CN116790388A - Rhizopus oryzae strain capable of producing acidic starch saccharification enzyme in high yield and yeast making method thereof - Google Patents
Rhizopus oryzae strain capable of producing acidic starch saccharification enzyme in high yield and yeast making method thereof Download PDFInfo
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Abstract
The invention belongs to the fields of microorganisms and fermentation brewing engineering, and particularly relates to a rhizopus strain capable of producing acidic starch saccharification enzymes in high yield and a yeast making method. The strain is rhizopus oryzaeRhizopus oryzae) XB-1 is preserved in China general microbiological culture Collection center (CGMCC) for 6-month 2 of 2023, and has a preservation address of CGMCC No.40646, which is a institute of microbiological study, national academy of sciences, no. 3, beijing, chaoyang, 1, and a high yield of acid amyloglucosidase, and acid protease, and can effectively improve activity and quality of bran kojiThe amount stability is improved, and the raw material utilization rate of the solid table vinegar is improved obviously.
Description
Technical Field
The invention belongs to the fields of microorganisms and fermentation brewing engineering, and particularly relates to rhizopus oryzae capable of producing acidic amylosaccharifying enzyme with high yield and a screening method thereof, and a starter propagation method of rhizopus oryzae.
Background
Vinegar is an indispensable seasoning in people's life and is also the most widely used acid seasoning in the world, chinese vinegar is mainly prepared by fermenting grains such as sorghum, rice, glutinous rice and the like as raw materials, and main flavor substances in vinegar comprise organic acids, amino acids, saccharides, polyphenols, flavone, alcohols, esters, aldehydes, ketones and other compounds; in the vinegar fermentation process, the traditional brewing process mainly uses experience, and has the problems of low standardization degree, low raw material utilization rate and the like, so that the fermentation process in the vinegar industry needs to be improved; bran koji is a prominent representation of vinegar koji, is taken as a microbial preparation and an enzyme preparation to participate in the formation of flavor substances in the brewing process, and mold is one of main strains in the koji, can secrete various functional enzymes such as saccharifying enzyme, protease and the like, and can promote the decomposition and utilization of raw materials, so that the bran koji has important significance in screening and applying multifunctional mold to vinegar brewing.
At present, in order to improve the flavor and quality of the solid state fermentation vinegar, the following modes are mainly adopted: (1) a multi-strain mixed fermentation technology (2) is adopted to change the post-maturation aging study of the vinegar brewing raw material (3).
It has been reported that a strain of aspergillus tubingensis with high yield of saccharifying enzyme, alpha-amylase and acid protease is provided in the invention patent 201410185333.6, and has better raw material utilization capability, however, the application object of the strain of aspergillus tubingensis is the white spirit industry, and the main flavor substances of the aspergillus tubingensis are greatly different from those of table vinegar; the invention patent 201410014845.6 discloses a rhizopus chinensis strain with high acid protease activity, and describes a culture method and application thereof in detail, but the rhizopus chinensis strain is mainly applied to brewing by preparing bran koji, which is different from the application object of the patent; the invention patent 201910393499.X mentions a strain of rhizopus oryzae with high yield of saccharifying enzyme, which is derived from highland barley small yeast, the saccharifying enzyme activity is 1400-1500U/g, the acid protease activity is 60-100U/g, which is different from the source of the strain of the invention, and the saccharifying enzyme activity and the protease activity are lower than the invention.
Disclosure of Invention
The invention aims to screen and obtain high-yield acid starch saccharifying enzyme mould, prepare bran koji for vinegar fermentation by using the strain, complete decomposition of starchiness and proteins in the koji making process, and simultaneously form some important flavor substances such as amino nitrogen and ester substances so as to achieve the purposes of improving the raw material utilization rate of vinegar in the fermentation process, enriching the flavor substances and improving the product quality, thereby solving the problems in the background technology.
In order to achieve the above purpose, the present invention adopts the following technical scheme.
Different brewing Daqus are selected, including Angel Xiangba, angel fermented starter, angel sweet fermented starter, angel small yeast, angel rhizopus, etc., and 6 rhizopus strains are obtained from the yeast sample by adopting the traditional mould separation and purification means.
The determination of the activity of the saccharifying enzyme of the mould bran koji is referred to the method in QB/T4257-2011 general analysis method of Daqu for brewing.
