CN111979122B - Ultrasonic crusher with tooth-shaped structure - Google Patents
Ultrasonic crusher with tooth-shaped structure Download PDFInfo
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- CN111979122B CN111979122B CN202010826485.5A CN202010826485A CN111979122B CN 111979122 B CN111979122 B CN 111979122B CN 202010826485 A CN202010826485 A CN 202010826485A CN 111979122 B CN111979122 B CN 111979122B
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- 238000012545 processing Methods 0.000 claims abstract description 30
- 238000012360 testing method Methods 0.000 claims abstract description 23
- 239000007788 liquid Substances 0.000 claims abstract description 12
- 239000012141 concentrate Substances 0.000 claims abstract description 4
- 230000005855 radiation Effects 0.000 claims description 4
- 230000000694 effects Effects 0.000 abstract description 13
- 210000003781 tooth socket Anatomy 0.000 abstract description 12
- 239000002699 waste material Substances 0.000 abstract description 3
- 239000000523 sample Substances 0.000 description 47
- 239000012472 biological sample Substances 0.000 description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 239000000498 cooling water Substances 0.000 description 4
- 238000000034 method Methods 0.000 description 4
- 238000002525 ultrasonication Methods 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 238000002604 ultrasonography Methods 0.000 description 2
- 235000019047 Crassocephalum crepidioides Nutrition 0.000 description 1
- 244000287353 Crassocephalum crepidioides Species 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 238000004891 communication Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 230000000875 corresponding effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 230000001066 destructive effect Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
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- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
- C12M35/04—Mechanical means, e.g. sonic waves, stretching forces, pressure or shear stimuli
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M45/00—Means for pre-treatment of biological substances
- C12M45/02—Means for pre-treatment of biological substances by mechanical forces; Stirring; Trituration; Comminuting
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Abstract
The invention discloses an ultrasonic crusher with a tooth-shaped structure, which comprises an ultrasonic transducer (1), a tooth-shaped energy concentrator (2) and a sample processing tank (3), wherein the ultrasonic transducer (1) is arranged at the lower part of the sample processing tank (3), and the tooth-shaped energy concentrator (2) is arranged inside the sample processing tank (3) so as to concentrate sound energy. The tooth-shaped energy concentrator (2) comprises a plate-shaped part and a tooth-shaped part, the bottom surface of the plate-shaped part is fixedly connected with the inner bottom surface of the sample processing groove (3), the number of teeth of the tooth-shaped part is two or the tooth-shaped part is a tooth-shaped array consisting of more than two teeth, the cross section of each tooth is triangular, rectangular or trapezoidal, and a chamfer or fillet structure is arranged on each tooth. The ultrasonic crusher with the tooth-shaped structure provided by the invention utilizes the reflection of sound waves between the test tube support (or the liquid level) and the tooth-shaped energy concentrator and the energy-collecting effect of the tooth-shaped structure, improves the local sound intensity of the tooth socket position, reduces the waste of sound energy and improves the utilization rate.
Description
Technical Field
The invention relates to the technical field of biological sample processing devices, in particular to a non-contact type tooth-shaped structure ultrasonic crusher.
Background
Under normal temperature and normal pressure, ultrasonic waves emitted by the ultrasonic transducer are transmitted in liquid, and when the amplitude of the sound waves is larger than a cavitation threshold value, negative pressure is formed in the liquid, so that acoustic cavitation is excited. When acoustic cavitation occurs, a large number of bubbles vibrate and collapse in the liquid: micro-flow and mechanical friction are generated around the cavitation bubbles when the cavitation bubbles vibrate, micro-jet flow and shock wave equivalent effects are generated when the cavitation bubbles collapse, and the effects have certain destructive effects on biological particles, so that the cavitation bubbles can be used for crushing biological samples.
