CN109897799A - One plant of production γ-aminobutyric acid lactic acid bacteria strains and its screening technique and the preparation method rich in γ-aminobutyric acid mesona Yoghourt - Google Patents
One plant of production γ-aminobutyric acid lactic acid bacteria strains and its screening technique and the preparation method rich in γ-aminobutyric acid mesona Yoghourt Download PDFInfo
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- CN109897799A CN109897799A CN201910169739.8A CN201910169739A CN109897799A CN 109897799 A CN109897799 A CN 109897799A CN 201910169739 A CN201910169739 A CN 201910169739A CN 109897799 A CN109897799 A CN 109897799A
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- aminobutyric acid
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- acid bacteria
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Abstract
The invention discloses one plant to produce γ-aminobutyric acid lactic acid bacteria strains and its screening technique and preparation method rich in γ-aminobutyric acid mesona Yoghourt, feature is that the lactic acid bacteria classification naming is Lactobacillus brevis PDD-2 plants, China Committee for Culture Collection of Microorganisms's common micro-organisms center was preserved on 06 19th, 2018, deposit number is CGMCC No.15954, and method for preparing sour milk is the step of the first leavening and the second leavening is accordingly made in the streptococcus thermophilus after activating and Lactobacillus brevis；The step of red bean bud powder, skimmed milk powder and sucrose add the first leavening and the second leavening after homogeneous sterilizes again is added after salt algae enzymolysis liquid and mesona solution are mixed；Heat-preservation fermentation, cooling, after-ripening, which are made, again is rich in γ-aminobutyric acid mesona sour milk product, and advantage is rich in γ-aminobutyric acid, beta carotene, polypeptide, polysaccharide, flavones, ursolic acid, vitamin and the dietary cellulosic isoreactivity factor.
Description
Technical field
The present invention relates to a kind of method for preparing sour milk, more particularly, to a kind of production γ-aminobutyric acid lactic acid bacteria strains and richness
The preparation method of the Yoghourt of mesona containing γ-aminobutyric acid.
Background technique
γ-aminobutyric acid (GABA) is a kind of nonprotein amino acid, in vivo by Pidolidone (L-Glu) or
L-sodium (L-MSG) passes through the effect of glutamate decarboxylase, sloughs a molecule carboxyl and is formed.γ-aminobutyric acid is one
Kind inhibitory neurotransmitter, can effectively alleviate depression, additionally it is possible to β cytothesis is stimulated, to effectively treat diabetes.
In last century the eighties, Robert group finds the presence of γ-aminobutyric acid first in animal brain leaching liquor, then exists
γ-aminobutyric acid is listed in new resource food within 2008, then has started the upsurge for studying γ-aminobutyric acid.Currently, synthesis γ-
The method of aminobutyric acid is divided into two major classes: chemical method and bioanalysis.Chemical method, process is complicated and uses more not environment friendly
There is organic reagent residual without that can add in food in type reagent, end product.Bioanalysis includes plant method and micro-
Bioanalysis, the brown rice of germination and beans contain a large amount of γ-aminobutyric acid；Microorganism is since growth cycle is short, reproduction speed
Fastly, it was applied to production γ-aminobutyric acid extensively in recent years.Wherein, lactic acid bacteria is generally considered micro- life of aliment security level
Object, therefore it is significant for screening highly producing gamma-aminobutyric acid and the excellent lactic acid bacteria of resistance.
Red bean, respiration enhances during sprouting, and proteolytic enzyme is activated, therefore soluble protein content is aobvious
It writes and increases, a variety of anti-nutritional factors such as phytic acid, protease inhibitors is degraded, healthcare function ingredient such as γ-aminobutyric acid, flavones
Class, phenols, vitamin, dietary fiber and saponin(e all increased.Glutamic acid decarboxylase enzyme activity, the substrate Pidolidone of enzyme effect
Or L-sodium has all been largely fixed the amount of γ-aminobutyric acid, therefore, utilizes the coenzyme 5 ' of glutamate decarboxylase
Phosphopyridoxal pyridoxal phosphate (PLP) and the substrate (MSG) of enzyme effect stimulation red bean germinate, to accumulate more γ-aminobutyric acids.Red bean
It is sweet natured, there is special fragrance, by liking for consumer, since it there are many prebiotic functions, exploitation is rich in γ-ammonia
The red bean flavor yoghourt of base butyric acid is of great significance.
Mesona also known as Mesona chinensis Benth, celestial being's jelly, Chinese mesona herb, are Lamiaceae plant Chinese mesona herb (Mesona chinensis
Benth. herb), resourceful distribution is wide, and the ground such as Guangdong and Guangxi Provinces area, Zhejiang, Jiangxi, Fujian, Hainan, Yunnan and Taiwan have
It produces.Its is cold in nature for Chinese mesona herb, puckery, sweet, have clear heat quench one's thirst, cool blood the effect of, active constituent has polysaccharide, anthocyanidin, amino acid
And the ingredients such as triterpenes.Polysaccharide in Chinese mesona herb has anti-oxidant, immunological regulation, the different physiological roles such as hypoglycemic, triterpenes
In ursolic acid and oleanolic acid have the bioactivity such as anti-inflammatory, antibacterial, antiallergic action, antitumor, antiviral, flavonoids
Object is closed, there is the multiple biological activities such as anticancer, antiulcer, anti-oxidant, antiviral, anti-inflammatory, cancer prevention, comfortable decompression, town
The pharmacological actions such as pain.
The natural carotenoid contained in salt algae not only includes content full cis-beta-carotene, and there are also 9 cis- β-carrots
Element, there are also alpha-carotene, lutein, luteole, gamma carotenes etc. in addition to this.Beta carotene in salt algae, selenium,
The antioxidations such as vitamin E can remove free radical, have strong antioxidant action, make cell from the infringement of free radical, protect
Cell membrane is protected, the permeability of cell is enhanced, also there are the 18 kind amino acid and nutrient etc. beneficial to human body in salt algae.Acid
Milk is then even more the healthy food being favored by people, although however Yoghourt contains the nutritional ingredients such as protein abundant, current city
It is all relatively simple to sell yoghurt function, but lacks the specific function factor.If being rich in a certain amount of specific function factor in common sour milk,
By the product is made not only there is the mouthfeel of common sour milk can also play the role of certain nutrition and health care.