Determination of the protease activity of mould moldy bran is referred to SB/T10317-1999 protease activity assay.
Obtaining a high-yield acid amylosaccharifying enzyme with higher acid starch productionMould of powder saccharifying enzyme and acid proteinase, no XB-1, is identified as rhizopus oryzae by ITS sequencingRhizopus oryzae) The method comprises the steps of carrying out a first treatment on the surface of the The mould XB-1 is cultivated on a solid culture medium, and at the initial stage of cultivation, the nutrition hypha is white and creeping on the culture medium; in the middle culture period, hyphae are cotton-like and are fully distributed on the flat plate; in the later stage of culture, black spores appear at the top of aerial hyphae, and bacterial colonies are large and loose; the mould XB-1 can directly observe creeping hyphae and rhizoids of mould under a low-power mirror, the cyst stems have no branches, and the tail ends of the spore stems are connected with circular and dark-color sporangia.
The results of the spore production curve and environmental tolerance test on rhizopus oryzae XB-1 in a bran culture medium show that the strain XB-1 has strong temperature tolerance capability and good growth activity at a high temperature of 36 ℃ when being cultured for 3d as optimal spore production time and 4d as optimal saccharifying enzyme production time, and ethanol has certain influence on the growth of the strain and saccharifying enzyme activity and good growth performance and saccharifying enzyme activity within 1% of ethanol content.
The present invention has the following advantages.
The rhizopus oryzae starter propagation provided by the invention has the acid-producing starch saccharifying enzyme activity of 2057.63 +/-149.35U/g dry mash, the acid-producing starch saccharifying enzyme activity of 1310.81 +/-100.27U/g dry mash and the acid-producing protease activity of 428.27 +/-25.45U/g dry mash, and the saccharifying enzyme activity and the protease activity of the rhizopus oryzae starter propagation are obviously higher than those of the traditional Daqu starter propagation, so that the rhizopus oryzae starter propagation can be applied to a solid state fermentation edible vinegar starter propagation process, and the bran starter enzyme activity and the quality stability are effectively improved.
The rhizopus oryzae strain provided by the invention is used for fermenting bran koji in industrial production, has developed hypha, and is more suitable for fermenting bran koji.
The invention adds rhizopus oryzae to the mixtureRhizopus oryzae) The XB-1 is used for preparing bran koji with aromatic koji flavor.
Biological preservation information.
Rhizopus oryzaeRhizopus oryzae) XB-1 is preserved in China general microbiological culture Collection center (CGMCC) for 6 months and 2 days of 2023, with a preservation address of CGMCC No.4064, which is the institute of microbiological study, national academy of sciences No. 3, national institute of sciences, no. 1, north China, chenglu, korea, beijing, and a collection position of China6。
Drawings
FIG. 1 is a screen of rhizopus oryzae XB-1 producing an acidic amyloglucosidase.
FIG. 2 is a screen for acid producing amylosaccharifying enzymes produced by Rhizopus oryzae XB-1.
FIG. 3 is a screen of rhizopus oryzae XB-1 acid protease producing mold.
FIG. 4 is a Rhizopus oryzae XB-1 phylogenetic tree.
FIG. 5 shows the spore production curve and the enzyme activity of Rhizopus oryzae XB-1.
Detailed Description
Example 1 screening and identification of high yield acid saccharifying enzyme mould.
And (5) separating and purifying the mould.
Weighing 5 g yeast sample, adding into a triangular flask containing 45 ml sterile distilled water, adding several glass beads, shaking in 150rpm/min shaking table for 30 min, and making the obtained liquid into different dilution gradients 10 -3 、10 -4 、10 -5 、10 -6 The preparation method comprises the steps of weighing 200-g potatoes, cleaning, peeling, cutting, adding 1000-ml of water, boiling for half an hour, filtering with gauze, adding 10-20-g of glucose and 15-20-g of agar, fully dissolving, split charging, sterilizing at 121 ℃ for 20 minutes, culturing for 24-h, selecting strains with a mould typical colony form, purifying for multiple times, and preserving in an inclined plane PDA culture medium.
And (5) screening the acid-producing amylosaccharifying enzyme mould.
(1) Preparation of bran koji.