Two common approaches to ultrasonication are: one is contact ultrasonic crushing, in which an ultrasonic transducer with an amplitude transformer is inserted into a sample container for crushing. Another is non-contact ultrasonication: and arranging an ultrasonic transducer at the bottom of the water tank, and putting the test tube containing the sample in the ultrasonic water tank for sample crushing. The non-contact ultrasonication has the advantages that multiple samples can be processed simultaneously, and the test tube can be sealed in the processing process, so that the pollution of the samples is avoided.
Generally, the capacity and the sound intensity of ultrasonic crushing are related to the frequency, and the effect of ultrasonic crushing can be directly improved by increasing the sound intensity of the ultrasonic. However, for the conventional ultrasonic crusher, most of the sound energy is distributed in the whole container, and if the local sound intensity at the crushing position is increased, the sound intensity in the whole container needs to be increased, so that the waste of sound energy is caused, and the loss of equipment is increased.
Therefore, it is necessary to provide an ultrasonograph which can efficiently collect sound waves, improve the utilization efficiency of sound energy, and have a good ultrasonograph effect.
Disclosure of Invention
In order to overcome the above problems, the present inventors have made intensive studies to design an ultrasonicator having a tooth-shaped structure, which improves the local sound intensity at a sample position by using the reflection of ultrasonic waves between a test tube holder and the tooth-shaped structure and the energy-collecting effect of the tooth-shaped structure, and realizes ultrasonication with high sound energy utilization, thereby completing the present invention.
In particular, the invention aims to provide an ultrasonic crusher with a tooth-shaped structure, wherein the ultrasonic crusher comprises an ultrasonic transducer 1, a tooth-shaped energy concentrator 2 and a sample processing groove 3,
wherein the ultrasonic transducer 1 is arranged at the lower part of the sample processing groove 3,
a toothed concentrator 2 is provided inside the sample processing well 3 to concentrate the acoustic energy.
Wherein, the ultrasonic transducer 1 is one or more, and the radiation surface thereof is fixedly connected with the bottom surface of the sample processing groove 2.
Wherein the toothed concentrator 2 comprises a plate-shaped portion and a toothed portion,
the bottom surface of the plate-shaped part is fixedly connected with the inner bottom surface of the sample processing groove 3, and the number of the teeth-shaped parts is multiple.
The number of teeth of the tooth-shaped part is two or the tooth-shaped part is a tooth-shaped array consisting of more than two teeth.
The tooth of the tooth-shaped part is triangular, rectangular or trapezoidal in cross section, and a chamfer or fillet structure is arranged on the tooth.
When the tooth-shaped part is in a tooth-shaped array structure consisting of more than two teeth, the cross sections of the teeth are the same.
Wherein, the upper part of the tooth-shaped energy concentrator 2 and the inside of the sample processing groove 3 are also provided with a test tube bracket 4 for fixing the sample containing tube 5 and reflecting the ultrasonic waves emitted from the upper surface of the tooth-shaped energy concentrator 2.
Wherein the test tube holder 4 comprises a vertical plate and a horizontal plate, both having sound reflection capability;
an opening is arranged on the horizontal plate to place the sample containing tube 5.
Wherein, a gap is arranged between the bottom of the sample containing pipe 5 and the bottom of the tooth groove.
Wherein, the sample in the sample containing pipe 5 is liquid, and the height of the liquid level is not higher than the vertical height of the tooth socket.
The invention has the advantages that:
(1) the ultrasonic crusher with the tooth-shaped structure provided by the invention is simple to operate, wide in application range and strong in universality;
(2) according to the ultrasonic crusher with the tooth-shaped structure, the reflection of sound waves between the test tube support and the tooth-shaped energy concentrator and the energy concentrating effect of the tooth-shaped structure are utilized, the local sound intensity of the tooth socket position is improved, the waste of sound energy is reduced, and the utilization rate is improved;
(3) according to the ultrasonic crusher with the tooth-shaped structure, the sound flow generated in the upper area of the tooth socket can play a role in stirring liquid in the sample containing pipe, and the crushing uniformity of the sample is improved.
