KR20210107435A - Method of preparing lava seawater natural mineral coating probiotics and spray-drided formulations of lava seawater natural mineral coating probiotics thereby - Google Patents

Abstract

The present invention uses a method of adding lava seawater so that a concentration of lava seawater in a medium becomes 30-70% (v/v) for culturing lactic acid bacteria, thereby precipitating natural minerals contained in lava seawater and proteins on the medium of lactic acid bacteria in a salted out state, and adjusting the salinity of the medium to the concentration at which lactic acid bacteria can grow. In addition, as the lactic acid bacteria are cultured, chaperones, a stress-responsive protein, are generated in the cells due to the stress caused by salt on the salt concentration-controlled medium. By concentrating the culture solution after incubation so that the salted out protein generated during medium sterilization is evenly coated on the outside of the cells, the thermal stability of the cells is significantly increased inside and outside to suppress the death of lactic acid bacteria due to the generation of high temperature during spray drying. Accordingly, it is possible to derive a method of preparing probiotics containing the effect of remarkably improving the storage stability of the spray-dried lactic acid bacteria over time and the effect of withstanding the continuous pH change of the digestive tract of stomach and bile acids in the body. According to the present invention, natural mineral-coated probiotics derived from lava seawater with excellent storage stability against external temperature changes can be produced and effectively used as biomaterials in food, health functional food and pharmaceutical fields.

Description

Method of preparing lava seawater natural mineral coating probiotics and spray-drided formulations of lava seawater natural mineral coating probiotics thereby}

The present invention uses lava seawater as culturing water without separate pretreatment to produce natural mineral-coated lactic acid bacteria derived from lava seawater, thereby simultaneously increasing the survival rate, storage stability over time, and intake stability during spray drying, and furthermore, causes electrolyte imbalance through natural minerals It relates to a method for producing natural mineral-coated probiotics derived from lava seawater that can maximize the efficacy of intestinal probiotics by balancing diarrhea, etc.

Probiotics refer to functional lactic acid bacteria in powder form that play a role in helping the smooth bowel movement and proliferation of lactic acid bacteria in the intestine, mainly among health functional foods, unlike the manufacturing method of yoghurt like general yogurt. In particular, in 2002, the World Health Organization (WHO/FAO) defined probiotics as microorganisms that benefit the health of the host when consumed in an appropriate amount as live cells, and became popular. In Korea, it is listed as a raw material for health functional food by the Ministry of Food and Drug Safety, and the daily intake of live probiotics for intestinal health is defined as more than 100 million CFU/day (CFU: Colony Forming Unit).

In recent years, the incidence of colon diseases due to westernized eating habits and a lot of stress is increasing, and in particular, the prevalence of irritable bowel syndrome and inflammatory bowel disease is increasing. One of the characteristics of these intestinal diseases is diarrhea, which disrupts the electrolyte balance in the large intestine, resulting in diarrhea accompanied by water. In this case, it becomes difficult for probiotics input from the outside to settle in the normal intestine. Therefore, the use of probiotics in people with these diseases is necessary to primarily restore electrolyte balance to show the effect of probiotics.

In order to have a smooth intestinal probiotic effect, it is necessary to ingest more than a certain number of live bacteria. In most cases, it is difficult to expect the effect because the number of bacteria reaching the intestine is insufficient as the number of bacteria reaching the intestine is insufficient during passage through the gastrointestinal tract. Therefore, in the industry, there was an effort to overcome the method of increasing the survival rate of lactic acid bacteria during powder manufacturing as an important issue along with the development of a method for culturing lactic acid bacteria with a high concentration of bacteria. For the production of live cell powder formulations, various industrialized cell culture methods, tablet recovery processes, coating methods, etc. have been continuously optimized to increase the survival rate of lactic acid bacteria, which is related to the shelf life and stability of the product.

As a technology for high concentration of lactic acid bacteria in culture technology, in order to stably manufacture more than 100 million CFU/day (CFU: Colony Forming Unit) of live probiotics, it is necessary to culture the number of bacteria in the culture medium at a high concentration of 5 billion CFU/ml or more. , as a prior art, there is a technology for enhancing stability through the addition of proline to increase the number of lactic acid bacteria (Korean Patent Registration No. 10-1605516). However, this method has been pointed out as a problem that it is difficult to use for mass production because the effect of this method is insignificant compared to the economical efficiency as proline amino acid is input in ton unit production.

As another prior art field, a coating technology can be exemplified as a method of increasing the survival rate of lactic acid bacteria during powder manufacturing. As a conventional technique, a quadruple coating lactic acid bacteria manufacturing technology (Korean Patent Registration No. 10-1280232) is representative.

The coating technology in the prior art of lactic acid bacteria is to laminate a coating agent on the lactic acid bacteria exhibiting anaerobic characteristics, thereby increasing storage stability by extending the shelf life. Therefore, when the coating layer collapses due to the uniformity of the coating and external moisture inflow, it is inevitably vulnerable to exposure to external air, resulting in the death of lactic acid bacteria exhibiting anaerobic properties. As such, the prior art focuses only on storage stability by forming a protective film through the coating agent in the culture process step and post-treatment process step, rather than improving the resistance ability of the lactic acid bacteria itself to external aggressors such as air, temperature, stomach acid, and bile acid. However, the durability of the lactic acid bacteria itself is not secured when symptoms that cause intestinal electrolyte imbalance such as diarrhea occur or when the coating film is peeled off during continuous passage through the gastrointestinal tract. It has been pointed out that there are technical limitations in overcoming the stress of the environment.

As a method of overcoming stress by enhancing the vitality of lactic acid bacteria itself, stress such as heat shock, osmotic shock, and acid shock was presented as an alternative to overcoming anaerobic conditions by short-term treatment to the critical condition for survival. However, in industrial production conditions, it is difficult physically and mechanically to apply rapid stress to lactic acid bacteria for a short time within a few minutes, and commercialization has not been achieved due to cell death during stress treatment and loss of cell mass in the process of removing stress factors.

