TWI434932B - Rhizopus spp., the culture method and the application thereof - Google Patents

Description

Rhizopus and its culture method and application

The present invention relates to a rhizopus and a culture method and application thereof, in particular to a rhizopus mutant strain for reducing sporangia production, a method for cultivating a spore-forming cyst of a rhizopus, and an application thereof.

Rhizopus spp. is a fungus commonly found in nature. It produces liquefied enzymes and saccharification enzymes and is widely used in the food fermentation industry. In addition, Rhizopus growth is extremely rapid, and the composition of its cell wall also contains chitin components.

The research literature proves that chitin and chitosan have biocompatibility, biodegradability, biological activity and other characteristics, and have the effect of promoting wound healing and hemostasis, so it is very suitable for use as biomedical materials. source. With the advancement of biotechnology, the processing technology for making biomedical materials using shells rich in chitin-like crustaceans has been developed to replace traditional natural plant fibers or animal hair dressings.

However, the production of chitin-containing biomedical materials from the outer shell of crustacean organisms must be extracted and extracted with high-temperature and high-concentration acid and alkali, and then bleached, dissolved, drawn and braided. As a porous film of a biomedical material, it can be seen that the overall processing process is cumbersome and time-consuming to manufacture, and in addition, a large amount of industrial wastewater generated in the production process is also very difficult to handle, and the cost is naturally expensive.

Although the technology of using the fungus-like Ganoderma lucidum waste residue to make chitin-containing biomedical materials has been developed, the growth time of Ganoderma lucidum is longer, and the preparation time is at least 15 days, so it is difficult to effectively Mass production of biomedical materials containing chitin. As for the aforementioned Rhizopus, although it grows rapidly, the mature Rhizopus hyphae has black sporangia, which may affect the appearance of biomedical materials. Therefore, it is generally considered that Rhizopus is not suitable for making biomedical materials, nor is it Rhizopus. Producing biomedical materials for raw materials.

In view of this, how to use the advantage of Rhizopus to find the rhizopus with lower sporangia yield and its culture conditions, thereby developing a biomedical material that has a good effect on wound healing and simplifying the process of the biomedical material, Shortening the production capacity at the same time during the production period is an urgent problem to be solved.

The main purpose of the present invention is to provide a method for cultivating Rhizopus by adding an additional monosaccharide to a medium to prepare a selective medium, so that the ratio of the carbon source of the selective medium is higher than the ratio of the nitrogen source, thereby delaying the rhizopus spores. The production of the sac and promote the production of mycelium; in addition, the present invention also provides a mutant strain of Rhizopus which has a low amount of sporangia, and the bacterium is cultured by the method of stimulating the growth of sporangia to produce mycelium for application to the bacterium. Preparation of medical materials.

In order to achieve the above object, a broader aspect of the present invention provides a method for delaying the growth of spore sac of Rhizopus, which is applied to the preparation of a biomedical material, the method comprising: adjusting the carbon source and the proportion of the nitrogen source of the medium, The proportion of the carbon source is higher than the nitrogen source; the rhizopus is inoculated in the medium; the medium inoculated with the rhizopus is cultured in an environment having a temperature range of substantially 25-32 ° C; and the culture time is controlled to be substantially 6 -9 days.

According to the concept of the present invention, the medium is a selective medium selected from the group consisting of potato dextrose broth and millet medium, and an additional 2% of monosaccharide is additionally added, and the monosaccharide is preferably glucose.

According to the concept of the present case, the Rhizopus genus is a mutant Litchi rhizopus strain, and it has the identification feature of the strain deposited in the Hsinchu Food Industry Development Research Institute of Taiwan and registered as BCRC930114.

According to the concept of the present invention, the culture temperature is substantially 29 ° C and the culture time is substantially 8 days.

In order to achieve the above object, another broad aspect of the present invention provides a method for preparing a biomedical material, which comprises the steps of: adjusting the culture conditions and medium of Rhizopus to reduce the sporangia production of Rhizopus, cultivating and collecting Mycelium of Rhizopus; removal of protein of mycelium; and adjustment of the pH value of mycelium to make it substantially neutral, and the use of Rhizopus mycelium as a raw material for simple processing to obtain the biomedical material .

According to the concept of the present invention, the protein of the mycelium is heat treated with an alkali solution.

