Culture medium and application thereof in mucor culture

Abstract

The invention discloses a culture medium and application thereof in mucor culture. The invention provides a culture medium containing fulvic acid, tyrosine, phenylalanine, sodium alginate, disodium hydrogen phosphate, sodium dihydrogen phosphate monohydrate and sodium chloride, aiming at the defects that the metabolic potential of mucor cannot be fully exerted and the biomass of the mucor cannot be obviously improved in the conventional culture medium. When the culture medium is used for culturing the mucor, the growth of the mucor can be remarkably promoted, and the biomass (yield) of the mucor can be improved, so that the quantity of mucor metabolites, such as the quantity of enzymes generated by metabolism of the mucor, can also be improved. The method is beneficial to culture and utilization of mucor and is beneficial to development of the mucor-based enzyme preparation production industry.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation and cultivation, and more specifically, relates to a culture medium and application thereof in cultivating Mucor. Background Art

[0002] Mucor is a common fungus in the field of food fermentation. It is often used to ferment tofu and fermented black beans. It can produce protease, decompose soy protein, and enhance the flavor and nutritional value of food. In addition to being used to ferment soy products, Mucor can also be used to produce enzyme preparations. For example, Mucor rouxianus can produce xylanase, Mucor indicus can produce rennet, and Mucor circinelloides can produce fatty acid desaturase.

[0003] However, the existing culture medium for culturing Mucor cannot fully exert the metabolic potential of Mucor, resulting in low growth rate and product generation efficiency, which affects its biomass and product yield. Therefore, it is necessary to provide a culture medium that can effectively promote the growth of Mucor, increase its biomass, and thus increase its product yield. Although there are reports on fermentation culture medium for increasing the yield of γ-linolenic acid produced by Mucor circinelloides, the biomass of Mucor circinelloides obtained by culturing the culture medium is still not high enough, and its maximum biomass does not exceed 20 g / L. Summary of the invention

[0004] The present invention aims at the above problems existing in the prior art and provides a culture medium, which can promote the growth of Mucor and significantly increase its biomass.

[0005] The first object of the present invention is to provide a culture medium.

[0006] The second object of the present invention is to provide use of the culture medium in culturing Mucor.

[0007] The third object of the present invention is to provide application of the culture medium in increasing the biomass of Mucor.

[0008] The fourth object of the present invention is to provide the use of the culture medium in increasing the yield of metabolites of Mucor.

[0009] The fifth object of the present invention is to provide a culture method for increasing the yield of Mucor or the yield of its metabolites.

[0010] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0011] The present invention provides a culture medium for culturing Mucor, which is capable of fully exerting the metabolic potential of Mucor and causing the deficiency of low biomass. The culture medium of the present invention can effectively increase the biomass of Mucor to a level higher than 20 g / L. Therefore, the present invention claims protection for the culture medium.

[0012] Specifically, the culture medium contains 30-70 mM fulvic acid, 8-12 mM tyrosine, 8-12 mM phenylalanine, 15-25 mM sodium alginate, 3-10 mM disodium hydrogen phosphate, 20-30 mM sodium dihydrogen phosphate monohydrate and 80-100 mM sodium chloride.

[0013] More specifically, the culture medium consists of 30-70 mM fulvic acid, 8-12 mM tyrosine, 8-12 mM phenylalanine, 15-25 mM sodium alginate, 3-10 mM disodium hydrogen phosphate, 20-30 mM sodium dihydrogen phosphate monohydrate, 80-100 mM sodium chloride and water.

[0014] Preferably, the concentration of fulvic acid is 50-70 mM. In the culture medium of the present invention, the concentration of fulvic acid has a greater impact on the biomass of Mucor. When the concentration of fulvic acid is 50-70 mM, the biomass of Mucor can be higher than 25 g / L.

[0015] Preferably, in the culture medium of the present invention, the concentration of fulvic acid is 50-70 mM, the concentration of tyrosine is 9-11 mM, the concentration of phenylalanine is 9-11 mM, the concentration of sodium alginate is 28-22 mM, the concentration of disodium hydrogen phosphate is 3-7 mM, the concentration of sodium dihydrogen phosphate monohydrate is 23-27 mM, and the concentration of sodium chloride is 90-100 mM.

