CN109971657B - Rhizopus oryzae capable of producing saccharifying enzyme at high yield and application of rhizopus oryzae - Google Patents
Rhizopus oryzae capable of producing saccharifying enzyme at high yield and application of rhizopus oryzae Download PDFInfo
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Abstract
The invention discloses rhizopus oryzae for high-yield saccharifying enzyme and application thereof in optimizing a starter propagation process of highland barley Xiaoqu, belonging to the field of microorganism and fermentation brewing engineering. The rhizopus oryzae is obtained by separating highland barley koji from different regions of Tibetan in China, the screening method comprises the steps of collecting crushed highland barley koji, separating and purifying mould by using a culture medium method, measuring the activities of saccharifying enzyme, liquefying enzyme and protease of strains through primary screening, secondary screening and fermentation experiments, and selecting the strains with good fermentation performance and relatively high saccharifying enzyme activity; the strain can effectively improve the saccharification activity and quality stability of the highland barley koji with the characteristics in Tibet regions.
Description
Technical Field
The invention belongs to the field of microorganism and fermentation brewing engineering, and particularly relates to rhizopus oryzae with high glucoamylase yield and a screening method thereof, wherein the rhizopus oryzae with high glucoamylase yield is stored in Guangdong province microorganism strain collection center with the collection number of GDMCC NO.60628; the invention also relates to application of the rhizopus oryzae in optimizing a highland barley starter propagation process.
Background
The traditional highland barley wine is fermented wine with distinctive characteristics, which is prepared by taking highland barley which is a main grain crop of Tibet as a raw material and adding highland barley Xiaoqu for saccharification and fermentation, and is a representative of Tibet wine culture and has the characteristics of proper taste, low alcoholic strength, high nutritional efficacy and the like. Is the essential fine brewing for Tibetan people in the occasions of relatives and friends party, important festivals, welcome guests, weddings and funerals, and the like. It is closely related to the production and life of local people.
The highland barley koji is a wine-making leaven prepared by Tibet people in a traditional natural inoculation mode, contains rich microorganisms and enzymes generated by the microorganisms, participates in saccharification, liquefaction, alcohol fermentation and flavor formation in the wine-making process, has great influence on the wine yield and the wine body quality due to the quality, and is called as 'the bone of wine'. Therefore, the quality and the flavor of the highland barley wine are determined to a great extent by the microorganisms in the highland barley koji. At present, the quality of highland barley Xiaoqu is unstable due to the influence of factors such as geographical position, brewing process, starter propagation raw materials, environment and the like, so that the problems of low wine yield, generation of unpleasant flavor substances such as bitterness and the like, turbid wine body, large taste difference in different areas, difficulty in storage and the like of the highland barley wine are caused.
The saccharifying enzyme has the function of sequentially hydrolyzing alpha-1, 4-glucosidic bonds from the non-reducing end of starch to generate glucose, is applied to microbial fermentation to generate alcohol, provides raw materials and nutrient substances for subsequent fermentation, and is one of key enzymes in the fermentation process of the highland barley wine. In addition, the barley koji mainly contains mould which converts starch in the raw materials into reducing sugar for the growth and utilization of other microorganisms. The activity of the saccharifying enzyme is one of important indexes for evaluating the quality of the distiller's yeast, and the higher activity of the saccharifying enzyme is beneficial to fully utilizing raw materials and improving the liquor yield.
Therefore, the screening of the mould with high saccharifying enzyme activity, particularly the screening of the mould strain with good fermentation performance is one of the main measures for improving the saccharifying enzyme activity of the highland barley koji, producing the koji suitable for brewing the highland barley wine in Tibet areas and stabilizing and improving the quality of the highland barley wine.
Disclosure of Invention
One of the purposes of the invention is to provide rhizopus oryzae with high glucoamylase yield aiming at the current situation that the quality of highland barley koji in Tibet area is unstable, and the rhizopus oryzae strain capable of remarkably improving and stabilizing the glucoamylase activity of highland barley koji is of great significance for developing highland barley koji products with characteristics in Tibet area.
Another object of the present invention is to provide a method for screening Rhizopus oryzae as described above.
The invention also discloses a method for preparing the highland barley koji in Tibet region by using the rhizopus oryzae.
In order to achieve the above object, the present invention provides a Rhizopus oryzae strain with high glucoamylase yield, the taxonomic name of the fungus is Rhizopus oryzae, and the biological material is: rhizopus oryzae YC666, deposited in Guangdong province microbial culture Collection in 2019, 4/11, with the deposit number GDMCC NO.60628.
