A phenol red-containing trypsin solution with uniform color distribution after cryopreservation and its preparation method

Abstract

The present invention provides a method for preparing a phenol red-containing trypsin solution with uniform color distribution after cryopreservation, comprising the following steps: weighing buffer components and phenol red, mixing them, and ball-milling to obtain a pretreated buffer component powder; weighing the pretreated buffer component powder, trypsin powder, and ultrapure water, mixing them, stirring, and performing sterile filtration to obtain the phenol red-containing trypsin solution; wherein the passing rate of the pretreated buffer component powder through a 100-mesh sieve is ≥95%. By determining reasonable grinding conditions and adopting the method of grinding first and then dissolving, the present invention makes the material particle size uniform, the particle size small, and the dissolution speed faster, and it is easier to form a uniform solution during the dissolution and stirring process, ensuring that the prepared phenol red-containing trypsin solution remains a uniform light yellow color after being cryopreserved at -20°C for 24 hours.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological preparation configuration, and particularly relates to a phenol red-containing trypsin solution with uniform color distribution after cryopreservation and a preparation method thereof. Background Art

[0002] Trypsin belongs to the serine protease family and is a specific endopeptidase that can selectively cleave the peptide bonds formed at the carboxyl termini of lysine and arginine residues. It is usually prepared into a trypsin solution with PBS buffer for cell digestion (dispersion) of adherent cultured animal cells. The trypsin solution containing phenol red is a common animal cell dissociation reagent. Phenol red is an acid-base indicator with a color change range of pH: 6.8 (yellow) - 8.4 (red). The optimal pH value of trypsin is about 7.8 - 8.5. Within this pH range, the appearance color of the phenol red-containing trypsin solution is "pink". Due to changes in experimental environmental conditions, the pH of trypsin may deviate from the optimal pH. At this time, phenol red, as an acid-base indicator, can help experimental personnel quickly confirm and identify whether the current trypsin solution is at the optimal pH.

[0003] Generally, after the phenol red-containing trypsin solution (PBS buffer system) is prepared, it needs to be stored at -20°C for cryopreservation and standby. However, the current common problem in the industry is that once this solution is cryopreserved, its product appearance will undergo uncertain color changes, specifically manifested as different colors such as red, brown, and yellow, and the color distribution is also different. This problem seriously affects the product appearance, hinders the production enterprise's judgment of product quality stability, brings misunderstandings to consumers, reduces the credibility of product quality, and seriously damages the economic benefits of this product.

[0004] Phenol red is slightly soluble in water, has a very slow dissolution rate in water, and is easily soluble in sodium hydroxide solution. Due to the poor solubility of phenol red in water, problems such as "phenol red is difficult to dissolve completely" and "although the remaining phenol red solid particles are still slowly dissolving, it takes an extremely long time (>24h) to wait for them to completely dissolve" will occur when directly adding phenol red during the traditional solution preparation process. In the industry, phenol red powder is usually dissolved in sodium hydroxide solution to form a uniform phenol red mother liquor, and then the mother liquor is added to the product solution during the product production process to achieve rapid and complete dissolution of phenol red. However, although this traditional process solves the technical problem of difficult dissolution of phenol red, there is still a problem that "the product appearance color is uncertain after cryopreservation, manifested as different colors such as red, brown, and yellow, and the color distribution is uneven". Therefore, in the products made by the traditional process of "sodium hydroxide - phenol red mother liquor", the pigment molecule phenol red is not completely uniformly dispersed in the final product solution. More precisely, at least after cryopreservation, the pigment molecule phenol red in the products produced by the traditional process cannot maintain an overall uniform distribution, so color unevenness will occur. Summary of the Invention

[0005] In view of this, the present invention provides a trypsin solution which is "uniformly light yellow" after freezing and a preparation method thereof, which effectively solves the problem of abnormal color and uneven color distribution of a trypsin solution containing phenol red (PBS buffer system) after freezing, and not only does it not affect its cell digestion effect, does not increase cytotoxicity, and does not increase production costs, but simplifies the production process, saves time and economic costs, and ultimately eliminates the negative impact of the poor appearance of the product, thereby ensuring the economic benefits of the product.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A method for preparing a pancreatic enzyme solution containing phenol red with uniform color distribution after freezing, comprising the following steps: weighing a buffer component and phenol red, mixing, ball milling, and obtaining a pretreatment buffer component powder; weighing the pretreatment buffer component powder, trypsin powder, and ultrapure water, mixing, stirring, and aseptically filtering to obtain the pancreatic enzyme solution containing phenol red;

[0008] Wherein, the 100-mesh sieving rate of the pretreatment buffer component powder is ≥95%.

