CN115948298A - Production process of Mackanka agar medium - Google Patents
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- CN115948298A CN115948298A CN202211687634.XA CN202211687634A CN115948298A CN 115948298 A CN115948298 A CN 115948298A CN 202211687634 A CN202211687634 A CN 202211687634A CN 115948298 A CN115948298 A CN 115948298A
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Abstract
The invention relates to the technical field of culture medium production, in particular to a production process of a MacconKa agar culture medium. The production process comprises the following steps: ball-milling a preparation raw material of the MacConkey agar culture medium for 2-3 h to obtain the MacConkey agar culture medium; before ball milling, respectively carrying out crushing pretreatment on neutral red and crystal violet serving as raw materials, wherein the mass ratio of balls to the prepared raw materials is 1.0-1.5. The invention changes the granularity of the raw material with low dissolving speed through proper pretreatment, ball milling time and ball-material ratio, improves the dissolving speed, and can be synchronously dissolved with other raw materials finally in the preparation and use processes. Meanwhile, the provided production process parameters can improve the mixing uniformity of the raw materials of the Macconkey agar culture medium and the recovery rate of microorganisms, thereby improving the quality of the culture medium.
Description
Technical Field
The invention relates to the technical field of culture medium production, in particular to a production process of a MacconKa agar culture medium.
Background
The culture medium is artificially prepared and is suitable for the growth, separation and identification of microorganisms, and the development of the culture medium is an important content in the field of microbiology. It is widely applied in the fields of medicine and health, food, cosmetics, industry and agriculture, environmental protection and the like. In recent 20 years, the culture medium industry in China has been rapidly developed, so that the gap between the culture medium industry and developed countries is greatly shortened, and a certain rising space is provided. The main problems existing at present are: the slow dissolving speed of the MacconKa agar culture medium produced by the prior art causes insoluble substances after preparation, the mixing uniformity of the culture medium is low, and the recovery rate of microorganisms is low.
Disclosure of Invention
In order to solve the problems, the invention provides a production process of a MacconKa agar culture medium. The production process provided by the invention can accelerate the dissolution speed of related raw materials, solve the problem that insoluble substances exist after the culture medium is prepared, and simultaneously improve the mixing uniformity of the raw materials of the MacconKa agar culture medium and the recovery rate of microorganisms, thereby improving the quality of the culture medium.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides a production process of a MacconKa agar culture medium, which comprises the following steps:
ball-milling a preparation raw material of the MacConkey agar culture medium for 2-3 h to obtain the MacConkey agar culture medium;
during ball milling, the mass ratio of the balls to the preparation raw materials is 1.0-1.5;
the ball milling process comprises the following steps: respectively crushing neutral red and crystal violet in the prepared raw materials, and sieving by a 150-mesh sieve to obtain neutral red undersize and crystal violet undersize.
Preferably, the crystal violet undersize is mixed with peptone to obtain a crystal violet mixture; the weight ratio of the crystal violet undersize product to the peptone is 1 (9-99).
Preferably, the rotation speed of the ball mill is 30 to 50 revolutions per minute.
Preferably, the ball milling process also comprises the step of adjusting the pH value to be 6.9-7.3.
Preferably, the pH adjusting substance comprises sodium carbonate.
Has the advantages that:
the invention provides a production process of a Macconkey agar culture medium, which comprises the following steps: ball-milling a preparation raw material of the MacConkey agar culture medium for 2-3 h to obtain the MacConkey agar culture medium; during ball milling, the mass ratio of the balls to the preparation raw materials is 1.0-1.5; the ball milling process comprises the following steps: respectively crushing neutral red and crystal violet in the prepared raw materials, and sieving by a 150-mesh sieve to obtain a neutral red undersize material and a crystal violet undersize material. The invention changes the granularity of the raw material with slow dissolving speed through proper pretreatment, ball milling time and ball-material ratio, improves the dissolving speed, and can be finally dissolved with other raw materials synchronously in the preparation and use process. Meanwhile, the provided production process parameters can improve the mixing uniformity of the raw materials of the Macconkey agar culture medium and the recovery rate of microorganisms, thereby improving the quality of the culture medium.
