WO2020204615A1 - Method for producing lava seawater-derived natural mineral-coated probiotics and lava seawater-derived natural mineral-coated probiotics using same - Google Patents

Abstract

The present invention relates to a method for producing lava seawater-derived natural mineral-coated probiotics and lava seawater-derived natural mineral-coated probiotics using same. More specifically, the present invention uses a method for adding lava seawater to a medium for culturing lactobacilli so that the concentration of the lava seawater becomes 30-70% (v/v), thus causing minerals included in the lava seawater and proteins on the lactobacilli medium to precipitate in a mineral-protein salt state, thus inducing smooth culture of lactobacilli through the control of salinity on the medium, and while lactobacilli are cultured, chaperones, which are stress-responsive proteins, are produced in microbial bodies on a medium in which the concentration of salt is adjusted, due to salt-induced stress, and a culture broth after culture is concentrated so that the outer sides of the microbial bodies are uniformly coated with a mineral-protein salt generated when the medium is sterilized, and thus overall thermal stability of the inner and outer sides of the microbial bodies are remarkably enhanced. Through this, it is possible to obtain efficacy in remarkably enhancing the viability and storage stability over time when lactobacilli are lyophilized or spray-dried, efficacy in enduring continuous pH changes of gastric acid and bile acid in the digestive tract inside a body, and the like.

Description

Lava seawater-derived natural mineral coating probiotics manufacturing method and lava seawater-derived natural mineral coating probiotics using the same

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. In particular, when ingested in an appropriate amount as live cells by the World Health Organization (WHO/FAO) in 2002, it was widely popularized as probiotics were defined as microorganisms that help the health of the host. In Korea, 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).

Recently, colon diseases due to westernized eating habits and a lot of stress are increasing rapidly, and in particular, the prevalence of irritable bowel syndrome and inflammatory growth disease is increasing. One of the characteristics of such bowel disease is diarrhea, which causes the electrolyte balance in the large intestine to be broken, resulting in diarrhea accompanied by water. In this case, it becomes difficult for the probiotics introduced from the outside to settle the intestine normally. Therefore, the use of probiotics by people with these diseases is necessary to restore the electrolyte balance in the first place so that the probiotic effect can be exhibited.

In order to exhibit a smooth intestinal probiotic effect, it is necessary to consume more than a certain number of live bacteria, but in most cases, it is difficult to expect the effect as the number of bacteria reaching the intestine is insufficient due to death from gastric acid and bile acids during passage through the gastrointestinal tract. Therefore, in the industry, efforts were made to overcome the method of increasing the survival rate of lactic acid bacteria during powder manufacturing as an important matter, along with the development of a method for cultivating lactic acid bacteria with a high concentration of bacteria, especially from liquid fermentation broth with anaerobic properties that are killed when exposed to air. For the production of live bacteria powder formulations, various industrialized bacterial cell culture methods, tablet recovery processes, coating methods, etc., have been continuously optimized to increase the survival rate of lactic acid bacteria related to the shelf life and stability of the product.

As a technology for high concentration of lactic acid bacteria in the culture technology, it is necessary to cultivate the number of bacteria in the culture medium at a high concentration of 5 billion CFU/ml or more in order to stably produce probiotic live bacteria of 100 million CFU/day (CFU: Colon Forming Unit). , As a prior art, there is a technology for improving stability through the addition of proline to increase the number of lactic acid bacteria (Korean Patent No. 10-1605516). However, this method has been pointed out as a problem that it is difficult to use in mass production because proline amino acids are injected into the ton-unit production.

Another prior art field is coating technology as a typical method of increasing the survival rate of lactic acid bacteria when manufacturing powder. As a conventional technology, a four-coated lactic acid bacteria manufacturing technology (Korean Patent No. 10-1280232) 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. As described above, 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. However, when symptoms that cause intestinal electrolyte imbalance such as diarrhea occur, or when the coating film is peeled off during the continuous passage of the gastrointestinal tract, the durability of the lactic acid bacteria itself is not secured. It has been pointed out that there are technical limitations in overcoming the stress of the gastrointestinal environment.

As a method of overcoming stress by increasing the vitality of lactic acid bacteria itself, it has been suggested as an alternative to overcome anaerobicity by treating stress such as heat shock, osmotic shock, and acid shock for a short time to a critical condition for viability. However, in industrial production conditions, it is physically and mechanically difficult to apply rapid stress to lactic acid bacteria for a short time within a few minutes, and commercialization has not been made due to the death of the cells during stress treatment and the loss of the amount of cells in the process of removing the stress factor.

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. Among them, 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. In addition, these minerals are in an ionized state, and the ionized minerals are easily digested and absorbed by the human body or other animals. In addition, 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.

In particular, 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. However, when 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. In order to do so, water to which the demineralized water manufacturing method was introduced was used, such as in the prior art (Korean Patent No. 10-1347694). However, such prior art has economic/technical limitations such as incurring costs for desalting lava seawater and reducing mineral content in desalted lava seawater.

Therefore, in the present invention, by inducing mineral-protein salts of lactic acid bacteria that cannot survive at high concentration in a medium with a high salt concentration by using lava seawater that is useful for human consumption and contains minerals, 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. In addition, 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.

[Prior technical literature]

[Patent Literature]

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)

Republic of Korea Patent Registration No. 10-1605516 (Name of invention: method of increasing the survival rate, storage stability, acid resistance or bile resistance of lactic acid bacteria, Applicant: Chong Kun Dang Bio Co., Ltd., registration date: 10-1605516)

Republic of Korea Patent Registration No. 10-1347694 (Title of invention: Method for producing fermented product of desalted lava seawater, fermented product obtained by the method, and cosmetic composition using the fermented product, Applicant: Jeju Technopark Foundation, registration date: 2013 12 27th of month)

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)

Republic of Korea Patent Publication No. 10-2019-0008597 (Name of the invention: cultivation method of lactic acid bacteria using culture medium containing deep sea water and medium composition for cultivation of lactic acid bacteria, Applicant: Bifido and others, Publication date: January 25, 2019 Work)

[Non-patent literature]

LJM Linders, G. Meerdink, K. Van't Riet. (1997) Effect of growth parameters on the residual activity of Lactobacillus plantarum after drying. Journal of microbiology 82, 683-688.