[0016] Further preferably, in the culture medium of the present invention, the concentration of fulvic acid is 70 mM, the concentration of tyrosine is 10 mM, the concentration of phenylalanine is 10 mM, the concentration of sodium alginate is 20 mM, the concentration of disodium hydrogen phosphate is 5 mM, the concentration of sodium dihydrogen phosphate monohydrate is 25 mM, and the concentration of sodium chloride is 100 mM. When Mucor is cultured using this culture medium, the biomass of Mucor is the highest, which is higher than 28 g / L.

[0017] The present invention also claims to protect the use of the culture medium in culturing Mucor.

[0018] The present invention also claims to protect the use of the culture medium in increasing the biomass of Mucor.

[0019] The present invention also claims to protect the use of the culture medium in improving the yield of metabolites of Mucor.

[0020] The use of the culture medium of the present invention in preparing products for culturing Mucor, preparing products for increasing the biomass of Mucor, or preparing products for increasing the yield of Mucor metabolites should also be within the protection scope of the present invention.

[0021] Specifically, the Mucor is Mucor rouxii, Mucor circinelloides or Mucor indica.

[0022] The present invention also provides a culture method for increasing the yield of Mucor or the yield of its metabolites, the method comprising: using the culture medium of the present invention to culture Mucor.

[0023] Specifically, after inoculating Mucor into the culture medium of the present invention, the culture is firstly carried out at 30-35° C. and 120-150 rpm for 4-5 days, and then at 25-27° C. and 90-110 rpm for 4-24 hours.

[0024] More specifically, the culture is first performed at 30-32° C. and 140-150 rpm for 4-5 days, and then at 25-27° C. and 90-100 rpm for 4-24 hours.

[0025] Specifically, the Mucor is Mucor rouxii, Mucor circinelloides or Mucor indica.

[0026] The present invention has the following beneficial effects:

[0027] The present invention aims at the deficiency that the existing culture medium cannot give full play to the metabolic potential of Mucor and significantly improve its biomass, and provides a culture medium containing fulvic acid, tyrosine, phenylalanine, sodium alginate, disodium hydrogen phosphate, sodium dihydrogen phosphate monohydrate and sodium chloride. Cultivating Mucor with the culture medium of the present invention can significantly promote the growth of Mucor, improve the biomass or yield of Mucor, and thus can also increase the amount of Mucor metabolites, such as the amount of enzymes produced by Mucor metabolism. The present invention is helpful for the cultivation and utilization of Mucor, and is conducive to the development of the enzyme preparation production industry based on Mucor. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The growth curves of Mucor rouxii in potato dextrose liquid medium and the medium described in Example 1 are shown.

[0029] Figure 2 The results of the effects of different carbon sources on the biomass of Mucor; different letters in the figure indicate significant differences (p<0.05).

[0030] Figure 3 The results show the effects of different fulvic acid concentration gradients on the biomass of Mucor; different letters in the figure indicate significant differences (p<0.05).

[0031] Figure 4The results of the effects of different nitrogen sources on the biomass of Mucor; different letters in the figure indicate significant differences (p<0.05). DETAILED DESCRIPTION

[0032] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0033] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0034] The Mucor rouxianus used in the embodiment of the present invention is the Mucor rouxianus GDMCC 3.89 strain, the Mucor circinelloides used is the Mucor circinellcides CSC72.27 strain, and the Mucor indicus used is the Mucor indicus CBC226.29 strain.

[0035] Example 1 Preparation of culture medium and cultivation of Mucor

[0036] The preparation method of the culture medium described in this embodiment is as follows:

[0037] Accurately weigh 0.71 g of disodium hydrogen phosphate (Na2HPO4), 4.47 g of sodium dihydrogen phosphate monohydrate (NaH2PO4·H2O), 5.85 g of sodium chloride (NaCl), 21 g of fulvic acid, 4.8 g of sodium alginate, 1.81 g of tyrosine and 1.65 g of phenylalanine, dissolve them in distilled water and make up to 1 L; thereby preparing a culture medium containing 5 mM disodium hydrogen phosphate, 25 mM sodium dihydrogen phosphate monohydrate, 100 mM sodium chloride, 70 mM fulvic acid, 20 mM sodium alginate, 10 mM tyrosine and 10 mM phenylalanine.

