A medium and its use in culturing mucorales

Abstract

The application discloses a culture medium and application thereof in culturing Mucor. In view of the fact that the existing culture medium cannot fully exert the metabolic potential of Mucor and cannot significantly improve the biomass of Mucor, the application provides a culture medium containing fulvic acid, tyrosine, phenylalanine, sodium alginate, disodium hydrogen phosphate, monosodium phosphate monohydrate and sodium chloride. The culture medium is used for culturing Mucor, so that the growth of Mucor can be significantly promoted, the biomass (yield) of Mucor can be improved, and the amount of metabolic products of Mucor, such as the amount of enzymes produced by Mucor, can also be improved. The application is helpful for the culturing and utilization of Mucor and is favorable for the development of an enzyme preparation production industry based on Mucor.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation culture technology. More specifically, it relates to a culture medium and its application in culturing Mucor. Background Technology

[0002] Mucor is a commonly used fungus in the field of food fermentation, often used in the fermentation production of tofu and fermented black beans. It produces proteases that break down soybean protein, enhancing the flavor and nutritional value of the food. Besides fermenting soy products, Mucor can also be used in the production of enzymes. For example, *Mucor rouxianus* produces xylanase, *Mucorindicus* produces rennet, and *Mucor circinelloides* produces fatty acid desaturase.

[0003] However, existing culture media for culturing *Mucor* cannot fully realize its metabolic potential, resulting in low growth rates and product production efficiency, thus affecting its biomass and product yield. Therefore, there is a need for a culture medium that can effectively promote *Mucor* growth, increase its biomass, and consequently improve its product yield. While there are reports of fermentation media for increasing γ-linolenic acid production by *Mucor* truncatulae*, the biomass of *Mucor* truncatulae* cultured using these media is still insufficient, with the highest biomass not exceeding 20 g / L. Summary of the Invention

[0004] To address the problems existing in the prior art, the present invention provides a culture medium that can promote the growth of Mucor and significantly increase its biomass.

[0005] The first objective of this invention is to provide a culture medium.

[0006] A second objective of the present invention is to provide the application of the culture medium in the cultivation of Mucor.

[0007] A third objective of this invention is to provide the application of the culture medium in increasing Mucor biomass.

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

[0009] The fifth objective of this invention is to provide a cultivation method for increasing the yield of Mucor or its metabolites.

[0010] The above-mentioned objective of this invention is achieved through the following technical solution:

[0011] This invention addresses the shortcomings of existing culture media for culturing Mucor, which fail to fully utilize the metabolic potential of Mucor, resulting in low biomass. The invention provides a culture medium that effectively increases the biomass of Mucor to above 20 g / L. Therefore, this invention seeks protection for the described culture medium.

[0012] Specifically, the culture medium contains 30–70 mM humic 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 humic 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 fulvic acid concentration is 50–70 mM. In the culture medium described in this invention, the fulvic acid concentration has a significant impact on the biomass of Mucor; when the fulvic acid concentration is 50–70 mM, the biomass of Mucor can exceed 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] More preferably, the culture medium of the present invention contains 70 mM fulvic acid, 10 mM tyrosine, 10 mM phenylalanine, 20 mM sodium alginate, 5 mM disodium hydrogen phosphate, 25 mM sodium dihydrogen phosphate monohydrate, and 100 mM sodium chloride. When Mucor is cultured using this culture medium, the biomass of Mucor is the highest, exceeding 28 g / L.

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

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

[0019] The present invention also claims protection for the use of the culture medium in increasing the yield of metabolites from Mucor.

[0020] The application of the culture medium described in this invention in the preparation of products for culturing Mucor, products for increasing Mucor biomass, or products for increasing the yield of Mucor metabolites should also be within the scope of protection of this invention.

[0021] Specifically, the mold is *Mucor rumenella*, *Mucor rotundifolia*, or *Mucor indica*.

[0022] The present invention also provides a cultivation method for increasing the yield of Mucor or its metabolites, wherein the method comprises: culturing Mucor using the culture medium described in the present invention.

