CN109576182A - A kind of strong resistance Lactobacillus rhamnosus A-4 and application thereof - Google Patents
A kind of strong resistance Lactobacillus rhamnosus A-4 and application thereof Download PDFInfo
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- CN109576182A CN109576182A CN201811579430.8A CN201811579430A CN109576182A CN 109576182 A CN109576182 A CN 109576182A CN 201811579430 A CN201811579430 A CN 201811579430A CN 109576182 A CN109576182 A CN 109576182A
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
- A23L2/38—Other non-alcoholic beverages
- A23L2/382—Other non-alcoholic beverages fermented
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/175—Rhamnosus
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/225—Lactobacillus
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Polymers & Plastics (AREA)
- Food Science & Technology (AREA)
- Nutrition Science (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Have strong resistance the present invention relates to a kind of Lactobacillus rhamnosus (Lactobacillus rhamnosus) A-4, deposit number is CGMCC No.16550.The invention further relates to Lactobacillus rhamnosus A-4 to prepare the purposes in beverage.Lactobacillus rhamnosus A-4 of the present invention can tolerate 75 DEG C of high temperature, be up to 96.88% ± 1.4% through 2.0 acid processing 2h survival rate of pH；Under osmotic pressure stress, 8 times of culture mediums diluted the low nutrition stress of 4.5%NaCl, pH3.5 acid stress and 5 ~ 45 DEG C of temperature stress still can growth and breeding, be with a wide range of applications in terms of preparing room temperature viable type sour milk beverage.
Description
Technical field
The invention belongs to field of biotechnology.More particularly it relates to a kind of strong resistance Lactobacillus rhamnosus
(Lactobacillus rhamnosus) A-4, further relate to the purposes of Lactobacillus rhamnosus A-4.
Background technique
Lactic acid bacteria refers to that fermentable carbohydrate generates the general name of a kind of gram-positive bacterium of a large amount of lactic acid.
Lactic acid bacteria, which has, adjusts gastrointestinal bacterial flora balance, anti-lactose intolerance, the suction for promoting the nutriments such as albumen and microelement
Receipts, lowering blood pressure and blood fat, enhancing immunity of organisms, anti-aging extend service life and other effects, therefore are widely used in food processing.
In recent years, as epoch progress people move jump for the demand of food, beverage is palatable from past sweetness
Natural, nutrition and health care function is turned to, the fermenting beverage based on lactic acid bacteria has become the important directions of research and development.So
And lactacidase fermenting beverage needs 4 DEG C of cold chain storage and transportations, and the problems such as shelf-life is short, viable count decline is fast, sense organ is unstable is always
The tackling key problem bottleneck of industry.
Currently, regarding to the issue above CN108294109A, CN108094802A, CN106858260A,
The Invention Announces such as CN106550989A, CN106550991A, CN106417597A addition stabilizer, emulsifier, buffer salt
Ameliorative way.Although improving sour milk beverage stability to a certain extent, cold chain storage and transportation, shelf-life too short problem
Still it does not solve.
The unstable key problem of viable lactic acid bacteria type beverage be the strong acidic environment of product, storage and transportation temperature, nutrition at
Variation is divided to will have a direct impact on the growth and breeding for wherein adding bacterial strain.
Therefore, the present invention is to obtain the strong resistance bacterial strain for being resistant to the environment such as strong acid, low temperature, high temperature, low nutrition, Thief zone
For research direction, the present invention is completed on the basis of acquisition characteristic sample extensively, numerous studies screening.
Summary of the invention
Technical problems to be solved: the object of the present invention is to provide a kind of Lactobacillus rhamnosus for having strong resistance
(Lactobacillus rhamnosus) A-4.It is a further object to provide the use of the Lactobacillus rhamnosus A-4
On the way.
Technical solution: the present invention is achieved through the following technical solutions.
First aspect present invention be related to it is a kind of with strong resistance Lactobacillus rhamnosus (Lactobacillus rhamnosus) A-4, the bacterial strain is on September 29th, 2018 in the Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Chinese Academy of Sciences
The preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms center, institute of microbiology, deposit number CGMCC
No.16550。
Strong resistance Lactobacillus rhamnosus A-4 screening process of the present invention is as follows.
(1) low temperature resistant and high temperature resistant lactic acid bacteria screening.
Using conventional method from Burkina Faso ferment barley beverage, day basic annals state pickles, Mianyang, Sichuan pickles, Yunnan
Dali cream fan, separate in the Qula of Nagqu pastoral area 18 plants can 15 DEG C of low-temperature epitaxies lactic acid bacteria；Utilize 75 DEG C of differences
The method of time high temperature stress processing has carried out further high temperature resistant to them and has screened.