Summary of the invention
Technical problem to be solved by the invention is to provide one plant of production γ-aminobutyric acid lactic acid bacteria strains and its screening techniques
With the preparation method for being rich in γ-aminobutyric acid mesona Yoghourt, the mesona Yoghourt is rich in γ-aminobutyric acid, beta carotene, more
Peptide, polysaccharide, flavones, ursolic acid, vitamin and the dietary cellulosic isoreactivity factor have anti-oxidant, anti-inflammatory, immunological regulation, drop
Blood lipid alleviates the functions such as depression, defaecation and anti-aging.
The technical scheme of the invention to solve the technical problem is:
1, one plant production γ-aminobutyric acid lactic acid bacteria, the lactic acid bacteria classification naming be Lactobacillus brevis (Lactobacillus brevis) PDD-2 plants, it was preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms on 06 19th, 2018
The heart, deposit number are CGMCC No.15954.
2, the screening technique of the lactic acid bacteria of above-mentioned production γ-aminobutyric acid, comprising the following steps:
(1) it the preliminary screening of lactic acid bacteria and isolates and purifies
Selecting fresh milk is after sample dilutes, to be coated on the MRS solid medium tablets containing bromocresol purple, is stood, in 37-
43 DEG C of culture 36-48h, pick out the bacterium colony in yellow, and it is preliminary that screening is obtained the single bacterium colony with typical lactic acid bacteria feature
It is set to lactic acid bacteria；The bacterium colony primarily determined is crossed several times repeatedly in MRS solid medium tablets, so that further progress is pure
The purpose of change, until there is the single consistent bacterium colony of form, preservation of bacteria strain；
(2) screening of aimed strain
The strain of step (1) preservation is sent out by the liquid that 1% inoculum concentration of percent by volume is added to the L-sodium containing 1wt%
Ferment culture medium, after 37 DEG C of culture 48h；Supernatant is taken after fermentation liquid is centrifuged, while compound concentration is the standard items L- of 10g/L
Glutamic acid and γ-aminobutyric acid will take 2ul standard items and sample point sample on chromatographic paper respectively, chromatographed in chromatography cylinder,
Chromatographic paper is taken out after chromatography and is dried 80 minutes in 70 DEG C, the cream of γ-aminobutyric acid Pidolidone will be converted to
Sour bacterium carries out Gram's staining and physio-biochemical characteristics identification, then carries out 16srDNA and API50-CHL sugar fermenting experiment, choosing
Take Gram's staining for the positive, catalase experiment is negative single bacterium colony, then by the bacterial strain of γ-aminobutyric acid high yield
16srDNA and API50-CHL sugar fermenting experiment is carried out, the lactic acid bacteria of one plant of highly producing gamma-aminobutyric acid is obtained, lactic acid bacteria classification
Be named as Lactobacillus brevis (Lactobacillus brevis) PDD-2 plants, China Microbiological was preserved on 06 19th, 2018
Culture presevation administration committee common micro-organisms center, deposit number are CGMCC No.15954.
The preparation method of the MRS solid medium containing bromocresol purple is as follows: by MRS meat soup 52.24g, bromine first
Phenol violet 0.04g, agar powder 10g ~ 20g are dissolved in 1L distilled water, in 121 DEG C of sterilizing 20min；The chromatographic solution is positive
After butanol, glacial acetic acid and water 5:3:2 in mass ratio mixing, ninhydrin, which is added, makes its mass percent 1.2%, in advance in 30 DEG C
It is spare that 16-18h is balanced in water-bath.
3, the lactic acid bacteria preparation of above-mentioned production γ-aminobutyric acid is rich in the preparation method of γ-aminobutyric acid mesona Yoghourt, including
Following steps:
(1) activation of bacterial strain: under aseptic technique, by streptococcus thermophilus (BNCC187932 or BNCC186629 or
BNCC175966 or BNCC175937) and deposit number be CGMCC No.15954 Lactobacillus brevis PDD-2 be inoculated in MRS respectively
In fluid nutrient medium, cultivated 20-36 hours in 37-42 DEG C of incubator respectively；
(2) preparation of lactic acid bacteria fermenting agent: by after activation streptococcus thermophilus and Lactobacillus brevis under aseptic technique, respectively
The inoculum concentration of 4-6% is inoculated in the plain chocolate for being cooled to 37-42 DEG C after pasteurize by volume, in 37-42 DEG C of incubator
Middle culture 3-6h, is accordingly made the first leavening and the second leavening；
(3) preparation of salt algae enzymolysis liquid: Dunaliella salina cell algal gel is collected, after cleaning up, cell algal gel is suspended in aqueous solution
The algal gel cell suspending liquid that mass fraction is 10-15% is obtained, algal gel cell suspending liquid is first surpassed under 1000-2000bar pressure
High pressure is crushed 5-10min, the ultrasonic cell disruption instrument for being again then 550-650W with output power smudge cells in ice bath
6-10 min adjusts pH value to 5-6, and the vigor for being separately added into algae solution quality 0.05-0.10% is the cellulose of 2000-4000U/g
The vigor of enzyme and 0.05-0.10% are the flavor protease of 30-50U/mg, and heat preservation enzymatic hydrolysis adjusts after 3-8 hours at 45-55 DEG C
PH to 7.0 obtains chlorella mixed enzymolysis liquid；
(4) preparation of mesona solution: after mesona cleaning, crushing, the water that 2-5 times of mesona quality is added in mesona is mixed, then
Add the dietary alkali (Na of mesona and water gross mass 0.15-0.35%2CO3) be uniformly mixed, in 85-95 DEG C of Heat preservation 10-
30min, then ground 2 ~ 3 times with colloid mill, with the screen to filtrate of 100 ~ 150 mesh, mesona solution is made；
(5) ingredient: by salt algae enzymolysis liquid and mesona solution (1-3) in mass ratio: 1 ratio is mixed to get salt algae and mixes with mesona
Liquid, then add red bean bud powder 1.0-3.0%, skimmed milk powder 10.0-13.0% and sucrose 4.0- in mass ratio in this mixed liquor
7.0%, it is uniformly mixed homogeneous again；
(6) it sterilizes, is cooling, inoculation, canned, fermentation: the mixed liquor after homogeneous being heated to 85-95 DEG C, keeps the temperature 15-30 minutes
Afterwards, it is cooled to 40-43 DEG C, under conditions of sterile working, adds the first leavening 2.0-4.0% and the by quality or volume ratio
Two leavening 3.0-6.0%, being sufficiently stirred makes it after mixing, canned to Sour milk bottle or plastic cup with can packing machine, after sealing,
Be packed into frame of plastic, move into fermenting cellar, 37-43 DEG C heat-preservation fermentation 3-8 hours；
(7) cooling, after-ripening: above-mentioned Yoghourt after fermentation is moved into cooling, after-ripening in 0-4 DEG C of freezer and obtains and is rich in
γ-aminobutyric acid mesona sour milk product.