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.

Accordingly, in the production of probiotics raw powder through the present invention, a consistent process using lava seawater of a specific concentration is implemented in the process of the pre-process and post-process, and the coating of lactic acid bacteria is performed with mineral-protein salts formed by lava seawater. By doing so, it dramatically improves the survival rate and storage stability of lactic acid bacteria against temperature stress, which is an external threat factor during rapid freezing or spray drying, and ingestion against stress caused by sudden pH changes of gastric acid and bile acid among external threat factors during ingestion. It is possible to provide a method for producing probiotics coated with natural minerals derived from lava seawater that increases stability at the same time.

The present invention relates to a method for producing natural mineral coated probiotics derived from lava seawater.

Even more preferably, the manufacturing method,

(Step 1) preparing a culture medium for lactic acid bacteria prepared by using water containing 30-70% (v/v) lava seawater as culture water; And,

(Second step) obtaining a culture solution by inoculating and culturing the lactic acid bacteria in the culture medium for lactic acid bacteria;

It may include.

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.

Among them, it is more preferable to freeze-dry the precipitate obtained by centrifuging the culture solution. In addition, 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).

Even more preferably, 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 ℃, 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. To this end, 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.

In one example of the third step, when drying, centrifugation is preferably performed at 5,000 to 8,000 rpm. At this time, for smooth centrifugation, 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.

In the third step, for freeze-drying, a mixture of lactic acid bacteria cells and mineral-protein salts is stirred (self) to coat the cells with mineral-protein salts precipitated together. Thereafter, in order to stabilize the coating of the cells, hydroxypropylmethylcellulose (HPMC) as a binder and trehalose as a freeze-dried protective agent may be selected and added. At this time, hydroxypropylmethylcellulose and trehalose are preferably added sequentially.

That is, 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. After obtaining the cells coated with mineral-protein salt through this stirring process, when adding the binder and trehalose to the cells coated with the mineral-protein salt, 20 to 80% by weight of the cells coated with the mineral-protein salt, the binder 10 to 40% by weight (powder), and 10 to 40% by weight of trehalose may be prepared and mixed. As the binder, one or more of the group consisting of hydroxypropylmethylcellulose (HPMC), carboxymethylcellulose (CMC), polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), and chitosan may be added.

However, 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 .

In one example of the third step, when drying, the mineral-protein salt 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. After 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. However, 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.

Accordingly, 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.

The pharmaceutical 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. In the case of formulation, it is prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants that are usually used. 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. In addition to water and liquid paraffin, which are commonly used simple diluents, 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. As the 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.

In addition, 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. For example, 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. According to the formulation or purpose of use, 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.

In addition, 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. In more detail, 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. As the lactic acid bacteria are cultured, 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.

Through this method, 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.

Accordingly, through the present invention, 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.

To add a little more explanation, there are the following advantages to lactic acid bacteria when adding 30-70% (v/v) lava seawater to the medium during probiotic production.

First, 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.

Second, 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.

Third, minerals rich in lava seawater in the medium affect the proliferation of lactic acid bacteria. In general lactic acid bacteria culture, solid minerals that are separately added in the form of a nutrient medium are not ionized and have low growth availability, but are included in lava seawater. Since the mineral has already been ionized in a liquid phase, there is an advantage of inducing the growth of probiotics within a short time of about 24 hours when using the culture method of the present invention.

Fourth, 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.

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.

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.

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.

5 is a photograph taken with a scanning electron microscope (SEM) of the coating state of the powder recovered in the freeze-dried Example 2 and the freeze-dried Preparation Example 2.

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.

# 7 to 12 are experimental results confirming the efficacy of the spray-dried formulation prepared by the method of the present invention.

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.

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.

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.

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.

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.

In order to solve the above problems, the method of manufacturing the natural mineral coated probiotics derived from lava seawater of the present invention was determined including the following steps.

First, first, 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).

Second, 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).

In more detail, in the first lactic acid bacteria culture medium containing lava seawater, 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.

That is, 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. When 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. In addition, 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.

On the other hand, minerals are essential for the growth of lactic acid bacteria, but if they are supplied from lava seawater without adding them from the outside and used for the growth of lactic acid bacteria, depending on the amount of mineral-protein salt precipitation, the content of free minerals may be insufficient or excessively supplied. . Therefore, it is necessary to set an appropriate concentration section of lava seawater in the medium according to the degree of proliferation of lactic acid bacteria.

In addition, when lactic acid bacteria are cultured in a liquid culture medium containing lava seawater, pH 6.0-7.5 in the medium is maintained in the form of fed-batch culture, which is caused by various organic acids, which are metabolites generated while lactic acid bacteria grow. If the value is acidic between 3.0 and 5.5, the solubility of the water-soluble protein in the presence of lava seawater decreases and excessive precipitation makes it difficult to continuously supply the water-soluble protein necessary for the growth of lactic acid bacteria, and the growth is limited. Therefore, in the present invention, 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.

Second, 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).

At this time, for lyophilization, a binder (hydroxypropylmethylcellulose: HPMC) and a freeze-drying protectant (trehalose) may be sequentially added to prepare a powder, and for spray drying, a binder (hydroxypropylmethylcellulose: HPMC) can be added.

Hereinafter, a preferred embodiment of the present invention will be described in detail so that the present invention may be more easily understood. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, it is provided to sufficiently convey the spirit of the present invention to those skilled in the art so that the contents introduced herein are thorough and complete.

In addition, it is intended to prove that 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.

