CN110066749A - A kind of microbial composite bacteria preparation and its application - Google Patents
A kind of microbial composite bacteria preparation and its application Download PDFInfo
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- CN110066749A CN110066749A CN201910336614.XA CN201910336614A CN110066749A CN 110066749 A CN110066749 A CN 110066749A CN 201910336614 A CN201910336614 A CN 201910336614A CN 110066749 A CN110066749 A CN 110066749A
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- microorganism
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/004—Aftersun preparations
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
Abstract
The invention discloses a kind of microbial composite bacteria preparation and its preparation method and application, which includes at least Lactobacillus helveticus (Lactobacillus helveticus) NS8 tunning and lactobacillus delbruockii subspecies bulgaricus (Lactobacillus delbrueckii subsp.bulgaricus) NS12 tunning.The present invention mixes NS8 fermentation liquid and NS12 fermentation liquid; obtained microbial composite bacteria preparation has the purposes in the skin of skin injury caused by protectiving ultraviolet; candidate protection is provided to be irradiated with ultraviolet radiation the skin of damage, can be used for preparing skin products composition.
Description
Technical field
The present invention relates to biological medicine and food technology field more particularly to a kind of microbial composite bacteria preparation and its answer
With.
Background technique
Skin plays important as the effective barrier for resisting various environmental pressures in terms of adjusting internal environment balance of humanbody
Effect.Solar ultraviolet radiation (mainly UVA and UVB) has been widely regarded as the main reason for causing several skin diseases,
Such as erythema, drying, pigment anomaly, inflammation, aging and cutaneum carcinoma.It is continuously exposed to that cellular anti-oxidant can be reduced under ultraviolet radiation
The efficiency of enzyme system promotes the accumulation of free radical and reactive oxygen species (ROS).This photooxidation stress can be to skin and epidermis group
Cell DNA, protein, lipid and the carbohydrate knitted damage.Another importance, skin are exposed to ultraviolet light
Afterwards, melanin production is activated for a long time, causes melanin over-deposit.Melanin biosynthesis approach is during ultraviolet radiation
Active oxygen and hydrogen peroxide are generated, on the contrary, active oxygen mediation stress also cause melanogenesis in melanocyte to be lacked of proper care.This
In the case of, encouraging ultraviolet protection is the major health strategy used in skin nursing.
Common sun-prevention component, mostly from synthesis or part natural prodcuts, such as kojic acid, vitamin C, carotenoid
And polyphenol.These substances have that stability is poor, high cost practical application, it is therefore necessary to seek other alternative
Effective component.
In recent years, the function and mechanism that the probiotics such as lactobacillus Lactobacillus are promoted health are gradually by people
Concern and generally acknowledge, in terms of especially safeguarding and improving immunity of organisms, for example improve intestinal inflammatory, pre- anti-cancer etc..
Newest some clinical tests and laboratory research show that probiotics improves the effect not only office of immunity of organisms
It is limited to gastrointestinal function, can also improves or treat the symptoms such as allergic dermatitis, infant eczema, chronic cutaneous wound, this says
Bright probiotics is also largely effective for the immunological regulation of integumentary system.
Authorization Notice No. is that the patent of invention of CN101090706B discloses and a effective amount of belongs to lactobacillus paracasei or dry
At least one of Lactobacillus paracasei strain microorganism, its part or its metabolin belong to Bifidobacterium cream or bifid with a effective amount of
The combination of at least one of bacillus species microorganism, its part or its metabolin is in preparation for treating and/or preventing and do
Property skin is related or the skin composition of incoherent response type sensitive in application.
Authorization Notice No. be the patent of invention of CN102105132B disclose a effective amount of at least one probiotic microorganisms and/
Or part thereof and/or its metabolin as treating and/or preventing Oily or skin and phase with oiliness trend
Close the cosmetic applications of the reagent of skin disorder.
Therefore, it is necessary to candidate of the more multi-probiotics as skin sparing is developed, to maintain the health status of skin, and
Mitigate the dermal photodamage that ultraviolet light mediates.
