Preparation method and application of Rhizopus arrhizus powder

Abstract

The present invention belongs to the technical field of bacterial powder preparation, and specifically relates to a preparation method and application of Rhizopus arrhizus powder. The present invention provides a preparation method of Rhizopus arrhizus powder, comprising placing silica gel at the bottom and around the dried Rhizopus arrhizus culture and then performing vacuum drying to obtain Rhizopus arrhizus powder. The vacuum drying temperature of the present invention is 25°C to 30°C. The drying method is simple, does not require heating and cooling during the drying process, is easy to implement, has a short drying time, and has low energy consumption. The drying method does not affect the vitality of the growing cells of Rhizopus arrhizus. The results of the embodiment show that the Rhizopus arrhizus powder is prepared by the drying method of the present invention, and the moisture content of the Rhizopus arrhizus cake can be reduced from 48% to 10% in 2h of drying time, and the time efficiency ratio is high, and the Rhizopus arrhizus powder has high vitality and high saccharification and fermentation vitality.

Description

Technical Field

[0001] The invention belongs to the technical field of bacterial powder preparation, and particularly relates to a preparation method and application of Rhizopus arrhizus powder. Background Art

[0002] Rhizopus arrhizus is widely found in the natural environment, such as soil, plant organs, dead branches and leaves, human body and air. Rhizopus arrhizus can produce a variety of metabolites, including various enzymes such as amylase, lipase and pectinase, so Rhizopus arrhizus is often used in saccharification and fermentation in the food industry (see "Yao Lingdan, Ju Xiao, Timothy YJames, et al. Diversified growth kinetic model of Rhizopus arrhizus [J]. Acta Microbiologica Sinica, 2019, 46(1): 42-53.").

[0003] Preparing bacteria into powder is one of the methods for application in the food industry and preservation of bacteria. Powder has the advantages of convenient transportation, easy preservation, and high safety (see "Yu Ping, Hu Chunyu, Huang Xingxing, et al. Preparation process of inositol-producing Lactobacillus plantarum ZJ2868 powder [J]. Journal of Chinese Institute of Food Science and Technology, 2021, 21(9): 142-149."). The main process of preparing powder is to fix high-concentration bacterial liquid and bacteria through a drying process, so that the live bacteria can be preserved and applied in powdered form. At present, the commonly used forms of drying powder are mainly vacuum freeze drying, among which vacuum freeze drying is the mainstream drying technology for powder used in industry. However, live bacteria must be freeze-dried before they can be mixed with a protective agent to prevent the bacteria from dying during freezing, and the freeze drying time is generally ≥24h, which is time-consuming. The bacterial powder obtained by vacuum freeze drying has poor storage resistance and is easy to absorb water from the external environment and rehydrate (see "Wang Shumin, Sang Yue, Hou Caiyun, et al. Study on the storage activity of freeze-dried Bifidobacterium lactis A04 bacterial powder [J]. Journal of Chinese Institute of Food Science and Technology, 2021, 21(9): 193-202."). The recovery of the bacterial powder activity will be affected after rehydration. At present, there is an urgent need for a method for preparing Rhizopus arrhizus powder with high activity. Summary of the invention

[0004] The purpose of the invention is to provide a method for preparing Rhizopus arrhiza powder, and the prepared Rhizopus arrhiza powder has high activity.

[0005] The purpose of the present invention is to provide a method for preparing Rhizopus arrhizus powder, comprising placing silica gel at the bottom and around a dried Rhizopus arrhizus culture and then performing vacuum drying to obtain the Rhizopus arrhizus powder; the vacuum drying temperature is 25°C to 30°C; and the mass ratio of the silica gel to the mass of the Rhizopus arrhizus culture is (40-60):2.

[0006] Preferably, the vacuum drying pressure is -0.085 MPa to -0.098 MPa, and the vacuum drying time for every 1 to 2 g of Rhizopus arrhizus culture is 1 to 2 h; the water content of the Rhizopus arrhizus culture after drying is 9% to 12%.

[0007] Preferably, the Rhizopus arrhizus culture comprises a Rhizopus arrhizus cake.

[0008] Preferably, the preparation of the Rhizopus arrhizus cake comprises: mixing the culture solution of the Rhizopus arrhizus pellets and the culture material, and then culturing to obtain the Rhizopus arrhizus cake; the culture material comprises lotus root starch or rice flour.

[0009] Preferably, before mixing the culture solution of the Rhizopus arrhizus pellets with the adsorption culture material, the method further comprises crushing the culture solution of the Rhizopus arrhizus pellets.

[0010] Preferably, the preparation of the culture solution of the mycelial pellets of Rhizopus arrhizus comprises: activating the Rhizopus arrhizus to obtain a bacterial solution; and performing shake flask culture on the bacterial solution to obtain the culture solution of the mycelial pellets of Rhizopus arrhizus.

[0011] Preferably, the culture temperature is 30-37° C., and the culture time is 24-48 hours.

[0012] Preferably, during the mixing, the mass ratio of the crushed liquid of Rhizopus arrhizus pellets to the culture material is 1-1.5 mL:1 g; the mixing is carried out on a cell culture plate, which is padded with filter paper, and the filter paper covers the bottom of the culture plate wells and the periphery of the well walls.

[0013] Preferably, the Rhizopus arrhizus includes the Rhizopus arrhizus strain Ca-201613 with a preservation number of CGMCC NO.19912.

[0014] The present invention provides the application of the preparation method of the Rhizopus arrhizus powder described in the above technical scheme in improving the activity of the Rhizopus arrhizus powder or improving the saccharification and fermentation activity of the Rhizopus arrhizus powder.

[0015] Beneficial effects of the invention: The invention provides a method for preparing Rhizopus arrhiza powder, comprising placing silica gel at the bottom and around the dried Rhizopus arrhiza culture and then performing vacuum drying to obtain Rhizopus arrhiza powder; the vacuum drying temperature is 25°C to 30°C; the mass ratio of the silica gel to the mass of the Rhizopus arrhiza culture is (40-60) g:2g. The drying temperature is 25°C to 30°C, which will not affect the vitality of the growing cells of Rhizopus arrhiza during the drying process. The drying temperature is basically room temperature, and no heating or refrigeration is required during the drying process, and the energy consumption is low. Compared with the freeze-drying and hot air drying time, the drying time of this design is 2h, and the drying time is short. The results of the embodiment show that the vitality of the Rhizopus arrhiza powder prepared by the drying method of the invention is high. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0017] Figure 1 The activated Rhizopus arrhizus PDA plate of Example 1; Figure 2 The mycelial pellets of Rhizopus arrhizus and the crushed mycelial pellets prepared in the examples; Figure 3 For normal pipette tips and special pipette tips; Figure 4 The conditions before and after the cultivation of Rhizopus arrhizus in Example 2 and Comparative Examples 1-2; Figure 5 The surface changes of Rhizopus arrhizus before and after cultivation when rice flour is used as adsorption culture material in Example 4; Figure 6 Surface changes of Rhizopus arrhizus before and after cultivation when the sweet potato starch of Comparative Example 5 (upper row) and the lotus root starch of Example 3 (lower row) were used as adsorption culture materials; Figure 7 Surface changes of Rhizopus arrhizus before and after cultivation when cassava starch (upper row) of Comparative Example 3 and potato starch (lower row) of Comparative Example 4 were used as adsorption culture materials; Figure 8 The changes of color-changing silica gel before and after absorbing moisture from the mushroom cake; Fig. 9 The state of the rice flour cake before and after drying of Example 4; Fig.10 Example 3: The state of the lotus root starch cake before and after drying; Fig.11 Comparative Example 3: The state of cassava starch cake before and after drying; Fig.12 Comparative Example 4: The state of potato starch cake before and after drying; Fig.13 Comparative Example 5: The state of the sweet potato starch cake before and after; Fig.14 Specific states of the two paper padding methods of Example 7 and Comparative Example 10; the paper padding method of Example 7 is Method 2; the paper padding method of Comparative Example 10 is Method 1; Fig.15 The state of the Rhizopus arrhizus cakes of the two paper padding methods of Example 7 and Comparative Example 10 after 48 hours of culture; the paper padding method of Example 7 is method 2; the paper padding method of Comparative Example 10 is method 1; Fig.16 Comparative Example 10 Paper pad method 1 The morphology of the Rhizopus agenes cake before and after drying; Fig.17 Example 7 Paper pad method 2 The morphology of the Rhizopus arrhizus cake before and after drying; Fig.18 The morphology of Rhizopus arrhizus after 48 h of culture in two sample addition methods of Example 10 and Comparative Example 14; Fig.19 Example 11 Mode 5 Silica gel addition and cake state before vacuum drying; Fig. 20 Example 11: Morphology of dried mushroom cakes in five drying methods; Fig.21 Example 13 Vacuum silica gel drying verification test The state of the Rhizopus arrhizus cake before and after drying; Fig. 22Example 13 The surface of the Rhizopus arrhizus cake before and after drying under a dissecting microscope at a magnification of 10 times; Fig.23 Embodiment 13 The finished product of the powder of the Rhizopus arrhizus cake ground; Fig.24 Application Example 1: PDA plate status after 48h of culture of Rhizopus arrhizus powder and CK; CK represents CK1; Fig.25 Application Example 1: PDA plate status after 48h of culture of Rhizopus arrhizus powder dilution and CK; CK represents CK2; Fig.26 Application Example 1: The state of the Rhizopus arrhizus powder after storage for 10 days before and after cultivation on the PDA plate; Fig. 27 Application Example 1: The status of CK and Rhizopus arrhizus powder inoculated test tubes 1, 2, and 3 before and after fermentation; Fig.28 Application Example 1: The status of CK and Rhizopus arrhizus powder inoculated test tubes 4, 5, and 6 before and after fermentation treatment in the raw powder liquid treatment group; Fig.29 Application Example 1: Close-up of the CK and inoculated fermentation of the group treated with the gelatinized liquid of lotus root starch and the group treated with the raw liquid of lotus root starch; Fig.30 Application Example 1: The glucose standard curve obtained by drawing; Fig.31 Application Example 1: The state of the fermentation liquid of the group treated with the gelatinized liquid of lotus root starch and the group treated with the raw liquid of lotus root starch after being colored with the DNS reagent.