I. Preparation and efficacy experiment of lyophilized formulation

<Lyophilization Preparation Example 1: Lactic Acid Bacteria Basic Medium Preparation and Culture Method>

As a basic medium for culturing lactic acid bacteria in the present invention, glucose 3% (w/v) per 1 liter, yeast extract 2% (w/v), soypeptone 2% (w/v), casein 1% (w/v) Each component was dissolved in water and sterilized at 121-123°C for 30 minutes.

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.

<Lyophilization Preparation Example 2: Probiotic Powder Manufacturing Method>

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.

50% by weight of the thus stirred precipitate and 40% by weight of hydroxypropylmethylcellulose (HPMC) (dry weight, dissolved in 5 times the weight of water) were stirred for 20 minutes at 80 rpm, followed by 10% by weight of trehalose. After mixing, lyophilization was performed to obtain a dried probiotic powder.

<Lyophilization Example 1: Cultivation of lactic acid bacteria using lava seawater>

Water containing 10 to 90% (v/v) lava seawater, or lava seawater itself, was prepared as cultivation water, and the cultivation water was used instead of water under the conditions for preparing the basic medium used in Preparation Example 1.

Thus, the medium was sterilized as in Preparation Example 1 of freeze-drying using the culture water containing lava seawater at each concentration. On the other hand, when a sterilized medium is prepared using lava seawater, the minerals of lava seawater react with the proteins of the medium to produce a mineral-protein salt precipitated in a salt state.

Next, inoculate the lactic acid bacteria seed culture solution as in the freeze-dried preparation example 1 in the sterilized lava seawater medium, and cultivate it while adjusting the pH to 6.0-7.5 using a glucose-sodium hydroxide solution for 20 hours under conditions of 100 rpm and 37°C. I did.

<Lyophilization Example 2: Preparation of natural mineral coated probiotics derived from lava seawater>

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.

After the homogenization process was completed, 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.

<Lyophilization Experimental Example 1: Comparison of the amount of mineral-protein salt and the amount of protein according to the concentration of lava seawater in the medium before culture>

As already described in 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.

To measure this, only the sterilized freeze-dried base medium of Preparation Example 1 and the lava seawater-containing medium of Freeze-dried Example 1 were allowed to stand at 4° C. for 1 hour and then centrifuged at 6,000 rpm for 30 minutes to obtain mineral-protein The salt was recovered and the recovered product was lyophilized.

Next, 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.

The results of each experiment are shown in FIG. 1. According to the results of Figure 1, compared to the basic medium of lyophilized Preparation Example 1, mineral-protein salts in the lava seawater medium containing lava seawater by concentration, and the mineral-protein salt added amount of lava seawater 10 to 80% (v/v) Was found to increase in proportion to. Even at 90% (v/v) and 100% (v/v), the slight decrease in the amount of increase is believed to decrease the precipitation effect of protein salting out because the salt concentration in the medium is too high.

Through this, it is possible to check the amount of mineral-protein salts that can be used for the purpose of coating lactic acid bacteria when preparing a sterilized medium using lava seawater by concentration.

<Lyophilization Experimental Example 2: Confirmation of the number of lactic acid bacteria viable bacteria by lava seawater concentration in the medium>

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).

According to the results of Figure 2, compared to the number of lactic acid bacteria cultivated in lyophilized Preparation Example 1, the number of lactic acid bacteria cultivated in a medium to which 30% (v/v) lava seawater was applied in lyophilized Example 1 was explosively increased. It can be seen, and after that, the number of viable lactic acid bacteria appears to decrease. However, by confirming that the number of lactic acid bacteria in the culture medium increases by 5.0x10 ⁹ CFU/ml or more up to 70% (v/v) of lava seawater concentration, lava that can stably produce powders of 1.0x10 ⁸ CFU/g or more, which is the standard for viable bacteria of probiotics. The seawater concentration range could be confirmed.

It can be seen that the cultivation of lactic acid bacteria does not unconditionally increase even if the amount of lava seawater is increased, and it can be determined that the proper salt concentration in the lava seawater is an important factor in culturing lactic acid bacteria.

<Lyophilization Experimental Example 3: Comparison of the correlation between the dry weight of the mineral-protein salt and lactic acid bacteria cells in the culture medium according to the concentration of lava seawater and the number of lactic acid bacteria live 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.

According to the results of Figure 3, the dry weight of mineral-protein salts and lactic acid bacteria cells all look similar in the place containing 30-100% (v/v) lava seawater, and this result is 30-70% (v/v) lava In addition to the mineral-protein salts generated before cultivation in seawater culture medium, the number of viable cells exploded and the total weight became similar.

This result also proves that the dry weight of mineral-protein salts and lactic acid bacteria cells in the lava seawater culture medium of 80% (v/v) or more is similar to the weight of mineral-protein salts in the medium before cultivation, and that the number of viable cells is not increased.

Therefore, it will be recognized once again that 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.

<Lyophilization Experimental Example 4: Comparison of culture method using lava seawater (batch culture vs. Glucose/NaOH fed-batch culture)>

In the freeze-dried Preparation Example 1 and the freeze-dried Example 1, 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.

First, 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).

Thereafter, 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.

In addition, 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.

This is a freeze-dried Fed-batch culture (water) of Preparation Example 1, a Fed-batch culture of lyophilization Example 1 (lava seawater 30% (v/v)), and a batch culture of lyophilization Example 1 (lava seawater 30%). (v/v)) and shown in FIG. 4.

According to the results of FIG. 4, it can be seen that even if lava seawater was added, the strains hardly proliferated in batch culture. Therefore, it is understood that the components of the dried product recovered from batch culture are mostly mineral-protein salts in the medium, not lactic acid bacteria cells.

<Lyophilization Experimental Example 5: Confirmation of morphological characteristics of freeze-dried probiotic powder>

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.

Referring to Figure 5, 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.

