Method for storing wet paste strain and mold for manufacturing wet paste strain storage equipment
By using a wet paste bacterial strain preservation equipment made of crisp shells and molds, the problems of high storage temperature and equipment requirements in the existing technology are solved, and good preservation effect under mild conditions and convenient bacterial strain use are achieved, which simplifies the The recovery process.
Patent Information
- Application Number
- CN202411360838.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-27
- Publication Date
- 2025-01-03
AI Technical Summary
The existing bacterial strain preservation methods have high requirements for storage temperature and equipment, and the resuscitation process during use of bacterial strains is complicated, which affects convenience.
Use a crispy shell to fill wet paste bacteria and encapsulate it through a packaging bag with vacuum or filled with protective gas, reducing the requirements for ultra-low temperature storage temperature, and making wet paste bacteria storage equipment with good sealability through molds to simplify the use of bacteria process.
A good bacterial preservation effect is achieved under the conditions of -10℃~35℃, which simplifies the use of bacterial preservation process, improves convenience, and extends the life of the storage equipment.
Smart Images
Figure CN119242447A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of bacterial strain preservation methods, and in particular relates to a method for preserving wet paste bacterial strains and a mold for making wet paste bacterial strain preservation equipment. Background Art
[0002] Strain preservation technology is a technology that maintains the vitality and genetic traits of microbial strains. Its purpose is to keep the wild type isolated from nature or the mutant pure strain obtained through artificial selection alive, without loss, contamination of foreign bacteria, no or less mutation, and maintain the original characteristics and physiological activity of the strain. The basic principle of strain preservation is to keep the life activities of microorganisms in a semi-permanent dormant state, that is, to limit the metabolism of microorganisms to the lowest range. Dryness, low temperature and air isolation are the main measures to ensure this state. Targeted creation of dryness, low temperature and air isolation external conditions is the key to long-term preservation of microbial strains.
[0003] A Chinese utility model patent with an authorization announcement date of April 26, 2024 and an authorization announcement number of CN 220843905 U discloses a storage tank for maintaining the activity of liquid microorganisms. The storage tank is provided with a strain retrieval port and a liquid nitrogen injection port. When in use, the strain is first loaded into the storage tube, and then the storage tube is placed in the storage tank, and then liquid nitrogen is injected through the liquid nitrogen injection port to lower the internal temperature of the storage tank, thereby achieving ultra-low temperature storage of the strain.
[0004] The long-term preservation of existing strains generally relies on ultra-low temperature equipment such as liquid nitrogen storage tanks, which has high requirements for storage temperature and equipment. When the strains are used, the strains stored under ultra-low temperature conditions need to be revived to meet subsequent production and experimental needs, and the use process is relatively complicated. Summary of the invention
[0005] The purpose of the present invention is to provide a method for preserving wet paste-like bacterial strains, so as to solve the problems that the existing method has high requirements on the preservation temperature and preservation equipment and is inconvenient to use the bacterial strains.
[0006] The second object of the present invention is to provide a mold for making a wet paste-like bacteria preservation device, so as to obtain a wet paste-like bacteria preservation device that is well sealed, convenient and practical.
[0007] In order to achieve the above first purpose, the technical solution adopted by the present invention is:
[0008] A method for preserving wet paste bacteria comprises the following steps: placing the wet paste bacteria in a sealed crisp shell, then sealing the crisp shell containing the wet paste bacteria in a packaging bag, and filling the packaging bag with protective gas or keeping the packaging bag in vacuum.
[0009] The present invention is a pioneering invention, which uses a crispy shell to fill the wet paste strain and seal it for storage, and seals the crispy shell with a vacuum or protective gas-filled packaging bag, thereby creating a dry and air-isolated condition suitable for strain storage, and can reduce the requirement for ultra-low temperature storage temperature, and can achieve a good storage effect under the condition of -10°C to 35°C. In addition, since ultra-low temperature storage is not required, the strain recovery process can be omitted or simplified when the strain is used, thereby improving the convenience of strain use.
[0010] Preferably, the crispy shell is formed by a female half shell and a male half shell, and a wet paste bacteria filling hole is provided on the female half shell or the male half shell; the female half shell and the male half shell joint position and the filling hole position after the wet paste bacteria are filled are sealed with sealing materials.