[0018] Biological Deposit Description

[0019] Rhizopus arrhizus Ca-201613 was deposited in the China General Microbiological Culture Collection Center (CGMCC) on June 8, 2020, with the deposit number CGMCC No.19912. The address of the deposit unit is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. DETAILED DESCRIPTION

[0020] The invention provides a method for preparing Rhizopus arrhizus powder, comprising placing silica gel at the bottom and around the bottom of a dried Rhizopus arrhizus culture and then performing vacuum drying, wherein the vacuum drying temperature is 25°C to 30°C; the mass ratio of the silica gel to the mass of the Rhizopus arrhizus cake is (40 to 60): 2. The vacuum drying temperature of the invention is 25°C to 30°C, preferably 25°C to 28°C, and more preferably 25°C.

[0021] In the present invention, the vacuum drying pressure is preferably -0.085MPa to -0.098MPa, further preferably -0.090MPa to -0.098MPa, and more preferably -0.098MPa; the vacuum drying time for each 1 to 2g of Rhizopus arrhizus culture of the present invention is preferably 1 to 2h, and more preferably 2h. The amount of Rhizopus arrhizus culture of the present invention is related to the vacuum drying time. The more the amount of Rhizopus arrhizus culture, the longer the vacuum drying time. The vacuum drying time in the present invention is quantitatively operated based on 2.0g of Rhizopus arrhizus cake.

[0022] In the present invention, the role of the silica gel is to adsorb moisture in the Rhizopus arrhizus and moisture in the air. The vacuum freeze drying operation requirements in the prior art are relatively strict, with pre-freezing, sublimation dehydration and low-temperature rehydration steps, which have certain specificity for operators, and freeze drying must be maintained by refrigeration and sublimation dehydration at low temperatures, so the energy consumption is high, and the drying method described in the present invention is universal, easy to operate, low in energy consumption, and high in time efficiency. The dried Rhizopus arrhizus only needs to use culture dishes, silica gel and filter paper, and does not need to use special protective agents for Rhizopus arrhizus, and most technicians can implement the drying operation. The low temperature range of growth inhibition of Rhizopus arrhizus, the optimal growth temperature range, the high temperature range of growth inhibition and the lethal high temperature range are 4-9°C, 30-37°C, 40-49°C and 40-52°C, respectively. The drying temperature of the present application is 25°C-30°C, which is a mild temperature range for Rhizopus arrhizus, and the vitality of Rhizopus arrhizus will not be affected during the drying process. The drying temperature is basically room temperature, and no heating or cooling is required during the drying process, so the energy consumption is low. Compared with freeze drying and hot air drying, the drying time of this application is 2 hours, which is short and has a high time efficiency ratio.

[0023] The hot air drying in the prior art is relatively simple. Generally, the bacteria are prepared into mycelium balls or bacterial films, placed in a container, and dried in a drying oven with air blowing. The drying temperature is generally 30-40°C, and the time is generally 8 hours. The bacteria powder obtained by hot air drying generally has large particles or is difficult to crush, and the loss of bacteria powder mass during the drying process is large. The drying method of the present application reduces the loss of Rhizopus arrhizus bacteria and spores during the drying process. When the Rhizopus arrhizus cake is dried, the integrity of the cake can also be maintained.

[0024] In the present invention, the Rhizopus arrhizus preferably includes the Rhizopus arrhizus strain Ca-201613 with a deposit number of CGMCC NO.19912. The Rhizopus arrhizus strain Ca-201613 of the present invention was isolated and identified from cassava tubers by professional and technical personnel of the Cassava Research Institute of the Guangxi Zhuang Autonomous Region Subtropical Crops Research Institute. The basic morphological characteristics of Rhizopus arrhizus are creeping hyphae and rhizoids are brown, sporangiophores are brown, can grow to 600 μm, 5 to 15 μm in diameter, spherical or nearly spherical, with a diameter of 60 to 140 μm, and mature sporangia are black. Sporangiospores are single, spherical, with a diameter of 3.5 to 6um; or nearly spherical, ovoid, irregular: colorless or light gray, gray-brown when aggregated; there are inconspicuous stripes and edges on the surface. No zygospores were found.

[0025] The present invention places silica gel at the bottom and around the dried Rhizopus arrhizus culture before vacuum drying. When the present invention uses silica gel for drying, the mass ratio of silica gel to the mass of Rhizopus arrhizus culture is (40-60):2, preferably (40-50):2, and more preferably 40:2. The present invention places silica gel at the bottom and around the Rhizopus arrhizus culture at the same time to absorb the moisture of the Rhizopus arrhizus culture and the moisture in the air, which is beneficial to subsequent vacuum drying. The source of the silica gel described in the present invention is not particularly limited, and a conventional method can be used. The quality of the silica gel will affect the drying effect, and silica gel of sufficient quality must be placed.

[0026] The Rhizopus arrhizus culture of the present invention preferably comprises a Rhizopus arrhizus fungus cake.

[0027] The preparation of the Rhizopus arrhizus cake of the present invention preferably comprises: mixing the culture solution of the Rhizopus arrhizus pellet and the adsorption culture material, and then culturing to obtain the Rhizopus arrhizus cake. The preparation of the culture solution of the Rhizopus arrhizus pellet of the present invention preferably comprises: obtaining a bacterial solution after activating the Rhizopus arrhizus; and culturing the bacterial solution in a shake flask to obtain a mycelial pellet culture solution. In the present invention, the culture medium used in the activation is preferably PDA, the activation temperature of the present invention is preferably 30-37°C, more preferably 30-32°C, more preferably 32°C, and the activation time is preferably 24-72h, more preferably 48-72h, more preferably 72h. The PDA culture medium of the present invention preferably adopts a conventional commercially available potato dextrose agar culture medium, and the potato dextrose agar culture medium is prepared by adding a certain amount of water according to the instructions of the commercially available package, and the potato dextrose agar culture medium of the present invention is preferably produced by Hangzhou Baisi Biotechnology Co., Ltd.

[0028] After obtaining the bacterial liquid, the present invention preferably performs shake flask culture on the bacterial liquid to obtain a culture liquid of Rhizopus arrhizae. In the present invention, the culture medium used for the shake flask culture is preferably PDB, the temperature of the shake flask culture of the present invention is preferably 30-37°C, more preferably 30-32°C, more preferably 32°C, the rotation speed of the shake flask culture is preferably 120-180r / min, more preferably 120-150r / min, more preferably 150r / min, the time of the shake flask culture of the present invention is preferably 24-72h, more preferably 48-72h, more preferably 72h. The PDB culture medium of the present invention preferably adopts conventional commercially available potato glucose water culture medium, and the potato glucose water culture medium is prepared by adding a certain amount of water according to the instructions of the commercially available packaging. The PDB culture medium of the present invention is preferably produced by Qingdao Haibo Biotechnology Co., Ltd.

[0029] After obtaining the mycelial ball culture solution, the present invention preferably crushes the mycelial ball culture solution to obtain the mycelial ball crushed liquid of Rhizopus arrhiza. The crushing of the present invention preferably includes homogenization crushing, and the homogenization crushing time of the present invention is preferably 20 to 30 seconds, more preferably 25 seconds. In the present invention, there is no special limitation on the equipment and operating parameters of the crushing, and conventional crushing equipment and parameters are sufficient. The present invention preferably uses a small Joyoung household mill (model S1-M81) to crush the mycelial ball culture solution. The crushing of the present invention is preferably completed in a clean bench. The crushing of the present invention preferably includes the following steps: the crusher main machine and the stirring cup of the Joyoung household mill (model S1-M81) are first disinfected with 75% ethanol for 30 seconds, and then sterilized by ultraviolet irradiation on the clean bench for 30 minutes; then the mycelial ball culture solution (PDB culture solution containing mycelial balls) is poured into the mill, and the homogenization crushing is 20 to 30 seconds to obtain the mycelial ball crushed liquid of Rhizopus arrhiza. The invention uses the crushed liquid of Rhizopus arrhizae pellets for inoculation in order to improve the growth potential of Rhizopus arrhizae.

[0030] After obtaining the crushed liquid of Rhizopus arrhizae mycelial pellets, the present invention preferably mixes the crushed liquid of Rhizopus arrhizae mycelial pellets with an adsorption culture material and then cultures them to obtain a Rhizopus arrhizae cake.

[0031] The mass ratio of the crushed liquid of the Rhizopus arrhizus mycelium pellets to the culture material of the present invention is preferably 1-1.5 mL: 1 g, and more preferably 1 mL: 1 g. The selection of the mass ratio of the crushed liquid of the Rhizopus arrhizus mycelium pellets to the adsorption culture material of the present invention ensures the rapid growth of Rhizopus arrhizus while making the morphology of the Rhizopus arrhizus cakes before and after drying basically the same. The culture material of the present invention includes lotus root starch or rice flour.