The result shows that: 5 strains of lactic acid bacteria can be resistant to simultaneously high and low temperature stress, be respectively labeled as A-4, B-32, E-9, J-18,
L-13, wherein -4 resistance results of strains A are detailed in attached drawing 1.These bacterial strains are preserved in Jiangsu Heng Kang Biotechnology Co., Ltd bacterium
Kind collection.
(2) secondary screening of acidproof lactic acid bacteria.
High-low temperature resistant bacterial strain is activated by three generations using lactic acid bacteria conventional medium and conventional method, utilizes the sterile life of same volume
After reason salt water centrifugation (4 DEG C, 4000 r/min, 6min) washes twice, it is resuspended into bacteria suspension.1 mL bacteria suspension is drawn in 9 mL
In the MRS fluid nutrient medium of pH 1.5,2.0,2.5,6.8,37 ± 0.5 DEG C of shaking baths (150r/min) handle 2 h, and in 0,
2 h use GB 4789.35-2016 count plate, carry out acid resistance screening.
The result shows that: strains A -4 is up to 96.88% ± 1.4% through 2.0 acid processing 2h survival rate of pH, and viable count maintains
1.99×109CFU/mL(attached drawing 2).
(3) physio-biochemical characteristics and gene sequencing of strains A -4.
It is single according to " Berger's Handbook of Systemic Bacteriology " the 9th edition progress strains A -4
Cellular morphology observation, single colonie morphologic observation and analysis of physio biochemical characteristics；16S rDNA sequence point is carried out using conventional method
Analysis.
The result shows that: the single colonie form of strains A -4 is shown in that attached drawing 3, unicellular form are shown in that attached drawing 4, physiological and biochemical property are shown in
Attached drawing 5, the phylogenetic tree based on 16S rDNA genetic fragment are shown in attached drawing 6, strains A -4 can be classified as Lactobacillus rhamnosus
A-4。
(4) the resistance evaluation of Lactobacillus rhamnosus A-4.
Lactobacillus rhamnosus A-4 was activated into for two generations using lactic acid bacteria conventional medium and conventional method, by second-generation bacterial liquid
(OD600nm=1.7) (4 DEG C, 4000 r/min, 6min) abandoning supernatants are centrifuged, same volume physiological saline is added and mixes, according to above-mentioned ginseng
Number centrifugation, being resuspended after so cleaning 3 times is bacteria suspension (108CFU/mL).By Lactobacillus rhamnosus A-4 bacteria suspension with 2%(volume
Meter) inoculative proportion, carry out ordinary temperature stress, acid stress, Nutrient Stress, the evaluation of hyperosmotic stress resistance.
The result shows that: Lactobacillus rhamnosus A-4 can tolerate the osmotic pressure stress (attached drawing 10) of 4.5% NaCl and 8 times are trained
Base diluted low nutrition stress (attached drawing 9) is supported, in pH3.5 ~ 4.0(attached drawing 8) and 5 ~ 45 DEG C between (attached drawing 7) can still grow it is numerous
It grows.
Second aspect of the present invention is related to a kind of Lactobacillus rhamnosus A-4 bacteria preparation, and said preparation is by by above-mentioned rhamnose
Lactobacillus A-4(CGMCC No.16550) expand what culture was realized.
Lactobacillus rhamnosus A-4 of the present invention expands cultural method and is referred to rhamnose cream bar in the prior art
The expansion cultural method of bacterium can expand well and cultivate Lactobacillus rhamnosus A-4 of the invention.
Third aspect present invention further relates to the Lactobacillus rhamnosus A-4, prepared by Lactobacillus rhamnosus A-4 bacteria preparation
Purposes in beverage.
Preferably, the beverage is milk beverage or plant beverage, preferably, the beverage includes Lactobacillus rhamnosus
Milk beverage, Lactobacillus rhamnosus cereal beverage, Lactobacillus rhamnosus bean beverage, Lactobacillus rhamnosus potato beverage, rhamnose
Lactobacillus fruit beverage, Lactobacillus rhamnosus vegetable beverage, Lactobacillus rhamnosus coarse food grain beverage, Lactobacillus rhamnosus tea beverage.
Milk beverage of the present invention containing Lactobacillus rhamnosus A-4, cereal beverage, bean beverage, potato beverage, water
Fruit beverage, vegetable beverage, coarse food grain beverage, a kind of preferred production process of tea beverage are to carry out routine using Lactobacillus rhamnosus A-4
Fermentation, and be made with conventional ratio and conventional auxiliary material；Another preferred production process is by Lactobacillus rhamnosus A-4 bacterium system
Agent is directly added, and is made with conventional ratio and conventional auxiliary material.
