CN109749914A - It is a kind of for improving the biological reinforcing method of Shanxi mature vinegar alpha-aminobutyric acid content - Google Patents
It is a kind of for improving the biological reinforcing method of Shanxi mature vinegar alpha-aminobutyric acid content Download PDFInfo
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Abstract
The invention belongs to microorganisms technical fields, provide a kind of for improving the biological reinforcing method of Shanxi mature vinegar alpha-aminobutyric acid content.Addition is rich in the germinating barley of γ-aminobutyric acid in traditional Shanxi mature vinegar brewing materials；The alcoholic fermentation stage adds the compounding throw type leaven of saccharomyces cerevisiae CGMCC 15729, lactobacillus plantarum CGMCC 15731 system of monascus, the excellent highly producing gamma-aminobutyric acid of Shanxi mature vinegar original inhabitants；The acetic fermentation stage adds the compounding throw type leaven of Pasteur's acetobacter CGMCC 15730, lactobacillus plantarum CGMCC 15731 system of the excellent highly producing gamma-aminobutyric acid of Shanxi mature vinegar original inhabitants.Newly drench total acid 5.85g/100mL in vinegar, γ-aminobutyric acid 0.23mg/mL, total ester 4.58g/100mL improve 55.58%, 283.33%, 54.73% compared with the control, enrich organic acid and volatile aroma type and content.
Description
Technical field
The invention belongs to microorganisms technical fields, and in particular to one kind is for improving Shanxi mature vinegar γ-aminobutyric acid
(GABA) biological reinforcing method of content.
Background technique
Shanxi mature vinegar is one of traditional four big vinegar in China, wherein containing a variety of organic acids such as lactic acid, malic acid and
The aroma-producing substances such as ester, alcohol, ketone, aldehyde, the synergistic effect of these flavor substances impart Shanxi mature vinegar " silk floss, acid, perfume, sweet tea, fresh "
The features such as.Ligustrazine is physiological function substance important in Shanxi mature vinegar, in addition to this, in newly discovered Shanxi mature vinegar
Contain micro γ-aminobutyric acid.
γ-aminobutyric acid (GABA) be a kind of nonprotein composition natural amino acid and higher organism brain and
Widely distributed most important nerve inhibits mediator in spinal nerve system.It is hypoglycemic with control hypertension, antianxiety,
With fixed attention, neuropathy is treated, endocrine is adjusted, sharp kidney is good for liver, Control of asthma, and a series of health cares and the physiology function such as delay to decline
Energy.The principal synthetic route of GABA is de- by glutamic acid decarboxylase enzymatic Pidolidone in animal, plant and microbial body
Carboxylic, as a kind of new function factor, GABA is just gradually paid attention to by people.Since GABA is very low in animals and plants content,
Want to obtain GABA resource abundant out of nature biotechnology body relatively difficult.Now, realize GABA's with microbe fermentation method
Production mainly passes through the strain fermentations such as monascus parpureus Went, saccharomycete.
Publication No. is that CN101899382 A discloses a kind of preparation method of grain vinegar with high content of gamma-amino butyric.Its with
The red yeast rice of the Monascus preparation of high yield is saccharifying ferment, has inquired into the brewing condition of grain vinegar with high content, has been prepared
GABA content is up to the grain vinegar of 100mg/L.It is a kind of rich in γ-aminobutyric acid that publication No. is that CN104774734 A is disclosed
The preparation method of monascus vinegar.After adding Pasteur's acetobacter, lactobacillus plantarum and monascus, γ-in the monascus vinegar that is prepared
Gamma-propalanine content, which is compared with the traditional method, improves 120 ~ 230%.
The present invention produces the strain of GABA using more plant heights, and addition is rich in the germinating barley of GABA in the feed, multistage
Duan Qianghua, the method for improving Shanxi mature vinegar GABA content, the separation screening mainly from Shanxi mature vinegar fermentation process
The bacterial strain of high yield GABA out is of great significance for the nutritive physiology function and abundant flavor that improve Shanxi mature vinegar.
