CN107189958B - The streptomycete that one plant of marine source produces protease is used for the biological reinforced method of vinegar - Google Patents
The streptomycete that one plant of marine source produces protease is used for the biological reinforced method of vinegar Download PDFInfo
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Abstract
The invention discloses the streptomycetes that one plant of marine source produces protease to be used for the biological reinforced method of vinegar, belongs to food brewing technical field.The present invention has screened a streptomycete (Streptomyces sp.) CCTCC NO:M 2017210 from marine environment, which high temperature resistant (68 DEG C -70 DEG C) and can have high proteinase yield ability.The bacterial strain is fabricated to bacterium powder and is used to strengthen vinegar acetic fermentation, amino acid content in vinegar can be improved in this method, and can improve the product special flavour of vinegar and promote vinegar quality.
Description
Technical field
The present invention relates to the streptomycetes that one plant of marine source produces protease to be used for the biological reinforced method of vinegar, belongs to food
Brewing technology field.
Background technique
Traditional vinegar brewing with starch-containing more glutinous rice, wheat bran, etc. for raw material, wherein also there is protein component.These eggs
White matter is after cooking, the albumen enzymatic as secreted by aspergillus, in saccharification, alcoholic fermentation and in acetic fermentation each stage, gradually
Resolve into various amino acid and its mesostate.But producing protease merely with Institute of Micro-biology in Koji can't be fully hydrolyzed
Protein in raw material, in addition, there are also to be strengthened for vinegar flavor substance.Currently, it is smoked to mostly use high temperature to pour leaching in the industrial production
Unstrained spirits adds the methods of complex enzyme formulation, seasoning and flavour enhancing to improve amino acid content and its flavor substance in vinegar, although flavor
It makes moderate progress, but production cost significantly improves, the extensive use being unfavorable in production.So far, in studying both at home and abroad not yet
About the report that streptomycete is applied to amino acid and flavour enhanced aspect in vinegar brewing.
Therefore, using modern biotechnology, the microorganism of excellent performance is screened for producing high quality, high yield, wind
The unique high-quality vinegar of taste is of great significance.
Summary of the invention
It is an object of the invention to solve the deficiency of existing vinegar solid brewing technology, a kind of bacterium powder of function admirable is provided
(Streptomyces sp.CCTCC NO:M 2017210) is biological reinforced for carrying out in the fermentation process of solid brewing vinegar,
To improve the product special flavour of vinegar, vinegar quality is promoted, improves raw material availability, marine-derived microorganism is preferably played and is eating
Application in vinegar brewing.
The first purpose of the invention is to provide a streptomycete, the streptomycete is in preservation on April 26 in 2017
In China typical culture collection center, preservation address is Wuhan, China Wuhan University, and deposit number is CCTCC NO:M
2017210。
Streptomycete of the invention has following excellent performance:
(1) it is applied to Vinegar Fermentation system, does not interfere with the normal fermentation of vinegar；
(2) for strengthening Vinegar Fermentation, raw material availability not only can be improved but also vinegar flavor and quality can be promoted；
The content of free amino acid in vinegar fermented grain can reach the dry unstrained spirits of 1413.83mg/100g, carry out reinforcing control compared to streptomycete is not added
Group improves 14%, and 27% and 21% has been respectively increased than control group in the content of volatility esters and carboxylic-acid substance.
(3) it is to be separated from marine sediment, adapts to 68 DEG C -70 DEG C of hot environment growth, and white with laying eggs
The ability of enzyme, enzyme activity are up to 2036U/mL fermentation liquid.
Contain the micro- of 2017210 bacterial strain of streptomycete CCTCC NO:M a second object of the present invention is to provide described
Bacteria agent.
In one embodiment, the microbial bacterial agent contain 2017210 thallus of streptomycete CCTCC NO:M work it is thin
Born of the same parents, the streptomycete CCTCC NO for being freeze-dried obtained 2017210 dry mycelium of streptomycete CCTCC NO:M, immobilization:
M2017210 cell, the liquid bacterial agent of streptomycete CCTCC NO:M 2017210, streptomycete CCTCC NO:M 2017210 consolidate
Body microbial inoculum, or in the form of other are any existing for 2017210 bacterial strain of streptomycete CCTCC NO:M.
In one embodiment, the microbial bacterial agent is the freeze-dried powder of streptomycete CCTCC NO:M 2017210.
