KR102764644B1 - Stem fermentation extract for preventing and alleviating periodontal diseases - Google Patents

Abstract

The present invention relates to a steamed fermented extract for preventing and improving periodontal disease and a composition containing the same as an effective ingredient. The composition containing the bacopa extract of the present invention as an effective ingredient has the effects of simultaneously implementing radical scavenging activity, effects of improving polyphenol and flavonoid content, and anti-inflammatory and antibacterial activity.

Description

{STEM FERMENTATION EXTRACT FOR PREVENTING AND ALLEVIATING PERIODONTAL DISEASES}

The present invention relates to a steamed fermented extract for preventing and improving periodontal disease and a composition containing the same as an active ingredient, and more specifically, to a pharmaceutical composition or food composition containing an extract containing bacopa ( Bacopa monnieri ) as an active ingredient, which exhibits antibacterial, anti-inflammatory, and biofilm-suppressing effects against bacteria causing periodontal disease, thereby preventing or treating dental caries and periodontal disease or providing an effect of improving bad breath.

Representative diseases that can occur due to oral health problems include dental caries and periodontal disease. In particular, periodontal disease presents various clinical symptoms such as gingival bleeding and swelling, periodontal pocket formation, loss of attached gingiva, destruction of alveolar bone, and bad breath, and is the main cause of tooth loss (Ali RW et al., J. Clin. Periodontol. 1997 24:830-835; Socransky SS et al., J. Clin. Periodontol. 1998 15;440-444). Periodontal disease can be divided into gingivitis and periodontitis depending on the severity of the disease. Gingivitis is a relatively mild and quickly recoverable form of periodontal disease that is limited to the gums, i.e., soft tissues such as gingival bleeding, and when this inflammation progresses to the gums and the alveolar bone (alveolar bone) and absorption of the alveolar bone occurs, it is called periodontitis (Petersen and Ogawa, J Periodontol. 2005 76;2187-2193). Periodontal disease is one of the most common chronic diseases caused by infectious causes, and it is known that 10-60% of adults have it, depending on the diagnosis status.

More than 350 species of microorganisms live in the oral cavity, but bacteria involved in the progression of periodontal disease account for less than 5%. The oral cavity, which has little contact with oxygen and contains a large amount of dead epithelial cells, salivary proteins, and food debris, provides an environment in which specific microorganisms can live, and these microorganisms cause diseases. The major pathogens of periodontal disease include P orphyromonas gingivalis, Tannerella forsythia, Treponema denticola , and Prevotella intermedia , which belong to the group called the "red complex" among the microorganisms existing in the subgingival plaque located below the gingiva. In addition, Fusobacterium nucleatum is known to play an important role in the process in which early-formed plaque develops into late-formed plaque composed of various microorganisms that can cause diseases (Loomer PM et al., Periodontol 2000. 2004;34:49-56). These microorganisms produce toxins such as lipopolysaccharide, nuclease, leukotoxin, and hydrogen sulfide, which are metabolites or cell wall components, during the process of attaching to or penetrating host tissues and cells, thereby causing an inflammatory response in the surrounding periodontal tissues. At this time, cytokines produced in the host cells and immune system substances produced in response to cytokines further destroy periodontal tissues (Noda D et al. J Periodontal Res. 2007 42(6): 566-571; Dong Chen et al., 2008 Pharmacology 82(4): 264-269).

When an inflammatory response is induced in periodontal cells, the activity of matrix metalloproteinases (MMPs), including inactive collagenase, is increased or their secretion is stimulated. Matrix metalloproteinases are calcium- and zinc-dependent peptidases secreted by various cells such as polymorphonuclear leukocytes, macrophages, gingival fibroblasts, and osteocytes. They act at neutral pH and utilize various extracellular matrices as their substrates. Matrix metalloproteinases such as collagenase decompose collagen, which is the matrix of periodontal tissue, causing local tissue destruction and gingival recession (Jeng AY et al., Bioorg Med Chem Lett 1998 8: 897.; Emingil G et al., J Clin Periodontol 2008 35(3): 221-229). As this progresses, loss of connective tissue attachment and alveolar bone loss lead to tooth loss.

Periodontal disease occurs when various reactions such as the action of specific bacteria, the host's inflammatory response, MMP action, and bone loss become chronic. When general anti-inflammatory drugs or antibacterial agents are continuously taken to treat periodontal disease, various side effects such as digestive disorders, hypersensitivity, renal toxicity, oral toxicity, and tooth discoloration may occur. Therefore, research is actively being conducted to find natural substances with specific activity for periodontal disease and high safety.

Meanwhile, the search for new substances that can be used as health functional foods that can prevent and treat human diseases or improve various functions from natural products has been a continuous research subject. Through the development of such natural resources, more than 50% of the currently used medicines are derived from natural products, and there are about 120 kinds of natural product medicines approved by the US FDA, and their market size is about 10 trillion dollars. Although many natural products with anti-hepatotoxic activity are being searched and developed, only a few are actually being used as treatments or have undergone clinical trials.

Natural products related to dental health, such as extracts of the Chinese juniper tree, are known to be included in Donguibogam, the Encyclopedia of Natural Products, and other books.

Meanwhile, Bacopa ( Bacopa monnieri ) is a perennial creeping herb that grows in wetlands and muddy shores. It has been used in traditional Ayurvedic medicine for thousands of years for its purported anti-amnesiac, sedative, memory-enhancing, anti-epileptic, and anxiolytic effects.