Drawings
Fig. 1 is a schematic view showing the overall structure of a tooth-shaped ultrasonic crusher according to a preferred embodiment of the present invention;
fig. 2 is a schematic sectional view showing a structure of a tooth-shaped ultrasonic crusher according to a preferred embodiment of the present invention.
The reference numbers illustrate:
1-an ultrasound transducer;
2-a toothed concentrator;
3-a sample treatment tank;
4-test tube holder;
5-sample containing tube.
Detailed Description
The present invention will be described in further detail below with reference to the accompanying drawings and embodiments. The features and advantages of the present invention will become more apparent from the description. In which, although various aspects of the embodiments are shown in the drawings, the drawings are not necessarily drawn to scale unless specifically indicated.
The inventor researches and finds that the tooth-shaped structure has better energy gathering effect and can effectively reduce the loss of sound waves, so that the invention provides the ultrasonic crusher with the tooth-shaped structure, the ultrasonic crusher comprises an ultrasonic transducer 1, a tooth-shaped energy gathering device 2 and a sample processing groove 3,
wherein the ultrasonic transducer 1 is arranged at the lower part of the sample processing groove 3,
a toothed concentrator 2 is provided inside the sample processing well 3 to concentrate the acoustic energy, as shown in fig. 1.
In the present invention, the ultrasonic crusher having the tooth-shaped structure is suitable for processing biological samples, such as cells, biological tissues, nucleotide fragments, and the like.
According to a preferred embodiment of the present invention, the radiation surface of the ultrasonic transducer 1 is fixedly connected to the bottom surface of the sample processing well 2, and the fixed connection may be a connection commonly used in the art, such as adhesion.
In the present invention, the ultrasonic transducer is used to convert the input electric power into mechanical power, i.e. ultrasonic waves, and then transmit the mechanical power. The ultrasound transducers commonly used in the art may be selected, such as: a sandwich piezoelectric transducer.
The radiation surface of the ultrasonic transducer is fixedly connected with the bottom surface of the sample processing groove, so that the vibration of the ultrasonic transducer excites the vibration of the tooth-shaped energy concentrator in the sample processing groove.
In a further preferred embodiment, the ultrasonic transducers 1 may be one or more, and the models may be the same or different.
The number of the ultrasonic transducers is positively correlated with the working power of the ultrasonic transducers, and the working power is higher when the number is larger. According to the invention, different working powers are set according to different processing targets of the ultrasonic transducer.
In a further preferred embodiment, the operating frequency of the ultrasonic transducer is between 16kHz and 10 MHz.
In the present invention, the operating frequency of the ultrasonic transducer can be selected according to requirements, for example, a frequency of 20kHz to 3MHz can be selected.
According to a preferred embodiment of the invention, the toothed concentrator 2 comprises a plate-shaped portion and a toothed portion, as shown in figure 2,
the bottom surface of the plate-like portion is fixedly connected to the inner bottom surface of the sample treatment well 3 by means of a conventional connection means, such as adhesion.
In a further preferred embodiment, the number of teeth of the tooth-like portion is plural, preferably, the number of teeth is two, or
The tooth-shaped part is a tooth-shaped array formed by more than two teeth.
In the invention, the number of teeth of the tooth-shaped part can be adjusted according to requirements, and tooth grooves are formed among a plurality of teeth.
In a further preferred embodiment, the teeth of the tooth-like portion have a triangular, rectangular or trapezoidal cross-sectional shape.
Preferably, a chamfer or fillet structure is provided on the teeth.
According to a preferred embodiment of the present invention, when the tooth portion has a tooth array structure including two or more teeth, the plurality of teeth have the same cross-sectional shape.
In a further preferred embodiment, when the tooth portion has a tooth array structure of two or more teeth, the pitch between adjacent teeth is the same.