In the case of freeze-drying, in order to produce a current probiotic preparation, an upstream process of culturing in a large-capacity fermenter and a downstream process of recovering cells by centrifugation, etc., removing water from the recovered cells, and making dry powder (downstream process), at this time, since the upstream process is usually used in ton units, if the processing capacity of the downstream does not reach this level, if the processing capacity is 100 ~ 500L, when the ton unit culture is performed, it is all freeze-dried powder Even if it is difficult to produce with a refrigeration system, or freeze-drying, a production bottleneck that takes a long time occurs.

Therefore, as a way to solve the imbalance of the production bottleneck between the upstream process (cultivation process) and the downstream process (drying process), the production of live cells through spray drying can be considered. However, spray drying can be dried directly in the original volume without concentrating the culture solution, so time and production bottlenecks can be solved at once. Because it is a method to remove and dry the nozzle, the instantaneous temperature of the nozzle rises by more than 100°C, killing lactic acid bacteria with an average growth temperature of 30-50°C, and the survival rate is low.

As a conventional technique of spray-drying the production of lactic acid bacteria probiotics, Streptococcus thermophilus with excellent survival rate during spray-drying and its use (Korean Patent Registration No. 10-1699741) are suggested, but this is to discover strains with excellent thermal stability In addition to the use of Streptococcus thermophilus, which has higher stability than the low-temperature lactic acid bacteria of the Lactobacillus sp. family with relatively low thermal stability, the use of 16% reduced skim milk as a culture medium reduces during spray drying. As the protective effect of skim milk, only the survival rate during initial spray drying is indicated, and there is no mention about the stability over time according to temperature and time related to the shelf life. It cannot be said to be a fundamental solution because the range is limited for the purpose of using it for food yogurt, etc., which is not related to the standard range of 100 to 10 billion CFU/day.

Lava seawater is water that has been aged for a long time by flowing into the stratum as seawater is naturally filtered through the basalt and sanitary layers. It is a unique water resource unique to Jeju Island. has been actively used as a breeding water for halibut farms, and from 5 to 6 years ago, it has been used as water for saunas, etc. For lava seawater, demineralized lava seawater from which salt has been removed or used as it is may be used, depending on the purpose of use.

Lava seawater contains not only essential natural minerals such as sodium, magnesium, calcium, and potassium, but also general useful mineral components (iron, manganese, zinc, molybdenum, selenium, etc.) Among them, vanadium, which is known to stabilize insulin secretion or to improve diabetes, hyperlipidemia, etc., germanium, which promotes blood circulation, enhances immunity, and has anticancer action, suppresses oxidation of fat, synergistic effect to maintain the heart and liver, and eliminates radicals The content of selenium, which has the ability to improve anticancer, fertility, 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, phosphorus phosphate, phenols, etc. are not detected, and harmful components such as arsenic, mercury and cadmium are not detected or lead is detected in very small amounts, so there are obstacles to industrial application. It is a clean raw material.

In particular, natural minerals such as magnesium and calcium contained in lava seawater are essential for the growth of lactic acid bacteria, and are used as essential nutrients for humans as well. However, when lava seawater is applied as water for culturing lactic acid bacteria by itself, there is a technical limitation (LJM Linders et al., 1997) that makes it impossible to cultivate high concentration of lactic acid bacteria because the lactic acid bacteria are inhibited by the high concentration of salt in the lava seawater (LJM Linders et al., 1997). In order to do this, water in which the demineralized water manufacturing method was introduced was used as in the prior art (Korean Patent Registration No. 10-1347694). However, this prior art has economic/technical limitations such as the cost for desalting lava seawater and reduction of the mineral content in the desalted lava seawater.

Therefore, the present invention uses natural lava seawater that is edible and contains natural minerals beneficial to our body to induce salting out lactic acid bacteria that cannot survive at high concentrations in a medium with high salt concentration to control the salt concentration to enable rapid proliferation of cells. and chaperone, a stress-responsive protein, is generated inside and outside the cells due to the salt concentration, so survival stability and storage stability during distribution are increased even when spray drying is performed. Thus, a new form of probiotics was commercialized by devising a method to enhance stability during ingestion.

Republic of Korea Patent Registration No. 10-1280232 (Title of the invention: manufacturing method of quadruple-coated lactic acid bacteria and quadruple-coated lactic acid bacteria manufactured by the method, Applicant: Ildong Pharmaceutical Co., Ltd., registration date: June 25, 2013) Republic of Korea Patent Registration No. 10-1605516 (Title of the invention: method for increasing the survival rate, storage stability, acid resistance or bile resistance of lactic acid bacteria, Applicant: Chong Kun Dang Bio Co., Ltd., Registration date: March 16, 2016) Republic of Korea Patent No. 10-1347694 (Title of the invention: Method for manufacturing a fermented product of desalted lava seawater, a fermented product obtained by the method and a cosmetic composition using the fermented product, Applicant: Jeju Technopark Foundation, Registration date: December 2013 27th of month) Republic of Korea Patent No. 10-1927859 (Title of the invention: Method for increasing the stability and coating efficiency of probiotics using ultrasound and food composition containing freeze-dried probiotic powder prepared by the method as an active ingredient, Applicant: Korea Yakult Co., Ltd. , registration date: December 05, 2018) Korean Patent Laid-Open Patent No. 10-2002-0097237 (Title of the invention: Drying of cells by spray drying, Applicant: Biofermin Seiyaku Co., Ltd., Publication date: December 31, 2002) Republic of Korea Patent Registration No. 10-0530279 (Title of the invention: Manufacturing method of formulation containing lactic acid bacteria powder and novel use of lactic acid bacteria powder, Applicant: Jeong Myung-jun, Registration date: November 15, 2005) Republic of Korea Patent Publication No. 10-2019-0008597 (Title of the invention: culturing method of lactic acid bacteria using culture medium containing deep sea water and medium composition for culturing lactic acid bacteria, Applicant: Bifido Co., Ltd. and one other person, Publication date: January 25, 2019 Work) Republic of Korea Patent Registration No. 10-1699741 (Title of the invention: Streptococcus thermophilus with excellent survival rate during spray drying and use thereof, Applicant: Myung-Jun Jung, Registration date: 10-1699741)

L.J.M. 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 use lava seawater as culture water without separate pretreatment to coat lactic acid bacteria through salting out proteins of natural minerals and medium, and through chaperones generated in the cells during culture, survival rate during spray drying, storage stability over time , to provide a method for producing natural mineral-coated probiotics derived from lava seawater having the effect of simultaneously increasing intake stability, and a spray-dried formulation of natural mineral-coated probiotics derived from lava seawater using the same.