There has been no disclosure or publication regarding Bacopa ( Bacopa monnieri ) as the main ingredient that simultaneously provides prevention and improvement of periodontal disease.

In addition, Korean Patent Publication No. 2011-0011350 relates to a chewing gum containing a nutmeg extract for preventing and improving periodontal disease, wherein the composition comprises a single ingredient or a mixture thereof selected from the group consisting of nutmeg, rhubarb, rhizome, scutellaria baicalensis, rhizome of schisandra chinensis, peony root, and hornbeam, and suppresses the activity of prostaglandins and collagen-decomposing enzymes caused by an inflammatory reaction, while suppressing the formation of dental plaque, and is safe for the human body with no side effects even when used for a long period of time as a natural substance.

Accordingly, the inventors of the present invention completed the present invention by confirming that a mixture using Bacopa ( Bacopa monnieri ) and nutmeg can exhibit periodontal disease-improving effects by preventing or treating dental caries and periodontal disease or by providing an effect of improving bad breath while studying a composition that exhibits antibacterial, anti-inflammatory, and biofilm-inhibiting effects against the causative agent of periodontal disease.

The purpose of the present invention is to provide a composition that exhibits antibacterial, anti-inflammatory, and biofilm-suppressing effects against periodontal disease-causing bacteria by inoculating a solid steamed dried product containing Bacopa ( Bacopa monnieri ) with a fermented strain, or an extract thereof as an effective ingredient, thereby preventing or treating periodontal disease or improving bad breath.

The above objects and other objects of the present invention can all be achieved by the present invention described below.

In order to achieve the above purpose, the present invention

A composition for preventing and improving periodontal disease is provided, which comprises a powder of a steamed fermented extract obtained by inoculating a fermented strain into a solid steamed dried product using Bacopa ( Bacopa monnieri ) and nutmeg , or an extract thereof as an effective ingredient.

The above bacopa ( Bacopa monnieri ) and nutmeg (Nutmeg) can satisfy a weight ratio of Bacopa (a) : Nutmeg (b) of 1:0.25 to 2.

The above steamed fermented extract may be a fermented extract of a steamed product steamed at 80 to 110°C for 1 to 48 hours and dried at 35 to 75°C for 1 to 24 hours, and having a moisture content of 10% or less.

The above fermentation strain may be at least one selected from Bifidobacterium longum , Lactobacillus acidophilus , Leuconostoc mesenteroides , Lactobacillus plantarum , Lactobacillus bulgaricus , and Enterococcus faecium .

The above steamed fermented extract can be extracted using a reflux extraction solvent at 70 to 100°C for 2 to 5 hours under reflux.

The above reflux extraction solvent may contain at least one selected from chloroform, ethanol, methanol, water, ethyl acetate, hexane, and diethyl ether in an amount of 5 to 20 times the weight of the fermented product.

The above steamed dried product can be sequentially steamed at 30 to 45°C for 1 to 6 hours, steamed at a temperature higher than 45°C and lower than 55°C for 1 to 6 hours, and steamed at a temperature higher than 55°C and lower than 100°C for 1 to 6 hours.

According to the present invention, a composition for preventing and improving periodontal disease can be provided, which contains as an effective ingredient a steamed fermented extract derived from bacopa that simultaneously exhibits radical scavenging activity, effects of improving polyphenol and flavonoid content, and anti-inflammatory and antibacterial activities.

Figure 1 is a graph comparing the DPPH and ABTS radical scavenging activities of four types of samples (BN-1 to BN-3) obtained in Comparative Examples 1 to 3 with the sample (BN-4) obtained in Example 1.
Figure 2 is a graph comparing the total polyphenol and total flavonoid contents of four types of samples (BN-1 to BN-3) obtained in Example 1 and Comparative Examples 1 to 3.
Figure 3 is a graph comparing the anti-inflammation evaluation results for four types of samples (BN-1 to BN-3) obtained in Comparative Examples 1 to 3 with the sample (BN-4) obtained in Example 1.
Figure 4 is a graph comparing the cytotoxicity of four types of samples (BN-1 to BN-3) obtained in Comparative Examples 1 to 3 with the sample (BN-4) obtained in Example 1.

To help understand the present invention, the present invention will be described in more detail below.

Terms or words used in this specification and claims should not be interpreted as limited to their usual or dictionary meanings, but should be interpreted as meanings and concepts that conform to the technical idea of the present invention, taking into account that the inventor can appropriately define the concept of the term in order to explain the invention in the best way.

As confirmed in the following examples and experimental examples, the present invention provides a composition for preventing and improving periodontal disease by simultaneously exhibiting radical scavenging activity, effects of improving polyphenol and flavonoid content, and anti-inflammatory and antibacterial activity when an extract is produced by preparing a steamed extract using Bacopa monnieri and nutmeg and steaming and fermenting the extract using a fermentation strain.

Ethanol room temperature stirred extracts, ethanol fermented extracts, or ethanol steamed fermented extracts did not exhibit radical scavenging activity, improvement in polyphenol and flavonoid content, anti-inflammation, and antibacterial activity effects at the same time, and although not presented in the experimental examples below, when the content of Bacopa monnieri and the content of nutmeg did not satisfy the weight ratio of 1:0.25~2, radical scavenging activity, improvement in polyphenol and flavonoid content, anti-inflammation, and antibacterial activity effects did not exhibit at the same time or the effects were reduced.

