CN107648269B - Method for extracting antibacterial composition from body cavity fluid of stichopus japonicus, composition and application thereof - Google Patents
Method for extracting antibacterial composition from body cavity fluid of stichopus japonicus, composition and application thereof Download PDFInfo
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- 241000965254 Apostichopus japonicus Species 0.000 title claims abstract description 46
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- 238000000034 method Methods 0.000 title claims abstract description 23
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- 238000001914 filtration Methods 0.000 claims abstract description 18
- 210000001835 viscera Anatomy 0.000 claims abstract description 9
- 241000894006 Bacteria Species 0.000 claims abstract description 8
- 240000002900 Arthrospira platensis Species 0.000 claims abstract description 7
- 235000016425 Arthrospira platensis Nutrition 0.000 claims abstract description 7
- 229940082787 spirulina Drugs 0.000 claims abstract description 7
- 238000004659 sterilization and disinfection Methods 0.000 claims abstract description 7
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- 238000001035 drying Methods 0.000 claims abstract description 4
- 239000007788 liquid Substances 0.000 claims description 29
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- 241000251511 Holothuroidea Species 0.000 claims description 27
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 18
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 18
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims description 16
- 239000012141 concentrate Substances 0.000 claims description 15
- 230000005587 bubbling Effects 0.000 claims description 13
- 239000013535 sea water Substances 0.000 claims description 12
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 10
- 229910001873 dinitrogen Inorganic materials 0.000 claims description 9
- 229910052757 nitrogen Inorganic materials 0.000 claims description 9
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- 229930003268 Vitamin C Natural products 0.000 claims description 8
- 238000000527 sonication Methods 0.000 claims description 8
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- 239000011718 vitamin C Substances 0.000 claims description 8
- 239000011780 sodium chloride Substances 0.000 claims description 6
- 239000007787 solid Substances 0.000 claims description 6
- 239000008223 sterile water Substances 0.000 claims description 6
- 238000009210 therapy by ultrasound Methods 0.000 claims description 6
- 238000010521 absorption reaction Methods 0.000 claims description 5
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 5
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- 241000191967 Staphylococcus aureus Species 0.000 claims description 3
- 239000011592 zinc chloride Substances 0.000 claims description 3
- 241000351920 Aspergillus nidulans Species 0.000 claims description 2
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- 241000193755 Bacillus cereus Species 0.000 claims description 2
- 244000063299 Bacillus subtilis Species 0.000 claims description 2
- 235000014469 Bacillus subtilis Nutrition 0.000 claims description 2
- 241000222122 Candida albicans Species 0.000 claims description 2
- 241001515917 Chaetomium globosum Species 0.000 claims description 2
- 241000588724 Escherichia coli Species 0.000 claims description 2
- 241000588747 Klebsiella pneumoniae Species 0.000 claims description 2
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- 208000003455 anaphylaxis Diseases 0.000 abstract 1
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- 241000607618 Vibrio harveyi Species 0.000 description 2
- 241001148079 Vibrio splendidus Species 0.000 description 2
- 230000003385 bacteriostatic effect Effects 0.000 description 2
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- 239000002609 medium Substances 0.000 description 2
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- 241000965253 Apostichopus Species 0.000 description 1
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- 206010028980 Neoplasm Diseases 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
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- 241000519590 Pseudoalteromonas Species 0.000 description 1
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- 241000607142 Salmonella Species 0.000 description 1
- 241000878021 Shewanella baltica Species 0.000 description 1
- 230000002009 allergenic effect Effects 0.000 description 1
- 230000000172 allergic effect Effects 0.000 description 1
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 1
- 229960000723 ampicillin Drugs 0.000 description 1
- 230000002052 anaphylactic effect Effects 0.000 description 1
- 238000009360 aquaculture Methods 0.000 description 1
- 244000144974 aquaculture Species 0.000 description 1
- 208000010668 atopic eczema Diseases 0.000 description 1
- 239000012752 auxiliary agent Substances 0.000 description 1