The high-yield glucoamylase rhizopus oryzae provided by the invention has the advantages that bacterial colonies growing on a solid PDA culture medium for 72 hours are villous, are initially white, are changed into grey brown after being changed into grey brown, are dry, large, loose and opaque, are wound by hyphae, are easy to pick up, and are paved on a whole flat plate; the rhizopus oryzae glucoamylase provided by the invention has the enzyme activity of 1400-1500 mg/g.h, the liquefying enzyme activity of 1.3-1.4 g/g.h, the neutral protease activity of 800-900 mu g/g.min and the acidic protease activity of 60-100 mu g/g.min.
The invention provides a screening method of the rice-root enzyme, which comprises the following steps:
step 1) crushing a proper amount of highland barley Xiaoqu collected in different regions of Tibet, performing gradient dilution under aseptic operation, coating the crushed highland barley Xiaoqu in a separation culture medium, performing static culture in an incubator at 28 ℃ for 2-3 days, randomly selecting a single bacterial colony with typical mould bacterial colony characteristics, performing streak separation and purification for 2-3 times, and storing an inclined plane for later use;
and 2) carrying out primary screening, secondary screening and fermentation tests on the separated mould strain to obtain a mould strain with very high glucoamylase activity.
In some preferred embodiments of the present invention, the primary screening method in step 2) adopts a mold saccharifying enzyme transparent ring and protease transparent ring test, and specifically comprises the following steps: the method comprises the steps of adopting a spot grafting method, sequentially spot-grafting mould strains on a primary screening culture medium by using an aseptic toothpick, culturing for 72 hours at 28 ℃, measuring the diameter D of a bacterial colony, adding 4mL of dilute iodine solution into a culture dish, measuring the diameter D of a transparent ring after 5min, selecting the strain with a larger D/D ratio, spot-grafting the strain on the primary screening culture medium producing protease, culturing for 72 hours at 28 ℃, measuring the diameter D of the bacterial colony and the diameter D of the transparent ring, and selecting the strain with the larger D/D ratio. In some more preferred embodiments of the invention, the prescreening medium and protease-producing prescreening medium used comprise the components:
primary screening of a culture medium: 10g/L of soluble starch, 1.5g/L of yeast extract powder and NaNO 3 1.5g/L，K 2 HPO 4 1g/L，NaCl 0.5g/L，MgSO 4 0.5g/L，FeSO 4 0.01g/L of agar and 20g/L of agar;
primary screening culture medium for protease production: 5g/L beef extract, 10g/L peptone, 5g/L NaCl, 3g/L skimmed milk powder and 20g/L agar.
In some preferred embodiments of the present invention, the re-screening method in step 2) adopts fermentation performance (saccharifying power, liquefying power and protease activity) test of mold starter propagation, and comprises the following specific processes: suspending mould spore (spore number is 10) 6 About one seed/mL) is inoculated into a bran culture medium according to the inoculation amount of 10 percent, mixed evenly, cultured for 72 hours at the temperature of 28 ℃, and then the flask is buckled for continuous culture until the culture lasts for 6 days. Drying for 12h at 40 ℃ in a forced air drying oven; the saccharifying power and liquefying power of the mouldy bran are measured according to QB/T4257-2011, the protease activity is measured according to GB/T23527-2009, and strains with good fermentation performance are selected; in some more preferred embodiments of the present invention, the bran culture medium used is composed of the following specific processes: sieving testa Tritici with 80 mesh sieve, spraying water onto testa Tritici at water-to-material ratio of 40%, mixing, moistening for 30min, and packagingSterilizing 30min at 121 ℃ in a 500mL triangular flask with 30g per flask, and scattering for later use when the temperature is reduced to 30-35 ℃.
In some preferred embodiments of the present invention, the fermentation test in step 2) comprises the following specific steps:
(1) preparing a simulated highland barley Xiaoqu raw material: sieving semen Avenae Nudae powder with 80 mesh sieve, placing into a basin, covering the basin, and aging at 121 deg.C for 30min; after cooling, uniformly spraying water on the highland barley powder by a spray can according to the water-material ratio of 40%, uniformly stirring, and sieving by a 20-mesh sieve; sieving testa Tritici with 80 mesh sieve, spraying water onto testa Tritici at water-to-material ratio of 40%, mixing, and moistening for 30min; uniformly mixing highland barley powder and bran according to a ratio of 1;
(2) simulating fermentation of highland barley Xiaoqu: preparing spore suspension from the mold obtained by re-screening in claim 5, inoculating into highland barley flour bran culture medium according to the inoculation amount of 10%, mixing, culturing at 28 deg.C for 72h, buckling bottle, and continuously culturing to 6d; drying for 12h at 40 ℃ in a forced air drying oven; the saccharification capacity of the moldy bran is measured according to QB/T4257-2011, and a mold Rhizopus oryzae YC666 with relatively high saccharification capacity is selected.