The composition of the present invention, which simultaneously exhibits radical scavenging activity, an effect of improving the content of polyphenols and flavonoids, and an anti-inflammatory and antibacterial activity, is provided based on these experimental results. The composition for preventing and improving periodontal disease of the present invention is characterized in that it comprises, as an active ingredient, a steamed fermented extract obtained by mixing Bacopa (Bacopa monnieri ) and nutmeg in a weight ratio of 1:0.25 to 2 and then inoculating a fermenting strain into a steamed and dried solid phase product.

The composition for preventing and improving periodontal disease of the present invention is preferably characterized by containing, as an effective ingredient, a steamed fermented extract obtained by mixing Bacopa ( Bacopa monnieri ) and nutmeg in a weight ratio of 1:0.25 to 2, then inoculating a fermenting strain into the steamed dried product of the obtained solid phase product.

In this description, unless otherwise specified, the active ingredient means a component that exhibits the desired activity alone or can exhibit the activity together with a carrier that is inactive in itself.

In this description, unless otherwise specified, the extract refers to an extract obtained by using an extraction solvent from the solid substance itself as the target of extraction, or a fraction obtained by fractionating the extract, and the extraction method may be a method such as maceration, reflux, heating, ultrasonic radiation, supercritical extraction, or ultra-high pressure extraction, considering the polarity, degree of extraction, and degree of preservation of the active substance. In the case of a fractionated extract, it refers to a fraction obtained by suspending the extract in a specific solvent, mixing it with a solvent having a different polarity, and allowing it to settle, and a fraction obtained by adsorbing the crude extract on a column packed with silica gel or the like, and using a hydrophobic solvent, a hydrophilic solvent, or a mixed solvent thereof as a mobile phase.

In this description, the shape of the solid material is not particularly limited, and may be in the form of pills, granules, powder, etc.

The above solid material can be obtained in the form of a solid material by thoroughly washing Bacopa ( Bacopa monnieri ) and nutmeg . After thoroughly washing, it can be dried in any way, such as natural drying (shade drying) or hot air drying, to remove some of the moisture, and then crushed and used for steaming.

In the present invention, unless otherwise specified, vapoca and nutmeg mean extracts obtained by extraction from various organs or parts (e.g., leaves, branches, flowers, roots, stems, fruits, etc.), preferably extracts obtained from each leaf or fruit or root, and most preferably extracts obtained from each fruit and root.

In the present invention, nutmeg is a plant used as a herbal medicine ingredient, and in oriental medicine, it is dried and used as an aromatic tonic and tonic, and in the West, it is used as a flavoring agent together with mace. In the present invention, the nutmeg is effective in suppressing cariogenic bacteria (S. mutans) and periodontal disease bacteria (P. aingivalis), which are one of the causes of periodontal disease.

In addition, it can be used as is in the form of a pulverized product, or in a dried state after being steamed step by step, and when used in a dried state after being steamed step by step, it can provide the effect of increasing the usability of storage and the effectiveness of the active ingredients.

In the present invention, unless otherwise specified, the steamed extract may be a steamed dried product having a moisture content of 10% or less, obtained by steaming a steamed product at 30 to 110° C., or 95 to 105° C., for 1 to 48 hours, or 1 to 10 hours, and drying the steamed product at 35 to 75° C., or 40 to 60° C., for 1 to 24 hours, or 6 to 15 hours. In this case, it is preferable because it can provide an effect of increasing the effective ingredient.

As a specific example, the steamed dried product may be a steamed dried product having a moisture content of 10% or less obtained by sequentially performing steaming at 30 to 45°C for 1 to 6 hours, steaming at a temperature exceeding 45°C and below 55°C for 1 to 6 hours, and steaming at a temperature exceeding 55°C and below 100°C for 1 to 6 hours.

At this time, a conventional steaming device can be used for the steaming, a conventional drying device such as a hot air dryer can be used for drying, and a conventional drying device such as a freeze dryer can be used for drying to control moisture content.

The steamed extract of the present invention is preferably obtained by using the steamed and dried extract, the moisture content of which is adjusted to 20% or less through drying pretreatment.

The above steaming and drying can be performed in multiple stages as needed. For example, the steaming time in the first steaming and drying can be set to 6 to 15 hours, the steaming time in the second steaming and drying can be set to 8 to 15 hours, and the steaming time in the third steaming and drying can be set to 8 to 12 hours.

The above steamed dried product is inoculated with a fermentation strain.

In the present invention, the fermentation strain is preferably a mixture of Lactobacillus acidophilus (KACC 12419), Leuconostoc mesenteroides (KACC 12312), which is a mixture of Bifidobacterium longum (KACC 20597), L Bifidobacterium longum (KACC 20597), Lactobacillus acidophilus (KACC 12419), and Leuconostoc mesenteroides (KACC 12312) used in the examples below, in order to provide a composition for preventing pigmentation and protecting against ultraviolet rays. In addition, Lactobacillus plantarum, Lactobacillus bulgaricus, and Enterococcus faecium may also be used as a substitute or in combination, and the amount used is 10 ⁴ to 10 ⁷ CFG/g, specifically, 10 ⁵ to It can be in the range of ^10-6 CFG/g.

In the present invention, the culture after the inoculation can be performed at 15 to 45°C, or 25 to 45°C. If the culture temperature is lower than the above range, the fermentation rate may slow down or undesirable by-products may be generated, and if the culture temperature is higher than the above range, the fermentation rate may also slow down or undesirable fermentation by-products may be generated due to the death of fermentation microorganisms. The culture time can be performed for about 1 to 7 days, or about 2 to 5 days.