[0032] The lotus root starch of the present invention has a good absorption effect on the crushed liquid of the mycelium pellets of Rhizopus arrhizus, can improve the growth of Rhizopus arrhizus, generate a large number of hyphae and a large number of sporangia, which is suitable for the growth of Rhizopus, and the rice powder cake generates a large number of hyphae. The present invention has no special restrictions on the sources of the lotus root starch and rice powder, and conventional commercial products can be used.

[0033] The mixing of the present invention is preferably completed in a cell culture plate. During the mixing of the present invention, preferably, filter paper is placed in the hole of the cell culture plate, and the lotus root starch is placed on the filter paper and flattened, and then the crushed liquid of Rhizopus arrhizae is added to the surface of the lotus root starch. The addition method of the lotus root starch and the crushed liquid of Rhizopus arrhizae can improve the growth vitality of Rhizopus. During the mixing of the present invention, preferably, a pipette is used to absorb the crushed liquid of Rhizopus arrhizae and add it to the adsorption material. The tip of the pipette of the present invention is preferably a special tip or a normal tip. The preparation method of the special tip of the present invention is preferably: from a 1mL normal pipette tip, at a distance of 6.00±0.02mm from the tip of the gun, the tip of the gun is cut off at a 45° angle to obtain it.

[0034] When the filter paper is placed in the hole of the cell culture plate of the present invention, the filter paper preferably covers the bottom of the culture plate hole and the periphery of the hole wall, and more preferably, the filter paper completely covers the bottom of the culture plate hole and the periphery of the hole wall. The selection of the paper padding method of the present invention can increase the amount of hyphae and sporangia attached to the surface of the Rhizopus arrhizus cake.

[0035] After the crushed liquid of the Rhizopus arrhizus pellet is mixed with the adsorption culture material, the present invention preferably covers the lid of the cell culture plate for culture. The culture temperature of the present invention is preferably 30-37°C, more preferably 30-32°C, and more preferably 32°C; the culture time of the present invention is preferably 24-48h, more preferably 36-48h, and further preferably 40-48h, and more preferably 48h. When the culture time is 48h, the Rhizopus arrhizus cake already has a large amount of hyphae and sporangia, and has grown all over the surface. The choice of the culture time of 48h is because the Rhizopus arrhizus is in a period of vigorous growth during this period, and the growth activity of the Rhizopus arrhizus is high.

[0036] The crushed liquid of Rhizopus arrhiza pellets is mixed and cultured with an adsorption culture material to obtain a Rhizopus arrhiza culture. The present invention adopts the technical solution described in the above technical solution, that is, silica gel is placed at the bottom and around the Rhizopus arrhiza culture and then vacuum-dried for drying. The vacuum silica gel drying technical solution has been discussed above and will not be repeated here.

[0037] The present invention provides the application of the vacuum silica gel drying method described in the above technical scheme or the preparation method of Rhizopus arrhiza powder described in the above technical scheme in improving the activity of Rhizopus arrhiza powder or improving the saccharification and fermentation activity of Rhizopus arrhiza powder.

[0038] The Rhizopus arrhizus powder prepared by the invention has strong activation ability, strong storage resistance and strong saccharification and fermentation activity on starch substrates.

[0039] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0040] The mass of each Rhizopus arrhizus cake in the embodiment is 2 g.

[0041] Example 1 Preparation of Rhizopus arrhizus powder

[0042] The preparation method of Rhizopus arrhizus powder is as follows:

[0043] (1) Preparation of Rhizopus arrhizus liquid: Prepare potato dextrose agar medium (PDA medium, produced by Hangzhou Baisi Biotechnology Co., Ltd.), sterilize at 121℃ for 15 minutes, take out the medium and cool it to 50-60℃, pour 15-20mL of PDA medium into a 90mm culture dish, and wait for it to cool and solidify to form a PDA plate. Take the slope of a test tube containing the Rhizopus arrhizus strain (strain patent preservation number: CGMCC NO.19912), use an inoculation loop to pick up a loop of Rhizopus, draw a line on the PDA plate, place the plate in a 32℃ incubator for activation and culture for 72 hours, place the activated PDA plate on a clean bench, rinse the surface of the plate with 20mL of sterile water, and use an inoculation loop to continuously scrape the surface spores for 20 seconds. The liquid collected on the surface of the PDA plate is the Rhizopus arrhizus liquid. The activated PDA plate is shown in Figure 1 .

[0044] (2) Preparation of Rhizopus arrhiza pellet pulverized liquid: Prepare potato glucose liquid culture medium (PDB culture medium, produced by Qingdao Haibo Biotechnology Co., Ltd.), use a measuring cylinder to measure 50 mL of liquid culture medium, divide it into glass bottles, sterilize it at high temperature, take it out and cool it to room temperature, and use a pipette with a range of 1 mL on a clean bench to draw 1 mL of the prepared Rhizopus arrhiza pellet liquid, add it to a glass bottle containing sterilized PDB culture medium, and shake the bottle in a shaker for 72 h at a culture temperature of 32°C and a rotation speed of 150 r / min. After shaking out the large mycelium balls, take a small Joyoung household mill (model S1-M81) on the clean bench, first spray the main unit and the inside and outside of the mixing cup with 75% ethanol for 30 seconds, then sterilize with ultraviolet irradiation on the operating table for 30 minutes, pour the PDB culture solution containing mycelium balls into the sterilized and dried mixing cup of the mill, homogenize and crush for 20 to 30 seconds, pour the crushed liquid back into the original culture bottle, and the obtained culture solution containing bacteria is the crushed liquid of Rhizopus arrhizae mycelium balls. The number of mycelial activity of the crushed liquid of Rhizopus arrhizae mycelium balls is greater than or equal to 107 / mL order of magnitude. Figure 2 .

[0045] (3) Preparation of Rhizopus oligiformis cake: Take a 6-well cell culture plate (size specification is 126mm×83mm×22mm), and pad a circular filter paper with a radius of 30mm in the well of the culture plate, so that the filter paper can cover both the bottom of the well and the surrounding of the well wall. Weigh 2.000g of the adsorption culture material lotus root starch and pour it into the well padded with filter paper, and flatten the starch surface; according to the ratio of 1mL:1g of Rhizopus oligiformis mycelium pellet crushed liquid and lotus root starch, use a pipette to evenly add 2mL of mycelium pellet crushed liquid to the flattened lotus root starch surface in the well, and use a special pipette tip to absorb the Rhizopus oligiformis mycelium pellet crushed liquid. The special pipette tip is obtained by cutting off the tip of a 1mL normal pipette tip at a 45° angle at a distance of 6.00±0.02mm from the tip of the pipette, see Figure 3 After the inoculation of the crushed liquid of Rhizopus arrhizus pellets was completed, the cell culture plate was covered and cultured in an incubator at 32°C for 48 hours to obtain a Rhizopus arrhizus cake.

[0046] (4) Drying of Rhizopus oligiformis cake: First, a layer of 20 g of color-changing silica gel was placed on the lid of a 90 mm culture dish, and then a 9 cm filter paper was placed on it. The Rhizopus oligiformis cake was taken out from the hole of the cell culture plate, and after removing the bottom pad paper, it was placed in the center of the filter paper. 20 g of color-changing silica gel was poured around the Rhizopus oligiformis cake so that the silica gel surrounded the edge of the cake. The cake was then placed directly in a vacuum drying oven for drying. The vacuum drying oven was set to a vacuum pressure of -0.098 MPa, a drying temperature of 25 ° C, and a drying time of 2 h. The mass of each Rhizopus oligiformis cake was 2 g.

[0047] (5) After the drying operation of the Rhizopus arrhizus cake is completed, the dried Rhizopus arrhizus cake is placed in a sterilized mortar and ground for 10 seconds to obtain the Rhizopus arrhizus powder that is ground into powder and uniformly adsorbed with hyphae and spores.

[0048] Example 2

[0049] The preparation of the pulverized liquid of Rhizopus arrhizae mycelium pellet is the same as in Example 1. After preparing the pulverized liquid of Rhizopus arrhizae mycelium pellet, a 6-well cell culture plate (size specification is 126mm×83mm×22mm) is taken. A hole on the culture plate is selected and a circular filter paper with a radius of 16mm is placed at the bottom of the hole. 1.000g of rice flour is poured into the hole, and the rice flour surface is flattened with a tool. 1mL of mycelium pellet pulverized liquid is drawn with a special pipette tip on a 1mL range pipette, and slowly added to the rice flour. The liquid is evenly distributed on the surface of the rice flour. The preparation method of the special pipette tip is the same as in Example 1. After the inoculation is completed, the culture plate cover is covered, and the growth of the Rhizopus arrhizae mycelium pellet is observed in a 32°C incubator for 48h.

[0050] Comparative Example 1

[0051] Preparation of bacterial solution: Same as in Example 1, transfer the bacterial solution to a sterilized glass bottle and store in a refrigerator at 4°C for later use. Use a pipette to draw 1 mL of PDB culture solution and slowly add it to the rice flour to wet the surface of the rice flour so that the water content of the rice flour is consistent with that of the first well, then draw 0.5 mL of bacterial solution and add it to the rice flour. The remaining conditions are the same as in Example 2.

[0052] Comparative Example 2

[0053] The PDA plate of Rhizopus arrhizus obtained in the preparation of the Rhizopus arrhizus liquid in Example 1 was activated and cultured for 72 hours in a 32°C incubator, and a round agar block with bacteria growing on the surface was punched at the edge of the plate with a sterilized 6mm puncher, i.e., a bacterial block, and the diameter of the bacterial block was 6mm. 1mL PDB culture solution was sucked with a pipette and slowly added to the rice flour to moisten the surface of the rice flour so that the water content of the rice flour was consistent with that of the first hole, and a 6mm bacterial block was picked and inverted in the center of the hole surface. The remaining conditions were the same as in Example 2.

