CN110499261A - The preparation process of protein enzymatic hydrolyzate and its application in fermented and cultured - Google Patents

The preparation process of protein enzymatic hydrolyzate and its application in fermented and cultured Download PDF

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CN110499261A
CN110499261A CN201810472588.9A CN201810472588A CN110499261A CN 110499261 A CN110499261 A CN 110499261A CN 201810472588 A CN201810472588 A CN 201810472588A CN 110499261 A CN110499261 A CN 110499261A
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dregs
seed liquor
beans
seed
prepares
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杜鹏
高启超
梁晓娟
郄子玥
高雷
王斌
刘超
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Abstract

The invention belongs to fermentation technical fields, disclose the preparation process of protein enzymatic hydrolyzate comprising following steps: step 1) prepares dregs of beans culture solution, and step 2 prepares seed liquor, step 3) fermentation, and step 4) handles mycoprotein.Preparation process of the present invention is simple and feasible, low in cost, will be in protein enzymatic hydrolyzate application fermentation medium of the present invention, it is easier to be absorbed and utilized by bacterial strain.

Description

The preparation process of protein enzymatic hydrolyzate and its application in fermented and cultured
Technical field
The invention belongs to fermentation technical fields, and in particular to the preparation process of protein enzymatic hydrolyzate and its answering in fermented and cultured With.
Background technique
Dregs of beans is a kind of byproduct obtained after soybean extracting bean oil, is important vegetable protein source, also known as " soybean The dregs of rice ".Dregs of beans generally in irregular fragment shape, color be it is light yellow to light brown, taste is with roasting soybean fragrance.Dregs of beans it is main Ingredient are as follows: protein 40%~48%, lysine 2.5%~3.0%, tryptophan 0.6%~0.7%, methionine 0.5%~0.7%.About 85% dregs of beans is used for poultry and swine rearing, and a variety of amino acid that dregs of beans includes are suitable for the demand of poultry and pig to nutrition. Experiment shows that only amino acid contained in dregs of beans is just enough to balance house in the case where being not required to that animal protein additionally is added The nutrition of fowl and pig, to promote the nutrient absorption of livestock.In poultry and feeding live pig, dregs of beans has obtained maximum benefit With.Only when the unit-protein cost of Cottonseed Meal and peanut meal is taken into account use far below dregs of beans Shi Caihui.In fact, dregs of beans Have become the benchmark product that other protein sources compare.In the breeding process of milk cow, delicious flavour, dregs of beans easy to digest can Improve out milk amount.In the raising of stocker, dregs of beans is also one of most important oilseed extraction.Dregs of beans also be used to that pet food be made Product.Food is simply mixed and using food value having the same made of high animal protein in corn, dregs of beans.Dregs of beans is also wide It is applied in culture fishery generally.The a variety of amino acid contained in dregs of beans can sufficiently meet fish to the special need of amino acid It wants.
