Trichoderma atroviride powder

After the successful engineering transformation of Trichoderma atroviride, we first obtained plenty of Trichoderma powder by solid state fermentation. The Trichoderma atroviride is inoculated in a solid culture medium composed of agricultural miscellaneous materials such as straws, rice bran and the like, and is fermented under proper conditions. After fermentation, the culture medium is dehydrated and crushed to obtain the engineering Trichoderma powder. The effective ingredient of Trichoderma powder is spores. In addition, an appropriate amount of protective agents such as dextrin and trehalose were added to play a positive role in the preservation of Trichoderma atroviride. We counted spores of Trichoderma powder using blood cell technology panels and found that its spore content could reach an astonishing 1.48 billion per gram!

Figure 1. Appearance of Trichoderma powder, which looks green

TACE the carrier

To control rice sheath blight in paddy fields, the current method using Trichoderma is to directly spray the Trichoderma spore suspension on a large scale. However, directly spraying of spore suspension is an inefficient solution, which will cause dramatic changes in farmland ecology, and the actual utilization of spore is very low.

Figure 2. The matrix material of TACE 1.0 is starch grafted acrylate polymer, which is white granular. The matrix material of TACE2.0 is hypromellose, which is white powder.

In order to precisely block the transmission of Rhizoctonia solani sclerotia from aquatic interface and soil, we designed TACE. The full name of TACE is Trichoderma's acest carrier ever, which is used as encapsulation/adsorption carrier for Trichoderma atroviride spores. We first designed TACE1.0, and then iteratively launched TACE2.0 based on TACE1.0's shortcomings found in the experiment and the feedback from human practices.

TACE1.0

The first generation of TACE is small white particles with diameters of 40 mesh and 80 mesh. The matrix material of TACE1.0 is Starch Graft Acrylate Polymer, which has a strong water absorption and retention. Experiments showed that TACE 1.0 has the greatest absorption of deionized water, reaching 206.20g/g.

Figure 3. TACE 1.0 forms jelly-like particles when it absorbs water.

Firstly, the Trichoderma spore powder is added with water according to the dosing ratio of 1:200 to prepare the spore suspension. Then add TACE1.0 according to the liquid-said ratio of 45:1, wait for at least 2min for full imbibition. According to the proportion of 0.1g dry TACE1.0 per rice plant, the field dissemination will be carried out by UAV or manual.

Figure 4. TACE1.0 dyed red with safranine were adsorbed at the rice stem on the aquatic interface.

Once encounter water in paddy fields, TACE1.0 floats, being adsorbed to rice stems at the aquatic interface. The spores loaded in the TACE1.0 diffuse at the adsorption point, forming a continuous local high spore concentration environment at the rice stem, which could help Trichoderma atroviride colonize. After that, TACE1.0 will be degraded by microorganisms.

Iteration of TACE

After exploring the features of TACE1.0, we believe that using TACE has advantages over the direct spraying of Trichoderma. We took TACE1.0 to Huizhou for product promotion, and received feedback from agricultural governors, scientists, farmers and managers of enterprises. They pointed out the shortcomings of TACE 1.0, which would make it difficult to promote. Here are the comparisons of advantages and disadvantages.

In the experiments and product promotion of TACE 1.0, we found some problems that can not be ignored. Therefore, it is necessary for us to make a comprehensive upgrade of TACE. Thankfully, we have successfully iterated on TACE. TACE2.0 can better perform the function of blocking the two transmission routes of Rhizoctonia solani, superior to TACE1.0 in terms of industrial production, loading capacity, adsorption radius and so on.

See more about products promotion and interviews in iHP.

See more about experiments in Measurement.

TACE2.0

Inspired by pharmaceutics, we designed and developed the second generation of TACE. TACE2.0 is a hydrophilic sustained-release matrix preparation with Hypromellose (HPMC) as the main component and T.atroviride spores as the effective component.

Matrix preparation refers to the preparation in the form of lamellar, granular, lumpy or so made by mixing drugs and inert matrix. HPMC is a common material for hydrophilic sustained-release matrix preparation. The HPMC matrix acts as a reservoir and is primarily used to control the release of the formulation. HPMC is soluble in water, but its dissolution in water is a slow process due to its viscosity. The viscosity of HPMC used in TACE2.0 is 4000mPa`\cdot`s and 15000mPa`\cdot`s respectively. In general, the greater the viscosity of HPMC, the slower the dissolution.