Summary of the invention
The present invention provides a kind of for improving the biological reinforced side of Shanxi mature vinegar γ-aminobutyric acid (GABA) content
Method, technical scheme is as follows:
It is a kind of for improving the biological reinforcing method of Shanxi mature vinegar GABA content, the specific steps are as follows:
The screening of high yield GABA bacterial strain: monascus CGMCC 3.15546 and Shanxi mature vinegar alcoholic fermentation to laboratory purchase
42 saccharomycetes that the distiller's wort sample in stage and the vinegar fermented grain sample in acetic fermentation stage are isolated, 30 strains of lactic acid bacteria, 35 plants of acetic acid
Bacterium, 10 plants of moulds produce the measurement of GABA ability, finally screen the monascus CGMCC 3.15546, the ferment that obtain high yield GABA
Female bacterium 421, lactic acid bacteria 7 and acetic acid bacteria 2416, GABA yield be respectively 5.76mg/mL, 2.05mg/mL, 3.94mg/mL,
1.56mg/mL.Morphology and sequencing identification are carried out to saccharomycete 421, lactic acid bacteria 7 and acetic acid bacteria 2416, are identified as wine brewing ferment
Female (Saccharomyces cerevisiae), lactobacillus plantarum (Lactobacillus plantarum) and Pasteur's acetobacter
(Acetobacter pasteurianus).It is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms
The heart (CGMCC), address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, preservation date: on May 7th, 2018, deposit number
Respectively CGMCC 15729, CGMCC 15731, CGMCC 15730.
Monascus fermentation broth the preparation method comprises the following steps: be connected to PDA solid slope for 3.15546 points of monascus ruber CGMCC, 30
Activated slant strains are rinsed with sterile saline and obtain spore suspension by DEG C 35 d of culture, and filter paper is filtered to remove bacterium
Silk, is prepared into 106The Monascus spore suspension of cfu/mL；The red yeast rice of inoculation medium volume 10% in PDA fluid enlargement culture base
Mould spore suspension, adjusting pH is 5,30 DEG C, obtains Monascus fermentation broth after 180r/min culture 10d.
Compound throw type leaven the preparation method comprises the following steps:
(1) the saccharomyces cerevisiae CGMCC 15729 after activation is linked into PDA liquid medium according to 3% inoculum concentration, 30 DEG C quiet
Set culture culture for 24 hours；Lactobacillus plantarum CGMCC 15731, Pasteur's acetobacter CGMCC 15730 after activation is connect according to 3%
Kind amount is respectively connected to MRS fluid nutrient medium, and for 24 hours, Pasteur's acetobacter is at 30 DEG C for stationary culture at 37 DEG C for lactobacillus plantarum
180r/min cultivates 2d, and the cell concentration to culture solution reaches 108It after cfu/mL, is concentrated using hollow-fibre membrane, fermentation liquid
Sterile protective agent mixing is added after being concentrated into the 1/5 of original volume respectively, concentrated broth and protectant volume ratio are 1:3
V:v, with inlet air temperature for 120 DEG C, leaving air temp is 55 DEG C of low temperature spray dryings；
(2) mixing saccharomyces cerevisiae CGMCC 15729,15731 dry powder of lactobacillus plantarum CGMCC 1:1 in mass ratio is alcohol
The compounding throw type leaven of fermentation stage, by lactobacillus plantarum CGMCC 15731,15730 dry powder of Pasteur's acetobacter CGMCC
The mixing of 1:1 in mass ratio is the compounding throw type leaven in acetic fermentation stage, and viable count is 1011Cfu/g, cool place are dry
Storage period is 1 year under the conditions of dry.
The throw type leaven reinforcing that GABA strengthens is applied in Shanxi mature vinegar fermentation process method particularly includes:
Sorghum is crushed to four to six valves, and it is 50 55 DEG C that 60 70 kilograms of temperature, which are added, in 100 kilograms of sorghums and 5 kilograms of germinating barley
The sorghum moistened is steamed 2h, ceased fire when no sandwich tack-free by water material moistening 12h；The water that 300 kilograms of temperature are 80 DEG C is added, is stirred
It mixes uniformly leaching material, temperature and admixes 60 kilograms of yeast when being down to 25 33 DEG C, Monascus fermentation broth and alcoholic fermentation stage is added
It compounds throw type leaven (saccharomyces cerevisiae CGMCC 15729, lactobacillus plantarum CGMCC 15731), is transported to after mixing evenly
In ethanol fermentation tank, controlling temperature in fermentor is 25 33 DEG C, and alcoholic fermentation 8 10 days, first 2 days were open fermentation, Zhi Houwei
Sealing fermentation is added 150 kilograms of wheat brans and 100 kilograms of cavings, stirs evenly when alcoholic strength is 6%, and 10 kilograms of fiery unstrained spirits are added,
Add compounding throw type leaven (the lactobacillus plantarum CGMCC 15731, Pasteur's acetobacter CGMCC in acetic fermentation stage
15730), control fermentor in temperature be 24 47 DEG C progresss acetic fermentation 10 12 days, acidity be 4.66g/100g when, addition 10
Kilogram salt stops fermentation, obtains through smoked unstrained spirits, leaching vinegar link and newly drenches vinegar, measures the physical and chemical index of new leaching vinegar, organic acid and volatility
Fragrance component.