[0054] The conditions before and after the cultivation of the bacteria in Example 2 and Comparative Examples 1 to 2 are shown in Table 1 and Figure 4 According to Table 1 and Figure 4 It can be seen that under the same culture conditions, the inoculation methods of Example 2 and Comparative Examples 1-2 are different, and the growth potential of Rhizopus arrhizus is different. The growth potential of Rhizopus arrhizus is from strong to weak in the order of mycelial pellet crushed liquid> fungus block> fungus liquid.

[0055] Table 1 Changes before and after cultivation of three inoculation methods

[0056]

[0057] Example 3

[0058] The preparation of the crushed liquid of Rhizopus arrhizus mycelium pellets is the same as in Example 1. After the crushed liquid of Rhizopus arrhizus mycelium pellets is prepared, lotus root starch is selected as the adsorption culture material. A 6-well cell culture plate is taken, and a circular filter paper with a radius of 16 mm is first placed at the bottom of each well of the culture plate. 2.000 g of lotus root starch is weighed and poured into the well, the surface of the lotus root starch is flattened, and three parallel repeated experiments are set.

[0059] According to the ratio of 1:1 (mL / g) of the crushed liquid of Rhizopus arrhizus mycelium pellets to the weight of the culture material, lotus root starch, 2 mL of crushed liquid of Rhizopus arrhizus mycelium pellets was added to the adsorbed culture material in the well. After the inoculation, the culture plate cover was covered and cultured in a 32°C incubator for 48 hours to obtain the Rhizopus arrhizus cake, and the growth of the Rhizopus arrhizus mycelium on the cake was observed.

[0060] Example 4

[0061] The culture material was changed to rice flour, and the other conditions were the same as in Example 3.

[0062] Comparative Example 3

[0063] The adsorption culture material was cassava starch, and the other conditions were the same as those in Example 3.

[0064] Comparative Example 4

[0065] The adsorption culture material was potato starch, and the other conditions were the same as those in Example 3.

[0066] Comparative Example 5

[0067] The adsorption culture material is sweet potato starch, and the other conditions are the same as those in Example 3.

[0068] The sources and properties of the adsorption culture materials of Examples 3 to 4 and Comparative Examples 3 to 5 are shown in Table 2. The culture results of Rhizopus arrhizus of Example 3 and Comparative Examples 3 to 5 are shown in Table 3 and Figure 5 to Figure 7 .

[0069] According to Table 3 and Figure 5 to Figure 7 It can be seen that from the perspective of the difficulty of inoculating the crushed liquid of Rhizopus arrhizus mycelial pellets, potato starch is the easiest to inoculate, followed by lotus root starch and rice flour, and cassava starch is the most difficult to inoculate, which may be due to the low water content and small mesh size of cassava starch itself, which makes it difficult to rehydrate cassava starch; from the perspective of the absorption effect of the crushed liquid of mycelial pellets, for the same amount of liquid, lotus root starch has the best absorption effect, which has not reached the absorption saturation state, followed by rice flour, and potato starch, sweet potato starch and cassava starch are all oversaturated; from the perspective of the growth of the fungus, the mushroom cake made with lotus root starch has the best growth, with both a large number of hyphae and a large number of spores, which are suitable for the growth of Rhizopus, and the mushroom cake made with sweet potato starch has the weakest growth.

[0070] Table 2 Sources and properties of adsorption culture materials of Examples 3 to 4 and Comparative Examples 3 to 5

[0071]

[0072] Table 3 Cultivation results of Rhizopus arrhizus of Examples 3 to 4 and Comparative Examples 3 to 5

[0073]

[0074] The Rhizopus oligorrhiza cakes obtained in Examples 3-4 and Comparative Examples 3-5 were dried by low-temperature silica gel filter paper adsorption dehydration. The specific operation of drying was as follows: the adsorption culture materials (i.e., Rhizopus oligorrhiza cakes) with Rhizopus oligorrhiza of Examples 3-4 and Comparative Examples 3-5 were picked out from the hole together with the bottom filter paper, a set of 90 mm culture dishes was taken, 20 g of blue color-changing silica gel was poured into the dish cover, and evenly paved, a 9 cm dry filter paper was placed on the paved silica gel surface, and the bacteria cake with the bottom filter paper was placed on the plate. The cake is placed in the middle of the filter paper, and after it is placed, it is covered with a 9cm dry filter paper on the upper layer. 20g of blue color-changing silica gel is poured on top of this layer of filter paper, and the silica gel is evenly paved. The supporting culture dish bottom plate is placed on the silica gel surface and pressed. Finally, the entire culture dish is wrapped with plastic wrap and placed in a refrigerator at 8°C for 8h. The moisture content of the Rhizopus arrhizus cake obtained in Examples 3 to 4 and Comparative Examples 3 to 5 before drying is recorded, and the moisture content after drying is completed, and the difference between the two recorded values ​​before and after drying is calculated. The results are shown in Table 4 and Figure 8 to Figure 13 According to Table 4 and Figure 8 to Figure 13 It can be seen that under the same drying conditions, from the perspective of the moisture content of the dried cakes, cassava starch and sweet potato starch have the lowest moisture content, both around 9.70%, and lotus root starch has the highest moisture content, which is 18.55%. However, this moisture content is lower than 20%, which is still considered a relatively low moisture content level; from the perspective of the difference in moisture content before and after drying, sweet potato starch has the most dehydration, with a difference of 39.03, and lotus root starch has the least dehydration, with a difference of 30.17. The larger the difference, the higher the degree of dehydration, but the overall dehydration difference range of the five materials is in the range of 30-40, without too much fluctuation; from the perspective of the bacterial content of the dried cakes, the lotus root starch cake has the most hyphae and spore sacs attached to the surface, followed by the potato starch cake, while the sweet potato starch cake has the least bacterial content, with only a small amount of hyphae attached to the surface.

[0075] Table 4 Moisture content of the Rhizopus arrhizus cake obtained in Examples 3 to 4 and Comparative Examples 3 to 5 before and after drying

[0076]

[0077] Example 5 Screening of different culture times

[0078] Prepare the pulverized liquid of Rhizopus oligiformis mycelium ball according to the manner of Example 1. Take a 6-well cell culture plate, first pad a circular filter paper with a radius of 16mm at the bottom of the three holes in the same row of the culture plate, weigh 2.000g of lotus root starch and pour it into each hole, and flatten the starch surface. Each hole with lotus root starch added, according to the volume of mycelium ball pulverized liquid: starch weight is 1: 1 (mL / g) ratio to culture material in the hole to add 2mL Rhizopus oligiformis mycelium ball pulverized liquid. After inoculation, cover the culture plate cover, first record the weight of the whole culture plate before cultivation, then culture in 32 DEG C of incubators, culture 48h, obtain Rhizopus oligiformis cake, record the weight of the whole culture plate after cultivation, observe the growth of Rhizopus oligiformis on the cake.

[0079] Example 6

[0080] After the inoculation is completed, the cell culture plate cover is closed, and the weight of the entire culture plate before culture is recorded first, and then cultured in a 32° C. incubator for 24 hours. The other conditions are the same as in Example 5.

[0081] Comparative Example 6

[0082] After the inoculation is completed, the cell culture plate cover is closed, and the weight of the entire culture plate before culture is recorded first, and then cultured in a 32° C. incubator for 72 hours. The other conditions are the same as those in Example 5.

[0083] Comparative Example 7

[0084] After the inoculation is completed, the cell culture plate cover is closed, and the weight of the entire culture plate before culture is recorded first, and then cultured in a 32° C. incubator for 96 hours. The other conditions are the same as those in Example 5.

[0085] Comparative Example 8

[0086] After the inoculation is completed, the cell culture plate cover is closed, and the weight of the entire culture plate before culture is recorded first, and then cultured in a 32° C. incubator for 120 h. The other conditions are the same as those in Example 5.

[0087] Comparative Example 9

[0088] After the inoculation is completed, the cell culture plate cover is closed, and the weight of the entire culture plate before culture is recorded first, and then cultured in a 32° C. incubator for 144 h. The other conditions are the same as those in Example 5.

[0089] The weight changes of the culture plates before and after culture and the morphology of the bacterial cakes of Examples 5 to 6 and Comparative Examples 6 to 9 are shown in Table 5.

[0090] Table 5 Changes in weight of culture plates before and after culture and results of bacterial cake morphology of Examples 5 to 6 and Comparative Examples 6 to 9

[0091]

[0092]

[0093] According to Table 5, Rhizopus arrhizus can use its own amylase to decompose the dry lotus starch matrix into small molecular sugars, and at the same time use these sugars as available carbon sources for its own growth, resulting in a reduction in the weight of the starch matrix. Therefore, when the bacterial growth activity is more vigorous, the corresponding dry lotus starch consumption is more, and the matrix weight is reduced more. Under the same culture conditions, when the culture time is extended from 24h to 48h, the weight difference between the two time periods is the largest, indicating that the Rhizopus in this time period is in a vigorous growth period and has good growth activity. When the culture time is extended, the difference between the two adjacent time periods gradually decreases; from the growth trend of the bacteria, when the culture time is 48h, the bacterial cake already has a large number of hyphae and sporangia, and has grown all over the surface. After the culture is further cultivated, the bacterial growth activity is maintained for a period of time before the bacterial aging begins. Therefore, based on the comprehensive culture situation, the effect of 48h culture time when preparing the Rhizopus arrhizus cake is the best.

