WO2020204615A1 - Procédé de production de probiotiques enrobés de minéraux naturels issus de d'eau de mer de lave et probiotiques enrobés de minéraux naturels issus d'eau de mer de lave l'utilisant - Google Patents

Procédé de production de probiotiques enrobés de minéraux naturels issus de d'eau de mer de lave et probiotiques enrobés de minéraux naturels issus d'eau de mer de lave l'utilisant Download PDF

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WO2020204615A1
WO2020204615A1 PCT/KR2020/004489 KR2020004489W WO2020204615A1 WO 2020204615 A1 WO2020204615 A1 WO 2020204615A1 KR 2020004489 W KR2020004489 W KR 2020004489W WO 2020204615 A1 WO2020204615 A1 WO 2020204615A1
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Prior art keywords
lava seawater
lactic acid
mineral
culture
acid bacteria
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PCT/KR2020/004489
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English (en)
Korean (ko)
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최지휘
김두성
김경민
양서진
이창완
이승훈
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에스케이바이오랜드 주식회사
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Priority claimed from KR1020190039918A external-priority patent/KR102084350B1/ko
Priority claimed from KR1020200022437A external-priority patent/KR102305076B1/ko
Application filed by 에스케이바이오랜드 주식회사 filed Critical 에스케이바이오랜드 주식회사
Publication of WO2020204615A1 publication Critical patent/WO2020204615A1/fr

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • 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

Definitions

  • the present invention uses lava seawater as cultivation water without separate pretreatment to increase the survival rate, storage stability over time, and intake stability during freeze-drying at the same time, and further balance diarrhea, a disease that causes electrolyte imbalance through minerals, to prevent intestinal probiotics.
  • the present invention relates to a method of manufacturing a natural mineral coated probiotics derived from lava seawater that can maximize efficacy, and a natural mineral coated probiotics derived from lava seawater using the same.
  • Probiotics refers to functional lactic acid bacteria in powder form that play a role in smooth bowel movement and proliferation of lactic acid bacteria in the intestine, mainly among health functional foods, unlike the manufacturing method of yogurt like regular yogurt.
  • WHO/FAO World Health Organization
  • probiotics were defined as microorganisms that help the health of the host.
  • WHO/FAO World Health Organization
  • it is registered as a health functional food notified raw material by the Ministry of Food and Drug Safety, and the daily intake of probiotics for intestinal health is defined as more than 100 million CFU/day (CFU: Colony Forming Unit).
  • Another prior art field is coating technology as a typical method of increasing the survival rate of lactic acid bacteria when manufacturing powder.
  • a four-coated lactic acid bacteria manufacturing technology is representative.
  • the coating technology of the prior art of lactic acid bacteria is to laminate a coating agent on the lactic acid bacteria exhibiting anaerobic characteristics, thereby increasing the shelf life and increasing storage stability. Therefore, when the coating layer collapses due to the uniformity of the coating and the inflow of external moisture, it is inevitable to be vulnerable to exposure to the outside air, resulting in the death of lactic acid bacteria that exhibit anaerobic properties.
  • conventional prior technologies focus only on storage stability by forming a protective film through a coating agent in the cultivation and post-treatment stages rather than improving the resistance of lactic acid bacteria itself to external attack factors such as air, temperature, gastric acid, and bile acids.
  • Lava seawater is a water that has been aged for a long period of time as seawater flows into the strata as it is naturally filtered through the basalt and sandy layers, and is a unique water resource possessed by Jeju Island.Since the 1980s, lava seawater in Jeju is characterized by the low temperature and cleanliness of lava seawater. It has been actively used as breeding water for flounder farms, and from 5 to 6 years ago, it has been used as water for saunas in terms of health and beauty, and is gaining much attention. Depending on the purpose of use, desalted lava seawater from which salt has been removed is used or as it is collected.
  • Lava seawater contains more essential minerals such as sodium, magnesium, calcium, and potassium, as well as general useful minerals (iron, manganese, zinc, molybdenum, selenium, etc.) than general seawater, deep water, and Samdawater.
  • vanadium which is known to stabilize insulin secretion or improve diabetes and hyperlipidemia, promotes blood circulation, enhances immunity
  • germanium which has anticancer activity, inhibits oxidation of fat, synergistic effect to maintain the heart and liver, scavenging radicals.
  • the content of selenium which has the effect of improving ability, anticancer, infertility, aging and cholesterol levels, is a characteristic of lava seawater that has never been reported in deep ocean water.
  • these minerals are in an ionized state, and the ionized minerals are easily digested and absorbed by the human body or other animals.
  • lava seawater is a clean groundwater resource in which E. coli, nitrate nitrogen, phosphate phosphorus, phenols, etc. are not detected, and harmful components such as arsenic, mercury, cadmium, etc. are not detected, or lead is detected in a very small amount, which is an obstacle to industrial application. There is no clean raw material.
  • magnesium and calcium minerals contained in lava seawater are essential for the proliferation of lactic acid bacteria and are also used as essential nutrients for humans.
  • lava seawater is applied as a water for cultivating lactic acid bacteria by itself, there is a technical limitation in which it is impossible to cultivate a high concentration of lactic acid bacteria (LJM Linders et al., 1997) because the lactic acid bacteria are inhibited by high concentration salts in the lava seawater.
  • water to which the demineralized water manufacturing method was introduced was used, such as in the prior art (Korean Patent No. 10-1347694).
  • such prior art has economic/technical limitations such as incurring costs for desalting lava seawater and reducing mineral content in desalted lava seawater.
  • the salt concentration is adjusted to enable high concentration culture. It increases the durability against stress of the lactic acid bacteria itself and coats the mineral-protein salt obtained from the salting-out of lava seawater on the lactic acid bacteria as a protective film of the strain, so that the survival stability after freeze drying or spray drying, and the storage stability during distribution are improved.
  • a novel form of probiotics was commercialized by devising a method to enhance stability during ingestion by inhibiting the loss of the coating film while passing through the gastrointestinal tract in vivo.
  • Korean Patent Registration No. 10-1280232 Name of the invention: manufacturing method of 4-coated lactic acid bacteria and 4-coated lactic acid bacteria produced by the method, Applicant: Ildong Pharmaceutical Co., Ltd., registration date: June 25, 2013)
  • Korean Patent Registration No. 10-1927859 (Title of invention: A method of increasing the stability and coating efficiency of probiotics using ultrasonic waves, and a food composition containing freeze-dried powder of probiotics manufactured by the method as an active ingredient, Applicant: Korea Yakult Co., Ltd. , Registration date: December 5, 2018)
  • An object of the present invention is to increase the survival rate, storage stability over time, and intake stability at the same time by coating the lactic acid bacteria through the formation of mineral-protein salts in the medium by using lava seawater as culture water without separate pretreatment.