Beverage made of of the present invention, it is characterised in that: the effective component of the beverage is Lactobacillus rhamnosus A-4
Or Lactobacillus rhamnosus A-4 bacteria preparation；The production method of the beverage is to be fermented using Lactobacillus rhamnosus A-4 or directly added
Add Lactobacillus rhamnosus A-4 bacteria preparation；Lactobacillus rhamnosus A-4 viable bacteria when the characteristics of beverage is normal temperature storage 6 months
Number is still greater than 106CFU/mL。
The above description is only an overview of the technical scheme of the present invention, is not construed as limiting the invention, in order to more clear
Chu understands technological means of the invention, and can be implemented according to the content of specification, below with the preferred embodiment of the present invention
And it is equipped with attached drawing detailed description is as follows.
The utility model has the advantages that the present invention provides one kind to have strong resistance Lactobacillus rhamnosus A-4 and its bacteria preparation, it can be extensive
Production for milk beverage and plant beverage.Lactobacillus rhamnosus A-4 of the present invention has high-low temperature resistant, acidproof, resistance to low battalion
Support the characteristics such as stress；The beverage produced using the bacterial strain, its viable count is still greater than 10 at normal temperature storage 6 months6CFU/mL.This hair
Bright very good solution the problems such as lactic bacteria activity beverage needs low temperature storage and transportation, viable count is low, has great application potential.
Detailed description of the invention
Attached drawing is used to provide further understanding of the present invention, and constitutes part of specification, with reality of the invention
It applies example to be used to explain the present invention together, not be construed as limiting the invention.
Fig. 1 is 75 DEG C of high temperature stress to Lactobacillus rhamnosus A-4 Survival Effects.
Fig. 2 is the influence that acid stress survives to Lactobacillus rhamnosus A-4.
Fig. 3 is Lactobacillus rhamnosus A-4 single colonie form (× 2 times).
Fig. 4 is the unicellular form of Lactobacillus rhamnosus A-4 Gram's staining (× 1000 times).
Fig. 5 is the physiological and biochemical property of Lactobacillus rhamnosus A-4.
Fig. 6 is the phylogenetic tree of Lactobacillus rhamnosus A-4.
Fig. 7 is the growth characteristics of Lactobacillus rhamnosus A-4 under temperature stress.
Fig. 8 is the growth characteristics of Lactobacillus rhamnosus A-4 under acid stress.
Fig. 9 is the growth characteristics that low nutrition coerces lower Lactobacillus rhamnosus A-4.
Figure 10 is the growth characteristics of Lactobacillus rhamnosus A-4 under hyperosmotic stress.
In the attached drawing 1,2,8,9,10 capitalization difference indicate significant difference (p< 0.05).
Specific embodiment
It will be better understood that the present invention by following embodiments, but do not limit the present invention.
Experimental method in following embodiments is unless otherwise specified conventional method.
Test material as used in the following examples is unless otherwise specified commercially available.
Percentage composition is mass percentage unless otherwise instructed.
Embodiment 1: separation, screening and the identification of Lactobacillus rhamnosus A-4.
(1) processing of low temperature resistant and high temperature resistant lactic acid bacteria screening sample: weighing 5 g solid samples, (fluid sample is drawn
5 mL) it is added in the homogenizing bag equipped with 45 mL physiological saline, utilize 5 min(velocity of flapping of Patting type homogenizer homogeneous: 6 times/
Second) obtain sample suspension；Enrichment: it draws in the skimmed milk of 2 mL sample suspensions addition, 18 mL and is placed in 15 DEG C of progress samples
The first time of suspension is enriched with (7d), using MRS broth bouillon, carries out second according to above-mentioned condition of culture and is enriched with；Separation
It lines with purifying: second of pregnant solution of picking containing 7.5 ‰ CaCO3MRS agar medium (15 DEG C, 14d), every 48h protects
Deposit the bacterial strain with transparent molten calcium circle, Gram-positive and negative catalase.
Above-mentioned Low Temperature-Resistant Strain is activated into three generations using lactic acid bacteria conventional method using MRS broth bouillon, draws 1 mL
Three generations's test bacteria liquid is in 9 mL MRS broth bouillons, 75 DEG C of water-bath stewing process, and GB is used when 0,15,30,60min
4789.35-2016 count plate carries out high temperature resistant screening.