It is applied in Shanxi mature vinegar production using GABA forced fermentation agent reinforcing, total acid content is in obtained new leaching vinegar
5.85g/100mL, GABA content 0.23mg/mL, total ester content 4.58g/100mL are respectively increased compared with the control
55.58%, 283.33%, 54.73%, in addition, enriching the type and content of organic acid and volatile aroma.
The fire unstrained spirits is previous the 2nd day vinegar fermented grain of wholesale ferment.The sterility protection agent prescription are as follows: A: 10 g of skimmed milk power,
Distilled water 100 mL, 115 DEG C of 15 min of sterilizing；B: 1.5 g of trehalose, 0.5 g of glycerol, 2 g of sorbierite, 1 g of maltodextrin,
Distilled water 100 mL, 121 DEG C of 15 min of sterilizing；After A and B is sterilized separately, being cooled to room temperature mixing is protective agent.
Detailed description of the invention
The colonial morphology and cellular morphology of Fig. 1 high yield GABA bacterial strain；A is the bacterium colony of saccharomyces cerevisiae CGMCC 15729 in figure
Aspect graph；A be saccharomyces cerevisiae CGMCC 15729 1000 × under cellular morphology figure；B is lactobacillus plantarum CGMCC 15731
Colonial morphology figure；B be lactobacillus plantarum CGMCC 15731 1000 × under cellular morphology figure；C is Pasteur's acetobacter
The colonial morphology figure of CGMCC 15730；C be Pasteur's acetobacter CGMCC 15730 1000 × under cellular morphology figure.
Specific embodiment
Technical solution of the present invention is described further below by specific embodiment.
Embodiment 1: the screening of high yield GABA bacterial strain
To the monascus CGMCC 3.15546 and the distiller's wort sample and acetic acid in Shanxi mature vinegar alcoholic fermentation stage of laboratory purchase
10 plants of moulds that the vinegar fermented grain sample of fermentation stage is isolated, 42 saccharomycetes, 30 strains of lactic acid bacteria, 35 plants of acetic acid bacterias, use fermentation
Culture medium culture bacterial strain, to after fermentation, 8000 r of fermentation material/min is centrifuged 20 min, takes supernatant spare.Using thin
The content of layer chromatography (TLC) and reversed-phase high performance liquid chromatography (RP-HPLC)-OPA pre-column derivatization detection GABA, finishing screen
Choosing obtains monascus CGMCC 3.15546, saccharomycete 421, lactic acid bacteria 7 and the acetic acid bacteria 2416 of high yield GABA, GABA yield
Respectively 5.76mg/mL, 2.05mg/mL, 3.94mg/mL, 1.56mg/mL.
Above-mentioned fermentation medium the preparation method comprises the following steps:
(1) fermentation medium of mould: rice meal 2%, glucose 4%, monosodium glutamate (MSG) 1%, NaNO3 0.5%,
KH2PO4 0.15%, MgSO4·7H26.0,121 DEG C of sterilizing 20min of O 0.1%, distilled water 1000mL, pH are spare；
(2) fermentation medium of saccharomycete: glucose 30g, NaNO33g, yeast extract 1g, K2HPO41g, MgSO4·7H2O
0.5g, KCl 0.5g, FeSO4·7H2O 0.01g, Pidolidone 0.5g, 6.0,121 DEG C of distilled water 1000mL, pH sterilizings
20min is spare；
(3) fermentation medium of lactic acid bacteria: peptone 10g, beef extract 10g, yeast extract 5g, K2HPO42g, sodium acetate 2g, lemon
Lemon acid triamine 2g, glucose 20g, Tween 80 1mL, MgSO40.2g, MnSO40.05g, sodium glutamate 10g, distilled water
6.6,121 DEG C of sterilizing 20min of 1000mL, pH6.2 are spare；
(4) fermentation medium of acetic acid bacteria: glucose 20g, yeast extract 10g, Carbon Dioxide calcium 10g, sodium glutamate 10g, distillation
6.6,121 DEG C of sterilizing 20min of water 1000mL, pH6.2, the dehydrated alcohol for being cooled to 60 DEG C of additions 4% are spare.