<Lyophilization Experimental Example 6: Comparison of freeze-drying survival rate of natural mineral coated probiotics derived from lava seawater>

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.

division	Binder mixture (CFU/g)	Probiotics powder (CFU/g)	Survival rate (%)
Manufacturing Example 2	1.3x10 10	3.6x10 9	28
Example 2	4.1x10 11	3.2x10 11	78

According to the results of Table 1, it was confirmed that 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.

<Lyophilization Experimental Example 7: Intake stability of freeze-dried preparation of natural mineral coated probiotics derived from lava seawater>

Freeze-drying Experimental Example 7-1: Acid resistance (alone)

Using hydrochloric acid, the pH of 100 ml of Lactobacilli MRS broth (BD) was adjusted to 3.0 and then sterilized, and 0.4 g of pepsin (Sigma P7000, 250 Units/mg) was mixed with 100 ml of sterilized MRS broth to prepare an artificial gastric juice medium.

0.5 g of 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.

Thereafter, 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.

Each result is shown in Table 2 below.

division	Initial number of probiotics (CFU/g)	Acid resistance
		Initial (CFU/g)	After culture (CFU/g)	Survival rate (%)
Manufacturing Example 2	3.6 x 10 9	1.6 x 10 7	2.0 x 10 6	12.5
Example 2	3.2 x 10 11	1.0 x 10 9	4.8 x 10 8	48.0

According to the results of Table 2, it can be seen that the natural mineral-coated probiotic powder derived from lava seawater of lyophilized Example 2 has very better resistance to acidity than the probiotic powder of lyophilized Preparation Example 2 (about 50% survival rate confirmed, 35.5% increase compared to Preparation Example 2).

Freeze-drying Experimental Example 7-2: Bile acidity (alone)

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).

Thereafter, 0.5 g of the probiotic powders of Preparation Example 2 and Freeze-dried Example 2 were mixed in 100 ml of the artificial bile broth prepared afterwards, respectively, and the initial number of viable cells / number of viable cells after stationary culture for 2 hours were freeze-dried Experimental Example 7-1 It was confirmed as in, and it is shown in Table 3.

division	Initial number of probiotics (CFU/g)	Bile acid resistance
		Initial (CFU/g)	After culture (CFU/g)	Survival rate (%)
Manufacturing Example 2	3.6x10 9	2.2x10 7	3.1x10 7	141
Example 2	3.2x10 11	9.8x10 8	2.4x10 9	244

According to the results of Table 3, it is shown that the natural mineral coated probiotic powder derived from lava seawater of lyophilized Example 2 has about 103% better bile resistance than the probiotic powder of lyophilized Preparation Example 2.

Freeze-drying Experimental Example 7-3: Acid resistance-bile acid resistance (continuous)

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.

division	Initial number of probiotics (CFU/g)	Acid resistance-bile acid resistance
		Initial (CFU/g)	After culture (CFU/g)	Survival rate (%)
Manufacturing Example 2	3.6x10 9	1.6x10 7	1.2x10 7	75
Example 2	3.2x10 11	1.0x10 9	2.3x10 9	230

According to the results of Table 4, it was found that the natural mineral coated probiotic powder derived from lava seawater of lyophilized Example 2 has a very excellent survival rate difference of 155% or more because it has stronger acid resistance-bile resistance than the probiotic powder of lyophilized Preparation Example 2. It is possible to prove the excellence of the method for producing natural mineral coated probiotics derived from lava seawater of the present invention.

<Lyophilization Experimental Example 8: Storage stability of a freeze-dried formulation of natural mineral coated probiotics derived from lava seawater (comparison of stability over time according to temperature)>

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℃	15℃	25℃	35℃
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℃	15℃	25℃	35℃
Day 0 (CFU/g)	3.2x10 11	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 11	2.7x10 11	2.3x10 11	5.3x10 10
90 days (CFU/g)	3.0x10 11	2.5x10 11	2.1x10 11	4.8x10 10
120 days (CFU/g)	2.9x10 11	2.3x10 11	2.0x10 11	4.1x10 10
Survival rate (%)	90.6	71.9	62.5	12.8

According to the results of Table 5, it was confirmed that the natural mineral coated probiotic powder derived from lava seawater of lyophilized Example 2 has higher storage stability within 120 days than the probiotic powder of lyophilized Preparation Example 2.

<Lyophilization Experimental Example 9: Natural mineral content of freeze-dried formulation of natural mineral coated probiotics derived from lava seawater>

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.

division	Magnesium (mg/100g)	Calcium (mg/100g)
Lava seawater	116	42
Manufacturing Example 2	0.3	0.4
Example 2	182	55

According to the results of Table 6, it can be seen that magnesium and calcium were well transferred to the natural mineral coated probiotic powder derived from lava seawater of Example 2 of lyophilization due to the addition of lava seawater during culture. In addition, magnesium and calcium are representative components of natural minerals, and since this content value can represent the value of the content of natural mineral coated probiotics derived from lava seawater, additional experiments were repeatedly conducted. Natural mineral coated probiotics derived from lava seawater It can be understood that the mineral content of the powder is 0.1~0.5g/100g. At this time, it was also confirmed that magnesium was 0.07 ~ 0.4g / 100g, calcium 0.02 ~ 0.09g / 100g individually.

<Lyophilization Experimental Example 10: Confirmation of manufacturing conditions of natural mineral coated probiotics freeze-dried formulation derived from lava seawater by strain>

When lactic acid bacteria were cultivated under the conditions of lyophilization Example 1, it was confirmed that the lactic acid bacteria seed culture solution in addition to the Lactobacillus rhamnosus strains, as well as other types of lactic acid bacteria, were all smoothly cultured in the lava seawater addition medium.

At this time, as comparative conditions, the culture solution prepared by the cultivation method of lyophilization Preparation Example 1 was presented.

This result is shown in Figure 6, compared to the freeze-dried Preparation Example 1 culture medium, it can be seen that the number of lactic acid bacteria viable cells in all the culture solutions of the experiment was significantly increased under the culture conditions of the lava seawater addition medium of the freeze-dried Example 1.