According to the concept of the present case, the medium can be placed in a flat plate for flat plate culture.

According to the concept of the present invention, the biomedical material is a porous membrane formed by Rhizopus mycelium, which can be used as a dressing.

In order to achieve the above purposes, another broad aspect of the present invention provides a Rhizopus strain with low sporulation and a strain of Rhizopus oryzae, which is deposited in the Hsinchu Food Industry Development Research Institute of Taiwan. It is the identification feature of the strain of BCRC930114.

Some exemplary embodiments embodying the features and advantages of the present invention are described in detail in the following description. It is to be understood that the present invention is capable of various modifications in the various aspects of the present invention, and the description and illustration are in the nature of

This case utilizes the characteristics of Rhizopus spp., which is commonly found in nature, and its cell wall contains the characteristics of chitin and polysaccharides, and with its rapid and safe growth, its mycelium forms a cotton-like natural woven porous. The film is used as a raw material for biomedical materials, such as wound dressings or masks, but not limited thereto. However, due to the wide variety of Rhizopus , it is an example in which Rhizopus stonolnifer is gradually distributed and white in the early stage of growth, followed by sporangia and gradually changed to brown, and the spores are adjusted by adjusting the culture conditions. The low-yield lychee strains produce the light-colored mycelium as a raw material, which is beneficial to the application of biomedical materials. However, it should be understood that those with similar characteristics to the lychee rhizopus should also be protected by the case. .

As mentioned above, since Rhizoctonia solani produces brown sporangia with increasing culture time, in order to prevent the appearance of black sporangia from affecting the appearance of biomedical materials, this case first adjusts the culture conditions of Litchi rhizogenes to delay Sporangia production and increased mycelial yield.

Please refer to the first figure, which is a flow chart of a preferred embodiment of the method for delaying the growth of the rhizoctonia spore sac of the present invention. As shown in the first figure, first adjust the carbon source and nitrogen source of a medium, increase the content of the carbon source, and make the proportion of the carbon source higher than the nitrogen source (step S11) to form a stagnation of the rhizopus sporangia. Selective medium. In the present embodiment, the medium is preferably a liquid medium, which is a millet medium or a medium containing potato starch and dextrose, such as potato dextrose broth (PDB), and is added substantially It is a 2% monosaccharide to increase the carbon source content, and is prepared into the selective medium. As for the source of the monosaccharide, glucose is preferred, but not limited thereto.

After the medium has been sterilized, Rhizopus , such as Rhizopus stonolnifer , is grown on potato dextrose agar (PDA) slant medium, for example at 30 ° C, to substantially 1 per ml. spores ^{(1 × 10 7 spores / ml} ) × 10 7 were seeded in selective medium of the step S11 (step S12), after which the medium was inoculated root stolonifer aerobic disposed substantially mildew of 25-32 ℃ In the environment (step S13), the culture time is controlled to be substantially cultured for 6-9 days (step S14), and the culture condition is preferably cultured at 29 ° C for 8 days, since it is slightly higher than the growth temperature suitable for the fungus. (25 ° C), so the mycelium yield is higher, and the risk of bacterial contamination of the medium at high temperatures can be avoided.

However, since the color of Rhizoctonia solani gradually changes from white to brown or even black with the increase of sporangia, the production of Litchi rhizopus can be achieved by the above-mentioned culture method for delaying the formation of spore sac of Rhizoctonia solani. Shallow mycelium for the preparation of biomedical materials.

Please refer to the second figure, which is a flow chart of a preferred embodiment of the method for preparing a biomedical material in the present case, wherein the step S21 of the second figure uses the steps shown in the first figure to delay the sporangia of the rhizosphere of Litchi generate. As shown in the second figure, first in step S21, the medium is first adjusted to increase the carbon source ratio of the medium to become a selective medium for delaying the growth of the rhizopus spore sac, and after sterilization, for example, growing at 30 ° C. Rhizopus oryzae on potato dextrose agar slant medium was inoculated into the selective medium at a dose of 1 × 10 ⁷ spores per ml (1 × 10 ⁷ spores / ml), wherein the composition of the selective medium was extra A potato dextrose culture solution or a millet medium containing substantially 2% of a monosaccharide is added, and the added monosaccharide is preferably glucose, but not limited thereto. Because the carbon source content of the selective medium is larger than the nitrogen source content, in addition to the function of the sporangia which delays the rhizosphere of Litchi, it can also promote the formation of the mycelium of the rhizopus. In addition, since Rhizopus is required to grow in an aerobic environment, in step S21, the selective medium can be accommodated in a tray to perform flat plate culture in a subsequent step, and the selective medium can be large in area. The ground is exposed to an aerobic environment, so that the mycelium of the root-cutting fungus has a large growth area.