  • the aim is to provide a method for producing natural mineral coated probiotics derived from lava seawater and natural mineral coated probiotics derived from lava seawater using the same.
  • the present invention relates to a method for producing natural mineral coated probiotics derived from lava seawater.
  • Step 1 preparing a culture medium for lactic acid bacteria prepared by using water containing 30-70% (v/v) lava seawater as culture water;
  • the present invention may include a step of drying the precipitate obtained by centrifuging the culture medium of the second step after (third step), or concentrating and drying the culture medium of the second step.
  • the drying may be performed through freeze drying or spray drying.
  • the concentrate obtained by concentrating the culture solution is preferably spray-dried.
  • the medium of the first step may preferably be any medium capable of culturing lactic acid bacteria, but more preferably, as a constituent, glucose, yeast extract, soypeptone, casein are included, and the constituent is lava seawater It may be a culture medium for lactic acid bacteria prepared by dissolving and sterilizing in water containing 30 to 70% (v/v).
  • the medium of the first step is based on a total volume of 1 liter, glucose 1-5% (w/v), yeast extract 0.5-5% (w/v), soypeptone 0.5-5% (w/v) ), it may be a culture medium for lactic acid bacteria prepared by dissolving each component in water containing 30-70% (v/v) lava seawater and sterilizing it so that the casein becomes 0.5-3% (w/v).
  • the sterilization of the medium is preferably performed at 100 to 125°C for 20 to 40 minutes.
  • the pressure at this time is preferably 0.13 ⁇ 0.17Mps, and most preferably, it can be carried out for 30 minutes at 0.15Mps, 121 °C, the optimal sterilization conditions.
  • Lactic acid of the second step is Lactobacillus genus (Lactobacillus sp.), Bifidobacterium (Bifidobacterium sp.), Streptococcus genus (Streptococcus sp.), Lactococcus genus (Lactococcus sp.), Enterococcus genus (Enterococcus sp.), Pediococcus sp., and Weissella sp. may be selected from the group consisting of.
  • the lactic acid bacteria culture condition of the second step is preferably 18 to 24 hours at 70 to 150 rpm and 30 to 37°C.
  • the culture of the lactic acid bacteria in the second step is characterized in that it is performed by a fed-batch culture method.
  • a method of maintaining a pH of 6.0 to 7.5 during culture by dropping a mixed solution of 10 to 50% (w/v) glucose and 10 to 50% (w/v) sodium hydroxide may be used.
  • the mixed solution may be obtained by mixing a glucose solution and a sodium hydroxide solution in a volume ratio of 1:0.5 to 1:2, respectively.
  • centrifugation when drying, centrifugation is preferably performed at 5,000 to 8,000 rpm.
  • the culture solution may be cooled and allowed to stand at 3 to 5° C. for 1 to 24 hours and then centrifuged.
  • the preferred time is 20 minutes or more and 24 hours or less is sufficient.
  • a mixture of lactic acid bacteria cells and mineral-protein salts is stirred (self) to coat the cells with mineral-protein salts precipitated together.
  • hydroxypropylmethylcellulose (HPMC) as a binder and trehalose as a freeze-dried protective agent may be selected and added.
  • hydroxypropylmethylcellulose and trehalose are preferably added sequentially.
  • the sediment obtained by centrifugation in the third step for freeze-drying is in a state in which mineral-protein salts precipitated due to the addition of lava seawater during the preparation of the medium in the first step are unevenly mixed with the cells (simple mixing process). ), it is stirred at a speed of 30 ⁇ 100rpm to perform a homogenization coating process so that the mineral-protein salt can be coated on the cells.
  • the homogenization time of the coating process is preferably performed for 30 minutes or more and 50 minutes or less.
  • the binder 10 to 40% by weight (powder), and 10 to 40% by weight of trehalose may be prepared and mixed.
  • the binder one or more of the group consisting of hydroxypropylmethylcellulose (HPMC), carboxymethylcellulose (CMC), polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), and chitosan may be added.
  • the binder when the binder is mixed with the mineral-protein salt-coated cells, it is recommended to add the binder after dissolving it in water (culture water) containing 30 to 70% (v/v) lava seawater.
  • the amount of water containing 30 to 70% (v/v) lava seawater for dissolving the binder is not limited, but it is preferably 3 to 7 times the weight of the binder. At this time, it is better to first add a binder solution to the mineral-protein salt coated cells, stir, mix trehalose, and freeze-dry, more preferably, mineral-protein salt coated cells, and a binder solution of 30 to 100 rpm. , After stirring for 10 to 30 minutes, it is recommended to mix trehalose and freeze-dry .
  • the mineral-protein salt when drying, is coated on the cells by concentrating the culture solution of the lactic acid bacteria in the second step, and it is easy to obtain only the cells while removing the culture solution.
  • the volume of the concentrate obtained by concentration is reduced to 1/5 ⁇ 1/15 volume compared to the culture medium before concentration. That is, while the culture solution is concentrated in the third step, the mineral-protein salt may be coated on the outside of the cells through homogenization.
  • concentration hydroxypropylmethylcellulose (HPMC), carboxymethylcellulose (CMC), polyvinylpyrrolidone (PVP) as a binder, which is an amphiphilic material that can be electrostatically used for both positive and negative, to stabilize the coating of cells after concentration.
  • polyvinyl alcohol (PVA), and one or more of the group consisting of chitosan may be added. That is, a culture (simple mixed culture) in which the mineral-protein salt precipitated due to the addition of lava seawater during the medium preparation is mixed with the cells through the first stage of culture (simple mixed culture) through this concentration process. It is possible to obtain a concentrate containing the mineral-protein salt-coated cells, and at this time, a binder is added to the mineral-protein salt-coated cells for coating safety. Mineral-protein salt coated cells 100 parts by weight compared to 50 to 200 parts by weight of a binder (powder) can be prepared and mixed.
  • the binder when the binder is mixed with the mineral-protein salt coated cells, the binder can be added after dissolving it in pure water or lava seawater as a solvent in water (culture water) containing 30 to 70% (v/v). .