The prior art, which has had, to be digested dregs of beans to be used for the research of strain fermentation, and enzymatic isolation method degrading soybean protein makes Animal protease has: pepsin, trypsase；Plant rennet: bromelain, ficin, pawpaw egg White enzyme etc.；Microbial protease: bacillus subtilis, bacillus licheniformis, aspergillus niger, actinomyces etc..Document is " using finishing red ferment Corn flour-dregs of beans medium optimization of mother's production single cell protein, Chinese feed 2013 " utilizes enzymatic isolation method by corn flour and beans The dregs of rice are hydrolyzed, then by single factor experiment and optimization of orthogonal test fermentation medium, using Pichia pastoris as starting strain, Single cell protein is produced using corn flour and dregs of beans as carbon source and nitrogen source；This method ferment effect is good, but needs to consume big The enzyme preparation of amount, wherein alkali protease enzyme concentration is 2%, and the enzyme concentration of neutral proteinase is 3%, the enzyme concentration of compound protease It is 4%, entreprise cost increases.Document " research of saccharomycete single bacterium solid state fermentation dregs of beans, Chinese grain and oil journal 2009 " passes through ferment Female fermentation process dregs of beans, trypsin ihhibitor content are remarkably decreased, degradation rate up to 56.2% (, small peptide content improves 4.3 Times, the raw dregs of beans energy obvious degradation anti-nutritional factors of microbial fermentation, so as to improve its Forage quality.The prior art is to use mostly The mode of enzymatic hydrolysis handles dregs of beans for also having part research to improve by strain fermentation dregs of beans feeding in feed or small peptide production Quality, but few research and utilization bacterial strain carries out the product of fermentation enzymatic hydrolysis to dregs of beans as fermentation medium component.Applicant Patented technology " preparation process of dregs of beans enzymolysis liquid and its purposes in fermentation medium preparation ", digested using four kinds of bacterial strains Dregs of beans, mutually antagonism, mutually collaboration symbiosis can not digest dregs of beans effectively, generate and be suitble to strain growth each bacterial strain in mixed culture Nutriment, save entreprise cost.But above-mentioned patented technology does not carry out effective hydrolysis process to mycoprotein, The protein ratio of middle macromolecule is still higher, and the present invention has made it further to change on the basis of above-mentioned patented technology Into to further increase ferment effect.
Summary of the invention
The purpose of the present invention is further being improved prior art, provide protein enzymatic hydrolyzate preparation process and its Application in fermented and cultured.
The present invention is achieved by the following technical solution:
The preparation process of protein enzymatic hydrolyzate comprising following steps: step 1) prepares dregs of beans culture solution, and step 2 prepares seed Liquid, step 3) fermentation, step 4) handle mycoprotein.
Further,
The technique includes the following steps:
Step 1) prepares dregs of beans culture solution: dregs of beans and water being mixed, then 121 DEG C of steam treatment 10min naturally cool to room Temperature obtains dregs of beans culture solution；
Step 2 prepares seed liquor: by bacillus licheniformis, Cellumomonas flavigena, aspergillus niger and aspergillus oryzae respectively according to normal Rule culture obtains seed liquor；
Step 3) fermentation: by bacillus licheniformis seed liquor, Cellumomonas flavigena seed liquor, aspergillus niger seed liquor and meter Qu Mould seed liquor mixing, obtains seed mixture liquid；Then seed mixture liquid is linked into according to 10% inoculum concentration containing dregs of beans culture Carry out enzymatic production in the fermentor of liquid, temperature is 35 DEG C, and tank pressure is 0.03MPa, air quantity 500L/h, and the enzymatic production time is 60h；
Step 4) handles mycoprotein: ultrasonic treatment is then heated to 55 DEG C, and adding sulfuric acid adjustment pH is 6, is separately added into lysozyme And acid protease, under heat-retaining condition, digest 12 hours；Then 121 DEG C of steam treatment 10min, cooled to room temperature obtain Protein enzymatic hydrolyzate.
Preferably,
In the step 1), dregs of beans and water are mixed according to the mass ratio of 1:1.
Preferably,
In the step 3), by bacillus licheniformis seed liquor, Cellumomonas flavigena seed liquor, aspergillus niger seed liquor and rice Aspergillus seed liquor is mixed according to the volume ratio of 2:2:1:1.
Preferably,
In the step 3), fermentating enzyme-producing condition are as follows: temperature is 35 DEG C, and tank pressure is 0.03MPa, air quantity 500L/h, and fermentation produces The enzyme time is 60h.
Preferably,
The condition of the ultrasonic treatment are as follows: supersonic frequency 25kHz, ultrasonic time 30min.
Preferably,
The additive amount of the lysozyme is 20,000 U:1L solution, and the additive amount of acid protease is 10,000 U:1L solution.
