CN105754975A - Extraction of salt-resistant protease and application method for shortening fish sauce fermentation time - Google Patents

Extraction of salt-resistant protease and application method for shortening fish sauce fermentation time Download PDF

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CN105754975A
CN105754975A CN201610205362.3A CN201610205362A CN105754975A CN 105754975 A CN105754975 A CN 105754975A CN 201610205362 A CN201610205362 A CN 201610205362A CN 105754975 A CN105754975 A CN 105754975A
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高向阳
谢莉
肖运柱
陈瑜珠
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South China Agricultural University
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Abstract

The invention provides extraction of salt-resistant protease and an application method for shortening fish sauce fermentation time.Penicillium citrinum YL-1 screened from fish sauce fermentation liquid and used for producing salt-resistant protease is adopted, rough protease liquid obtained through fermentation is subjected to precipitation and dialysis with ammonium sulfate and subjected to chromatography with a DEAE cellulose-52 ion exchange column so that three-step purification can be achieved, and obtained protease has the total activity of 19940 U, the total protein of 180.17 mg, the specific activity increased to 110.68 U/mg from 23.74 U/mg, the purification multiple of 4.66 times and the total recovery rate of 48.7%.Serine type protease with protease molecular amount of 32 KDa is measured through an SDS-PAGE electrophoresis method.The protease is good in salt resistance and low in production cost; by adding the protease in the primary stage of fish sauce fermentation, the fish sauce fermentation speed can be accelerated, the fish sauce flavor can not be influenced or changed, and enzymic preparations for shortening fish sauce fermentation time can be developed.

Description

The extraction of a kind of salt-tolerant protease and the application process of shortening fish sauce fermentation time
Technical field
The present invention relates to technical field of enzyme engineering, be specifically related to the extraction of a kind of salt tolerance serine albuminoid enzyme also Utilize its method shortening fish sauce fermentation time.
Background technology
Fish sauce is China's tradition aquatic products flavouring, is that the leftover bits and pieces of the fishes and shrimps relatively low with economic worth and processing of aquatic products is Raw material, mainly utilizes protease and other enzymes that fish body self contains, and multiple salt tolerant and halophilic microorganism at certain bar Under part, common effect is formed.But the conventionally produced cycle of China's fish sauce is longer at present, production cost is high, produces and lacks Perfect quality evaluation and control system, its production technology also needs to be improved with technology.And under natural surroundings, fish body divides Solution is relatively slow, typically adds saturated common salt, the suppression putrefactive microorganisms growth of this hypersaline environment.Therefore, shorten under hypersaline environment Fermentation time, carries out Rapid Fermentation and is always the research emphasis of fish sauce zymotechnique.
The research of the domestic and international Rapid Fermentation to fish sauce is in addition to heat-preservation fermentation, external enzyme fermentation and composite fermentation, and some grind Sight is concentrated on the microbe to screen in fish sauce zymotic fluid and application by the person of studying carefully.Thailand scholar in 2008 again from fermentation one month Thailand's fish sauce in be separated to virgibacillus sp. SK33(Sinsuwan et al., 2008).Japanese researchers 2004 Year isolates the bacterial strain of the substantial amounts of alkali protease that can grow on soy peptone culture medium from Vietnam's fish sauce Bacillus subtilisCN2(Uchida et al., 2004).At home, researcher is from the self-control fermentation fish of 3 months Dew filters out bacterial strain Ao Mo Kodak yeast M8(Yang Li elder brother etc. with degradation biological amine ability, 2012)；And send out from tradition The Chaozhou-Shantou region fish sauce of ferment is isolated lactic acid bacteria (Zhang Hao etc., 2013).The most increasing scholar isolated one from fish sauce The microorganism with salt tolerance of a little halophagias includes Bacillus sp., Pseudomonas sp., Filobacillus sp., Micrococcus sp., Staphylococcus sp., Virgibacillus sp.,Halobacillus sp., Halococcus sp., H. salinarum, Halobacterium cutirubrum, H. salinarium(Kawasaki Fukami et al., 2004；Akolkar et al., 2010；Lopetcharat et al., 2001；Namwong et al., 2006；Hiraga et al., 2005).Another research emphasis of fish sauce Rapid Fermentation is from tradition fish sauce from now on Sweat separates salt tolerant and addicted to salt high proteinase yield microorganism, identify the character of its protease, and carry out fish sauce The research of Rapid Fermentation, finds and can significantly shorten fish sauce fermentation period, can produce again the bacterial classification of excellent flavor, and can answer For large-scale industrial production.But have not yet to see salt tolerant and addicted in salt high proteinase yield microbe application to actual production Obtain the report of better effects, more rarely have report Penicillium citrinum and the salt-tolerant protease produced.