  • the content of the solvent for dissolving the binder is not largely limited, but it is preferably 3 to 7 times the weight of the binder powder.
  • the present invention provides a natural mineral coated probiotics derived from lava seawater prepared by the above manufacturing method.
  • the mineral content of the natural mineral-coated probiotic powder derived from lava seawater is 0.1 to 0.5% by weight.
  • the present invention can provide various functional pharmaceutical compositions containing natural mineral coated probiotics derived from lava seawater prepared by the above method.
  • the natural mineral coated probiotics derived from lava seawater may be added in an amount of 0.001 to 100% by weight to the pharmaceutical composition of the present invention.
  • compositions may be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injectable solutions, respectively, according to conventional methods.
  • Carriers, excipients and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl Cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oils.
  • Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and these solid preparations are at least one excipient, such as starch, calcium carbonate, for the natural mineral coated probiotics derived from lava seawater of the present invention. , Sucrose or lactose, gelatin, etc. are mixed and prepared. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.
  • Liquid preparations for oral use include suspensions, liquid solutions, emulsions, syrups, etc.
  • various excipients such as wetting agents, sweetening agents, fragrances, and preservatives may be included.
  • Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories.
  • non-aqueous solvent and suspending agent propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used.
  • As a base for suppositories witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like may be used.
  • the dosage of the pharmaceutical composition of the present invention will vary depending on the age, sex, and weight of the subject to be treated, the specific disease or pathology to be treated, the severity of the disease or pathology, the route of administration, and the judgment of the prescriber. Dosage determination based on these factors is within the level of one of skill in the art, and dosages generally range from 0.01 mg/kg/day to approximately 2,000 mg/kg/day. A more preferred dosage is from 1 mg/kg/day to 500 mg/kg/day. Administration may be administered once a day, or may be divided several times. The above dosage does not in any way limit the scope of the present invention.
  • the pharmaceutical composition of the present invention can be administered to mammals such as mice, livestock, and humans by various routes. All modes of administration can be expected and can be administered, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or cerebrovascular injection.
  • the natural mineral-coated probiotics derived from lava seawater of the present invention have little toxicity and side effects, so they can be safely used even when taken for a long time for prophylactic purposes.
  • the present invention provides a variety of health functional foods including the natural mineral-coated probiotics derived from lava seawater and food supplementary additives that are acceptable food.
  • the health functional food can be applied to various foods to which probiotics can be applied, and can be used for the purpose of helping to proliferate intestinal lactic acid bacteria, inhibit harmful bacteria in the intestine, and facilitate bowel activity.
  • the natural mineral coated probiotics derived from lava seawater may be added in an amount of 0.001 to 100% by weight to the health functional food of the present invention.
  • the health functional food of the present invention includes the form of tablets, capsules, pills, liquids, etc., and foods to which the natural mineral coated probiotics derived from lava seawater of the present invention can be added include, for example, various drinks, meat, Sausages, breads, candies, snacks, noodles, ice cream, dairy products, soups, ion drinks, beverages, alcoholic beverages, gum, tea and vitamin complexes.
  • the present invention can also provide a cosmetic containing a natural mineral coated probiotics derived from lava seawater prepared by the above method.
  • the cosmetic may contain all commonly used ingredients.
  • it may contain general auxiliary ingredients such as emulsifiers, thickeners, emulsions, surfactants, lubricants, alcohols, water-soluble polymers, gelling agents, stabilizers, vitamins, inorganic salts, emulsifiers, and fragrances.
  • the amount of the ingredients may be selected within a range that does not impair the inherent effect of the cosmetic.
  • the amount of the ingredients added may be, for example, 0.1 to 10% by weight, preferably 0.1 to 6% by weight, based on the total weight of the composition, but is not limited thereto.
  • the type of cosmetic is not particularly limited, and for example, lotion, emulsion, gel, cream, essence, pack, ampoule, lotion, detergent, soap, body products, skin care cosmetics such as soap, oil, lipstick, Makeup cosmetics such as foundation, cosmetics for hair, and the like, and the formulation is not particularly limited.
  • the present invention relates to a method for producing natural mineral coated probiotics derived from lava seawater and to a natural mineral coated probiotic using the same.
  • the present invention uses a method of adding lava seawater so that the concentration of lava seawater is 30-70% (v/v) in the medium for cultivation of lactic acid bacteria, and thus minerals and minerals contained in the lava seawater
  • the protein on the lactic acid bacteria medium is precipitated in a mineral-protein salt state, and a growth environment suitable for the lactic acid bacteria is created by controlling the salinity of the medium. Using these conditions and fed-batch culture, the optimum conditions for lactic acid bacteria growth are implemented, and the culture is made with the number of viable bacteria suitable for probiotic standards.
  • the salt concentration is controlled Stress causes a stress-responsive protein, chaperone, to occur in the cells. Thereafter, the precipitated mineral-protein salt and the cultured lactic acid bacteria are recovered through centrifugation of the culture medium and homogenized through stirring so that the mineral-protein salt is coated on the lactic acid bacteria, or the mineral-protein salt is applied to the lactic acid bacteria through a membrane separation method. Let it be coated.
  • the overall thermal stability of the cells inside and outside the cells is remarkably increased, the efficacy of dramatically improving the survival rate and storage stability over time during freeze-drying or spray-drying of lactic acid bacteria, and withstands continuous pH changes in the digestive tract of the body of stomach and bile acids. Efficacy can be derived.
  • natural mineral coated probiotics derived from lava seawater having excellent storage stability against changes in external temperature can be produced and effectively used as biomaterials in the fields of foods, health functional foods, and pharmaceuticals.
  • the osmotic concentration outside the cells is higher than the inside of the cells, resulting in a decrease in moisture in the cell membrane of the cells, and thus the volume of the cells can be kept low. If the volume of the cytosol in the cells is kept low until freeze-drying, it will not damage the inner cell membrane due to volume expansion when the ice crystal is generated in the cells, thus contributing to the survival rate.
  • a separate coating solvent such as soy protein and milk protein is added when the lactic acid bacteria are freeze-dried by precipitating proteins that can protect lactic acid bacteria through minerals in lava seawater in a mineral-protein salt state by salting out in the pre-fermentation stage. Since probiotics can be coated using self-produced mineral-protein salts, the manufacturing stability, intake stability, and storage stability of probiotics can be dramatically improved.