Next, the flat plate containing the medium inoculated with Rhizopus oryzae is placed in an aerobic environment of substantially 25-32 ° C, and the flat plate culture is carried out for about 6-9 days, and the root mold of the lychee is collected on the surface of the selective medium. The produced hyphal membrane block, wherein the culture condition is further cultured at 29 ° C for 8 days, and the produced hyphal membrane block is the mycelium of the root litchi, and the appearance is cotton-like and naturally entangled to form A mycelial membrane block similar to a sponge pad. Further, in the present embodiment, since the Rhizopus oryzae system accommodates a large amount of culture by using a flat disk method, the recovery of the mycelial membrane block is more than that of the flask. (flask) is easy, and the appearance is flat and beautiful (as shown in the third figure).

Please refer to the second figure. After step S21, the collected hyphal membrane block is filtered by a sieve to remove the residual selective medium, and the mycelial membrane block is washed three times with the secondary pure water to remove the hyphae. The body is dried (step S22), which can be dried by a freeze dryer, but is not limited thereto. Next, the dried mycelium is subjected to a step of removing protein (step S23). In the present embodiment, the solution is heated by a 1 M potassium hydroxide (KOH) alkali solution, and heat-treated at 70 ° C for 5 hours (or 85 ° C). The water bath is heated for 2 hours to remove the protein, but not limited thereto. In other words, any method that does not damage the structure of the mycelium and can remove the protein can be used in the present case.

Then adjusting the pH value of the mycelium to make it substantially neutral (step S24). In this step, the mycelium can be washed with a large amount of water, and the pH value of the litmus paper or other is used to determine the pH value ( The reagent or instrument of pH) tests the pH value of the mycelium to ensure that the mycelium has no residual alkaline substances. After that, the mycelium is further washed with secondary pure water for about three times and dried (step S25), thereby obtaining a porous film containing polysaccharide and chitin, since the porous membrane is mycelium of Rhizopus, Name it RHIZOCHITIN.

Please refer to Table 1, which is based on the preferred culture conditions (8 days culture at 29 ° C) in this case, using the method shown in the second figure of the present case to produce RHIZOCHITIN from the mycelium of Litchi. The number of sporangia, the dry weight of the mycelium and the recovery rate of the mycelia of the root litchi bacteria obtained from the potato glucose culture solution without additional monosaccharide were compared.

It can be seen from the data in Table 1 that the addition of substantially 2% monosaccharide, such as glucose, to the culture medium can indeed delay the rhizoctonia spore sac under the same culture conditions by changing the ratio of the carbon source to the nitrogen source. The growth and the growth amount of the mycelium are increased, so that the obtained porous membrane RHIZOCHITIN has a higher yield and a lighter color than the mycelium produced without using the selective medium.

In order to further obtain the genus Rhizopus genus with lower sporulation, this case further mutated the wild strain of Rhizopus genus to screen the mutant strain with the lowest sporangia production. The screening method for the mutation in this case will be further elaborated as follows:

(1) First, the number of spores of the spore suspension of the wild plant of Rhizoctonia solani was counted by a micro-counting disk, and it was diluted to make a spore suspension of 1 × 10 ⁶ /ml to be inoculated on the culture dish.

(2) Next, ultraviolet light was irradiated, the irradiation intensity was 15 W, and the irradiation distance was 44 cm. The sample was taken for every 15 seconds of irradiation, and 200 μl of the spore suspension was taken away from the light at the time of the timer. After calculation, the appropriate amount of spore suspension was applied to potato dextrose agar medium, cultured at room temperature, the number of growing colonies was observed by a dissecting microscope and recorded, and the time required for 5% survival was determined by using the residual curve of the colony. Take 5% of the remaining points for mutation screening.