Summary of the invention
The present invention provides a kind of microbial composite bacteria preparation and its application, which has protection purple
Purposes in the skin of skin injury caused by outside line provides candidate protection to be irradiated with ultraviolet radiation the skin of damage
Object can be used for preparing skin products composition.
Specific technical solution is as follows:
A kind of microbial composite bacteria preparation, the tunning of tunning and microorganism B including at least microorganism A；
The microorganism A is Lactobacillus helveticus (Lactobacillus helveticus) NS8；
The microorganism B is lactobacillus delbruockii subspecies bulgaricus (Lactobacillus delbrueckii
subsp.bulgaricus)NS12；
The tunning is the without fermented liquid that acquisition is filtered after microbial fermentation.
Lactobacillus helveticus (Lactobacillus helveticus) NS8 and lactobacillus delbruockii subspecies bulgaricus
(Lactobacillus delbrueckii subsp.bulgaricus) NS12 is isolated from the Koumiss in Siklingelei area,
It is separated by Chinese Academy of Sciences mental health key lab behavioural biology laboratory and identifies and save, be commercial product, no
It is related to the preservation of strain.
Lactobacillus delbruockii subspecies bulgaricus (Lactobacillus delbrueckii in the present invention
Subsp.bulgaricus) NS12 plays synergy to Lactobacillus helveticus (Lactobacillus helveticus) NS8
Effect, further increases microbial composite bacteria preparation and is repairing the function and effect in skin injury caused by ultraviolet light.
It is found by cytologic experiment, the mix preparation of NS8 and NS12 tunning removing free radical with higher, suppression
Melanin production processed and the activity for improving lipid and cholesterol metabolic.
Further across skin test, the combination of NS8 and NS12 compound bacteria can be with effective protection skin as caused by ultraviolet light
Erythema, dermal melanin deposition, skin dehydration equivalent damage.Pass through molecular biology method, it has been found that compound bacteria combination can
Effectively to inhibit the tyrosinase activity of ultraviolet induction, and influence of the ultraviolet light to skin Antioxidant Enzyme Systems is protected simultaneously.
Further, the present invention provides a kind of specific microbial composite bacteria preparation, by microorganism A tunning and
The tunning of microorganism B forms；
In terms of the volume of microbial composite bacteria preparation, the volume fraction of microorganism A tunning is 50~90%, microorganism
The volume fraction of B fermentation product is 10~50%.
Preferably, the volume fraction of microorganism A tunning is 75%, the volume fraction of microorganism B fermentation product is
25%.
Further, the preparation method of the microorganism A tunning or microorganism B fermentation product, comprising:
(1) microorganism A or microorganism B is taken, after activation, seed culture is carried out, obtains seed culture fluid；
(2) seed culture fluid is seeded in fermentation medium and carries out fermented and cultured, obtain tunning；
(3) tunning is centrifuged, takes supernatant, after filtering, obtain sterile microorganism A fermentation liquid or microorganism
B fermentation liquid.
Preferably, skimmed milk aqueous solution of the seed culture medium used when the seed culture for 10%；With volume point
Number meter, the inoculum concentration that seed culture fluid is seeded to fermentation medium is 5%；
The fermentation medium is the skimmed milk aqueous solution of volume fraction 10%；The temperature of fermented and cultured is 35~45 DEG C,
Time is 10~15h.
The preparation method of microbial composite bacteria preparation of the present invention be microorganism A fermentation liquid and microorganism B fermentation liquid are pressed than
Example mixing, obtains microbial composite bacteria preparation.
The present invention also provides the microbial composite bacteria preparations to prepare skin injury caused by protectiving ultraviolet
Application in skin products composition.
Further, the application metering of the microbial composite bacteria preparation is 50~150 μ L/cm2。
Further, the present invention is found by experiment that, the microbial composite bacteria preparation is by inhibiting UV-induced
Epidermis thickens skin injury caused by protectiving ultraviolet.