Summary of the invention
It is an object of the invention to provide the extracting method of salt tolerance serine albuminoid enzyme and add fish sauce fermentation to In can shorten the application of fermentation time, it uses the Penicillium citrinum bacterium bacterium producing salt-tolerant protease of screening from fish sauce zymotic fluid Strain, extracts through solid state fermentation and obtains salt tolerance serine albuminoid enzyme, and it has preferable salt tolerance, permissible for fish sauce fermentation Shorten fermentation time, and local flavor is similar to the local flavor of the fish sauce high-grade products of spontaneous fermentation, will not produce bad flavor.
For achieving the above object, the technical scheme is that the extracting method of a kind of salt tolerance serine albuminoid enzyme, Being to utilize the Penicillium citrinum YL-1 filtered out from fish sauce zymotic fluid to ferment, the crude enzyme liquid obtained is again Obtain through ammonium sulfate precipitation, dialysis and DEAE cellulose-52 ion-exchange chromatography.Described Penicillium The screening technique of citrinum YL-1 comprises the following steps:
(1) by the proportioning preparation isolation medium of following weight portion: potato 200 parts, glucose sugar 20 parts, NaCl 100 parts, 20 parts of agar；
(2) take in fish sauce zymotic fluid sterilized water and dilute 10 times, successively by ten times of differential dilutions, take different dilution factor sample liquid, Being respectively coated the separation flat board that isolation medium is cultivated, same dilution factor repeats three flat boards, is then inverted in 30 DEG C of incubators Middle cultivation, observes once every 24 h；
(3) using flat board transparent circle method to carry out bacterial strain primary dcreening operation, used medium is the solid medium containing skimmed milk, Preliminary screening Produce protease strain；
(4) by good for the fungal strain growth selection of isolated, the significantly single bacterium colony of form, dibbling is in degreasing milk medium On, after 30 DEG C are cultivated 5 d, by its hydrolysis circle screening bacteria produced proteinase strain, it is inoculated on slant medium in 30 DEG C of cultivations, treats 4 DEG C of refrigerator cold-storages are put into after bacterium length is good.
More specifically embodiment is: the extracting method of a kind of salt tolerance serine albuminoid enzyme, comprises the following steps:
A, by weight percentage KH2PO4 0.2%、MgSO4·7H2O 0.2%, NaCl 10% mixing is made into 1000mL liquid base Culture medium, adjusts pH to 5.5, then weighs the dry wheat bran of 6g in every 250mL triangular flask, adds the mixing of 9mL liquid basal medium, With 121 DEG C, 20min high-temperature heat sterilization, obtain basic solid medium；
B, taking spore density is 106Individual/mL Penicillium citrinum YL-1 bacterial strain spore suspension joins basis On solid medium, it is mixed evenly rear 30 DEG C of quiescent culture, the solid culture medium after fermentation ends will add 0.1 Mol/L NaCl solution, smashs to pieces and stirs, and 30 DEG C of shaking tables shake 1 h, then by filtered through gauze, filtrate under the conditions of 4 DEG C, 5000 r/min are centrifuged 5 min, take supernatant, and supernatant is crude enzyme liquid；
C, under the conditions of 4 DEG C, in crude enzyme liquid add ammonium sulfate so that it is become the saturated solution of ammonium sulfate, 12000r/min be centrifuged 20min, abandons supernatant, collects precipitation, with 0.01 mol/L pH 7.0 phosphate buffer dissolution precipitation, the egg after being saltoutd White enzyme solutions, then dialyses；
D, the protease sample concentrated by dialysis desalting, utilize DEAE 52-Cellulose ion-exchange chromatography to divide further From purifying, chromatographic column is with 0.01mol/L pH 7.0 phosphate buffer linear gradient elution out, collects and has enzymatic activity Eluting peak part, merges, freeze-drying, obtains salt tolerance serine albuminoid enzyme enzyme powder.