This proves that the culture solutions for each strain under the above conditions are finally suitable conditions for producing natural mineral coated probiotics derived from lava seawater.

<Lyophilization Experimental Example 11: Generation of mineral-protein salts in desalted lava seawater medium and confirmation of the number of viable bacteria in the lactic acid bacteria culture medium>

Under the conditions of preparing the basic medium of Freeze-dried Preparation Example 1, a medium was prepared using desalted lava seawater (using the method of Example 1 of Korean Patent Registration No. 10-0947637) instead of water as culture water, and precipitated minerals by centrifugation- The dry weight of the protein salt was confirmed and shown in Table 7.

Culture water added to the medium	Mineral-protein salt (mg/ml)
Lava seawater 30% (v/v) (freeze drying Example 1 condition)	16
Desalted lava seawater 100(v/v)	8

In addition, the number of viable lactic acid bacteria in the culture medium obtained by culturing Lactobacillus rhamnosus spp. in the medium of Table 7 was measured. These values are shown in Table 8.

Culture medium	Lactobacillus viable cell count (CFU/ml)
Cultured with 30% (v/v) of lava seawater (freeze-dried Example 1 condition) as culture water	2 x 10 10
Cultured with 100% (v/v) desalted lava seawater as culture water	9 x 10 8

Checking the results in Tables 7 and 8, the result of using desalted lava seawater is similar to that of lyophilization Preparation Example 1 using water as culture water, and has little effect on the production of mineral-protein salts and proliferation of lactic acid bacteria. It is understood to be.

II. Preparation and efficacy test of spray-dried formulation

<Spray drying Preparation Example 1: Preparation and culture method of lactic acid bacteria basic medium for spray drying>

As a basic medium for culturing lactic acid bacteria in the present invention, glucose 3% (w/v) per 1 liter, yeast extract 2% (w/v), soypeptone 2% (w/v), casein 1% (w/v) Each component was dissolved in water so as to be sterilized at 121°C for 30 minutes.

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).

<Spray-dried manufacturing example 2: Probiotics spray-dried powder manufacturing method>

Spray-drying 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㎛, 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.

<Spray drying Example 1: Culture of lactic acid bacteria using lava seawater>

Water containing 10 to 90% (v/v) lava seawater, or lava seawater itself, was prepared as cultivation water, and the cultivation water was used instead of water in the basic medium preparation conditions used in Spray-drying Preparation Example 1.

In this way, the medium was sterilized as in Preparation Example 1 by spray drying using the culture water containing lava seawater for each concentration. On the other hand, when 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.

Next, 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.

<Spray drying Example 2: Preparation of spray-dried probiotics with natural mineral coating derived from lava seawater>

Spray drying 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. After obtaining the concentrate, 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. At this time, in the process of concentration, the mineral-protein salt coats the outside of the cells, and hydroxypropylmethylcellulose acts as a binder that makes the coating more robust.

<Spray drying Experimental Example 1: Comparison of mineral-protein salt amount and protein amount according to sterilization temperature and lava seawater concentration in the medium before culture>

Spray drying Experimental Example 1-1: Lava seawater concentration

As already described in 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.

To measure this, as a sterilized medium before cultivation of lactic acid bacteria, 400 ml of only the basic medium of Spray-drying Preparation Example 1 and the medium containing lava seawater of Spray-drying Example 1 were dispensed into separate containers, respectively, and 1 hour at 4°C. After standing, the mineral-protein salt was recovered by centrifugation at 6,000 rpm for 30 minutes, and the recovered product was spray-dried.

Next, 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.

The results of each experiment are shown in Table 9.

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)
0	0.4	0.1
10	3.2	1.6
20	5.2	2.5
30	11.8	5.9
40	13.2	6.4
50	16.5	7.7
60	17.4	8.3
70	18.4	9.4
80	18.2	9.3
90	17.9	8.7
100	17.7	8.5

According to the results of Table 9, mineral-protein salts in the lava seawater medium containing lava seawater by concentration compared to the basic medium of Spray-drying Preparation Example 1, and the mineral-protein salt added amount of lava seawater 10 to 70% (v/v) Was found to increase in proportion to. When the lava seawater concentration is 80% (v/v), the amount of increase is stagnant, or even when the amount of lava seawater is added at 90% (v/v) and 100% (v), the increase in the amount decreases. It is believed to have not increased or decreased.

Through this, when preparing a sterilized medium using lava seawater for each concentration, it is possible to check the amount of mineral-protein salt that can be used for coating purposes of lactic acid bacteria, and to determine an appropriate concentration as culture water (lava seawater 30~ 70% (v/v) is appropriate).

Spray drying Experimental Example 1-2: Sterilization temperature

In order to compare the amount of mineral-protein salt production according to the sterilization temperature, 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 (℃)	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℃	30	3.5	1.9
105℃	30	5.4	2.9
110℃	30	7.4	3.1
115℃	30	9.8	3.8
121℃	30	11.8	5.9
121℃	0	0.5	0.2

Looking at the results of Table 10, when the amount of pure protein in the mineral-protein salt was also used at 30 (v/v)% of lava seawater as the culture water, the amount of pure protein in the mineral-protein salt in the medium sterilized at 121°C. It is the most common, and it can be seen that mineral-protein salts are formed even at temperatures above 100°C.

However, in the case of using water (lava seawater concentration of 0%), it was found that a sufficient amount of mineral-protein salt required for coating was not generated even when sterilized at 121°C.

<Spray drying Experimental Example 2: Confirmation of the number of lactic acid bacteria viable bacteria by lava seawater concentration in the medium>

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).