In one embodiment, the preparation of the freeze-dried powder: multistage activation culture is first carried out using LB culture medium and is obtained
Seed liquor is then centrifuged for, washs and repeatedly obtain bacterium mud；Then it uses and is dissolved containing the freeze drying protectant of skimmed milk powder and trehalose
Bacterium mud, pre-freeze, then using being lyophilized in freeze dryer, freeze temperature is -72 DEG C, and pressure position 0.1MPa takes out after freeze-drying.
In one embodiment, the protective agent usage amount is the 10% of seed liquor.
Third object of the present invention is to provide 2017210 bacterial strain of streptomycete CCTCC NO:M or microbial bacterias
The application of agent.
In one embodiment, the application refers to for brewing technology field.
In one embodiment, the brewing is vinegar brewing.
In one embodiment, the application is for promoting vinegar flavor or raw material availability.
Fourth object of the present invention is to provide a kind of method that vinegar is biological reinforced, and the biological reinforcing method is to use
Streptomycete CCTCC NO:M 2017210 ferments after directly mixing with wine vinegar raw material.
In one embodiment, the biological reinforcing method is directly mixed with wine vinegar raw material using the bacterium powder of streptomycete
After ferment.
In one embodiment, the biological reinforcing method is to originate in fermentation by streptomycete CCTCC NO:M
2017210 bacterium powder ferment after mixing in fermentation vat with raw materials such as distiller's wort, wheat brans according to the inoculum concentration of 0.8-1.2%, send out
The technique that ferment expands culture using traditional layer-by-layer unstrained spirits.
Beneficial effects of the present invention:
(1) present invention screens the high temperature bacterium of high proteinase yield from marine environment, is then based on microorganism enhanced technology, right
High proteinase yield bacterium powder is added during vinegar acetic fermentation, is improved the percent hydrolysis of protein in raw material and is further increased vinegar
Amino acid content in liquid, while nutritive value, delicate flavour, color and the mouthfeel of vinegar are improved, reach amino in raising vinegar
Acid and volatile matter content and promotion vinegar quality purpose.
(2) after being strengthened using streptomycete, the content of the free amino acid in vinegar fermented grain can reach the dry unstrained spirits of 1413.83mg/100g,
Improve 14% compared to the control group, the content of volatility esters and carboxylic-acid substance have significantly, are respectively increased than control group
27% and 21%.
Biomaterial preservation
One streptomycete (Streptomyces sp.), taxology is named as streptomycete PRO1Streptomyces
Sp.PRO1, in being preserved in China typical culture collection center on April 26th, 2017, preservation address is the streptomycete
Wuhan, China Wuhan University, deposit number are CCTCC NO:M 2017210.
Detailed description of the invention
Total amino acid content situation of change in Fig. 1: addition Streptomyces sp. experimental group and control group；
Volatile materials total content situation of change in Fig. 2: addition Streptomyces sp. experimental group and control group.
Specific embodiment
The detection of vinegar physical and chemical index: total acid is detected according to GB/T 5009.41 (2003) NaOH neutralisation；Reduction
Sugar determination is measured according in GB/T 5009.7 (2008) using direct titrimetric method；Amino acid is detected using HPLC；Wind
Taste substance classes are detected using gas chromatography mass spectrometry instrument.
Embodiment 1: the screening of high proteinase yield bacterial strain in marine sediment
(1) marine sediment samples pre-process
Deposit uses direct method of dilution butteron on plate, and scene is coated on casein seawater plate, 25 DEG C of culture 20d.
Seawater casein culture medium: NaH2PO4·7H2O 1.07g, KH2PO40.36g, casein 4g, agar 15g, Chen Haishui
1000mL。
(2) plate transparent circle primary dcreening operation
Single colonie dibbling on casein seawater plate is selected to on screening and culturing medium, is measured after 25 DEG C of culture 48h transparent
Loop diameter (Rt) and colony diameter (Rs) calculate circle diameter ratio K, K=Rt/Rs.The bigger bacterium colony of selection circle diameter cross pure
Change 2-3 times, microscopy is seeded to the preservation of 4 DEG C of inclined-plane, -20 DEG C of preservations in bacteria suspension and 10% glycerol after determining pure culture.
Screening and culturing medium: peptone 5g, yeast extract 1g, ferric phosphate 0.1g, skimmed milk power 20g, Chen Haishui 1000mL, agar
Powder 15g, pH 8.0.