Above-mentioned thin-layer chromatography (TLC) method method particularly includes: it is water-soluble to prepare 10 500 mg/L GABA series standards
Liquid, takes the GABA standard solution and fermentation liquid of 2 μ L various concentrations respectively, point sample in thin plate and be unfolded (solvent is 95% ethyl alcohol:
25% ammonium hydroxide=4:1), it opens up away from 10 cm.It being dried up with postposition thin plate in draught cupboard, 90 DEG C of baking ovens keep the temperature 30 min and wave most ammonia, with
0.2% ethanol solution of ninhydrin is spraying, 90 DEG C of 10 min of colour developing, comparative sample and standard specimen spot size and shade, analysis
The content of GABA in sample.
Above-mentioned reversed-phase high performance liquid chromatography (RP-HPLC)-OPA pre-column derivatization method particularly includes: 134 mg OPA
It is dissolved in 10mL dehydrated alcohol, sodium tetraborate (pH=9.6) solution of 200 μ L 2-MCE and 40mL 0.1mol/L is added, is made into
OPA derives liquid.Fermentation liquid is through 0.1mol/L K2CO3Buffer solution dilution, 10000 r/min are centrifuged 20 min, supernatant mistake
0.45 μm of filter membrane.The derivative liquid of 500 μ LOPA is added in 1mL fermentating liquid filtrate, mixes gently, is stored at room temperature 2 min, 20 μ L of sample introduction.
The GABA series standard liquid that 10 μ g/L, 10 mg/L is prepared with 0.1 mol containing 50% methanol/L potassium carbonate buffer, through same
Condition is derivative and sample introduction, draw standard curve.Chromatographic condition are as follows: chromatographic column be Kromasil C18(5 μm, 250. 0 × 4.6
Mm)；Mobile phase is kaliumphosphate buffer (0.1 mol/L, pH 6.0): methanol: acetonitrile volume ratio is 6:3:1；Detector is glimmering
Photodetector；Detection wavelength Ex=340 nm, Em=455 nm；Flow velocity 0.6mL/min.
Embodiment 2: the identification of high yield GABA saccharomycete 421, lactic acid bacteria 7 and acetic acid bacteria 2416
Morphological Identification: saccharomycete 421 forms bacterium colony in regular circle shapes on PDA plate, and white is opaque, smooth, there is light
Pool, surface elevation, colony diameter 1.5mm, cellular morphology are in ellipticity；Bacterium colony of the lactic acid bacteria 7 on MRS culture medium is in white
Color, opaque, bacterium colony protrusion moisten, flush edge, and colony diameter is respectively 0.6 0.8mm, it is thin under the microscope for optical microphotograph
Born of the same parents' form, Gram's staining are in G+, thallus is in small rod-short；Acetic acid bacteria 2416 forms bacterium colony on acetic acid bacteria plating medium
Diameter 0.8mm, regular circle shapes, milky is translucent, smooth, glossy, the very flat bacterium colony in surface；Its cellular morphology: gram
Dyeing is in G-, it is in small rod-short, sees Fig. 1.
Be identified as saccharomycete 421 be saccharomyces cerevisiae (Saccharomyces cerevisiae), deposit number CGMCC
15729, lactic acid bacteria 7 be lactobacillus plantarum (Lactobacillus plantarum), deposit number is CGMCC 15731, acetic acid
Bacterium 2416 be Pasteur's acetobacter (Acetobacter pasteurianus), deposit number is CGMCC 15730.It is deposited in China
Microbiological Culture Collection administration committee common micro-organisms center (CGMCC), address: BeiChen West Road, Chaoyang District, BeiJing City 1
Institute 3, preservation date: on May 7th, 2018.
Above-mentioned PDA solid medium the preparation method comprises the following steps: take potato 20g, glucose 2g, peptone 0.2g, KH2PO4
0.2g, MgSO4·2H2O 0.1g, agar 2g add distilled water 100mL, 121 DEG C of sterilizing 20min.
Above-mentioned MRS culture medium the preparation method comprises the following steps: glucose 20g, peptone 10g, beef extract 10g, yeast extract
5g, dipotassium hydrogen phosphate 2g, Triammonium citrate 2g, sodium acetate 2g, Tween 80 1g, anhydrous magnesium sulfate 0.2g, manganese sulfate
6.2 6.4,121 DEG C of 0.05g, distilled water 1L, pH, 20min sterilizing.
Above-mentioned acetic acid bacteria solid medium the preparation method comprises the following steps: glucose 20g, yeast extract 10g, Carbon Dioxide calcium 10g,
Agar 20g, distilled water 1000mL adjust 6.6,121 DEG C of sterilizing 20min of pH6.2, and the dehydrated alcohol for being cooled to 60 DEG C of additions 4% is standby
With.