As optimization, in step b, the addition of described 0.1 mol/L NaCl solution and the volume of basis solid medium Quality proportioning is 100ml:10-20g.
As optimization, in step c, the concentration of described ammonium sulfate saturated solution is 80%.
As optimization, in step c, described precipitation temperature is 4 DEG C, time 24 more than h, centrifugation rotating speed 12000 r/ Min, time 20min.
Salt-tolerant protease provided by the present invention shortens the application process of fish sauce fermentation time, including: carried above-mentioned The salt tolerance serine albuminoid enzyme the taken ratio with volume as 1:3-5 is added in 6 opah wine with dregs of spontaneous fermentation, is subsequently adding Salt makes fish sauce zymotic fluid maintain saturation state, stirs, and enzyme reaction temperature 35-45 DEG C ferments 12-16 days, by zymotic fluid Filter.
As optimization, described salt tolerance serine albuminoid enzyme is 1:4 with the volume ratio of fish wine with dregs, and fermentation temperature 40 DEG C is sent out 15 days ferment time.
The bacterial strain of heretofore described product salt-tolerant protease is Penicillium citrinum bacterium Penicillium citrinum YL-1, It is to be screened by primary dcreening operation and multiple sieve from fish sauce zymotic fluid, carries out morphology and molecular biology identification the most again.Described Strain morphology feature is as follows: colony colour surface is cyan, and reverse side is faint yellow, fine hair shape.This bacterium conidia chain of microscopy is Clear and definite dispersion column, conidiophore betides matrix, and wall smooths；The length betiding aerial hyphae person on a small quantity is less；Broom Shape branch two-wheel is raw, has single-wheel raw；Metulae often takes turns 3-5, and 8.0-16 × 2.5-3.0um, top presents cryptomere and expands；Bottle stalk 8.0- 9.0 × 2.0-2.5um, ampuliform, stalk neck is short；Conidium presents spherical or subsphaeroidal, 2.5-3.2um, and wall is smooth or is bordering on flat Sliding.After using molecular biology method that the DNA genomic samples of mould YL-1 carries out PCR amplification order-checking, amplified production is carried out Electrophoresis detection understands obvious bright band between 500-750bp, has obtained the DNA band of expection length.By expanded to ITS gene order carries out tetraploid rice with the known array in GenBank database.Qualification result finds, bacterial strain YL-1 Penicillium citrinum similarity reaches 99%, in conjunction with above YL-1 Morphological observation result, looks into " China fungi Will " (mould and relevant epigamous belong to) thereof understand, and preliminary assert that YL-1 belongs to Penicillium citrinum.
The present invention is to separate a strain Penicillium citrinum bacterium Penicillium citrinum YL-from fish sauce zymotic fluid 1, optimize its solid state fermentation conditions, and the protease being produced its solid state fermentation carries out isolated and purified and zymologic property research, The product protease optimum temperature gone out is 40 DEG C, and optimal pH is 8.0, belongs to alkali protease, under the conditions of pH5.0 and 6.0, stability Preferably, the enzyme activity of its protease still keeps 81.33% and 83.56%, illustrates under the conditions of fish sauce zymotic fluid pH5.5, should Enzyme still retains greater activity.Recording protease molecule amount by SDS-PAGE electrophoresis is 32KDa, uses different protease Its enzyme activity is affected by inhibitor, finds that serpin (PMSF) has significant inhibition to protease, says Bright its is serine albuminoid enzyme.This protease has certain enzyme to live and stability at 0 ~ 30%NaCl, will purify enzyme in high salt Degree is to preserve 1h under the conditions of 30%, records its relative enzyme and lives and remain 20%, and prolongation in time, enzyme activity does not has substantially Change, be demonstrated by preferable salt tolerance.