Lava seawater concentration (%, v/v)	Viable bacteria count in spray-dried powder (×10 9 CFU/g)
0	11
10	15
20	34
30	212
40	164
50	131
60	122
70	106
80	47
90	26
100	24

According to the results of Table 11, compared to the number of lactic acid bacteria cultivated in Spray-drying Preparation Example 1, the number of lactic acid bacteria cultivated in a medium to which 30% (v/v) lava seawater was applied in spray-drying Example 1 was explosively increased. It can be seen, and after that, the number of viable lactic acid bacteria appears to decrease.

However, up to 70% (v/v) concentration of lava seawater, 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.

In addition, it can be seen that the cultivation of lactic acid bacteria does not unconditionally increase even if the amount of lava seawater is increased, and it can be determined that the proper salt concentration in the lava seawater is an important factor in culturing the lactic acid bacteria.

In addition, through these results, it can be confirmed that the mineral-protein salt has a heat protection function.

<Spray drying Experimental Example 3: Comparison of the correlation between the spray-dried weight of mineral-protein salts and lactic acid bacteria cells in the culture medium according to the concentration of lava seawater and the number of lactic acid bacteria viable cells>

Spray-drying 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.

According to the results of Figure 7, the dry weight of'mineral-protein salts and lactic acid bacteria cells' looks similar in all places where 30-100% (v/v) of lava seawater is included, and this result is 30-70% (v/v ) In addition to the mineral-protein salts produced before cultivation under the condition of lava seawater culture, the number of viable bacteria exploded and the total weight became similar.

This result also proves that the dry weight of mineral-protein salts and lactic acid bacteria cells in the lava seawater culture medium of 80% (v/v) or more is similar to the weight of mineral-protein salts in the medium before cultivation, and that the number of viable cells is not increased.

Therefore, once again that 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.

<Spray drying Experimental Example 4: Comparison of culture method using lava seawater (batch culture vs. Glucose/NaOH fed-batch culture)>

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.

First, in the spray-drying Example 1, 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) and 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).

Thereafter, 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. In addition, 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㎛, manufacturer DOW separation systems, Serial No.546/57 205-92.) After obtaining the concentrate, 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.

This is spray-dried Fed-batch culture (water) of Preparation Example 1, Fed-batch culture of Spray-drying Example 1 (lava seawater 30% (v/v)), and Spray-drying Example 1 batch culture (lava seawater 30%) (v/v)) and shown in FIG. 8.

According to the results of FIG. 8, it can be seen that even if lava seawater was added, the strains hardly proliferated in batch culture. Therefore, it is understood that the components of the dried product recovered from batch culture are mostly mineral-protein salts in the medium, not lactic acid bacteria cells.

A picture of each cell was taken using a Scanning Electron Microscope (SEM), but the bacillus state was taken rather than the surface of the cell. As shown in FIG. 9, it was confirmed that the mineral salt was bound to the cell of the spray-dried powder. I can.

<Spray drying Experimental Example 5: Comparison of spray drying survival rate of natural mineral coated probiotics derived from lava seawater>

Spray drying In Preparation Example 2 and Spray Drying Example 2, ① a mixture of a binder (hydroxypropylmethylcellulose) before spray drying was performed; ② After spray drying, the number of viable bacteria of each lactic acid bacteria in probiotics powder; was compared to confirm the spray drying survival rate by the natural mineral coating derived from lava seawater.

At this time, in order to confirm whether large-capacity production is possible, the culture medium cultured in a mass of 500 L was concentrated and tested using a membrane filtration concentrator.

The experimental results are shown in Table 12 by measuring the solid content of each binder mixture by the loss-on-drying method to calculate the accurate survival rate and converting it to the number of lactic acid bacteria viable bacteria in the binder mixture to obtain the survival rate.

division	Spray-drying Preparation 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

According to the results of Table 12, 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.

Therefore, 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.

<Spray drying Experimental Example 6: Intake stability of spray-dried formulation of natural mineral coated probiotics derived from lava seawater>

Spray drying Experimental Example 6-1: Acid resistance (alone)

Using hydrochloric acid, the pH of 100 ml of Lactobacilli MRS broth (BD) was adjusted to 3.0 and then sterilized, and 0.4 g of pepsin (Sigma P7000, 250 Units/mg) was mixed with 100 ml of sterilized MRS broth to prepare an artificial gastric juice medium. .

0.5 g of 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.

After that, 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.

Each result is shown in Table 13 below.

division	Initial number of probiotics (CFU/g)	Acid resistance
		Initial (CFU/ml)	After incubation (CFU/ml)	Survival rate (%)
Manufacturing Example 2	1.1×10 10	5.0×10 8	3.1×10 7	6.2
Example 2	2.1×10 11	1.2×10 9	5.9×10 8	49.1

According to the results of Table 13, it can be seen that the natural mineral-coated probiotic powder derived from lava seawater of spray-dried Example 2 has much better resistance to acidity than the probiotic powder of Spray-dried Preparation Example 2 (about spray drying Example 2 Confirmation of survival rate of 49%, 7.9 times increase compared to Preparation Example 2).

Spray drying Experimental Example 6-2: Bile acidity (alone)

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).

Subsequently, 0.5 g of the probiotic powders of Preparation Example 2 and Spray Drying Example 2 were mixed in 100 ml of the prepared artificial bile broth, respectively, and the initial viable cell count/the viable cell count after stationary culture for 2 hours was spray-dried Experimental Example 6- It was confirmed as in 1, and it is shown in Table 14.

division	Initial number of probiotics (CFU/g)	Bile acid resistance
		Initial (CFU/g)	After culture (CFU/g)	Survival rate (%)
Manufacturing Example 2	1.1×10 10	5.0×10 8	4.1×10 8	82.0
Example 2	2.1×10 11	1.2×10 9	1.9×10 9	158.3

According to the results of Table 14, it was found that the natural mineral coated probiotic powder derived from lava seawater of spray-dried Example 2 had 1.9 times better bile acid resistance than the probiotic powder of Spray-dried Preparation Example 2.