Embodiment 3: the preparation of Monascus fermentation broth
PDA solid slope, 30 DEG C of 3 ~ 5 d of culture, by activated inclined-plane bacterium are connected to by 3.15546 points of monascus ruber CGMCC
Kind, lower spore is rinsed with sterile saline, is transferred in the sterile triangular flask with bead, vibrates, sufficiently break up spore, use
Filter paper is filtered to remove mycelia, is prepared into 106The Monascus spore suspension of cfu/mL；The PDA liquid of 85mL liquid amount is taken to expand training
Base is supported, 10% Monascus spore suspension is inoculated with, adjusting pH is 5,30 DEG C, obtains Fermentation Condition of Monascus spp bacterium after 180r/min culture 10d
Kind.
Above-mentioned expansion culture medium the preparation method comprises the following steps: 10% glucose, 1% peanut powder, 1% KH2P04, 0.5%
MgS04, 87.5% PDA liquid medium.
Embodiment 4: the preparation in alcoholic fermentation stage and acetic fermentation stage compounding throw type leaven
(1) preparation of alcoholic fermentation stage compounding throw type leaven: by the saccharomyces cerevisiae CGMCC 15729 after activation according to 3%
Inoculum concentration be linked into PDA liquid medium, 30 DEG C of stationary culture cultures are for 24 hours；By the lactobacillus plantarum CGMCC after activation
15731 are respectively connected to MRS fluid nutrient medium according to 3% inoculum concentration, 37 DEG C of stationary cultures for 24 hours, the cell concentration to culture solution
Reach 108After cfu/mL, be concentrated using hollow-fibre membrane, fermentation liquid be concentrated into respectively original volume 1/5 after add it is sterile
Protective agent mixing, concentrated broth and protectant volume ratio are 1:3(v:v), fermentation liquid is carried out by low temperature spray drying
After processing, it is alcohol that saccharomyces cerevisiae CGMCC 15729,15731 dry powder of lactobacillus plantarum CGMCC 1:1 in mass ratio, which are mixed,
Fermentation stage compounds throw type leaven.
(2) preparation of acetic fermentation stage compounding throw type leaven: by the lactobacillus plantarum CGMCC after activation
15731, Pasteur's acetobacter CGMCC 15730 is respectively connected to MRS fluid nutrient medium according to 3% inoculum concentration, and lactobacillus plantarum exists
For 24 hours, Pasteur's acetobacter 180r/min at 30 DEG C cultivates 2d to stationary culture, and the cell concentration to culture solution reaches at 37 DEG C
108After cfu/mL, be concentrated using hollow-fibre membrane, fermentation liquid be concentrated into respectively original volume 1/5 after add sterile protective agent
Mixing, concentrated broth and protectant volume ratio are 1:3(v:v), after being handled by low temperature spray drying fermentation liquid,
It is acetic fermentation that lactobacillus plantarum CGMCC 15731, Pasteur's acetobacter CGMCC 15730 dry powder 1:1 in mass ratio, which are mixed,
Stage compounds throw type leaven.
Above-mentioned protective agent is the preparation method comprises the following steps: A: 10 g of skimmed milk power, distilled water 100 mL, 115 DEG C of 15 min of sterilizing；
B: 1.5 g of trehalose, 0.5 g of glycerol, 2 g of sorbierite, 1 g of maltodextrin, distilled water 100 mL, 121 DEG C of 15 min of sterilizing；
After A, B are sterilized separately, it is cooled to room temperature mixing, as protective agent.
Above-mentioned low temperature spray drying technological parameter are as follows: leaving air temp is 55 DEG C, and inlet air temperature is 120 DEG C, drying time
6min, moisture content are≤5%.The compounding throw type leaven of preparation, viable count are 1010Cfu/g, under shady and cool drying condition
Storage period is 1 year.
Embodiment 5: the throw type leaven reinforcing that GABA strengthens is applied in Shanxi mature vinegar fermentation process
Sorghum is crushed to four to six valves, and it is 50 55 DEG C of water material moistening 12h that 60 70 kilograms of temperature, which are added, in 100 kilograms of sorghums, will moisten
Sorghum steam 2h, cease fire when no sandwich tack-free, adding 300 kilograms of temperature is 80 DEG C of water, stirs evenly leaching material, temperature drop
The yeast that 60 kilograms are admixed when to 25 33 DEG C, is transported in ethanol fermentation tank after mixing evenly, controls temperature in fermentor and is
25 33 DEG C, alcoholic fermentation 8 10 days, first 2 days were open fermentation, are later sealing fermentation, when alcoholic strength is 6%, it is public to be added 150
Jin wheat bran and 100 kilograms of cavings, stir evenly, and 10 kilograms of fiery unstrained spirits (vinegar fermented grain that last consignment of ferments the 2nd day), control fermentation is added
In tank temperature be 24 47 DEG C progresss acetic fermentation 10 12 days, when acidity is 4.66g/100g, be added 10 kilograms of salt, stop sending out
Ferment obtains through smoked unstrained spirits, leaching vinegar link and newly drenches vinegar, measure new leaching vinegar physical and chemical index (pH, total acid, reduced sugar, total ester, fixed acid,
Ammonia nitrogen, Baume degrees, ligustrazine) (the results are shown in Table 1), organic acid (the results are shown in Table 2) and aroma volatile (the results are shown in Table
3).