Therefore, in the production of raw probiotics through the present invention, a consistent process using lava seawater of a specific concentration is implemented in the process of the pre-process and the post-process, and the outer coating of the cell body and the inside of the cell body of the salted-out protein formed by the lava seawater The generated stress-responsive protein, chaperone, dramatically improves the survival rate and storage stability over time against high-temperature stress, which is a peripheral factor of lactic acid bacteria during spray drying, It is possible to provide a method for manufacturing natural mineral-coated probiotics derived from lava seawater that simultaneously increases the stability of intake against stress.

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 medium for culturing lactic acid bacteria prepared by using water containing 30-70% (v/v) lava seawater as culture water;

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

(Third step) spray-drying the concentrate obtained by concentrating the culture solution; may include.

The medium of the first step may be any medium capable of culturing lactic acid bacteria, but more preferably, glucose, yeast extract, soipeptone, and casein are included as components, and the components are lava seawater It may be a medium for culturing lactic acid bacteria prepared by dissolving in water containing 30 to 70% (v/v) and sterilizing.

Even more preferably, the medium of the first step is based on a total volume of 1 L, glucose 1-5% (w/v), yeast extract 0.5-5% (w/v), soy peptone 0.5-5% (w/v) ), it may be a medium for culturing lactic acid bacteria prepared by dissolving each component in water containing 30 to 70% (v/v) of lava seawater and sterilizing it so that it becomes 0.5 to 3% (w/v) of casein.

The medium sterilization is preferably carried out at 100 ~ 125 ℃ for 20 ~ 40 minutes. At this time, the pressure is preferably 0.13 to 0.17 Mps, and most preferably 0.15 Mps, which is the optimum sterilization condition, can be performed at 121° C. for 30 minutes.

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.), the genus Pediococcus ( Pediococcus sp.) and the genus Weissella ( Weissella sp.) may be selected from the group consisting of. The lactic acid bacteria culture conditions of the second step are preferably 18 to 24 hours at 70 to 150 rpm and 30 to 37 ° C. The lactic acid bacteria culture of the second step is characterized in that it is performed by a fed-batch culture method. To this end, a method of maintaining the pH at 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 a mixture of a glucose solution and a sodium hydroxide solution in a volume ratio of 1:0.5 to 1:2, respectively.

By concentrating the lactic acid bacteria culture solution in the third step, the salting-out protein is coated on the cells, 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 solution before concentration. That is, the salt-out protein may be coated on the outside of the cells through homogenization while the culture solution is concentrated in the third step.

After concentration, hydroxypropylmethylcellulose (HPMC), carboxymethylcellulose (CMC), polyvinylpyrrolidone (PVP) as an amphiphilic binder that can be used for both positive and negative electrostatically in order to stabilize the coating of the cells. ), polyvinyl alcohol (PVA), and at least one from the group consisting of chitosan may be added.

That is, the culture (simple mixed culture) in which the salted-out protein precipitated due to the addition of lava seawater during medium preparation is non-uniformly mixed with the cells through the first-stage culture is subjected to this concentration process to obtain the salted-out protein A concentrate containing the coated cells can be obtained, and at this time, hydroxypropylmethyl cellulose is added to the salted-out protein-coated cells for coating safety. 50 to 200 parts by weight of hydroxypropylmethylcellulose (powder) can be prepared and mixed with 100 parts by weight of the salt-out protein-coated cells.

However, when hydroxypropylmethylcellulose is mixed with the cells coated with salted-out protein, it is recommended to dissolve the hydroxypropylmethylcellulose in water (culture water) containing 30~70% (v/v) lava seawater before adding it. good. The content of water containing 30 to 70% (v/v) lava seawater for dissolution of hydroxypropylmethylcellulose is not particularly limited, but is preferably 3 to 7 times the weight of hydroxypropylmethylcellulose powder.

The present invention provides 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 characterized in that it 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 lava seawater-derived natural mineral coating probiotics may be added to the pharmaceutical composition of the present invention in an amount of 0.001 to 100% by weight.

The pharmaceutical composition may be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., solutions, suppositories, and sterile injection 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, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, it is prepared using diluents or excipients, such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations contain at least one excipient, for example, starch, calcium carbonate, to the natural mineral-coated probiotics derived from lava seawater of the present invention. , sucrose or lactose, gelatin, etc. are mixed and prepared. Lubricants such as magnesium stearate and talc are also used as simple excipients. Liquid formulations for oral use include suspensions, internal solutions, emulsions, syrups, etc. Commonly used simple diluents such as water and liquid paraffin may include various excipients, for example, wetting agents, sweeteners, fragrances, and preservatives. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like can 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 pathological condition to be treated, the severity of the disease or pathological condition, the route of administration, and the judgment of the prescriber. Dosage determination based on these factors is within the level of one of ordinary 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 1 mg/kg/day to 500 mg/kg/day. Administration may be administered once a day, or may be administered in several divided doses. The above dosage does not limit the scope of the present invention in any way.

The pharmaceutical composition of the present invention may be administered to mammals such as mice, livestock, and humans by various routes. Any mode of administration can be envisaged, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebrovascular injection. Since the natural mineral-coated probiotics derived from lava seawater of the present invention have almost no toxicity and side effects, they can be safely used even when taken for a long period of time for the purpose of prevention.