[0038] In this example, Mucor rouxii was used as the experimental bacteria to culture Mucor. The culture method is as follows: take the activated Mucor rouxii seed liquid, inoculate it in 1L of the prepared culture medium at a 5% (v / v) inoculation amount; first ferment and culture for 120 hours at 30°C and 150rpm to maximize the yield of the bacteria, then adjust the temperature to 27°C and the rotation speed to 100rpm, and then culture for another 48h to aggregate the bacteria for better separation; after the culture is completed, remove the culture medium by filter paper or centrifugation, collect the bacteria, and measure the biomass after drying at 105°C to constant weight.

[0039] The biomass of Mucor rouxii obtained by culturing using the culture medium and method described in this example was 28.89 g / L.

[0040] This embodiment is a culture of Mucor under laboratory conditions. In order to explore the highest yield of Mucor, the fermentation time was extended to 48h in the late stage, and filtration or centrifugation was added. However, in actual batch production, the steps of filtering or centrifuging to collect the bacteria can be omitted. The purpose of adding sodium alginate to the culture medium of the present invention is also to make the bacterial colony better aggregate. In addition, combined with the growth curve of Mucor, it can be seen that the actual cultivation of 4 to 5 can maximize the yield of the bacteria. After reaching the maximum yield, culture at 27°C and 100rpm for 4 to 24h to aggregate the bacterial colony for better separation.

[0041] Example 2 Preparation of culture medium and cultivation of Mucor

[0042] The difference between the culture medium described in this example and the culture medium described in Example 1 is that the concentration of fulvic acid is 60 mM.

[0043] In this example, Mucor rouxii was used as the experimental bacteria to culture Mucor. The culture method was the same as in Example 1. After the culture was completed, the culture medium was removed by filter paper or centrifugation, the bacteria were collected, and the biomass was measured after drying at 105° C. to constant weight. The biomass of Mucor rouxii obtained by culturing using the culture medium described in this example was 26.77 g / L.

[0044] Example 3 Preparation of culture medium and cultivation of Mucor

[0045] The difference between the culture medium described in this example and the culture medium described in Example 1 is that the concentration of fulvic acid is 50 mM.

[0046] In this example, Mucor rouxii was used as the experimental bacteria to culture Mucor. The culture method was the same as in Example 1. After the culture was completed, the culture medium was removed by filter paper or centrifugation, the bacteria were collected, and the biomass was measured after drying at 105°C to constant weight. The biomass of Mucor rouxii obtained by culturing using the culture medium and method described in this example was 25.80 g / L.

[0047] Example 4 Preparation of culture medium and cultivation of Mucor

[0048] The difference between the culture medium described in this example and the culture medium described in Example 1 is that the concentration of fulvic acid is 40 mM.

[0049] In this example, Mucor rouxii was used as the experimental bacteria to culture Mucor. The culture method was the same as in Example 1. After the culture was completed, the culture medium was removed by filter paper or centrifugation, the bacteria were collected, and the biomass was measured after drying at 105° C. to constant weight. The biomass of Mucor rouxii obtained by culturing using the culture medium and method described in this example was 23.80 g / L.

[0050] Example 5 Preparation of culture medium and cultivation of Mucor

[0051] The difference between the culture medium described in this example and the culture medium described in Example 1 is that the concentration of fulvic acid is 30 mM.

[0052] In this example, Mucor rouxii was used as the experimental bacteria to culture Mucor. The culture method was the same as in Example 1. After the culture was completed, the culture medium was removed by filter paper or centrifugation, the bacteria were collected, and the biomass was measured after drying at 105°C to constant weight. The biomass of Mucor rouxii obtained by culturing using the culture medium and method described in this example was 23.10 g / L. Comparative Example 1 Effect of different culture media on the biomass of Mucor

[0053] The present invention uses Mucor rouxii as the experimental bacteria, and cultured Mucor rouxii using the culture medium described in Example 1 and the conventional potato glucose liquid culture medium, respectively. The culture method is the same as that in Example 1, and the biomass of Mucor is measured every day, and its growth curves in different culture media are plotted.