Further, the present invention is found by experiment that, the microbial composite bacteria preparation is UV-induced by alleviating
Skin injury caused by skin dehydration and/or erythema protectiving ultraviolet.
Further, the present invention is found by experiment that, the microbial composite bacteria preparation is UV-induced by reducing
Skin injury caused by melanin deposition protectiving ultraviolet.
Further, the skin composition includes the skincare product composition and medical supplies that adult or children use
Composition, such as sunscreen cosmetic and skin care product etc.；
Wherein, when being used to prepare sunscreen cosmetic, emulsion, face can be used for using microbial composite bacteria preparation as ingredient
Film, creme, spray etc. are mainly used for the Protection Product of skin surface.
Wherein, when being used to prepare skin care product, emulsion, face can be used for using microbial composite bacteria preparation as ingredient
Film, creme, spray etc. are mainly used for care product or daily nursing product after the solarization of skin surface.
Compared with prior art, the invention has the following advantages:
(1) of the invention by Lactobacillus helveticus (Lactobacillus helveticus) NS8 fermentation liquid and Lactobacillus delbrueckii
Subspecies bulgaricus (Lactobacillus delbrueckii subsp.bulgaricus) NS12 fermentation liquid is mixed, is obtained
To microbial composite bacteria preparation have protectiving ultraviolet caused by skin injury skin in purposes, to be shone by ultraviolet light
The skin for penetrating damage provides candidate protection, can be used for preparing skin products composition.
(2) microbial composite bacteria preparation of the present invention is caused by inhibiting UV-induced epidermis to thicken, alleviating ultraviolet light
Skin dehydration and erythema, the mode for reducing UV-induced melanin deposition, improve skin and ultraviolet light make it
At damage.
(3) ability of microbial composite bacteria system removing free radical of the present invention is suitable with vitamin C, and stability is then obvious excellent
In vitamin；It inhibits melanin production effect kojic acid and other effects quite, and safety is better than kojic acid.
(4) preparation method of microbial composite bacteria of the present invention is simple, at low cost, is conducive to the utilization of its industry；And its
Matter has the characteristics that natural, safe and nontoxic, pollution-free from food, can be used for preparing in cosmetics or skin care product,
It is widely used, Social benefit and economic benefit is significant.
Specific embodiment
The invention will be further described combined with specific embodiments below, and what is be exemplified below is only specific implementation of the invention
Example, but protection scope of the present invention is not limited only to this.
(Lactobacillus helveticus) NS8 of Lactobacillus helveticus involved in the following example and Lactobacillus delbrueckii
NS12 is with being isolated from Siklingelei for subspecies bulgaricus (Lactobacillus delbrueckii subsp.bulgaricus)
The Koumiss in area is separated by Chinese Academy of Sciences mental health key lab behavioural biology laboratory and is identified and save, is
Commercial product is not related to the preservation of strain.
The preparation of 1 microbial composite bacteria preparation of embodiment
1, Lactobacillus helveticus (Lactobacillus helveticus) NS8
Specific steps are as follows:
1) Lactobacillus helveticus (Lactobacillus helveticus) NS8 that -80 DEG C freeze is inoculated into MRS culture medium
Then single clone is inoculated into seed culture medium, carries out seed fermentation, obtain highdensity seed liquor by middle recovery；
The ingredient (g/L) of MRS culture medium:
The condition of seed fermentation: cultivation temperature is 37 DEG C, and incubation time is 20 hours.
2) seed liquor is seeded in the aqueous solution of (volume fraction) 10% skimmed milk in the ratio that mass fraction is 5%,
40 DEG C ferment 12 hours, carry out expanding fermented and cultured for the first time, then will in the ratio that mass fraction is 5% in identical condition
Seed liquor is seeded in the aqueous solution of 10% skimmed milk, and 40 DEG C ferment 24 hours, is carried out second and is expanded fermented and cultured, is sent out
Ferment product；
3) tunning is centrifuged 10 minutes in 3,000g, the supernatant after transfer centrifugation；
4) fermented supernatant fluid is sterile filtered by the filter membrane in 0.22 μm of aperture, obtains sterile Lactobacillus helveticus
(Lactobacillus helveticus) NS8 fermentation liquid.