In addition, the present invention provides a variety of health functional food containing the natural mineral-coated probiotics derived from the lava seawater and food pharmaceutically acceptable food supplement additives.

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 the growth of intestinal lactic acid bacteria, inhibition of intestinal harmful bacteria, and smooth bowel movements.

The natural mineral coated probiotics derived from lava seawater may be added to the health functional food of the present invention in an amount of 0.001 to 100% by weight. The health functional food of the present invention includes the form of tablets, capsules, pills or liquids, and the food 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, bread, candy, snacks, noodles, ice cream, dairy products, soups, ionized beverages, beverages, alcoholic beverages, gum, tea, and vitamin complexes.

The present invention can also provide a cosmetic containing natural mineral-coated probiotics derived from lava seawater prepared by the above method. The cosmetic may include all commonly used ingredients. For example, it may include general auxiliary ingredients such as emulsifiers, thickeners, emulsifiers, 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 can be selected within a range that does not impair the intrinsic effect of the cosmetic. The amount of the components 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 cosmetics is not particularly limited, and for example, lotion, emulsion, gel, cream, essence, pack, ampoule, lotion, cleaning agent, soap, body products, skin care cosmetics such as soap and oil, lipstick, and makeup cosmetics such as foundation, and cosmetics for hair, and the formulation is not particularly limited.

The present invention uses a method of adding lava seawater so that the concentration of lava seawater is 30 to 70% (v/v) in the medium for culturing lactic acid bacteria, thereby increasing the natural minerals and lactic acid bacteria contained in the lava seawater. A method is used in which the salinity on the medium is adjusted to a concentration at which lactic acid bacteria can grow by precipitating the protein in a salted-out state.

In addition, as the lactic acid bacteria are cultured, chaperones, a stress-responsive protein, are generated in the cells due to the stress caused by salt on the salt concentration-controlled medium. By uniformly coating on the surface, thermal stability for the inside and outside of the cells is significantly increased as a whole, and it suppresses the death of lactic acid bacteria due to the high temperature during spray drying. It is possible to derive a probiotic manufacturing method containing the effect of remarkably improving the storage stability of the spray-dried lactic acid bacteria over time, and the effect of resisting the continuous pH change of the digestive tract of stomach and bile acids in the body.

Therefore, through the present invention, natural mineral-coated probiotics derived from lava seawater with excellent storage stability against external temperature changes can be produced and effectively used as biomaterials in food, health functional food and pharmaceutical fields.

To add a little more explanation, when lava seawater is added to the medium at 30-70% (v/v) in the production of probiotics, lactic acid bacteria have the following advantages.

First, it is possible to increase the thermal stability of the cells by forming a coating layer on the outside of the cells, so that it is possible to reduce the manufacturing time and reduce the manufacturing cost by using the spray drying method, unlike the freeze-drying used in the production of conventional probiotics.

Second, by precipitating the protein that can protect the lactic acid bacteria through the natural minerals of lava seawater in a salting out state, the salted out protein produced by itself is used without adding a separate coating solvent such as soy protein or milk protein during spray drying of lactic acid bacteria. Thus, the probiotics can be coated, so the manufacturing stability, intake stability, and storage stability of probiotics can be dramatically improved.

Third, the natural minerals abundant in the lava seawater in the medium affect the growth of lactic acid bacteria. In general, the solid minerals added in the form of a nutrient medium in the form of a nutrient medium are not ionized and have poor growth availability, but Since the included natural minerals are already ionized in liquid phase, there is an advantage in that the growth of probiotics can be induced within a quick time of about 24 hours when using the culture method of the present invention.

Fourth, because lava seawater is used in both the pre- and post-process of culturing lactic acid bacteria and spray drying (powdering) process, the lactic acid bacteria survive and adapt to the high-concentration natural mineral salts of lava seawater during the manufacturing process, which is the lava seawater. It continuously applies osmotic stress to lactic acid bacteria to form proteins in cells that respond to stress, contributing to increasing the survival rate. The protein produced for survival due to changes in the external environment is called a chaperone protein. Therefore, probiotics cultured in lava seawater can exhibit strong resistance to temperature (storage stability) and pH (intake stability) stress, thereby realizing all effects that cannot be achieved with conventional lactic acid bacteria culture technology.

1 is a graph comparing the correlation between the dry weight of salted out protein and lactic acid bacteria cells in the culture solution and the number of live lactic acid bacteria according to the lava seawater concentration.
Figure 2 is a culture method to which lava seawater is applied, and is a graph comparing the fed-batch culture and batch culture conditions of Example 1;
3 is a photograph taken with a transmission electron microscope (TEM) of the coating state of the spray-dried probiotic powder recovered in Example 2 and Preparation Example 2. FIG.
4 is a graph showing the results of culturing lactic acid bacteria by type in the lava seawater-added medium culture conditions of Example 1 or Preparation Example 1.
5 is a 2D-SDS PAGE result photograph showing that a new protein was formed in the cells in the culture medium of Example 1 or Preparation Example 1.
6 is an agaroge gel electrophoresis result photograph showing that the chaperone protein gene was expressed in the cells in the culture medium of Example 1 or Preparation Example 1.

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

First, first, lactic acid bacteria are cultured in liquid phase in a lactic acid bacteria culture medium containing lava seawater (a pre-process of lava seawater).

Second, the salted out protein and lactic acid bacteria produced through the first culturing step are recovered, coated and dried to obtain lava seawater mineral-coated probiotic powder (lava seawater post-process).

To explain this in more detail, in the first lactic acid bacteria culture medium containing lava seawater, salted out protein is precipitated by adding lava seawater instead of water to the basic medium for lactobacillus culture by concentration, and through this, the salinity of the medium is adjusted, thereby proliferating lactic acid bacteria. This is a step to determine the appropriate concentration of lava seawater in which the salting-out protein to be used for coating in the post-process is possible.