The result shows that: 5 strains of lactic acid bacteria can be resistant to simultaneously high and low temperature stress, be respectively labeled as A-4, B-32, E-9, J-18,
L-13, wherein -4 resistance results of strains A are detailed in attached drawing 1.These bacterial strains are preserved in Jiangsu Heng Kang Biotechnology Co., Ltd bacterium
Kind collection.
(2) secondary screening of acidproof lactic acid bacteria: strains A -4, B-32, E-9, J-18, L-13 are utilized using MRS broth bouillon
Lactic acid bacteria conventional method activates three generations, is centrifuged (4 DEG C, 4000 r/min, 6min) washings two using same volume sterile saline
After secondary, it is resuspended into bacteria suspension.1 mL bacteria suspension is drawn in the MRS fluid nutrient medium of 9 mL pH 1.5,2.0,2.5,6.8,37
± 0.5 DEG C of shaking bath (150r/min) handles 2 h, and uses GB 4789.35-2016 count plate in 0,2 h, carries out resistance to
Acidity screening.The calculating of survival rate Sr is carried out using following formula, a is test strain viable count/CFU through acid processing 2h in formula
∙mL-1, b is test strain viable count/CFU mL without acid processing-1.The result shows that: strains A -4 is through 2.0 acid processing 2h of pH
Survival rate is up to 96.88% ± 1.4%, and viable count maintains 1.99 × 109CFU/mL(attached drawing 2).
(3) physio-biochemical characteristics and gene sequencing of strains A -4: according to " Berger's Handbook of Systemic
Bacteriology " the 9th edition carry out the unicellular morphologic observation of strains A -4, single colonie morphologic observation, analysis of physio biochemical characteristics
It is analyzed with 16S rDNA genetic fragment.
The gene sequencing: the DNA of strains A -4, which is extracted, refers to TIANGEN Biotech (Beijing) Co., Ltd. bacterium
Step carries out the extraction of total DNA in genome DNA extracting reagent kit specification；With F5'-AGAGTTTGATCCTGGCTCAG3',
R5'-CCGTCAATTCCTTTGAGTTT3' is that primer carries out 16S rDNA fragment amplification, and the product after taking 5 μ L to expand is used
1.0% agarose gel electrophoresis detection, if there is clearly amplified band at 1500 bp, no nonspecific products show that PCR expands
Increase successfully；Entrust Sangon Biotech (Shanghai) Co., Ltd.) limited liability company to pcr amplification product be sequenced；Sequencing result utilizes
After the two-way splicing of SeqMan in DNAStar software, homology sequence retrieval, selection are carried out using BLAST in GeneBank
Adjacent method (Neighbor-Joining) in Mega7.0 carries out the higher strain sequence of homology together with test strain sequence
After 1000 (Bootstrap) inspections of bootstrapping, drawing system development tree.
The result shows that: the single colonie form of strains A -4 is shown in that attached drawing 3, unicellular form are shown in that attached drawing 4, physiological and biochemical property are shown in
Attached drawing 5, the phylogenetic tree based on 16S rDNA genetic fragment are shown in attached drawing 6, strains A -4 can be classified as Lactobacillus rhamnosus
A-4.(4) the resistance evaluation of Lactobacillus rhamnosus A-4: Lactobacillus rhamnosus A-4 is used and is utilized using MRS broth bouillon
Lactic acid bacteria conventional method activated for two generations, by second-generation bacterial liquid (OD600nm=1.7) (4 DEG C, 4000 r/min, 6min) abandoning supernatants are centrifuged
Liquid is added same volume physiological saline and mixes, is centrifuged according to above-mentioned parameter, and being resuspended after so cleaning 3 times is bacteria suspension (108CFU/
ML).By Lactobacillus rhamnosus A-4 bacteria suspension in terms of 2%(volume) inoculative proportion, carry out ordinary temperature stress, acid stress, nutrition
Stress, the evaluation of hyperosmotic stress resistance.
The temperature stress: in terms of 2%(volume) inoculative proportion by Lactobacillus rhamnosus A-4 bacteria suspension access MRS broth cultivation
Base is supported, 5 DEG C, 15 DEG C, 25 DEG C, 35 DEG C, 45 DEG C of cultures is placed in, respectively in 1d, 2d, 3d, 6d, 9d, detects it using microplate reader
OD600nmValue.
The acid stress: in terms of 2%(volume) inoculative proportion by Lactobacillus rhamnosus A-4 bacteria suspension access pH2.5,
The MRS broth bouillon of pH3.0, pH3.5, pH4.0, pH6.8 are detected after being placed in 37 ± 0.5 DEG C of culture 48h using microplate reader
Its OD600nmValue.