(3) Then, the mutant strain was picked from the potato dextrose agar medium for culture, and the growth weight and the amount of sporangia of the wild strain and the mutant strain were compared to select the number of mutants of the rhizoctonia genus having the lowest sporangia yield. The line name is Rhizopus stolonifer F6 (hereinafter referred to as Rhizopus rhizogenes mutant RSF.6), which was deposited on October 17, 1997 in Hsinchu Food Industry Development Research Institute, Taiwan, under the registration number BCRC930114.

The Rhizoctonia solani mutant strain RSF.6 and the wild Litchi rot fungi strain are coated with the biomedical material as shown in the second figure of the present invention, and the spores of the obtained porous membrane (RHIZOCHITIN) formed by the mycelium are obtained. The comparison of the number of capsules, the dry weight of mycelium and the recovery rate are shown in Table 2.

Table 2 Comparison of Rhizopus oryzae mutant strain RSF.6 and wild strain under the same culture conditions

It can be seen from Table 2 that under the same culture conditions, the Rhizoctonia solani mutant RSF.6 (Accession No. BCRC930114) can produce less sporangia than the wild strain, and the yield of its mycelium is not sub- In the wild strain.

The porous membrane RHIZOCHITIN produced by the above method and the strain was acid-hydrolyzed, and then determined by thin layer chromatography (TLC) and quantified by the Elson-Morgan coloring method. It contains 27% of a sugar amine and has a component of N-acetyl-D-glucosamine, which is a cycline monomer. Because ketone has water absorption, bacteriostasis, good biocompatibility, biodegradability and biological activity, and can activate polymorphic white blood cells to enhance immunity, promote fibroblast growth and keratinocyte proliferation, beneficial In the healing of the wound, it is known that the porous membrane (RHIZOCHITIN) obtained by the method and the strain of the present invention is very suitable for preparing biomedical materials, for example, dressings for wound wounds, skin care, such as healing of acne. The effect of RHIZOCHITIN on wound healing in this case is as follows.

Example 1 RHIZOCHITIN promotes wound healing

Male Wister rats, eight weeks old and weighing more than 300 g, were opened with a 6 mm Φ wound on their back, and gauze (negative control group) and RHIZOCHITIN (experimental group) of the case were used as wound dressings before the wound was completely healed. When the dressing is dry or shedding, a new dressing is added, and during the wound healing process, the wound area is healed by the PIA software, and the average and standard deviation of the three repeated calculations are made, and the wound area is plotted.

Please refer to the fourth figure, which is a comparison of wound area changes in the case of RHIZOCHITIN in this case and gauze as a wound dressing for rats. As shown in the fourth figure, the wounds of the rats were covered with RHIZOCHITIN in this case. On the fourth, eighth and fourth days, the wound healing rate was faster than that of the negative control group and the area was more condensed. The wound was almost completely healed in fourteen days, which was 35% faster than the gauze negative control.

Example 2 RHIZOCHITIN promotes the secretion of growth factors

After the animal experiment, on the first, third, seventh, ninth and twelfth days, the 6mmΦ wound center opened by the original center is taken as the center, and then the 8mmΦ hole puncher is used to align the center around the wound and extract the tissue. Protein is included to determine the total protein concentration, and the growth factor TGF-β (tumor necrosis factor beta) is used as an indicator enzyme, and a commercially available enzyme immunoassay technique kits (Mouse/Rat/Porcine TGF-βimmunoassay, RD) The content of TGF-β (tumor necrosis factor beta) was determined, and the standard curve was drawn to interpolate the concentration of TGF-β to evaluate the effect of RHIZOCHITIN on wound healing through TGF-β content.

In general, the growth factor TGF-β can be secreted by platelet alpha particles, neutrophils, and mononuclear cells, attracting neutrophils and mononuclear cells to secrete various cytokines to attract more cells to the wound. For example, fibroblasts in the dermis near the wound begin to activate, migrate to the wound site and secrete extracellular matrix containing a large amount of collagen, so TGF-β accelerates wound healing.