That is, the concentration of lava seawater 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 their solubility in water in general water, while their solubility decreases in water with a high salt concentration, such as lava seawater, and precipitates as salted out proteins. When the concentration of external ions increases when the salt concentration increases, the ions attract water molecules distributed on the hydrophobic part of the protein surface, exposing the hydrophobic part of the protein surface to the outside, and the protein aggregation due to the hydrophobic bond between the proteins. because the phenomenon occurs. In addition, the salted-out protein precipitated through this salting-out phenomenon acts as a coating agent that can protect the lactic acid bacteria from the harsh external stress environment.

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, the content of free minerals may be insufficient or excessively supplied depending on the amount of salted out protein precipitation. 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 culturing lactic acid bacteria in liquid phase in a lactic acid bacteria culture medium containing lava seawater, pH 6.0-7.5 conditions are maintained in the medium in the form of fed-batch culture. When the acidity value is 3.0~5.5, the solubility of the water-soluble protein in the presence of lava seawater decreases and excessive precipitation causes it to become difficult to continuously supply the water-soluble protein necessary for the growth of lactic acid bacteria, thereby limiting the growth. Therefore, in the present invention, the lactic acid bacteria growth restriction problem that may occur when sticking to the batch culture method is solved by designing the lactic acid bacteria culture process in a fed-batch culture form that maintains the pH at 6.0 to 7.5 with sugars, rather than the general batch culture method. did.

Second, the salt-out protein and lactic acid bacteria produced through the first culturing step are concentrated so that the salted-out protein is coated on the lactic acid bacteria cells, and then spray-dried to obtain a natural mineral-coated probiotic powder derived from lava seawater (lava seawater). post process).

At this time, a binder (hydroxypropylmethylcellulose:HPMC) may be added before spray drying.

Hereinafter, preferred embodiments 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 so that this disclosure will be thorough and complete, and will fully convey the spirit of the invention to those skilled in the art.

<Preparation Example 1: Preparation and culture method of lactic acid bacteria basic medium>

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), soipeptone 2% (w / v), casein 1% (w / v) Each component was dissolved in water and sterilized at 121°C for 30 minutes.

In this sterilized basic medium, the lactic acid bacteria seed culture medium was inoculated into the sterile medium of the fermenter and cultured for 20 hours at 100 rpm and 37 ° C., 40% (w / v) glucose and 40% (w / v) sodium hydroxide were 1: An aqueous solution (hereinafter, referred to as a glucose-sodium hydroxide solution) mixed in a volume ratio of 1 was dripped at regular time intervals while maintaining the pH at 6.0-7.5 (Fed-batch culture performed).

* Lactobacillus seed culture medium: Lactobacilli MRS Broth (BD) cultured for 24 hours at 37°C, hereinafter referred to as lactobacillus seed culture medium.

<Preparation Example 2: Probiotic powder manufacturing method>

The culture medium 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 μm, manufacturer DOW separation systems, Serial No. 546/57 205-92.) to obtain a concentrate. , 100 g of the concentrate was mixed with a solution in which 100 g of hydroxypropylmethyl cellulose (HPMC) powder was dissolved in 500 g of water, and spray-dried to obtain a dried probiotic powder.

<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 culture water, and the culture water was used instead of water in the basic medium preparation conditions used in Preparation Example 1.

In this way, the culture medium containing lava seawater for each concentration was sterilized as in Preparation Example 1. On the other hand, when a sterilized medium is prepared using lava seawater, natural minerals from lava seawater react with proteins in the medium to produce salted-out proteins precipitated in a salt state.

Next, the lactic acid bacteria seed culture solution was inoculated into a sterilized lava seawater medium as in Preparation Example 1, and the pH was corrected to 6.0-7.5 using a glucose-sodium hydroxide solution at 100 rpm and 37° C. for 20 hours and cultured.

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

After culturing the lactic acid bacteria in a medium prepared using 30% (v/v) lava seawater among the conditions of Example 1, concentrate to 1/10 volume of the original culture solution as in the method of Preparation Example 2 to obtain a concentrate , 100 g of the concentrate was mixed with a solution in which 100 g of hydroxypropylmethyl cellulose (HPMC) powder was dissolved in 500 g of water, and spray-dried to obtain a dried probiotic powder. At this time, during the concentration process, the salted out protein coats the outside of the cells, and hydroxypropylmethylcellulose acts as a binder to make the coating stronger.

<Experimental Example 1: Comparison of salted out protein amount and protein amount according to sterilization temperature and lava seawater concentration in the medium before culture>

Experimental Example 1-1: Lava Seawater Concentration

As already described in Example 1, when a sterilized medium is prepared using lava seawater, natural minerals in lava seawater react with proteins of the medium components to produce salted-out proteins precipitated in a salt state. Although this salted-out protein is partially produced in the basal medium used in Preparation Example 1, the production amount is significantly increased in the medium containing lava seawater.

In order to measure this, as a sterilized medium before culturing lactic acid bacteria, 400 ml of only the basic medium of Preparation Example 1 and the medium containing lava seawater of Example 1 were dispensed in separate containers, respectively, and left at 4° C. for 1 hour, after which 6,000 The salted-out protein was recovered by centrifugation at rpm for 30 minutes, and the recovered product was spray-dried.

Next, the dry weight of the salted out protein and the amount of protein in the salted out protein were checked. This amount of protein was measured by BCA protein assay to confirm the protein content contained in the salted-out protein.

The results of each experiment are shown in Table 1.

According to the results of Table 1, compared to the basic medium of Preparation Example 1, the salt-out protein in the lava seawater medium containing lava seawater by concentration, the salt-out protein increases in proportion to the amount of lava seawater added by 10 to 70% (v/v). appear. If the lava seawater concentration is stagnant at 80% (v/v), or the increase is slightly decreased even at 90% (v/v) and 100% (v) of lava seawater addition, the salt concentration in the medium is too high, so the precipitation effect of protein salting out is significantly higher. It is found not to increase or decrease.