[0094] Example 7

[0095] Prepare the crushed mycelium pellets of Rhizopus arrhizus in the manner of Example 1. Take a 6-well cell culture plate, and place a circular filter paper with a radius of 30 mm in the wells of the culture plate. The filter paper can cover the bottom of the well and the periphery of the well wall. Take three wells in the same row of each plate as repeated tests. Weigh 2.000g of lotus root starch and pour it into each well, flatten the surface of the starch, and add 2mL of crushed mycelium pellets to the culture material in the well at a ratio of 1:1 (mL / g) in terms of the volume of the crushed mycelium pellets of Rhizopus arrhizus: the weight of starch. After the inoculation is completed, cover the culture plate with the cover, first record the weight of the entire culture plate before culture, then culture in a 32°C incubator for 48 hours, record the weight of the culture plate and observe the growth of the bacteria, and at the same time calculate the difference in the weight recorded values ​​before and after culture. See the paper padding method of Example 7. Fig.14 Method 2 in .

[0096] Comparative Example 10

[0097] A circular filter paper with a radius of 16 mm was placed in the well of the culture plate, and the filter paper only covered the bottom of the well. Three wells in the same row of each plate were used as repeated tests; the other conditions were the same as those in Example 7, and the paper padding method of Comparative Example 10 was shown in Fig.14 Method 1 in.

[0098] The weight changes of the culture plates before and after the cultivation of Rhizopus arrhizus in Example 7 and Comparative Example 10 are shown in Tables 6 and Fig.15 ,in Fig.15 The paper pad method 1 is the culture result of comparative example 10, Fig.15 The paper pad method 2 is the culture result of Example 7.

[0099] Table 6 Changes in weight of culture plates before and after culture with different paper padding methods in Example 7 and Comparative Example 10

[0100]

[0101] The Rhizopus arrhiza cakes obtained in Example 7 and Comparative Example 10 were dried in the same manner as in Example 3, and were placed in a refrigerator at 8°C for 8 hours. The moisture content of the cakes was recorded before drying, and the moisture content of the cakes was recorded after drying, and the difference between the two recorded values ​​before and after drying was calculated. The results are shown in Tables 7 and Figure 16-17 .

[0102] Table 7 Moisture changes and sampling difficulty before and after drying with different padding methods in Example 7 and Comparative Example 10

[0103]

[0104] According to Table 7 and Figure 16-17 It can be seen that from the perspective of the difficulty of sampling and drying, the paper pad method 2 of Example 7 is more convenient to pick up the cake, and the cake can be quickly taken out by directly extracting the filter paper around the cake that is higher than the surface of the cake, while the paper pad method 1 of Comparative Example 10 requires the assistance of tools to pick up the cake, which may damage the surface of part of the cake; from the perspective of the moisture content of the cake after drying, under the same drying conditions, there is no significant difference in the moisture content of the cake after drying in the two methods; on the surface, the paper pad method 2 of Example 7 has more hyphae and spore sacs attached to the surface of the cake.

[0105] Example 8

[0106] Prepare the crushed liquid of mycelium pellets of Rhizopus arrhizus in the manner of Example 1. Take a 6-well cell culture plate, first put a circular filter paper with a radius of 30mm on the well pad of the culture plate, so that the filter paper can cover the bottom of the well and the periphery of the well wall. Weigh 2.000g of lotus root starch and pour it into the well of the culture plate, and flatten the surface of the starch. The weight ratio of the crushed liquid of mycelium pellets of Rhizopus arrhizus to lotus root starch is 1mL:1g. Add 2mL of mycelium pellet crushed liquid to the well of lotus root starch in the culture plate, and take three wells in the same row of each plate as repeated tests. After the inoculation is completed, cover the culture plate cover, first record the weight of the entire culture plate before culture, then culture in a 32°C incubator for 48h, record the weight of the culture plate and observe the growth of the bacteria, and calculate the difference between the weight records before and after culture.

[0107] Example 9

[0108] The weight ratio of the crushed mycelium pellets of Rhizopus arrhizus to the lotus root starch was set to 1.5:1 (mL / g), 3 mL of the crushed mycelium pellets were added to the wells of the culture plate with the lotus root starch, and three wells in the same row of each plate were used as repeated tests. The other conditions were the same as those in Example 8.

[0109] Comparative Example 11

[0110] The weight ratio of the crushed mycelium pellets of Rhizopus arrhizus to the lotus root starch was set to 0.5:1 (mL / g), 1 mL of the crushed mycelium pellets was added to the wells of the culture plate with the lotus root starch, and three wells in the same row of each plate were used as replicate tests. The other conditions were the same as those in Example 8.

[0111] Comparative Example 12

[0112] The weight ratio of the crushed mycelium pellets of Rhizopus arrhizus to the lotus root starch was set to 2:1 (mL / g), 4 mL of the crushed mycelium pellets were added to the wells of the culture plate with the lotus root starch, and three wells in the same row of each plate were used as replicate tests. The remaining conditions were the same as those in Example 8.

[0113] Comparative Example 13

[0114] The weight ratio of the crushed mycelium pellets of Rhizopus arrhizus to the lotus root starch was set to 2.5:1 (mL / g), 5 mL of the crushed mycelium pellets was added to the wells of the culture plate with the lotus root starch, and three wells in the same row of each plate were used as replicate tests. The other conditions were the same as those in Example 8.

[0115] The results of the weight change of the culture plate before and after the culture of the culture plate of different mycelial ball crushing liquid addition ratios of Examples 8 to 9 and Comparative Examples 11 to 13 are shown in Table 8. According to Table 8, from the weight difference before and after the culture plate is cultured, under the same culture conditions, when the ratio of the mycelial ball crushing liquid addition amount is increased from 0.5:1 to 1:1, the weight difference is significantly increased from 0.582g to 0.703g, and the increase is 0.121g; when the liquid addition ratio continues to increase from 1:1, the weight difference increase trend gradually slows down, and when the liquid addition ratio is increased from 2:1 to 2.5:1, the weight difference increases by 0.023g. Therefore, the mycelial ball liquid addition ratio of 1:1 in this test is a suitable liquid addition amount for the growth of Rhizopus.

[0116] Table 8 Changes in weight of culture plates before and after culture at different ratios of mycelial pellet crushing liquid addition

[0117]

[0118] The Rhizopus arrhiza cakes of Examples 8 to 9 and Comparative Examples 11 to 13 were placed in a refrigerator at 8°C for 8 hours according to the low-temperature silica gel filter paper adsorption dehydration method of Example 3, and the moisture content of the cakes before drying was recorded. After drying, the moisture content of the cakes was recorded, and the difference between the two recorded values ​​before and after drying was calculated, as shown in Table 9. According to Table 9, from the perspective of the morphology before and after drying and the moisture content after drying, the water content of the cakes with a liquid addition ratio of 0.5:1 was the lowest, which was 16.24%, followed by the liquid addition ratio of 1:1, which had a water content of 19.20%, and the morphology of the cakes with the two ratios before and after drying was basically the same. Therefore, considering the comprehensive culture effect and drying effect, this experiment selected a liquid addition ratio of 1:1 (mL / g) for the volume of the mycelial pellet crushing liquid and the weight of the lotus root starch as the liquid addition ratio of the mycelial pellet crushing liquid in the later optimization experiment.

[0119] Table 9 Moisture changes and cake morphology before and after drying with different paper padding methods

[0120]

[0121]

[0122] Example 10

[0123] Prepare the pulverized liquid of mycelium pellets of Rhizopus arrhizae in the manner of Example 1. Take a 6-well cell culture plate, first pad the hole of the culture plate with a circular filter paper with a radius of 30 mm, so that the filter paper can cover the bottom of the hole and the periphery of the hole wall, first add 2.000g of lotus root starch to the hole of the culture plate padded with the filter paper and flatten it, add 2mL of mycelium pellet pulverized liquid to the culture material in the hole according to the ratio of the volume of mycelium pellet pulverized liquid and the weight of starch of 1:1 (mL / g), cover the culture plate cover after the inoculation is completed, first record the weight of the entire culture plate before culture, then culture in a 32°C incubator for 48h, record the weight of the culture plate and observe the growth of the bacteria, and calculate the difference between the weight records before and after culture.

[0124] Comparative Example 14

[0125] Prepare the pulverized liquid of Rhizopus arrhizae mycelium ball according to the manner of Example 1. Take a 6-well cell culture plate, first pad the hole of the culture plate with a circular filter paper with a radius of 30mm, so that the filter paper can cover the bottom of the hole and the periphery of the hole wall, first add 1.000g of lotus root starch to the hole of the culture plate padded with filter paper and flatten, according to the volume of mycelium ball pulverized liquid: lotus root starch weight is 1: 1 (mL / g) ratio to culture material in the hole to add 1mL mycelium ball pulverized liquid, stand for 10min, cover the hole with 1.000g of lotus root starch again, after flattening, according to the same liquid addition amount, add 1mL mycelium ball pulverized liquid, cover the culture plate cover after inoculation, and the remaining conditions are the same as Example 10.