[0054] The growth curves of Mucor rouxii in potato dextrose liquid medium and the medium described in Example 1 are shown in FIG. Figure 1 As shown. Figure 1 It can be seen that compared with the traditional potato glucose liquid culture medium, the culture medium of the present invention can significantly promote the growth of Mucor and increase the biomass of Mucor.

[0055] Comparative Example 2 Effects of different carbon sources and nitrogen sources on the biomass of Mucor

[0056] Since different fungi grow differently in the same culture medium, and different carbon source and nitrogen source combinations have different effects on the growth of the same fungi, there is no culture medium that can significantly increase the biomass of Mucor. Therefore, the present invention selects a variety of different carbon sources and nitrogen sources, and on this basis, compound other components, and obtains the culture medium of the present invention through continuous attempts and optimization. The formula of some culture media tested by the present invention using Mucor rouxii, Mucor circinelloides and Mucor indica and the results of their effects on the biomass of different Mucor are as follows.

[0057] 1. Effects of different carbon sources on the biomass of Mucor

[0058] Culture media containing different single carbon sources were prepared according to Table 1, and the prepared culture media were used to culture Mucor rouxii, Mucor circinelloides and Mucor indica, respectively (each group was repeated 3 times).

[0059] Table 1

[0060]

[0061]

[0062] The culture method is as follows: take activated Mucor rouxii / Mucor circinelloides / Mucor indica seed liquid, inoculate it into 1L prepared culture medium at an inoculum size of 5% (v / v), first ferment and culture at 30°C and 150rpm for 24 hours; then ferment and culture at 27°C and 100rpm for 60 hours; after terminating the culture, harvest the bacteria and remove the culture medium using filter paper method (or centrifugation), dry at 105°C to constant weight, then measure the biomass and perform data analysis (one-way analysis of variance (ANOVA) compares whether the biomass of the same Mucor strain is significantly different for different carbon sources; Tukey HSD (post hoc test) clarifies the significance of the differences between different carbon sources; use R language to analyze the data and draw a chart of the average biomass and standard error of each carbon source group).

[0063] The effects of different carbon sources on the biomass of Mucor Figure 2 As shown in the figure, different letters indicate significant differences (p<0.05). Figure 2 It can be seen that when fulvic acid is used as a single carbon source, the three Mucor species showed the highest average yield (Mucor rouxii 28.49 g / L, Mucor circinelloides 26.77 g / L, Mucor indica 26.28 g / L), indicating that fulvic acid has superiority over the other three carbon sources in the yield of Mucor. The data were analyzed by ANOVA, and the significance ranking was clarified by Tukey analysis. Among them, fulvic acid showed the most significant difference with the other three carbon sources, indicating that fulvic acid has a significant advantage in increasing the biomass of Mucor and fulvic acid is universal as a carbon source for Mucor.

[0064] 2. Effects of different fulvic acid concentration gradients on Mucor biomass

[0065] Referring to medium No. 1 in Table 1, culture media with fulvic acid concentrations of 10, 20, 30, 40, 50, 60, and 70 mM were prepared and cultured with Mucor rouxii, Mucor circinelloides, and Mucor indica, respectively (each group was repeated 3 times). The culture method and data analysis method were the same as above.

[0066] The results of the effects of different fulvic acid concentration gradients on the biomass of Mucor are shown in Figure 3 As shown in the figure, different letters indicate significant differences (p<0.05). Figure 3It can be seen that when the concentration of fulvic acid is 70mM, the average yield of the three types of Mucor reaches the maximum (Mucor rouxii 28.6g / L, Mucor circinelloides 27.3g / L, Mucor indica 26.6g / L). Among them, the reason why the biomass of the 10mM fulvic acid group is higher than that of the 20mM group and equal to that of the 30mM group is that when the fulvic acid is 10mM, in order to make up the overall TOC (TOC is total organic carbon, maintaining the overall TOC concentration at 8000mg / L, which can reflect the influence of the concentration gradient of the type of fulvic acid as a carbon source, so when the fulvic acid concentration TOC is less than 8000mg / L, glucose carbon is used to make up to 8000mg / L), a higher concentration of glucose is used for making up, and the participation of fulvic acid as a carbon source is low. This phenomenon is similar to the result of glucose as a carbon source in this comparative example 1. The results of ANOVA significant difference analysis and Tukey post hoc test showed that the dosage of fulvic acid in the range of 50-70mM had a high difference according to the concentration gradient, among which 70mM showed the highest difference, indicating that the biomass of Mucor was significantly improved when the concentration of fulvic acid was 50-70mM, and the highest biomass was reached when the concentration of fulvic acid was 70mM. The differences between the three types of Mucor in the group were maintained at the same level, indicating that the growth performance of Mucor production in the concentration range of 30-70mM showed stability.