In some embodiments of the present invention, the isolation medium in step 1) is a PDA (chloramphenicol) -containing medium, specifically, the PDA (chloramphenicol) -containing medium comprises: 3g/L of potato extract powder, 20g/L of glucose, 20g/L of agar and 0.1g/L of chloramphenicol.
In some embodiments of the present invention, the slant storage medium in step 1) is PDA medium, which comprises the following components: 3g/L of potato soaking powder, 20g/L of glucose and 20g/L of agar.
In some embodiments of the present invention, the mold spore suspension is prepared by the following steps:
pouring 10mL of sterile normal saline into the activated strain slant, scraping hyphae on the slant by using an inoculating needle, scattering and uniformly mixing spores in the test tube, counting by using a blood counting chamber, and diluting the number of the spores to about 106/mL.
In some embodiments of the invention, the specific process for preparing the bran culture medium used is: sieving testa Tritici with 80 mesh sieve, spraying water onto testa Tritici at water-to-material ratio of 40%, mixing, moistening for 30min, subpackaging in 500mL triangular bottles, each bottle containing 30g, sterilizing at 121 deg.C for 30min, and scattering when the temperature is reduced to about 30 deg.C.
The invention provides a highland barley starter propagation process, which adopts the Rhizopus oryzae YC666 provided by the invention to carry out highland barley starter propagation process; in some embodiments of the present invention, the Rhizopus oryzae rhizozae YC666 provided by the present invention is prepared into spore suspension, and then is produced according to the fermentation process of the highland barley Xiaoqu.
Compared with the prior art, the invention has the following beneficial effects:
1. the activity of the saccharifying enzyme of the optimized highland barley koji provided by the invention is 1482 mg/g.h, compared with the commercialized rhizopus oryzae, the activity of the saccharifying enzyme is improved by 15%, and compared with the traditional highland barley koji, the activity of the saccharifying enzyme is improved by 67%; secondly, the liquefying enzyme activity of the rhizopus oryzae is 1.3-1.4 g/g.h, the neutral protease activity is 800-900 mug/g.min, and the acidic protease activity is 60-100 mug/g.min, so that the rhizopus oryzae can be used as a saccharifying bacterium to be applied to the optimization of the characteristic highland barley koji making process in Tibet regions, and the saccharifying activity and the quality stability of the highland barley koji with the characteristics in Tibet regions are effectively improved.
2. The rhizopus oryzae strain provided by the invention is used for fermentation of highland barley koji in industrial production, has developed hypha and is more suitable for fermentation of highland barley koji; secondly, the color of the strain provided by the invention is white, which is closer to the color of the highland barley Xiaoqu.
3. The highland barley koji with aromatic koji flavor is prepared by adding Rhizopus oryzae YC666.
Detailed Description
The Rhizopus oryzae provided by the invention is classified and named as Rhizopus oryzae, and the biological material is as follows: rhizopus oryzae YC666 was deposited in Guangdong province culture Collection on 11.4.2019 with the deposit number GDMCC NO.60628.
The present invention will be described in detail with reference to specific examples.
The methods in the following examples are all conventional methods unless otherwise specified.
The preparation of the simulated highland barley Xiaoqu raw material in the following embodiment comprises the following specific processes:
sieving semen Avenae Nudae powder with 80 mesh sieve, placing into enamel basin, covering the basin, and aging at 121 deg.C for 30min. After cooling, uniformly spraying water on the highland barley powder by a spray can according to the water-material ratio of 40%, uniformly stirring, and sieving by a 20-mesh sieve; in addition, the bran is sieved by a sieve of 80 meshes, water is sprayed on the bran according to the water-material ratio of 40 percent, and the mixture is evenly mixed and moistened for 30min. Uniformly mixing the highland barley powder and the bran according to a ratio of 1.
The components and preparation of the bran culture medium in the following examples are:
sieving testa Tritici with 80 mesh sieve, spraying water onto testa Tritici at water-to-material ratio of 40%, mixing, moistening for 30min, subpackaging in 500mL triangular bottles, each bottle containing 30g, sterilizing at 121 deg.C for 30min, and scattering when the temperature is reduced to about 30 deg.C.