The present invention serine albuminoid enzyme extracted is added to fish sauce fermentation initial stage can substantially shorten send out The ferment time, and local flavor is similar to the local flavor of the fish sauce high-grade products that Traditional Method ferments, and will not produce bad flavor, can be developed into Shorten the enzyme preparation of fish sauce fermentation time.
Accompanying drawing explanation
Fig. 1 a is the NaCl impact on proteinase activity power
Fig. 1 b is the impact on protease stability of Fig. 1 b. variable concentrations NaCl solution
Fig. 2 is that protease is to Fish protein hydrolysis result
Fig. 3 is to add the change of AAN content in fish sauce ferments of serine albuminoid enzyme.
Detailed description of the invention
Embodiments of the invention are given below, to describe the present invention in detail.
Embodiment 1: the screening of Penicillium citrinum bacterium Penicillium citrinum YL-1 bacterial strain, concrete grammar and step are such as Under:
(1) five kinds of mould isolation medium (see Table 1) are prepared
(2) dilution spread flat band method is used.Shantou, Guangdong Province fish sauce Co., Ltd., Factory fish zymotic fluid 1 mL will be taken from and join 9 ML sterilized water dilutes 10 times, i.e. 10-1.Successively by ten times of differential dilutions, obtain a series of dilution sample liquid.Take 0.1 mL not Same dilution factor sample liquid, is respectively coated the separation flat board that A, B, C, D, E five kinds cultivates, and same dilution factor repeats three flat boards.So After be inverted in 30 DEG C of incubators cultivation, every 24 h observe once.Observe colonial morphology, including bacterium colony size, color, tissue State, surface configuration etc..
(3) flat board transparent circle method is used to carry out bacterial strain primary dcreening operation.Good, the form by the fungal strain growth selection of isolated The most single bacterium colony, dibbling is on degreasing milk medium, and the bacterial strain producing protease can specifically decompose skim milk, in choosing Periphery of bacterial colonies on selecting property skim milk culture medium produces obvious hydrolysis, can produce protease strain with Preliminary screening. After 30 DEG C are cultivated 5 d, by its hydrolysis circle screening bacteria produced proteinase strain, it is inoculated on slant medium in 30 DEG C of cultivations, treats bacterium 4 DEG C of refrigerator cold-storages are put into after length is good.
(4) the bacteria produced proteinase strain that primary dcreening operation is screened by application foundation solid fermentation culture medium carries out multiple sieve, in 30 DEG C After cultivating a period of time, extract crude enzyme liquid, measure proteinase activity in the case of pH5.5, pH7.0 and pH8.5, filter out high yield Protease strain Penicillium citrinum YL-1 carries out next step research.