Figure 5. Hypromellose (HPMC).

In TACE2.0, spores will be uniformly dispersed as effective ingredients in the HPMC matrix, slowly released in water. In addition, glucose was added to TACE2.0 to inhibit the suicide switch of engineered T.atroviride.

TACE2.0 can be prepared by tableting mixed powders. Because the preparation is simple, and traditional tabletting process and equipment can be used to manufacture TACE2.0. Thanks to the availability and cheapness of raw materials, TACE2.0 has unique advantages in industrial production.

Figure 6. TACE2.0 of different particle sizes.

Ingredient

Compared to TACE1.0, one of the features of TACE2.0 is that the composition is clear. That is to say, we can accurately adjust the prescription of TACE2.0 and change the release rate, release amount of spores and other properties according to the actual needs. After a lot of experiments and calculations, we have come up with a recommended prescription.

Table1. Recommended prescription for TACE 2.0.

In the prefabrication stage, the factory uses Trichoderma atroviride spore powder, glucose, HPMC4000, HPMC15000 and magnesium stearate as raw materials to prepare mixed powder according to the recommended prescription for TACE 2.0. Then TACE2.0 is made into tablets through modern pharmaceutical process.

Functioning

1.Dissemination

After purchasing TACE2.0 products, users can spray 3mm TACE2.0 particles evenly in the field by means of UAV or manual fertilizer pump. After comprehensive consideration, we recommend that the dosage of 3mm TACE2.0 is 1.33kg/mu (1mu equals 666.67㎡), an average of two grains per rice plant.

Figure 7.Functioning process of TACE2.0.

2.Floatage and Adhesion

The dry TACE2.0 has a measured density of about 0.87 g/ml and can suspend on the water surface. TACE2.0 is able to float directionlessly on the aquatic surface due to agitation caused by field breezes or water currents, combined with a weak driving force generated when HPMC is dissoluted. TACE2.0 can actively approach rice stems. Because the surface of TACE2.0 is gelatinized after absorbing water, the sticky gel layer can help TACE2.0 adhere to the surface of rice stalks, and has good adhesion performance so that it is not easy to be peeled off.

3.Diffusion

When the TACE2.0 is exposed to water, the outer layer is hydrated into gel, and the spores and glucose on the surface are released. With the further penetration of water, the gel layer becomes thicker and thicker, which blocks the release of spore from the matrix. At the same time, HPMC is also dissolved, but the dissolution of HPMC matrix is quite a slow process because of the viscosity of HPMC. Therefore, the release of spores is precisely the combined result of the soluble diffusion of spores and the dissolution of HPMC matrix.

TACE2.0 formed a continuous local high concentration environment of T. atroviride spores at the aquatic interface and below, which could help T.atroviride to colonize the rice stem.

Figure 8.Functioning process of TACE2.0

4.Sedimentation

In the original design, the matrix material of TACE2.0 was pure HPMC4000. It was found that the adhesion was completely dissolved and no sedimentation occurred. We know that the R.solani sclerotia buried in soil can directly infect rice roots, and if TACE does not sedimentate to help T.atroviride colonize the roots, rice may still be infected.

To solve this problem, we added 10% HPMC15000 to the TACE2.0 prescription. Because the dissolution of HPMC15000 is slower than that of HPMC4000, the HPMC15000 matrix remains after the complete dissolution of HPMC4000. Due to the higher density of the HPMC 15000 material, sedimentation occurs. TACE2.0 falls on the soil surface over the root system. The residue still contains Trichoderma atroviride, and the hyphae in the residue can directionally grow towards the rice root system due to chemotaxis, as to block the soil transmission of R.solani.

5.Dissolution

The dissolution of TACE2.0 is divided into two process: HPMC4000 and HPMC15000. It was determined that the HPMC4000 matrix of 6mm TACE2.0 completely dissolved after 1~2 days, and the residual HPMC15000 was completely dissolved after another day. The HPMC matrix of TACE2.0 with a diameter of 3 mm was completely dissolved after about 8 hours, and the residual HPMC 15000 was completely dissolved after another 4 hours. That is, TACE2.0 will not leave any residue in the soil.