Embodiment 6: being reference examples with embodiment 5, is added to 5 public affairs in raw material (100 kilograms of sorghums) according to the method described above
The germinating barley of jin.
Above-mentioned germinating barley the preparation method comprises the following steps: screen, removing wheat skin, stone, mildew and prematurity particle, then first
By the dry barley of 500g in large beaker 7 DEG C of 6 h of immersion, after paddy after immersion is laid in be covered with 8 layers of gauze pallet (1200 ×
In 1000mm), 800mL distilled water is added and (contains 1g CaCl2, 6g sodium glutamate), 30 DEG C of heat and moisture preserving culture 3d, 40 DEG C of bakings
It does to constant weight.
Embodiment 7: being reference examples with embodiment 5, is added to 5 kilograms of hair in 100 kilograms of sorghums according to the method described above
Malting barley；While the alcoholic fermentation stage 60 kilograms of yeast being added, Monas cuspurpureus Went fermentation liquid and the sorghum of sorghum weight 2% are added
The 0.8% alcoholic fermentation stage of weight compounds throw type leaven (saccharomyces cerevisiae CGMCC 15729, lactobacillus plantarum CGMCC
15731) it, is transported in ethanol fermentation tank after mixing evenly, alcoholic fermentation 8 10 days.
The preparation method of above-mentioned alcoholic fermentation stage compounding throw type leaven: 1g compounding throw type leaven is dissolved into
It is spare after 30 DEG C of activation 30min in 10mL sterile water.
Embodiment 8: being reference examples with embodiment 5, is added to 5 kilograms of hair in 100 kilograms of sorghums according to the method described above
Malting barley；While the alcoholic fermentation stage 60 kilograms of yeast being added, Monas cuspurpureus Went fermentation liquid and the sorghum of sorghum weight 2% are added
The 0.8% alcoholic fermentation stage of weight compounds throw type leaven (saccharomyces cerevisiae CGMCC 15729, lactobacillus plantarum CGMCC
15731) it, is transported in ethanol fermentation tank after mixing evenly, alcoholic fermentation 8 10 days；10 kilograms of fire are added in the acetic fermentation stage
While unstrained spirits, 0.8% acetic fermentation stage of sorghum weight compounding throw type leaven (lactobacillus plantarum CGMCC is added
15731, Pasteur's acetobacter CGMCC 15730), it carries out acetic fermentation 10 12 days.
The throw type leaven reinforcing strengthened using GABA is applied in Shanxi mature vinegar production, and the total of new leaching vinegar is improved
Acid, GABA, total ester and organic acid content enrich the volatile aroma spectrogram of new leaching vinegar.Embodiment 8 is significantly better than embodiment
4(reference examples), 8 total acid content of embodiment is 5.85g/100mL, GABA content 0.23mg/mL, total ester content 4.58g/
55.58%, 283.33%, 54.73%(table 1 has been respectively increased in 100mL compared with the control)；Organic acid total content is by 29.8663g/L
44.8895g/L is risen to, wherein lactic acid, acetic acid content significantly improve, and 77.74%, 43.01%(table has been respectively increased than control group
2)；Aroma volatile detects 40 kinds of (18 kinds of esters, 4 kinds of alcohols, 3 kinds of acids, 5 kinds of ketone, 7 kinds of aldehydes, phenols 1 altogether
Kind, other 2 kinds), esters total content is 4.26g/100mL, alcohols total content is 0.99g/100mL, acids total content is
3.86g/100mL, aldehydes total content be 16.93g/100mL；Wherein have fruity ethyl acetate (1.08g/100mL),
Ethyl caprilate (0.38g/100mL) with brandy fragrance, in miel ethyl phenylacetate (0.32g/100mL), be in coconut palm
Sub fragrant ethyl caprate (0.08g/100mL), the benzyl carbinol (0.51g/100mL) with soft sweet rose fragrant are to mature vinegar flavor
Provide certain contribution (table 3).