Compared with prior art, the beneficial effect that the present invention obtains mainly includes but is not limited to the following aspects:
Aspergillus oryzae and aspergillus niger can generate neutral proteinase, acid protease, sugar under the conditions of independent koji-making and mixed culture Change enzyme；Cellumomonas flavigena can generate cellulase, be digested the fibre fractionation in dregs of beans to obtain reduced sugar；Lichens Bacillus generates alpha-amylase and protease；Carbohydrate in dregs of beans can under the action of amylase and carbohydrase, Enzymatic hydrolysis obtains saccharic composition；Part albumen in dregs of beans together with fiber interweaving, common protease be difficult to by albumen completely with Fiber separation, the present invention generate cellulase using Cellumomonas flavigena and digest to fibre fractionation, cooperate with other enzymes, Albumen can also be separated from fiber, improve the hydrolysis result of albumen by both available reduced sugars；The present invention utilizes Ultrasonic wave cavitation, generate partial high pressure high temperature, somatic cells impacted, somatic cells is caused to deform and broken It splits, facilitates thallus broken wall, then use enzymolysis processing, improve the enzymatic hydrolyzation of albumen；The present invention is using four kinds of bacterial strain enzymatic hydrolysis Dregs of beans, the mutual not antagonism of each bacterial strain, mutually collaboration symbiosis, so that co-culture system is in an equilibrium-like in mixed culture State；Enzymolysis product of the invention uses bean pulp fermentation zymolyte and mycoprotein zymolyte to obtain enzymolysis liquid for primary raw material, can It is full of nutrition with the higher beef extract of price in substitutive medium or yeast extract component, it is cheap, reduce entreprise cost.
Detailed description of the invention
Fig. 1: the measurement result of main active principle in enzymolysis liquid；
Fig. 2: influence of the different strains compatibility to protein hydrolysis degree.
Specific embodiment
Those skilled in the art can use for reference present disclosure, be suitably modified realization of process parameters.In particular, it should be pointed out that All similar substitutions and modifications are apparent to those skilled in the art, they are considered as being included in this hair It is bright.Product and method of the invention is described by preferred embodiment, and related personnel can obviously not depart from this hair Product as described herein and method are modified in bright content, spirit and scope or appropriate changes and combinations, to realize and answer Use the technology of the present invention.For a further understanding of the present invention, the following describes the present invention in detail with reference to examples.
Embodiment 1
The preparation process of protein enzymatic hydrolyzate comprising following steps:
Dregs of beans and water are mixed according to the mass ratio of 1:1,121 DEG C of steam treatment 10min, then cooled to room temperature, obtains Dregs of beans culture solution；
Bacillus licheniformis, Cellumomonas flavigena, aspergillus niger and aspergillus oryzae are obtained into seed liquor according to routine culture；
Bacillus licheniformis seed liquor, Cellumomonas flavigena seed liquor, aspergillus niger seed liquor and aspergillus oryzae seed liquor are pressed According to the volume ratio mixing of 2:2:1:1, seed mixture liquid is obtained；Then seed mixture liquid is linked into according to 10% inoculum concentration and is contained Have and carry out enzymatic production in the fermentor of dregs of beans culture solution, temperature is 35 DEG C, and tank pressure is 0.03MPa, air quantity 500L/h, fermentation The producing enzyme time is 60h, ultrasonic treatment, supersonic frequency 25kHz, ultrasonic time 30min, broken when stopping (broken wall, stopping) every time Between be 4s, be then heated to 55 DEG C, add sulfuric acid adjustment pH be 6, be separately added into lysozyme and acid protease, lysozyme adds Dosage is 20,000 U:1L solution, and the additive amount of acid protease is 10,000 U:1L solution, under heat-retaining condition, is digested 12 hours；Then 121 DEG C of steam treatment 10min, cooled to room temperature obtain protein enzymatic hydrolyzate.