2, lactobacillus delbruockii subspecies bulgaricus (Lactobacillus delbrueckii subsp.bulgaricus)
NS12
1) lactobacillus delbruockii subspecies bulgaricus (the Lactobacillus delbrueckii frozen -80 DEG C
Subsp.bulgaricus) NS12 is inoculated into MRS culture medium and recovers, and is then inoculated by the inoculum concentration that mass fraction is 5%
In seed culture medium, seed fermentation is carried out, highdensity seed liquor is obtained；
The component and dosage of MRS culture medium: ibid.
The condition of seed fermentation: cultivation temperature is 37 DEG C, and incubation time is 20 hours.
2) seed liquor is seeded to (volume fraction or mass fraction) 10% skimmed milk in the ratio that mass fraction is 5%
Aqueous solution in, 40 DEG C ferment 12 hours, carry out for the first time expand fermented and cultured, be then by mass fraction by identical condition
Seed liquor is seeded in the aqueous solution of 10% skimmed milk by 5% ratio, and 40 DEG C ferment 24 hours, is carried out second and is expanded fermentation
Culture, obtains tunning；
The component and dosage of fermentation medium: the aqueous solution of 10% skimmed milk.
3) tunning is centrifuged 10 minutes in 3,000g, the supernatant after transfer centrifugation；
4) fermented supernatant fluid is sterile filtered by the filter membrane in 0.22 μm of aperture, obtains sterile Lactobacillus delbrueckii and protects
Add Leah subspecies (Lactobacillus delbrueckii subsp.bulgaricus) NS12 fermentation liquid.
3, microbial composite bacteria preparation
Sterile Lactobacillus helveticus (Lactobacillus helveticus) the NS8 tunning A and step that step 1 is obtained
Rapid 2 sterile lactobacillus delbruockii subspecies bulgaricus (the Lactobacillus delbrueckii obtained
Subsp.bulgaricus) NS12 tunning B is mixed, and obtains microbial composite bacteria preparation；
Wherein, with the total volume meter of microbial composite bacteria preparation, the volume fraction of tunning A is 75%, tunning B
Volume fraction be 25%.
2 microbial composite bacteria preparation of embodiment is in the effect for inhibiting UV-induced epidermis to thicken
1) (8 week old, divide three groups: control group/UVB irradiation group/UVB irradiation+microorganism is compound for female SKH-1 hairless mouse
Bacteria preparation group；Every group 10), feeding standard diet and tap water, and under the conditions of 23 DEG C, 12 small time/12 hour dark circulations
It is maintained in SPF environment；
2) by using ultraviolet radiator (peak value radiation is 313nm), animal is exposed to three-times-weekly under ultraviolet light irradiation；It will
The predose of UVB is set as 36mJ/cm2, then increase by 1/3 irradiation weekly, until total radiation dosage reaches 1.8J/cm2
(in total 8 weeks).
3) it is administered, is given in 2 square centimeters of skin of back with microbial composite bacteria preparation prepared by 200 μ L embodiments 1 daily
The medicine time is to irradiate previous hour.
4) after putting to death animal with difluoromethane and dislocation of cervical vertebra, it is rapidly separated skin of back.
5) skin samples are fixed in 4% paraformaldehyde, and routine paraffin wax slice, hematoxylin/Yihong (H&E) dyeing is made.
6) micro-image being sliced is captured by Pannoramic scanner (U.S. Perkinelmer), selects each sample
Four different zones, measure sample mesocuticle thickness.