- 239000000022 bacteriostatic agent Substances 0.000 description 1
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- 201000011510 cancer Diseases 0.000 description 1
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- 230000002401 inhibitory effect Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
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- 239000002994 raw material Substances 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical compound O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/56—Materials from animals other than mammals
- A61K35/616—Echinodermata, e.g. starfish, sea cucumbers or sea urchins
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Zoology (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Marine Sciences & Fisheries (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention discloses a method for extracting an antibacterial composition from body cavity fluid of stichopus japonicus, the composition and application thereof. The method comprises the following steps: (1) providing fresh Stichopus japonicus, washing gently, feeding with Spirulina, and stopping feeding for 12-24 hr; (2) carrying out viscera discharge on the stichopus japonicus obtained in the step 1 through temperature change, water salinity change, slight electrical stimulation or viscera taking; (3) collecting the feeding water body after the stichopus japonicus is dirtied, filtering, collecting filtrate, and concentrating to obtain concentrated solution; (4) extracting the concentrated solution and drying to obtain the product. The extraction method has reasonable steps, advanced technology and feasible operation. The prepared composition can be widely used for killing bacteria or fungi, has better antibacterial effect and lower anaphylactic reaction, and can be widely used for wound disinfection and the like.
Description
Technical Field
The invention relates to a medical preparation derived from materials of other animals except mammals and birds, in particular to a method for extracting an antibacterial composition from body cavity fluid of stichopus japonicus, the composition and application thereof.
Background
Stichopus japonicus selenka (A)Apostichopus japonicus) Belonging to the echinoderm phylum (Echinodemata) Sea cucumber class (1)Holothuroidea) Tenons eyes (eyes)Aspidochirotidae) Apostichopus japonicus Temminck et SchlegelStichopodidae) Sea cucumber of genus (A)Apostichopus) Is an important aquaculture variety in China. The body cavity liquid of the stichopus japonicus is rich in various bioactive substances with physiological effects of inhibiting cancer cells, resisting bacteria and inflammation, resisting oxidation and the like. The study finds that the coelomic fluid supernatant of the stichopus japonicus is to the vibrio harveyi ((V))Vibrio harveyi) Vibrio splendidus (E)Vibrio splendidus) Shewanella bacteria (Shewanella)Shewanella baltica) Pseudoalteromonas (Pseudoalteromonas nigrifacien) Staphylococcus aureus (1)Staphyloccocus aureus) And the like has better antibacterial effect.
Because of the lack of a technology for efficiently extracting and preparing coelomic fluid, the development of the coelomic fluid of the stichopus japonicus is not sufficient. At present, no report about the extraction of bacteriostatic substances from body cavity fluid of oplopanax elatus nakai is found.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention aims to provide a method for extracting an antibacterial composition from the body cavity fluid of the stichopus japonicus, the composition and the application thereof, which have reasonable steps, advanced technology and feasible operation. The method for extracting the body cavity liquid of the stichopus japonicus can be used for the large-scale extraction and preparation of the high-activity body cavity liquid for cultivating the stichopus japonicus, and the prepared active body cavity liquid has obvious antibacterial activity and can be used for developing antibacterial preparations or antibacterial biological preparations as raw materials of the antibacterial biological preparations.
The technical scheme adopted by the invention for solving the technical problems is as follows: the method for extracting the antibacterial composition from the body cavity fluid of the oplopanax elatus nakai is characterized by comprising the following steps of: which comprises the following steps:
(1) providing fresh Stichopus japonicus, washing gently, feeding with Spirulina, and stopping feeding for 12-24 hr;
(2) carrying out viscera discharge on the stichopus japonicus obtained in the step 1 through temperature change, water salinity change, slight electrical stimulation or viscera taking;
(3) collecting the feeding water body after the stichopus japonicus is dirtied, filtering, collecting filtrate, and concentrating to obtain concentrated solution;
(4) extracting the concentrated solution and drying to obtain the product.