Detailed Description
The invention provides a production process of a MacconKa agar culture medium, which comprises the following steps:
ball-milling a preparation raw material of the MacConkey agar culture medium for 2-3 h to obtain the MacConkey agar culture medium;
during ball milling, the mass ratio of the balls to the preparation raw materials is 1.0-1.5;
the ball milling process comprises the following steps: respectively crushing neutral red and crystal violet in the prepared raw materials, and sieving by a 150-mesh sieve to obtain a neutral red undersize material and a crystal violet undersize material.
In the invention, the preparation raw materials of the MacconKa agar medium preferably comprise the following components in parts by weight: 10 to 20 portions of peptone, 1 to 4 portions of sodium deoxycholate, 15 to 20 portions of lactose, 3 to 8 portions of sodium chloride, 10 to 15 portions of agar, 0.01 to 0.05 portion of neutral red passing through a 150-mesh screen and 0.05 to 0.25 portion of crystal violet mixture; preferably comprises 15.00 parts of peptone, 2.75 parts of sodium deoxycholate, 16.50 parts of lactose, 5.00 parts of sodium chloride, 10.62 parts of agar, 0.03 part of neutral red with the granularity of more than or equal to 150 meshes and 0.10 part of crystal violet mixture; the crystal violet mixture preferably comprises the following components in parts by weight: 1 part of crystal violet and 9-99 parts of peptone which pass through a 150-mesh screen.
In the invention, the ball milling process comprises the following steps: respectively crushing the neutral red and the crystal violet, and sieving by a 150-mesh sieve to obtain a neutral red undersize material and a crystal violet undersize material. According to the invention, the particle components with large particle size and slow dissolution speed are crushed and sieved by a 150-mesh sieve, so that the particle size of the large particle components is reduced, the subsequent ball milling is facilitated, and the dissolution speed of the large particle components can be increased.
After neutral red undersize and crystal violet undersize are obtained, the crystal violet undersize and peptone are preferably uniformly mixed to obtain a crystal violet mixture; the weight ratio of the crystal violet undersize product to the peptone is preferably 1 (9-99), and more preferably 1:99. the present invention does not require any particular mixing means, as would be known to one skilled in the art. Because the percentage content of the crystal violet in the MacconKa agar medium is lower, the crystal violet and the peptone are mixed firstly, and the crystal violet mixture and other raw materials are ball-milled, so that the crystal violet can be mixed more uniformly.
After obtaining the crystal violet mixture, the preparation raw materials are ball-milled for 2-3 h to obtain the Macconkey agar culture medium. In the invention, the ball milling time is 2-3 h, preferably 2.5h; the mass ratio of the ball to the preparation raw material is 1.0-1.5, preferably 1.2; the rotation speed of the ball mill is preferably 30 to 50 rpm, more preferably 35 to 40 rpm, and still more preferably 36 rpm. The ball milling device has no special requirement, and the device known by the technical personnel in the field can be adopted. The ball mill used in the examples and comparative examples of the present invention is preferably available from Nantong Youbang mechanical Co., ltd., model number OM-200.
The crystal violet and the neutral red are crushed to change the particle size and improve the dissolving speed, and finally can be synchronously dissolved with other raw materials in the preparation and use processes.
The invention determines the optimal ball milling time and ball-to-material ratio process parameters in the industrial production process of the MacconKa agar medium through experiments. The culture medium prepared by the production process provided by the invention can reduce the granularity difference of the culture medium raw materials, improve the sensory degree, improve the physical properties of the culture medium, improve the mixing uniformity of the culture medium raw materials, solve the problem that insoluble substances exist after the culture medium is prepared, and improve the microbial recovery rate of the culture medium.
In the invention, the ball milling process preferably also comprises the steps of adjusting the pH value to 6.9-7.3, more preferably adjusting the pH value to 7.1-7.3; the substance for adjusting the pH value preferably comprises sodium carbonate; the frequency of the adjustment is preferably adjusted every 0.5h.
The Mackanka agar culture medium prepared by the production process can improve the mixing uniformity, has short time for dissolving the materials completely after being prepared with water, and has high microbial recovery rate.
In order to further illustrate the present invention, the following examples are given to describe the production process of the Macconyya agar medium of the present invention in detail, but they should not be construed as limiting the scope of the present invention.