Test result:
1. mouse skin thickness of table (unit: μm)
| Processing | Epidermal thickness (μm) |
| Control | 21.1023±1.2099 |
| UVB | 96.0328±11.1482 |
| UVB+ microbial composite bacteria preparation | 49.3251±8.1999 |
As shown in table 1, microbial composite bacteria preparation can be significantly inhibited by UV-induced epidermis thickening phenomenon.
3 microbial composite bacteria preparation of embodiment alleviates UV-induced skin dehydration function
1) (8 week old, divide three groups: control group/UVB irradiation group/UVB irradiation+microorganism is compound for female SKH-1 hairless mouse
Bacteria preparation group；Every group 10), feeding standard diet and tap water, and under the conditions of 23 DEG C, 12 small time/12 hour dark circulations
It is maintained in SPF environment.
2) by using ultraviolet radiator (peak value radiation is 313nm), animal is exposed to three-times-weekly under ultraviolet light irradiation.It will
The predose of UVB is set as 36mJ/cm2, then increase by 1/3 irradiation weekly, until total radiation dosage reaches 1.8J/cm2
(in total 8 weeks).
3) it is administered daily with 200 μ L microbial composite bacteria preparations in 2 square centimeters of skin of back, administration time is before irradiating
One hour.
4) after irradiation cycle, using multiprobe adaption system MPa5 (German Courage-Khaz aka)TM300 probe measures the transepidermal water loss amount (TEWL) of skin of living body.
Test result:
2. mouse skin transepidermal water loss amount (unit: g/m of table2/h)
| Processing | Transepidermal water loss amount (g/m2/h) |
| Control | 18.9564±5.9723 |
| UVB | 51.7600±15.2326 |
| UVB+ microbial composite bacteria preparation | 23.5320±4.0354 |
As shown in table 2, microbial composite bacteria preparation can be significantly inhibited is lost by UV-induced moisture of skin.
4 microbial composite bacteria preparation of embodiment alleviates the effect of UV-induced rubefaction (erythema)
1) (8 week old, divide three groups: control group/UVB irradiation group/UVB irradiation+microorganism is compound for female SKH-1 hairless mouse
Bacteria preparation group；Every group 10), feeding standard diet and tap water, and under the conditions of 23 DEG C, 12 small time/12 hour dark circulations
It is maintained in SPF environment.
2) by using ultraviolet radiator (peak value radiation is 313nm), animal is exposed to three-times-weekly under ultraviolet light irradiation.It will
The predose of UVB is set as 36mJ/cm2, then increases by 1/3 irradiation weekly, until total radiation dosage reaches 1.8J/cm2
(in total 8 weeks).
3) it is administered daily with 200 μ L microbial composite bacteria preparations in 2 square centimeters of skin of back, administration time is before irradiating
One hour.
4) after irradiation cycle, using multiprobe adaption system MPa5 (German Courage-Khazaka)18 probe of MX measures the erythema coefficient of skin of living body.
Test result:
3. mice skin erythema of table (unit: erythema coefficient)
As shown in table 3, microbial composite bacteria preparation can be significantly inhibited is showed by UV-induced rubefaction or erythema
As.
5 microbial composite bacteria preparation of embodiment alleviates UV-induced dermal melanin deposition
1) ultraviolet irradiation experiment is carried out to 10 brown cavys (female, 300-350g)；Illumination procedure and local administration
It is similar to experiment (that is: the experiment of embodiment 1) of SKH-1 mouse, but the predose of UVB is set as 120mJ/cm2, and continue
Until there is apparent pigmentation (in total 3 weeks).
2) before first time UVB irradiation, cavy is placed under chloraldurate (250mg/kg) deep anaesthesia, is got a haircut with baby
Back hair is picked out three hairless regions by device, and (2 square centimeters, respectively irradiated site (that is: UVB), irradiated site+microorganism is compound
Bacteria preparation defence area (that is: UVB+ microbial composite bacteria preparation) ,+5% kojic acid area of irradiated site (that is: UVB+ kojic acid)), it reserves simultaneously
Region blank control (that is: Control) of 1 hairiness.