[0011] Further preferably, the crispy shell is made of a water-soluble material; when the bacteria are used, the crispy shell containing the wet paste bacteria is placed in water or a water-containing matrix, and the water-soluble material dissolves in water to release the bacteria.
[0012] Further preferably, the crispy crust is mainly made by mixing flour, eggs, vegetable oil and milk in a mass ratio of 1-2:2-3:0.5-1:0.5-1 and then baking; and the sealing material is selected from cocoa butter or cocoa butter substitute.
[0013] Further preferably, the female half shell and the male half shell are both hemispherical.
[0014] Preferably, the inner wall of the crispy shell is also coated with animal fat. Applying animal fats such as lard and butter on the inner wall of the crispy shell can increase the life of the strain storage device (i.e. the crispy shell) and reduce the water loss of the wet paste strain.
[0015] Preferably, the packaging bag is sealed with the crispy shell containing the wet paste-like bacteria and then stored at -10°C to 35°C.
[0016] In order to achieve the above second purpose, the technical solution adopted by the present invention is:
[0017] A mold for making a wet paste-like culture preservation device comprises a mother mold, a male half-shell mold and a female half-shell mold, wherein the mother mold is provided with a shell molding pit, and the male half-shell mold and the female half-shell mold both have a shell molding part corresponding to the shell molding pit, and the shell molding part is inserted into the shell molding pit during molding and has a gap with the shell molding pit, and the gap forms a molding cavity for molding the male half-shell or the female half-shell; the mother mold is provided with a limiting structure for limiting the male half-shell mold and the female half-shell mold in a molding position; the male half-shell mold or the female half-shell mold is also provided with an opening structure for molding a filling hole.
[0018] The mold for making wet paste-like bacteria preservation equipment of the present invention can conveniently produce a female half shell and a male half shell, which can be assembled into a preservation equipment suitable for wet paste-like bacteria preservation after being sealed and matched, thereby reducing the requirements of the bacteria on the preservation temperature and preservation equipment, and facilitating the reuse of the bacteria.
[0019] Preferably, the limiting structure includes a limiting cavity connected to the upper end of the shell forming pit, the inner diameter of the limiting cavity is larger than the inner diameter of the shell forming pit, the shell forming pit is located on the inner side of the limiting cavity, and a limiting step is formed at the junction of the shell forming pit and the limiting cavity; the male half-shell sub-mold and the female half-shell sub-mold are both provided with limiting surfaces that cooperate with the limiting step.
[0020] Further preferably, the shell molding pit is hemispherical, and the limiting cavity is cylindrical; the male half-shell mold and the female half-shell mold both include a hemispherical shell molding part and a cylindrical guide part connected to the upper end of the hemispherical shell molding part, the cylindrical guide part is adapted to the limiting cavity, the cylindrical guide part has an annular end face surrounding the hemispherical shell molding part, and the annular end face constitutes the limiting surface; the opening structure includes a cylindrical protrusion arranged on the hemispherical shell molding part, the cylindrical protrusion contacts with the corresponding shell molding pit during molding, and a wet paste-like culture filling hole is formed at the position of the cylindrical protrusion after molding. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Figure 1 The structure of the male half shell and the female half shell made by the mold is shown in FIG.
[0022] Figure 2 This is a schematic diagram of the structure of a baking tray mold for making a wet paste-like bacteria preservation device according to Example 1 of the present invention;
[0023] Figure 3 This is a schematic diagram of the structure of the male half shell loading mold for making a wet paste-like bacteria preservation device according to Example 1 of the present invention;
[0024] Figure 4 This is a schematic diagram of the structure of the female half-shell loading mold for making a wet paste-like bacteria preservation device according to Example 1 of the present invention;
[0025] Among them, 1-male half shell, 2-male half shell sealing end face, 3-female half shell, 4-filling hole, 5-female half shell sealing end face; 6-baking tray mold, 7-half shell mold pit, 8-mold pit hemispherical section, 9-limiting platform, 10-mold pit cylindrical limiting section; 11-male half shell load mold, 12-male half shell load hemispherical section, 13-male half shell load cylindrical section, 14-male half shell load handle; 15-female half shell load mold, 16-filling hole opening cylinder, 17-female half shell load hemispherical section, 18-female half shell load cylindrical section, 19-female half shell load handle. DETAILED DESCRIPTION
[0026] The present invention proposes a method for preserving wet paste strains and a mold for making wet paste strain preservation equipment, wherein the wet paste strains are water-rich paste strains obtained by removing free water from the culture solution by methods such as centrifugation, filtration, and natural sedimentation. The wet paste strains are highly active and can be used directly without resuscitation. However, the wet paste strains are rich in water and difficult to preserve. The storage time at room temperature is extremely short, generally not exceeding 48 hours. In order to extend the storage time, they need to be frozen in a sterile environment below -20°C. The storage time is generally no more than 6 months. At the same time, they need to be thawed and resuscitated before use.