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

Lava seawater concentration (%, v/v) sediment (salted out protein)
dry weight (mg/ml) Amount of net protein in sediment (salted out protein) (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

Experimental Example 1-2: Sterilization temperature

In order to compare the production of salted-out protein according to the sterilization temperature, prepare water containing 30% (v/v) lava seawater for culture and dissolve by adding 3 g of glucose, 2 g of yeast extract, and 2 g of soy peptone when the entire medium is 100 ml. After that, it was sterilized for 30 minutes at a temperature of 5°C in the range from 100 to 121°C, respectively. After cooling the sterilized medium, 400 ml of each concentration of lava seawater was dispensed into separate containers. Each stationary solution was centrifuged at 6,000 rpm for 30 minutes to recover the salted out protein, dried to determine 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 was treated under the same conditions and compared, and it is shown in Table 2.

Sterilization temperature (℃) Lava Seawater Concentration
(%, v/v) sediment (salted out protein)
dry weight (mg/ml) Amount of net protein in sediment (salted out protein) (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 in Table 2, when the amount of pure protein in salted out protein was also 30 (v/v)% when lava seawater was used as culture water, the amount of pure protein in salted out protein was the highest in the medium sterilized at 121 ° C, 100 It can be confirmed that the salting-out protein is formed even at a temperature of ℃ or higher.

However, it was found that when water (lava seawater concentration 0%) was used, a sufficient amount of salted out protein required for coating was not produced even after sterilization at 121°C.

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

1 g of the spray-dried powder of Preparation Examples 2 and 2 was diluted with physiological saline, 1 ml of the diluted solution was dispensed in petridish, and 20 ml of sterilized Lactobacilli MRS Agar (BD) was added to harden. The number of viable cells was confirmed by counting the colonies of Petridish cultured at 37° C. in a political incubator for 48 hours, and it is shown in Table 3 (hereinafter referred to as lactic acid bacteria viable cell count measurement method).

Lava seawater concentration (%, v/v) Number of viable cells 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 3, compared to the number of viable lactic acid bacteria cultured in Preparation Example 1, it can be seen that the number of viable lactic acid bacteria cultured in the medium to which 30% (v / v) lava seawater in Example 1 is applied explosively increases significantly, After that, the number of lactic acid bacteria viable cells appears to decrease.

However, up to the lava seawater concentration of 70% (v/v), the number of viable lactic acid bacteria in the culture medium is measured as a very high value, and ^{1.0×10 11} CFU/g or more, which is significantly higher than the value of 1.0×10 ^{8 CFU/g or more, which is the standard for the number of viable bacteria for probiotics.} It can be confirmed that the probiotic powder can be stably manufactured.

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

In addition, these results confirm that the salt-out protein has a heat-protective function.

<Experimental Example 3: Comparison of the correlation between the dry weight of salted out protein and lactic acid bacteria cells in the culture solution according to the lava seawater concentration and the number of live lactic acid bacteria>

400 ml of each of the lactic acid bacteria cultured in each lava seawater medium of Example 1 was dispensed in a separate sterilized container, and cooled 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 Preparation Example 1 was treated under the same conditions and compared, and this is shown in FIG. 1 . At this time, the results of the dry weight were compared with the number of live lactic acid bacteria.

According to the results of Figure 1, the dry weight of 'salted out protein and lactic acid bacteria' seems to be similar in all places where 30-100% (v/v) of lava seawater is included, and this result is 30-70% (v/v) lava. This is because, in addition to the salted out protein produced before culturing in the seawater culture medium, the number of viable cells increased explosively and the total weight became similar.

This result also demonstrates that the number of viable cells hardly increased when the dry weight of salted out protein and lactic acid bacteria in the 80% (v/v) or more lava seawater culture was similar to the weight of salted out protein in the medium before culturing.

Therefore, it can be understood once again that the conditions of lava seawater in the medium that most affect the culture of lactic acid bacteria are 30-70% (v/v) lava seawater culture medium conditions in which the number of viable cells is significantly increased compared to the culture conditions of Preparation Example 1. A better condition is when using a lava seawater concentration of around 30-40% (v/v) as culture water, and the best condition in this experiment is culture water with a lava seawater concentration of 30% (v/v). It can be confirmed that it is time to culture lactic acid bacteria.

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

The conditions for culturing lactic acid bacteria in Preparation Example 1 and Example 1 can be said to be fed-batch culture conditions using glucose-sodium hydroxide solution (Glucose/NaOH). In order to compare this culture condition with batch culture, the following experiment was performed.

First, the lava seawater 30% (v / v) medium culture conditions in Example 1 (Example 1 or Example 2 in the following experimental examples corresponds to the lava seawater 30% (v / v) medium culture conditions) Lactic acid bacteria were cultured under the fed-batch culture conditions of Preparation Example 1, and lactic acid bacteria were cultured under 30% (v/v) lava seawater 30% (v/v) medium culture conditions of Example 1 under comparative conditions in which only glucose-sodium hydroxide solution was added. was cultured (referred to as the comparative conditions of Example 1, cultured without pH/glucose correction for 20 hours).

Thereafter, the number of viable cells in each culture medium cultured for 20 hours was measured by the lactic acid bacteria live cell count method. In addition, each culture solution is 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.) to obtain a 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.

Fed-batch culture of Preparation Example 1 (water), Fed-batch culture of Example 1 (lava seawater 30% (v/v)), Batch culture of Example 1 (lava seawater 30% (v/v)) It is shown in FIG. 2 by dividing by .

According to the result of FIG. 2, it can be seen that the strain hardly proliferated in the batch culture even when lava seawater was added. Therefore, it is understood that most of the components of the dried product recovered from batch culture are salted-out proteins in the medium, not lactic acid bacteria.

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

In Preparation Examples 2 and 2, ① a mixture of binder (hydroxypropylmethyl cellulose) before spray drying; ② After spray-drying, the number of lactic acid bacteria in each of the probiotic powders was compared to confirm the spray-drying survival rate by natural mineral coating derived from lava seawater.