The red bean bud powder the preparation method is as follows: choose each 100, red bean of different cultivars, weigh respectively, with 75%
Ethyl alcohol, which impregnates 15 minutes, to be sterilized, and is then transferred in vessel and a certain proportion of water immersion 8h is added, weigh again after immersion, will
The incubator that seed after immersion is placed in 30 DEG C carries out being protected from light culture, uses 0.0015g/L every 30 minutes during germination
5 ' phosphopyridoxal pyridoxal phosphates and 2g/L L-sodium spray, obtain the highest seed of germination percentage be rich in γ-aminobutyric acid
Then red bean bud is put in -80 DEG C of refrigerator pre-cooling 2h, 20-28h then is lyophilized in -80 DEG C of freeze driers, by the red bean of freeze-drying
Bud crushes after taking out and obtains red bean bud powder, and it is spare to cross 200 meshes.
Compared with the prior art, the advantages of the present invention are as follows: present invention firstly discloses one plant of highly producing gamma-aminobutyric acid creams
Sour bacterium and its screening technique and preparation method rich in γ-aminobutyric acid mesona Yoghourt, including producing γ-aminobutyric acid lactic acid bacteria
Separation screening, the preparation rich in γ-aminobutyric acid germination red bean powder, the bead rich in the function factors such as beta carotene and polypeptide
Algae zymolyte and rich in function factors such as polysaccharide, flavones, ursolic acid mesona solution preparation, through mixture prepare, it is filling, hair
The technical process such as ferment, cooling, after-ripening obtain a kind of containing γ-aminobutyric acid, beta carotene, polypeptide, polysaccharide, flavones, black bearberry
Acid, vitamin and the dietary cellulosic isoreactivity factor have anti-oxidant, anti-inflammatory, immunological regulation, reducing blood lipid, alleviate depression, defaecation
With the sour milk product of the functions such as anti-aging.The functions such as addition germination red bean powder, salt algae zymolyte, mesona solution in the sour milk product
Property ingredient contain in salt algae zymolyte rich in the function factors such as γ-aminobutyric acid, dietary fiber, vitamin in the red bean powder that germinates
The function factors such as beta carotene, polypeptide, dietary fiber, vitamin, the function such as content polysaccharide, flavones, ursolic acid in mesona solution
The energy factor, they have anti-oxidant, anti-inflammatory, immunological regulation, reducing blood lipid, alleviate the plurality kinds of health care function such as depression, defaecation and anti-aging
Can, mesona solution is added, the gel characteristic of Yoghourt can be significantly improved, prevent moisture wash rice from going out.Pass through the Lactobacillus brevis of separation screening
PDD-2 fermentation and red bean germination generate the function factors such as γ-aminobutyric acid；Pass through super-pressure and ultrasonication and cellulase
The synergistic effect of enzymatic hydrolysis promotes the broken of Dunaliella salina cell cell wall, releases beta carotene and protein, improve its utilization
Value and bioactivity under their synergistic effect, promote to match by the enzymatic hydrolysis of flavor protease and the fermentation of lactic acid bacteria
The decomposition of salt algae in material, red bean albumen, lactoprotein generates more free amino acids and polypeptide, with single lactobacillus-fermented or
Enzymatic hydrolysis is compared, and the content of amino acid and polypeptide improves 40-50% in product, and antioxidant activity improves 200-300%, γ-ammonia
Base butyric acid, beta carotene, flavones, ursolic acid, polyoses content respectively reach 530~850 μ g/mL, 350-450 μ g/mL, 350-
450 μ g/mL, 75-305 μ g/mL, 180-720 μ g/mL, 110-405mg/mL improve the flavor and nutritive value of product, increase
Its functional characteristic is added.The Yoghourt that the present invention is prepared, not only unique flavor, full of nutrition, and has anti-oxidant, anti-
Inflammation, immunological regulation, alleviates the functions such as depression, defaecation and anti-aging at reducing blood lipid, provides for the functional yoghourt exploitation of special flavor
Certain thinking and inspiration, also for effect of the raw materials such as salt algae, mesona in development functionality food explore one it is feasible
By way of.
The lactic acid bacteria of one plant of production γ-aminobutyric acid, the lactic acid bacteria classification naming Lactobacillus brevis (Lactobacillus brevis) PDD-2 plants, it was preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms on 06 19th, 2018
The heart, deposit number are CGMCC No.15954, and preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, the Chinese Academy of Sciences
Institute of microbiology.
Specific embodiment
Present invention is further described in detail with reference to embodiments.