In a preferred aspect of the invention, the method is carried out by the steps of:
(1) providing fresh stichopus japonicus, transferring to seawater at 12-18 ℃ after filtration sterilization according to the density of 80-100 heads/200L, and carrying out mild washing for 1-2 times; introducing air into seawater to maintain dissolved oxygen content of seawater at 5-8mg/L, adding fresh Spirulina 1-2 times according to 5-10% of total Stichopus japonicus weight, and performing water change by 10-15% within 8-12 hr or using circulating water filtration system; then stopping feeding for 12-24 hours;
(2) transferring the stichopus japonicus obtained in the step (1) to 12-18 ℃ light saline water with the concentration of 1.8-3.3%, increasing the temperature to 28-30 ℃ at the speed of 5-10 ℃/h to enable the sea cucumbers to have a dirt spitting behavior, transferring the dirty sea cucumbers and spit internal organs out until 70-80% of the sea cucumbers have the dirt spitting, and taking out the rest of the sea cucumbers;
(3) collecting the light salt water obtained after the sea cucumber is taken out in the step 2, cooling to 12-18 ℃, adding the filtered sterile water according to the volume ratio of 1:1-2.6%, roughly filtering by using a 50-100-mesh bag filter to remove solid particles such as the feces, the visceral tissues and the like of the sea cucumber, removing grease on the liquid surface by using a diaphragm filter or oil absorption paper and the like, centrifuging for 30-60min by using a pipeline centrifuge 8000-12000rpm, and concentrating to 5-10% of the original volume; continuously concentrating to 0.5-1% of the original liquid volume by using a desalting concentration column; the liquid temperature is kept between 12 and 18 ℃ in the operation process;
(4) and (4) freezing and drying the concentrated solution obtained in the step (3) to obtain a product, and storing at the temperature of between 80 ℃ below zero and 20 ℃ below zero.
In a preferred aspect of the present invention, between the steps 3 and 4, the method further comprises the steps of bubbling the concentrated solution at 5-10 ℃ by using nitrogen and carrying out ultrasonic treatment.
In a preferred aspect of the present invention, between steps 3 and 4, the step of bubbling with nitrogen and sonicating comprises: adding 1.5-3.5% of ethanol, 3-5% of sodium carbonate and 0.05-0.25% of vitamin C by weight into the concentrated solution, blowing nitrogen bubbles into the concentrated solution at the speed of 150-300 mL/L concentrated solution per minute, and carrying out ultrasonic treatment; the nitrogen bubbling and sonication were maintained for 10-15 minutes.
In a preferred aspect of the present invention, between steps 3 and 4, 3% by weight of ethanol, 4% by weight of sodium carbonate and 0.15% by weight of vitamin C are also added to the concentrate, and the concentrate is bubbled with nitrogen gas at a rate of 240mL per minute per liter of concentrate and sonicated; the bubbling of nitrogen gas and sonication were maintained for 12 minutes.
In a preferred aspect of the invention, said step 4 is followed by dissolving the product in water and adding ZnCl2To 100-.
In a preferred aspect of the invention, said step 4 is followed by dissolving the product in water and adding ZnCl2To 150 mM or NaCl to 150 mM.
In other aspects of the invention, there is provided an antibacterial composition prepared according to the steps described in any of the preceding paragraphs.
In other aspects of the invention, there is also provided the use of the above antibacterial composition in the manufacture of a medicament for killing bacteria or fungi.
In a preferred aspect of the invention, the bacteria or fungi are selected from the group consisting of Bacillus subtilis, Bacillus cereus, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Salmonella marcescens, Klebsiella pneumoniae, Aspergillus terreus, Aspergillus nidulans, Chaetomium globosum, Candida albicans, Saccharibacillus kuuerlensis.