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

The experimental results are shown in Table 4 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 viable lactic acid bacteria in the binder mixture.

division Preparation Example 2
Water culture spray dried powder Example 2
Lava Seawater Culture Spray Dried Powder 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 4, the natural mineral-coated probiotic powder derived from lava seawater of Example 2 is significantly higher than the probiotic powder of Preparation Example 2 by about 67%, compared to about 6.7%, and it is confirmed that it is 10 times higher. .

Therefore, natural mineral-coated probiotics derived from lava seawater were subjected to a high-concentration growth and coating process through a post-process while being stressed at a certain salt concentration from the pre-process culture stage. have.

<Experimental Example 6: Ingestion stability of natural mineral-coated lactic acid bacteria derived from lava seawater>

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. .

In 100 ml of the prepared artificial gastric juice medium, 0.5 g of the probiotic powder of Preparation Example 2 and Example 2 was mixed, respectively. Each mixed solution was immediately taken, diluted with physiological saline, 1ml of the diluted solution was dispensed in a Petridish, and 20ml of sterilized Bromo cresol purple agar (BD) medium was added to harden. The initial number of viable cells of each probiotic powder was confirmed by counting yellow colonies of Petridish cultured for 48 hours in a stationary incubator at 37°C.

After that, each mixed solution was cultured at 37°C for 2 hours, and the cultured solution was diluted with physiological saline, 1ml of the diluted solution was dispensed in a Petridish, and 20ml of sterilized Bromo cresol purple agar (BD) medium was added. hardened 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 5 below.

division probiotics
Initial number of bacteria (CFU/g) acid resistance Initial (CFU/ml) After incubation (CFU/ml) Survival rate (%) Preparation 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 5, it can be seen that the natural mineral-coated probiotic powder derived from lava seawater of Example 2 has very good resistance to acidity than the probiotic powder of Preparation Example 2 (confirmation of a survival rate of about 49% in Example 2, 7.9 times increase compared to Preparation Example 2).

Experimental Example 6-2: Bile acidity (alone)

An artificial bile medium was prepared by mixing 0.3 ml of sterilized oxgall solution with 100 ml of sterilized Lactobacilli MRS broth (BD).

Then, 0.5 g of each of the probiotic powders of Preparation Example 2 and Example 2 were mixed in 100 ml of the prepared artificial bile medium, and the number of live cells in the initial stage / the number of viable cells after 2 hours of stationary culture was confirmed as in Experimental Example 6-1. Table 6 shows.

division probiotics
Initial number of bacteria (CFU/g) bile acid resistance Initial (CFU/g) After incubation (CFU/g) Survival rate (%) Preparation 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 6, it was found that the natural mineral-coated probiotic powder derived from lava seawater of Example 2 had 1.9 times better bile acid resistance than the probiotic powder of Preparation Example 2.

Experimental Example 6-3: Acid resistance-Bile acid resistance (continuous)

For each of the probiotic powders of Preparation Example 2 and Example 2, an acid resistance confirmation test was performed according to the method of Experimental Example 6-1, and the culture medium was centrifuged to remove the supernatant, and live cells were recovered. The recovered live cells were continuously subjected to a bile acid resistance confirmation test according to the method of Experimental Example 6-2 to confirm the results, and are shown in Table 7.

division probiotics
Initial number of bacteria (CFU/g) acid resistance - bile acid resistance Initial (CFU/g) After incubation (CFU/g) Survival rate (%) Preparation 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 7, it can be seen that the natural mineral-coated probiotic powder derived from lava seawater of Example 2 has a very excellent survival rate difference of 133% or more because it has stronger acid resistance and bile resistance than the probiotic powder of Preparation Example 2 The superiority of the manufacturing method of natural mineral coating probiotics derived from lava seawater is demonstrated.

<Experimental Example 7: Storage stability of natural mineral-coated probiotics derived from lava seawater (comparison of stability over time)>

The probiotic powder of Preparation Example 2 and Example 2 is divided into 5 g each in an airtight storage container and stored at 4 ℃, 15 ℃, 25 ℃, 35 ℃ thermo-hygrostat (humidity 75%) for 4 months, and the number of live lactic acid bacteria in units of 1 month Based on the measurement method, the number of viable cells was measured and shown in Table 8.

Preparation 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 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 8, it is confirmed that the natural mineral-coated probiotic powder derived from lava seawater of Example 2 has higher storage stability within 120 days than the probiotic powder of Preparation Example 2.

<Experimental Example 8: Mineral content of natural mineral coating probiotics derived from lava seawater>

Lava seawater (lava seawater itself without culturing strains), Magnesium and calcium content in the probiotic powder of Preparation Examples 2 and Example 2 were analyzed based on the Magnesium and Calcium test method of the Ministry of Food and Drug Safety, and naturally derived from lava seawater. The amount of probiotics contained in the mineral component was compared in Table 9.

division Magnesium (mg/100g) Calcium (mg/100g) lava seawater
(The lava seawater itself without culturing the strain) 114.2 40.1 Probiotic powder of Preparation Example 2 0.2 0.3 Probiotic powder of Example 2 179.5 54.2

According to the results of Table 9, it can be confirmed that magnesium and calcium were well introduced into the natural mineral-coated probiotic powder derived from lava seawater of Example 2 due to the addition of lava seawater during culturing.

On the other hand, magnesium and calcium are representative components of natural minerals, and these content values become values representing the content values of natural mineral-coated probiotics derived from lava seawater. It was confirmed that the mineral content of 0.1 ~ 0.5g / 100g. At this time, magnesium was individually 0.07 to 0.4 g/100 g, and calcium was 0.02 to 0.09 g/100 g.

<Experimental Example 9: Confirmation of manufacturing conditions of natural mineral-coated probiotics derived from lava seawater by strain>

When culturing lactic acid bacteria under the conditions of Example 1, it was confirmed whether all other types of lactic acid bacteria in addition to the Lactobacillus rhamnosus strain were cultured smoothly in the lava seawater-added medium as the lactic acid bacteria seed culture medium.