One, experimental determining method
1, aimed strain produces the measurement of γ-aminobutyric acid ability
(1) whether paper chromatography qualitative determination lactic acid bacteria produces γ-aminobutyric acid
For the lactic acid bacteria of preliminary screening after isolating and purifying, 1% inoculum concentration is added to liquid fermentation medium in 37 DEG C of culture 48h, liquid
Body culture medium contains 1% L-sodium.Supernatant is taken after fermentation liquid is centrifuged, while preparing standard items glutamic acid and γ-ammonia
Standard items and sample are taken 2ul point sample on chromatographic paper respectively, are chromatographed in chromatography cylinder by base butyric acid, concentration 10g/L,
Chromatographic solution is about 1.5cm high.Chromatographic paper is taken out after chromatography and is dried 80 minutes in 70 DEG C.According to γ-ammonia in fermentation liquid
Whether there is or not tentatively to judge that bacterial strain produces γ-aminobutyric acid with the spot depth with the spot of corresponding standard items for base butyric acid and Pidolidone.
(2) alpha-aminobutyric acid content measurement in the drafting of γ-aminobutyric acid standard curve and fermentation liquid:
γ-aminobutyric acid standard items are each configured to 0,0.5,1,1.5,2,2.5,5,10,15,20g/mL solution, it is different
The solution of concentration draws 2ul point sample on chromatographic paper respectively, every being spaced 1.5cm between sample, 70 DEG C of drying after chromatography,
γ-aminobutyric acid spot is cut, spot is shredded to be placed in eluent elutes, eluent is 0.6% Salzburg vitriol: 75% second
Alcohol=2:38, revolving speed 50rpm, eluting temperature are 40 DEG C, and the time is 60 minutes.Finally extinction will be measured at eluent and 512nm
Value.Make linear regression, obtain equation: y=0.047x-0.0085, coefficient R2γ-aminobutyric acid measures in=0.996. fermentation liquid
Using same method.
2, in red bean bud and Yoghourt alpha-aminobutyric acid content measurement
The bean sprouts of sprouting is first freeze-dried, and freeze-drying bean sprouts crushed 200 meshes, stayed overnight with 0.02mol/L HCl extract, leaching
Supernatant is taken after extract filtering.By three bean sprouts supernatants after the Yoghourt of after-ripening or extraction respectively with 3,5 disulfo salicylic acid 1:1
Mixing, 10000rpm are centrifuged 10 minutes with protein precipitation.Standard items mark and γ-aminobutyric acid standard items 1 are mixed using amino acid:
1 mixing, is analyzed using amino-acid analyzer.
3, polysaccharide and ursolic acid content measurement
(1) determination of polysaccharide: using phend-sulphuric acid (it is strong etc. according to fourth steel, food polyoses content different measuring methods
Research, practical preventive medicine, 2000, the 5th phase of volume 7,325-327 pages)
(2) ursolic acid content measures: taking 100 grams of Yoghourts to be freeze-dried to obtain frozen dried sour milk powder, takes 5 grams of frozen dried sour milk powders, be placed in
In stuffed conical flask, 50 mL, 95% ethyl alcohol is added in precision, beyond the Great Wall glass stopper, 30 min of ultrasonic extraction.It lets cool to room temperature, with
0.45 μm of filter membrane filtration, takes filtrate to carry out liquid phase analysis (Feng Bairu etc., the extraction work of ursolic acid and oleanolic acid in Chinese mesona herb
Skill research, study of pharmacy, volume 2018,37, the 9th phase, 503-505 pages).
4, determining content of peptides
(1) production of standard curve: take the volumetric flask of ten 10ml successively to prepare 0.0 with 5% TCA, 0.2,0.4,0.6,
0.8, then 1.0,1.2,1.4,1.6 and 1.8mg/ml Gly-Gly-Tyr-Arg tetrapeptide standard solution takes 6.0ml respectively
Standard solution is added 4.0ml biuret reagent and stands 10min 2000r/min centrifugation in uniformly mixed on whirlpool mixed instrument
10min takes supernatant to measure OD value (doing blank control using the first pipe) under 540nm using the concentration of peptide as abscissa X (mg/
Ml) OD value is that ordinate Y makes standard curve, obtains regression equation y=0.3681x+0.0013(R2=1)；
(2) measurement of content of peptides: take 5ml yoghourt pulp that trichloroacetic acid (TCA) aqueous solution of 5ml 10% (W/V) is added in whirlpool
It is uniformly mixed to stand 10min and then be centrifuged 15min at 4000r/min on mixed instrument and supernatant is transferred completely into 50ml
Scale is settled in volumetric flask and with 5% TCA shaking up and then take the above-mentioned solution of 6.0ml to set in another test tube, biuret is added
Reagent 4.0ml (sample liquid: biuret reagent=3:2) (V/V) stands 10min, 2000r/ in uniformly mixed on whirlpool mixed instrument
Min is centrifuged 10min, takes supernatant to measure OD value reference standard curve under 540nm and acquires peptide concentration C in sample solution
(mg/ml) and then content of peptides in sample can be acquired.
5, in YoghourtβCarotene carotene content measurement
Using high effective liquid chromatography for measuring [Ren Ke, Li Qingping, Zhang Hairong.8 kinds of different types of high effective liquid chromatography for measuring
In health foodβCarrotene, 2018, food safety quality testing journal, 2018,9 (11): 2585-2589].
6, the measurement of Flavonoids content
(1) drafting of standard curve: precision weighs control substance of Rutin 200mg, adds 70%(v/v) ethyl alcohol dissolution after, be settled to
100mL.Precision draws 10mL, is settled to 100mL with distilled water, obtains the standard solution of 0.2 mg/mL.Accurate absorption standard
Solution 0,0.4,0.8,1.2,1.6,2.0,2.4mL, are respectively placed in 10mL measuring bottle, add distilled water to 2.4mL, it is sub- to be added 5%
Sodium nitrate solution 0.4mL is mixed, and places 6 min, and 10% aluminum nitrate 0.40mL is added, shakes up, and places 6 min, and 4.3% hydrogen is added
Sodium oxide molybdena 4.0mL, then plus distilled water to scale, shake up, place 15min, be control with reagent blank.In 500 nm wavelength
Place's measurement trap A, is abscissa by ordinate, concentration C of absorbance A, draws rutin concentration versus absorbance scale directrix curve, make
Linear regression obtains equation: -0.7302(r=0.9991 C=78.52A)；
(2) flavonoid content measures: accurately weighing a certain amount of yoghurt example, is put into beaker, is added a certain amount of
70% ethyl alcohol stirs into starchiness with glass bar, is put into beaker of the 500ml with plug, cleans beaker and glass with 70% ethyl alcohol
Stick, washing lotion are also poured into beaker, then plus 70% ethyl alcohol to 250mL, then with ultrasonic treatment 20 minutes, then pipette is used in filtering
It draws 10mL filtrate and measures absorbance value according to the above method with 70% ethyl alcohol constant volume to 100mL, calculate Huang with mark curve
The content of ketone compounds.