The Nutrient Stress: in terms of 2%(volume) inoculative proportion by Lactobacillus rhamnosus A-4 bacteria suspension access MRS broth cultivation
It supports the dilution after base dilutes 0,1,2,4,8 times and detects its OD using microplate reader after being placed in 37 ± 0.5 DEG C of culture 48h600nmValue.
The hyperosmotic stress: in terms of 2%(volume) inoculative proportion by the access of Lactobacillus rhamnosus A-4 bacteria suspension containing 0,1.5%,
2.5%, the MRS broth bouillon of 3.5%, 4.5% NaCl detects it using microplate reader after being placed in 37 ± 0.5 DEG C of culture 48h
OD600nmValue.
The result shows that: Lactobacillus rhamnosus A-4 can tolerate the osmotic pressure stress (attached drawing 10) of 4.5% NaCl and 8 times are trained
Base diluted low nutrition stress (attached drawing 9) is supported, in pH3.5 ~ 4.0(attached drawing 8) and 5 ~ 45 DEG C between (attached drawing 7) can still grow it is numerous
It grows.
Embodiment 2: the preparation method of Lactobacillus rhamnosus A-4 bacteria preparation.
(1) actication of culture: the Lactobacillus rhamnosus A-4 that one cyclic glycerol of picking saves lines MRS agar medium,
48h is cultivated at 37 ± 0.5 DEG C；Picking single colonie is inoculated at 37 ± 0.5 DEG C of MRS broth bouillon and cultivates 22h, obtains the first generation
Culture；Viable count detection is carried out using GB 4789.35-2016, first generation culture contains 1~3 × 109CFU/mL sandlwood
Sugared lactobacillus A-4 living cells.
(2) expand culture: according to 2% inoculum concentration of MRS broth bouillon stereometer, the first generation culture that step (1) is obtained
Object is inoculated in MRS broth bouillon, cultivates 22h at 37 ± 0.5 DEG C, obtains second generation culture；Using GB 4789.35-
2016 carry out viable count detection, and second generation culture contains 1~3 × 109CFU/mL Lactobacillus rhamnosus A-4 living cells.
By second generation culture, 2% inoculum concentration is inoculated in barley culture medium by volume, the high density at 37 ± 0.5 DEG C
Ferment 20h, obtains third generation culture；Viable count detection is carried out using GB 4789.35-2016, third generation culture contains 7 ~
9×109CFU/mL Lactobacillus rhamnosus A-4 living cells.
The barley culture medium prescription are as follows: 0.48% sodium citrate is dissolved in 5 ~ 5.5BoBarley matrix, pH6.8,115 DEG C go out
Bacterium 20min.
(3) it is centrifugated: 4min will be centrifuged under third generation culture 6000r/min that step (2) obtains, discarded supernatant,
With equivalent sterile saline centrifuge washing (6000r/min, 4min) 3 times, the sediment washed is obtained sediment
Lactobacillus rhamnosus A-4 bacterium mud.
(4) add protective agent: adding protective agent in the Lactobacillus rhamnosus A-4 bacterium mud that step (3) obtains, wherein with gram
It is 1:10 that bacterium mud, which is counted, with ratio protectant in terms of milliliter, and vortex mixes.
The protection agent prescription are as follows: 10% skimmed milk, 3% trehalose, cysteine salt 0.5%.
(5) it is spray-dried: protectant bacteria suspension will be contained obtained by step (4) with the speed of 6mL/min by doing by spraying
Dry machine is dried, and inlet temperature is 145 DEG C, and outlet temperature is 75 DEG C；It can be obtained Lactobacillus rhamnosus A-4 after spray drying
Bacteria preparation.
Viable count detection is carried out according to standard GB/T 4789.35-2016, contains 5 ~ 7 × 10 in the bacteria preparation10CFU/g
Lactobacillus rhamnosus A-4 living cells.
Embodiment 3: Lactobacillus rhamnosus A-4 leben.
(1) raw material preparation: 11% skimmed milk, 10% carboxymethyl cellulose, pectin 5%, sucrose 10% are dissolved in purified water,
After mixing evenly, homogeneous 5min(pressure 23MPa after feed liquid being warming up to 60 DEG C).
(2) it sterilizes: 45 DEG C will be cooled to after 90 DEG C of processing 6min of step (1) feed liquid.
(3) inoculation fermentation: with Lactobacillus rhamnosus A-4 bacteria preparation in 2% ratio access embodiment 2,37 ± 0.5 DEG C of constant temperature
Ferment 18 ~ 20h.
