阅读说明：本技术 提高微生物生产有机酸效率的外源添加物及其应用 (Exogenous additive for improving efficiency of producing organic acid by microorganisms and application thereof ) 是由 佟毅 徐晴 章佳慧 吴娜 欧文 李义 彭超 陶进 于 2021-09-24 设计创作，主要内容包括：本发明公开了一种提高微生物生产有机酸效率的外源添加物及其应用,该微生物为根霉菌,在根霉菌发酵生产有机酸过程中,取多肽类物质加至根霉菌发酵培养基,用于提高根霉菌发酵生产有机酸的效率。多肽类物质改变根霉菌发酵培养基的碳氮源组成和比例,本发明通过外源添加缩短发酵周期,显著提高有机酸生产效率。(The invention discloses an exogenous additive for improving the efficiency of producing organic acid by microorganisms and application thereof. The polypeptide substance changes the composition and proportion of carbon and nitrogen sources of the rhizopus fermentation medium, and the invention shortens the fermentation period by exogenous addition and obviously improves the production efficiency of organic acid.)

1. The exogenous additive for improving the efficiency of producing organic acid by microorganisms is characterized in that the microorganisms are rhizopus, and the exogenous additive is a polypeptide substance.

2. The exogenous additive for improving the efficiency of microbial production of an organic acid according to claim 1, wherein the rhizopus is rhizopus oryzae or rhizopus arrhizus.

3. The exogenous additive for improving the efficiency of producing an organic acid by a microorganism according to claim 1, wherein the organic acid is malic acid or fumaric acid.

4. The exogenous additive for improving the efficiency of producing organic acids by microorganisms according to claim 1, 2 or 3, wherein the polypeptide substance is elastin peptide, fibroin peptide or collagen tripeptide.

5. Use of an exogenous additive for increasing the efficiency of microbial production of organic acids according to any one of claims 1 to 4 for addition to a rhizopus fermentation medium during the fermentative production of organic acids by rhizopus.

6. The use of the exogenous additive for improving the efficiency of producing organic acid by microorganisms according to claim 5, wherein the mass-to-volume ratio of the exogenous additive to the rhizopus fermentation medium is 1-5g: 1L.

7. The use of the exogenous additive for improving the efficiency of producing organic acid by a microorganism according to claim 5 or 6, wherein the rhizopus fermentation medium is a liquid medium.

8. The use of the exogenous additive for improving the efficiency of producing organic acids by microorganisms according to claim 5 or 6, wherein the conditions for the fermentation of the rhizopus are as follows: the temperature is 30-35 ℃, the time is 36-72h, and the stirring speed is 180-220 rpm.

Technical Field

The invention belongs to the technical field of bioengineering, and relates to an exogenous additive for improving the production efficiency of microorganisms and application thereof, in particular to an exogenous additive for improving the production efficiency of microorganisms for producing organic acid and application thereof.

Background

The organic acid refers to an acidic organic compound (excluding amino acids) containing a carboxyl group widely present in living organisms, and includes malic acid, fumaric acid, lactic acid, and the like. The organic acids are mostly involved in the life process of animal and plant metabolism, can be used as metabolic intermediate products or have remarkable biological activity, are important raw materials for organic synthesis and medical product development, have wide application in the fields of food, medicines, chemical industry and the like, and have great market application value.

At present, the traditional method for producing organic acid is a chemical synthesis method, the raw material source of the method is petroleum-based compounds, the resource is short, and the production process brings certain environmental problems. Since most organic acids are intermediate products in microbial metabolic pathways, the process for producing organic acids by fermentation using renewable biomass resources as raw materials by using microorganisms has a great development prospect. Rhizopus is considered to be an ideal microorganism for producing organic acids because it has a simple nutrient source required for the fermentation process and can significantly reduce the production cost.

However, the production of organic acids by fermentation with rhizopus still has the problems of long fermentation period and low production efficiency, and the existing method for solving the problem mainly modifies the rhizopus by means of gene editing and is limited by the application of organic acids in the food industry, and the method for solving the production efficiency of the organic acids by modifying the rhizopus by means of gene editing such as molecular biology and the like is not ideal.

In part of microbial fermentation processes, exogenous additives are added to effectively improve the production efficiency, the method does not pass through gene editing and has low cost, but the method for improving the efficiency of producing organic acid by rhizopus has not been reported at present.