  • lava seawater can be used not only in the pre-process of the cultivation and drying (powdering) process of lactic acid bacteria, but also in the post-process if necessary, so that the lactic acid bacteria survive and adapt to the high concentration mineral salts of the lava seawater during the manufacturing process. It acts to contribute to increasing the survival rate by continuously applying osmotic stress to lactic acid bacteria to form proteins in cells that respond to stress.
  • the protein produced for survival due to changes in the external environment is called chaperone protein.
  • the chaperone protein expressed by a specific stress exerts an effect that can withstand the stresses of other environments. Therefore, probiotics cultured with lava seawater can exhibit strong resistance to temperature (storage stability) and pH (intake stability) stress, and thus can realize all effects that cannot be achieved with conventional lactic acid bacteria culture techniques.
  • # 1 to 6 are experimental results confirming the efficacy of the freeze-dried preparation prepared by the method of the present invention.
  • Example 1 is a graph showing the dry weight of the mineral-protein salt and the amount of protein in the mineral-protein salt in the sterilized lyophilized base medium of Preparation Example 1 and the lava seawater-containing medium of lyophilized Example 1 before culturing lactic acid bacteria.
  • FIG. 2 is a graph showing the results of measuring the number of viable lactic acid bacteria in the culture medium of lyophilized Preparation Example 1 and the culture medium containing lava seawater of lyophilization Example 1;
  • 3 is a graph showing the results of comparing the correlation between the dry weight of the mineral-protein salt and lactic acid bacteria cells and the number of live lactic acid bacteria in the culture medium according to the concentration of lava seawater.
  • Example 4 is a graph showing a result of comparing fed-batch culture and batch culture conditions of lyophilized Example 1 as a culture method using lava seawater.
  • FIG. 6 is a graph showing the results of culturing lactic acid bacteria for each type under the culture conditions of lava seawater addition medium of lyophilized Example 1 or Freeze-dried Preparation Example 1.
  • FIG. 6 is a graph showing the results of culturing lactic acid bacteria for each type under the culture conditions of lava seawater addition medium of lyophilized Example 1 or Freeze-dried Preparation Example 1.
  • # 7 to 12 are experimental results confirming the efficacy of the spray-dried formulation prepared by the method of the present invention.
  • FIG. 7 is a graph showing the results of comparing the correlation between the dry weight of the mineral-protein salt and lactic acid bacteria cells and the number of live lactic acid bacteria in the culture medium according to the concentration of lava seawater.
  • Example 8 is a graph showing a result of comparing fed-batch culture and batch culture conditions of spray-dried Example 1 as a culture method using lava seawater.
  • TEM 9 is a photograph taken with a transmission electron microscope (TEM) of the coating state of the spray-dried powder of probiotics recovered in the spray drying Example 2 and the spray drying Preparation Example 2.
  • TEM transmission electron microscope
  • FIG. 10 is a graph showing the results of culturing lactic acid bacteria for each type under the culture conditions of the lava seawater addition medium of spray-dried Example 1 or Spray-dried Preparation Example 1.
  • FIG. 10 is a graph showing the results of culturing lactic acid bacteria for each type under the culture conditions of the lava seawater addition medium of spray-dried Example 1 or Spray-dried Preparation Example 1.
  • FIG. 11 is a 2D-SDS PAGE result photograph showing that a new protein was formed in the cells in the spray-dried Example 1 or Spray-dried Preparation Example 1 culture.
  • FIG. 12 is a photograph of agaroge gel electrophoresis results showing that the chaperone protein gene was expressed in the cells in the spray-dried Example 1 or Spray-dried Preparation Example 1 culture.
  • the method of manufacturing the natural mineral coated probiotics derived from lava seawater of the present invention was determined including the following steps.
  • the lactic acid bacteria are cultured in a liquid phase in a lactic acid bacteria culture medium containing lava seawater (the whole process of lava seawater).
  • the mineral-protein salt and lactic acid bacteria generated through the first culturing step are recovered, coated, and dried to obtain a natural mineral coated probiotic powder derived from lava seawater (lava seawater post-process).
  • mineral-protein salts are precipitated and produced by adding lava seawater by concentration instead of water to the basic culture medium for lactic acid bacteria culture. This is the step of determining the appropriate concentration of lava seawater in which lactic acid bacteria can grow and mineral-protein salts to be used for coating in the post-process are present.
  • the lava seawater concentration at this time should be a condition that does not inhibit the growth of lactic acid bacteria by high concentration salts by inducing salting out of protein components in the medium.
  • Polyelectrolyte components such as proteins increase in water solubility in normal water, whereas in water with a high salt concentration such as lava seawater, solubility decreases and precipitates in a mineral-protein salt state.
  • solubility decreases and precipitates in a mineral-protein salt state.
  • the salt concentration increases, when the concentration of external ions increases, the water molecules distributed in the hydrophobic part of the protein surface are attracted by the ions, and the hydrophobic part of the protein surface is exposed, and the protein is aggregated by hydrophobic bonds between proteins. This is because a phenomenon occurs.
  • the mineral-protein salt precipitated through the salting-out phenomenon acts as a coating agent that can protect lactic acid bacteria from harsh external stress environments.
  • the lactic acid bacteria cultivation process is designed in the form of fed-batch culture that maintains the pH of 6.0-7.5 with sugars, not the general batch culture method, thereby solving the problem of limiting lactic acid bacteria growth that may occur when adhering to the batch culture method I did.
  • the mineral-protein salt and lactic acid bacteria cells generated through the first culturing step are collected as a precipitate by centrifugation, and the recovered precipitate is separately stirred so that the mineral-protein salt is coated on the lactic acid bacteria cells. , Dry this to obtain a natural mineral coated probiotic powder derived from lava seawater (post-process lava seawater for freeze drying),
  • the mineral-protein salt and lactic acid bacteria cells generated through the first culturing step are concentrated to coat the mineral-protein salts on the lactic acid bacteria cells, and then spray-dried to obtain a natural mineral coated probiotic powder derived from lava seawater (spray drying For lava seawater post process).
  • a binder hydroxypropylmethylcellulose: HPMC
  • a freeze-drying protectant trehalose
  • HPMC hydroxypropylmethylcellulose
  • the method of the present invention is a method of preparing a novel type of probiotic preparation by repeating the same experiment by preparing a freeze-dried preparation and a spray-dried preparation for each subsequent experiment.