(4) mixing: 0.15% yoghurt flavours, 5% xylitol, 0.05 ‰ potassium sorbates.
(5) after quality inspection is qualified, filling storage.
Embodiment 4: Lactobacillus rhamnosus A-4 fermentation foodstuff beverage.
Lactobacillus rhamnosus A-4 fermentation barley beverage.
(1) by 5 ~ 6 BoThe empty fermentor to disappear is added in barley juice, carries out 100 DEG C, the bacteria removing of 90min.
(2) after 0.48% sodium citrate, 7.36% isomalt, potassium sorbate 0.005% being prepared, through 121 DEG C,
20min sterilization；Fermentor in (1) is added.
(3) 0 .2% sodium alginate is added to 50 times of 40 ~ 50 DEG C of warm water, after mixing, 30min is swollen, is first added
60% water waits for that water temperature is raised to 40 DEG C or so, successively adds sodium alginate mixed liquor, 0 .05% sodium carboxymethylcellulose and 0
.05% guar gum, after mixing, using sterile water constant volume, 115 DEG C, Quench after 20min sterilization.
(4) glue in feed liquid in (2) and (3) is uniformly mixed.
(5) 40 DEG C are down to hereinafter, with Lactobacillus rhamnosus A-4 in 2 ‰ ratios access embodiment 2 to temperature in fermentor
Bacteria preparation.
(6) fermentation temperature is 37 ± 0.5 DEG C, when pH reaches 4.0 ± 0.1, terminates fermentation.
(7) Sucralose and edible essence of 0.22 μm of filtration sterilization are mixed into.
(8) after quality inspection is qualified, filling storage.
Lactobacillus rhamnosus A-4 fermented soy milk beverage.
(1) it prepares soya-bean milk: the purified water that 3 times of volumes are added in the soybean after cleaning is impregnated into 12h；80 DEG C of defibrator process；10 layers of yarn
100 DEG C of sterilization 30min after cloth filtering；It is cooling.
(2) it is inoculated with: with Lactobacillus rhamnosus A-4 bacteria preparation in 2% ratio access embodiment 2.
(3) ferment: 37 ± 0.5 DEG C of ferment at constant temperature 16h, after-ripening for 24 hours, obtains fermented soybean milk under the conditions of 4 DEG C.
Lactobacillus rhamnosus A-4 fermentation purple potato drink.
(1) purple sweet potato powder: being mixed into the purified water of 1 times of quality by feedstock processing, after mixing 100 DEG C of processing 30min；To cold
When but to 60 DEG C, 0.06% citric acid color protection, 0.1% pectase, heat preservation enzymatic hydrolysis 2h is added.
(2) 0.05% alpha-amylase is added, 95 DEG C of liquefaction 60min are warming up to.
(3) it is saccharified: step (2) enzymatic hydrolysis purple sweet potato liquid is cooled to 75 DEG C, adjust material liquid pH to 4 .5,0.05% saccharification of addition
Enzyme, be saccharified 90min.
(4) it is inoculated with: with Lactobacillus rhamnosus A-4 bacteria preparation in 2% ratio access embodiment 2.
(5) it ferments: 37 ± 0.5 DEG C of ferment at constant temperature 12h.
(6) impregnation: it is mixed into 0.25% xanthan gum.
(7) after quality inspection is qualified, filling storage.
Lactobacillus rhamnosus A-4 fermentation Job's tears seed beverage.
(1) gross mass 6 feedstock processing: is added in 50 parts of adlay rice flour, 8 parts of glutinous rice flours, 8 parts of fructus hordei germinatus powder after mixing
Water again, 100 DEG C of saccharification 1h.
(2) it is inoculated with: with Lactobacillus rhamnosus A-4 bacteria preparation in 2% ratio access embodiment 2.
(3) one-step fermentation: 37 ± 0.5 DEG C of 16 ~ 18h of ferment at constant temperature.
(4) two step tastes fermentation: in one-step fermentation liquid and seasoning slurries 1:1 ratio mixing, 37 ± 0.5 DEG C of ferment at constant temperature 16
~18h.The seasoning slurry formula is 45 portions of bitter oranges, 25 parts of green apples, 5 portions of fructus choerospondiatis, 6 portions of hawthorn, 7 parts of Rhizoma Chuanxiongs.
(5) impregnation: it is mixed into 0.2% xanthan gum.
(6) fermentation liquid obtained by step (5) and the purified water epoxy glue of its 8 times of quality are ground.
(7) after quality inspection is qualified, filling storage.