Spray drying Experimental Example 6-3: Acid resistance-bile acid resistance (continuous)

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.

division	Initial number of probiotics (CFU/g)	Acid resistance-bile acid resistance
		Initial (CFU/g)	After culture (CFU/g)	Survival rate (%)
Manufacturing Example 2	1.1×10 10	5.0×10 8	1.1×10 8	22.0
Example 2	2.1×10 11	1.2×10 9	1.6×10 9	133.3

According to the results of Table 15, it was found that the natural mineral coated probiotic powder derived from lava seawater of spray-dried Example 2 has a very excellent survival rate difference of 133% or more because it has stronger acid resistance-bile resistance than the probiotic powder of Spray-dried Preparation Example 2. It is possible to prove the excellence of the method for producing natural mineral coated probiotics derived from lava seawater of the present invention.

<Spray drying Experimental Example 7: Storage stability of spray-dried formulation of natural mineral coated probiotics derived from lava seawater (comparison of stability over time according to temperature)>

Spray-drying Preparation 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℃	15℃	25℃	35℃
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℃	15℃	25℃	35℃
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)	2.1×10 11	1.7×10 11	1.4×10 11	9.7×10 10
90 days (CFU/g)	2.0×10 11	1.5×10 11	1.1×10 11	4.5×10 10
120 days (CFU/g)	2.0×10 11	1.5×10 11	1.0×10 11	2.2×10 9
Survival rate (%)	95.2	71.4	47.6	10.4

According to the results of Table 16, it was confirmed that the natural mineral coated probiotic powder derived from lava seawater of spray-dried Example 2 has higher storage stability within 120 days than the probiotic powder of Spray-dried Preparation Example 2.

<Spray drying Experimental Example 8: Mineral content of spray-dried formulation of natural mineral coated probiotics derived from lava seawater>

The contents of magnesium and calcium in the probiotic powders of Lava Seawater (Lava Seawater without culturing the strain itself), spray-dried Preparation Example 2, and Spray-dried Example 2 were analyzed according to the Magnesium and Calcium Test Method of the Health Functional Food Code of the Ministry of Food and Drug Safety. Table 17 shows the amount of probiotics contained in natural mineral components derived from lava seawater.

division	Magnesium (mg/100g)	Calcium (mg/100g)
Lava seawater (lava seawater itself without cultivating strains)	114.2	40.1
Probiotic powder of spray-dried preparation example 2	0.2	0.3
Spray-dried probiotic powder of Example 2	179.5	54.2

According to the results of Table 17, it can be seen that magnesium and calcium were well transferred to the natural mineral coated probiotic powder derived from lava seawater of Example 2 of spray drying due to the addition of lava seawater during culture.

On the other hand, 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.

<Spray drying Experimental Example 9: Confirmation of the manufacturing conditions of a spray-dried formulation of natural mineral coated probiotics derived from lava seawater by strain>

When culturing lactic acid bacteria under the conditions of spray-drying Example 1, it was confirmed that the lactic acid bacteria seed culture solution in addition to the Lactobacillus rhamnosus strains, as well as other types of lactic acid bacteria, were all cultured smoothly in the lava seawater addition medium.

At this time, a culture solution prepared by the cultivation method of Spray-drying Preparation Example 1 was presented as a comparative condition.

This result is shown in Fig. 10, compared with the spray-dried Preparation Example 1 culture solution, it can be seen that the number of lactic acid bacteria viable bacteria in all the culture solutions of the experiment is significantly increased under the culture conditions of the lava seawater-added medium of spray-dried Example 1.

This proves that the culture solutions for each strain under the above conditions are finally suitable conditions for producing natural mineral coated probiotics derived from lava seawater.

<Spray drying Experimental Example 10: Generation of mineral-protein salts in desalted lava seawater medium and confirmation of the number of viable bacteria in the lactic acid bacteria culture medium>

Under the conditions of preparing the basic medium of Spray-drying Preparation Example 1, a medium was prepared using desalted lava seawater (using the method of Example 1 of Korean Patent Registration No. 10-0947637) instead of water as culture water, and the mineral precipitated by centrifugation- The dry weight of the protein salt was confirmed and shown in Table 18.

Culture water added to the medium	Mineral-protein salt (mg/ml)
Lava seawater 30% (v/v) (spray drying Example 1 condition)	11.8
100% desalted lava seawater	0.4

In addition, 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.

Culture medium	Lactobacillus powder viable cell count (CFU/g)
Cultured using 30% (v/v) of lava seawater (spray-dried Example 1 condition) as culture water	2.1 × 10 11
Cultured with 100% (v/v) desalted lava seawater as culture water	1.2 × 10 10
Cultured with water for cultivation using constant 100% (v/v) (Spray-dried Preparation Example 1 condition)	1.1 × 10 10

Checking the results in Tables 18 and 19, the result of using desalted lava seawater is similar to that of Spray-drying Preparation Example 1 using water as culture water, and has little effect on the production of mineral-protein salts and proliferation of lactic acid bacteria. It is understood to be.

<Spray drying Experimental Example 11: Confirmation of the formation of self-defense protein inside the cells by lava seawater addition culture>

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.

Specifically, 100 mg of the cells of the spray-dried Preparation Example 1 and 100 mg of the cells of the spray-drying Example 1 were collected, washed three times with PBS (phosphate buffered saline, pH 7.2), and then 500 µl lysis buffer (50 mM Tris-HCl). , 0.3% SDS, 200 mM DTT, 0.4 mM PMSF, pH 7.5). The lactic acid bacteria suspension was transferred to a 2 ml micro cap tube containing 250 mg zirconium bead, and the cells were crushed at 4°C to obtain a cell lysate. After heat treatment at 95° C. for 10 minutes from the cell lysate, it was centrifuged again at 4° C. and 12,000 rpm to obtain only the supernatant. From the supernatant containing protein, remove salt through a desalting column, precipitate only protein with acetone, wash with ethanol, dry at room temperature, and IEF (isoelectric focusing) solution (8M urea, 0.12%(w/v) destreak solution). , 4% (w/v) CHAPS, 2% (w/v) IPG buffer), and cup loading 100-150 ㎍ of protein into an immobiline dry strip in the range of pI 3-10 to perform IEF, and after completion, The strip was gel-imaged by SDS-PAGE to confirm that there was a new protein expressed by the lava seawater culture method (FIG. 11), and the gene expression change of the representative chaperone protein in the following experiment to clarify whether the chaperone protein increased due to stress. Was analyzed.