The invention establishes the process for extracting and preparing the body cavity liquid of the stichopus japonicus, which has reasonable steps, advanced technology and feasible operation. The invention analyzes the antibacterial activity of the active coelomic fluid, and the prepared composition can be widely used for killing bacteria or fungi, has better antibacterial effect and lower anaphylactic reactivity, and can be widely used for wound disinfection and other purposes.
Detailed Description
The present invention will be further described with reference to the following examples.
Example 1
A method for extracting an antibacterial composition from a body cavity fluid of an oplopanax elatus nakai comprises the following steps:
(1) pretreatment of fresh and alive cultured stichopus japonicus
Taking healthy fresh and alive cultured stichopus japonicus, transferring into seawater at 15 ℃ after filtration and sterilization according to 90 heads/200L, and carrying out mild washing for 2 times to remove most mucus and attached organisms or impurities on the surfaces of the fresh and alive stichopus japonicus. Aerating to maintain dissolved oxygen in seawater to 6.5mg/L, adding fresh Spirulina for 2 times within 12 hr according to 7% of total weight of Stichopus japonicus, and changing water according to 12% or maintaining with circulating water filtration system. Stopping feeding for 18h, increasing the water change amount to 16%, and promoting defecation.
(2) Obtaining of active body cavity fluid
Transferring the treated healthy stichopus japonicus to 15 ℃ fresh salt water with the final concentration of 2.5%, increasing the temperature to 29 ℃ at the speed of 7 ℃/h, wherein the sea cucumber is spitted dirty due to the drastic change of the environmental temperature, and removing the spitted sea cucumber and spitted viscera in time. Until 70-80% of the sea cucumber has spitted.
(3) Extraction of active coelomic fluid
And (3) mixing the obtained sea cucumber active coelomic fluid stock solution according to the ratio of 1: adding sterile water after filtration according to the volume ratio of 1.8 until the salt concentration is reduced to 0.9%, roughly filtering with a 80-mesh bag filter to remove solid particles such as stichopus japonicus feces and visceral tissues remained in the liquid, removing grease on the liquid surface with a diaphragm filter or oil absorption paper, centrifuging at 9600rpm for 45min with a pipeline centrifuge, and concentrating to 7% of the original volume; concentration was continued to 0.8% of the original liquid volume using a desalting concentration column. The liquid temperature was maintained at 15 ℃ during the operation.
(4) Preparation of active cavity liquid dry powder
The active cavity liquid dry powder is prepared by adopting a low-temperature freeze drying technology. The preparation temperature was-15 ℃. The prepared lyophilized powder is stored at-33 ℃.
Example 2
A method for extracting an antibacterial composition from a body cavity fluid of an oplopanax elatus nakai comprises the following steps:
(1) pretreatment of fresh and alive cultured stichopus japonicus
Taking healthy fresh and alive cultured stichopus japonicus, transferring into seawater at 18 ℃ after filtration sterilization according to 80 heads/200L, and washing gently for 2 times to remove most mucus and attached organisms or impurities on the surfaces of the fresh and alive stichopus japonicus. Aerating to maintain dissolved oxygen in seawater to 5mg/L, adding fresh Spirulina for 2 times within 8 hr according to 10% of total weight of Stichopus japonicus, and changing water according to 15% or maintaining with circulating water filtration system. Stopping feeding for 12h, increasing water exchange amount to 20%, and promoting defecation.
(2) Obtaining of active body cavity fluid
Transferring the treated healthy stichopus japonicus into 18 ℃ light salt brine with the final concentration of 3.3%, increasing the temperature to 28 ℃ at the speed of 10 ℃/h, wherein the sea cucumber is spitted dirty due to the drastic change of the environmental temperature, and removing the spitted sea cucumber and the spitted viscera in time. Until 70-80% of the sea cucumber has spitted.