Embodiment 2: the extracting method of salt tolerance serine albuminoid enzyme, concrete grammar and step are as follows:
(1) preparation basis solid medium: KH2PO40.2%, MgSO4·7H2O 0.2%, NaCl 10% mixing is made into 1000mL Liquid basal medium, adjusts pH to 5.5, then weighs the dry wheat bran of 6g in every 250mL triangular flask, adds 9mL fluid nutrient medium Mixing, with 121 DEG C, 20min high-temperature heat sterilization；
(2) taking spore density is 106The embodiment 1 gained Penicillium citrinum YL-1 bacterial strain spore suspension of individual/mL Liquid joins on the solid medium of basis, is mixed evenly rear 30 DEG C of quiescent culture.By the solid culture medium after fermentation ends Middle addition 0.1 mol/L NaCl solution, smashs to pieces and stirs, and 30 DEG C of shaking tables shake 1 h, and then by filtered through gauze, filtrate exists Under the conditions of 4 DEG C, 5000 r/min are centrifuged 5 min, take supernatant, and supernatant is crude enzyme liquid.Described 0.1 mol/L NaCl is molten The addition of liquid is 100mL:10g with the volume mass proportioning of basis solid medium；
(3) under the conditions of 4 DEG C, take crude enzyme liquid 100ml, calculate according to ammonium sulfate saturation computation formula required optimal saturated molten Degree be the quality of the ammonium sulfate of 80% be 56.1g, make it fully dissolve its finely ground being slowly added in enzyme liquid, 4 DEG C place 24 h After, 12000 r/min are centrifuged 20 min, remove its supernatant, collect precipitation, molten with 0.01 mol/L pH 7.0 phosphate buffer Solve precipitation, the protein enzyme solution after being saltoutd, then dialyse.
(4) protease sample concentrated through dialysis desalting, utilizes DEAE 52-Cellulose ion-exchange chromatography to enter one Walking isolated and purified, chromatographic column is with 0.01mol/L pH 7.0 phosphate buffer linear gradient elution out, and collection has enzyme and lives Property eluting peak part, merge, freeze-drying.
After three main separating steps, specific activity of enzyme is brought up to 11.1 ten thousand U/g by 2.3 ten thousand U/g, total purifying of enzyme Multiple reaches 4.66 times.
Embodiment 3: the salt tolerance serine albuminoid enzyme that the present invention extracts is on the impact experiment of fish sauce ferment effect
The penicillium bacterial strain Penicillium citrinum YL-1 of high proteinase yield salt tolerance is filtered out from fish sauce zymotic fluid, Its produced protease has certain salt tolerance, and also can play preferable enzymatic activity under the conditions of fish sauce yeasting, can use Solid state fermentation mode carries out fermenting and producing, through ammonium sulfate precipitation, dialysis and DEAE cellulose-52 ion-exchange chromatography It is 19940U that three steps purifying obtain the total activity of enzyme, and total protein is 180.17mg, and Rate activity is brought up to by 23.74U/mg 110.68U/mg, purification reaches 4.66 times, and overall recovery is 48.7%.Its low production cost, adds this enzyme to fish The initial stage of dew fermentation, can accelerate the speed of fish sauce fermentation but not interfere with change Flavor of Fish Sauce.For verifying that fish sauce is sent out by it The effect of ferment, the present invention has made following experiment:
(1) NaCl is on prolease activity and the impact of stability
Pure enzyme liquid is mixed with variable concentrations NaCl solution, to make the NaCl concentration in reaction system be finally 0%, 5%, 10%, 15%, 20%, 25%, 30%, according to proteinase activity power assay method, measure the vigor of protease.Same primary condition, in room temperature Lower insulation, takes out liquid of protease the most respectively and carries out enzyme activity determination, and the enzyme activity adding NaCl enzyme liquid with the first beginning and end is 100%, determine that the NaCl of variable concentrations is on prolease activity and the impact of stability.
Fish sauce fermentation is carried out under hypersaline environment, is mixed with the NaCl solution of variable concentrations respectively by pure enzyme liquid, room Temperature is placed, and measures proteinase activity power and stability.Such as Fig. 1 a and 1b.
As shown in Figure 1a, in the range of NaCl concentration is 0 ~ 30%, along with the rising of NaCl concentration, proteinase activity power by Gradually reduce；Reacting under the conditions of NaCl concentration is 30%, protease enzyme activity is still more than 20%.From Fig. 1 b, purify After enzyme preserves 1 h respectively under 5%, 10%, 15%, 20%, 25% and 30%NaCl solution, record its enzyme activity and be reduced to respectively 70.16%, 53.83%, 40.9%, 31.36%, 23.08% and 19.17%, then as the prolongation of time, enzyme activity is basic Do not change, and with the increase of NaCl concentration, proteinase activity power is gradually lowered similar to Fig. 1 a result.Therefore this protease Can not only have the ability of liquid fish under low-salt environment, and still have in the case of high salinity.