[0009] Furthermore, three types of grinding beads, small, medium and large, are added to the ball mill, the number ratio of the small, medium and large grinding beads is (120-140):(20-25):(6-9), the total volume of the grinding beads to the total volume of the buffer solution components and phenol red is (0.5-3.0):1, the ball mill rotation speed is 70-110rpm, and the ball mill time is ≥1h.

[0010] Furthermore, the diameter ratio of the three types of grinding beads, small, medium and large, is 1:2:3.

[0011] In some specific embodiments, preferably, the diameters of the small, medium and large grinding beads are 0.5 cm, 1.0 cm and 1.5 cm respectively.

[0012] In some specific embodiments, preferably, the ratio of the number of small, medium and large grinding beads is 120:25:6, the ratio of the total volume of the grinding beads to the total volume of the buffer solution components and phenol red is 1:1, the ball milling speed is 110 rpm, and the ball milling time is 1 h.

[0013] In some specific embodiments, preferably, the ratio of the number of small, medium and large grinding beads is 140:20:9, the ratio of the total volume of the grinding beads to the total volume of the buffer solution components and phenol red is 2:1, the ball milling speed is 80 rpm, and the ball milling time is 1 h.

[0014] Furthermore, the stirring speed is 300 rpm, and the stirring time is 15 min.

[0015] Furthermore, the sterile filtration pore size is ≤0.22 μm.

[0016] The pancreatin solution prepared by the above preparation method.

[0017] The pancreatin solution is uniformly light yellow when stored at -20°C.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention determines the grinding bead type, the ratio of different types of grinding beads, the grinding speed, the grinding time and other conditions during the grinding process, and grinds the buffer component before preparing the solution, so that the 100-mesh screening rate is ≥95% and the turbidity (NTU) after dissolution is ≤1, so that the material has uniform particle size, fine particle size, and faster dissolution speed, and it is easier to form a uniform solution during the dissolution and stirring process, ensuring that the prepared phenol red-containing pancreatic enzyme solution is frozen at -20°C and still presents a uniform light yellow color after 24 hours. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The states of the phenol red-containing pancreatic enzyme solutions prepared in the examples and comparative examples after being frozen at -20°C for 24 hours; wherein the examples 1 to comparative examples 7 are numbered A to H in sequence.

[0021] Figure 2 The result diagrams of digesting Hacat cells with phenol red-containing trypsin solutions prepared in Example 1 and Comparative Example 1, respectively, and then photographing the cell status after subculture; wherein A corresponds to Example 1, and B corresponds to Comparative Example 1.

[0022] Figure 3 The result diagrams of digesting SV-HUC-1 cells with phenol red-containing trypsin solutions prepared in Example 1 and Comparative Example 1, respectively, and then photographing the cell status after subculture; wherein A corresponds to Example 1, and B corresponds to Comparative Example 1. DETAILED DESCRIPTION

[0023] The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand the present invention more clearly.

[0024] Example 1

[0025] This embodiment provides a phenol red-containing pancreatic enzyme solution with uniform color distribution after freezing, and the preparation method is as follows:

[0026] Weigh the following materials by weight parts: 2.0 parts of potassium chloride, 2.7 parts of potassium dihydrogen phosphate, 80.1 parts of sodium chloride, 11.36 parts of disodium hydrogen phosphate anhydrous, 2 parts of EDTA·2Na·2H2O, and 0.1 part of phenol red; place the above materials into a ball mill in sequence, and add 120 small beads, 25 medium beads, and 6 large beads to it. At this time, the total volume ratio of the grinding beads to the total volume of the buffer components and phenol red is about 1:1; start the ball mill and grind for 60 min at a rotation speed of 80 rpm to obtain the buffer component powder.

[0027] Take the following materials according to mass parts: 98.3 parts of the buffer component powder obtained in the above step, 25 parts of trypsin powder, and 10000 parts of ultrapure water; mix the above materials and stir to dissolve for 15 min at a rotation speed of 300 rpm, and then filter through a bacteriological filter with a pore size of 0.22 μm to obtain a phenol red-containing trypsin solution with uniform color distribution after freezing.

[0028] Among them, the parameters of the grinding beads are as shown in Table 1 below.