Please refer to the fifth figure, which compares the TGF-β content of RHIZOCHITIN in this case, the chitin-containing dressing from the outer shell of Ganoderma lucidum or crustacean, and the gauze as the wound dressing of rats. As shown in the fifth figure, RHIZOCHITIN is used as a wound dressing (R). The growth hormone TGF-β that can be stimulated during wound healing is more than the gauze (C) of the negative control group, while Ganoderma lucidum is chitin. Source dressings (S) and dressings based on the shell of crustaceans (B) can also stimulate the secretion of TGF-β, so it should be understood that RHIZOCHITIN does with other dressings containing chitin Generally, it has the ability to promote wound healing. In addition to the growth factor TGF-β, RHIZOCHITIN also has the function of promoting another growth factor VEGF (VEGF), so it can stimulate the secretion of VEGF to enlarge blood vessels, and attract immune cells and plasma proteins from microvessels into tissues. Remove dead cells or invading bacteria, viruses and activate endothelial cells, epithelial cells and lymphocytes for angiogenesis.

Example 3 RHIZOCHITIN inhibits the effect of matrix metalloproteinase

The tissue extract of the above-mentioned rat wounds was subjected to Zymography test, and the activity of matrix metalloproteinases (MMPs) was analyzed by electrophoresis. One of the causes of the wounds being difficult to heal due to the activity or content of matrix metalloproteinases was too high. Therefore, it is known through the detection of matrix metalloproteinase activity whether the wound dressing used is beneficial for wound healing.

Please refer to the sixth figure, which is the activity of matrix metalloproteinase in the wound healing process of RHIZOCHITIN in this case, chitin-containing dressing based on the outer shell of ganoderma lucidum or crustacean, and gauze as the wound dressing of rats. As shown in the sixth figure, the wounds of the rats covered with RHIZOCHITIN as the dressing (R), the matrix metalloproteinases MMP-2 (molecular weight 72KDa) and MMP-9 (molecular weight 92KDa) activity of the wound tissue were negative compared with gauze. The matrix metalloproteinase activity of the control group (C) is low, and as the number of days increases, the matrix metalloproteinase which can be inhibited is lower than the negative control group of the gauze (C), and the chitin-containing dressing derived from Ganoderma lucidum ( S) and chitin-containing dressings (B) derived from crustacean bio-shells, and excessive inhibition of matrix metalloproteinases can prevent excessive decomposition of matrix metalloproteinases, destroy cell matrix and growth factors, delay healing rate and repeat Repairing causes uneven accumulation of collagen. In other words, RHIZOCHITIN in this case not only promotes wound repair, but also avoids the phenomenon that matrix metalloproteinase acts on the wound and is not easy to heal. It also helps the beauty of scars.

Of course, the application of the porous membrane RHIZOCHITIN made by Rhizopus oryzae in this case is not limited to the above-mentioned wound dressing, since RHIZOCHITIN has polysaccharides and chitin components, and chitin has good biological activity. In addition to the above-mentioned application as a wound dressing, it is also possible to use a RHIZOCHITIN extra growth factor to produce a mask, etc., and it can be widely used in the pharmaceutical industry and the cosmetics industry.

In summary, the case uses the fast-growing Rhizopus rhizogenes to produce and promote the mycelium production by delaying the sporangia of Rhizoctonia solani by adjusting the carbon source content of the medium and the culture conditions. In addition, in order to obtain a strain with low sporangia, the case was further mutated with a wild Rhizopus strain, so that the strain number BCRC930114 was registered, making it easier to use the Rhizopus to produce biomedical materials.

Compared with the prior art, this method successfully adjusts the culture method of Rhizopus and delays the production of sporangia. It is a barrier that can not be used to make biomedical materials due to the sporangia caused by Rhizopus, so that it can only be used. Rhizopus for food fermentation can be more widely used. In addition, compared with the conventional biomedical materials that use the crustacean bioshell as a source of chitin, the production method of the biomedical material provided in the present invention is a membrane in which the mycelium is naturally woven by the characteristics of the growth of Rhizopus. It saves the conventional steps of treating crustacean shells with acid and alkali, and complicated steps such as bleaching, spinning, weaving, etc., thereby simplifying the manufacture of biomedical materials and saving costs, and the alkali solution used for removing proteins in this case. It can also be reused, so it is more environmentally friendly and can avoid the pollution caused by the manufacturing process of the prior art. Moreover, since the growth of Rhizopus is not limited by space, the size of the porous membrane produced by the mold is not limited, and the application of the biomedical material is further improved.