Accurately weighing 10 g bran in a triangular flask, adding 10 g distilled water, stirring uniformly, steaming at 121 ℃ under high pressure for sterilization for 20 min, scattering the material blocks after steaming, and cooling the bran for later use; culturing the mould obtained by screening on an inclined plane at 28 ℃ for 3d, eluting mould spores on the inclined plane into 5 mL spore eluent, taking 500 mu L of spore suspension in a bran culture medium, uniformly mixing, and culturing at 28 ℃ for 72 h, wherein each mould is parallel for 3 times.
(2) Determination of the saccharification force of the mycotic bran.
Saccharification force refers to the content of glucose generated by decomposing soluble starch by 1 h under the condition that the absolute dry yeast of 1 g is at 40 ℃ and the pH value is 4.6, and the measurement of the saccharification force of the bran is referred to QB/T4257-2011 'general analytical method for Daqu for brewing', and the moisture content of the bran is measured by a moisture rapid tester; the results of the measurement of the bran koji saccharification force are shown in figure 1, wherein the activity level of the saccharifying enzyme of the XB-1 mould bran koji in the measured strain is highest, and is 2057.63 +/-149.35U/g dry fermented grains, and the level of the bran koji saccharification force of the SFM-1 is next highest, and is 1840.51 +/-134.15U/g dry fermented grains.
Screening of acid-producing amylosaccharifying enzyme mould.
Raw starch saccharification force refers to the content of glucose generated by decomposing raw starch by 1 h under the conditions that the temperature of the absolute dry yeast of 1 g is 40 ℃ and the pH value is 4.6, and the measurement method is the same as that of the raw starch, and wheat starch is used for replacing soluble starch; the measurement results are shown in fig. 2, and the saccharification force level of raw starch is from high to low: XQ-1> SFM-1> XB-1> TQ-1> SGM-1> NQ-1.
Screening of acid protease-producing mould.
The casein was hydrolyzed at a temperature (40 ℃ C. Under 0.2 ℃) and at a corresponding pH (acid protease pH 3.0) to produce an enzyme amount equivalent to 1. Mu.g of a phenolic amino acid (expressed by tyrosine equivalent) within 1min, which is 1 enzyme activity unit, and the measurement method is described in SB/T10317-1997 protease activity assay, and the results are shown in FIG. 3, wherein the protease activities of three strains SFM-1, XB-1 and TQ-1, namely 458.5 + -11.13, 428.27 + -25.45 and 415.22 + -3.50U/g of dry fermented grains, respectively.
Taking the saccharifying enzyme activity as a main screening basis, combining the saccharifying enzyme activity and the protease activity of the strain, finally selecting mould XB-1 as a target strain, and carrying out strain identification, spore production curve and saccharifying enzyme activity condition analysis on the target strain.
Identification of strains.
(1) Morphological identification: carrying out morphological observation on the strain XB-1 on a solid PDA culture medium, wherein at the initial stage of culture, the nutritional hypha is white, and creeping growth is carried out on the culture medium; in the middle culture period, hyphae are cotton-like and are fully distributed on the flat plate; in the later stage of culture, black spores appear at the top of aerial hyphae, and bacterial colonies are large and loose; the creeping hyphae and the rhizomes of the mould can be directly observed under an optical microscope (10 multiplied by 10), the cyst stems have no branches, and the tail ends are connected with circular and dark sporocysts.
(2) Molecular biology identification: extracting the total DNA of the mould XB-1, amplifying by using a universal primer to obtain the ITS sequence, sequencing the ITS sequence, and comparing the sequence in an NCBI database.