7, oxidation resistance index determining
(1) measurement of total antioxidant capacity: fragrant tripe crude extract is added in oxidation reaction system, is produced using Fenton reaction system
Raw hydroxy radical measures light absorption value using ascorbic acid as positive control at 510nm after reaction；Total antioxidant capacity
It is calculated by following formula:
；
(2) measurement of ultra-oxygen anion free radical: in reaction system, every liter of sample reacts 40min at 37 DEG C and is inhibited super
The changing value for the ultra-oxygen anion free radical that the vitamin C that oxygen anion free radical is equivalent to 1mg is inhibited is a vigor list
Position,
OD1: the absorbance of control tube；OD2: measure the absorbance of pipe；OD3: the absorbance of standard pipe；
(3) measurement of hydroxy radical: Fenton reaction is the most common chemical reaction for generating hydroxy radical, H2O2Amount and
Fenton reaction generation hydroxy radical is directly proportional, after giving electron acceptor, with gress reagent colour development, forms red material,
The number of colour generation and hydroxy radical is proportional:
, standard pipe concentration is 8.824mmol/L；Sampling amount is 1mL；OD1: the absorbance of control tube；OD2: measure the absorbance of pipe；
OD3: the absorbance of standard pipe；OD4: the absorbance of blank tube.
8, immune indexes measure
(1) experimental animal grouping and stomach-filling
60 mouse are randomly divided into 3 groups, every group 20.Respectively Normal group, cyclophosphamide (CY) control group, CY+ perfume (or spice) tripe
Crude extract group (experimental group).It adapts to after a week, start stomach-filling, Normal group and the daily stomach-filling physiology salt of CY control group in mouse
Water 0.10ml/10g weight, the daily stomach-filling Yoghourt of experimental group slightly propose 0.10ml/10g weight, and continuous 30 days.At first 5 days of stomach-filling,
In addition to Normal group, the cyclophosphamide 100mg/kg weight of isometric(al) is injected intraperitoneally in cyclophosphamide (CY) control group daily；
(2) organ index calculation formula
Each group mouse is weighed afterwards for 24 hours in last dose, after tail vein takes blood, cervical dislocation to put to death mouse, takes liver, spleen and chest
Gland.Weighing on an electronic balance, which is blotted, with filter paper calculates spleen index and thymus index:
；
(3) phagocytic index measures
,,K: the finger of not calibrated phagocytosis
Number；OD1: blood specimen OD value at 2 minutes；OD2: blood specimen OD value at 20 minutes).
Two, specific embodiment
Embodiment 1
One plant production γ-aminobutyric acid lactic acid bacteria, the lactic acid bacteria classification naming be Lactobacillus brevis (Lactobacillus brevis) PDD-2 plants, it was preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms on 06 19th, 2018
The heart, deposit number are CGMCC No.15954, and preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, the Chinese Academy of Sciences
Institute of microbiology.
Embodiment 2
The screening technique of the production γ-aminobutyric acid lactic acid bacteria of above-described embodiment 1, comprising the following steps:
(1) it the preliminary screening of lactic acid bacteria and isolates and purifies
Selecting fresh milk is after sample dilutes, to be coated on the MRS solid medium tablets containing bromocresol purple, is stood, in 37-
43 DEG C of culture 36-48h, pick out the bacterium colony in yellow, and it is preliminary that screening is obtained the single bacterium colony with typical lactic acid bacteria feature
It is set to lactic acid bacteria；The bacterium colony primarily determined is crossed several times repeatedly in MRS solid medium tablets, so that further progress is pure
The purpose of change, until there is the single consistent bacterium colony of form, preservation of bacteria strain；The wherein MRS solid culture containing bromocresol purple
The preparation method of base is as follows: MRS meat soup 52.24g, bromocresol purple 0.04g, agar powder 10g ~ 20g is dissolved in 1L distilled water,
In 121 DEG C of sterilizing 20min；The chromatographic solution is addition after n-butanol, glacial acetic acid and water 5:3:2 in mass ratio mixing
Ninhydrin makes its mass percent 1.2%, and it is spare to balance 16-18h in 30 DEG C of water-baths in advance；
(2) screening of aimed strain
The strain of step (1) preservation is sent out by the liquid that 1% inoculum concentration of percent by volume is added to the L-sodium containing 1wt%
Ferment culture medium, after 37 DEG C of culture 48h；Supernatant is taken after fermentation liquid is centrifuged, while compound concentration is the standard items L- of 10g/L
Glutamic acid and γ-aminobutyric acid will take 2ul standard items and sample point sample on chromatographic paper respectively, chromatographed in chromatography cylinder,
Chromatographic solution is about 1.5cm high, takes out chromatographic paper after chromatography and dries 80 minutes in 70 DEG C, Pidolidone will be converted
Gram's staining and physio-biochemical characteristics identification are carried out at the lactic acid bacteria of γ-aminobutyric acid, then carries out 16srDNA and API50-
CHL sugar fermenting experiment, choosing Gram's staining is the positive, and catalase experiment is negative single bacterium colony, then by γ-ammonia
The bacterial strain of base butyric acid high yield carries out 16srDNA and API50-CHL sugar fermenting experiment, obtains the cream of one plant of highly producing gamma-aminobutyric acid
Sour bacterium, the lactic acid bacteria classification naming be Lactobacillus brevis (Lactobacillus brevis) PDD-2 plants, in 06 month 2018 19
It is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center day, deposit number is CGMCC No.15954.