In addition, the properties of the active ingredient can change depending on the temperature and pressure conditions of the extraction. For example, if the steamed fermented extract is extracted under low-temperature conditions, the solubility of the active ingredient decreases and the concentration of sugar in the extract increases, and even if the extraction is performed under high-temperature conditions suitable for the extraction conditions of the active ingredient, if the extract in the early part of the extraction is fractionated and separated, the concentration of the active ingredient will be higher than that of the extract in the latter part. In this extraction method, the content of free fatty acids and the content of active ingredients can be controlled by adjusting the fractionation time. In order to obtain an extract with a low free fatty acid content, that is, a low acid value, there is a method of lowering the concentration by mixing fraction sections with a low free fatty acid content of the raw material or a high content of active ingredients.

The free fatty acid content of raw materials varies depending on the origin or crop, and raw materials with low acid values are usually formed at high prices, which reduces their competitiveness. In addition, since there is no raw material without free fatty acids, it is not easy to meet the acid value standard while meeting the effective ingredient content above a certain concentration even if the standard is set and the product is made. Therefore, in order for the process of making a high-concentration fraction using a steamed fermented extract to have industrial stability, a technology is needed to remove free fatty acids by repeated extraction under reflux (also called reflux extraction).

The above-mentioned extraction of the refund can be performed repeatedly at 70 to 100°C, or at 85 to 95°C for 12 hours or less, or 3 to 10 hours. The number of times of the above-mentioned repeated extraction can be 5 times or less, and specifically 2 to 3 times.

In the case of the above reflux extraction, it is preferable to increase the processing efficiency by supplying an extraction solvent to the fermentation product. At this time, the usable extraction solvent may be at least one selected from chloroform, ethanol, methanol, water, ethyl acetate, hexane, and diethyl ether. At this time, the extraction solvent may be included in an amount of 5 to 20 times, or 5 to 10 times, the weight of the fermentation product.

A purification process is required that removes the above-mentioned free fatty acids without loss of effective ingredients and without affecting efficacy. To this end, a method of filtering using a microfiltration membrane and then concentrating under reduced pressure to remove free fatty acids is preferable. In order to provide better efficacy, it is more preferable to use a microfiltration membrane having a size of 1.0 ㎛ or less, preferably 0.45 ㎛ or less.

An effective extract can be obtained through the above-described purification process.

In this description, the effective extract includes a concentrated liquid extract or solid extract from which the reflux extraction solvent has been removed by a method such as freeze-drying, vacuum drying, hot air drying, spray drying, or concentration under reduced pressure.

In this description, the form of the resulting extract is not particularly limited and may be a powder or other extract form, including a pill form, a granule form, and a powder form.

The method for producing an extract according to the present invention may include, for example, a step of thoroughly washing Bacopa ( Bacopa monnieri ) and nutmeg, steaming and drying the obtained solid or dried product; and a step of inoculating the steamed and dried product with a fermenting strain.

In the present invention, the Bacopa monnieri and nutmeg can be obtained in a solid form after being thoroughly washed. After being thoroughly washed, they are dried in any manner, such as natural drying (shade drying) or hot air drying, to remove some of the moisture (after which the moisture content of the solid is reduced to 10% (w/w) or less), and then crushed into a pulverized form with a mesh size of 10 to 50 mesh, specifically, 30 to 50 mesh, to increase the surface area, and then can be used for pretreatment before fermentation.

If necessary, it can be dried at 95℃ or lower, or at 50 to 90℃ for 36 hours or longer, or for 48 hours or longer, and then pulverized.

The above solid material may undergo a fermentation extraction step prior to reflux extraction.

There is no special limitation on the solid used in this description, and the fermentation extraction step may include treatment to remove harmful substances such as microorganisms, residual pesticides, and preservatives on the surface of the solid or its dried product (also referred to as “dried product of solid”), treatment to increase the extraction area, etc.

In the case of the above fermentation extraction, at least one selected from among Bifidobacterium longum , Lactobacillus acidophilus , Leuconostoc mesenteroides , Lactobacillus plantarum , Lactobacillus bulgaricus , and Enterococcus faecium may be used as the fermentation strain.

Next, the fermentation extract can be repeatedly refluxed under reflux using a reflux extraction solvent. The reflux extraction solvent can be, for example, ethanol having a concentration of 70% or higher, preferably 80 to 98%.

At this time, the extraction can be repeated at a temperature of 70 to 100°C, specifically at a temperature of 85 to 95°C, for 12 hours or less, specifically 1 to 6 hours. The number of times the extraction can be repeated can be 5 times or less, specifically 2 to 3 times.

The above steamed fermented extract can be purified and concentrated under reduced pressure as described above to remove the reflux extraction solvent and separate the effective extract.

The composition of the present invention can be manufactured by including, in addition to the effective ingredient, a caries prevention active ingredient, an abrasive ingredient, a wetting agent ingredient, a binding agent ingredient, a foaming agent ingredient, a sweetener ingredient, a flavoring ingredient, a coloring ingredient, etc. known in the art to prevent or improve periodontal disease.

As the caries prevention active ingredient, at least one ingredient selected from the group consisting of sodium fluoride, sodium fluorophosphate, amine fluoride, tin fluoride, chlorhexidine, cetylpyridium chloride, triclosan, xylitol, bamboo salt, and propolis can be used.

As the above abrasive component, at least one component selected from the group consisting of precipitated silica, silica gel, zirconium silicate, calcium dihydrogen phosphate, anhydrous calcium dihydrogen phosphate, hydrous alumina, light calcium carbonate, heavy calcium carbonate, calcium pyrophosphate, insoluble metaphosphate, and aluminum silicate can be used.