[0126] The conditions before and after cultivation of Rhizopus arrhizus of Example 10 and Comparative Example 14 are shown in Tables 10 and Fig.18 According to Table 10 and Fig.18 It can be seen that the number of hyphae and sporangia on the surface of the cake of Comparative Example 14 is less than that of Example 10; from the weight difference before and after culture on the culture plate, the weight difference of Comparative Example 14 is higher than that of Example 10, indicating that the growth activity of Rhizopus in the cake of Example 10 is stronger than that of Comparative Example 14. Fig.18 Method 1 represents Comparative Example 14, and Method 2 represents Example 10.

[0127] Table 10 Weight changes of culture plates before and after culture in different sample addition methods in Example 10 and Comparative Example 14

[0128]

[0129] Embodiment 11

[0130] Prepare the pulverized liquid of Rhizopus spp. in the manner of Example 1. Take five 6-well cell culture plates, number them 1, 2, 3, 4, and 5 respectively, and first put a circular filter paper with a radius of 30 mm on the hole pad of the culture plate so that the filter paper can cover the bottom of the hole and the periphery of the hole wall. Weigh 2.000g of lotus root starch and pour it into the hole of the culture plate, flatten the surface of the starch, add 2mL of pulverized liquid of mycelium to the culture material in the hole according to the volume of pulverized liquid of Rhizopus spp.: starch weight of 1:1 (mL / g), cover the culture plate cover after the inoculation, first record the weight of the entire culture plate before culture, and then culture it in a 32°C incubator for 48h to obtain the Rhizopus spp. cake, record the weight of the five cell culture plates and observe the growth of the bacteria, and calculate the difference of the weight record value before and after culture. The specific conditions before and after culture of the five culture plates are shown in Table 11. According to Table 11, under the same culture conditions, there is no significant difference in the weight differences of the five cell culture plates, indicating that the growth activity of the Rhizopus strains in the five culture plates is at the same level.

[0131] Table 11 Weight changes of five cell culture plates before and after culture

[0132]

[0133] The lotus root starch (i.e., the Rhizopus arrhizus cake) prepared from the above cell culture plates 1, 2, 3, 4, and 5 was taken out for drying, and five different drying methods were set, and the cake in one culture plate corresponded to one drying method. The specific drying method operation is as follows:

[0134] The cell culture plate 1 is dried by method 1. The specific operation of method 1 is as follows: first, the low-temperature silica gel filter paper adsorption dehydration method of Example 3 is used to stand and dry in a refrigerator at 8°C for 4 hours, then taken out, the silica gel on the upper and lower surfaces that has absorbed water is poured out, and the upper and lower surfaces are replaced with new silica gel of the same weight, and after sealing with plastic wrap, it is stood and dried in a refrigerator at 8°C for 4 hours again;

[0135] The cell culture plate 2 is dried by method 2. The specific operation of method 2 is as follows: first, the low-temperature silica gel filter paper adsorption dehydration method of Example 3 is used to stand and dry in a refrigerator at 8°C for 4 hours, then taken out, the silica gel that has absorbed water on the upper and lower surfaces is poured out, and the upper and lower surfaces are replaced with new silica gel of the same weight. After sealing with plastic wrap, it is stood and dried in a refrigerator at 8°C for 4 hours again, and finally the silica gel is taken out and poured out, and the filter paper on the top is removed, and it is stood and air-cooled and dried in a refrigerator at 8°C for 4 hours;

[0136] The cell culture plate 3 is dried by the method 3, and the specific operation of the method 3 is as follows: the low-temperature silica gel filter paper adsorption dehydration method of Example 3 is used to stand and dry in a refrigerator at 8° C. for 8 hours;

[0137] The cell culture plate 4 was dried using method 4. The specific operation of method 4 was as follows: the bacterial cake and the bottom padding paper were placed in the middle of a sterile 9 cm dry filter paper, the filter paper was placed on the cover of a 90 mm culture dish, and finally directly placed in a refrigerator at 8°C for air cooling and drying for 40 hours;

[0138] The cell culture plate 5 was dried using method 5. The specific operation of method 5 was as follows: after removing the bottom pad paper of the bacterial cake, it was placed in the middle of a sterile 9cm dry filter paper, and a 90mm culture dish cover was taken. A layer of 20g of silica gel was first spread on the dish cover, and then the filter paper with the bacterial cake was placed on the silica gel. Finally, 20g of silica gel was poured around the bacterial cake, and it was placed in a vacuum drying oven (the conditions were vacuum pressure -0.098MPa, and culture temperature was 25°C) and dried for 1h.

[0139] In all drying processes of Methods 1 to 5, the moisture content of the Rhizopus arrhizus cake before drying was recorded first, and then the moisture content of the cake after drying was recorded, and the difference before and after drying was calculated. The drying effects of the Rhizopus arrhizus cake of Methods 1 to 5 are shown in Table 12 and Figure 19-20 According to Table 12 and Figure 19-20It can be seen that from the perspective of the moisture content of the dried mushroom cake, the moisture content of method 2 is the lowest, which is 12.73%, followed by method 4, and the moisture content of method 5 is the highest, which is 23.32%; from the perspective of the difference in moisture content before and after drying, method 2 also has the largest difference, which is 36.72, followed by method 4, and the moisture content of method 5 is the highest, which is 26.40, and the dehydration effect can only reach about 72% of that of method 2; from the above results, the drying effect of method 5 is poor, but from the perspective of time efficiency ratio, method 5 has the highest time efficiency ratio. Method 2 needs to spend 12 hours to achieve its optimal drying effect, method 4 needs 40 hours, and method 5 only takes 1 / 12 of the total time of method 2 to achieve 72% of the dehydration effect of this method, and takes 1 / 40 of the total time of method 4 to achieve 76% of the dehydration effect of this method. In addition, from the perspective of the integrity of the dried cake, the cake integrity of methods 4 and 5 is the best. The other methods have more or less bacteria and spores attached to the filter paper due to contact with the filter paper, resulting in a small loss. Therefore, considering the comprehensive time efficiency ratio and the effect of maintaining the integrity of the cake, method 5 is the best cake drying method.

[0140] Table 12 Changes in moisture before and after drying in different drying methods 1 to 5

[0141]

[0142] Example 12

[0143] According to the method of Example 11, a Rhizopus arrhiza cake was prepared, the weight of the culture plate was recorded, the growth of the bacteria was observed, and the difference in the weight recorded values ​​before and after the culture was calculated, as shown in Table 13. According to the data in Table 13, under the same culture conditions, there was no significant difference in the weight difference between the five culture plates, indicating that the growth activity of the Rhizopus strains on the five culture plates was at the same level.

[0144] Table 13 Weight changes of five culture plates before and after culture

[0145]

[0146] Take out the lotus root starch (i.e., the root mold cake with few roots) of the root mold prepared by the above-mentioned cell culture plates 1, 2, 3, 4, and 5 and dry them. The mass of each root mold cake with few roots is 2.0g. The culture dish cover of 90mm specification is covered with a layer of 20g silica gel, and then covered with 9cm specification filter paper. After removing the bottom pad paper of the bacteria cake, it is placed in the center of the filter paper. 20g silica gel is poured around the bacteria cake, and it is placed in a vacuum drying oven for drying. The vacuum drying oven is set to a vacuum pressure of -0.098MPa, and the drying temperature is 25°C. Under this culture condition, the vacuum drying time of the bacteria cake obtained by setting the cell culture plate 1 is 1h, the vacuum drying time of the bacteria cake obtained by the cell culture plate 2 is 2h, the vacuum drying time of the bacteria cake obtained by the cell culture plate 3 is 3h, the vacuum drying time of the bacteria cake obtained by the cell culture plate 4 is 4h, and the vacuum drying time of the bacteria cake obtained by the cell culture plate 5 is 5h. The drying effect under different vacuum drying time treatments is shown in Table 14. According to Table 14, when the vacuum drying time is in the range of 1h to 3h, the difference in moisture content gradually increases with time, and the difference increases most significantly from 25.04 to 37.88 when the time is extended from 1h to 2h; when the time is extended from 3h, the increase in the difference in moisture content is very small, and the change curve tends to be gentle; from the moisture content after drying, the moisture content after drying for 5h is the lowest, which is 3.75%, and the moisture content after drying for 2h is at a medium level, which is 10.19%. This moisture content value is relatively suitable, and it will not cause preservation difficulties due to being too wet, nor will it cause the cake to crack due to being too dry, and this moisture content is equivalent to the original moisture content of lotus root starch; from the perspective of the integrity of the dried cake, the cakes vacuum dried for 1h and 2h have the best integrity retention.

[0147] Table 14 Moisture changes and cake state before and after drying at different vacuum drying times

[0148]

[0149] Example 13

[0150] According to the method according to the previous step, the dry lotus root starch (i.e., cake) with root mold is prepared, and the mass of each root mold cake is 2.0g, and all the holes of 6-well cell culture plates 1 and 6-well cell culture plates 2 are selected as root mold culture holes. The root mold cake is prepared according to the method of Example 1, the weight of the culture plate is recorded, the growth of the thalline is observed, and the difference of the weight record value before and after the culture is calculated. The specific culture weight changes before and after are shown in Table 15. According to Table 15, from the weight difference before and after the culture plate is cultivated, under the same culture conditions, the weight difference of the two culture plates has no significant difference from each other, indicating that the growth vitality of the root mold strains of the two culture plates is at the same level.