Embodiment 3
γ-aminobutyric acid mesona Yoghourt is rich in using the lactic acid bacteria preparation that above-described embodiment 1 and embodiment 2 produce γ-aminobutyric acid
Preparation method, comprising the following steps:
(1) activation of bacterial strain: under aseptic technique, by streptococcus thermophilus (BNCC187932 or BNCC186629 or
BNCC175966 or BNCC175937) and deposit number be CGMCC No.15954 Lactobacillus brevis PDD-2 be inoculated in MRS respectively
In fluid nutrient medium, cultivated 28 hours in 40 DEG C of incubator respectively；
(2) preparation of lactic acid bacteria fermenting agent: by after activation streptococcus thermophilus and Lactobacillus brevis under aseptic technique, respectively
5% inoculum concentration is inoculated in the plain chocolate for being cooled to 40 DEG C after pasteurize by volume, is cultivated in 40 DEG C of incubators
The first leavening and the second leavening is accordingly made in 4h；
(3) preparation of salt algae enzymolysis liquid: Dunaliella salina cell algal gel is collected, after cleaning up, cell algal gel is suspended in aqueous solution
The algal gel cell suspending liquid that mass fraction is 12% is obtained, by the first high-pressure breaking under 1500bar pressure of algal gel cell suspending liquid
8min, the ultrasonic cell disruption instrument for being again then 600W with output power 8 min of smudge cells in ice bath adjust pH value to 5-
6, cellulase that the vigor for being separately added into algae solution quality 0.08% is 3000U/g and 0.08% vigor be 40U/mg flavor
Protease, heat preservation enzymatic hydrolysis adjusts pH to 7.0 at 50 DEG C after 6 hours, obtains chlorella mixed enzymolysis liquid；
(4) preparation of mesona solution: after mesona cleaning, crushing, the water that 2-5 times of mesona quality is added in mesona is mixed, then
Add the dietary alkali (Na of mesona and water gross mass 0.25%2CO3) be uniformly mixed, in 90 DEG C of Heat preservation 20min, then use colloid mill
Mesona solution is made with the screen to filtrate of 100 ~ 150 mesh in mill 2 ~ 3 times；
(5) ingredient: being mixed to get salt algae and mesona mixed liquor for salt algae enzymolysis liquid and mesona the solution ratio of 2:1 in mass ratio,
It adds red bean bud powder 2.0%, skimmed milk powder 12.0% and sucrose 6.0% in mass ratio in this mixed liquor again, is uniformly mixed again
Matter；Wherein red bean bud powder the preparation method is as follows: choose each 100, red bean of different cultivars, weigh, soaked with 75% ethyl alcohol respectively
It steeps 15 minutes and sterilizes, be then transferred in vessel and a certain proportion of water immersion 8h is added, weigh again after immersion, after immersion
Seed be placed in 30 DEG C of incubator and carry out being protected from light culture, every 30 minutes 5 ' phosphorus with 0.0015g/L during germination
The L-sodium of sour pyridoxal and 2g/L spray, obtaining the highest seed of germination percentage is the red bean bud rich in γ-aminobutyric acid,
Then -80 DEG C of refrigerator pre-cooling 2h are put in, 20-28h then is lyophilized in -80 DEG C of freeze driers, the red bean bud of freeze-drying is taken out
It crushes afterwards and obtains red bean bud powder, it is spare to cross 200 meshes；
(6) it sterilizes, is cooling, inoculation, canned, fermentation: the mixed liquor after homogeneous being heated to 90 DEG C, heat preservation is after twenty minutes, cooling
To 42 DEG C, under conditions of sterile working, the first leavening 3.0% and the second leavening 5.0% is added by quality or volume ratio, is filled
Point stirring makes it after mixing, canned to Sour milk bottle or plastic cup with can packing machine, after sealing, is packed into frame of plastic, moves into fermentation
Room, 40 DEG C heat-preservation fermentation 5 hours；
(7) cooling, after-ripening: above-mentioned Yoghourt after fermentation is moved into cooling, after-ripening in 0-4 DEG C of freezer and obtains and is rich in
γ-aminobutyric acid mesona sour milk product.
Embodiment 4
With above-described embodiment 1, difference is:
(1) activation of bacterial strain: under aseptic technique, by streptococcus thermophilus (BNCC187932 or BNCC186629 or
BNCC175966 or BNCC175937) and deposit number be CGMCC No.15954 Lactobacillus brevis PDD-2 be inoculated in MRS respectively
In fluid nutrient medium, cultivated 36 hours in 37 DEG C of incubator respectively；
(2) preparation of lactic acid bacteria fermenting agent: by after activation streptococcus thermophilus and Lactobacillus brevis under aseptic technique, respectively
4% inoculum concentration is inoculated in the plain chocolate for being cooled to 37 DEG C after pasteurize by volume, is cultivated in 37 DEG C of incubators
The first leavening and the second leavening is accordingly made in 6h；
(3) preparation of salt algae enzymolysis liquid: Dunaliella salina cell algal gel is collected, after cleaning up, cell algal gel is suspended in aqueous solution
The algal gel cell suspending liquid that mass fraction is 10% is obtained, by the first high-pressure breaking under 1000bar pressure of algal gel cell suspending liquid
10min, the ultrasonic cell disruption instrument for being again then 550W with output power 10 min of smudge cells in ice bath adjust pH value extremely
5-6, cellulase that the vigor for being separately added into algae solution quality 0.05% is 4000U/g and 0.05% vigor be 50U/mg wind
Taste protease, heat preservation enzymatic hydrolysis adjusts pH to 7.0 at 45 DEG C after 8 hours, obtains chlorella mixed enzymolysis liquid；
(4) preparation of mesona solution: after mesona cleaning, crushing, the water that 2-5 times of mesona quality is added in mesona is mixed, then
Add the dietary alkali (Na of mesona and water gross mass 0.15%2CO3) be uniformly mixed, in 85 DEG C of Heat preservation 30min, then use colloid mill
Mesona solution is made with the screen to filtrate of 100 ~ 150 mesh in mill 2 ~ 3 times；
(5) ingredient: being mixed to get salt algae and mesona mixed liquor for salt algae enzymolysis liquid and mesona the solution ratio of 1:1 in mass ratio,
It adds red bean bud powder 1.0%, skimmed milk powder 10.0% and sucrose 4.0% in mass ratio in this mixed liquor again, is uniformly mixed again
Matter；
(6) it sterilizes, is cooling, inoculation, canned, fermentation: is cooling after the mixed liquor after homogeneous is heated to 85 DEG C, heat preservation 30 minutes
To 40 DEG C, under conditions of sterile working, the first leavening 2.0% and the second leavening 3.0% is added by quality or volume ratio, is filled
Point stirring makes it after mixing, canned to Sour milk bottle or plastic cup with can packing machine, after sealing, is packed into frame of plastic, moves into fermentation
Room, 37 DEG C heat-preservation fermentation 8 hours.