(3) Extraction of active coelomic fluid
And (3) mixing the obtained sea cucumber active coelomic fluid stock solution according to the ratio of 1:1, adding filtered sterile water in a volume ratio of 1 until the salt concentration is reduced to 0.9%, roughly filtering by using a 100-mesh bag filter to remove solid particles such as stichopus japonicus feces, visceral tissues and the like remaining in the liquid, removing grease on the liquid surface by using a diaphragm filter or oil absorption paper and the like, centrifuging for 60min at 8000rpm by using a pipeline centrifuge, and concentrating to 10% of the original volume; concentration was continued to 1% of the original liquid volume using a desalting concentration column. The liquid temperature was maintained at 12 ℃ during the operation.
(4) Preparation of active cavity liquid dry powder
The active cavity liquid dry powder is prepared by adopting a low-temperature freeze drying technology. The preparation temperature was-18 ℃. The prepared lyophilized powder is stored at-80 deg.C.
Example 3
A method for extracting an antibacterial composition from a body cavity fluid of an oplopanax elatus nakai comprises the following steps:
(1) pretreatment of fresh and alive cultured stichopus japonicus
Taking healthy fresh and alive cultured stichopus japonicus, transferring into seawater at 12 ℃ after filtration and sterilization according to 100 heads/200L, and carrying out mild washing for 1 time to remove most mucus and attached organisms or impurities on the surfaces of the fresh and alive stichopus japonicus. Aerating to maintain dissolved oxygen in seawater to 8mg/L, adding 1 time fresh Spirulina according to 5% of total Stichopus japonicus weight within 12 hr, and changing water according to 10% or maintaining with circulating water filtration system. Stopping feeding for 24h, increasing the water change amount to 15%, and promoting defecation.
(2) Obtaining of active body cavity fluid
Transferring the treated healthy stichopus japonicus into 12 ℃ fresh salt water with the final concentration of 1.8%, increasing the temperature to 30 ℃ at the speed of 5 ℃/h, and then leading the sea cucumbers to spit dirty due to the drastic change of the environmental temperature, and removing the spitted sea cucumbers and spitted internal organs in time. Until 70-80% of the sea cucumber has spitted.
(3) Extraction of active coelomic fluid
And (3) mixing the obtained sea cucumber active coelomic fluid stock solution according to the ratio of 1: 2.6, adding the filtered sterile water in the volume ratio until the salt concentration is reduced to 0.9%, roughly filtering by using a 50-mesh bag filter to remove solid particles such as stichopus japonicus feces and visceral tissues remained in the liquid, removing grease on the liquid surface by using a diaphragm filter or oil absorption paper and the like, centrifuging for 30min at 12000rpm by using a pipeline centrifuge, and concentrating to 5% of the original volume; concentration was continued to 0.5% of the original liquid volume using a desalting concentration column. The liquid temperature was maintained at 18 ℃ during the operation.
(4) Preparation of active cavity liquid dry powder
The active cavity liquid dry powder is prepared by adopting a low-temperature freeze drying technology. The preparation temperature was-15 ℃. The prepared lyophilized powder is stored at-20 deg.C.
Example 4
The extraction method of this example is substantially the same as that of example 1, except that: between steps 3 and 4, 3% by weight of ethanol, 4% by weight of sodium carbonate and 0.15% by weight of vitamin C are also added to the concentrate, and the concentrate is bubbled with nitrogen at a rate of 240mL per minute per liter of concentrate and is sonicated; the bubbling of nitrogen gas and sonication were maintained for 12 minutes.
Example 5
The extraction method of this example is substantially the same as that of example 1, except that: between steps 3 and 4, ethanol, 5% sodium carbonate and 0.05% vitamin C, in a weight of 1.5% to the concentrate, were also added, and the concentrate was bubbled with nitrogen gas bubbles at a rate of 150mL per minute per liter of concentrate and sonicated; the bubbling of nitrogen gas and sonication were maintained for 15 minutes.