[0027] The technical concept of the present invention is to place the wet paste-like bacteria in an independent and isolated external environment, which is beneficial to maintaining its activity and avoiding contamination by foreign bacteria, thereby reducing the dependence of the bacteria preservation on the ultra-low temperature environment. When the bacteria are used again, the outer packaging can be removed and the wet paste-like bacteria together with its preservation shell can be directly dissolved in water, and the bacteria can be directly released for use without even the need for resuscitation.
[0028] The present invention uses a female half shell and a male half shell to assemble into a crispy shell as a preservation device for wet paste bacteria, uses cocoa butter or cocoa butter substitute to connect the sealing end surface of the male half shell and the sealing end surface of the female half shell to form a wet paste bacteria preservation container, the wet paste bacteria are injected into the container through a filling hole, and then the filling hole is blocked with cocoa butter or cocoa butter substitute, thereby ensuring the sealing of the preservation device, and the manufacturing process is simple and convenient, and the relevant raw materials are harmless to the bacteria, and the bacteria can also be used as nutrients when used.
[0029] Furthermore, brushing animal fat on the inner wall of the crispy shell can further increase the life of the bacteria preservation device and reduce the water loss of the wet paste bacteria.
[0030] The implementation process of the present invention is described in detail below in conjunction with specific embodiments.
[0031] 1. Specific embodiment of the mold for making wet paste-like bacteria preservation equipment of the present invention
[0032] Example 1
[0033] The mold for making the wet paste-like bacteria preservation device in this embodiment is mainly to ensure the good matching and assembly of the positive and negative hemispherical shells by constructing precise positive and negative hemispherical shells, thereby creating good conditions for forming a bacteria preservation device with good sealing performance. Figure 1 As shown, the male half shell 1 and the female half shell 3 are hollow hemispherical, the female half shell 3 has a filling hole 4 at the top, the large end surface of the male half shell 1 forms a male half shell sealing end surface 2, and the large end surface of the female half shell 3 forms a female half shell sealing end surface 5. The male half shell 1 and the female half shell 3 can be assembled into a complete sphere after being matched.
[0034] Figure 2 and Figure 3 In the figure, the baking tray mold 6 (i.e., the mother mold) and the male half-shell load mold 11 (i.e., the male half-shell sub-mold) form the male half-shell 1, and the baking tray mold 6 and the female half-shell load mold 15 (i.e., the female half-shell sub-mold) form the female half-shell 3. In other embodiments, the male half-shell 1 and the female half-shell 3 can also be square, cylindrical, conical or other structures.
[0035] Figure 2 The baking tray mold 6 is a constant temperature baking tray made of cast iron and can be used for crispy baking. A half-shell mold pit 7 is provided on the baking tray mold 6. The half-shell mold pit 7 is divided into two sections. The lower part is a mold pit hemispherical section 8 (i.e., a shell forming pit), and the upper part is a mold pit cylindrical limiting section 10 (i.e., a limiting cavity). The diameter of the mold pit cylindrical limiting section 10 is larger than the diameter of the mold pit hemispherical section 8. The junction of the mold pit hemispherical section 8 and the mold pit cylindrical limiting section 10 forms an annular limiting platform 9.