Embodiment 5
With above-described embodiment 1, difference is:
(1) activation of bacterial strain: under aseptic technique, by streptococcus thermophilus (BNCC187932 or BNCC186629 or
BNCC175966 or BNCC175937) and deposit number be CGMCC No.15954 Lactobacillus brevis PDD-2 be inoculated in MRS respectively
In fluid nutrient medium, cultivated 20 hours in 42 DEG C of incubator respectively；
(2) preparation of lactic acid bacteria fermenting agent: by after activation streptococcus thermophilus and Lactobacillus brevis under aseptic technique, respectively
6% inoculum concentration is inoculated in the plain chocolate for being cooled to 42 DEG C after pasteurize by volume, is cultivated in 42 DEG C of incubators
The first leavening and the second leavening is accordingly made in 3h；
(3) preparation of salt algae enzymolysis liquid: Dunaliella salina cell algal gel is collected, after cleaning up, cell algal gel is suspended in aqueous solution
The algal gel cell suspending liquid that mass fraction is 15% is obtained, by the first high-pressure breaking under 2000bar pressure of algal gel cell suspending liquid
5min, the ultrasonic cell disruption instrument for being again then 650W with output power 6 min of smudge cells in ice bath adjust pH value to 5-
6, cellulase that the vigor for being separately added into algae solution quality 0.10% is 2000U/g and 0.10% vigor be 30U/mg flavor
Protease, heat preservation enzymatic hydrolysis adjusts pH to 7.0 at 55 DEG C after 3 hours, obtains chlorella mixed enzymolysis liquid；
(4) preparation of mesona solution: after mesona cleaning, crushing, the water that 2-5 times of mesona quality is added in mesona is mixed, then
Add the dietary alkali (Na of mesona and water gross mass 0.35%2CO3) be uniformly mixed, in 95 DEG C of Heat preservation 10min, then use colloid mill
Mesona solution is made with the screen to filtrate of 100 ~ 150 mesh in mill 2 ~ 3 times；
(5) ingredient: being mixed to get salt algae and mesona mixed liquor for salt algae enzymolysis liquid and mesona the solution ratio of 3:1 in mass ratio,
It adds red bean bud powder 3.0%, skimmed milk powder 13.0% and sucrose 7.0% in mass ratio in this mixed liquor again, is uniformly mixed again
Matter；
(6) it sterilizes, is cooling, inoculation, canned, fermentation: is cooling after the mixed liquor after homogeneous is heated to 95 DEG C, heat preservation 15 minutes
To 43 DEG C, under conditions of sterile working, the first leavening 4.0% and the second leavening 6.0% is added by quality or volume ratio, is filled
Point stirring makes it after mixing, canned to Sour milk bottle or plastic cup with can packing machine, after sealing, is packed into frame of plastic, moves into fermentation
Room, 43 DEG C heat-preservation fermentation 3 hours.
Above description is not limitation of the present invention, and the present invention is also not limited to the example above.The art it is common
Within the essential scope of the present invention, the variations, modifications, additions or substitutions made also should belong to protection of the invention to technical staff
Range.
Claims (5)
1. the lactic acid bacteria of one plant of production γ-aminobutyric acid, it is characterised in that: the lactic acid bacteria classification naming is Lactobacillus brevis
(Lactobacillus brevis) PDD-2 plants, Chinese microorganism strain preservation management committee was preserved on 06 19th, 2018
Member's meeting common micro-organisms center, deposit number are CGMCC No.15954.
2. a kind of screening technique of the lactic acid bacteria described in claim 1 for producing γ-aminobutyric acid, it is characterised in that including following step
It is rapid:
(1) it the preliminary screening of lactic acid bacteria and isolates and purifies
Selecting fresh milk is after sample dilutes, to be coated on the MRS solid medium tablets containing bromocresol purple, is stood, in 37-
43 DEG C of culture 36-48h, pick out the bacterium colony in yellow, and it is preliminary that screening is obtained the single bacterium colony with typical lactic acid bacteria feature
It is set to lactic acid bacteria；The bacterium colony primarily determined is crossed several times repeatedly in MRS solid medium tablets, so that further progress is pure
The purpose of change, until there is the single consistent bacterium colony of form, preservation of bacteria strain；
(2) screening of aimed strain
The strain of step (1) preservation is sent out by the liquid that 1% inoculum concentration of percent by volume is added to the L-sodium containing 1wt%
Ferment culture medium, after 37 DEG C of culture 48h；Supernatant is taken after fermentation liquid is centrifuged, while compound concentration is the standard items L- of 10g/L
Glutamic acid and γ-aminobutyric acid will take 2ul standard items and sample point sample on chromatographic paper respectively, chromatographed in chromatography cylinder,
Chromatographic paper is taken out after chromatography and is dried 80 minutes in 70 DEG C, the cream of γ-aminobutyric acid Pidolidone will be converted to
Sour bacterium carries out Gram's staining and physio-biochemical characteristics identification, then carries out 16srDNA and API50-CHL sugar fermenting experiment, choosing
Take Gram's staining for the positive, catalase experiment is negative single bacterium colony, then by the bacterial strain of γ-aminobutyric acid high yield
16srDNA and API50-CHL sugar fermenting experiment is carried out, the lactic acid bacteria of one plant of highly producing gamma-aminobutyric acid is obtained, lactic acid bacteria classification
PDD-2 plants of Lactobacillus brevis are named as, it is common to be preserved in China Committee for Culture Collection of Microorganisms on 06 19th, 2018
Microorganism center, deposit number are CGMCC No.15954.