Disclosure of Invention

The invention aims to provide an exogenous additive for improving the organic acid production efficiency of microorganisms, and a polypeptide substance for improving the organic acid production efficiency of rhizopus is determined by analysis and screening.

The invention also aims to provide the application of the exogenous additive for improving the efficiency of producing the organic acid by the microorganisms, wherein in the process of producing the organic acid by fermenting the rhizopus, the polypeptide substance with specific concentration is added to change the nutrient composition of a fermentation culture medium of the rhizopus, and the production efficiency of the organic acid is improved by regulating and controlling a metabolic network.

In order to achieve the purpose, the invention adopts the technical scheme that:

an exogenous additive for improving the efficiency of producing organic acid by microbes is disclosed, the microbes are rhizopus, and the exogenous additive is a polypeptide substance.

As a limitation, the rhizopus is Rhizopus oryzae or Rhizopus arrhizus, both of which are Rhizopus fungi.

As another limitation, the organic acid is malic acid or fumaric acid.

Wherein the organic acid also comprises lactic acid and other organic acids which can be produced by fermentation of rhizopus.

As a third limitation, the polypeptide-based substance is elastin peptide, fibroin peptide or collagen tripeptide.

The invention also provides an application of the exogenous additive for improving the efficiency of producing the organic acid by the microorganism, which is used for being added into a rhizopus fermentation culture medium in the process of producing the organic acid by fermenting the rhizopus.

In the process of producing organic acid by fermenting rhizopus, after the rhizopus is cultured by seeds, the rhizopus is inoculated to a rhizopus fermentation culture medium to produce organic acid by fermentation; the seed culture medium is cultured at 30-35 deg.C for 24 hr under stirring speed of 200 rpm.

The seed culture medium and the fermentation culture medium according to the invention are understood to be the culture media suitable for the growth or fermentation of the rhizopus which have been sterilized and cooled in the prior art, the culture media each containing the nutrients necessary for the growth or fermentation of this species of rhizopus, including carbon sources, nitrogen sources, inorganic salts and moisture.

As a limitation, the mass-volume ratio of the exogenous additive to the rhizopus fermentation medium is 1-5g: 1L.

As another limitation, the rhizopus fermentation medium is a liquid medium. The liquid culture medium can be directly used as fermentation liquor for fermentation production.

As a further limitation, the conditions under which the rhizopus is fermented are: the temperature is 30-35 ℃, the time is 36-72h, and the stirring speed is 180-220 rpm.

The fermentation conditions can be adjusted during production in combination with the common knowledge in the art, for example, the characteristics of the particular Rhizopus fungus, such as fermentation at the optimum temperature, to achieve better results.

Due to the adoption of the technical scheme, compared with the prior art, the invention has the technical progress that:

the invention adopts a non-genetic engineering method, and by adding polypeptide substances with a certain concentration from an external source, the composition and the proportion of a carbon-nitrogen source in a rhizopus fermentation medium are changed, the glycolysis path is obviously enhanced, the fermentation period of a target product is shortened to a certain extent, and the efficiency of producing organic acid by fermenting rhizopus is improved;

the method has simple steps and convenient operation, only needs to add the polypeptide substances with low cost into the rhizopus fermentation culture medium, and is suitable for large-scale green and high-efficiency industrial production of the organic acid.

The invention is described in detail below with reference to the figures and the embodiments.

Drawings

FIG. 1 is a graph showing the results of a verification experiment in example 7 in which the efficiency of producing organic acids by microorganisms was improved by the exogenous addition of polypeptides;

FIG. 2 is a graph showing the results of the experimental verification for improving the efficiency of producing organic acids by microorganisms by adding exogenously added polypeptides in example 7.

Detailed Description

The present invention is described in further detail below with reference to specific examples and figures, it being understood that the examples are illustrative only and are not limiting upon the present invention.

The materials, reagents and the like used in the examples of the present invention are commercially available unless otherwise specified. The experimental procedures, in which specific conditions are not indicated in the examples, are generally carried out under conventional conditions or conditions recommended by the manufacturer.

The strains adopted by the invention are the typical representative strains of rhizopus arrhizus NRRL 1526 and rhizopus oryzae ATCC 20344 commonly used in industrial production and can be purchased from commercial sources.