[0029] Table 1 Specific parameters of each grinding bead

[0030] Type Diameter (cm) <![CDATA[Volume (cm 3 )]]> Mass (g) <![CDATA[Density (g / cm 3 )]]> Small bead 0.5 0.52 4.2 7.9 Medium bead 1.0 4.19 33.2 7.9 Large bead 1.5 14.13 111.8 7.9

[0031] Example 2

[0032] This example provides a phenol red-containing trypsin solution with uniform color distribution after freezing. Its raw materials and preparation method are basically the same as those in Example 1, except that: add 140 small beads, 20 medium beads, and 9 large beads to the ball mill. At this time, the total volume ratio of the grinding beads to the total volume of the buffer components and phenol red is about 2:1; the grinding rotation speed is 80 rpm, and the rest remain unchanged.

[0033] Comparative Example 1

[0034] This comparative example provides a phenol red-containing trypsin solution, which adopts a conventional preparation method, specifically as follows:

[0035] S1. Prepare a 10 mg / mL phenol red solution: Weigh 10 g of phenol red solid and dissolve it in 1 L of 2 mol / L sodium hydroxide solution.

[0036] S2. Accurately weigh the following materials: 2.0 parts of potassium chloride, 2.7 parts of potassium dihydrogen phosphate, 80.1 parts of sodium chloride, 28.7 parts of disodium hydrogen phosphate dodecahydrate, 2 parts of EDTA·2Na·2H2O. Add the above materials to 8000 parts of ultrapure water in sequence and stir while adding until the solution is clear. The stirring time is 30 min.

[0037] S3. Weigh 25 parts of trypsin powder and add it to the mixture in step S2. Stir while adding until the solution is clear. The stirring time is 30 min.

[0038] S4. Then add a certain amount of 10 mg / mL phenol red solution so that the phenol red content accounts for 0.1 part. After the color is evenly distributed, make up the volume to the volume corresponding to 10,000 parts of ultrapure water.

[0039] S5. Use 2 mol / L hydrochloric acid solution to adjust the pH of the solution to 7.1 - 7.3. After sterile filtration, the phenol red-containing trypsin solution is obtained.

[0040] Comparative Example 2

[0041] This comparative example provides a phenol red-containing trypsin solution, whose raw materials and steps are basically the same as those in Example 1, except that: only 482 small beads are added to the ball mill. At this time, the total volume ratio of the grinding beads to the total volume of the buffer components and phenol red is about 1:1.

[0042] Comparative Example 3

[0043] This comparative example provides a phenol red-containing trypsin solution, whose raw materials and steps are basically the same as those in Example 1, except that: only 60 medium beads are added to the ball mill. At this time, the total volume ratio of the grinding beads to the total volume of the buffer components and phenol red is about 1:1.

[0044] Comparative Example 4

[0045] This comparative example provides a phenol red-containing trypsin solution, whose raw materials and steps are basically the same as those in Example 1, except that: only 18 large beads are added to the ball mill. At this time, the total volume ratio of the grinding beads to the total volume of the buffer components and phenol red is about 1:1.

[0046] Comparative Example 5

[0047] This comparative example provides a phenol red-containing trypsin solution, whose raw materials and steps are basically the same as those in Example 1, except that: 283 small beads, 15 medium beads and 3 large beads are added to the ball mill. At this time, the total volume ratio of the grinding beads to the total volume of the buffer components and phenol red is about 1:1.

[0048] Comparative Example 6

[0049] This comparative example provides a phenol red-containing trypsin solution, whose raw materials and steps are basically the same as those in Example 1, except that: the rotational speed of the ball mill is 130 rpm, and the rest remain unchanged.

[0050] Comparative Example 7

[0051] This comparative example provides a phenol red-containing trypsin solution, whose raw materials and steps are basically the same as those in Example 1, except that: the grinding time of the ball mill is 20 min, and the rest remain unchanged.

[0052] Furthermore, in order to understand the physical properties of the phenol red-containing trypsin solutions prepared in the above examples and comparative examples, the following tests were also carried out.

[0053] Sieving rate detection: Take 100 g of the ground powder, sieve it using a 100-mesh sieve, collect the sieved powder, weigh it, and record its mass as X. Among them, the sieving rate = X÷100×100%.

[0054] Turbidity test: Take the liquid sample to be tested, pour it into a turbidity colorimetric flask until the volume reaches the standard calibration line of the colorimetric flask, put the colorimetric flask containing the liquid to be tested into a turbidimeter, and take the reading. Among them, the model of the turbidimeter used is "Thermo ScientificTM OrionTM AQUAfast AQ3010 turbidimeter".

[0055] Appearance detection: Freeze each phenol red trypsin solution at -20°C for 24 hours, then take it out and observe the appearance of the product. See Figure 1 。

[0056] The specific test results are shown in Table 2.