[0067] 3. Effects of different nitrogen sources on the biomass of Mucor

[0068] Referring to medium No. 1 in Table 1, the nitrogen sources (tyrosine + phenylalanine) were replaced with the following nitrogen source combinations of the same concentration to prepare culture media, and the prepared culture media were used to culture Mucor rouxii, Mucor circinelloides and Mucor indica (each group was repeated 3 times), and the culture method and data analysis method were the same as above.

[0069] The results of the effects of different nitrogen sources on the biomass of Mucor are shown in Figure 4 As shown in the figure, different letters indicate significant differences (p<0.05). Figure 4 It can be seen that the nitrogen source combination of tyrosine + phenylalanine showed the highest average biomass of Mucor (Mucor rouxii 28.76g / L, Mucor circinelloides 27.3g / L, Mucor indica 26.5g / L). The results of variance analysis and post hoc test showed that the yield of the nitrogen source combination of tyrosine and phenylalanine was most significantly improved. At the same time, this combination showed stability among different Mucor, reflecting the universality of this combination. The yield differences among different Mucor of other nitrogen source combinations were large.

[0070] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A culture medium, characterized in that The culture medium contains 30-70 mM fulvic acid, 8-12 mM tyrosine, 8-12 mM phenylalanine, 15-25 mM sodium alginate, 3-10 mM disodium hydrogen phosphate, 20-30 mM sodium dihydrogen phosphate monohydrate and 80-100 mM sodium chloride.

2. The culture medium according to claim 1, characterized in that The culture medium consists of 30-70 mM fulvic acid, 8-12 mM tyrosine, 8-12 mM phenylalanine, 15-25 mM sodium alginate, 3-10 mM disodium hydrogen phosphate, 20-30 mM sodium dihydrogen phosphate monohydrate, 80-100 mM sodium chloride and water.

3. The culture medium according to claim 2, characterized in that The concentration of fulvic acid is 50-70 mM.

4. The culture medium according to claim 3, characterized in that The concentration of tyrosine is 9-11 mM, the concentration of phenylalanine is 9-11 mM, the concentration of sodium alginate is 28-22 mM, the concentration of disodium hydrogen phosphate is 3-7 mM, the concentration of sodium dihydrogen phosphate monohydrate is 23-27 mM, and the concentration of sodium chloride is 90-100 mM.

5. The culture medium according to claim 4, characterized in that The concentration of fulvic acid was 70 mM, the concentration of tyrosine was 10 mM, the concentration of phenylalanine was 10 mM, the concentration of sodium alginate was 20 mM, the concentration of disodium hydrogen phosphate was 5 mM, the concentration of sodium dihydrogen phosphate monohydrate was 25 mM, and the concentration of sodium chloride was 100 mM.

6. Use of the culture medium according to any one of claims 1 to 5 in culturing Mucor.

7. Use of the culture medium according to any one of claims 1 to 5 in increasing the biomass of Mucor.

8. Use of the culture medium according to any one of claims 1 to 5 for increasing the yield of metabolites of Mucor.

9. A method for increasing the yield of Mucor or its metabolites, characterized in that: The method comprises culturing Mucor using the culture medium described in any one of claims 1 to 5.

10. The method according to claim 9, characterized in that: First, culture at 30-35°C and 120-150 rpm for 4-5 days, and then culture at 25-27°C and 90-110 rpm for 4-24 hours.

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