As the above humectant component, at least one selected from the group consisting of glycerin, sorbitol, xylitol, polyethylene glycol, and propylene glycol may be used. This humectant component is an essential base component for making a paste formulation, and prevents the toothpaste from drying and solidifying when exposed to the air, provides gloss to the surface of the toothpaste, and, depending on the type, provides a sweetening effect when brushing the teeth.

Any type of water-soluble polymer can be used as the binder component, but sodium carboxymethyl cellulose, carrageenan, xanthan gum, etc. can be preferably used. The binder acts to prevent the solid powder component and the liquid component from separating.

As the foaming agent component, it is preferable to use sodium lauryl sulfate, which is an anionic surfactant, and, depending on the characteristics of the formulation, nonionic surfactants such as a polyoxyethylene-polyoxypropylene copolymer (poloxamer), polyoxyethylene hydrogenated castor oil, and polyoxyethylene sorbitan fatty acid ester can be used as auxiliary agents. Such foaming agent components enhance the feeling of use of the product, help with the cleansing action, speed up the dispersion and penetration of other medicinal ingredients, and reduce interfacial tension to easily remove foreign substances in the oral cavity.

The above-mentioned flavoring ingredients, sweetener ingredients, coloring ingredients, etc. are intended to improve the usability of the composition. It is preferable to use edible ingredients as the flavoring ingredients. Examples of such ingredients include menthol, peppermint oil, spearmint oil, sage, eucalyptol, methyl salicylate, or fruit extracts.

It is preferable to use sodium saccharin as the sweetener component, and it is preferable to use food coloring as the coloring component.

These ingredients may be included one or more together with the effective ingredient in the composition of the present invention.

The composition of the present invention may contain an effective ingredient in any amount (effective amount) as long as it can exhibit an antioxidant effect, whitening effect, etc. depending on the product type, product formulation, etc., and the effective amount may be determined within a range of 0.001 to 15 wt% based on the total weight of the composition.

In the present invention, unless otherwise specified, the effective amount refers to the amount of an effective ingredient included in the composition of the present invention that can exhibit intended functional/medical effects, such as nursing function, hangover relief function, etc., when the composition of the present invention is administered to a mammal, preferably a human, which is the subject of application, during an administration period as recommended by a medical professional, etc. This effective amount can be experimentally determined within the scope of a person skilled in the art.

The composition of the present invention, in a specific embodiment, can be considered a food composition.

The food composition of the present invention can be manufactured in any form, and for example, can be manufactured in the form of beverages such as drinks, tea, juice, carbonated beverages, and ionic beverages, processed dairy products such as milk and yogurt, foods such as gum, rice cakes, Korean traditional confectionery, bread, cookies, and noodles, and health functional food preparations such as tablets, capsules, pills, granules, liquid, powder, flakes, paste, syrup, gel, jelly, and bars.

In addition, the food composition of the present invention may have any product classification as long as it complies with the laws and regulations in effect at the time of manufacturing/distribution in terms of functional classification. For example, it may be a health functional food according to the Health Functional Food Act, or a soy milk product, a fermented beverage, a special-purpose food, etc. according to each food type according to the Food Code of the Food Sanitation Act (Food and Drug Administration Notice, Food Standards and Specifications).

The food composition of the present invention may contain food additives in addition to its effective ingredients. Food additives are generally understood as substances that are added to, mixed with, or infiltrated into food during the manufacturing, processing, or preservation of food, and their safety must be guaranteed because they are taken daily and for a long period of time with food. The Food Additive Code (Food and Drug Administration Notice, Food Additive Standards and Specifications) according to the Food Sanitation Act restrictively stipulates food additives whose safety is guaranteed by classifying them into chemical synthetic products, natural additives, and mixed preparations.

These food additives can be classified into sweeteners, flavoring agents, preservatives, emulsifiers, acidulants, and thickeners in terms of functionality.

Sweeteners may be used in an amount that provides the food with an appropriate sweetness, and may be natural or synthetic. Preferably, natural sweeteners are used, and examples of natural sweeteners include sugar sweeteners such as corn syrup solids, honey, sucrose, fructose, lactose, and maltose.

Flavoring agents can be used to improve taste or aroma, and both natural and synthetic ones can be used. It is preferable to use natural ones. When using natural ones, in addition to flavor, the purpose of enhancing nutrition can also be used. Natural flavoring agents can be obtained from apples, lemons, tangerines, grapes, strawberries, peaches, etc., or from green tea leaves, dandelion leaves, bamboo leaves, cinnamon, chrysanthemum leaves, jasmine, etc. In addition, those obtained from ginseng (red ginseng), bamboo shoots, aloe vera, ginkgo nuts, etc. can be used. Natural flavoring agents can be liquid concentrates or solid extracts.

Synthetic flavoring agents may also be used, including esters, alcohols, aldehydes, and terpenes.

Preservatives that can be used include sodium calcium sorbate, sodium sorbate, potassium sorbate, calcium benzoate, sodium benzoate, potassium benzoate, and EDTA (ethylenediaminetetraacetic acid), and emulsifiers that can be used include acacia gum, carboxymethyl cellulose, xanthan gum, and pectin.

Acidulants that can be used include lactic acid, malic acid, fumaric acid, adipic acid, phosphoric acid, gluconic acid, tartaric acid, ascorbic acid, acetic acid, and phosphoric acid. In addition to the purpose of enhancing taste, acidulants can be added to ensure that the food composition has an appropriate acidity for the purpose of inhibiting the growth of microorganisms.

