CN115491315B

CN115491315B - Rhizopus oryzae for space breeding and application thereof in preparation of bran koji and brewing

Info

Publication number: CN115491315B
Application number: CN202211420749.2A
Authority: CN
Inventors: 丁子元; 刘沛通; 孙玉婷; 郑晓卫; 邹斐; 余冰; 冯亮; 陈晓园; 张无疾
Original assignee: Jiugui Liquor Co ltd; Cofco Nutrition and Health Research Institute Co Ltd
Current assignee: Jiugui Liquor Co ltd; Cofco Nutrition and Health Research Institute Co Ltd
Priority date: 2022-11-15
Filing date: 2022-11-15
Publication date: 2023-04-25
Anticipated expiration: 2042-11-15
Also published as: CN115491315A

Abstract

The invention relates to the technical field of biology, in particular to rhizopus oryzae for space breeding and application thereof in preparation of bran koji and brewing. The preservation number of the rhizopus oryzae is CGMCC No.40234. The bran koji prepared by the strain has high saccharification force, the content of the isoamyl acetate in the white wine brewed by the bran koji is obviously improved, the wine has pleasant fruit fragrance, and the wine body has excellent smell, flavor and texture, can improve the yield and the quality of the white wine, has important significance for improving the economic benefit of white wine enterprises, and has good application prospect.

Description

Rhizopus oryzae for space breeding and application thereof in preparation of bran koji and brewing

Technical Field

The invention relates to the field of microorganisms, in particular to rhizopus oryzae, a microbial agent, bran koji and a preparation method thereof, application of rhizopus oryzae, microbial agent and bran koji in production of fermented foods, and wine and a preparation method thereof.

Background

Rhizopus oryzae is an important strain in the process of brewing white wine, has the characteristics of producing lipase, amylase, saccharifying enzyme, pectase and the like, and many distiller's yeasts find Rhizopus oryzae. Rhizopus oryzae has strong amylase production capacity and is widely used in starter propagation and brewing production. During the fermentation of distiller's yeast, rhizopus oryzae can produce aromatic flavoring substances such as ethyl acetate, ethyl propionate, isoamyl acetate, propanol, isoamyl alcohol, acetaldehyde, etc. The rhizopus oryzae cells also contain a small amount of enzyme system for producing organic acid such as lactic acid, so that the wine fermented by rhizopus oryzae has unique flavor. The rhizopus oryzae can be used for preparing pure rhizopus oryzae, and the pure rhizopus oryzae has the advantages of high saccharification power, short preparation period, low production cost and the like, and the existing winery partly using small starter uses pure rhizopus oryzae to produce white wine.

The microgravity effect, extreme temperature difference, high vacuum, high-energy particle radiation, weak magnetic field and the like in the space environment can generate mutagenesis effect on space microorganisms, and the gene mutation frequency is obviously improved, so that the biological properties (including colony characteristics, physiological and biochemical characteristics, individual morphology and the like) and fermentation production performance (including enzyme activity, aroma producing capability and the like) of the microorganisms are changed. At present, research on space breeding microorganisms at home and abroad is focused on space pathogenic bacteria, microbiological pharmacy and the like, but no related report on the preparation of bran koji and white spirit by space breeding rhizopus oryzae exists in China. The rhizopus oryzae with high saccharification capacity and excellent aroma producing performance is obtained by utilizing spatial breeding and screening, and has important significance for improving the yield and the quality of white spirit and improving the economic benefit of white spirit enterprises.

Disclosure of Invention

The invention aims to provide rhizopus oryzae with high saccharification power and excellent aroma producing performance, and bran koji prepared by using the rhizopus oryzae has the advantages of high saccharification power, high isoamyl acetate content and excellent flavor and texture of white spirit when being used for brewing the white spirit, and can improve the yield and the quality of the white spirit.

To achieve the above object, a first aspect of the present invention provides a rhizopus oryzae strainRhizopus oryzaeThe preservation number of the rhizopus oryzae is CGMCC No.40234.

In a second aspect the invention provides a microbial agent comprising rhizopus oryzae as described above.

In a third aspect, the present invention provides a method for preparing a bran koji, comprising: rhizopus oryzae as described above was inoculated into a bran koji medium for cultivation.

In a fourth aspect the present invention provides a bran koji obtainable by the process as described above.