Wherein the pitch between the adjacent teeth refers to a distance between tooth tops of the adjacent teeth.
In the present invention, the vibration of the ultrasonic transducer excites the vibration of the toothed concentrator so that ultrasonic waves are emitted from the upper surface of the concentrator. In order to ensure the emission effect of the ultrasonic waves, the toothed concentrator is preferably arranged directly above the ultrasonic transducer.
Preferably, when the number of the ultrasonic transducers is 1, the ultrasonic transducers are arranged coaxially with the tooth-shaped transducer;
when the ultrasonic transducer is a plurality of ultrasonic transducers, the ultrasonic transducers are uniformly distributed at the lower part of the tooth-shaped transducer.
In the invention, the length of the tooth-shaped energy concentrator (the length of the plate-shaped part), the number and the size of the teeth can be adjusted according to actual requirements, and the number, the size, the arrangement mode, the working frequency and the like of the ultrasonic transducers can also be selected according to actual requirements.
For example: in fig. 1 and 2, the tooth-shaped energy concentrator is provided with four teeth to form three tooth sockets, three rows of samples can be processed simultaneously, and two transducers of the same type are arranged below the tooth-shaped energy concentrator to perform excitation.
According to a preferred embodiment of the present invention, a test tube holder 4 is further provided at the upper portion of the toothed concentrator 2, inside the sample processing bath 3, to fix the sample holding tube 5 and to reflect the ultrasonic waves emitted from the upper surface of the toothed concentrator 2.
Wherein, the test tube bracket can be movably or fixedly arranged in the sample processing groove.
In a further preferred embodiment, the test tube holder 4 comprises a vertical plate and a horizontal plate, both having acoustic reflection capability;
preferably comprising two oppositely disposed vertical plates and a horizontal plate connected between the two vertical plates,
wherein, the vertical plates are arranged at both sides of the tooth-shaped energy concentrator 2 and arranged along the direction of increasing the number of teeth;
the horizontal plate is disposed above the teeth as shown in fig. 2.
Wherein a gap is formed between the vertical plate and the tooth-shaped energy concentrator.
In a further preferred embodiment, openings are provided in the horizontal plate for the placement of sample receiving tubes 5;
preferably, the arrangement of the holes on the horizontal plate is matched with the arrangement of the tooth sockets, so that the test tube 5 is vertically arranged in the tooth sockets, and the corresponding holes above each tooth socket can be one or more, preferably in-line distribution.
In the invention, the ultrasonic waves are emitted from the upper surface of the energy concentrator and are reflected by the horizontal plate and the vertical plate of the test tube support, so that a larger local standing wave sound field is generated in the tooth grooves, the utilization rate of sound energy is improved, and simultaneously, a biological sample is crushed by utilizing the cavitation effect generated by large sound pressure.
According to a preferred embodiment of the present invention, the sample holding tube 5 may be a test tube, a centrifuge tube, or the like, and has a gap between the bottom and the bottom of the tooth socket, wherein the gap is 0mm to 10mm, but not 0 mm.
The present inventors found that the ultrasonic crushing effect is weakened by an excessively large clearance, and therefore the distance between the sample-holding tube and the bottom of the tooth socket is set to the above range.
Wherein, the upper portion of sample splendid attire pipe is fixed on test tube holder, sets up the clearance fit that proper height realized sample splendid attire socle bottom and tooth tank bottom through test tube holder. The sample holding tube and the test tube holder can be matched by the common technology in the field, for example, the sample holding tube adopts a centrifuge tube.
Preferably, the distance between the outer wall of the sample containing tube and the adjacent teeth is 2mm to 10 mm.
In a further preferred embodiment, the sample-containing tube 5 is located on the central plane of the tooth grooves, which is perpendicular to the direction in which the number of teeth increases.
In a further preferred embodiment, the sample in the sample holding tube 5 is liquid, and the liquid level is not higher than the vertical level of the gullet.