[0036] Figure 3 The male half shell load mold 11 includes three parts. The lower part is the male half shell load hemispherical section 12. The hemispherical diameter of the male half shell load hemispherical section 12 is smaller than the hemispherical diameter of the mold pit hemispherical section 8. The difference between the two is the wall thickness of the male half shell 1; the middle part is the male half shell load cylindrical section 13. The male half shell load cylindrical section 13 can be tightly inserted into the cylindrical limiting section 10 of the mold pit. The cylindrical diameters of the two are equivalent. The connecting platform between the male half shell load cylindrical section 13 and the male half shell load hemispherical section 12 fits tightly with the annular limiting platform 9; the upper part is the male half shell load handle 14, which can be cylindrical or in other shapes, so as to facilitate the use of the male half shell load mold 11.
[0037] Figure 4The female half shell load mold 15 includes four parts, the bottom of which is a filling hole opening cylinder 16, the diameter of the filling hole opening cylinder 16 is 2 to 10 mm, and the height is equivalent to the thickness of the female half shell 3. Connected to the filling hole opening cylinder 16 is the female half shell load hemispherical segment 17, which is a spherical body that matches the spherical surface of the mold pit hemispherical segment 8. The hemispherical diameter of the female half shell load hemispherical section 17 is the same as the hemispherical diameter of the male half shell load hemispherical section 12, and both are smaller than the hemispherical diameter of the mold pit hemispherical section 8. The difference between the hemispherical diameters of the female half shell load hemispherical section 17 and the mold pit hemispherical section 8 is the wall thickness of the female half shell 3; above the female half shell load hemispherical section 17 is the female half shell load cylindrical section 18, and the cylindrical diameter of the female half shell load cylindrical section 18 is the same as that of the male half shell load cylindrical section 13, and both can be tightly inserted into the mold pit cylindrical limiting section 10, and the connecting platform between the female half shell load cylindrical section 18 and the female half shell load hemispherical section 17 is tightly fitted with the circular limiting platform 9; the uppermost part is the female half shell load handle 19, which can be cylindrical or in other shapes, so as to facilitate the use of the male half shell load mold 15.
[0038] The baking tray mold 6 is combined with the male half-shell load mold 11, and an appropriate amount of crispy slurry is filled into the mold pit hemispherical section 8. The male half-shell load cylindrical section 13 is tightly inserted into the mold pit cylindrical limit section 10 and stably stays on the limit platform 9. The male half-shell load mold 11 plays the role of a core. It has a certain weight and can apply a stable load downward by its own weight. The mold pit hemispherical section 8 and the male half-shell load hemispherical section 12 form a stable molding cavity. After the crispy slurry fills the molding cavity formed by the half-shell mold pit hemispherical section 8 and the male half-shell load hemispherical section 12, it is directly baked in the oven to complete the molding process, thereby ensuring the formation of a high-quality male half-shell 1 with the expected shape.
[0039] The baking tray mold 6 is combined with the female half-shell load mold 15, and an appropriate amount of crispy slurry is filled into the mold pit hemispherical section 8. The female half-shell load cylindrical section 18 is tightly inserted into the mold pit cylindrical limit section 10 and stably stays on the limit platform 9. The female half-shell load mold 15 plays the role of a core. It has a certain weight and can apply a stable load downward by its own weight. The mold pit hemispherical section 8 and the female half-shell load hemispherical section 17 form a stable molding cavity. After the crispy slurry fills the molding cavity formed by the half-shell mold pit hemispherical section 8 and the female half-shell load hemispherical section 17 by itself, it is directly baked in the oven to complete the molding process, thereby ensuring the formation of a high-quality female half-shell 3 with the expected shape.
[0040] In a specific implementation, the diameter of the mold pit cylindrical limiting section 10 is 1 to 20 mm larger than the diameter of the mold pit hemispherical section 8, the height of the mold pit cylindrical limiting section 10 is 5 to 50 mm, and the hemispherical diameter of the male half shell load hemispherical section 12 is 2 to 25 mm smaller than the hemispherical diameter of the mold pit hemispherical section 8. The diameter of the male half shell 1 thus formed can be 2 to 25 mm.
[0041] In other embodiments, the limiting structure may take other forms. For example, the half-shell mold pit 7 only retains the molding section, and an annular protrusion is set around the half-shell mold pit 7, or corresponding protrusions are set around the half-shell mold pit 7, and guide grooves are set on the protrusions or protrusions. The male half-shell load mold 11 is added with corresponding guide columns on the basis of retaining only the solid hemisphere, and the solid hemisphere of the male half-shell load mold 11 is limited to the molding position through the cooperation of the guide columns and the guide grooves.