Suspending agents, precipitating agents, gelling agents, and bulking agents can be used as thickening agents.

In addition to the aforementioned food additives, the food composition of the present invention may contain physiologically active substances or minerals known in the art and guaranteed to be safe as food additives for the purpose of supplementing and reinforcing functionality and nutrition.

Such physiologically active substances include catechins contained in green tea, vitamins such as vitamin B1, vitamin C, vitamin E, and vitamin B12, tocopherol, and dibenzoylthiamine, and minerals include calcium preparations such as calcium citrate, magnesium preparations such as magnesium stearate, iron preparations such as ferrous citrate, chromium chloride, potassium iodine, selenium, germanium, vanadium, and zinc.

The food composition of the present invention may contain the aforementioned food additives in an appropriate amount that can achieve the purpose of addition depending on the product type. With regard to other food additives that may be contained in the food composition of the present invention, reference may be made to the Food Codex or the Food Additives Codex.

The composition of the present invention, in a specific embodiment, can be considered a pharmaceutical composition.

The pharmaceutical composition of the present invention can be prepared as an oral formulation or a parenteral formulation according to the administration route by a conventional method known in the art, including a pharmaceutically acceptable carrier in addition to an effective ingredient.

In the present invention, pharmaceutically acceptable means that, unless otherwise specified, it does not inhibit the activity of the active ingredient and does not have toxicity exceeding the adaptable target of application (prescription).

When the pharmaceutical composition of the present invention is prepared as an oral dosage form, it can be prepared in the form of powder, granules, tablets, pills, sugar-coated tablets, capsules, liquids, gels, syrups, suspensions, wafers, etc., using a suitable carrier and a method known in the art.

Suitable pharmaceutically acceptable carriers include, for example, sugars such as lactose, glucose, sucrose, dextrose, sorbitol, mannitol, and xylitol; starches such as corn starch, potato starch, and wheat starch; cellulosics such as cellulose, methylcellulose, ethyl cellulose, sodium carboxymethylcellulose, and hydroxypropylmethylcellulose; polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate, mineral oil, malt, gelatin, talc, polyols, and vegetable oils. If necessary, the composition may be formulated by including diluents and/or excipients such as fillers, bulking agents, binders, wetting agents, disintegrating agents, and surfactants.

When the pharmaceutical composition of the present invention is prepared as a parenteral formulation, it can be formulated into the form of an injection, a transdermal administration agent, a nasal inhaler, and a suppository according to a method known in the art together with a suitable carrier.

When formulated as an injection, suitable carriers include sterile water, ethanol, polyols such as glycerol or propylene glycol, or mixtures thereof, and preferably, Ringer's solution, phosphate buffered saline (PBS) containing triethanolamine, sterile water for injection, or an isotonic solution such as 5% dextrose can be used.

When formulated as a transdermal agent, it can be formulated in the form of ointment, cream, lotion, gel, external preparation, paste, liniment, aerosol, etc.

For nasal inhalation, it can be formulated in the form of an aerosol spray using a suitable propellant such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, or carbon dioxide.

The suppository base that can be used is witepsol, Tween 61, polyethylene glycol, cocoa butter, laurin butter, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene stearate, sorbitan fatty acid ester, etc.

Formulation of pharmaceutical compositions is well known in the art, and reference can be made to reference to literature [Remington's Pharmaceutical Sciences (19th ed., 1995)], etc., which is considered a part of this specification.

The preferred dosage of the pharmaceutical composition of the present invention may be in the range of 0.001 mg/kg to 10 g/kg per day, preferably in the range of 0.001 mg/kg to 1 g/kg, depending on the patient's condition, weight, sex, age, severity of the patient, and route of administration. Administration may be performed once a day or divided into several times. Such dosage should not be construed as limiting the scope of the present invention in any way.

The food composition and pharmaceutical composition of the present invention can be manufactured according to a manufacturing method of the composition commonly practiced in the art, except that they contain an effective ingredient known in the art.

The composition of the present invention can be used as a composition for preventing or treating periodontal disease, wherein the periodontal disease is a relatively mild and quickly recoverable form of periodontal disease, such as gingivitis limited to the gums, i.e., soft tissues such as gingival bleeding, or periodontitis in which such inflammation progresses to the gums and the surrounding gum bone (alveolar bone) and absorption of the alveolar bone occurs.

In describing the composition for preventing and improving periodontal disease of this invention, it is stated that other conditions or equipment, etc. that are not explicitly described can be appropriately selected within the scope commonly practiced in the art and are not particularly limited.

Hereinafter, embodiments of the present invention will be described in detail so that those with ordinary skill in the art can easily implement the present invention. However, the present invention may be implemented in various different forms and is not limited to the embodiments described herein.

[Example]

<Example 1> Preparation of steamed fermented bacopa extract

(1) Bacopa ( Bacopa monnieri ) and nutmeg were thoroughly washed, dried at 70℃ for 48 hours, and ground to a size of 40 mesh or less. The ground raw materials were mixed to a constant weight (100 g: 100 g).

(2) The mixed raw materials were steamed at 50℃, 60℃, and 90℃ for 2 hours each, then steamed at 100℃ for 3 hours, and then naturally dried for 12 hours. The above steaming process was repeated 5 times.

(3) The mixed steamed dried material was inoculated with Bifidobacterium longum (KACC 20597), Lactobacillus acidophilus (KACC 12419), and Leuconostoc mesenteroides (KACC 12312) at a concentration of 10 ⁶ CFU/g each, fermented at 37°C for 3 days, and then centrifuged to collect the supernatant.