See more about experiments about TACE2.0 in Measurements

Usage and Storage

We searched the existing commercial Trichoderma powder in the market and investigated the traditional usage of commercial Trichoderma powder, calculating the spore concentration under the application method. We will use the spore concentration as a reference to better design the usage of TACE2.0.

Table 2. Traditional usage of Trichoderma spore powder

The traditional application dosage of the Trichoderma are as follows: spraying, 200-400g/mu(1mu equals 666.67㎡), diluting the microbial inoculum by 200-300 times, and then uniformly spraying in paddy fields. We have measured that the spore content of commercial Trichoderma powder was 1.48 billion per gram. According to our calculation, the traditional total usage of Trichoderma spore suspension is 40~60L per mu, and the spore concentration of spraying is 4.94 ~ 7.42 ×`10 ^ 6ml^-1`.

Then an ideal TACE2.0 should have the following characteristics: Environmental-friendly, the spore powder of TACE2.0 used per mu is less than or equal to 200g; High efficiency, the local high concentration of the TACE2.0 should be at least higher than 4.94×`10^6 ml^-1`. For this reason, we designed the recommended usage of TACE2.0 according to the prescription for TACE 2.0 and the traditional usage of spore suspension.

Table 3. Recommended usage of 3mm TACE2.0

According to the traditional usage of 200 g Trichoderma spore powder and the recommended prescription of TACE2.0, we calculated that the usage of 3mm TACE2.0 was 1.33 kg/mu, an average of two grains per rice plant. That is to say, 1.33 kg of TACE2.0 can reach the spore carrying capacity of 40~60 kg of spore suspension, which is more conducive to UAV dissemination.

Figure 9. TACE2.0 adhered to rice stem on the aquatic surface

We communicated with Trichoderma powder manufacturers and learned that temperature had the greatest impact on the preservation, followed by protective agents and moisture, and matrix material had a relatively small impact on it. The optimum conditions for preservation of Trichoderma were as follows: temperature 5°C, moisture content 7%, and dextrin as protective agent. When stored under the above condition, the proportion of effective spores was 76% of the initial value after 8 months of storage. At 25°C, the proportion of effective spores was about 56% after 3 months.

Costing

Table 4. Costing of TACE2.0

We investigated the market price of raw materials and calculated the cost price of the TACE2.0, which is merely 4.27 dollars/kg. Moreover, we've learnt that the price of UAV spraying is 1.83 dollars per mu, combined with the recommended TACE2.0 dosage of 1.33kg per mu, we calculated that the total cost of application is 7.51 dollars per mu, which is economical for farmers.

Advantages of TACE2.0

We carefully compared the first and second generation of TACE and found that TACE 2.0 is superior to TACE 1.0 in most aspects. 2022 SZU-China carefully summarizes the advantages of TACE2.0.

Besides what described above, Trichoderma spores were released slowly after TACE2.0 adhered to rice stems. The release center of Trichoderma spores is actually the adhesion point of rice stem. At this point, TACE2.0 produced a locally high spore concentration that was far higher than 4.94~7.42* `10^6 ml^-1`. And the other tillers of rice in the water area near this point can also be continuously exposed to the high concentration of spores. In addition, compared with direct spraying, TACE2.0 provides a more stable inner environment for spores, while the activity of sprayed spores may be reduced due to external factors such as sunlight. By using TACE2.0, we can get a better colonization effect of T.atroviride.

Vedio Functioning Process of 3mm TACE2.0

In summary, TACE2.0 is a highly successful iteration that will play an important role in the prevention phase of the project. 2022 SZU-China has high expectations for TACE2.0.

ENose

Introduction

Electronic nose (ENose) can be applied as a rapid, cost-effective option for several applications. ENose, as a daily detection device for Rice Sheath Blight disease (ShB) in the field, can monitor environmental data such as gas, temperature and humidity in the rice field through sensors and uploads the data, and then analyze the disease situation in the rice field through the ShB detection algorithm on the server. Finally, the server returns the analysis results to the client. Users can easily understand the disease situation of the rice field, so as to treat the affected area.

Figure 10. Schematic diagram of the electronic nose system

Features

• Early detection: By detecting volatile organic compounds (VOCs) in rice fields, ENose enables early detection of ShB, that is, when the rice plant phenotype has not changed.
• Low cost:Compared to one-off test strips, ENose enables one device to cover a monitoring area, and the cost is greatly reduced.
• Convenience: After placing the ENose device on the field, the user can view the environmental data of the rice field and the disease analysis results through the Smart Farm APP.
• Accuracy: The detection algorithm of ENose is based on convolutional neural networks and achieves an average detection accuracy of 99.87% in the experimental environment.
• Stability: We developed a stable ENose system after fully considering the real environmental situation in the field.