In a fifth aspect the present invention provides the use of rhizopus oryzae, a microbial agent or a bran koji as described above for the production of a fermented food product.

In a sixth aspect the present invention provides a method of brewing wine, the method comprising: the bran koji as described above is inoculated into a brewing clinker for brewing.

In a seventh aspect the present invention provides wine brewed by the method of brewing as described above.

The rhizopus oryzae HTJG5-5 with high saccharification capacity and high fruity flavor is obtained through space mutagenesis, and the number is BC70162. The bran koji prepared by using the HTJG5-5 strain has high saccharification power, the content of the isoamyl acetate in the white wine brewed by using the bran koji is obviously improved, the wine has pleasant fruit fragrance, and the wine body has excellent smell, flavor and texture, can improve the yield and the quality of the white wine, has important significance for improving the economic benefit of white wine enterprises, and has good application prospect.

Preservation of organisms

The classification of rhizopus oryzae provided by the invention is named rhizopus oryzaeRhizopus oryzaeThe microbial strain is preserved in China general microbiological culture collection center (CGMCC) for the year 6 and 29 of 2022, the preservation number is CGMCC No.40234, and the preservation address is North Chenxi Lu No. 1 and No. 3 of the Korean region of Beijing city.

Drawings

FIG. 1 is a schematic view of the appearance of rhizopus oryzae according to the present invention;

FIG. 2 is a microscopic view of rhizopus oryzae according to the present invention;

FIG. 3 is a radar chart of sensory evaluation of the fermentation experiments of the different Rhizopus oryzae moldy bran brewing.

Detailed Description

The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.

In a first aspect, the invention provides a rhizopus oryzae strainRhizopus oryzaeThe preservation number of the rhizopus oryzae is CGMCC No.40234.

According to the invention, space mutagenesis strain is carried out on the initial strain rhizopus oryzae JG5, and then the rhizopus oryzae of the invention is obtained through separation, purification and screening after mutagenesis. The mutagenized strains are carried out in a manner conventional in the art.

According to a preferred embodiment of the present invention, the purification culture may be repeated a plurality of times according to the actual situation in order to secure the separation and purification effect.

Wherein the isolation and purification may be performed in a solid medium conventionally used in the art, preferably, the solid medium is PDA solid medium. During the isolation and purification process, single colonies with typical mold colony characteristics are selected.

In the invention, screening can be divided into multiple rounds of screening, for example, strains with excellent saccharification performance can be screened out through preliminary screening, and then saccharification force re-screening is carried out.

The primary screening method can be a conventional method in the art, preferably, the separated mould strain is inoculated on a saccharifying enzyme activity screening culture medium plate by a spot planting method, and the primary screening of the high-yield saccharifying enzyme activity mould is performed by a transparent circle method. Wherein, the primary screening is carried out by comparing the sizes of transparent circles, and the strain with the large transparent circles is the strain with high saccharifying enzyme activity.

In a preferred embodiment of the invention, the mould strain is inoculated on a saccharifying enzyme activity screening culture medium by a spot grafting method sequentially by using a sterile inoculating needle, the mould strain is cultivated for 48 to 84 hours at the temperature of 25 to 35 ℃ in a positive way, iodine solution is added into a culture dish, the diameter of a transparent circle is measured after 2 to 8 minutes, and the strain with the large transparent circle is the strain with high saccharifying enzyme activity.

Wherein, the saccharifying enzyme activity screening culture medium can be PDA culture medium.

The means of re-screening may be conventional in the art and may include, for example, preparation of the bran koji and saccharification force assays and optionally sensory assays.

Wherein, the preparation method of the bran koji preferably comprises inoculating rhizopus oryzae into bran koji culture medium for culturing.

The bran koji culture medium may be a bran koji culture medium conventional in the art, preferably, the bran koji culture medium comprises bran and water.

Preferably, the bran content in the bran koji culture medium is 50-70 wt%.

The preparation method of the bran koji culture medium may be a method conventional in the art, for example, the preparation of the bran koji culture medium includes: weighing 30-50g bran, placing into 500mL triangular flask with thickness of about 2-4 cm, and adding water to make bran content in bran koji culture medium 50-70 wt% (preferably 55-65 wt%). And (5) sterilizing at high temperature and high pressure. After sterilization, the mixture is gently shaken while hot, the caking is broken up, the condensed water on the bottle wall and the bran fall into a culture medium, and the mixture is cooled to 30-35 ℃ for standby.