The specific procedure for the preparation of the mold spore suspension in the following examples was:
pouring 10mL of sterile normal saline into the activated strain slant, scraping off hyphae on the slant with an inoculating needle, scattering and mixing spores in the test tube, counting with a blood counting plate, and diluting the number of the spores to 10 6 About one/mL.
The formulation of the primary screening medium and protease-producing primary screening medium used in the following examples was as follows:
primary screening of culture medium: 10g/L of soluble starch, 1.5g/L of yeast extract powder and NaNO 3 1.5g/L，K 2 HPO 4 1g/L，NaCl0.5g/L，MgSO 4 0.5g/L，FeSO 4 0.01g/L of agar and 20g/L of agar;
primary screening culture medium for protease production: 5g/L of beef extract, 10g/L of peptone, 5g/L of NaCl, 3g/L of skimmed milk powder and 20g/L of agar.
The PDA media formulations in the following examples are as follows:
3g/L of potato extract powder, 20g/L of glucose and 20g/L of agar.
The PDA (Chloramphenicol) medium formulation in the following examples is as follows:
PDA (chloramphenicol) -containing medium: 3g/L of potato extract powder, 20g/L of glucose, 20g/L of agar and 0.1g/L of chloramphenicol.
Example 1 isolation, purification and characterization of high-yielding saccharifying enzyme mold
21 mould strains separated from highland barley Xiaoqu collected from different regions of Tibet are separated, identified and screened. The separation method adopts a plate dilution coating method to pick a single colony and carry out continuous purification twice, and the identification method adopts the traditional morphology and combines with ITS region sequence analysis.
The strain is identified as a new strain of rhizopus oryzae by morphology and ITS region sequence analysis, and the specific identification result is as follows:
a virtual form observation method and results: after 72h culture, the colony growing on PDA (chloramphenicol) -containing culture medium is villous, initially white, then grey brown, dry, large, loose, opaque, and easy to pick up, and hyphae intertwine, and spread over the whole plate.
Method and results of analysis of the capsule wall ITS zone sequence:
the extraction of the mould genome DNA adopts a kit method, and the specific method is carried out according to the instruction of a plant genome DNA extraction kit.
Secondly, the amplification of the specific fragment of the mold ITS region is synthesized by the company of the Pythium biological engineering (Shanghai) by adopting universal primers ITS1/ITS 4; PCR amplification conditions of 95 ℃ pre-denaturation 3min,95 ℃ denaturation 45s,53 ℃ annealing 30s,72 ℃ extension 45s (35 cycles), and finally 72 ℃ extension 8min; the composition of the PCR amplification system (25 uL) was: 10 XPCR Buffer 2.5 uL, d NTPs 2 uL, DNA template 1uL, taq enzyme 0.3 uL, ITS 1uL, ITS4 uL, using dd H 2 And supplementing O to 25 mu L, and mixing uniformly. The PCR amplification products were then detected: 5uL of PCR amplification product is mixed with 1uL and spotted on 1% agarose gel. Electrophoresis buffer solution is 1 XTAE, electrophoresis is carried out for 20min under the voltage of 120V, a gel imaging system is used for photographing an electrophoresis pattern and analyzing, the size of a PCR amplification product is judged by adopting 2000bp DNAmarker, and the result shows that the specific fragment of the mould is longThe degree is about 600bp.
(3) Analysis of the ITS sequence of the mould: the PCR amplification product was sequenced by Biotechnology engineering (Shanghai) Co., ltd, and based on the sequencing result, homology sequence search was performed from Genbank nucleic acid sequence database using BLAST software, and the degree of similarity between the test strain and the corresponding sequence of the known mold strain was compared.
From the current view point of the field of research in molecular systems of moulds: namely, strains with the same or similar ITS sequences (the sequence homology is more than or equal to 99 percent) belong to the same species, and the strains with the sequence homology of more than or equal to 99 percent with the gene bank reference strains are determined to be rhizopus oryzae according to the sequence analysis result.
Example 2 screening of high-yield saccharifying enzyme Rhizopus oryzae
Performing transparent ring primary screening, secondary screening and fermentation test on the identified strains to obtain a plurality of mould strains with excellent brewing characteristics, and inoculating the mould strains with 10% of inoculum size (spore number: 10) in a simulated highland barley Xiaoqu starter-making raw material 6 seed/mL), fermenting at 28 deg.C for 6 days, measuring diastase activity of semen Avenae Nudae Xiaoqu after fermentation, and evaluating sensory index.