  • glucose 3% (w/v) per 1 liter glucose 3% (w/v) per 1 liter
  • yeast extract 2% w/v
  • soypeptone 2% w/v
  • casein 1% w/v
  • the lactic acid bacteria seed culture solution was inoculated into the sterilized medium of the fermentation tank in this sterilized basic medium, and cultured for 20 hours at 100 rpm and 37°C, but 40% (w/v) glucose and 40% (w/v) sodium hydroxide were 1: An aqueous solution mixed at a volume ratio of 1 (hereinafter referred to as glucose-sodium hydroxide solution) was incubated while maintaining the pH at 6.0-7.5 while dropping at regular time intervals (Fed-batch culture performed).
  • Lactobacillus seed culture solution Lactobacilli MRS broth (BD) cultured for 24 hours at 37°C for 24 hours, hereinafter referred to as lactic acid bacteria seed culture solution.
  • the culture solution cultured in Freeze-dried Preparation Example 1 was centrifuged at 6,000 rpm for 30 minutes. After removing the supernatant and recovering the precipitate, the recovered precipitate was homogenized with a stirrer at 50 rpm for 30 minutes.
  • HPMC hydroxypropylmethylcellulose
  • the medium was sterilized as in Preparation Example 1 of freeze-drying using the culture water containing lava seawater at each concentration.
  • the minerals of lava seawater react with the proteins of the medium to produce a mineral-protein salt precipitated in a salt state.
  • Example 2 Freeze-dried Under the conditions of Example 1, 4 tons of culture solution obtained by culturing lactic acid bacteria in a medium prepared using 30% (v/v) lava seawater was centrifuged to recover a precipitate in which mineral-protein salts and lactic acid bacteria cells were mixed. The recovered precipitate (mineral-protein salt and lactic acid bacteria cell mixture) was homogenized with a stirrer for 30 minutes at 50 rpm, so that the mineral-protein salt was coated on the cells.
  • hydroxypropylmethylcellulose and trehalose were added to the freeze-dried preparation example 2 to the cells coated with mineral-protein salts to prepare a probiotic powder, but instead of dissolving hydroxypropylmethylcellulose in water, 30% (v /v) was dissolved in lava seawater (culture water) to obtain probiotic powder.
  • Example 1 of lyophilization when a sterilized medium is prepared using lava seawater, minerals of lava seawater react with proteins of the medium components to produce mineral-protein salts precipitated in a salt state.
  • This mineral-protein salt is partially produced in the basic medium used in Preparation Example 1, but the amount of production is remarkably increased in the medium containing lava seawater.
  • the dry weight of the mineral-protein salt and the amount of protein in the mineral-protein salt were confirmed.
  • the amount of this protein was measured by BCA protein assay to confirm the protein content of the mineral-protein salt.
  • Freeze-dried Preparation Example 1 and freeze-dried Example 1 The culture solution cultured for 20 hours was taken, diluted with physiological saline, 1 ml of the diluted solution was dispensed into Petridish, and 20 ml of sterilized Lactobacilli MRS Agar (BD) were mixed and solidified.
  • the number of lactic acid bacteria viable cells was confirmed by counting colonies cultured for 48 hours in a 37° C. stationary incubator, and this is shown in FIG. 2 (hereinafter referred to as a method for measuring the number of lactic acid bacteria viable cells).
  • Lactobacillus culture solutions cultured in each lava seawater medium of lyophilization Example 1 were dispensed into separate sterilized containers, respectively, and cooled at 4°C for 1 hour. Each stationary solution was centrifuged at 6,000 rpm for 30 minutes to recover mineral-protein salts and lactic acid bacteria, and freeze-dried to check the dry weight of the dried product. For comparison, the culture solution of lyophilized Preparation Example 1 was treated and compared under the same conditions, and it is shown in FIG. 3. At this time, the result of the dry weight was shown in comparison with the number of live lactic acid bacteria.
  • the lava seawater condition in the medium that most affects the culture of lactic acid bacteria is 30-70% (v/v) lava seawater culture medium condition in which the number of viable bacteria is significantly increased compared to the culture condition of lyophilized Preparation Example 1.
  • the best condition is when using a lava seawater concentration of around 30-40% (v/v) as the culture water, and the best condition in this experiment is a culture with a lava seawater concentration of 30% (v/v). It can be seen that it is time to cultivate lactic acid bacteria with water.
  • the conditions for culturing the lactic acid bacteria may be referred to as fed-batch culture conditions using a glucose-sodium hydroxide solution (Glucose/NaOH). This culture condition is compared with the batch culture. For this, the following experiment was performed.
  • the culture conditions of 30% (v/v) lava seawater in lyophilized Example 1 (referred to as lyophilized Example 1 or lyophilized Example 2 in the subsequent experimental examples is the lava seawater 30% (v/v) Corresponds to the medium culture conditions) and lyophilized lactic acid bacteria in the fed-batch culture conditions of Preparation Example 1, and glucose-sodium hydroxide solution in the culture conditions of the lava seawater 30% (v/v) medium of the freeze-dried Example 1
  • the lactic acid bacteria were cultured under comparative conditions in which only the addition conditions were not performed (referred to as comparative conditions of lyophilized Example 1, cultured without pH/glucose correction for 20 hours).
  • the number of viable cells was measured for each culture solution cultured for 20 hours by a method for measuring viable cells of lactic acid bacteria.
  • each culture solution was dispensed into a separate sterilized container and allowed to stand at 4°C for 1 hour, and the stationary solution was centrifuged at 6,000 rpm for 30 minutes to recover lactic acid bacteria cells or mineral-protein salts and lactic acid bacteria cell precipitates.
  • the recovered cells or precipitates were lyophilized to perform weight measurement.
  • the coating state of the probiotic powder recovered in the freeze-dried Example 2 and the freeze-dried Preparation Example 2 was photographed with a Scanning Electron Microscope (SEM) to confirm the morphological characteristics, which are shown in FIG. 5.
  • SEM Scanning Electron Microscope
  • the mineral-protein salt is coated on the surface of the probiotics of Example 2 to increase the size of the lactic acid bacteria cells, and hydroxyproxymethylcellulose binds the cells so that the mineral-protein salt coating is not detached. Can be observed.
  • Example 2 In the freeze-dried Preparation Example 2 and the freeze-dried Example 2, 1 a binder mixture before performing freeze-drying (conditions after sequentially mixing hydroxypropylmethylcellulose and trehalose); 2 After freeze-drying, the number of live bacteria of each lactic acid bacteria of probiotics powder; was compared to confirm the freeze-drying survival rate by the natural mineral coating derived from lava seawater. In order to accurately calculate the survival rate, the solid content of each binder mixture was measured by the loss-on-drying method, and the survival rate was calculated by converting the number of lactic acid bacteria in the binder mixture to the number of viable bacteria.