The strain XB-1 is subjected to ITS sequence determination to obtain the following sequences:
CCTTCCGTAGGGGAACCTGCGGAAGGATCATTAATTATGTTAAAGCGCCTTACCTTAGGGTTTCCTCTGGGGTAAGTGATTGCTTCTACACTGTGAAAATTTGGCTGAGAGACTCAGACTGGTCATGGGTAGACCTATCTGGGGTTTGATCGATGCCACTCCTGGTTTCAGGAGTACCCTTCATAATAAACCTAGAAATTCAGTATTATAAAGTTTAATAAAAAACAACTTTTAACAATGGATCTCTTGGTTCTCGCATCGATGAAGAACGTAGCAAAGTGCGATAACTAGTGTGAATTGCATATTCAGTGAATCATCGAGTCTTTGAACGCAGCTTGCACTCTATGGTTTTTCTATAGAGTACGCCTGCTTCAGTATCATCACAAACCCACACATAACATTTGTTTATGTGGTGATGGGTCGCATCGCTGTTTTATTACAGTGAGCACCTAAAATGTGTGTGATTTTCTGTCTGGCTTGCTAGGCAGGAATATTACGCTGGTCTCAGGATCTTTTTTTTTGGTTCGCCCAGGAAGTAAAGTACAAGAGTATAATCCAGTAACTTTCAAACTATGATCTGAAGTCAGGTGGGATTACCCGCTGAACTTAAGCATATCAATAAGCGGAGGAA。
the result shows that the homology of the strain and rhizopus oryzae reaches 99%, the sequence of the strain is uploaded to NCBI, and a phylogenetic tree is constructed for the strain and the strain with higher sequence similarity by using MEGA7.0 software (figure 4); as can be seen from FIG. 4, the bacterium and LC514299.1Rhizopus oryzaeThe affinity of UICC 8 is the closest.
Comprehensive morphological and molecular biological test results, mold XB-1 was identified asRhizopus oryzaeThe strain is preserved in China general microbiological culture Collection center (CGMCC) with a preservation address of CGMCC No.40646, which is the national institute of microbiology, national academy of sciences, national institute of sciences, no. 1, north Chen, west Lu, korea, beijing, and a preservation number of China.
Example 2 rhizopus oryzae XB-1 spore production curve and saccharifying enzyme activity.
The culture medium of the triangular flask bran is prepared for standby, and the spore production characteristics and the saccharifying enzyme activity condition of rhizopus oryzae XB-1 are studied according to the culture time (0-6 d), the culture temperature (10, 20, 28, 36, 40, 45 ℃) and the ethanol addition amount (0%, 0.5%, 1.0%, 1.5%, 2.0% and 3.0%).
According to the conditions, rhizopus oryzae XB-1 is inoculated in a bran culture medium, the saccharification power and biomass of the bran koji are measured at different time points, and the influence of the culture time on the activity and biomass of saccharifying enzyme is explored; as shown in FIG. 5, rhizopus oryzae XB-1 produced spores in 3d cultivation is maximized and tends to be stable, and the activity of saccharifying enzyme in 4d cultivation is highest, 2428.99 + -6.27U/g, and the tendency of decreasing with time is observed, so that the optimal cultivation time is 4 d.
Through culturing rhizopus oryzae XB-1 in bran culture medium at different temperatures, the rhizopus oryzae XB-1 is found to have strong tolerance to temperature, and has strong growth capacity at 36 ℃ and saccharifying enzyme activity of 2601.84 +/-18.46U/g; meanwhile, the ethanol has a certain negative effect on the growth of the strain and the activity of the saccharifying enzyme, and has better growth performance and saccharifying enzyme activity within 1% of the ethanol content, the 0 ethanol group saccharifying enzyme activity is 2557.93 +/-2.42U/g, and the saccharifying enzyme activity is reduced to 1503.82 +/-3.83U/g when 3% of ethanol is added.
The foregoing is only a preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art, who is within the scope of the present invention, should be covered by the protection scope of the present invention by making equivalents and modifications to the technical solution and the inventive concept thereof.
Claims (4)
1. Rhizopus oryzae strain capable of producing acid starch saccharifying enzyme in high yieldRhizopus oryzae) XB-1 is preserved in China general microbiological culture Collection center (CGMCC) for 6 months and 2 days of 2023, the preservation address is CGMCC No.40646, and rhizopus oryzae XB-1 is mainly used in food fermentation.
2. Use of rhizopus oryzae according to claim 1, characterized in that: the food fermentation is solid fermentation process of wines or vinegar.
3. A starter propagation method comprising rhizopus oryzae according to claim 1, characterized in that: can effectively improve the activity and quality stability of the bran koji enzyme and remarkably improve the utilization rate of the solid state fermentation raw materials.
4. A method for making koji according to claim 3, wherein: the optimal culture time of the bran koji is 4d, and the optimal culture temperature is 36 ℃.
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