[0151] Table 15 Weight changes of two culture plates before and after culture

[0152]

[0153] A total of 12 Rhizopus agenes cakes prepared by 6-well cell culture plates 1 and 6-well cell culture plates 2 were dried using the specific parameters of the vacuum silica gel drying process, that is, a layer of 20g silica gel was laid on the cover of a 90mm culture dish, and then a 9cm filter paper was laid on it. After removing the bottom pad paper from the cake, it was placed in the center of the filter paper. 20g silica gel was poured around the cake and placed in a vacuum drying oven for drying. The vacuum drying oven was set to a vacuum pressure of ~0.098MPa, a drying temperature of 25°C, and a drying time of 2h. Among the 12 Rhizopus agenes cakes, the growth surface of the Rhizopus of two cakes was placed upright on a dissecting microscope before drying, and the observation magnification was 10 times the eyepiece × 1 times the objective lens. After the bacterial body field in the eyepiece was clear, the image was captured using the instrument's built-in camera. After drying, the cakes were observed again at the same observation magnification and the image was captured; the moisture content of the remaining ten cakes was recorded before drying, and the moisture content of the cakes was recorded after drying, and the difference between the two recorded values ​​before and after drying was calculated. The results are shown in Table 16 and Figure 21 to Figure 23 At the same time, put the ten dried mushroom cakes into a sterile dry mortar and grind them for 10 seconds. When all of them are ground into powder, weigh the total weight of the powder obtained by grinding, and then store it in a triangular bottle. Figure 21 to Figure 23 It can be seen that under the same culture and drying conditions of the Rhizopus spp. cakes, the moisture content of the ten Rhizopus spp. cakes after drying and the moisture content difference before and after drying did not fluctuate significantly, and were basically at the same numerical level; the Rhizopus spp. cakes before and after drying remained intact, and under the observation of the 10-fold magnification of the dissecting microscope, the morphology of the hyphae and sporangium was consistent and undamaged, indicating that this experiment has good repeatability and good bacterial integrity. In addition, the total weight of the powder after grinding the ten cakes was 17.607g, and the amount of powder prepared was large, while a total of 12mL of mycelial pellet crushing liquid was required for ten cakes, which shows that a small volume of mycelial pellet crushing liquid can obtain a large amount of powder after adsorption culture and drying.

[0154] Table 16 Moisture changes of mushroom cake before and after drying

[0155]

[0156]

[0157] Application Example 1: Bacterial powder activity test

[0158] 1. Test on activation ability of bacterial powder inverted into PDA culture medium

[0159] Take four complete sets of 90 mm culture dishes, pour 15-20 mL of high temperature sterilized PDA culture medium into the dishes, and wait for the PDA culture medium plate to cool and solidify before use. Weigh 0.05 g of each of the two portions of Rhizopus arrhizus powder prepared in Example 1.

[0160] Treatment 1: Take 0.05g of Rhizopus powder and pour it into the center of PDA plate a and flatten it slightly; weigh 0.05g of lotus root starch and inoculate it into PDA plate b in the same way as CK1;

[0161] Treatment 2: Take PDA plate c, place a sterile standard Oxford cup (cup size 7.8mm×6mm×10mm) in the center of PDA plate c, take another 0.05g of Rhizopus powder and pour it into a sterilized 10mL centrifuge tube, add 3mL of sterile water to dilute, cover the lid and shake for 10s to mix, then use a pipette to draw 0.15mL of the powder dilution and add it to the Oxford cup on PDA plate c;

[0162] Take PDA plate d, place a sterile standard Oxford cup (cup size 7.8mm×6mm×10mm) in the center of PDA plate d, take 0.05g of lotus root starch and pour it into a sterilized 10mL centrifuge tube, add 3mL of sterile water to dilute, cover the lid and shake for 10s to mix, then use a pipette to absorb 0.15mL of bacterial powder dilution and add it to the Oxford cup on PDA plate d as CK2.

[0163] Then, the PDA plates a, b, c and d were placed in a 32°C incubator for 48 h to observe the growth of Rhizopus arrhizus and the changes in CK1 and CK2. Figure 24-25 , Fig.24 CK in the figure represents CK1. Fig.25 CK in the figure represents CK2. Figure 24-25 It can be seen that the prepared Rhizopus arrhizus powder and its 60-fold diluted solution, both of which were inoculated on PDA plates a and PDA plates c, can grow Rhizopus arrhizus under the same culture conditions, compared with CK1 and CK2, and the fungus growth is more vigorous, which indicates that the prepared bacterial powders have good activation ability.

[0164] 2. Storage activity test of Rhizopus arrhizus powder

[0165] Weigh the Rhizopus oligorrhiza powder prepared in Example 1, weigh a small amount of Rhizopus oligorrhiza powder and place it in a culture dish of 90 mm, then place the culture dish with a half-open lid in an incubator (storage temperature 32 ° C, relative humidity 55%) for 10 d, measure the moisture content of the Rhizopus oligorrhiza powder before storage, after the storage time is up, take out the Rhizopus oligorrhiza powder and measure the moisture content of the Rhizopus oligorrhiza powder after storage, in addition, pick a little of the Rhizopus oligorrhiza powder after storage with a sterile spoon and sprinkle it on a solid plate equipped with 15 mL PDA culture medium, and place it in a 32 ° C incubator and cultivate for 48 h, observe the growth of the Rhizopus oligorrhiza body, and the moisture change and activation ability change of the Rhizopus oligorrhiza powder before and after storage are shown in Table 17 and Fig.26 According to Table 17 and Fig.26 It can be seen that the moisture content of Rhizopus arrhizus powder before and after storage was 10.86% and 10.62% respectively. From the two values, the moisture content of Rhizopus arrhizus powder did not change significantly during storage, indicating that Rhizopus arrhizus powder has good water retention; after 10 days of storage, Rhizopus arrhizus powder was inoculated on PDA plates, and Rhizopus cells with good growth were grown. The above results all show that the prepared Rhizopus arrhizus powder is resistant to storage and has good storage vitality.

[0166] Table 17 Moisture changes of Rhizopus powder before and after storage for 10 days

[0167]

[0168] 3. Test of saccharification and fermentation activity of bacterial powder:

[0169] Take 4 50mL glass round-bottom test tubes with scales, add 1.0g of lotus root starch to each test tube, prepare MS culture solution and boil it, then add 20mL MS culture solution to the test tube while it is hot, so as to prepare 5% (g / mL) lotus root starch gelatinization solution, cover it with a silicone plug, sterilize it at high temperature, take it out and cool it for use, and mark these 4 lotus root starch paste solution test tubes 1, 2, 3, CK as lotus root starch gelatinization solution treatment groups;

[0170] Take 4 50mL glass round-bottom test tubes with scales and prepare MS culture medium. First, add 20mL MS culture medium to each test tube, cover with silicone stopper, sterilize it at high temperature, take it out and cool it to room temperature, and then add 1.0g of lotus root starch to each test tube to prepare a 5% (g / mL) lotus root starch powder solution. The 4 lotus root starch powder solution test tubes 4, 5, 6, and CK are marked as lotus root starch powder solution treatment groups.

[0171] Take the lotus root starch gelatinization liquid treatment group, add 0.1g of Rhizopus arrhizus powder prepared in Example 1 to each of the three starch gelatinization liquid test tubes 1, 2, and 3, and stir each test tube with a sterilized glass rod for 20s to fully disperse the fungus powder in the starch gelatinization liquid. At this time, the inoculation concentration of the fungus powder is equivalent to 0.5% (g / mL). In addition, 0.1g of lotus root starch is added to the other one starch gelatinization liquid test tube CK as CK;

[0172] Take the lotus root starch powder liquid treatment group, add 0.1g of Rhizopus arrhizus powder prepared in Example 1 to each of the three starch powder liquid test tubes 4, 5, and 6, and stir each test tube with a sterilized glass rod for 20s to fully disperse the bacterial powder in the starch powder liquid. At this time, the inoculation concentration of the bacterial powder is equivalent to 0.5% (g / mL). In addition, 0.1g of lotus root starch is added to the other starch powder liquid test tube CK as CK.

[0173] Then the two treatment groups, the hydrophilic lotus root starch gelatinized liquid treatment group and the hydrophilic lotus root starch raw powder liquid treatment group, were placed in a 32°C incubator for 48 hours of static fermentation. Figure 27 to Figure 29 .according to Figure 27 to Figure 29 It can be seen that the test tubes 1, 2, 3 of the group treated with the hydrophilic lotus starch gelatinized liquid and the test tubes 4, 5, and 6 of the group treated with the hydrophilic lotus starch raw powder liquid all had Rhizopus growth, while the CK of the two groups had no growth. In addition, the liquid of the CK tube of the hydrophilic lotus starch gelatinized liquid group still had the viscosity characteristics of starch gelatinized liquid and poor fluidity. The liquid of the test tubes 1, 2, and 3 of the hydrophilic lotus starch gelatinized liquid group connected to the tube treated with the powder lost its viscosity characteristics and improved its fluidity. The above results all indicate that the prepared Rhizopus arrhizus powder consumes and decomposes the starch matrix in the fermentation solution.

[0174] The reducing sugar content of the fermentation liquid of the group treated with the gelatinized liquid of lotus root starch and the group treated with the raw liquid of lotus root starch was determined by the 3,5-dinitrosalicylic acid method. Before the measurement, a glucose standard curve was drawn first. The specific operation was as follows: prepare 3,5-dinitrosalicylic acid reagent (DNS reagent for short), take a small amount of glucose and place it in an oven at 80°C to dry to constant weight, accurately weigh 0.1g of constant weight glucose in a beaker, dissolve it with 10mL of distilled water, transfer it to a 100mL volumetric flask, wash the beaker with water and dilute to the scale, and shake it well to obtain a 1mg / mL glucose standard mother solution. According to the reagents listed in Table 18, add each reagent from top to bottom to a 25mL graduated test tube, shake the mixed solution in each tube, heat it in a boiling water bath for 5min, take it out and put it in ice water to cool to room temperature, wait for the color to develop completely, add distilled water to dilute to the 25mL scale line, cover the stopper and mix well. Determine the absorbance value at a wavelength of 540nm, and adjust the zero with the blank tube as a reference. The absorbance values ​​of glucose standard tubes of various masses are shown in Table 18, and the standard curve is drawn with the absorbance value as the ordinate and the glucose mass as the abscissa to obtain the linear regression equation. Fig.30 .