The bacillus licheniformis seed liquor the preparation method comprises the following steps: bacillus licheniformis is inoculated on LB solid medium Culture, obtains single colonie；Picking single colonie is inoculated into primary-seed medium and is cultivated, and then carries out secondary seed medium Culture obtains bacillus licheniformis seed liquor；The component of the primary-seed medium and secondary seed medium is equal are as follows: grape Sugared 20g/L, yeast extract 10g/L, epsom salt 2g/L；
The Cellumomonas flavigena seed liquor is trained the preparation method comprises the following steps: Cellumomonas flavigena is inoculated on slant medium It supports, obtains single colonie；Picking single colonie is inoculated into primary-seed medium and is cultivated, and then carries out secondary seed medium training It supports, obtains Cellumomonas flavigena seed liquor；The slant medium component are as follows: yeast extract 60g/L, glucose 20g/L, agar 15g/L；The component of the primary-seed medium and secondary seed medium is equal are as follows: corn flour 30g/L, glucose 20g/L, ferment Female cream 1g/L, potassium dihydrogen phosphate 0.1g/L, dipotassium hydrogen phosphate 0.1g/L, epsom salt 0.05g/L, ferrous sulfate heptahydrate 0.01g/L；
The aspergillus niger seed liquor the preparation method comprises the following steps: aspergillus niger streak inoculation is cultivated on slant medium, obtain single bacterium It falls；Picking single colonie is inoculated into primary-seed medium and is cultivated, and then carries out secondary seed medium culture, obtains black song Mould seed liquor；The slant medium component are as follows: potato 200g/L, sucrose 30g/L, agar 20g/L；The first order seed training The component for supporting base and secondary seed medium is equal are as follows: corn flour 60g/L, sucrose 10g/L, ammonium sulfate 5g/L, potassium dihydrogen phosphate 1g/ L, dipotassium hydrogen phosphate 1g/L；
The aspergillus oryzae seed liquor the preparation method comprises the following steps: aspergillus oryzae streak inoculation is cultivated on PDA solid medium, obtain list Bacterium colony；Picking single colonie, which is inoculated into PDA liquid medium, is cultivated, and aspergillus oryzae seed liquor is obtained.
Embodiment 2
The dregs of beans group phase-splitting difference that various soybean species generate is little, and the present invention selects belt leather dregs of beans, each main component content are as follows: Protein 45%, ash content 7%, fiber 20%, moisture 10%, fat 2%, carbohydrate 14%, other is inorganic mineral.
Main indicator detection method: Kjeldahl nitrogen determination total protein；SDS-PAGE distinguishes survey to molecular weight of albumen It is fixed；The measurement of content of reducing sugar: direct titrimetric method, referring to GB/T5009.7-2008；Conventional H PLC detects free amino acid, widow Peptide and content of peptides；Protein hydrolysis degree measuring method measures degree of hydrolysis using ninhydrin.
The effect that group verifies each bacterial strain is set, wherein control group 1 is bacillus licheniformis, produces yellowish fiber unit cell Three kinds of bacterium compatibilities of bacterium and aspergillus oryzae, remaining is the same as embodiment 1；Control group 2 is bacillus licheniformis, Cellumomonas flavigena and black song Mould, remaining is the same as embodiment 1；Control group 3 is bacillus licheniformis, aspergillus oryzae and aspergillus niger, remaining is the same as embodiment 1；Control group 4 is Cellumomonas flavigena, aspergillus oryzae and aspergillus niger, remaining is the same as embodiment 1.
1. the protein component of enzymolysis liquid measures, concrete outcome is shown in Table 1:
Table 1
Index Low molecular protein ratio (10KD or less) % Middle molecule protein ratio (10-60KD) % Macromolecule protein ratio (being greater than 60KD) %
Embodiment 1 96.9 1.8 1.3
Control group 1 90.5 7.6 2.9
Control group 2 88.1 6.8 5.1
Control group 3 91.8 5.4 2.8
Control group 4 84.2 10.8 5.0
As shown in table 1, the present invention use four kinds of bacterium reasonable compatibilities, producing enzyme of mutually promoting, can fully degraded dregs of beans albumen, then By ultrasonic treatment and enzymatic treatment, it can degrade to mycoprotein, improve the content of low molecular weight protein, ratio reaches To 96.9%, it is better than control group 1-4；As shown in Figure 1, free amino acid, oligopeptides (2-9 amino in enzymolysis liquid in embodiment 1 Acid), polypeptide (10-50 amino acid) and restore saccharic composition be above the control group 1-4 only with three kinds of bacterial strains；Embodiment 1 The protein hydrolysis degree (the ratio between the peptide bond number being cleaved in protein hydrolytic process and total peptide bond number of given protein) of group reaches 58%, also above other groups (as shown in Figure 2).The above results illustrate enzymatic hydrolysis of the present invention using four kinds of bacterial strain compatibilities to albumen Then better effect uses ultrasonic wave added lysozyme, in conjunction with acid protease, can degrade mycoprotein, increase in solution Free amino acid, small peptide molecule content, will be in protein enzymatic hydrolyzate application fermentation medium of the present invention, it is easier to be absorbed by bacterial strain It utilizes.