[0042] The difference between the female half shell loading mold 15 and the male half shell loading mold 11 is the filling hole opening cylinder 16, that is, the filling hole 4 of the female half shell 3 can be formed through the filling hole opening cylinder 16, and the wet paste bacteria can be added into the final spherical crispy hollow shell through the filling hole 4. Figure 4 As shown, a filling hole opening cylinder 16 is provided at the top of the female half shell load hemispherical section 17, and the filling hole opening cylinder 16 is in close contact with the pit wall surface of the mold pit hemispherical section 8 during molding, and finally forms a filling hole 4. The height of the filling hole 4 is consistent with the wall thickness of the female and male half shells.
[0043] like Figure 1 As shown, the male half shell 1 and the female half shell 3 are of equal shape and size, and a filling port is provided on the female half shell 3. The male half shell sealing end face 2 of the male half shell 1 and the female half shell sealing end face 5 of the female half shell 3 can overlap seamlessly. By applying melted cocoa butter or cocoa butter substitute on the male half shell sealing end face 2 and the female half shell sealing end face 5, the male half shell 1 and the female half shell 3 can be connected together to form a hollow spherical device with a filling hole 4. The wet paste bacteria can enter the internal space of the spherical device through the filling hole 4, and the wet paste bacteria can occupy part or all of the internal space. After the filling is completed, the filling hole 4 can be sealed with melted cocoa butter or cocoa butter substitute, thereby completing the sealing of the crispy shell after the wet paste bacteria are filled.
[0044] The molding process of the mold for making wet paste-like bacteria preservation equipment of the present invention is as follows: flour, eggs, vegetable oil, milk, etc. are made into crispy slurry, and then the crispy slurry is added to each half-shell mold pit 7, and the added volume of the crispy slurry is controlled to be 1.05 to 1.15 times the volume of the molding cavity. After the male half-shell load mold 11 or the female half-shell load mold 15 is embedded in the half-shell mold pit 7, the crispy slurry fills the molding cavity by itself, and then the whole is placed in an oven for baking to obtain the yin and yang crispy hemispheres. After trimming the two yin and yang crispy hemispheres, melted cocoa butter or cocoa butter substitute is used to connect the yin and yang hemispheres together to form a hollow spherical equipment with a filling hole 4.
[0045] 2. Specific embodiments of the method for preserving wet paste strains of the present invention
[0046] Example 2
[0047] The method for preserving wet paste-like bacteria in this embodiment comprises the following steps:
[0048] (1) After the female half shell and the male half shell are made by using the mold of the above embodiment 1, melted cocoa butter or cocoa butter substitute is applied to the sealing end surfaces of the female half shell and the male half shell, and a hollow spherical device is obtained after solidification. The wet paste-like bacteria are introduced into the hollow spherical device from the filling port, and after it is filled, the opening is sealed with melted cocoa butter or cocoa butter substitute.
[0049] The raw materials of the crispy slurry used for the female half shell and the male half shell are flour, eggs, vegetable oil, and milk. The mass ratio of flour, eggs, vegetable oil, and milk is 2:3:1:1. The raw materials are evenly mixed and stirred to emulsify to obtain the crispy slurry. The crispy slurry is baked in an oven at 125-180° C. for 0.2-1.5 hours to obtain a crispy shell. In other implementation situations, additives such as butter and sugar can be further added. The mass of the additives is more than 2-20% of the total mass of the crispy slurry. After adding the above additives, the prepared strain preservation equipment can have better moisture retention and is beneficial to the strain activity.
[0050] In this embodiment, the wet paste bacteria are composed of bacteria, water, and a small amount of bacteria nutrients. In other implementation situations, the wet paste bacteria suitable for the present invention can also be fungi, etc.
[0051] (2) The sealed spherical device is individually packaged in an independent sealed plastic bag, filled with nitrogen, and then stored at -10℃ to 35℃.
[0052] In other implementations of this embodiment, an aluminum foil packaging bag may be used for packaging, and the bag may be filled with nitrogen. A vacuuming method may also be used to keep the bag in a vacuum negative pressure state.
