | 2017-03-16 | 2017-07-21 | 江南大学 | The method of microbial compound preparation efficient degradation salted fish nitrite |
| CN109645403A (en) * | 2018-11-30 | 2019-04-19 | 四川东坡中国泡菜产业技术研究院 | The Lactobacillus brevis of low temperature resistant antiphagin and its application in pickles |
| CN109645403B (en) * | 2018-11-30 | 2022-05-31 | 四川东坡中国泡菜产业技术研究院 | Low-temperature-resistant and bacteriophage-resistant lactobacillus brevis and application thereof in pickle |
| CN113416668A (en) * | 2021-06-08 | 2021-09-21 | 大连工业大学 | Lactobacillus brevis for reducing nitrite content and method and application thereof |
| CN113416668B (en) * | 2021-06-08 | 2023-06-06 | 大连工业大学 | Lactobacillus brevis for reducing nitrite content, and method and application thereof |
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