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

This result is shown in Figure 4, compared with the culture solution of Preparation Example 1, it can be confirmed that the number of live lactic acid bacteria in all the cultures of the test subjects in the lava seawater addition medium culture conditions of Example 1 is significantly increased.

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

<Experimental Example 10: Production of salted out protein in desalted lava seawater medium and confirmation of the number of viable cells in lactic acid bacteria culture medium>

Under the conditions of preparing the basic medium of Preparation Example 1, a medium was prepared using demineralized lava seawater (using the method of Example 1 of Korean Patent No. 10-0947637) instead of water for culture, followed by centrifugation to dry the precipitated salted out protein. The weight was confirmed and shown in Table 10.

Water for culture added to the medium Salted out protein (mg/ml) Lava seawater 30% (v / v) (Example 1 conditions) 11.8 100% desalted lava seawater 0.4

In addition, the culture solution obtained by culturing the Lactobacillus rhamnosus seed culture in the medium of Table 10 was prepared by the method of Example 2, and the number of viable lactic acid bacteria was measured (spray drying). These values are shown in Table 11.

culture medium Lactobacillus powder viable cell count (CFU/g) Cultured using lava seawater 30% (v/v) (Example 1 conditions) as culture water 2.1 X 10 ¹¹ Cultured using 100% (v/v) desalted lava seawater as culture water 1.2 X 10 ¹⁰ Cultured using constant 100% (v/v) (condition of Preparation Example 1) as culture water 1.1 X 10 ¹⁰

Checking the results of Tables 10 and 11, it is understood that the use of desalted lava seawater has similar results to Preparation Example 1 using water as culture water, and has little effect on the production of salted-out proteins and the growth of lactic acid bacteria. .

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

In order to check whether the self-defense protein (chaperone) in the stress-responsive cells that can withstand the spray-drying conditions in the culturing process through lava seawater is formed, a two-dimensional electrophoresis method was used before and after the reaction in constant water and lava seawater. Changes in protein expression levels were compared with gel images.

Specifically, 100 mg of the cells of Preparation Example 1 and the cells of Example 1 were recovered, washed 3 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 ㎖ micro cap tube containing 250 mg zirconium bead, and the cells were disrupted at 4°C to obtain a cell culture product. After heat treatment at 95° C. for 10 minutes from the cell lysate, centrifugation was performed again at 4° C. at 12,000 rpm to obtain only the supernatant. In the supernatant with protein, salt is removed through desalting column, protein is precipitated with acetone, washed with ethanol, dried 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 μg of protein in an immobiline dry strip with a pI of 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 lava seawater culture method (FIG. 5), and to clarify whether the chaperone protein increased due to stress, the gene expression change of the representative chaperone protein in the following experiment was analyzed.

As a self-defense protein (chaperone) in stress-responsive cells that can withstand spray-drying conditions in the culturing process through lava seawater, cDNA synthesis method and electrophoresis method were used to check whether the expression of GroEL gene, which promotes representative cell division and defense mechanisms, is increased. Using the electrophoresis method, changes in mRNA expression levels before and after reaction in constant water and lava seawater were compared with gel images.

Specifically, 100 mg of the cells of Preparation Example 1 and the cells of Example 1 were recovered, washed 3 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 at 4°C and 12,000 rpm to remove the supernatant, 1 ml of QIAzol (QIAzol, QIAGEN) was added to separate and quantify RNA according to Qiagen's RNA isolation 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). The primers used for PCR were synthesized and used by Macrogen, and the primer sequences are shown in Table 12 below.

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

Referring to FIG. 6, as the mRNA expression of the GroEL gene in the Example 1 cultured cells increased compared to the Preparation Example 1 cultured cells, the synthesized cDNA band was found to be significantly thicker, and accordingly, through the lava seawater culture of Example 1 It is judged that the expression of the defense protein (chaperone) is increased to have thermal stability during spray drying.

As described above, the present invention has been described with reference to the preparation examples, examples and experimental examples, but these are merely exemplary, and those of ordinary skill in the art to which the present invention pertains to various modifications and equivalents therefrom. It will be appreciated that other embodiments are possible. Therefore, the true technical protection scope of the present invention will have to be determined by the technical matters of the appended claims.

Claims (10)

(Step 1) preparing a medium for culturing lactic acid bacteria prepared by using water containing 30-70% (v/v) lava seawater as culture water;
(Second step) obtaining a culture solution by inoculating and culturing the lactic acid bacteria in the culture medium for lactic acid bacteria; and,
(Third step) A method for producing natural mineral-coated probiotics derived from lava seawater, comprising: spray-drying the concentrate obtained by concentrating the culture solution. According to claim 1,
The medium of the first stage contains glucose, yeast extract, soipeptone, and casein as components, and the components are dissolved in water containing 30 to 70% (v/v) lava seawater and sterilized to salt out protein Method for producing natural mineral-coated probiotics derived from lava seawater, characterized in that the formed lactic acid bacteria culture medium. According to 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. A method of producing lava seawater mineral coating probiotics, characterized in that selected from the group consisting of. According to claim 1,
Lava seawater-derived natural mineral-coated probiotics, characterized in that the second step of culturing lactic acid bacteria is performed by a fed-batch culture method. According to claim 1,
The method for producing natural mineral-coated probiotics derived from lava seawater, characterized in that the culture solution obtained in the second step is a mixture of salted out proteins and cells. 6. The method of claim 5,
A method for producing natural mineral-coated probiotics derived from lava seawater, characterized in that the salt-out protein is coated on the outside of the cells through homogenization while the culture solution is concentrated in the third step. A natural mineral-coated probiotics derived from lava seawater prepared by the method of claim 1. 8. The method of claim 7,
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. A food containing the natural mineral-coated probiotic powder derived from lava seawater of claim 7. 10. The method of claim 9,
The food is a food, characterized in that it is a health functional food for the growth of intestinal lactic acid bacteria, for inhibiting harmful bacteria in the intestine, or for promoting bowel movements.

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