  • the natural mineral coated probiotic powder derived from lava seawater of lyophilized Example 2 had a significantly higher lyophilization survival rate of about 60% than the probiotic powder of lyophilized Preparation Example 2.
  • Lava seawater-derived natural mineral coating probiotics can be seen to have durability that can overcome the temperature, which is a stress between different kinds, because the coating process through high-concentration growth and post-process while receiving stress from a certain salt concentration from the pre-culture stage.
  • probiotic powders of lyophilized Preparation Example 2 and lyophilized Example 2 were mixed in 100 ml of the prepared artificial gastric juice medium. Each mixture was immediately taken, diluted with physiological saline, 1 ml of the diluted solution was dispensed into Petridish, and 20 ml of sterilized Bromo cresol purple agar (BD) medium was added to solidify. Yellow colonies of Petridish cultured for 48 hours in a stationary incubator at 37°C were counted to confirm the initial viable cell count of each probiotic powder.
  • BD Bromo cresol purple agar
  • each mixture was incubated at 37°C for 2 hours, the cultured culture was taken, diluted with physiological saline, 1 ml of the diluted solution was dispensed into Petridish, and 20 ml of sterilized Bromo cresol purple agar (BD) medium was added to solidify. The number of viable cells was confirmed by counting yellow colonies of Petridish cultured for 48 hours in a stationary incubator at 37°C.
  • BD Bromo cresol purple agar
  • An artificial bile broth medium was prepared by mixing 0.3 ml of sterilized oxgall solution in 100 ml of sterilized Lactobacilli MRS broth (BD).
  • Freeze-dried Preparation Example 2 and freeze-dried probiotic powder of Example 2 were each subjected to an acid resistance test according to the method of Experimental Example 7-1, and the culture solution was centrifuged to remove the supernatant, and then live cells were recovered. The recovered live cells were continuously subjected to a test for confirming bile acid resistance by the method of lyophilization Experimental Example 7-2, and the results are shown in Table 4.
  • Freeze-dried Preparation Example 2 and freeze-dried probiotics powder of Example 2 were divided into 5 g each in an airtight container and stored in a thermo-hygrostat (75% humidity) for 4 months at 4°C, 15°C, 25°C, and 35°C.
  • the number of viable cells was measured based on the method for measuring the number of viable lactic acid bacteria and is shown in Table 5.
  • Spray drying Manufacturing Example 2 4°C 15°C 25°C 35°C Day 0 (CFU/g) 3.6x10 9 3.6x10 9 3.6x10 9 3.6x10 9 30 days (CFU/g) 3.2x10 9 1.2x10 9 1.2x10 9 2.4x10 8 60 days (CFU/g) 2.2x10 9 8.7x10 8 3.3x10 8 4.3x10 7 90 days (CFU/g) 1.3x10 9 4.2x10 8 1.5x10 8 1.1x10 7 120 days (CFU/g) 1.2x10 9 3.2x10 8 6.2x10 7 5.5x10 6 Survival rate (%) 33.0 8.9 1.7 0.2 Spray drying Example 2 4°C 15°C 25°C 35°C Day 0 (CFU/g) 3.2x10 11 3.2x10 11 3.2x10 11 30 days (CFU/g) 3.2x10 11 2.8x10 11 2.4x10 11 7.2x10 10 60 days (CFU/g) 3.1x10
  • Lava seawater (lava seawater without cultivation of the strain itself), freeze-dried Preparation Example 2, and the content of magnesium and calcium in the probiotic powder of Freeze-dried Example 2 were analyzed according to the Magnesium and Calcium Test Method of the Health Functional Food Code Table 6 shows the amount of probiotics contained in natural mineral components derived from lava seawater.
  • glucose 3% (w/v) per 1 liter glucose 3% (w/v) per 1 liter
  • yeast extract 2% w/v
  • soypeptone 2% w/v
  • casein 1% w/v
  • the lactic acid bacteria seed culture solution was inoculated into the sterilized medium of the fermentation tank in this sterilized basic medium, and cultured for 20 hours at 100 rpm and 37°C, but 40% (w/v) glucose and 40% (w/v) sodium hydroxide were 1: An aqueous solution mixed at a volume ratio of 1 (hereinafter referred to as glucose-sodium hydroxide solution) was incubated while maintaining the pH at 6.0-7.5 while dropping at regular time intervals (Fed-batch culture performed).
  • the culture solution cultured in Preparation Example 1 was concentrated to 1/10 volume of the original culture solution using a membrane filtration concentrator (filtration membrane size 0.1 ⁇ m, manufacturer DOW separation systems, Serial No.546/57 205-92.) After obtained, 100 g of the concentrate was mixed with a solution in which 100 g of hydroxypropylmethylcellulose (HPMC) powder was dissolved in 500 g of water, and spray-dried to obtain a dried probiotic powder.
  • HPMC hydroxypropylmethylcellulose
  • the medium was sterilized as in Preparation Example 1 by spray drying using the culture water containing lava seawater for each concentration.
  • a sterilized medium is prepared using lava seawater, natural minerals of lava seawater react with proteins of the medium to produce mineral-protein salts precipitated in a salt state.
  • the lactic acid bacteria seed culture solution was inoculated into the sterilized lava seawater medium as in Preparation Example 1, and the pH was calibrated to 6.0-7.5 using a glucose-sodium hydroxide solution for 20 hours at 100 rpm and 37°C. I did.
  • the culture solution obtained by culturing lactic acid bacteria in a medium prepared using 30% (v/v) of lava seawater among the conditions of Example 1 was concentrated to 1/10 volume of the original culture solution as in the spray drying method of Preparation Example 2.
  • 100 g of the concentrate was mixed with a solution in which 100 g of hydroxypropylmethylcellulose (HPMC) powder was dissolved in 500 g of water, and spray-dried to obtain a dried probiotic powder.
  • HPMC hydroxypropylmethylcellulose
  • the mineral-protein salt coats the outside of the cells, and hydroxypropylmethylcellulose acts as a binder that makes the coating more robust.
  • Example 1 of spray drying when a sterilized medium is prepared using lava seawater, the natural minerals of the lava seawater react with the proteins of the medium to produce a mineral-protein salt precipitated in a salt state.
  • This mineral-protein salt is partially produced in the basic medium used in Spray-drying Preparation Example 1, but the amount of production is remarkably increased in the medium containing lava seawater.