Table 1: using the measurement result of new leaching vinegar physical and chemical index obtained by compounding throw type leaven production Shanxi mature vinegar
Table 2: using the measurement result of organic acid in new leaching vinegar obtained by compounding throw type leaven production Shanxi mature vinegar
Table 3: using the measurement knot of aroma volatile in new leaching vinegar obtained by compounding throw type leaven production Shanxi mature vinegar
Fruit
The pH measurement of above-mentioned Shanxi mature vinegar is directly measured with pH meter；The reduced sugar of Shanxi mature vinegar, total ester are referring to GBT
Method specified in 19777-2013 " geography symbol product Shanxi mature vinegar " is measured；The fixed acid of Shanxi mature vinegar is joined
It is measured according to the formula way of distillation is singly boiled in GB 18187-2000 " making vinegar "；The ammonia nitrogen of Shanxi mature vinegar is referring to GBT
5009.39.2003 method specified in " vinegar sanitary standard analytic approach " is measured；The Baume degrees of Shanxi mature vinegar, which measures, to be used
Baume hydrometer directly measures.
The specific measuring method of above-mentioned HPLC method measurement organic acid content are as follows: sample 5g is taken, is settled to 50mL with ultrapure water,
12000r/min centrifugation 5min takes supernatant, with 0.22 μm of filtering with microporous membrane, loading.Chromatographic determination condition are as follows: liquid phase systems
UItimate 3000；Chromatographic column C18 4.6 × 150mm, 5 μm；Mobile phase 20mmol/L NaH2PO4, pH=2.7；20 μ of sample volume
L；Flowing velocity 0.8mL/min；Detector UV210nm；Column temperature: room temperature.
The specific measuring method of above-mentioned GC-MS method measurement aroma volatile are as follows: using headspace solid-phase microextraction to sample
Product are extracted, and using the type and content of direct its volatile aroma of internal mark method determination.Chromatographic condition: chromatographic column VF-
5MS(30 × 0.25mm × 0.25mm), carrier gas: helium, purity 99.999%, flow 1mL/min are not shunted.Temperature programming: it rises
40 DEG C of beginning temperature, 3min is kept, 160 DEG C is risen to the speed of 4 DEG C/min, keeps 1min.It is risen to again with the speed of 10 DEG C/min
270 DEG C of holding 5min.Mass Spectrometry Conditions: 280 DEG C of interface temperature, 280 DEG C of ion source temperature, electron energy 70eV, scanning quality model
Enclose 41 500amu.
Claims (7)
1. a kind of for improving the biological reinforcing method of Shanxi mature vinegar alpha-aminobutyric acid content, it is characterised in that: sorghum and hair
Malting barley is the ratio mixing of 20:1 as Shanxi mature vinegar fermentation raw material by weight, using sorghum weight as calculating benchmark,
Alcoholic fermentation stage, saccharomyces cerevisiae, the plant cream bar of addition monascus, the excellent highly producing gamma-aminobutyric acid of Shanxi mature vinegar original inhabitants
The compounding throw type leaven of bacterium preparation；In the acetic fermentation stage, the excellent highly producing gamma-aminobutyric acid of Shanxi mature vinegar original inhabitants is added
Pasteur's acetobacter, lactobacillus plantarum preparation compounding throw type leaven；Wherein: the monascus strain purchase is micro- in China
Biological inoculum preservation administration committee common micro-organisms center, deposit number CGMCC3.15546；Saccharomyces cerevisiae
(Saccharomyces cerevisiae), lactobacillus plantarum (Lactobacillus plantarum), Pasteur's acetobacter
(Acetobacter pasteurianus), depositary institution: in China Committee for Culture Collection of Microorganisms's common micro-organisms
The heart (CGMCC), address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, preservation date are as follows: on May 7th, 2018, preservation are compiled
Number it is respectively as follows: CGMCC 15729, CGMCC 15731, CGMCC 15730.
2. it is according to claim 1 a kind of for improving the biological reinforcing method of Shanxi mature vinegar alpha-aminobutyric acid content,
It is characterized by: the Monascus fermentation broth the preparation method comprises the following steps: being connected to PDA solid for 3.15546 points of monascus ruber CGMCC
Activated slant strains are rinsed with sterile saline and obtain spore suspension by inclined-plane, 30 DEG C of 35 d of culture, filter paper filtering
Mycelia is removed, is prepared into 106The Monascus spore suspension of cfu/mL；Inoculation medium volume 10% in PDA fluid enlargement culture base
Monascus spore suspension, adjust pH be 5,30 DEG C, 180r/min culture 10d after obtain Monascus fermentation broth.
