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

An extraction method and a fermentation time technology, applied in the field of enzyme engineering to achieve the effect of shortening the fermentation time

Inactive Publication Date: 2016-07-13
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are still no reports on the application of salt-tolerant and halophilic high-yielding protease microorganisms to actual production to achieve better results, and there are even fewer reports on Penicillium citrinum and the produced salt-tolerant protease

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028]Embodiment 1: the screening of Penicillium citrinumYL-1 bacterial strain, concrete method and steps are as follows:

[0029] (1) Prepare five kinds of mold isolation medium (see Table 1)

[0030]

[0031] (2) Use the dilution coating plate method. Add 1mL of squid fermentation broth from Guangdong Shantou Fish Sauce Factory Co., Ltd. to 9mL sterile water and dilute 10 times, that is, 10 -1 . Dilute in ten-fold steps successively to obtain a series of dilutions of the sample solution. Take 0.1mL of different dilutions of the sample solution, and spread them on the separation plates of the five cultures of A, B, C, D, and E, and repeat the same dilution on three plates. Then place them upside down in a 30°C incubator and observe them every 24 hours. Observe the colony morphology, including colony size, color, tissue state, surface shape, etc.

[0032] (3) The strains were initially screened by the flat plate transparent circle method. Select a single colony with g...

Embodiment 2

[0034] Embodiment 2: the extraction method of salt-tolerant serine protease, specific method and steps are as follows:

[0035] (1) Preparation of basic solid medium: KH 2 PO 4 0.2%, MgSO 4 ·7H 2 O0.2%, NaCl10% were mixed to make 1000mL liquid basal medium, adjusted to pH 5.5, then weighed 6g of dry bran in each 250mL Erlenmeyer flask, added 9mL liquid medium and mixed evenly, heated at 121°C for 20min at high temperature sterilization;

[0036] (2) Take the spore density as 10 6 The spore suspension of the PenicilliumcitrinumYL-1 strain obtained in Example 1 per mL was added to the basic solid medium, mixed evenly, and cultured at 30°C. Add 0.1mol / L NaCl solution to the solid enzyme-producing medium after fermentation, mash it and stir it evenly, shake it on a shaker at 30°C for 1 hour, then filter it with gauze, centrifuge the filtrate at 5000r / min for 5min at 4°C, and take The supernatant, the supernatant is the crude enzyme solution. The volume and mass ratio of the...

Embodiment 3

[0040] Example 3: Experiment on the influence of the salt-tolerant serine protease extracted by the present invention on the fermentation effect of fish sauce

[0041] A Penicillium citrinum YL-1 strain with high protease production and salt tolerance was screened from the fish sauce fermentation broth. Fermentation production by solid-state fermentation, three-step purification of ammonium sulfate precipitation, dialysis and DEAEcellulose-52 ion exchange column chromatography, the total activity of the enzyme is 19940U, the total protein is 180.17mg, and the specific activity is increased from 23.74U / mg to 110.68U / mg, the purification factor reached 4.66 times, and the total recovery was 48.7%. Its production cost is low, and adding this enzyme to the initial stage of fish sauce fermentation can speed up the fermentation of fish sauce without affecting the flavor of fish sauce. For verifying its effect to fish sauce fermentation, the present invention has done following exp...

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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

technical field [0001] The invention relates to the technical field of enzyme engineering, in particular to a method for extracting salt-tolerant serine protease and using it to shorten the fermentation time of fish sauce. Background technique [0002] Fish sauce is a traditional aquatic condiment in my country. It is made of fish and shrimp with low economic value and the leftovers of aquatic product processing. formed under certain conditions. However, at present, the traditional process of fish sauce in my country has a long production cycle, high production cost, lack of a perfect quality assessment and control system, and its production process and technology also need to be improved. Moreover, in a natural environment, the decomposition of the fish body is slow, and saturated salt is generally added. This high-salt environment inhibits the growth of spoilage microorganisms. Therefore, shortening the fermentation time and carrying out rapid fermentation in a high-salt ...

Claims

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Application Information

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IPC IPC(8): C12N9/58C12N1/14A23L27/50C12R1/80
CPCC12N1/14C12N9/58
Inventor 高向阳谢莉肖运柱陈瑜珠
Owner SOUTH CHINA AGRI UNIV
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