Through repeated screening, verification and brewing research, the rhizopus oryzae with high saccharifying enzyme activity and good fermentation performance is obtained by separating the small highland barley koji in Tibet region, the small highland barley koji brewed by the strain has high and stable saccharifying enzyme activity, white hypha grows on the surface of the small highland barley koji brewed by the strain and is uniformly distributed, and the small highland barley koji has no infectious microbes, strong koji flavor and no foreign flavor.
The specific screening process is as follows:
the transparent circle capability test of product that the machine was used to mould: adopting a point inoculation method, using an aseptic toothpick to inoculate a mould strain on a primary screening culture medium of a diastase-producing strain, culturing for 72 hours at 28 ℃, measuring the diameter D of a bacterial colony, adding 4mL of diluted iodine solution into a culture dish, measuring the diameter D of a transparent ring after 5min, selecting the strain with larger ratio D/D to inoculate on the primary screening culture medium of the diastase, culturing for 72 hours at 28 ℃, measuring the diameter D of the bacterial colony and the diameter D of the transparent ring, and selecting the strain with larger ratio D/D.
Testing the fermentation performance of the capsule wall mildew: inoculating 10% of mould spore suspension into bran culture medium, mixing, culturing at 28 deg.C for 72h, buckling bottle, and culturing for 6d. Drying for 12h at 40 ℃ in a forced air drying oven; and (3) measuring the saccharifying power and liquefying power of the mouldy bran according to QB/T4257-2011, measuring the protease activity according to GB/T23527-2009, and selecting a strain with good fermentation performance.
\9354: preparing spore suspension from mould, inoculating into highland barley flour bran culture medium at an inoculum size of 10%, mixing, culturing at 28 deg.C for 72 hr, buckling bottle, and culturing for 6 days. Drying for 12h at 40 ℃ in a forced air drying oven; and (4) measuring the saccharifying power of the mouldy bran according to QB/T4257-2011, and selecting rhizopus oryzae with highest saccharifying power.
Comparative example 1 isolation of commercial Rhizopus oryzae
Diluting and coating sweet distiller's yeast purchased from Angel Yeast GmbH in gradient, standing and culturing in 28 deg.C incubator for 3d, selecting single colony with Rhizopus oryzae colony characteristic, streaking, separating and purifying for 2-3 times, and storing on inclined plane for use.
Example 3 comparative test for miniature koji-making
Example 2 comparison of the production of high-yield glucoamylase Rhizopus oryzae with the production of commercial Rhizopus oryzae in a small starter propagation in highland barley.
Respectively filling 500g highland barley flour bran culture medium into 3 5L wide-mouth bottles, respectively adding screened high-yield rhizopus oryzae, commercialized rhizopus oryzae and highland barley Xiaoqu in a certain production area of Tibet region to make the final concentration of spore suspension reach 10 6 About one/mL, and producing according to a highland barley Xiaoqu fermentation test process. And (4) measuring the activity of the saccharifying enzyme and evaluating sensory indexes after fermentation.
TABLE 1 results of saccharifying power determination of two Rhizopus oryzae strains and highland barley Xiaoqu in Tibet region
Table 2 results of sensory index evaluation of two Rhizopus oryzae strains and production of highland barley Xiaoqu from highland barley Xiaoqu in Tibet region
The highland barley koji prepared by adding high-yield saccharifying enzyme Rhizopus oryzae has highest saccharifying power of 1482 + -77.38 mg/g.h, uniform surface hypha, white surface, and no mixed bacteria. Therefore, the screened strains meet the requirements of industrial fermentation, and simultaneously, the saccharifying enzyme activity of the screened strains is higher than that of commercial rhizopus oryzae and highland barley koji in a certain production area of Tibet; the fermented highland barley Xiaoqu has strong yeast flavor and no peculiar smell, and is more beneficial to preparing the Tibetan special highland barley wine product.
Claims (3)
1. A rhizopus oryzae capable of producing glucoamylase with high yield is classified and namedRhizopus oryzaeAnd the accession number is GDMCC NO.60628.
2. The use of Rhizopus oryzae as claimed in claim 1 in the process of making koji from highland barley koji.
3. The use as claimed in claim 2, wherein the Rhizopus oryzae is prepared into spore suspension, and then the fermentation process is carried out according to highland barley koji fermentation process.
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