The rhizopus oryzae may be inoculated in a manner conventional in the art, such as in the form of a spore suspension, or by picking a loop of strain from a rhizopus oryzae inclined plane.

Preferably, the rhizopus oryzae is inoculated in the form of a spore suspension with an inoculum size of 10 ⁶ -10 ⁸ The spores/g bran koji medium.

According to a preferred embodiment of the present invention, the conditions of the culture include: the temperature is 25-35 ℃ and the time is 40-90 h. More preferably, the conditions of the culture include: the temperature is 28-32 ℃ and the time is 48-72 h.

After the cultivation is finished, the product can be dried in a drying oven at 35-45 ℃ and the moisture content in the bran koji is controlled to be 8-12 wt%.

The bran koji sensory evaluation result prepared by rhizopus oryzae HTJG5-5 comprises the following steps: the bran koji is compact and elastic, has no heartburn and dry skin, no black spores and no foreign flavor.

And selecting rhizopus oryzae strains with high saccharifying enzyme activity according to the detection result.

According to the invention, saccharification force refers to evaluation of saccharification force of bran koji, and reference is made to QB/T4257-2011; the saccharification capacity is the saccharification capacity of the strain on the starch raw material, and is characterized by the size of the reducing sugar content.

Among them, PDA medium is commercially available, and one skilled in the art can prepare a solid medium according to a preparation method conventional in the art.

The form of the microbial agent may be a form of microbial agent commonly known in the art, such as a solid or liquid microbial agent. Preferably, the microbial agent is in the form of at least one of a koji, a bran koji, a wheat koji, a mycelium and a spore suspension.

The preparation methods of the Xiaoqu, the bran koji and the wheat koji can be prepared by referring to the preparation methods conventional in the art, and are not described herein. Wherein, wheat Qu Baokuo is raw wheat starter and cooked wheat starter.

The preparation method of the mycelium and spore suspension can be a preparation method conventional in the art, and is not described herein.

The preparation method of the bran koji can be referred to in the first aspect, and is not described herein.

The fermented food can be any fermented food which can be prepared by rhizopus oryzae fermentation, and the fermented food comprises, but is not limited to, fermented foods such as wine (including white wine, yellow wine, rice wine and the like), vinegar, soy sauce, fermented bean curd, fermented glutinous rice, fermented soya beans and the like.

The person skilled in the art can produce the fermented food according to methods conventional in the art and will not be described in detail here.

The preparation method of the bran koji can be referred to as the above, and is not described herein.

According to the invention, the preparation method of the brewing clinker preferably comprises the following steps: soaking and steaming the starchy raw material to obtain the brewing clinker.

The starchy material may be any starchy material commonly used in the art including, but not limited to, sorghum, rice, glutinous rice, corn, wheat, etc., preferably, the starchy material is selected from at least one of sorghum, rice, glutinous rice, corn, and wheat.

The content of the components in the starchy material can be selected within a wide range.

According to a preferred embodiment of the present invention, the starchy material comprises sorghum, rice, glutinous rice and corn, wherein the starch material comprises 60 to 80% by weight of sorghum, 5 to 20% by weight of rice, 10 to 20% by weight of glutinous rice and 1 to 10% by weight of corn.

One or more of the above-mentioned starchiness raw materials can be selected to prepare the brewing clinker, when a plurality of starchiness raw materials are selected, the starchiness raw materials can be soaked and steamed according to the difference of the types, and then the steamed starchiness raw materials are uniformly mixed to obtain the brewing clinker.

The soaking time can be selected in a wider range, and the soaking time of different starchy raw materials is different, for example, the soaking time of sorghum is 12-36h, and the soaking time of glutinous rice, corn and the like is 0.2-3 h.

Wherein, the cooking time is different according to the type of the starchy raw material. For example, sorghum is cooked for 4-5 h, and glutinous rice, corn, etc. is cooked for 1-2 h.

Preferably, the inoculation amount of the bran koji is 3-6 wt% based on the weight of the brewing clinker.

The brewing temperature may vary according to the type of wine, for example, when the brewed wine is white wine, the brewing temperature may be 25-35 ℃.

The brewing time can be selected within a wide range, and a person skilled in the art can select a suitable brewing time according to needs, for example, when the brewed wine is white wine, the brewing time is preferably 24-72 hours.

The present invention will be described in detail by examples.