Example 1
The Mackanka agar medium is prepared by the following steps:
1) Respectively crushing neutral red and crystal violet by a high-efficiency crusher until the neutral red and the crystal violet can pass through a 150-mesh screen for later use;
2) Diluting and mixing the crushed crystal violet and soytone according to the weight ratio of 1;
3) According to parts by weight, adding 15.00 parts of soytone, 2.75 parts of sodium deoxycholate, 16.50 parts of lactose, 0.03 part of neutral red, 5.00 parts of sodium chloride, 10.62 parts of agar and 0.10 part of crystal violet mixture into a ball milling tank filled with 60 parts of stainless steel balls (namely the ball-to-material ratio is 1.2; the pH value is adjusted by sodium carbonate every 0.5h in the production process, and the pH value is controlled to be 7.1-7.3.
Example 2
A mecnkia agar medium similar to that of example 1, with the only difference that the ball milling time in step 3) is 2h.
Example 3
A mecnkia agar medium similar to that of example 1, with the only difference that the ball milling time in step 3) is 3h.
Comparative example 1
A mecnkaya agar medium similar to that of example 1, with the only difference that step 1) is: respectively crushing the neutral red and the crystal violet by a high-efficiency crusher until the neutral red and the crystal violet can pass through a sieve with 50 meshes and can not pass through a sieve with 100 meshes for standby.
Comparative example 2
A mecanka agar medium similar to that of example 1, with the only difference that step 1) is: respectively crushing the neutral red and the crystal violet by a high-efficiency crusher until the neutral red and the crystal violet can pass through a 100-mesh sieve and do not pass through a 150-mesh sieve for later use.
Comparative application example 1
The dissolution rate of the mcnkaya agar media prepared in example 1, comparative example 1 and comparative example 2 was determined by the following specific method:
weighing 5g of sample, adding 100mL of purified water, heating to boil, cooling to 60 ℃, pouring the sample into a culture dish, and observing whether the crystal violet and the neutral red are completely dissolved or not after the sample is solidified; the boiling time is 1min, 3min and 5min respectively, if the crystal violet and the neutral red are completely dissolved after the sample which is boiled for 1min is solidified, the experiment of boiling for 3min and 5min is not needed, and if the crystal violet and the neutral red are completely dissolved after the sample which is boiled for 3min is solidified, the experiment of boiling for 5min is not needed. The results are shown in Table 1.
TABLE 1 solubility results for different samples
Note: the "-" in Table 1 indicates that no subsequent experiment was performed.
As can be seen from Table 1, the pH indicator is neutral red and the bacteriostatic agent crystal violet is crushed by a high-speed crusher to accelerate the dissolution speed; the neutral red and crystal violet can be dissolved synchronously with other raw materials after being prepared by using a finished product culture medium prepared from the neutral red and the crystal violet with the crushed particle size of 150 meshes.
Comparative example 3
A macconka agar medium similar to that of example 1, the only difference being that 60 parts of stainless steel balls in step 3) were replaced with 25 parts of stainless steel balls, i.e. the ball to feed ratio was 0.5.
Comparative example 4
A macconka agar medium similar to that of example 1, the only difference being that 60 parts of the stainless steel balls in step 3) were replaced with 95 parts of stainless steel balls, i.e. the ball to feed ratio was 1.9.
Comparative application example 2
The particle size distribution and the weight ratio of the number of meshes passing through each zone of the MacConkey agar medium prepared in example 1, comparative example 3 and comparative example 4 were measured.
The determination method comprises the following steps: 200g of a sample is weighed according to Chinese pharmacopoeia (particle size and particle size distribution determination method), placed in a sieve with a specified number (a receiving container is arranged below the sieve in a sealing manner), and covered on the sieve. The sieve was shaken in a horizontal direction for 5 minutes with occasional taps in the vertical direction. Weighing the sieved particles and powder, and calculating the ratio (%) of the particles and powder.
The measurement results are shown in Table 2, and the calculation results are shown in tables 3 and 4.