According to a preferred embodiment of the present invention, the sample treatment well 3 is further provided with a water inlet end and a water outlet end for inflow and discharge of cooling water;
wherein the cooling water is used for cooling the biological sample to be processed.
According to the ultrasonic crusher with the tooth-shaped structure, the tooth socket position generates larger sound pressure by utilizing the convergence effect of the tooth-shaped energy concentrator on sound waves, the utilization efficiency of sound energy is improved, meanwhile, sound flow generated in the upper area of the tooth socket can play a role in stirring liquid in a test tube, and the uniform degree of sample crushing is improved.
The working process of the ultrasonic crusher with the tooth-shaped structure comprises the following steps: the number, the arrangement mode, the working frequency, the power and the like of the ultrasonic transducers, the number, the size, the shape and the like of teeth of the tooth-shaped energy concentrator are set according to actual requirements; then, a proper amount of biological samples to be processed are placed in the sample containing tube and fixed in the test tube support, so that the samples are positioned in the grooves, the ultrasonic transducer is started to carry out ultrasonic crushing work, the vibration of the ultrasonic transducer excites the vibration of the tooth-shaped energy concentrator, ultrasonic waves are emitted from the upper surface of the energy concentrator and are reflected by the test tube support, so that standing waves are formed between the test tube support and the energy concentrator, larger sound pressure can be generated in the tooth space area, and the biological samples are crushed by utilizing the cavitation effect generated by the large sound pressure; in the working process, cooling water is pumped in from one end of the sample processing groove by a water pump, and the cooling water is discharged from the other end of the sample processing groove so as to cool the processed biological sample.
Examples
The present invention is further described below by way of specific examples, which are merely exemplary and do not limit the scope of the present invention in any way.
Example 1
In the embodiment, the cross section of the teeth of the tooth-shaped energy concentrator is triangular, the tooth height is 20mm, the tooth width is 10mm, the tooth space is 15mm, the length of the tooth-shaped energy concentrator is 60mm, 4 teeth are provided, 1 to 9 samples can be simultaneously crushed, the number of the ultrasonic transducers is 1, the model is HNC-4SS-3833 of Suzhou Hainan technology, the factory resonance frequency of the transducers is 33kHz and 67kHz, the maximum power is 60W, the active electric power of the transducers is 10W during actual work, and the working frequency is about 65.27 kHz.
The tooth-shaped energy concentrator with the structure is adopted to crush a DNA sample (10.4kbp provided by Ebolo biotechnology Co., Ltd. of the Zhuhai), the total crushing time is 20min, ON/OFF is 30s/30s, the length distribution of the DNA sample is detected by a PCR electrophoresis glue running method, about 70 percent of obtained DNA fragments are distributed between target lengths of 200bp-300bp, and the result shows that the ultrasonic crusher with the tooth-shaped structure can effectively crush the DNA sample.
In the description of the present invention, it should be noted that the terms "upper", "lower", "inner", "outer", etc. indicate orientations or positional relationships based on an operation state of the present invention, and are only for convenience of describing the present invention and simplifying the description, but do not indicate or imply that the referred device or element must have a specific orientation, be constructed in a specific orientation, and be operated, and thus, should not be construed as limiting the present invention.
In the present invention, unless otherwise specifically stated or limited, the terms "connected" and "connected" are to be understood broadly, and may be, for example, a fixed connection, a detachable connection, or a unitary connection; can be mechanically or electrically connected; the connection may be direct or indirect via an intermediate medium, and may be a communication between the two elements. The specific meanings of the above terms in the present invention can be understood in specific cases to those skilled in the art.
The present invention has been described above in connection with preferred embodiments, but these embodiments are merely exemplary and merely illustrative. On the basis of the above, the invention can be subjected to various substitutions and modifications, and the substitutions and the modifications are all within the protection scope of the invention.