(2) Fish protein hydrolysis result is measured by protease
Take a certain amount of Tilapia mossambica flesh of fish and add 0.1mol/L, pH 5.5 phosphate buffer of precooling with the ratio of 1:5, homogeneous, Then at 4 DEG C, 30 min are stirred, homogenate.Under the conditions of 4 DEG C, 12000 r/min are centrifuged 10 min.Take isopyknic supernatant Liquid is separately added into the NaCl of different quality, 4 DEG C of stirring 12 h, make the concentration of NaCl solution finally reach 0%, 5%, 10%, 15%, 20%, 25%, 30%, as the substrate solution of enzyme reaction.Liquid of protease is mixed with variable concentrations NaCl substrate solution, makes reaction NaCl concentration in system is finally 0%, 5%, 10%, 15%, 20%, 25%, 30%, acts on 40 min at 40 DEG C, after the time arrives, enters 2 The solution of trichloroacetic acid of mL 0.4 mol/L terminates reaction, and insulation stands 30 min, then filters.Use Lowry method to supernatant TCA-solubility oligopeptides in liquid is measured, and with the reaction group without NaCl for blank group, TCA-solubility oligopeptides content is 100%。
Utilize this protease hydrolytic Tilapia mossambica protein, the TCA-solubility oligopeptides content in hydrating solution is surveyed Fixed, result is as shown in Figure 2.
From the graph it can be seen that this protease has certain degradation to Tilapia mossambica protein.At NaCl concentration it is In the range of 5%～30%, along with the increase of NaCl concentration, the TCA-solubility oligopeptides content in each reaction group is gradually reduced, explanation Along with the increase of NaCl concentration, the degradation of Tilapia mossambica protein is gradually weakened by this protease.But it is 5% at NaCl concentration Time, the TCA-solubility oligopeptides content in reaction group exceeds 12% than blank group, illustrates that the NaCl of low concentration is to this proteasome degradation Tilapia mossambica protein has certain facilitation.Also illustrating under the conditions of the NaCl of high concentration, this protease is to Rofe simultaneously Fish protein also has degradation, but degradation effect is suppressed by certain.Therefore this protease under high low-salt environment all There is the ability of liquid fish.
(3) salt tolerant serine albuminoid enzyme enzymolysis shortens the mensuration of fish sauce fermentation time
This experiment is that the salt tolerant serine protease that separated by interpolation can be at short notice in enzymolysis high salt fish sauce zymotic fluid Fish protein, make the content of amino-acid nitrogen (AAN) significantly higher than without blank group of enzyme.Concrete operation method is as follows: Enzyme is added to spontaneous fermentation 6 opah wine with dregs for the 23.74U/mg salt tolerant serine protease ratio with volume as 1:4 than living In, it is subsequently adding salt and makes fish sauce zymotic fluid maintain saturation state, stir, keep the optimum temperature 40 DEG C of this enzyme reaction, It addition, with the fish sauce zymotic fluid without enzyme liquid for blank group, ferment 15 days, observe the feelings that fish sauce is fermented by serine protease Condition, by filtering fermentation liquor, (filter method can use any conventional filtering method) obtains filtrate.Then according to conventional method and basis Invention is added the method gained fish sauce of enzyme and is compared they content (such as Fig. 3) as the amino-acid nitrogen (AAN) of nutrients.
From Fig. 3, result can be seen that and compares through two weeks fermentation, and the change of the AAN of blank group is little, but The present invention with the addition of AAN content in the experimental group of serine protease substantially increase, than the amino-acid nitrogen of blank group Content is high, and AAN content has just had more 32% than blank group.Thus may determine that, add salt tolerant serine egg according to the present invention The method that white enzyme ferments to fish sauce, can significantly reduce fish sauce fermentation time and have additional nutrients thing content.