[0053] Example 3
[0054] The method for preserving wet paste-like bacteria in this embodiment is different from that in Embodiment 2 in that animal fats such as lard and butter are applied to the inner wall of the hollow spherical device. Specifically, after melting animal fats such as lard and butter, they are evenly applied to the inner walls of the yin and yang hemispheres, which can increase the life of the bacteria preservation device and reduce the water loss of the wet paste-like bacteria.
[0055] After the wet paste bacteria of this embodiment is stored at 25°C for 180 days, the bacteria activity is still maintained. When in use, the packaging bag is opened and the spherical device is directly placed in water, and the spherical device dissolves in water to release the bacteria. The entire bacteria storage process has low requirements on environmental conditions and is easy to implement, and the bacteria use process is simpler and more convenient.
Claims
1. A method for preserving wet paste-like bacterial strains, characterized in that: The following steps are involved: The wet paste-like spawn is placed in a sealed crisp shell, and then the crisp shell containing the wet paste-like spawn is sealed in a packaging bag, and the packaging bag is filled with protective gas or maintained in a vacuum.
2. The method for preserving wet paste-like bacteria according to claim 1, characterized in that: The crisp shell is formed by a female half shell and a male half shell, and a wet paste bacteria filling hole is provided on the female half shell or the male half shell; the female half shell and the male half shell are in contact with each other, and the filling hole position after the wet paste bacteria is filled is sealed with a sealing material.
3. The method for preserving wet paste-like bacteria as claimed in claim 2, characterized in that: The crisp shell is made of water-soluble materials. When the bacteria are used, the crisp shell containing the wet paste bacteria is placed in water or a water-containing matrix, and the water-soluble materials dissolve in water to release the bacteria.
4. The method for preserving wet paste-like bacteria according to claim 2 or 3, characterized in that: The crispy crust is mainly made by mixing flour, eggs, vegetable oil and milk in a mass ratio of 1-2:2-3:0.5-1:0.5-1 and then baking; the sealing material is selected from cocoa butter or cocoa butter substitute.
5. The method for preserving wet paste-like bacteria as claimed in claim 2, characterized in that: The female half shell and the male half shell are both hemispherical.
6. The method for preserving wet paste-like bacteria according to claim 1, 2 or 3, characterized in that: The inner wall of the crispy shell is also coated with animal fat.
7. The method for preserving wet paste-like bacteria as claimed in claim 1, characterized in that: After the packaging bag seals the crispy shell containing the wet paste-like bacteria, it is stored at -10°C to 35°C.
8. A mold for making a wet paste-like bacterial culture preservation device, characterized in that: It includes a mother mold, a male half-shell mold and a female half-shell mold, wherein the mother mold is provided with a shell molding pit, and the male half-shell mold and the female half-shell mold both have a shell molding part corresponding to the shell molding pit, and the shell molding part is inserted into the shell molding pit during molding and has a gap with the shell molding pit, and the gap forms a molding cavity for molding the male half-shell or the female half-shell; the mother mold is provided with a limiting structure for limiting the male half-shell mold and the female half-shell mold in the molding position; the male half-shell mold or the female half-shell mold is also provided with an opening structure for molding a filling hole.
9. The mold for making a wet paste-like bacteria preservation device according to claim 8, characterized in that: The limiting structure includes a limiting cavity connected to the upper end of the shell forming pit, the inner diameter of the limiting cavity is larger than the inner diameter of the shell forming pit, the shell forming pit is located on the inner side of the limiting cavity, and a limiting step is formed at the junction of the shell forming pit and the limiting cavity; the male half-shell mold and the female half-shell mold are both provided with limiting surfaces that cooperate with the limiting step.
10. The mold for making a wet paste-like bacteria preservation device according to claim 9, characterized in that: The shell forming pit is hemispherical, and the limiting cavity is cylindrical; the male half shell sub-mold and the female half shell sub-mold each include a hemispherical shell forming portion and a cylindrical guide portion connected to the upper end of the hemispherical shell forming portion, the cylindrical guide portion is adapted to the limiting cavity, and the cylindrical guide portion has an annular end surface surrounding the hemispherical shell forming portion, and the annular end surface constitutes the limiting surface; The opening structure includes a cylindrical protrusion arranged on the hemispherical shell forming part, and the cylindrical protrusion contacts the corresponding shell forming pit during molding, and a wet paste bacteria filling hole is formed at the position of the cylindrical protrusion after molding.