(3) shaking flask secondary screening
Inclined plane inoculating seed culture medium, 25-28 DEG C, 200rpm is cultivated for 24 hours, and 2% inoculum concentration is forwarded to basal fermentation culture
25 DEG C of base, 200rpm cultivates 48h, takes fermented supernatant fluid, measures prolease activity in fermentation liquid.
(4) proteinase activity measures
Reference protein enzyme preparation national standard GB/T23527-2009.Protease hydrolyzes junket egg under the conditions of certain temperature and pH
White background object generates the amino acid (such as: tyrosine, tryptophan) containing phenolic group, restores Folin reagent in alkaline condition, generates
Molybdenum blue and tungsten blue, are measured with spectrophotometry, calculate its enzyme activity.
Proteinase activity definition: enzyme amount needed for 1 μ g tyrosine of caseinhydrolysate generation per minute is defined as one at 30 DEG C
A protease activity unit of force.
According to transparent circle size in primary dcreening operation culture medium, and combine protease in the fermentation liquid after the 48h that ferments in shaking flask secondary screening
Screening active ingredients go out the bacterial strain of 1 plant of high proteinase yield, which is up to 2036U/mL fermentation liquid, and the identified bacterial strain tethers is mould
Pseudomonas (Streptomyces sp.).The bacterium is deposited in China typical culture collection center, deposit number is CCTCC NO:
M2017210。
Embodiment 2: the preparation of 2017210 freeze-dried powder of high proteinase yield bacterial strain Streptomyces sp.CCTCC NO:M
(1) activation culture of bacteria produced proteinase strain Streptomyces sp.CCTCC NO:M 2017210
The bacterium powder of bacterial strain Streptomyces sp.CCTCC NO:M 2017210 is accessed into 150mL liquid activation medium
In, 37 DEG C of shaking table culture 48-50h obtain primary seed solution；First order seed is accessed into 400mL liquid according to 6% (v/v) inoculum concentration
In body culture medium, 37 DEG C of shaking table culture 48-50h obtain secondary seed solution；Secondary seed solution is accessed according to 8% (v/v) inoculum concentration
In 6000mL fluid nutrient medium, 37 DEG C of shaking table culture 50-55h obtain three-level seed liquor.
Seawater LB culture medium: peptone 10g, yeast powder 5g, Chen Haishui 1000mL.
(2) preparation of 2017210 freeze-dried powder of Streptomyces sp.CCTCC NO:M
Three grade fermemtation liquid is poured into 500mL Centrifuge Cup (using after being dried with 75% alcohol wipe), 4500r/min centrifugation
15min discards supernatant liquid, washes twice bacterium mud with sterile saline, with same pelleted by centrifugation, discards supernatant liquid, precipitating
The as bacterium mud of bacterial strain Streptomyces sp.CCTCC NO:M 2017210.It weighs 125g skimmed milk powder and is dissolved in 900mL water
In, 105 DEG C of sterilizing 10min；10g trehalose is dissolved in 100mL water, and 115 DEG C of sterilizing 20min are mixed the two using preceding.With jelly
Dry protective agent dissolves bacterium mud, and protective agent usage amount is the 10% of three-level seed liquor, fullys shake, is subsequently poured into sterile petri dish
In, liquid height is 1cm or so, and pre-freeze 3h in -80 DEG C of refrigerators is put into after being sealed with preservative film.The good concentrate of pre-freeze is rapid
It is transferred in freeze dryer and is lyophilized, freeze temperature is -72 DEG C, pressure position 0.1MPa, and taking-up is broken encapsulation into pieces and put after 48h is lyophilized
Enter -20 DEG C to save for use.
Embodiment 3: Vinegar Fermentation process it is biological reinforced
(1) raw material
Fermentation vinegar raw material proportioning selected by the present embodiment are as follows:
Distiller's wort: 130kg/ cylinder (wine degree: 9.6, total acid: 4.4g/L)；Wheat bran: 50kg/ cylinder；Big chaff: 24kg/ cylinder；Water:
15kg/ cylinder；NaCl:3kg/ cylinder.
(2) traditional vinegar brewing process
[1] 130kg distiller's wort and 50kg wheat bran are added into cylinder, stirs and evenly mixs.
[2] 8kg kind unstrained spirits is added into cylinder.
[3] mention the hot stage: the appropriate drift ice on unstrained spirits adds chaff, and carries out turning over unstrained spirits, but does not wait in product temperature toward incision bran
Rise to 40 DEG C or more.