TABLE 2 weight of the number of meshes of the samples passing through each zone for different ball-to-feed ratios
TABLE 3 weight ratio of mesh number of screen passing each zone for samples of different ball-to-material ratios
TABLE 4 influence of different ball ratios on the particle size distribution by weight (unit:%)
As can be seen from tables 3 and 4, the proportion of particles that do not pass through the 60 mesh screen is maximum and 5.2% in the sample prepared at a ball to material ratio of 0.5, the proportion of particles that pass through the 100 mesh screen does not pass through the 200 mesh screen is 0.5, the proportion of particles that pass through the 100 mesh screen is 70.8%, the proportion of particles that pass through the 1.2. Comparing the particle size distributions of two samples prepared with a ball-to-feed ratio of 1.2 and a ball-to-feed ratio of 1.9, the particle sizes are less than 0.25mm, less than 0.18mm, and the occupation ratios of less than 0.15mm are substantially the same; the percent by weight of the sample ball-to-material ratio 1.2 with the particle size of less than 0.125mm is 77.7%, the percent by weight of 1.9 is 63.6%, the percent by weight of the sample ball-to-material ratio 1.2 with the particle size of less than 0.095mm is 33.1%, the percent by weight of 1.1 is 22.1%, and the grinding capacity of the ball-to-material ratio 1.9 is not as good as the grinding capacity of the ball-to-material ratio 1.2, and the sample is ground finer at the ball-to-material ratio 1.2. The reason for this may be that the balls in the tank are too many, and the powder cannot be uniformly dispersed, which affects the grinding effect and the mixing uniformity. According to the particle size distribution detection result, selecting a ball-to-material ratio of 1.2 as a production process parameter.
On the basis of selecting the ball-to-material ratio of 1.2 as a production process parameter, properly widening the process parameter range, and increasing the particle size and particle size distribution measurement of samples prepared under the following parameters of 1.0 ball-to-material ratio and 1.5. The test method is the same as above.
Example 4
A mecnkaya agar medium similar to that of example 1, the only difference being that 60 parts of stainless steel balls in step 3) are replaced by 50 parts of stainless steel balls, i.e. the ball to feed ratio is 1.0.
Example 5
A mecnkaya agar medium similar to that of example 1, the only difference being that 60 parts of stainless steel balls in step 3) are replaced by 75 parts of stainless steel balls, i.e. the ball to feed ratio is 1.5.
The particle size distributions of example 1, example 4 and example 5 were measured by the method in comparative application example 2, and the results are shown in Table 5.
TABLE 5 influence of the ball ratio on the particle size distribution (weight ratio) (unit:%)
As can be seen from table 5, after the sample prepared with the ball/material ratio of 1.0.
Comparative application example 3
According to the microbial limit inspection of non-sterile products of the general rule 1106 in the four departments of the 2020 edition of Chinese pharmacopoeia: the microbial recovery rates of the macconka agar media prepared in example 1 (pellet ratio 1.2.
TABLE 6 sample microorganism test results Table
Remarking:
the four rules for the culture medium suitability inspection in the "Chinese pharmacopoeia" 2020 edition: is detected and fixedThe ratio of the average number of colonies on the bulk medium to the average number of colonies on the control medium (recovery) should be in the range of 0.5 to 2.0, and the number of two colonies in parentheses is the result of two parallel experiments, and the following table is the same.
As can be seen from table 4, the recovery rates of escherichia coli CMCC (B) 44102 were 0.87,0.93,0.99,0.76,0.53, respectively, for five samples prepared by a pellet-to-pellet ratio of 0.5; the recovery rate of the first example (ball to feed ratio of 1.2) is higher than that of the second example 4 (ball to feed ratio of 1.0; the recovery rates of the salmonella paratyphi B CMCC (B) 50094 are respectively 0.94,1.13,1.17,1.21 and 0.86; the recovery rate of example 5 (pellet to pellet ratio of 1.5) is higher than that of example one (pellet to pellet ratio of 1.2; the samples of example 1, example 4, example 5, comparative example 3 and comparative example 4 all inhibited staphylococcus aureus CMCC (B) 26003 with the same inhibitory capacity. The microorganism recovery rate results corresponding to culture media prepared by different ball-material ratios are different, and the analysis reason that the ball-material ratio can influence the particle size distribution of materials, and the particle size distribution influences the mixing uniformity of the materials, thereby influencing the microorganism recovery rate. Through the results, the ball-to-material ratio is selected to be 1.0-1.5 as the production process parameter in combination with the results of the particle size distribution.
Comparative example 5
A mecnkaya agar medium similar to that of example 1, with the only difference that the ball milling time in step 3) is 0.5h.