  • the dry weight of the mineral-protein salt and the amount of protein in the mineral-protein salt were confirmed.
  • the amount of this protein was measured by BCA protein assay to confirm the protein content of the mineral-protein salt.
  • water containing 30% (v/v) of lava seawater is prepared for cultivation, and when the entire medium is 100 ml, 3 g of glucose, 2 g of yeast extract, and 2 g of soypeptone are used. After dissolution, the mixture was sterilized for 30 minutes at a temperature range of 100 to 121°C at 5°C. After cooling the sterilized medium, 400 ml of the medium for each concentration of lava seawater was dispensed into separate containers. Each stationary solution was centrifuged at 6,000 rpm for 30 minutes to recover mineral-protein salts and dried to check the dry weight, and the amount of protein in the dried product was measured by Bradford assay to confirm the protein amount. For comparison, the sterilized medium of Comparative Example 1 spray-dried under the same conditions was treated and compared, and are shown in Table 10.
  • Sterilization temperature (°C) Lava seawater concentration (%, v/v) Precipitate (mineral-protein salt) dry weight (mg/ml) The amount of pure protein in the precipitate (mineral-protein salt) (mg/ml) 100°C 30 3.5 1.9 105°C 30 5.4 2.9 110°C 30 7.4 3.1 115°C 30 9.8 3.8 121°C 30 11.8 5.9 121°C 0 0.5 0.2
  • Spray-drying 1 g of the spray-dried powders of Preparation Example 2 and Spray-drying Example 2 were taken, diluted with physiological saline, 1 ml of the diluted solution was dispensed into petridish, and 20 ml of sterilized Lactobacilli MRS Agar (BD) was added to solidify. The number of Petridish colonies cultured at 37°C for 48 hours in a stationary incubator was counted to check the number of viable cells, and this was shown in Table 11 (hereinafter referred to as a method for measuring the number of viable bacteria).
  • the number of lactic acid bacteria in the culture medium is measured as a very high value, and 1.0 ⁇ 10 11 CFU/g or more is significantly higher than the value of 1.0x10 8 CFU/g or more, which is the standard for probiotics. It can be seen that the probiotic powder can be stably manufactured.
  • the mineral-protein salt has a heat protection function.
  • Example 2 Each 400 ml of the lactic acid bacteria culture solution cultured in each lava seawater medium of Example 1 was dispensed into a separate sterilized container and allowed to cool at 4° C. for 1 hour. Each stationary solution was concentrated and spray-dried as in Example 2 to check the dry weight of the dried product. For comparison, the culture solution of Spray-dried Preparation Example 1 was treated and compared under the same conditions, and it is shown in FIG. 7. At this time, the result of the dry weight was shown in comparison with the number of live lactic acid bacteria.
  • the lava seawater conditions in the medium that most affect the cultivation of lactic acid bacteria are 30-70% (v/v) lava seawater culture medium conditions in which the number of viable bacteria is significantly increased compared to the culture conditions of spray-dried Preparation Example 1. It can be understood, and a better condition is when using a lava seawater concentration of around 30 ⁇ 40% (v/v) as culture water, and the best condition for this experiment is when the lava seawater concentration is 30% (v/v). It can be seen that it is time to cultivate lactic acid bacteria with cultivation water.
  • the conditions for culturing the lactic acid bacteria in Spray-drying Preparation Example 1 and Spray-drying Example 1 may be referred to as fed-batch culture conditions using a glucose-sodium hydroxide solution (Glucose/NaOH), which are compared with batch culture. For this, the following experiment was performed.
  • Glucose/NaOH glucose-sodium hydroxide solution
  • the lava seawater 30% (v/v) culture conditions in the following experimental examples, referred to as spray-drying Example 1 or spray-drying Example 2, the lava seawater 30% (v/v) Corresponds to the medium culture conditions
  • the lactic acid bacteria in the fed-batch culture conditions of Spray-dried Preparation Example 1, and the glucose-sodium hydroxide solution in the 30% (v/v) medium culture conditions of the spray-dried lava seawater of Example 1
  • the lactic acid bacteria were cultured under comparative conditions in which only the addition conditions were not performed (referred to as comparative conditions of spray drying Example 1, cultured without pH/glucose correction for 20 hours).
  • each culture solution was dispensed into a separate sterilized container and concentrated to 1/10 volume of the original culture solution using a membrane filtration concentrator (filtration membrane size 0.1 ⁇ m, manufacturer DOW separation systems, Serial No.546/57 205-92.)
  • a membrane filtration concentrator filtration membrane size 0.1 ⁇ m, manufacturer DOW separation systems, Serial No.546/57 205-92.
  • 100 g of the concentrate was mixed with a solution in which 100 g of hydroxypropylmethylcellulose (HPMC) powder was dissolved in 500 g of water, and spray dried to measure the total weight and the number of viable cells.
  • HPMC hydroxypropylmethylcellulose
  • the culture medium cultured in a mass of 500 L was concentrated and tested using a membrane filtration concentrator.
  • Example 2 Water culture spray-dried powder Spray-dried lava seawater culture spray-dried powder of Example 2 After spray drying ( ⁇ 10 9 CFU/g) 11 212 Before spray drying ( ⁇ 10 9 CFU/g) 163 318 Survival rate (%) 6.7 66.6
  • the natural mineral coated probiotic powder derived from lava seawater of spray-dried Example 2 compared with the spray-drying survival rate of about 6.7%, compared to the probiotic powder of Spray-dried Preparation Example 2, about 67% significantly more than 10 times. It is confirmed to be high.
  • the natural mineral coating probiotics derived from lava seawater were subjected to high-concentration growth and the coating process through the post-process while being under stress from a certain salt concentration from the pre-cultivation stage, so it can be seen that it has the durability to overcome the high temperature, which is a stress between different kinds. have.
  • probiotic powders of Spray-drying Preparation Example 2 and Spray-drying Example 2 were mixed in 100 ml of the prepared artificial gastric juice medium. Each mixture was immediately taken, diluted with physiological saline, 1 ml of the diluted solution was dispensed into Petridish, and 20 ml of sterilized Bromo cresol purple agar (BD) medium was added to solidify. Yellow colonies of Petridish cultured for 48 hours in a stationary incubator at 37°C were counted to confirm the initial viable cell count of each probiotic powder.