Example 6
The extraction method of this example is substantially the same as that of example 1, except that: between steps 3 and 4, 3.5% by weight of ethanol, 3% by weight of sodium carbonate and 0.25% by weight of vitamin C are also added to the concentrate, and the concentrate is bubbled with nitrogen gas at a rate of 300mL per minute per liter of concentrate and is sonicated; the bubbling of nitrogen gas and sonication were maintained for 10 minutes.
Comparative example 1
The extraction method of this comparative example is essentially the same as example 4, with the difference that: 3% ethanol, 4% sodium carbonate, but no vitamin C was added.
Comparative example 2
The extraction method of this comparative example is essentially the same as example 4, with the difference that: no sonication and bubbling operations were performed.
Comparative example 3
The extraction method of this comparative example is essentially the same as example 4, with the difference that: no sodium carbonate was added.
Comparative example 4
The extraction method of this comparative example is essentially the same as example 4, with the difference that: no ethanol was added.
First, the antibacterial effect test of the composition extracted in example 1 of the present invention is performed as follows
The test strains stored on the slant were picked up and spread evenly on an LB solid medium plate, and a sterilized 0.5 cm-diameter filter paper sheet was placed on the medium surface.
10 mul (1 mg/mL) of the composition dry powder solution of example 1 was prepared, spotted on the surface of a culture medium in which bacteria were cultured, and subjected to inverted culture at 37 ℃ for 18-20 hours to observe whether a zone of inhibition was formed. The results are shown in Table 1. Two parallel runs were also conducted, each of which was made by adding 150 mM ZnCl to the dry powder solution of the composition of example 12Or 150 mM NaCl.
As can be seen from Table 1, ZnCl was present at 150 mM in comparison with sterile water (without any antibacterial activity)2Or 150 mM NaCl solution. This indicates that a certain concentration of salt can increase its antibacterial activity. Table 1: antibacterial ability test statistics
Note: -: no obvious bacteriostatic circle is seen; +: the bacteriostatic circle is 1-3 mm; ++: zone of inhibition 3-10mm, + ++: the inhibition zone is more than 10 mm. IS: clinical isolate, DR: ampicillin and penicillin resistant strains.
Second, the compositions extracted in examples 1 to 6 of the present invention and comparative examples 1 to 4 were subjected to the allergy test
The compositions of examples 1 to 6 of the present invention and comparative examples 1 to 4 were subjected to the allergy test. The composition solution was tested for hypersensitivity by back-hand coating. Specifically, the stichopus japonicus homogenate was smeared on 4 spots on the back of the hand, the inner side of the upper arm of the subject using a cotton swab, and observation was performed. If 3 or 4 hives or red swelling appear within 30 minutes, the test subject is admitted. In this way 60 people (divided into 6 groups of 10 people each) were recruited who were allergic to the homogenate of stichopus japonicus. Please refer to the above subjects who applied the composition solutions of examples 1-6 of the present invention (1 mg/mL) to 4 spots on the inner sides of the back and upper arms of the subject, respectively, and observed. If one test site of one subject had urticaria or red swelling (i.e., a visible allergy) within 30 minutes, it was scored as 5 points. The allergy scores of the kelp fermentation liquors of each group are shown in table 2.
Table 2: allergy score of composition solutions of examples 1-6 and comparative examples 1-4
| Group number | Allergy score |
| Example 1 | 35 |
| Example 2 | 30 |
| Example 3 | 40 |
| Example 4 | 0 |
| Example 5 | 5 |
| Example 6 | 5 |
| Comparative example 1 | 15 |
| Comparative example 2 | 10 |
| Comparative example 3 | 25 |
| Comparative example 4 | 15 |
As can be seen from table 2, the compositions of examples 4-6 of the present invention have significantly lower allergenic properties through additional processing steps. The effect cannot be realized by only adding the auxiliary agent or only carrying out bubbling ultrasonic operation.