(4) sensory evaluation
Additionally, according in interpolation serine protease in the present invention to the fish sauce zymotic fluid of fermentation half a year, carry out sensory evaluation knot Fruit is as follows:
Outward appearance: supernatant liquid；
Color and luster: amber or brownish red；
Local flavor: strong ferment fragrance is without any bad smell and peculiar smell, close with the quality of senior fish sauce；
Mouthfeel: tasty mouthfeel, aftertaste is mellow, ooze that to prolong sense strong；
By the isolated and purified extracting method obtaining salt tolerant serine protease of the present invention, and it is added in fish sauce fermentation, Can substantially shorten fish sauce fermentation time, and the fish sauce made in the short time is equivalent to the fish sauce of Traditional Method, is commented by sense organ Fixed similar to the local flavor of fish sauce high-grade products, bad flavor will not be produced.
From the foregoing, it will be observed that this serine protease can accelerate liquid fish under hypersaline environment, add fish sauce fermentation to In, only by the fermentation time that two weeks is identical, AAN content has just had more 32% than blank group, can dramatically increase it Nutrient content, shortens fish sauce fermentation time, and does not change the traditional excellent local flavor of fish sauce itself, thus save production cost, carry High economic benefit, can be developed into the commercial enzyme preparation of fish sauce Rapid Fermentation.

Claims (8)

1. the extracting method of a salt tolerance serine albuminoid enzyme, it is characterised in that utilize and filter out from fish sauce zymotic fluid The Penicillium citrinum bacteria strain fermented crude enzyme liquid that obtains of Penicillium citrinum YL-1 producing salt-tolerant protease, then warp Ammonium sulfate precipitation, dialysis and DEAE cellulose-52 ion-exchange chromatography and obtain.
The extracting method of a kind of salt tolerance serine albuminoid enzyme, it is characterised in that described product is resistance to The Penicillium citrinum bacteria strain Penicillium citrinum YL-1 of salt protease is screened by the method comprised the following steps Arrive:
(1) by the proportioning preparation isolation medium of following weight portion: potato 200 parts, glucose sugar 20 parts, NaCl 100 parts, 20 parts of agar；
(2) take in fish sauce zymotic fluid sterilized water and dilute 10 times, successively by ten times of differential dilutions, take different dilution factor sample liquid, Being respectively coated the separation flat board that isolation medium is cultivated, same dilution factor repeats three flat boards, is then inverted in 30 DEG C of incubators Middle cultivation, observes once every 24 h；
(3) using flat board transparent circle method to carry out bacterial strain primary dcreening operation, used medium is the solid medium containing skimmed milk, Preliminary screening Produce protease strain；
(4) the fungal strain growth selection by step (3) isolated is good, the significantly single bacterium colony of form, and dibbling is in skimmed milk On culture medium, after 30 DEG C are cultivated 5 d, by its hydrolysis circle screening bacteria produced proteinase strain, it is inoculated on slant medium in 30 DEG C Cultivate, after bacterium length is good, puts into 4 DEG C of refrigerator cold-storages.