Embodiment 5: Lactobacillus rhamnosus A-4 fermentation beverage made of fruits or vegetables.
Lactobacillus rhamnosus A-4 fermentation watermelon juice beverage.
(1) feedstock processing: watermelon is cleaned, drain, remove the peel, takes flesh；By the melon pulp using being carried out after broken crusher machine
High-pressure pulse electric handles 10min, obtains watermelon just juice.
(2) it is crushed: juice partial size at the beginning of watermelon being milled to 120 μm hereinafter, high-pressure pulse electric processing 3min, obtains using colloid mill
To watermelon juice.
(3) it digests: 0.1 ‰ cellulases and 0.15 ‰ protease, 50 DEG C of enzymatic hydrolysis 2h being added in step (2) watermelon juice；
By 95 DEG C of heat preservation 5min of enzymolysis liquid, it is cooled to room temperature immediately and carries out enzyme deactivation, obtains to fermentation liquid.
(4) it is inoculated with: with Lactobacillus rhamnosus A-4 bacteria preparation in 3% ratio access embodiment 2.
(5) it ferments: 37 ± 0.5 DEG C of 18 ~ 20h of ferment at constant temperature.
(6) filtering and mixing: fermentation liquid in step (5) is subjected to 80 mesh filterings, 0.25% sodium alginate, 0.05 ‰ is added
Potassium sorbate.
(7) after quality inspection is qualified, filling storage.
Lactobacillus rhamnosus A-4 ferment carrot juice beverage.
(1) carrot the preparation of carrot juice: is selected into cleaning；Broken, squeezing；Cross 40 mesh filter coarse filtration；2 times of bodies are added
Product axenic purification water；High-pressure pulse electric handles 10min, obtains carrot just juice.
(2) it deploys: 5% sucrose, 0.5% sodium citrate and 3% isomalt is added in step (1) carrot just juice.
(3) sterilize: 80 DEG C of processing 10min are rapidly cooled to 37 DEG C.
(4) it is inoculated with: with Lactobacillus rhamnosus A-4 bacteria preparation in 3% ratio access embodiment 2.
(5) it ferments: 37 ± 0.5 DEG C of 18 ~ 20h of ferment at constant temperature.
(6) filtering and mixing: by step (5) fermentation liquid carry out 80 mesh filterings, be added 0.25% sodium carboxymethylcellulose,
0.05 ‰ potassium sorbates.
(7) after quality inspection is qualified, filling storage.
Embodiment 6: Lactobacillus rhamnosus A-4 fermented black tea beverage.
(1) prepared by tea liquid: 0.6% black tea powder is added in 90 DEG C of purified waters, after heat-insulation soaking 20min, carry out it is cooling,
Centrifugation and filtering.
(2) adjust sugar: 4% sucrose and 5% oligofructose, stirring and dissolving is added in tea liquid in step (1).
(3) it sterilizes: being rapidly cooled to room temperature after carrying out 95 DEG C of processing 5min to the alternative in step (2).
(4) it is inoculated with: with Lactobacillus rhamnosus A-4 bacteria preparation in 5% ratio access embodiment 2.
(5) it ferments: 37 ± 0.5 DEG C of 18 ~ 20h of ferment at constant temperature.
(6) 0.05% pectin, 0.05 ‰ potassium sorbates, 1% concentrated apple juice mixing: is added in step (5) fermentation liquid.
(7) after quality inspection is qualified, filling storage.
3,4,5, the 6 ferment beverage of embodiment: carrying out viable count detection according to standard GB/T 4789.35-2016,
In contain 1 × 108The Lactobacillus rhamnosus A-4 living cells of CFU/mL or more.
In the embodiment 3,4,5,6 when fermented beverage normal temperature storage wherein Lactobacillus rhamnosus A-4 in low temperature, acidity
In the Product environment of stress and low nutrition stress still can slow growth and breeding, thus Lactobacillus rhamnosus A- when guaranteeing 6 months
4 viable counts are still greater than 106CFU/mL。
Fermented beverage in the embodiment 3,4,6 can carry out 40 ~ 60 DEG C of heat treatment according to hobby before consumption,
The high-temperature stability of middle Lactobacillus rhamnosus A-4 makes its survival rate be greater than 90%.