Claims (6)
1. An ultrasonic crusher with a tooth-shaped structure is characterized by comprising an ultrasonic transducer (1), a tooth-shaped energy concentrator (2) and a sample processing groove (3),
wherein the ultrasonic transducer (1) is arranged at the lower part of the sample processing groove 3,
the tooth-shaped energy concentrator (2) is arranged inside the sample processing groove (3) to concentrate sound energy;
the toothed concentrator (2) comprises a plate-shaped part and a toothed part,
the bottom surface of the plate-shaped part is fixedly connected with the inner bottom surface of the sample processing groove (3), and the number of teeth of the toothed part is multiple;
a test tube bracket (4) is arranged at the upper part of the tooth-shaped energy concentrator (2) and inside the sample processing groove (3) so as to fix the sample containing tube (5) and reflect ultrasonic waves emitted by the upper surface of the tooth-shaped energy concentrator (2);
the test tube support (4) comprises a vertical plate and a horizontal plate, and both have sound reflection capacity;
the horizontal plate is provided with an opening for placing a sample containing tube (5);
the ultrasonic transducers (1) are one or more, and the radiation surface of the ultrasonic transducers is fixedly connected with the bottom surface of the sample processing groove (3).
2. The ultrasonograph of claim 1, wherein the number of teeth of the tooth portion is two or a tooth array of two or more teeth of the tooth portion.
3. The ultrasonic crusher with a tooth profile structure as claimed in claim 1, wherein the tooth of the tooth-shaped portion has a triangular, rectangular or trapezoidal cross-sectional shape, and a chamfer or fillet structure is provided on the tooth.
4. The ultrasonic disintegrator of tooth profile structure according to claim 2, wherein when the tooth profile is a tooth array structure composed of two or more teeth, the plurality of teeth have the same cross-sectional shape.
5. The ultrasonic crusher of a tooth profile structure as claimed in claim 1, wherein the bottom of the sample holding tube (5) has a gap with the bottom of the tooth bottom.
6. The ultrasonic crusher of a tooth profile structure as claimed in claim 1, wherein the sample in the sample holding tube (5) is a liquid, and the height of the liquid surface is not higher than the vertical height of the tooth grooves.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN202010826485.5A CN111979122B (en) | 2020-08-17 | 2020-08-17 | Ultrasonic crusher with tooth-shaped structure |
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| Application Number | Priority Date | Filing Date | Title |
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| CN202010826485.5A CN111979122B (en) | 2020-08-17 | 2020-08-17 | Ultrasonic crusher with tooth-shaped structure |
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| CN111979122A CN111979122A (en) | 2020-11-24 |
| CN111979122B true CN111979122B (en) | 2021-11-30 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| EP2419705B1 (en) * | 2009-04-14 | 2020-12-09 | Biocartis NV | Treatment of a sample with focused acoustic energy |
| CN202610237U (en) * | 2012-06-18 | 2012-12-19 | 陈智毅 | Operating platform beneficial for ultrasound mediating gene transfection |
| US20140038257A1 (en) * | 2012-08-01 | 2014-02-06 | Anuradha Subramanian | Methods of using ultrasound in tissue culture and tissue engineering |
| ES2661402T3 (en) * | 2013-03-15 | 2018-03-28 | Merck Patent Gmbh | Device for sonication application |
| US10809166B2 (en) * | 2017-01-20 | 2020-10-20 | Matchstick Technologies, Inc. | Ultrasound system for shearing cellular material in a microplate |
| CN213012895U (en) * | 2020-08-17 | 2021-04-20 | 珠海艾博罗生物技术股份有限公司 | Ultrasonic crusher |
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Denomination of invention: A toothed structure ultrasonic crusher Granted publication date: 20211130 Pledgee: Zhuhai Branch of China Construction Bank Co.,Ltd. Pledgor: ZHUHAI APPLETREE BIOTECHNOLOGY CO.,LTD. Registration number: Y2024980013033 |
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