Example 1A method for improving L-malic acid production efficiency of Rhizopus by exogenously adding elastin peptide

The method in the embodiment comprises the following steps of:

(1) preparation of a culture medium:

preparing a seed culture medium: mixing glucose 20kg, urea 2kg and KH₂PO₄ 0.3kg、MgSO₄·7H₂O 0.5g、ZnSO₄·7H₂O 0.02kg、FeSO₄·7H₂O 0.01kg、CuSO₄·5H₂0.03kg of O and 1000L of water;

preparing a fermentation culture medium: mixing glucose 80kg, (NH)₄)₂SO₄ 0.2kg、KH₂PO₄ 0.6kg、ZnSO₄·7H₂O 0.02kg、FeSO₄·7H₂O 0.01kg、MgSO₄·7H₂O 0.5kg、CaCO₃60kg and 1000L of water;

sterilizing the culture medium at 115 deg.C for 30min, and cooling to obtain seed culture medium and fermentation culture medium.

(2) Seed culture:

culturing Rhizopus arrhizus NRRL 1526 in solid culture medium, scraping with sterile water, and diluting to 1 × 10⁷And (4) obtaining rhizopus arrhizus NRRL 1526 spore suspension.

Inoculating 1L of rhizopus arrhizus NRRL 1526 spore suspension into the seed culture medium, and culturing at 30 ℃ and 200rpm for 24h to obtain seed liquid.

(3) Fermentation culture: adding 3kg of elastin peptide into fermentation medium to make its concentration be 3g/L, obtaining improved fermentation medium, labeled as P1, inoculating 5L of seed liquid, fermenting at 35 deg.C and stirring rate of 200rpm, stopping fermentation when residual sugar content of culture medium P1 reaches single digit, and obtaining L-malic acid fermentation liquid.

Examples 2-3 method for improving L-malic acid production efficiency by Rhizopus by exogenous addition of polypeptide substances

Examples 2 to 3 are methods for improving the efficiency of producing L-malic acid by rhizopus by exogenously adding a polypeptide, and the steps are substantially the same as those in example 1, except that the process parameters, the type and the amount of the polypeptide are different, as detailed in table 1:

table 1 summary of parameters of examples 2-3

The contents of other portions of examples 2 to 3 are the same as those of example 1.

Example 4A method for improving the efficiency of fumaric acid production by Rhizopus by exogenously adding elastin peptide

The method in the embodiment comprises the following steps of:

(1) preparation of a culture medium:

preparing a seed culture medium: mixing glucose 20kg, urea 1kg and KH₂PO₄ 0.3kg、MgSO₄·7H₂O 0.25g、ZnSO₄·7H₂O 0.066kg、FeSO₄·7H₂0.01kg of O and 1000L of water;

preparing a fermentation culture medium: mixing glucose 80kg, (NH)₄)₂SO₄ 0.2kg、KH₂PO₄ 0.15kg、ZnSO₄·7H₂O 0.05kg、FeSO₄·7H₂O 0.068kg、MgSO₄·7H₂O 0.1kg、CaCO₃60kg and 1000L of water;

sterilizing the culture medium at 115 deg.C for 30min, and cooling to obtain seed culture medium and fermentation culture medium.

(2) Seed culture:

culturing Rhizopus oryzae ATCC 20344 in solid culture medium, scraping with sterile water, and diluting to concentration of 1 × 10⁷Per mL, resulting in a suspension of Rhizopus oryzae ATCC 20344 spores.

Inoculating 1L rhizopus oryzae ATCC 20344 spore suspension into the seed culture medium, and culturing at 35 deg.C and 200rpm for 24h to obtain seed solution.

(3) Fermentation culture: adding 4kg of elastin peptide into fermentation medium to make its concentration be 4g/L to obtain improved fermentation medium, labeled as P1, inoculating 5L of seed liquid, and stopping fermentation when the residual sugar content of culture medium P1 reaches single digit, to obtain L-malic acid fermentation liquid.

Examples 5-6 method for improving fumaric acid production efficiency by Rhizopus by exogenously adding polypeptide-like substance

Examples 5 to 6 are methods for improving the efficiency of fumaric acid production by rhizopus by exogenously adding polypeptides, and the steps are substantially the same as those in example 4, except that the process parameters, the types and the amounts of the polypeptides are different, as detailed in table 2:

table 2 summary of parameters of examples 5-6

The contents of other portions of examples 5 to 6 are the same as those of example 4.