Comparative example 6
A mecnkia agar medium similar to that of example 1, the ball milling time in step 3) being 1.5h.
Comparative application example 4
The weight ratio of the mesh number of the sieves in each zone of the MacConk agar medium prepared in example 1, example 2, example 3, comparative example 5 and comparative example 6 was measured by the method of comparative application example 2, and the measurement results are shown in Table 5.
TABLE 5 weight ratio of the number of meshes of the sample passing through the screen in each zone for different ball milling times
As can be seen from Table 5, when the ball milling is carried out for 0.5h, large particles which do not pass through a 35-mesh (0.50 mm-diameter) screen are present in the sample; ball milling a sample which passes through a 100-mesh sieve and does not pass through a 200-mesh sieve for 0.5h to 63.6%, ball milling for 1.5h to 69.3%, ball milling for 2h to 2.5h, wherein the ball milling for 3.0h is more than 70%, the ball milling for 2h to 74.8%, the ball milling for 2.5h to 78.8% and the ball milling for 3h to 78.8%; the particle size distribution of the samples after ball milling for 2.5h and 3h is more concentrated.
Comparative application example 5
According to the microbial limit inspection of non-sterile products of the general rule 1106 in the four departments of the 2020 edition of Chinese pharmacopoeia: the microbial recovery rates of the Macconyya agar media prepared in examples 1, 2, 3, 5 and 6 were measured by the controlled bacteria assay, and the results are shown in Table 6.
TABLE 6 sample microorganism test results Table
As can be seen from Table 6, the recovery rate of microorganisms of the sample prepared by ball milling for 0.5h was overall low; the recovery rate of the sample CMCC (B) 44102 prepared by ball milling for 1.5h, the recovery rate of the Salmonella paratyphi B CMCC (B) 50094 is less than 1, and the recovery rate of the sample microorganism prepared by ball milling for 2h,2.5h and 3h is more than 1.
Visual inspection can observe that the samples of example 1 (ball milling for 2.5 h), example 2 (ball milling for 2 h), example 3 (ball milling for 3 h) are more uniform in particle size and better in sensory index than those of comparative example 5 (ball milling for 0.5 h) and comparative example 6 (ball milling for 1.5 h). And the same amount of the three samples of example 1, comparative example 5 and comparative example 6 were added simultaneously to the same amount of purified water, the total dissolution rate of example 1 was faster. Therefore, ball milling for 2.5h is selected as a production process parameter.
The ball milling time of the process can be selected from 2h to 3h by considering the factors of energy conservation, environmental protection, cost reduction and the like.
In conclusion, the Macconkey agar culture medium prepared by the production process provided by the invention can reduce the granularity difference of the culture medium raw materials, improve the organoleptic degree and the physical properties of the culture medium, can improve the mixing uniformity of the culture medium raw materials, solve the problem of insoluble substances after the culture medium is prepared and improve the microbial recovery rate of the culture medium.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (5)
1. A production process of a MacconKa agar culture medium is characterized by comprising the following steps:
ball-milling a preparation raw material of the MacConkey agar culture medium for 2-3 h to obtain the MacConkey agar culture medium;
during ball milling, the mass ratio of the balls to the preparation raw materials is 1.0-1.5;
the ball milling device comprises the following components: respectively crushing neutral red and crystal violet in the prepared raw materials, and sieving by a 150-mesh sieve to obtain a neutral red undersize material and a crystal violet undersize material.
2. The process according to claim 1, wherein the undersize of crystal violet is mixed with peptone to obtain a crystal violet mixture; the weight ratio of the crystal violet undersize product to the peptone is 1 (9-99).
3. The process according to claim 1, wherein the ball mill rotates at a speed of 30 to 50 rpm.
4. The process according to claim 1 or 3, wherein the ball milling process further comprises adjusting the pH to 6.9 to 7.3.
5. The process of claim 4 wherein the pH adjusting substance comprises sodium carbonate.
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| CN117887642A (en) * | 2024-03-14 | 2024-04-16 | 山东格研生物技术有限公司 | Wheat Conkai agar culture medium plate capable of being subjected to irradiation sterilization and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN117887642A (en) * | 2024-03-14 | 2024-04-16 | 山东格研生物技术有限公司 | Wheat Conkai agar culture medium plate capable of being subjected to irradiation sterilization and preparation method thereof |
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