As for the biomedical materials made by other fungi, the rhizopus used in this case grows rapidly and is easy to be obtained, so that the production efficiency can be improved and the economic production efficiency can be improved, and the cultivation of rhizopus and its application can be applied. The preparation of medical materials is more complete, and the case is extremely novel and progressive, and the application is made according to law.

The present invention has been described in detail by the above-described embodiments, and may be modified by those skilled in the art, without departing from the scope of the appended claims.

S11-S14. . . The method for delaying the growth of the spore sac of Rhizoctonia solani

S21-S25. . . The process of making biomedical materials in this case

First Figure: This is a flow chart of a preferred embodiment of the method for delaying the growth of the rhizoctonia spore sac of the present invention.

Second: It is a flow chart of a preferred embodiment of a method for producing biomedical materials in this case.

Fig. 3: The mycelium obtained by cultivating the root cultivar of the genus Lychee by the method shown in the second figure.

Figure 4: This is a comparison of wound area changes with RHIZOCHITIN in this case and gauze as a wound dressing for rats.

Figure 5: RHIZOCHITIN in this case, dressing containing chitin from the outer shell of Ganoderma lucidum or crustacean, and comparison of TGF-β content with gauze as a wound dressing for rats.

Figure 6: It is the activity of matrix metalloproteinase during the wound healing process of RHIZOCHITIN in this case, chitin-containing dressing from the outer shell of Ganoderma lucidum or crustacean, and gauze as the wound dressing of rats.

S21-S25. . . The process of making biomedical materials in this case

Claims (20)

A method for delaying the growth of a mold spore sac, the method comprising: adjusting a carbon source and a nitrogen source ratio of a medium to make a ratio of a carbon source higher than a nitrogen source; inoculating a lychee rhizogenes in the medium; The medium inoculated with the lychee rhizogenes is cultured in an environment having a temperature range of 25 to 32 ° C; and a culture time is controlled to be 6 to 9 days. The method of claim 1, wherein the medium is a selective medium. The method of claim 2, wherein the selective medium is selected from the group consisting of potato dextrose broth and millet medium, and an additional 2% monosaccharide is added. The method of claim 3, wherein the monosaccharide is a glucose. The method of claim 1, wherein the root rot fungus is a mutant strain, which is deposited in the Hsinchu Food Industry Development Research Institute of Taiwan, and is registered as a strain of BCRC930116. The method of claim 1, wherein the temperature is 29 ° C and the incubation time is 8 days. A method of making a biomedical material, comprising the steps of: (a) cultivating and collecting the method as described in claim 1 a mycelium of Rhizopus oryzae; (b) a protein that removes the mycelium; and (c) adjusting the pH value of the mycelium to make it neutral. The method of claim 7, wherein the step (a) further comprises: (a1) washing the mycelium; and (a2) drying the mycelium. The method of claim 7, wherein the step (b) is to remove the protein of the mycelium by heating with an alkali solution. The method of claim 7, wherein the step (c) further comprises: (c1) washing the mycelium; and (c2) drying the mycelium. The method of claim 7, wherein the Rhizopus oryzae is a mutant strain by ultraviolet mutagenesis, which is deposited in the Hsinchu Food Industry Development Research Institute of Taiwan, and is registered as a strain of BCRC930116. The method of claim 7, wherein the medium of the step (a) is a selective medium. The method of claim 12, wherein the selective medium is selected from the group consisting of potato dextrose broth and millet medium, and additionally adds 2% glucose. The method of claim 7, wherein the step (a) is advantageous Flat plate culture was carried out using a flat plate. The method of claim 7, wherein the temperature of the step (a) is 29 ° C and the incubation time is 8 days. The method of claim 7, wherein the biomedical material is a dressing. A biomedical material comprising a porous membrane formed by a cell wall of a lychee mycelium, wherein the biomedical material is produced by the method as described in claim 7 of the patent application. The biomedical material according to claim 17, wherein the Rhizopus oryzae is a mutant strain by ultraviolet mutagenesis, which is deposited in the Hsinchu Food Industry Development Research Institute of Taiwan, and the strain numbered BCRC930116 . The biomedical material described in claim 17 is a dressing. A Rhizopus mutant strain is used to prepare a biomedical material, and is deposited in the Hsinchu Food Industry Development Research Institute of Taiwan, and is registered as a strain of BCRC930116.