Claims (6)
1. A method for extracting an antibacterial composition from a body cavity fluid of an oplopanax elatus nakai is characterized by comprising the following steps: the method comprises the following steps:
(1) providing fresh stichopus japonicus, transferring to seawater of 12-18 ℃ after filtration sterilization according to the density of 80-100 heads/200L, and washing for 1-2 times; introducing air into seawater to maintain dissolved oxygen content of seawater at 5-8mg/L, adding fresh Spirulina 1-2 times according to 5-10% of total Stichopus japonicus weight, and performing water change by 10-15% within 8-12 hr or using circulating water filtration system; then stopping feeding for 12-24 hours;
(2) transferring the stichopus japonicus obtained in the step (1) to 12-18 ℃ light saline water with the concentration of 1.8-3.3%, increasing the temperature to 28-30 ℃ at the speed of 5-10 ℃/h to enable the sea cucumbers to have a dirt spitting behavior, transferring the dirty sea cucumbers and spit internal organs out until 70-80% of the sea cucumbers have the dirt spitting, and taking out the rest of the sea cucumbers;
(3) collecting the light salt water obtained after the sea cucumber is taken out in the step 2, cooling to 12-18 ℃, adding the filtered sterile water according to the volume ratio of 1:1-2.6%, roughly filtering by using a 50-100-mesh bag filter to remove solid particles such as the feces, the visceral tissues and the like of the sea cucumber, removing the grease on the liquid surface by using a diaphragm filter or oil absorption paper, centrifuging for 30-60min by using a pipeline centrifuge 8000-12000rpm, and concentrating to 5-10% of the original volume; continuously concentrating to 0.5-1% of the original liquid volume by using a desalting concentration column; the liquid temperature is kept between 12 and 18 ℃ in the operation process;
(4) freezing and drying the concentrated solution obtained in the step 3 to obtain a product, and storing at the temperature of between 80 ℃ below zero and 20 ℃ below zero; wherein,
between the steps (3) and (4), the method further comprises the step of bubbling the concentrated solution with nitrogen and carrying out ultrasonic treatment at the temperature of 5-10 ℃, wherein the step of bubbling with nitrogen and carrying out ultrasonic treatment comprises the following steps: adding 1.5-3.5% of ethanol, 3-5% of sodium carbonate and 0.05-0.25% of vitamin C by weight into the concentrated solution, blowing nitrogen bubbles into the concentrated solution at the speed of 150-300 mL/L concentrated solution per minute, and carrying out ultrasonic treatment; the nitrogen bubbles are blown in and the ultrasonic treatment is maintained for 10 to 15 minutes;
after the step (4), dissolving the product in water and adding ZnCl2To 100-.
2. The method for extracting an antibacterial composition from a body cavity fluid of an oplopanax elatus as claimed in claim 1, wherein: between steps (3) and (4), adding 3% by weight of ethanol, 4% by weight of sodium carbonate and 0.15% by weight of vitamin C to the concentrate, and bubbling nitrogen gas bubbles into the concentrate at a rate of 240mL per minute per liter of concentrate, and performing sonication; the bubbling of nitrogen gas and sonication were maintained for 12 minutes.
3. The method for extracting an antibacterial composition from a body cavity fluid of an oplopanax elatus as claimed in claim 1, wherein: after said step (4), the product is dissolved in water and ZnCl is added2To 150 mM or NaCl to 150 mM.
4. An antimicrobial composition prepared according to the method of any one of claims 1-3.
5. Use of an antibacterial composition prepared according to the process of any one of claims 1 to 3 in the manufacture of a medicament for killing bacteria or fungi.
6. Use according to claim 5, characterized in that: the bacteria or fungi are selected from Bacillus subtilis, Bacillus cereus, Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Serratia marcescens, Klebsiella pneumoniae, Aspergillus terreus, Aspergillus nidulans, Chaetomium globosum, Candida albicans, and Saccharibacillus kuuerlensis.
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