In the examples below, the starting materials and reagents used were all obtained commercially unless otherwise specified.

Potato Dextrose Agar (PDA) in the following examples is as follows:

12.0 g/L of potato extract powder, 20.0 g/L of glucose, 14.0 g/L of agar and pH value of 5.6+/-0.2 (25 ℃); and autoclaving at 115℃for 15 min.

Sensory evaluation and saccharification force measurement were performed according to QB/T4257-2011 standard.

The sensory evaluation of the wine was performed with reference to GB 12313-1990 standard.

Example 1

This example is used to demonstrate the selection of strains with excellent saccharification properties in Rhizopus oryzae by space mutagenesis.

(1) Strain mutagenesis, isolation and purification

The space mutation strain carried back by the test ship of the new generation manned spacecraft launched by the initial strain rhizopus oryzae JG5 through the carrier rocket of the carrier rocket number five B is marked as space mutation rhizopus oryzae HTJG5.

Placing the space mutation rhizopus oryzae HTJG5 glycerol bacteria-preserving tube frozen at-80 ℃ at 4 ℃ for slow freezing, inoculating into a PDA culture medium in an inoculum size of 2.0% by volume percent in a sterile workbench, culturing at 28 ℃ for 48 and h, randomly selecting single bacterial colonies with typical mould colony characteristics, streaking, separating and purifying for 3 times to obtain single pure bacterial colonies, observing the shapes of hyphae, aerial hyphae and spore filaments of the separated and purified bacterial strains by an optical microscope, and preserving the PDA for standby.

As shown in FIG. 1, the grown rhizopus oryzae HTJG5 was grown in PDA medium at 28deg.C, and the colony was fluffy, initially white, and turned to grey brown after drying, large, loose, opaque, intertwined hyphae, easy to pick up, and spread over the whole plate. As shown in FIG. 2, the form of Rhizopus oryzae HTJG5 appears creeping under a microscope (400 times), the pseudoroot is developed, and the branches appear root-like and brown. The cyst peduncles stand upright or are slightly bent, and the cyst spores are elliptic or spherical and are inconsistent in size.

(2) Preliminary screening of strains with excellent saccharification properties

The mould strain is inoculated on a PDA culture medium (three parallel) sequentially by a sterile inoculating needle in a spot grafting way, the mould strain is cultivated for 72 hours at the temperature of 28 ℃, 4mL of dilute iodine solution is added into a culture dish, the diameter of a transparent circle is measured after 5min, the strain with the large transparent circle is the strain with high saccharifying enzyme activity, 10 space-induced rhizopus oryzae HTJG5 strains with excellent saccharifying performance are screened out, the strains are marked as HTJG5-1 to HTJG5-10 (table 1), and the strain is preserved on an inclined plane for standby.

TABLE 1

(3) Saccharification force re-screening device

Preparing a bran culture medium: weighing 45g of bran, putting into a 500mL triangular flask, adding 30g of distilled water, sterilizing at 121 ℃ for 30 min under high pressure, cooling to 30+/-2 ℃, and scattering for later use. And (3) respectively picking one loop from the inclined planes of 10 alternative strains by using an inoculating loop, inoculating the inoculating loop to a bran culture medium, and uniformly mixing. Culturing at 30deg.C for 3 days, packaging into aseptic kraft paper bag under aseptic condition, and oven drying at 40deg.C in a drying oven for 24h with water content controlled at about 10%. The bran koji obtained was subjected to sensory evaluation and screening according to the following criteria: the bran koji is compact and elastic, has no heartburn and dry skin, no black spores and no foreign flavor. The saccharification force of the bran koji was measured, and a space-mutated rhizopus oryzae strain HTJG5-5 having high saccharifying enzyme activity was selected based on the result (Table 2).

TABLE 2

Example 2

This example is used to demonstrate the evaluation of the fermentation capacity of the space-mutated Rhizopus oryzae HTJG 5-5.

Weighing a proper amount of glutinous rice, washing twice, and filtering the water by using a drain basin. Adding the washed raw rice and distilled water into a sterile aluminum box, wherein the weight ratio of the raw rice to the distilled water is 1:1.5, ensuring that the rice is uniformly distributed, and steaming for 60 min. The lost weight after cooking is complemented with cold boiled water. 40g of steamed rice is weighed and placed in a sterile aluminum box. And (3) when the temperature of the cooked rice is reduced to 30-35 ℃, uniformly scattering the bran koji prepared in the step (2) in different culture time on the cooked rice and uniformly stirring, wherein the adding amount of the bran koji is 0.5 weight percent of the cooked rice. The rice is pulled to one side of the lunch box, uniformly pressed into an inclined plane, covered with a cover, put into a constant temperature incubator at 30 ℃, and taken out after 24h cultivation.