3) after irradiation cycle, using multiprobe adaption system MPa5 (German Courage-Khaz aka)18 probe of MX measures the melanin coefficient of skin of living body.
4. mouse melanin of table (unit: melanin coefficient)
| Processing | Melanin (melanin coefficient) |
| Control | 321.5000±60.6042 |
| UVB | 654.400±119.3470 |
| UVB+ microbial composite bacteria preparation | 420.8730±100.4512 |
| UVB+ kojic acid | 553.0279±98.1094 |
Test result:
As shown in table 4, microbial composite bacteria preparation can be significantly inhibited by UV-induced melanin deposition, and effect
It is better than reference substance kojic acid.
Claims (10)
1. a kind of microbial composite bacteria preparation, which is characterized in that the hair of tunning and microorganism B including at least microorganism A
Ferment product；
The microorganism A is Lactobacillus helveticus (Lactobacillus helveticus) NS8；
The microorganism B is lactobacillus delbruockii subspecies bulgaricus (Lactobacillus delbrueckii
subsp.bulgaricus)NS12；
The tunning is the without fermented liquid that acquisition is filtered after microbial fermentation.
2. microbial composite bacteria preparation as described in claim 1, which is characterized in that tunning and micro- life by microorganism A
The tunning of object B forms；
In terms of the volume of microbial composite bacteria preparation, the volume fraction of microorganism A tunning is 50~90%, microorganism B hair
The volume fraction of ferment product is 10~50%.
3. microbial composite bacteria preparation as described in claim 1, which is characterized in that tunning and micro- life by microorganism A
The tunning of object B forms；
In terms of the volume of microbial composite bacteria preparation, the volume fraction of microorganism A tunning is 75%, and microorganism B fermentation produces
The volume fraction of object is 25%.
4. microbial composite bacteria preparation as described in claim 1, which is characterized in that the microorganism A fermentation liquid or microorganism B
The preparation method of fermentation liquid, comprising:
(1) microorganism A or microorganism B is taken, after activation, seed culture is carried out, obtains seed culture fluid；
(2) seed culture fluid is seeded in fermentation medium and carries out fermented and cultured, obtain tunning；
(3) tunning is centrifuged, takes supernatant, after filtering, obtain sterile microorganism A fermentation liquid or microorganism B hair
Zymotic fluid.
5. as the described in any item microbial composite bacteria preparations of Claims 1 to 4 are preparing the damage of skin caused by protectiving ultraviolet
Application in the skin products composition of wound.
6. microbial composite bacteria preparation as claimed in claim 5 is used in the skin for preparing skin injury caused by protectiving ultraviolet
Application in product composition, which is characterized in that the application metering (or concentration) of the microbial composite bacteria preparation is 50~150 μ
L/cm2。
7. microbial composite bacteria preparation as claimed in claim 5 is used in the skin for preparing skin injury caused by protectiving ultraviolet
Application in product composition, which is characterized in that the microbial composite bacteria preparation is by inhibiting UV-induced epidermis to thicken
Skin injury caused by protectiving ultraviolet.
8. microbial composite bacteria preparation as claimed in claim 5 is used in the skin for preparing skin injury caused by protectiving ultraviolet
Application in product composition, which is characterized in that the microbial composite bacteria preparation is by alleviating UV-induced skin dehydration
And/or skin injury caused by erythema protectiving ultraviolet.
9. microbial composite bacteria preparation as claimed in claim 5 is used in the skin for preparing skin injury caused by protectiving ultraviolet
Application in product composition, which is characterized in that the microbial composite bacteria preparation is heavy by reducing UV-induced melanin
Skin injury caused by product protectiving ultraviolet.
10. application as described in any one of claims 1 to 9, which is characterized in that the skin composition includes adult or youngster
The skincare product composition and medical supplies composition that child uses.
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| WO2021236416A1 (en) * | 2020-05-21 | 2021-11-25 | Liberty Biosecurity, Llc | Compositions from a bacterial organism and uses thereof |
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