Embodiment 3
It is spray-dried and obtains dry powder (experimental group) after the enzymolysis liquid concentration prepared with embodiment 1, substitute the yeast in YPD culture medium Powder (control group), remaining components unchanged cultivate saccharomyces cerevisiae under the same conditions, evaluate and produce by comparing the growing state of cell The culture effect of product, is specifically shown in Table 2.
Table 2
12 hours (OD of saccharomyces cerevisiae culture600) 24 hours (OD of saccharomyces cerevisiae culture600)
Experimental group 0.642 1.916
Control group 0.614 1.851
As shown in table 3, compared with commercially available yeast powder, culture of the dry powder prepared by the present invention in culture yeasts bacterium more preferably, can be made For the substitute of commercially available yeast powder.
The above description of the embodiment is only used to help understand the method for the present invention and its core ideas.It should be pointed out that for this For the those of ordinary skill of technical field, without departing from the principle of the present invention, the present invention can also be carried out several Improvement and modification, these improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims (7)

1. the preparation process of protein enzymatic hydrolyzate comprising following steps: step 1) prepares dregs of beans culture solution, and step 2 prepares seed Liquid, step 3) fermentation, step 4) handle mycoprotein.
2. technique according to claim 1, which is characterized in that the technique includes the following steps:
Step 1) prepares dregs of beans culture solution: dregs of beans and water being mixed, then 121 DEG C of steam treatment 10min naturally cool to room Temperature obtains dregs of beans culture solution；
Step 2 prepares seed liquor: by bacillus licheniformis, Cellumomonas flavigena, aspergillus niger and aspergillus oryzae respectively according to normal Rule culture obtains seed liquor；
Step 3) fermentation: by bacillus licheniformis seed liquor, Cellumomonas flavigena seed liquor, aspergillus niger seed liquor and meter Qu Mould seed liquor mixing, obtains seed mixture liquid；Then seed mixture liquid is linked into according to 10% inoculum concentration containing dregs of beans culture Carry out enzymatic production in the fermentor of liquid, temperature is 35 DEG C, and tank pressure is 0.03MPa, air quantity 500L/h, and the enzymatic production time is 60h；
Step 4) handles mycoprotein: ultrasonic treatment is then heated to 55 DEG C, and adding sulfuric acid adjustment pH is 6, is separately added into lysozyme And acid protease, under heat-retaining condition, digest 12 hours；Then 121 DEG C of steam treatment 10min, cooled to room temperature obtain Protein enzymatic hydrolyzate.
3. technique according to claim 2, which is characterized in that in the step 1), by dregs of beans and water according to the quality of 1:1 Than mixing.
4. technique according to claim 2, which is characterized in that in the step 3), by bacillus licheniformis seed liquor, produce Yellowish fiber monad seed liquor, aspergillus niger seed liquor and aspergillus oryzae seed liquor are mixed according to the volume ratio of 2:2:1:1.
5. technique according to claim 2, which is characterized in that in the step 3), fermentating enzyme-producing condition are as follows: temperature 35 DEG C, tank pressure is 0.03MPa, and air quantity 500L/h, the enzymatic production time is 60h.
6. technique according to claim 2, which is characterized in that the condition of the ultrasonic treatment are as follows: supersonic frequency is 25kHz, ultrasonic time 30min.
7. technique according to claim 2, which is characterized in that the additive amount of the lysozyme is 20,000 U:1L solution, acid The additive amount of protease is 10,000 U:1L solution.
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