[0023] Specifically, after inoculating Mucor into the culture medium described in this invention, it is first cultured at 30-35°C and 120-150 rpm for 4-5 days, and then cultured at 25-27°C and 90-110 rpm for 4-24 hours.

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

[0025] Specifically, the mold is *Mucor rumenella*, *Mucor rotundifolia*, or *Mucor indica*.

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

[0027] This invention addresses the shortcomings of existing culture media in fully utilizing the metabolic potential of *Mucor* and significantly increasing its biomass. It provides a culture medium containing fulvic acid, tyrosine, phenylalanine, sodium alginate, disodium hydrogen phosphate, sodium dihydrogen phosphate monohydrate, and sodium chloride. Culturing *Mucor* using this medium significantly promotes its growth and increases its biomass or yield, thereby increasing the amount of its metabolic products, such as enzymes produced. This invention facilitates the cultivation and utilization of *Mucor* and promotes the development of the *Mucor*-based enzyme production industry. Attached Figure Description

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

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

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

[0032] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

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

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

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

[0036] The method for preparing 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 bring the volume to 1 L. This will prepare 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 embodiment, *Rhizopus rouxae* was used as the experimental strain for the culture of *Rhizopus rouxae*. The culture method is as follows: Activated *Rhizopus rouxae* seed culture was inoculated into 1L of prepared culture medium at a 5% (v / v) inoculation rate; fermentation was carried out at 30℃ and 150rpm for 120 hours to maximize the bacterial yield; then the temperature was adjusted to 27℃ and the rotation speed to 100rpm, and cultured for another 48 hours to allow the bacterial community to aggregate for better separation; after the culture was completed, the culture medium was removed by filter paper or centrifugation, the bacterial cells were collected, dried at 105℃ to constant weight, and the biomass was measured.

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

[0040] This embodiment describes the cultivation of *Mucor* under laboratory conditions. To explore the maximum yield of *Mucor*, the fermentation time in the later stage was extended to 48 hours, and filtration or centrifugation was also added. However, in actual mass production, the steps of filtration or centrifugation to collect the bacterial cells can be omitted. The purpose of adding sodium alginate to the culture medium in this invention is also to promote better aggregation of the bacterial community. Furthermore, based on the *Mucor* growth curve, it can be seen that 4-5 hours of cultivation is sufficient to achieve the maximum yield. After reaching the maximum yield, further cultivation at 27°C and 100 rpm for 4-24 hours allows the bacterial community to aggregate for better separation.

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

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

[0043] In this embodiment, *Rhizopus rouxae* was used as the experimental strain for *Rhizopus* culture. The culture method was the same as in Example 1. After culture, the culture medium was removed by filter paper or centrifugation, the bacterial cells were collected, dried at 105°C to constant weight, and the biomass was measured. The biomass of *Rhizopus rouxae* cultured using the culture medium described in this embodiment was 26.77 g / L.

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

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

[0046] In this embodiment, *Rhizopus rouxae* was used as the experimental strain for *Rhizopus* culture. The culture method was the same as in Example 1. After culture, the culture medium was removed by filter paper or centrifugation, the bacterial cells were collected, dried at 105°C to constant weight, and the biomass was measured. The biomass of *Rhizopus rouxae* cultured using the culture medium and method described in this embodiment was 25.80 g / L.

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

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

[0049] In this embodiment, *Rhizopus rouxae* was used as the experimental strain for *Rhizopus* culture. The culture method was the same as in Example 1. After culture, the culture medium was removed by filter paper or centrifugation, the bacterial cells were collected, dried at 105°C to constant weight, and the biomass was measured. The biomass of *Rhizopus rouxae* cultured using the culture medium and method described in this embodiment was 23.80 g / L.

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

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

[0052] In this embodiment, *Rhizopus rouxae* was used as the experimental strain for *Rhizopus* culture. The culture method was the same as in Example 1. After culture, the culture medium was removed by filter paper or centrifugation, the bacterial cells were collected, dried at 105°C to constant weight, and then the biomass was measured. The biomass of *Rhizopus rouxae* cultured using the culture medium and method described in this embodiment was 23.10 g / L. Comparative Example 1: Effect of different culture media on *Rhizopus* biomass.