(4) Then, 95% ethanol, 8 times the total weight of the steamed fermented product, was added and reflux extraction was performed three times at 90°C for 8 hours.

(5) The obtained extract was filtered through a 0.45 ㎛ membrane filter and then concentrated under vacuum at room temperature for 3 hours to prepare a steamed fermented bacopa extract (sample BN-4).

<Comparative example>

<Comparative Example 1> Preparation of Bacopa Extract by Reflux Extraction

(1) Bacopa ( Bacopa monnieri ) and nutmeg were thoroughly washed, dried at 70℃ for 48 hours, and ground to a size of 40 mesh or less. The ground raw materials were mixed to a constant weight (100 g: 100 g).

(4') Then, 95% ethanol, 8 times the total weight of the mixed dry matter, was added and reflux extraction was performed three times at 90°C for 8 hours.

(5) The obtained extract was filtered through a 0.45 ㎛ membrane filter and then concentrated under vacuum and at room temperature for 3 hours to prepare a reflux-extracted bacopa extract (sample BN-1).

<Comparative Example 2> Preparation of steamed bacopa extract

(1) Bacopa ( Bacopa monnieri ) and nutmeg were thoroughly washed, dried at 70℃ for 48 hours, and ground to a size of 40 mesh or less. The ground raw materials were mixed to a constant weight (100 g: 100 g).

(2) The mixed raw materials were steamed at 50℃, 60℃, and 90℃ for 2 hours each, then steamed at 100℃ for 3 hours, and then naturally dried for 12 hours. The above steaming process was repeated 5 times.

(4) Then, 95% ethanol, 8 times the total weight of the steamed dried product, was added and reflux extraction was performed three times at 90°C for 8 hours.

(5) The obtained extract was filtered through a 0.45 ㎛ membrane filter and then concentrated under vacuum at room temperature for 3 hours to prepare a steamed bacopa extract (sample BN-2).

<Comparative Example 3> Preparation of fermented bacopa extract

(1) Bacopa ( Bacopa monnieri ) and nutmeg were thoroughly washed, dried at 70℃ for 48 hours, and ground to a size of 40 mesh or less. The ground raw materials were mixed to a constant weight (100 g: 100 g).

(3') The mixed dry material was inoculated with Bifidobacterium longum (KACC 20597), Lactobacillus acidophilus (KACC 12419), and Leuconostoc mesenteroides (KACC 12312) at a concentration of 10 ⁶ CFU/g each, fermented at 37°C for 3 days, and then centrifuged to collect the supernatant.

(4) Then, 95% ethanol, 8 times the total weight of the steamed fermented product, was added and reflux extraction was performed three times at 90°C for 8 hours.

(5) The obtained extract was filtered through a 0.45 ㎛ membrane filter and then concentrated under vacuum at room temperature for 3 hours to prepare a fermented bacopa extract (sample BN-3).

<Experimental example>

<Experimental Example 1> Antioxidant activity test 1

ABTS method and DPPH method radical scavenging ability experiments were performed to evaluate the radical scavenging ability of the samples obtained in Example 1 and Comparative Examples 1 to 3 as antioxidant tests.

(1-1) ABTS radical scavenging activity

Each sample (BN-1 to BN-4) was diluted in water to prepare test solutions having concentrations of 100 μg/mL, 250 μg/mL, 500 μg/mL, and 1000 μg/mL. 7 mM ABTS and 2.45 mM potassium persulfate were mixed and reacted in a dark room at room temperature for 12 hours to form ABTS cations. Afterwards, ethanol was added to adjust the absorbance value at 734 nm to 0.70 ± 0.02.

Add 100 ㎕ of the test solution and 100 ㎕ of the prepared ABTS solution to a 96-well plate, react at room temperature for 7 minutes, and measure at 734 nm. Compare with the blank test solution, and the ABTS removal rate is calculated as a percentage (%) as follows and is shown in Figure 1 below.

ABTS radical scavenging ability (%) = [Control - (Sample - Blank)]/Control Υ 100

(Control: Absorbance of ABTS reagent, Sample: Absorbance of Sample + ABTS reagent, Blank: Absorbance of Sample + Blank)

(1-2) DPPH radical scavenging activity

Each of the samples (BN-1 to BN-4) was diluted in water to prepare test solutions having concentrations of 100 μg/mL, 250 μg/mL, 500 μg/mL, and 1000 μg/mL. 100 μl of the test solution and 100 μl of 0.2 mM DPPH were added to a 96-well plate, and the absorbance was measured at 517 nm using a microplate reader after 30 minutes. The DPPH radical scavenging rate was calculated as a percentage (%) by comparison with the blank test solution, and is shown in Figure 1 below.

DPPH radical scavenging ability (%) = [Control - (Sample - Blank)]/Control Υ 100

(Control: Absorbance of DPPH reagent, Sample: Absorbance of Sample + DPPH reagent, Blank: Absorbance of Sample + Blank)

As shown in Figure 1 below, the ABTS radical analysis results showed that the radical scavenging activity increased as the concentration increased. In the case of the sample (BN-4) according to Example 1, it was confirmed to be the highest at 66.2% at 1000 ㎍/mL, followed by the sample (BN-3) according to Comparative Example 3, the sample (BN-2) according to Comparative Example 2, and the sample (BN-1) according to Comparative Example 1, which showed 51.3%, 32.0%, and 26.8% at 1000 ㎍/mL, respectively.