[4] it spends the spoon stage: after proposing heat, according to product temperature, to incision bran, drift ice plus chaff, turning over unstrained spirits daily.Spoon is crossed to start
After a couple of days, start a point cylinder.In this stage, by water and chaff, all addition is finished.
[5] the show-through stage: after crossing spoon, beginning is show-through, i.e., daily carries out the vinegar fermented grain for having turned to bottom all turning over unstrained spirits.
The hydracid daily in measurement lower layer's thick gravy determines the envelope unstrained spirits date according to measured value simultaneously.
[6] it seals the unstrained spirits stage: after show-through, vinegar fermented grain being compacted, upper cover plastic foil tightens plastic film edge, then on side
One layer of salt is uniformly sprinkled at edge.Sealing the unstrained spirits duration is 7 days.
(3) strengthen strategy
Control group: it ferments according to the technique in the present embodiment (2)；
2017210 bacterium powder reinforcing group (i.e. experimental group) of high temperature bacterium Streptomyces sp.CCTCC NO:M: being by this reality
(2) [1] adjustment is applied in example are as follows: [1] 130kg distiller's wort and 50kg wheat bran are added into cylinder, stirs and evenly mixs；By 0.8-1%'s
Streptomyces sp. bacterium powder is uniformly mixed with raw material；Other techniques are constant.
Embodiment 4: 2017210 bacterium powder strengthening effect of high temperature bacterium Streptomyces sp.CCTCC NO:M analysis
After Streptomyces sp.CCTCC NO:M 2017210 is added, total acid and reduced sugar are differed not with control group
It is more, it is analyzed using 18 kinds of amino acid contents in HPLC method Dichlorodiphenyl Acetate fermentation process vinegar fermented grain, the total amount of amino acid is whole
Body is (Fig. 1) in rising trend；The amino of 2017210 experimental group of Streptomyces sp.CCTCC NO:M is added after fermentation 4 days
Sour total content variation is apparently higher than control group；It is dry to reach up to 1413.83mg/100g for the total content of amino acid when fermenting 12 days
Unstrained spirits, (the dry unstrained spirits of 1221.28mg/100g) improves 14% compared to the control group, and the total content of each amino acid is also universal in experimental group
Higher than the content (Tables 1 and 2) of amino acid in control group.
The situation of change of amino acid during 1 control group acetic fermentation of table
| The dry unstrained spirits of mg/100g | 0d | 2d | 4d | 6d | 8d | 10d | 12d | 14d | 18d |
| Aspartic acid | 29.01 | 50.68 | 54.82 | 59.79 | 70.32 | 75.91 | 85.21 | 82.69 | 78.17 |
| Glutamic acid | 35.15 | 123.45 | 159.30 | 170.47 | 199.98 | 218.34 | 239.84 | 234.53 | 203.29 |
| Serine | 0.02 | 0.12 | 0.31 | 0.19 | 0.24 | 0.45 | 0.29 | 4.47 | 3.19 |
| Histidine | 2.24 | 5.98 | 8.20 | 9.86 | 12.38 | 18.44 | 16.39 | 36.99 | 29.05 |
| Glycine | 13.33 | 30.19 | 38.19 | 38.14 | 43.57 | 46.99 | 51.99 | 50.01 | 47.76 |
| Tyrosine | 6.23 | 25.21 | 30.61 | 32.77 | 38.69 | 43.13 | 46.94 | 45.32 | 41.15 |
| Arginine | 16.84 | 32.87 | 66.68 | 50.51 | 61.24 | 70.86 | 84.03 | 82.26 | 77.00 |
| Alanine | 23.50 | 85.36 | 108.89 | 114.31 | 140.83 | 157.31 | 178.78 | 181.21 | 154.83 |
| Threonine | 4.34 | 18.05 | 25.11 | 27.82 | 33.19 | 34.46 | 38.78 | 34.33 | 31.90 |
| Cystine | 0.27 | 2.10 | 1.66 | 1.53 | 2.38 | 2.00 | 2.48 | 1.88 | 1.70 |
| Valine | 18.17 | 59.69 | 71.07 | 75.42 | 88.71 | 98.59 | 111.37 | 107.88 | 96.14 |
| Methionine | 4.37 | 15.81 | 18.92 | 21.02 | 24.65 | 26.08 | 28.44 | 26.01 | 20.81 |
| Phenylalanine | 13.75 | 50.91 | 57.78 | 54.94 | 58.67 | 60.94 | 68.77 | 61.30 | 59.58 |
| Isoleucine | 7.56 | 33.36 | 40.23 | 44.96 | 53.19 | 58.30 | 65.38 | 63.61 | 54.31 |
| Leucine | 24.72 | 100.74 | 129.42 | 129.89 | 151.57 | 164.12 | 184.02 | 176.95 | 158.10 |
| Lysine | 8.20 | 8.02 | 11.09 | 8.89 | 12.01 | 14.36 | 18.57 | 19.99 | 18.56 |
| Proline | 11.90 | 43.91 | 42.45 | 48.12 | 53.33 | 67.15 | 65.31 | 67.07 | 60.57 |
The situation of change of amino acid during the experimental group acetic fermentation of 2 Streptomyces sp. of table