CDNA synthesis method and electrophoresis to confirm whether the expression of GroEL gene that promotes typical cell division and defense mechanisms as a stress-responsive microbial self-defense protein (chaperone) that can withstand spray drying conditions in the process of cultivation through lava seawater is increased. The change in the level of mRNA expression before and after the reaction in constant water and lava seawater was compared with gel images using the Yeongdong method.

Specifically, 100 mg of the cells of the spray-dried Preparation Example 1 and 100 mg of the cells of the spray-drying Example 1 were collected, washed three times with PBS (phosphate buffered saline, pH 7.2), and then 500 µl lysis buffer (50 mM Tris-HCl). , 0.3% SDS, 200 mM DTT, 0.4 mM PMSF, pH 7.5). After centrifugation again at 4°C and 12,000 rpm to remove the supernatant, 1 ml of quiazole (QIAzol, QIAGEN) was added to isolate and quantify RNA according to Qiagen's RNA separation method, and then cDNA was synthesized with 1 μg of RNA. The band amplification pattern was confirmed by electrophoresis on 1.5% agarose gel (Fig. 6). Primers used for PCR were synthesized and used by Macrogen, and the primer sequences are shown in Table 20 below.

gene	Forward	Reverse
GroEL	5’-GAAGGTATGAAGAACGTC-3’	3’-CTTGTTCTAAGCACCGTG-5’

Referring to FIG. 12, as the mRNA expression of the GroEL gene in the spray-dried Example 1 increased than the spray-dried Preparation Example 1 cultured cells, 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.

As described above, the preparation examples, examples, and experimental examples have been referenced and described through freeze-drying and spray-drying for the present invention, but these are merely exemplary, and those of ordinary skill in the art pertaining to the present invention If so, it will be understood that various modifications and equivalent other embodiments are possible therefrom. Therefore, the true technical protection scope of the present invention should be determined by the technical details of the appended claims.

Claims (15)

1. (Step 1) preparing a culture medium for lactic acid bacteria prepared by using water containing 30-70% (v/v) lava seawater as culture water; And,

   (Second step) obtaining a culture solution by inoculating and culturing the lactic acid bacteria in the culture medium for lactic acid bacteria;

   Lava seawater-derived natural mineral coating method for producing probiotics comprising a.
2. The method of claim 1,

   (3rd step) drying the precipitate obtained by centrifuging the culture solution of the second stage, or concentrating and drying the culture solution of the second stage;

   Lava seawater-derived natural mineral coating method for producing probiotics comprising a.
3. The method of claim 2,

   The drying is a method for producing natural mineral coated probiotics derived from lava seawater, characterized in that the drying is carried out through freeze drying or spray drying.
4. The method of claim 1,

   The medium of the first step contains, as constituents, glucose, yeast extract, soypeptone, and casein, and the constituents are dissolved in water containing 30 to 70% (v/v) of lava seawater and sterilized to mineral- A method for producing natural mineral coated probiotics derived from lava seawater, characterized in that it is a medium for culturing lactic acid bacteria in which protein salts are formed.
5. The method of claim 1,

   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.
6. The method of claim 1,

   The second step of culturing the lactic acid bacteria is a method for producing natural mineral coated probiotics derived from lava seawater, characterized in that it is carried out by a fed-batch culture method.
7. The method of claim 2,

   The sediment recovered through centrifugation in the third step is a mixture of mineral-protein salts and cells, and the process of obtaining the mineral-protein coated cells is further performed through homogenization according to the stirring process after the sediment is recovered. A method for producing natural mineral coated probiotics derived from lava seawater.
8. The method of claim 7,

   In order to stabilize the coating of cells coated with mineral-protein salts,

   A binder selected from the group consisting of hydroxypropylmethylcellulose (HPMC), carboxymethylcellulose (CMC), polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), and chitosan; And,

   Trehalose; method for producing a natural mineral coated probiotics derived from lava seawater, characterized in that the addition.
9. The method of claim 8,

   The binder is in the form of a solution dissolved in water containing 30 to 70% (v/v) of lava seawater, and a binder solution is first added to the cells coated with mineral-protein salts, stirred, and then trehalose is mixed and dried. A method for producing natural mineral coated probiotics derived from lava seawater.
10. The method of claim 2,

    When the concentration in the third step, the culture medium is concentrated while homogenizing the mineral-protein salt is coated on the outside of the cells, characterized in that the production method of natural mineral coated probiotics derived from lava seawater.
11. The method of claim 10,

    After concentration, in order to stabilize the coating of the cells, as a binder, from the group consisting of hydroxypropylmethylcellulose (HPMC), carboxymethylcellulose (CMC), polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA) and chitosan Method for producing a natural mineral coated probiotics derived from lava seawater, characterized in that at least one is added.
12. Lava seawater-derived natural mineral coated probiotic powder prepared by the method of claim 1.
13. The method of claim 13,

    Natural mineral coated probiotics derived from lava seawater, characterized in that the mineral content of the natural mineral coated probiotics powder derived from lava seawater is 0.1 to 0.5% by weight.
14. A food containing the natural mineral coated probiotic powder derived from lava seawater of claim 13.
15. The method of claim 14,

    The food is a food, characterized in that for the growth of lactic acid bacteria in the intestine, for suppressing harmful bacteria in the intestine, or for promoting bowel activity.

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