  • BD Bromo cresol purple agar
  • each mixed solution was incubated at 37°C for 2 hours, and the cultured solution was taken, diluted with physiological saline, 1 ml of the diluted solution was dispensed in Petridish, and 20 ml of sterilized Bromo cresol purple agar (BD) medium was added. Solidified. The number of viable cells was confirmed by counting yellow colonies of Petridish cultured for 48 hours in a stationary incubator at 37°C.
  • BD Bromo cresol purple agar
  • An artificial bile broth medium was prepared by mixing 0.3 ml of sterilized oxgall solution in 100 ml of sterilized Lactobacilli MRS broth (BD).
  • the probiotic powders of Spray-drying Preparation Example 2 and Spray-drying Example 2 were subjected to an acid resistance test according to the method of Spray-drying Experimental Example 6-1, respectively, and the culture solution was centrifuged to remove the supernatant, and then live bacteria were recovered.
  • the recovered live bacteria were continuously subjected to a test for confirming bile acid resistance according to the method of spray drying Experimental Example 6-2, and the results are shown in Table 15.
  • Example 2 Spray-drying
  • the probiotic powder of Example 2 is subdivided into a closed storage container by 5 g each, and stored in a thermo-hygrostat (75% humidity) for 4 months at 4°C, 15°C, 25°C, and 35°C.
  • the number of viable cells was measured based on the method for measuring the number of viable lactic acid bacteria and is shown in Table 16.
  • Spray drying Manufacturing Example 2 4°C 15°C 25°C 35°C Day 0 (CFU/g) 1.1 ⁇ 10 10 1.1 ⁇ 10 10 1.1 ⁇ 10 10 1.1 ⁇ 10 10 30 days (CFU/g) 1.0 ⁇ 10 10 4.1 ⁇ 10 9 1.1 ⁇ 10 9 8.3 ⁇ 10 8 60 days (CFU/g) 9.1 ⁇ 10 9 1.4 ⁇ 10 9 6.6 ⁇ 10 8 1.9 ⁇ 10 8 90 days (CFU/g) 8.8 ⁇ 10 9 6.7 ⁇ 10 8 2.3 ⁇ 10 8 7.5 ⁇ 10 7 120 days (CFU/g) 7.4 ⁇ 10 9 3.3 ⁇ 10 8 1.3 ⁇ 10 8 1.2 ⁇ 10 7 Survival rate (%) 67.2 3.0 1.1 0.1 Spray drying Example 2 4°C 15°C 25°C 35°C Day 0 (CFU/g) 2.1 ⁇ 10 11 2.1 ⁇ 10 11 2.1 ⁇ 10 11 2.1 ⁇ 10 11 30 days (CFU/g) 2.1 ⁇ 10 11 1.9 ⁇ 10 11 1.7 ⁇ 10 11 1.2 ⁇ 10 11 60 days (CFU/g) 2.1 ⁇
  • magnesium and calcium are representative components of natural minerals, and this content value is a value representing the content value of natural mineral coated probiotics derived from lava seawater, and additional experiments are conducted based on this, and natural mineral coated probiotic powder derived from lava seawater. It was confirmed that the mineral content of 0.1 ⁇ 0.5g / 100g. At this time, respectively, magnesium was 0.07 ⁇ 0.4g / 100g, calcium was 0.02 ⁇ 0.09g / 100g.
  • a culture solution obtained by culturing Lactobacillus rhamnosus spp. in the medium of Table 18 was prepared by the method of Example 2, and the number of lactic acid bacteria viable cells was measured (spray-dried). These values are shown in Table 19.
  • a two-dimensional electrophoresis method is used to confirm whether the self-defense protein (chaperone) is formed in the bacteria by a stress response that can withstand the spray drying conditions in the cultivation process through lava seawater.
  • the change in the protein expression level was compared with a gel image.
  • the synthesized cDNA band was confirmed to be significantly thicker, and accordingly, the spray-dried Example 1 It is believed that the expression of self-defense protein (chaperone) is increased through lava seawater cultivation, and thus thermal stability can be obtained when spray-dried.
  • self-defense protein chaperone

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Abstract

La présente invention concerne un procédé de production de probiotiques enrobés de minéraux naturels issus d'eau de mer de lave et des probiotiques enrobés de minéraux naturels issus d'eau de mer de lave l'utilisant. Plus spécifiquement, la présente invention utilise un procédé pour ajouter de l'eau de mer de lave à un milieu pour cultiver des lactobacilles de sorte que la concentration d'eau de mer de lave soit entre 30 à 70 % (v/v), amenant ainsi des minéraux inclus dans l'eau de mer de lave et des protéines sur le milieu lactobacille pour précipiter à l'état de sel de protéine minérale, induisant ainsi une culture lisse de lactobacilles par le contrôle de la salinité sur le milieu. Pendant que les lactobacilles sont cultivés, des chaperons, qui sont des protéines sensibles au stress, sont produites dans des corps microbiens sur un milieu dans lequel la concentration de sel est ajustée, en raison d'une contrainte induite par un sel, et un bouillon de culture après culture est concentré de telle sorte que les côtés externes des corps microbiens sont uniformément revêtus d'un sel de protéine minérale généré lorsque le milieu est stérilisé, ainsi la stabilité thermique globale des côtés interne et externe des corps microbiens est remarquablement améliorée. Par conséquent, il est possible d'obtenir une efficacité d'amélioration remarquable de la viabilité et de la stabilité au stockage dans le temps lorsque les lactobacilles sont lyophilisés ou séchés par pulvérisation, une efficacité de changements de pH continus de l'acide gastrique et de l'acide biliaire dans le tube digestif à l'intérieur d'un corps et analogues.
PCT/KR2020/004489 2019-04-05 2020-04-02 Procédé de production de probiotiques enrobés de minéraux naturels issus de d'eau de mer de lave et probiotiques enrobés de minéraux naturels issus d'eau de mer de lave l'utilisant WO2020204615A1 (fr)

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KR101927859B1 (ko) * 2018-07-17 2018-12-11 주식회사한국야쿠르트 초음파를 이용한 프로바이오틱스의 안정성과 코팅효율을 증가시키는 방법 및 그 방법으로 제조된 프로바이오틱스 동결건조분말을 유효성분으로 함유하는 식품조성물

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR102323673B1 (ko) * 2021-04-12 2021-11-08 주식회사 현대바이오랜드 HtrA 샤페론 프로바이오틱스의 제조방법 및 이를 통해 제조된 장 마이크로바이옴 조절 효능이 있는 염증성 장질환 치료용 조성물

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