3. it is according to claim 1 a kind of for improving the biological reinforcing method of Shanxi mature vinegar alpha-aminobutyric acid content,
It is characterized by: it is described compounding throw type leaven the preparation method comprises the following steps:
(1) the saccharomyces cerevisiae CGMCC 15729 after activation is linked into PDA liquid medium according to 3% inoculum concentration, 30 DEG C quiet
Set culture culture for 24 hours；Lactobacillus plantarum CGMCC 15731, Pasteur's acetobacter CGMCC 15730 after activation is connect according to 3%
Kind amount is respectively connected to MRS fluid nutrient medium, and for 24 hours, Pasteur's acetobacter is at 30 DEG C for stationary culture at 37 DEG C for lactobacillus plantarum
180r/min cultivates 2d, and the cell concentration to culture solution reaches 108It after cfu/mL, is concentrated using hollow-fibre membrane, fermentation liquid
Sterile protective agent mixing is added after being concentrated into the 1/5 of original volume respectively, concentrated broth and protectant volume ratio are 1:3
V:v, with inlet air temperature for 120 DEG C, leaving air temp is 55 DEG C of low temperature spray dryings；
(2) mixing saccharomyces cerevisiae CGMCC 15729,15731 dry powder of lactobacillus plantarum CGMCC 1:1 in mass ratio is alcohol
The compounding throw type leaven of fermentation stage, by lactobacillus plantarum CGMCC 15731,15730 dry powder of Pasteur's acetobacter CGMCC
The mixing of 1:1 in mass ratio is the compounding throw type leaven in acetic fermentation stage, and viable count is 1011Cfu/g, cool place are dry
Storage period is 1 year under the conditions of dry.
4. according to claim 1 a kind of for improving the biological reinforcing method of Shanxi mature vinegar alpha-aminobutyric acid content
The method for making mature vinegar, it is characterised in that: the specific method for strengthening brewing mature vinegar are as follows:
Sorghum is crushed to four to six valves, and 60 70 kilograms of 50 55 DEG C of water material moistenings are added in 100 kilograms of sorghums and 5 kilograms of germinating barley
The sorghum moistened is steamed 2h, ceased fire when no sandwich tack-free by 12h；The water for adding 300 kilograms 80 DEG C stirs evenly leaching material, temperature
Degree admixes 60 kilograms of yeast when being down to 25 33 DEG C, Monascus fermentation broth is added and the compounding in alcoholic fermentation stage is directly putting type fermented
Agent is transported in ethanol fermentation tank after mixing evenly, control fermentor in temperature be 25 33 DEG C, alcoholic fermentation 8 10 days, preceding 2
It is open fermentation, and 150 kilograms of wheat brans and 100 kilograms of cavings is added when alcoholic strength is 6% for sealing fermentation later, stirs
Uniformly, 10 kilograms of fiery unstrained spirits are added, add the compounding throw type leaven in acetic fermentation stage, controlling temperature in fermentor is
24 47 DEG C progress acetic fermentation 10 12 days, when acidity is 4.66g/100g, be added 10 kilograms of salt, stop fermentation, through smoked unstrained spirits,
Leaching vinegar link, which obtains, newly drenches vinegar.
5. according to claim 4 a kind of for improving the biological reinforcing method of Shanxi mature vinegar alpha-aminobutyric acid content
The method for making mature vinegar, it is characterised in that: total acid content is 5.85g/100mL in prepared new leaching vinegar, and GABA content is
0.23mg/mL, total ester content 4.58g/100mL have been respectively increased 55.58%, 283.33%, 54.73% compared with the control.
6. according to claim 4 a kind of for improving the biological reinforcing method of Shanxi mature vinegar alpha-aminobutyric acid content
The method for making mature vinegar, it is characterised in that: the fire unstrained spirits is previous the 2nd day vinegar fermented grain of wholesale ferment.
7. according to claim 3 a kind of for improving the biological reinforcing method of Shanxi mature vinegar alpha-aminobutyric acid content
The method for making mature vinegar, it is characterised in that: the sterility protection agent prescription are as follows: A: 10 g of skimmed milk power, 100 mL of distilled water,
115 DEG C of 15 min of sterilizing；B: 1.5 g of trehalose, 0.5 g of glycerol, 2 g of sorbierite, 1 g of maltodextrin, 100 mL of distilled water,
121 DEG C of 15 min of sterilizing；After A and B is sterilized separately, being cooled to room temperature mixing is protective agent.
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| CN112899117A (en) * | 2021-03-01 | 2021-06-04 | 山西农业大学 | Method for co-fermenting high gamma-aminobutyric acid red date and pearl barley vinegar by combining monascus with lactobacillus and spore bacteria |
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