Sequence table
<120>a kind of strong resistance Lactobacillus rhamnosus A-4 and application thereof
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<170> SIPOSequenceListing 1.0
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tagggaatct tccacaatgg acgcaagtct gatggagcaa cgccgcgtga gtgaagaagg 360
ctttcgggtc gtaaaactct gttgttggag aagaatggtc ggcagagtaa ctgttgtcgg 420
cgtgacggta tccaaccaga aagccacggc taactacgtg ccagcagccg cggtaatacg 480
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gcagaagagg acagtggaac tccatgtgta gcggtgaaat gcgtagatat atggaagaac 660
accagtggcg aaggcggctg tctggtctgt aactgacgct gaggctcgaa agcatgggta 720
gcgaacagga ttagataccc tggtagtcca tgccgtaaac gatgaatgct aggtgttgga 780
gggtttccgc ccttcagtgc cgcagctaac gcattaagca ttccgcctgg ggagtacgac 840
cgcaaggttg aaactcaaag gaattgacgg gggcccgcac aagcggtgga gcatgtggtt 900
taattcgaag caacgcgaag aaccttacca ggtcttgaca tcttttgatc acctgagaga 960
tcaggtttcc ccttcggggg caaaatgaca ggtggtgcat ggttgtcgtc agctcgtgtc 1020
gtgagatgtt gggttaagtc ccgcaacgag cgcaaccctt atgactagtt gccagcattt 1080
agttgggcac tctagtaaga ctgccggtga caaaccggag gaaggtgggg atgacgtcaa 1140
atcatcatgc cccttatgac ctgggctaca cacgtgctac aatggatggt acaacgagtt 1200
gcgagaccgc gaggtcaagc taatctctta aagccattct cagttcggac tgtaggctgc 1260
aactcgccta cacgaagtcg gaatcgctag taatcgcgga tcagcacgcc gcggtgaata 1320
cgttcccggg ccttgtacac accgcccgtc acaccatgag agtttgtaac acccgaagc 1379
Claims (6)
1. one kind have strong resistance Lactobacillus rhamnosus (Lactobacillus rhamnosus) A-4, Lactobacillus rhamnosus
A-4 bacteria preparation and application thereof, the bacterial strain is on September 29th, 2018 in section, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 China
The preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms center, institute of microbiology, institute, deposit number are
CGMCC No.16550。
2. Lactobacillus rhamnosus A-4 according to claim 1 is it is characterized by: rhamnose is newborn as a preferred method,
Bacillus A-4 can tolerate 75 DEG C of high temperature, be up to 96.88% ± 1.4% through 2.0 acid processing 2h survival rate of pH；4.5% NaCl's
It can still be given birth under the diluted low nutrition stress of osmotic pressure stress, 8 times of culture mediums, pH3.5 acid stress and 5 ~ 45 DEG C of temperature stress
Long breeding.
3. the bacteria preparation of Lactobacillus rhamnosus A-4 according to claim 1, it is characterised in that: the bacteria preparation be pass through by
Lactobacillus rhamnosus A-4 expands what culture was realized.
4. Lactobacillus rhamnosus A-4 according to claim 1 or Lactobacillus rhamnosus A-4 bacterium as claimed in claim 3
Preparation is preparing the purposes in beverage.
5. beverage according to claim 4, it is characterised in that: the beverage be milk beverage or plant beverage, preferably,
The beverage includes Lactobacillus rhamnosus milk beverage, Lactobacillus rhamnosus cereal beverage, Lactobacillus rhamnosus bean beverage, sandlwood
Sugared lactobacillus potato beverage, Lactobacillus rhamnosus fruit beverage, Lactobacillus rhamnosus vegetable beverage, Lactobacillus rhamnosus coarse food grain drink
Material, Lactobacillus rhamnosus tea beverage.
6. beverage according to claim 5, it is characterised in that: the effective component of the beverage is described in claim 1
Lactobacillus rhamnosus A-4 or Lactobacillus rhamnosus A-4 bacteria preparation as claimed in claim 3；A kind of preferred life of the beverage
Production mode is to be fermented using Lactobacillus rhamnosus A-4 described in claim 1, and another preferably production method is direct
Add Lactobacillus rhamnosus A-4 bacteria preparation as claimed in claim 3；Sandlwood when the characteristics of beverage is normal temperature storage 6 months
Sugared lactobacillus A-4 viable count is still greater than 106CFU/mL。
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| CN113491331A (en) * | 2021-06-30 | 2021-10-12 | 华南理工大学 | Algal polysaccharide composition and composite fruit juice mixed fermentation powder as well as preparation method and application thereof |
| WO2022015248A1 (en) * | 2020-07-16 | 2022-01-20 | National University Of Singapore | A tea-based beverage |
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| CN113491331A (en) * | 2021-06-30 | 2021-10-12 | 华南理工大学 | Algal polysaccharide composition and composite fruit juice mixed fermentation powder as well as preparation method and application thereof |
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