[0175] Table 18 Reagent volume and OD value required for drawing glucose standard curve

[0176]

[0177] 5 mL of liquid was drawn from each test tube of the two treatment groups, the hydrophilic lotus root starch gelatinized liquid treatment group and the hydrophilic lotus root starch raw powder liquid treatment group, into a 10 mL centrifuge tube, and centrifuged at 10,000 r / min for 5 min at 25°C. A certain amount of supernatant was drawn from each tube and added to a 25 mL graduated test tube in sequence according to the reagent list in Table 19. The mixed liquid in each tube was shaken evenly, heated in a boiling water bath for 5 min, and then cooled to room temperature in ice water after being taken out. After the color development is complete, distilled water was added to make up to the 25 mL scale line, and the stopper was covered and mixed evenly. The absorbance was measured at a wavelength of 540 nm, and the blank tube was used as a reference for zeroing. The color of each tube after color development can be seen in Fig.31 In addition, the absorbance values ​​of the blank tube and each sample tube are shown in Table 19. According to the corresponding absorbance values, the corresponding amount of glucose is found on the standard curve, and the reducing sugar content in the fermentation broth is calculated according to the formula, expressed in mg / mL. The calculation formula of the reducing sugar content is as follows:

[0178] Reducing sugar content (mg / mL) = (m×v×n) / (v s ×v)(1)

[0179] In formula (1), m is the mass of glucose obtained from the standard curve, in mg; v is the total volume of the fermentation broth, which is 20 mL in this experiment; n is the dilution factor of the fermentation broth, which is 50 for starch gelatinized broth and 25 for starch powder broth; v s The volume of sample solution during determination is 2 mL in this test.

[0180] Table 19 The amount of reagents, OD values ​​and reducing sugar content required for the determination of reducing sugar in the two treatment groups

[0181]

[0182] According to Table 19 and Fig.31 It can be seen that the prepared starch gelatinized liquid and starch raw powder liquid have saccharification and fermentation capabilities, among which the saccharification and fermentation effect of starch gelatinized liquid is better than that of starch raw powder liquid. The average reducing sugar content of the three inoculated tubes in the gelatinized liquid treatment group is 21.931 mg / mL, and the average reducing sugar content of the three inoculated tubes in the raw powder liquid treatment group is 14.185 mg / mL, that is, the saccharification ability of the starch gelatinized liquid of the bacterial powder is 1.5 times that of the starch raw powder liquid. The above results all show that the saccharification and fermentation activity of the Rhizopus arrhizus powder prepared by the present invention is very strong.

[0183] Embodiment 14

[0184] The crushed liquid of Rhizopus arrhizus mycelial pellets was prepared according to the method of Example 1, and two culture drying methods were set for comparative experiments. The specific setting operations are as follows:

[0185] Treatment 1: Take a 6-well cell culture plate and place a circular filter paper with a radius of 30 mm on each well of the culture plate so that the filter paper can cover the bottom of the well and the periphery of the well wall. Weigh 2.000 g of lotus root starch and pour it into the well of the culture plate, flattening the starch surface. Add 2 mL of mycelial pellet crushing liquid to the lotus root starch in the well at a ratio of 1:1 (mL / g). After the inoculation is completed, cover the culture plate and culture in a 32°C incubator for 48 hours to obtain the Rhizopus arrhizus cake and observe the growth of the bacteria.

[0186] The lotus root starch (i.e., cake) with Rhizopus was dried by the vacuum silica gel drying method of method 5 of Example 9, wherein the vacuum drying oven was set to a vacuum pressure of -0.098MPa, a drying temperature of 25°C, and a drying time of 2h. The moisture content of the cake was recorded before and after drying, and the dried cake was ground into powder in a sterilized mortar, and the total weight of the cake after grinding into powder was recorded. A small amount of powder was picked and sprinkled on the PDA culture medium and cultured at 32°C for 48h.

[0187] Treatment 2: Conventional method: Use biofilm culture and hot air drying. First, take 12 culture bottles, each bottle is filled with 20mL PDB liquid culture medium, sterilized at 121℃ for 20min, taken out and cooled to room temperature, and each bottle of PDB culture solution is added with 2mL of mycelial pellet crushing liquid in a ratio of 1:10 (mL / mL) of mycelial pellet crushing liquid and PDB culture liquid, and then cultured in a 32℃ culture shaker. Observe the growth of Rhizopus every 48h, and take another bottle of sterilized 20mL PDB liquid culture medium and add 2mL PDB culture liquid as a blank control (CK). After the culture time is up, pick out the biofilm, place it on the cover of the culture dish, and dry it in a hot air drying oven at 35℃ for 6h. Record the moisture content of the biofilm before and after drying, pick a small amount of biofilm fragments and sprinkle them on the PDA culture medium, and culture it at 32℃ for 48h.

[0188] The specific culture drying and activation conditions of the two methods are shown in Table 20. According to Table 20, for the same bottle of mycelial pellet crushing liquid, the culture time of the Rhizopus sparse root mold cake of the present invention is shorter than that of the Rhizopus sparse root mold film, and the difference between the two times is 1 times; from the perspective of moisture content before drying, the moisture content of the Rhizopus sparse root mold cake obtained by the culture method of the present invention is significantly lower than that of the film, which is beneficial to the later drying operation; from the perspective of moisture content after drying, the moisture content of the cake dried by the vacuum silica gel drying method of the present invention is higher than that of the dried film by hot air drying, but is still at a relatively dry moisture level; from the perspective of morphology after drying, the morphology of the cake dried by the vacuum silica gel drying method of the present invention remains intact and is easy to sample and crush, while the film dried by hot air drying in treatment 2 is difficult to sample and is not easy to grind, and can only be cut; from the perspective of the amount of samples prepared, the weight of the dried bacterial powder obtained by the vacuum silica gel drying method of the present invention is significantly better than the amount of the film obtained by hot air drying in treatment 2, and the weight of the bacterial powder obtained by grinding 6 cakes is 10.63 times the weight of 12 films; from the perspective of activation ability, the growth of bacterial powder and bacterial film is consistent with the same culture time. Based on the above effects, the cell plate culture and vacuum drying method of the present invention is significantly better than the biofilm culture and hot air drying method.

[0189] Table 20 Comparison of the two methods of cultivation and drying

[0190]

[0191] In summary, the Rhizopus arrhizus powder prepared by the present invention has high activity and storage resistance, and the Rhizopus arrhizus powder has strong saccharification and fermentation activity. The vacuum silica gel drying method used in the preparation of the Rhizopus arrhizus powder only requires a culture dish, silica gel and filter paper, and the bacterial cake does not require a special protective agent during drying. Most technicians can implement the drying operation, and the time efficiency ratio is high.

[0192] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing Rhizopus arrhizus powder, characterized in that, The preparation method comprises the following steps: placing silica gel at the bottom and around the dried Rhizopus arrhizus culture and then performing vacuum drying to obtain Rhizopus arrhizus powder; the vacuum drying temperature is 25°C to 30°C; the mass ratio of the silica gel to the mass of the Rhizopus arrhizus culture is (40 to 60):2; the vacuum drying pressure is -0.085MPa to -0.098MPa, and the vacuum drying time for each 1 to 2g of the Rhizopus arrhizus culture is 1 to 2h; the water content of the Rhizopus arrhizus culture after drying is 9% to 12%; the Rhizopus arrhizus culture is a Rhizopus arrhizus cake; the preparation of the Rhizopus arrhizus cake comprises the following steps: firstly crushing the culture solution of the Rhizopus arrhizus pellet and then mixing it with the culture material, and then culturing to obtain the Rhizopus arrhizus cake; the culture material is lotus root starch; the Rhizopus arrhizus is the Rhizopus arrhizus strain Ca-201613, and the preservation number of the Rhizopus arrhizus strain Ca-201613 is CGMCC NO.19912.

2. The preparation method according to claim 1, characterized in that: The preparation of the culture solution of the mycelial pellets of Rhizopus arrhizus comprises: obtaining a bacterial solution after activating the Rhizopus arrhizus; and obtaining the culture solution of the mycelial pellets of Rhizopus arrhizus by performing shake flask culture on the bacterial solution.

3. The preparation method according to claim 1, characterized in that: The culture temperature is 30-37° C., and the culture time is 24-48 hours.

4. The preparation method according to claim 1, characterized in that: The culture temperature is 32° C., and the culture time is 48 h.

5. The preparation method according to claim 1, characterized in that: During the mixing, the mass ratio of the crushed liquid of Rhizopus arrhizus mycelium pellets to the culture material is 1-1.5 mL:1 g; the mixing is carried out on a cell culture plate, which is padded with filter paper, and the filter paper covers the bottom of the culture plate wells and the periphery of the well walls.

6. Application of the preparation method of the Rhizopus arrhizus powder according to any one of claims 1 to 5 in improving the activity of the Rhizopus arrhizus powder.

7. Application of the preparation method of the Rhizopus arrhizus powder according to any one of claims 1 to 5 in improving the saccharification and fermentation activity of the Rhizopus arrhizus powder.

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