Users and application scenarios

After investigation, we were surprised to find that the existing rice pathogen detection methods are either time-consuming and laborious, or expensive, requiring professional personnel and precision instruments (such as electron microscopy, fluorescence immunoassay, fluorescence quantitative PCR, etc.), which are not suitable for the target population farmers. Therefore, we designed an electronic nose device, which enables farmers to easily understand the rice disease situation in the rice field through our APP (Smart Farm).

Figure 11. The hardware design diagram of ENose

In addition, ENose realizes the early detection of ShB, and analyzes the disease situation through the detection algorithm. After farmers learn the abnormal situation in the rice field through the APP, they can confirm the condition by on-site viewing or using LAMP-LFP technology. What's more, ENose has realized the detection of a variety of rice diseases.

Application

Placement

1. Two types of devices

Enose devices are divided into acquisition devices and management devices. The acquisition device is used to collect environmental data and transmit the data to the management device. The management device is a data transfer station, which connects to the Internet through WiFi and upload environmental data to the server, and can also receive instructions from the server to manage the access to the acquisition device.

2. Way of placing

Both the ENose acquisition device and the management device have a 2.17 m high fixed rod. All devices need to be fully charged before fixation. For fixation, the fixed rod needs to be inserted about 50cm below the soil.

Figure 12. Schematic diagram of fixed rod inserted into soil

3. Distance of placing

It is recommended that the ENose collection device cover a radius of 5m. If the radius exceeds this value, the detection accuracy may decrease. The recommended radius of the ENose management device is 45 meters. If the radius exceeds this threshold, the collection device may fail to be connected.

4. Device connection

After the E-Nose device is placed, the ENose management device automatically scans the surrounding ENose collection devices and connects them. Users can view device information through the Smart Farm APP. After that, users need to connect the ENose management device through a USB cable and configure network information as prompted by the APP.

5. Data review

Users should download and install the Smart Farm APP. Users can view environment data and device information through the Monitoring module, as well as historical environment data and disease conditions through the Early Warning module.

Figure 13. The Monitoring module and Early Warning module pages of Smart Farm APP (Left: environmental data; Right: disease situation and historical data）.

Control

Users can check the operation of the device and control the switch of the device through the Smart Farm APP. For example, we can turn on or off data acquisition equipment called DAE1 by clicking a switch.

Figure 14. ENose device management interface (Left: turn on the device named DAE1; Right: turn off the device named DAE1)

Maintenance

Due to the complexity of the real environment of rice fields, ENose will inevitably break down. We provide users with a maintenance reservation module in the Smart Farm APP, which provides users with convenience. The user only needs to fill in the product information and address information, and will receive the repaired equipment within a week after sending the equipment.

Figure 15. Maintenance reservation interface

Note

• 1. Do not open the shell of the device by non-professional personnel to avoid manual damage.
• 2. If the power of the device is lower than 20%, charge the device in time.
• 3. Please pay attention to strong electromagnetic sources such as communication base stations, substations and high-power antennas. Signal interference may lead to disconnection of equipment.
• 4. If the device is abnormal, press the reboot button to reboot it. If the problem cannot be solved, connect with professionals through the APP.

LAMP-LFD

Introduction

At present, there is no accurate field detection method for rice sheath blight. LAMP-LFD detection breaks this bottleneck, which can accurately identify Rhizoctonia solani, the pathogen of rice sheath blight through amplified DNA fragments.

Our LAMP system has the following advantages:

• Simple operation. The primer labeled with biotin is used for amplification at 65°C for 60 mins, and then hybridized to a target-specific probe labeled with fluorescein amide. Finally, the mixture is diluted and loaded to the LFD detection strip by drop.
• Strong specificity. Specific primers are used to amplify the ITS (ITS1&5.8 SrRNA) sequences of Rhizoctonia solani AG-1 and AG-3 but not the DNA fragments of other organisms.
• The product is easy to detect. LAMP amplification reaction is very sensitive, with the lowest detectable content of `10^-7ng(10^-1fg)` DNA samples in our experiments.
• PCR equipment and expensive reagents are not required in LAMP detection system, which has a wide range of application prospects. If lyophilization technology is used to prepare the reaction reagents into freeze-drying products, the detection equipment and reagents can even be collected into portable kits and taken to rice fields for field detection.