3. a kind of screening technique of lactic acid bacteria for producing γ-aminobutyric acid according to claim 2, it is characterised in that described
The preparation method of MRS solid medium containing bromocresol purple is as follows: by MRS meat soup 52.24g, bromocresol purple 0.04g, agar
Powder 10g ~ 20g is dissolved in 1L distilled water, in 121 DEG C of sterilizing 20min；The chromatographic solution is n-butanol, glacial acetic acid and water
After the mixing of 5:3:2 in mass ratio, ninhydrin, which is added, makes its mass percent 1.2%, balances 16- in 30 DEG C of water-baths in advance
18h is spare.
4. the lactic acid bacteria preparation using the production γ-aminobutyric acid of any of claims 1-3 is celestial rich in γ-aminobutyric acid
The preparation method of oxalic acid milk, it is characterised in that the following steps are included:
(1) activation of bacterial strain: being the short of CGMCC No.15954 by streptococcus thermophilus and deposit number under aseptic technique
Lactobacillus PDD-2 is inoculated in respectively in MRS fluid nutrient medium, is cultivated 20-36 hours in 37-42 DEG C of incubator respectively；
(2) preparation of lactic acid bacteria fermenting agent: by after activation streptococcus thermophilus and Lactobacillus brevis under aseptic technique, respectively
The inoculum concentration of 4-6% is inoculated in the plain chocolate for being cooled to 37-42 DEG C after pasteurize by volume, in 37-42 DEG C of incubator
Middle culture 3-6h, is accordingly made the first leavening and the second leavening；
(3) preparation of salt algae enzymolysis liquid: Dunaliella salina cell algal gel is collected, after cleaning up, cell algal gel is suspended in aqueous solution
The algal gel cell suspending liquid that mass fraction is 10-15% is obtained, algal gel cell suspending liquid is first surpassed under 1000-2000bar pressure
High pressure is crushed 5-10min, the ultrasonic cell disruption instrument for being again then 550-650W with output power smudge cells in ice bath
6-10 min adjusts pH value to 5-6, and the vigor for being separately added into algae solution quality 0.05-0.10% is the cellulose of 2000-4000U/g
The vigor of enzyme and 0.05-0.10% are the flavor protease of 30-50U/mg, and heat preservation enzymatic hydrolysis adjusts after 3-8 hours at 45-55 DEG C
PH to 7.0 obtains chlorella mixed enzymolysis liquid；
(4) preparation of mesona solution: after mesona cleaning, crushing, the water that 2-5 times of mesona quality is added in mesona is mixed, then
Addition mesona is uniformly mixed with the dietary alkali of water gross mass 0.15-0.35%, in 85-95 DEG C of Heat preservation 10-30min, then uses glue
Body mill mill 2 ~ 3 times, with the screen to filtrate of 100 ~ 150 mesh, mesona solution is made；
(5) ingredient: by salt algae enzymolysis liquid and mesona solution (1-3) in mass ratio: 1 ratio is mixed to get salt algae and mixes with mesona
Liquid, then add red bean bud powder 1.0-3.0%, skimmed milk powder 10.0-13.0% and sucrose 4.0- in mass ratio in this mixed liquor
7.0%, it is uniformly mixed homogeneous again；
(6) it sterilizes, is cooling, inoculation, canned, fermentation: the mixed liquor after homogeneous being heated to 85-95 DEG C, keeps the temperature 15-30 minutes
Afterwards, it is cooled to 40-43 DEG C, under conditions of sterile working, adds the first leavening 2.0-4.0% and the by quality or volume ratio
Two leavening 3.0-6.0%, being sufficiently stirred makes it after mixing, canned to Sour milk bottle or plastic cup with can packing machine, after sealing,
Be packed into frame of plastic, move into fermenting cellar, 37-43 DEG C heat-preservation fermentation 3-8 hours；
(7) cooling, after-ripening: above-mentioned Yoghourt after fermentation is moved into cooling, after-ripening in 0-4 DEG C of freezer and obtains and is rich in
γ-aminobutyric acid mesona sour milk product.
5. the preparation method according to claim 4 rich in γ-aminobutyric acid mesona Yoghourt, it is characterised in that step (5)
Described in red bean bud powder the preparation method is as follows: choose each 100, red bean of different cultivars, weigh, soaked with 75% ethyl alcohol respectively
It steeps 15 minutes and sterilizes, be then transferred in vessel and a certain proportion of water immersion 8h is added, weigh again after immersion, after immersion
Seed be placed in 30 DEG C of incubator and carry out being protected from light culture, every 30 minutes 5 ' phosphorus with 0.0015g/L during germination
The L-sodium of sour pyridoxal and 2g/L spray, obtaining the highest seed of germination percentage is the red bean bud rich in γ-aminobutyric acid,
Then -80 DEG C of refrigerator pre-cooling 2h are put in, 20-28h then is lyophilized in -80 DEG C of freeze driers, the red bean bud of freeze-drying is taken out
It crushes afterwards and obtains red bean bud powder, it is spare to cross 200 meshes.
Priority Applications (1)
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| CN111733078A (en) * | 2020-07-24 | 2020-10-02 | 塔里木大学 | Method for producing gamma-aminobutyric acid by fermenting indigenous strains in sailima raw material |
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| CN115530325A (en) * | 2022-10-20 | 2022-12-30 | 江南大学 | Chickpea powder rich in gamma-aminobutyric acid and application thereof |
| CN115530325B (en) * | 2022-10-20 | 2024-02-27 | 江南大学 | Chickpea flour rich in gamma-aminobutyric acid and application thereof |
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