The extracting method of salt tolerance serine albuminoid enzyme the most according to claim 1, it is characterised in that include following step Rapid:
A, by weight percentage KH2PO4 0.2%、MgSO4·7H2O 0.2%, NaCl 10% mixing is made into 1000mL liquid base Culture medium, adjusts pH to 5.5, then weighs the dry wheat bran of 6g in every 250mL triangular flask, adds the mixing of 9mL fluid nutrient medium, uses 121 DEG C, 20min high-temperature heat sterilization, obtain total amount of binder culture medium；
B, to take spore density be 106Individual/mLPenicillium citrinumYL-1 bacterial strain spore suspension joins basis solid On culture medium, it is mixed evenly rear 30 DEG C of quiescent culture, the solid culture medium after fermentation ends will add 0.1 mol/L NaCl solution, smashs to pieces and stirs, and 30 DEG C of shaking tables shake 1 h, then by filtered through gauze, filtrate under the conditions of 4 DEG C, 5000 R/min is centrifuged 5 min, takes supernatant, and supernatant is crude enzyme liquid；
C, under the conditions of 4 DEG C, in crude enzyme liquid add ammonium sulfate so that it is become the saturated solution of ammonium sulfate, 12000r/min be centrifuged 20min, abandons supernatant, collects precipitation, with 0.01 mol/L pH 7.0 phosphate buffer dissolution precipitation, the egg after being saltoutd White enzyme solutions, then dialyses；
D, the protease sample concentrated by dialysis desalting, utilize DEAE 52-Cellulose ion-exchange chromatography to divide further From purifying, chromatographic column is with 0.01mol/L pH 7.0 phosphate buffer linear gradient elution out, collects and has enzymatic activity Eluting peak part, merges, freeze-drying, obtains salt tolerance serine albuminoid enzyme enzyme powder.
The extracting method of salt tolerance serine albuminoid enzyme the most according to claim 3, it is characterised in that in step b, institute Stating the addition of 0.1 mol/L NaCl solution with the volume mass proportioning of basis solid medium is 100ml:10-20g.
The extracting method of salt tolerance serine albuminoid enzyme the most according to claim 3, it is characterised in that in step c, institute Stating the optimal saturation degree of ammonium sulfate is 80%.
The extracting method of salt tolerance serine albuminoid enzyme the most according to claim 3, it is characterised in that in step c, institute Stating precipitation temperature is 4 DEG C, time 24 more than h, centrifugation rotating speed 12000 r/min, time 20min.
7. the application process of a salt-tolerant protease shortening fish sauce fermentation time, it is characterised in that including: by salt tolerant sex pilus ammonia The acids protease ratio with volume ratio as 1:3-5 is added in 6 opah wine with dregs of spontaneous fermentation, is subsequently adding salt and makes fish sauce send out Ferment liquid maintains saturation state, stirs, and enzyme reaction temperature 35-45 DEG C ferments 12-16 days, by filtering fermentation liquor.
Salt-tolerant protease the most according to claim 7 shortens the application process of fish sauce fermentation time, it is characterised in that institute The volume ratio stating salt tolerance serine albuminoid enzyme and fish wine with dregs is 1:4, fermentation temperature 40 DEG C, fermentation time 15 days.
CN201610205362.3A 2016-03-17 2016-04-05 Extraction of salt-resistant protease and application method for shortening fish sauce fermentation time Pending CN105754975A (en)

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CN109463717A (en) * 2018-12-06 2019-03-15 丹寨县金海绿色食品有限公司 A kind of production method of fishes and shrimps perfume (or spice) sauce acid
CN111227216A (en) * 2020-01-13 2020-06-05 江苏大学 Method for improving fermentation quality of low-salt fish sauce by utilizing planococcus sp mixed strain

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YUN-ZHU XIAO ET AL.: "statistical optimization of alkaline protease production from penicillium citrinum YL-1 under solid-state fermentation", 《PREPARATIVE BIOCHEMISTRY & BIOTECHNOLOGY》 *
晁岱秀等: "鱼露快速发酵研究的探讨", 《现代食品科技》 *
谢莉: "高盐条件下橘青霉产蛋白酶作用机理及在鱼露发酵中应用", 《中国优秀硕士学位论文全文数据库》 *

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109463717A (en) * 2018-12-06 2019-03-15 丹寨县金海绿色食品有限公司 A kind of production method of fishes and shrimps perfume (or spice) sauce acid
CN111227216A (en) * 2020-01-13 2020-06-05 江苏大学 Method for improving fermentation quality of low-salt fish sauce by utilizing planococcus sp mixed strain
CN111227216B (en) * 2020-01-13 2021-10-12 江苏大学 Method for improving fermentation quality of low-salt fish sauce by utilizing planococcus sp mixed strain

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