[0053] In this invention, *Rhizopus ruthenicus* was used as the experimental fungus. *Rhizopus ruthenicus* was cultured using the culture medium described in Example 1 and conventional potato dextrose liquid medium, respectively. The culture method was the same as in Example 1. The biomass of *Rhizopus ruthenicus* was measured daily, and its growth curves in different culture media were plotted.

[0054] The growth curves of *Rhizopus roux* in potato dextrose liquid medium and the medium described in Example 1 are as follows: Figure 1 As shown. By Figure 1 It is evident that, compared to the traditional potato glucose liquid culture medium, the culture medium described in this invention can significantly promote the growth of Mucor and increase Mucor biomass.

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

[0056] Because different fungi exhibit varying growth patterns in the same culture medium, and different combinations of carbon and nitrogen sources also have varying effects on the growth of the same fungi, and currently there is no culture medium that can significantly increase the biomass of *Mucor*. Therefore, this invention selects a variety of different carbon and nitrogen sources, and on this basis, combines other components, and through continuous experimentation and optimization, obtains the culture medium described in this invention. The formulations of some culture media tested using *Mucor* rumeni, *Mucor* truncatulata*, and *Mucor* indica, and the results of their effects on the biomass of different *Mucor* species are as follows.

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

[0058] Prepare culture media containing different single carbon sources according to Table 1, and culture Mucor rhubarb, Mucor truncatula, and Mucor ainula using the prepared culture media (each group was repeated 3 times).

[0059] Table 1

[0060]

[0061]

[0062] The cultivation method is as follows: Activated *Rhizopus rouxii* / *Rhizopus chinensis* / *Rhizopus ininus seed culture was inoculated into 1L of prepared culture medium at a 5% (v / v) inoculation rate. Fermentation was carried out at 30℃ and 150rpm for 24 hours, followed by fermentation at 27℃ and 100rpm for 60 hours. After terminating the culture, the mycelium was harvested, and the culture medium was removed using the filter paper method (or centrifugation). The mycelium was dried at 105℃ to constant weight, and the biomass was measured and data analyzed (one-way ANOVA was used to compare whether the biomass differences of different carbon sources on the same *Rhizopus* strain were significant; Tukey HSD (post-hoc test) was used to clarify the significance of differences between different carbon sources; R language was used to analyze the data and plot the mean biomass and standard error graphs for each group of carbon sources).

[0063] The effects of different carbon sources on Mucor biomass are as follows: Figure 2 As shown in the figure, different letters indicate significant differences (p<0.05). Figure 2 It was found that when fulvic acid was used as the sole carbon source, the three *Mucor* species exhibited the highest average yield (*Mucor rouxieri* 28.49 g / L, *Mucor truncatula* 26.77 g / L, and *Mucor amurensis* 26.28 g / L), indicating that fulvic acid is superior to the other three carbon sources in terms of *Mucor* yield. ANOVA analysis of the data and Tukey analysis clarified the ranking of significance. Fulvic acid showed the most significant difference compared to the other three carbon sources, indicating that fulvic acid has a significant advantage in increasing *Mucor* biomass and that fulvic acid is a universally applicable carbon source for *Mucor*.

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

[0065] Referring to culture medium No. 1 in Table 1, culture media with humic acid concentrations of 10, 20, 30, 40, 50, 60, and 70 mM were prepared and cultured separately for *Rhizopus rouxae*, *Rhizopus leuciscus*, and *Rhizopus indicus* (each group was repeated 3 times). The culture method and data analysis method were the same as above.