[0057] Table 2 Relevant parameters of each phenol red trypsin solution

[0058]

[0059]

[0060] It can be seen from Table 2 that: in Examples 1 and 2, the 100-mesh sieving rate of the buffer component powder can reach 95%, and the turbidity can reach below 1.0, which means that the particle size of the material before dissolution is uniform, the particle size is small, the dissolution speed is faster, and it is easier to form a uniform solution during the dissolution stirring process; therefore, even when frozen at -20°C for 24 hours, it still shows a uniform light yellow color. For Comparative Example 1 with a conventional configuration, its turbidity is relatively high (>2.5). Compared with Examples 1 and 2, the solution formed in the comparative example is not dissolved thoroughly enough. In Comparative Examples 2-7, due to changing parameters such as the type of grinding beads, the ratio of grinding beads, the grinding speed, and the grinding time, the 100-mesh sieving rate and the solution turbidity indexes cannot meet the requirements, resulting in the color changing from light yellow to yellowish brown and uneven after being frozen at -20°C for 24 hours. It can be seen that in the present invention, the grinding conditions have a significant relationship with the performance of the final product, and are caused by multiple factors during grinding.

[0061] Furthermore, in order to understand the cell digestion performance of the prepared phenol red trypsin solution, the following tests were also carried out.

[0062] The well-conditioned Hacat and SV-HUC-1 cells (both purchased from Wuhan Pusai Biotechnology Co., Ltd.) were seeded in 6-well plates. When the cells grew to about 80%, they were digested with the solutions of Example 1 and Comparative Example 1, and the digestion time was recorded. Then they were subcultured, and the subculture was repeated 5 times. The growth state of the cells was photographed and recorded. The results are shown in Figure 2 and 3 .

[0063] It can be seen from Figure 2 and 3 that there is no obvious difference between the cells treated by the above two methods. Most cells became round after 5 minutes, and basically all became round and could be digested smoothly after 7 minutes. There was no obvious change in morphology and state after continuous subculture for 3 to 5 generations. Therefore, the improvement of the preparation process of the present invention has no negative impact on the performance of the product (the state of the cells after digestion and subculture is not affected), and at the same time, it well solves the problems of abnormal color and uneven color distribution after freezing of the phenol red-containing trypsin solution (PBS buffer system).

[0064] In the present invention, the specific raw materials not described are existing substances and can be directly purchased from the market.

[0065] The above is only the preferred implementation of the present invention and is not used to limit the protection scope of the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing a phenol red-containing trypsin solution with uniform color distribution after cryopreservation, characterized in that, It includes the following steps: Weigh buffer components and phenol red, mix them, and ball-mill to obtain pretreated buffer component powder; weigh the pretreated buffer component powder, trypsin powder, and ultrapure water, mix them, stir, and perform sterile filtration to obtain the phenol red-containing trypsin solution. Among them, the sieving rate of the pretreated buffer component powder through a 100-mesh sieve is ≥95%. Three types of grinding beads, small, medium, and large, are added during the ball-milling. The number ratio of the small, medium, and large grinding beads is (120 - 140):(20 - 25):(6 - 9). The total volume ratio of the grinding beads to the total volume of the buffer components and phenol red is (0.5 - 3.0):

1. The ball-milling speed is 70 - 110 rpm, and the ball-milling time is ≥60 min. The diameter ratio of the small, medium, and large grinding beads is 1:2:

3. The diameters of the small, medium, and large grinding beads are 0.5 cm, 1.0 cm, and 1.5 cm respectively.

2. The preparation method according to claim 1, characterized in that, The number ratio of the small, medium, and large grinding beads is 120:25:

6. The total volume ratio of the grinding beads to the total volume of the buffer components and phenol red is 1:

1. The ball-milling speed is 110 rpm, and the ball-milling time is 1 h.

3. The preparation method according to claim 1, wherein The number ratio of the small, medium, and large grinding beads is 140:20:

9. The total volume ratio of the grinding beads to the total volume of the buffer components and phenol red is 2:

1. The ball-milling speed is 80 rpm, and the ball-milling time is 1 h.

4. The preparation method according to claim 1, characterized in that, The stirring speed is 300 rpm, and the stirring time is 15 min.

5. The preparation method according to claim 1, characterized in that The pore size of the sterile filtration is ≤0.22 µm.

6. The preparation method according to any one of claims 1-5, characterized in that, When the trypsin solution prepared by the method is stored at -20°C, it shows a uniform light yellow color.

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