Users and application scenarios

Through multiple surveys in Human Practice, we have learned that in China, scientific research institutions guide most of the pesticide use and sowing techniques in rice fields, and farmers also trust scientific research institutions. Therefore, LAMP-LFD detection targets farmers and researchers. Farmers can make an appointment for LAMP-LFD detection service on our APP "SmartFarm". Researchers can detect rice crops sent by farmers or when they visit the field.

Figure 16. Users and application scenarios

LAMP-LFD technology not only detects rice sheath blight in the high-incidence period (such as high temperature, high humidity climate and rainy season), or further detects the abnormalities detected by Enose, in order to determine whether there are Rhizoctonia solani in the field, which achieves accurate detection. In addition, after the usage of our Trichoderma spore sustained release tablets and nanomaterials-shRNA preparations to control rice sheath blight, farmers and researchers take use of LAMP-LFD detection to examine whether there are still residues of Rhizoctonia solani in the field to evaluate the control effect.

Figure 17. Detection time

Application

Laboratory testing

Laboratory testing procedure:

• 1. The LAMP amplification.
  The optimum concentration of Mg (2+) in LAMP system is 7 mM (optimum concentration). The reaction proceeds at 65°C for 60 mins.
• 2. Electrophoresis analysis of amplification products.
  (1) When subsequent detection is not required on the Lateral Flow Device (LFD): The amplification products are incubated at 80°C for 5 min to terminate Bst DNA polymerase activity, then analyzed by electrophoresis on 1.3% agarose gels.
  (2) When subsequent detection is required on Lateral Flow Device (LFD): LAMP reactions are performed using the same procedure as normal LAMP, but with 5' biotin-labeled primers. The probes are labeled with FAM at the 5' end.
• 3. Analysis of loop-mediated isothermal amplification products.
  For implementation in LFD, using the generic LFD, the device has anti-fluorescein antibodies to detect double-stranded amplicons labeled with fluorescein amide (FAM). The biotin-labeled LAMP products are hybridized with the FAM-labeled probes, and 25 μL of the hybridized LAMP products are diluted to 100 μL with Tris-HCl 0.05M, pH8.0. Load 100 μL of this mixture to the sample well of LFD strips. Test results will appear within 2-10 minutes.

Instructions

Please read the instructions below for more details.

Laboratory testing

In order to make field testing more convenient for researchers, we conceived a portable field testing kit based on lyophilized microsphere technology. This kit integrates all the devices and reagents required for field testing. Researchers can just carry this kit to conduct field tests on rice crops. If properly operated, the test results will appear within about 1.5 hours, which quickly diagnose whether there is rice sheath blight in the paddy field.

Lyophilized microsphere technology is a new type of lyophilized technology. Through the biopharmaceutical lyophilization equipment, the activities of enzymes and proteins can be maintained, and the lyophilized spheres have loose network structure and rapid resolution. Lyophilized microsphere technology can transform unstable chemical reagents at room temperature into high-quality, stable lyophilized spheres that can be stored for a long time, which can be stored and transported at room temperature.

Lyophilized microsphere technology has the following advantages:

• One microsphere corresponds to one reaction in order to avoid contamination or error caused by multiple reagent additions;
• The performance is stable, unlike the liquid reagents, the performance of many times caused by freezing and thawing;
• The lyophilized microspheres can be stored for a long time and have a porous network structure;
• It can be stored at room temperature and transported, which reduces transportation costs.
• Freeze-dried microspheres with different components and sizes can be customized flexibly to meet various requirements.

Figure 18. Product form

The portable field testing kit contains the following devices and reagents:

• LAMP amplification - PCR tubes (each contains a lyophilized microsphere), primers and probes (each about 100 mL) of ITS sequence of Rhizoctonia solani, portable electric kettle.
• LFD detection - 15ml centrifuge tubes each containing 4 mm steel balls (numbers: 4-6), LFD test strips, diluent (Tris-HCl 0.05M, pH8.0, 100 mL).
• Other devices - sterilized water (about 200 mL), ice packs, pipettes (2.5 μL, 10 μL, 100 μL) and tips.