In addition, analyzing the volatile flavor component of vinegar fermented grain during acetic fermentation by GC-MS method, adding
60 kinds of volatile flavor substances are detected in 2017210 experimental group of Streptomyces sp.CCTCC NO:M and control group,
The total content for adding volatile materials in 2017210 experimental group of Streptomyces sp.CCTCC NO:M is 3144.27 μ g/
The dry unstrained spirits of 100g improves 23% compared to control group (the 2413.28 dry unstrained spirits of μ g/100g).Content of the Ester in experimental group
Increase rate is relatively large, and the total content of esters is 1656.94 μ g/100g dry unstrained spirits when fermentation ends, compared to control group
(the 1197.65 dry unstrained spirits of μ g/100g) improves 27.7%；Carboxylic-acid substance, content when experimental group fermentation ends are 302.62 μ g/
100g improves 21% compared to the control group, and the total content of other class volatile materials also has different degrees of raising (Fig. 2).
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill
The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention
Enclosing subject to the definition of the claims.
Claims (9)
1. a streptomycete (Streptomyces sp.) PRO1, which is characterized in that the streptomycete is in April 26 in 2017
It is preserved in China typical culture collection center day, preservation address is Wuhan, China Wuhan University, and deposit number is CCTCC NO:
M 2017210。
2. the microbial bacterial agent containing streptomycete described in claim 1.
3. microbial bacterial agent according to claim 2, which is characterized in that the microbial bacterial agent is streptomycete CCTCC
The liquid bacterial agent or solid fungicide of NO:M 2017210.
4. microbial bacterial agent according to claim 2, which is characterized in that the microbial bacterial agent is streptomycete CCTCC
The freeze-dried powder of NO:M 2017210.
5. streptomycete described in claim 1 is in the application of brewing technology field.
6. application according to claim 5, which is characterized in that the brewing is vinegar brewing.
7. a kind of method that vinegar is biological reinforced, which is characterized in that the biological reinforcing method is using streptomycete CCTCC NO:
M 2017210 ferments after directly mixing with wine vinegar raw material.
8. the method according to the description of claim 7 is characterized in that the biological reinforcing method is straight using the bacterium powder of streptomycete
It connects and ferments after being mixed with wine vinegar raw material.
9. the method according to the description of claim 7 is characterized in that the biological reinforcing method, is to originate in fermentation by strepto-
After 2017210 bacterium powder of bacterium CCTCC NO:M mixes in fermentation vat with distiller's wort, bran feedstock according to the inoculum concentration of 0.8-1.2%
It ferments, the technique that fermentation expands culture using traditional layer-by-layer unstrained spirits.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101979526A (en) * | 2010-11-05 | 2011-02-23 | 山西三盟实业发展有限公司 | Method for preparing complex enzyme preparation for vinegar |
| CN102226142A (en) * | 2011-05-06 | 2011-10-26 | 江南大学 | Bio-augmentation technology for fermentation process of solid-state brewing vinegar |
-
2017
- 2017-04-28 CN CN201710290238.6A patent/CN107189958B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101979526A (en) * | 2010-11-05 | 2011-02-23 | 山西三盟实业发展有限公司 | Method for preparing complex enzyme preparation for vinegar |
| CN102226142A (en) * | 2011-05-06 | 2011-10-26 | 江南大学 | Bio-augmentation technology for fermentation process of solid-state brewing vinegar |
Non-Patent Citations (2)
| Title |
|---|
| Dynamics and diversity of microbial community succession in traditional fermentation of Shanxi aged vinegar;Zhiqiang Nie等;《Food Microbiology》;20141118;第47卷;第62-68页，参见全文 * |
| 酱香型大曲中产蛋白酶放线菌的分离及产酶条件研究;于华等;《中国酿造》;20170313;第36卷(第2期);第64-68页，参见全文 * |
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