The appearance of the fermentation product was observed, and the mixture of the fermentation product (the mixture of the fermented glutinous rice and juice) was subjected to sugar detection and sensory evaluation, and the results are shown in table 3.

TABLE 3 Table 3

Example 3

This example is for explaining a method of brewing white spirit by preparing bran koji from Rhizopus oryzae HTJG5-5 by space mutagenesis.

(1) Selecting starchiness raw materials: the starchy material comprises 70% by weight of sorghum, 10% by weight of rice, 15% by weight of glutinous rice and 5% by weight of corn.

(2) Pretreatment of raw materials: soaking sorghum for 24 hours, and steaming for 5 hours; mixing and soaking glutinous rice, rice and corn, and steaming for 2h for 2 h; and uniformly mixing the materials after the steaming and boiling to obtain the brewing clinker.

(3) Inoculating bran koji and brewing: the brewing clinker was equally divided into 4 parts and inoculated with space-mutated Rhizopus oryzae HTJG5-5 bran koji, JG5 bran koji and commercial bran koji (laugh godet sweet distiller's yeast from Guizhou Ligao light industry technology development Co., ltd.) obtained from example 2 for 3 days, respectively, in an amount of 5% by weight of the brewing clinker.

Fermenting at 30deg.C for 48 h.

The saccharification force of the bran koji was measured, and the results are shown in Table 4.

TABLE 4 Table 4

(4) Flavor substance detection and analysis: the fermented brewing materials are subjected to flavor substance detection analysis, and the results are shown in Table 5. As can be seen by comparison, the yield of isoamyl acetate from space-mutated Rhizopus oryzae HTJG5-5 is higher.

TABLE 5

(5) Sensory evaluation: sensory evaluation was performed on the brewing materials during and after the fermentation, and the results are shown in Table 6.

TABLE 6

And specifically, sensory evaluation is carried out according to the intensity of the flavor dimensions such as fruit flavor, green flavor, flower flavor and the like (see GB/T12313-1990), the result is shown in figure 3, and compared with the original strain and the commercial rhizopus oryzae, the space mutation rhizopus oryzae HTJG5-5 has prominent fruit flavor, the number of the space mutation rhizopus oryzae HTJG5-5 is BC70162 strain, the space mutation rhizopus oryzae HTJG5-5 is preserved in the China general microbiological culture center (abbreviated as CGMCC) in the month 29 of 2022, the preservation number of the space mutation rhizopus oryzae is CGMCC No.40234, and the preservation address of the space mutation rhizopus oryzae is North Chen Xideu No. 1 hospital No. 3 in the Korean of Beijing city.

The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, a number of simple variants of the technical solution of the invention are possible, including combinations of the individual technical features in any other suitable way, which simple variants and combinations should likewise be regarded as being disclosed by the invention, all falling within the scope of protection of the invention.

Claims

1. Rhizopus oryzae strainRhizopusoryzaeThe rhizopus oryzae has a preservation number of CGMCC No.40234.

2. A microbial agent comprising rhizopus oryzae of claim 1.

3. A method for preparing a bran koji, comprising: inoculating rhizopus oryzae of claim 1 into a bran koji culture medium for culturing;

wherein the rhizopus oryzae is inoculated in the form of spore suspension with an inoculum size of 10 ⁶ -10 ⁸ The spores/g bran koji medium;

wherein the bran koji culture medium comprises bran and water, and the content of the bran is 50-70 wt% in the bran koji culture medium;

wherein the culturing conditions include: the temperature is 28-30deg.C, and the time is 40-90 h.

4. A bran koji produced according to the method of claim 3.

5. Use of rhizopus oryzae of claim 1, a microbial agent of claim 2 or a bran koji of claim 4 in the production of a fermented food product.

6. A method of brewing wine, the method comprising: inoculating the bran koji of claim 4 into a brewing clinker for brewing;

wherein, the inoculation amount of the bran koji is 3-6 per mill by taking the weight of the brewing clinker as the reference.