Figure 19. LAMP-LFD product collection

RNAi

Introduction

After detecting rice sheath blight, we can use RNAi products to specifically kill R.solani. Our RNAi products have the following characteristics:

• Good specificity. Our RNAi molecules have all passed the biosafety inspection, which has proved that they will not affect other species in the field, but specifically target R.solani.
• High safety. RNA molecules will be degraded by RNase in the air. When it finishes killing R.solani, it will not stay on the surface of rice and will not have any impact on the normal growth of rice.
• Strong sustainability. Experiments have proved that our RNAi product can provide at least 5 days of continuous shRNA release and continuous silencing of target genes, ensuring that the product can also play a good role in the field environment.
• Eco-friendly. At present, the effect of biological pesticides such as antibiotics has declined year by year, and they are likely to be directly eliminated in the future. Our RNAi products directly target the genes of pathogenic fungi, without considering the generation of fungal drug resistance. In addition, unlike other pesticides, RNAi pesticide will not remain in the environment and damage the ecology. It is green and friendly to the environment.
• Wide application prospect. Our RNAi products can be used not only to treat rice sheath blight, but also to treat other diseases on rice and other crops. In the shRNA expression system, our target sequence elements can be replaced according to the desired pathogenic fungi to achieve the effect of simultaneously treating multiple diseases.

Production process

The following is the production process of our RNAi products.

• (1)Transforming the constructed shRNA expression plasmid into E. coli HT115(DE3).
• (2)Adding IPTG to induce Escherichia coli to produce shRNA molecules.
• (3)After obtaining enough shRNA, arabinose is added to induce the production of R-body.
• (4)Add sufficient CO2 to acidify the bacterial culture medium to a pH below 6.5, so that E.coli can be cracked.
• (5)The shRNA isolated from E.coli was obtained by centrifugation and incubated with CNT for 30 min to obtain shRNA CNT products.
• (6)The shRNA-CNT produced will be packaged in spray bottles.
• (7)The spray bottle is assembled on the UAV to complete the large area spraying of RNAi products in the field.

Figure 20. RNAi drug production

Application

We treated each leaf of rice with 100uL of 10ug shRNA solution.

After a period of time after the infection experiment was carried out, we observed the growth of the mycelium on the rice leaves under the stereo microscope, and obtained the extension length of the mycelium on the leaves under different shRNA treatments (Fig. 21). Similarly, after binding CNT, we also tested the effect of shRNA spraying on hyphal elongation (Fig. 22).

Compared with the control group and the group without CNT binding, the mycelia bound with CNT and smeared with CNT showed obvious inhibition. We can draw a conclusion that our CNT promoted the inhibition of shRNA against R.solani. Therefore, in actual application, shRNA combined with CNT will be used for spraying.

Figure 21. Effect of spraying different shRNA on mycelial elongation without binding CNT

Figure 22. Effect of spraying different shRNA after binding CNT on mycelial elongation

From the distribution of disease spots, spraying siRNA can reduce the incidence of leaves to a certain extent, but the effect is not obvious. The spraying effect of shRNA was better than that of siRNA, but it could not inhibit the infection of Rhizoctonia solani. After spraying shRNA bound with CNT, it can be found that the incidence of leaves has been greatly reduced, indicating that shRNA CNT can well inhibit and kill Rhizoctonia solani.

Figure 23. Distribution of disease spots on infected rice.

References

[1] Mehlman K. The Art of Pharmacy[J]. The Senior Care Pharmacist, 2022, 37(5): 169-170.
[2] Wang L, Liu X. Sustained release technology and its application in environmental remediation: a review[J]. International Journal of Environmental Research and Public Health, 2019, 16(12): 2153.
[3] Guarve K, Kriplani P. HPMC-A Marvel Polymer for Pharmaceutical Industry-Patent Review[J]. Recent Advances in Drug Delivery and Formulation: Formerly Recent Patents on Drug Delivery & Formulation, 2021, 15(1): 46-58.
[4] Zheng J, Wang B, Xiang J, et al. Controlled release of curcumin from HPMC (hydroxypropyl methyl cellulose) co-spray-dried materials[J]. Bioinorganic chemistry and applications, 2021, 2021.
[5] Al-Tabakha M M. HPMC capsules: current status and future prospects[J]. Journal of Pharmacy & Pharmaceutical Sciences, 2010, 13(3): 428-442.