[0066] The effects of different fulvic acid concentration gradients on Mucor biomass are as follows: Figure 3 As shown in the figure, different letters indicate significant differences (p<0.05). Figure 3It can be seen that the average yield of the three Rhizopus species reached its maximum when the fulvic acid concentration was 70 mM (Rhizopus rouxeris 28.6 g / L, Rhizopus spp. 27.3 g / L, Rhizopus indicus 26.6 g / L). The reason why the biomass of the 10 mM fulvic acid group was higher than that of the 20 mM group and equal to that of the 30 mM group is that when the fulvic acid concentration was 10 mM, a higher concentration of glucose was used to supplement the overall TOC (Total Organic Carbon, which is maintained at 8000 mg / L to reflect the influence of the concentration gradient of fulvic acid as a carbon source). Therefore, when the TOC concentration of fulvic acid was less than 8000 mg / L, glucose was used to supplement it to 8000 mg / L. The participation of fulvic acid as a carbon source was relatively low, a phenomenon similar to the results of glucose as a carbon source in Comparative Example 1. ANOVA and Tukey post-hoc tests showed that fulvic acid dosage exhibited high variability across the 50–70 mM concentration range, with the highest variability observed at 70 mM. This indicates that controlling fulvic acid concentration within the 50–70 mM range significantly increased Rhizopus biomass, reaching its peak at 70 mM. The differences among the three Rhizopus species within the same group remained relatively consistent, suggesting that fulvic acid's effect on Rhizopus yield increase was stable within the 30–70 mM concentration range.

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

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

[0069] The effects of different nitrogen sources on Mucor biomass are as follows: Figure 4 As shown in the figure, different letters indicate significant differences (p<0.05). Figure 4 It was found that the nitrogen source combination of tyrosine and phenylalanine exhibited the highest average biomass of *Mucor* (28.76 g / L for *Mucor leucocephala*, 27.3 g / L for *Mucor leucocephala*, and 26.5 g / L for *Mucor arachidii*). Analysis of variance and post-hoc tests showed that the nitrogen source combination of tyrosine and phenylalanine significantly increased yield. This combination also showed stability among different *Mucor* species, demonstrating its universality. Other nitrogen source combinations showed significant yield differences among different *Mucor* species.

[0070] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A culture medium for increasing Mucor yield, characterized in that, The culture medium consists of fulvic acid, tyrosine, phenylalanine, sodium alginate, disodium hydrogen phosphate, sodium dihydrogen phosphate monohydrate, sodium chloride, and water; the concentration of fulvic acid is 9–21 g / L, the concentration of tyrosine is 8–12 mM, the concentration of phenylalanine is 8–12 mM, the concentration of sodium alginate is 3.6–6 g / L, the concentration of disodium hydrogen phosphate is 3–10 mM, the concentration of sodium dihydrogen phosphate monohydrate is 20–30 mM, and the concentration of sodium chloride is 80–100 mM; the *Mucor* is *Mucor rumeni*, *Mucor rotundi*, or *Mucor indica*.

2. The culture medium according to claim 1, characterized in that, The concentration of fulvic acid is 15–21 g / L.

3. The culture medium according to claim 2, characterized in that, The concentrations of tyrosine, phenylalanine, sodium alginate, disodium hydrogen phosphate, sodium dihydrogen phosphate monohydrate, and sodium chloride were 90–100 mM.

4. The culture medium according to claim 3, characterized in that, The concentrations of fulvic acid, tyrosine, phenylalanine, sodium alginate, disodium hydrogen phosphate, sodium dihydrogen phosphate monohydrate, and sodium chloride were 21 g / L, 10 mM, 10 mM, 4.8 g / L, 5 mM, 25 mM, and 100 mM, respectively.

5. The use of the culture medium according to any one of claims 1 to 4 in increasing Mucor biomass.

6. A cultivation method for increasing Mucor yield, characterized in that, Mucor is cultured using the culture medium described in any one of claims 1 to 4, wherein the Mucor is Mucor rouxii, Mucor truncatula, or Mucor indica.

7. The method according to claim 6, characterized in that, The cultivation includes a first-stage cultivation and a second-stage cultivation; wherein... The conditions for the first stage of cultivation are: 30–35 ℃ and 120–150 rpm for 4–5 days; The conditions for the second stage of cultivation are: 25-27 ℃ and 90-110 rpm for 4-24 hours.

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