Example 7 validation experiment for improving efficiency of producing organic acid by microorganism by adding polypeptide substance exogenously

The time of fermentation culture (time from residual sugar amount to single digit) in examples 1-6 was recorded, and the change in glucose content during fermentation culture was examined to calculate the efficiency of producing organic acids.

Control group a: this group was the control group of examples 1 to 3, and organic acids were produced by fermentation in the same manner as in example 1 except that no polypeptide-like substance was added to the fermentation medium.

Control group B: this group was the control group of examples 4-6, and organic acids were produced by fermentation in the same manner as in example 4 except that no polypeptide was added to the fermentation medium.

The organic acid production and production efficiency of examples 1-6 and control A, B were compared to verify the effect of exogenous polypeptides on the efficiency of organic acid production by rhizopus.

Wherein, the results of the control group A and examples 1 to 3 are shown in FIG. 1 and Table 3; the results for control B and examples 4-6 are shown in FIG. 2 and Table 4.

TABLE 3 results of organic acid production and production efficiency

Group of	Control group A	Example 1	Example 2	Example 3
					Polypeptides	Is free of	Elastin peptides	Fibroin peptides	Collagen tripeptide
L-malic acid productionAmount (g/L)	20.86	21.95	21.58	20.12
					L-malic acid production efficiency (g/L/h)	0.29	0.61	0.60	0.48

As shown in figure 1 and table 3, the fermentation time of the control group A is 72h, the fermentation times of the examples 1-3 are 36h, 36h and 42h respectively, and the fermentation time is obviously shortened after the polypeptide substances are added to the fermentation medium; the production level of L-malic acid is shown in table 3, compared to the control group, the fermentation end point L-malic acid yield was not significantly different, but the production efficiency was significantly improved, wherein example 1 was improved by 110.34%, example 2 was improved by 106.90%, and example 3 was improved by 65.52%.

TABLE 4 results of organic acid production and production efficiency

Group of	Control group B	Example 4	Example 5	Example 6
					Polypeptides	Is free of	Elastin peptides	Fibroin peptides	Collagen tripeptide
Fumaric acid yield (g/L)	25.62	26.75	27.13	24.68
					Fumaric acid production efficiency (g/L/h)	0.36	0.64	0.75	0.51

As shown in fig. 2 and table 4, the fermentation time of the control group B was 72h, and the fermentation times of examples 4-6 were 42h, 36h and 48h, respectively, and the fermentation time was significantly shortened after the polypeptide substance was exogenously added to the fermentation medium; as shown in table 3, the fumaric acid production level was not significantly different from the control group in the fermentation end point fumaric acid production yield, but the production efficiency was significantly improved, wherein example 4 was improved by 77.78%, example 5 was improved by 108.33%, and example 6 was improved by 41.67%.

The result shows that the efficiency of producing organic acid by microorganisms can be obviously improved by adding polypeptide substances from external sources.

Examples 8-12 methods for increasing the efficiency of microbial production of organic acids by exogenous addition of Polypeptides

Examples 8 to 13 are each a method for improving the efficiency of producing organic acids by microorganisms by exogenously adding a polypeptide-like substance, and the steps of examples 8 to 10 are substantially the same as in example 1 or 4, and the steps of examples 11 to 13 are substantially the same as in example 4, except for the specific parameters, as detailed in Table 5:

TABLE 5 summary of parameters of examples 8-13

Comparative examples 8 '-13': comparative examples 8 ' -13 ' corresponding to the methods of examples 8-13 were designed, except that no polypeptide was added in comparative examples 8 ' -13 ', and the other parameters and methods were the same as those of the respective comparative examples, e.g., comparative example 8 ' was different from example 8 only in that no fibroin peptide was added, and the other parameters and methods were the same.

The product changes in the fermentation culture processes of each group are recorded, and the organic acid production efficiency is calculated, and the results are shown in table 6:

TABLE 6 organic acid production efficiency results table

As can be seen from the results in Table 6, the production efficiency of the organic acids in examples 8 to 13 is higher than that in the comparative example by 40%, and the results show that the production efficiency of the organic acids by the microorganisms can be remarkably improved by adding the polypeptide substances externally.

Although the present invention has been described in detail with reference to the above embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments described above, or equivalents may be substituted for elements thereof. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the claims of the present invention.