In addition, the DPPH radical analysis results showed the same trend as the ABTS analysis results described above. Specifically, the sample (BN-4) according to Example 1, the sample (BN-3) according to Comparative Example 3, the sample (BN-2) according to Comparative Example 2, and the sample (BN-1) according to Comparative Example 1 showed 60.7%, 44.3%, 30.9%, and 23.6%, respectively, at 1000 ㎍/mL.

From the above results, it was confirmed that the fermentation process affected the antioxidant capacity, and it was confirmed that the antioxidant capacity further increased through the subsequent steaming process.

<Experimental Example 2> Experimental study on total polyphenol and total flavonoid content

The polyphenol and flavonoid content experiments for the samples obtained in Example 1 and Comparative Examples 1 to 3 were analyzed using gallic acid and quercetin as standard substances, and the results are shown in Figure 2.

Specifically, the polyphenol content was analyzed as follows.

Take 100 mg of each of the mixed extracts of bacopa (BN-1~4) and dilute to 100 mL with 80% ethanol to prepare the test solution. Separately, take 100 mg of gallic acid and dilute to 100 mL with 80% ethanol. Take 0.1, 0.2, 0.5, and 1.0 mL of this solution and dilute to 5 mL to prepare the standard solution.

100 μL of the test solution and standard solution and 100 μL of 10% sodium carbonate were added to the e-tube, then 100 μL of Folin-Ciocalteu reagent was added and mixed using a vortex for 30 seconds. After leaving it in the dark for 30 minutes, the absorbance value of the reaction solution was measured at 750 nm.

The measured absorbance was used to draw a calibration curve for the standard solution, and the total polyphenol content was expressed in mg GAE/g as shown in Figure 2.

As shown in Fig. 2 below, the polyphenol content analysis results showed that the sample (BN-4) according to Example 1, which was fermented and steamed using lactic acid bacteria, had the highest content, which was confirmed to be 40.7 mg GAE/g. The polyphenol contents of the sample (BN-3) according to Comparative Example 3, the sample (BN-2) according to Comparative Example 2, and the sample (BN-1) according to Comparative Example 1 were analyzed to be 34.2 mg GAE/g, 26.2 mg GAE/g, and 24.2 mg GAE/g, respectively, and it was confirmed that the polyphenol content increased when the fermentation and steaming processes were added.

Furthermore, to analyze the total flavonoid content, take 100 mg of each of the bacopa mixed extracts (BN-1 to 4) and dilute to 100 mL with 80% ethanol to prepare a test solution. Separately, take 100 mg of quercetin and dilute to 100 mL with 80% ethanol. Take 0.1, 0.2, 0.5, and 1.0 mL of this solution and dilute to 5 mL to prepare a standard solution. Add 500 μL of the test solution and the standard solution to an e-tube, 100 μL of 10% aluminum nitrate, and 100 μL of 1 M potassium acetate, and mix. After 40 minutes of mixing, measure the absorbance at 415 nm using a UV-vis spectrophotometer. The measured absorbance was used as a calibration curve for the standard solution, and the total flavonoid content was expressed as mg QE/g in Fig. 2.

As shown in Fig. 2 below, the results of the flavonoid content analysis were consistent with the tendency for polyphenol content to increase. The sample (BN-4) according to Example 1 including the fermentation and steaming processes was analyzed to have the highest content of 57.6 mg QE/g, and the flavonoid contents of the sample (BN-3) according to Comparative Example 3, the sample (BN-2) according to Comparative Example 2, and the sample (BN-1) according to Comparative Example 1 were analyzed to be 52.9 mg QE/g, 43.7 mg QE/g, and 37.1 mg QE/g, respectively. The polyphenol and flavonoid contents showed similar tendencies depending on the extraction process, and it was confirmed that the content increased due to the fermentation process.

<Experimental Example 3> Anti-inflammatory activity evaluation

To evaluate the anti-inflammatory activity of the samples obtained in Example 1 and Comparative Examples 1 to 3, RAW 246.7 cells were used to confirm whether the NO concentration was reduced.

For reference, it is important to maintain the concentration of NO within the normal range because it can cause inflammatory diseases when the concentration of NO exceeds the normal range.

In this experiment, RAW 264.7 cells were cultured in DMEM (Dulbecco's modified Eagle's medium) containing 100 IU/mL penicillin, 100 IU/mL streptomycin, and 10% fetal bovine serum at 37°C in a 5% carbon dioxide incubator to confirm whether the produced NO was reduced by comparing the control group and the experimental group in which NO production was induced through LPS treatment.

RAW 264.7 cells were seeded in a 96-well plate at a density of ^3x104 cells/well and cultured in a 5% carbon dioxide incubator at 37°C for 12 hours to allow complete cell attachment. Then, 1 μg/mL of LPS, an inflammatory substance, and a mixed extract of bacopa (BN-1~4) were diluted in PBS (phosphate buffered saline) and treated at concentrations of 0, 100, 500, and 1,000 μg/mL and re-cultured for 24 hours.

The supernatant of the culture medium was obtained, and the same amount of Grease reagent (0.5% sulfanilamide, 2.5% phosphoric acid, and 0.5% naphthylethyleneamine) was added, reacted at room temperature for 10 minutes, and the absorbance was measured at 540 nm. The concentration of NO was calculated by comparison with the standard curve of nitrite, and the results are shown in Figure 3.

As shown in Figure 3 below, when the NO concentrations of the control group treated with LPS in Raw cell 246.7 cells and the samples according to the examples of the present invention and the samples (BN-1 to 4) according to Comparative Examples 1 to 3 were cultured for 24 hours, the NO concentration of the control group was analyzed to be the highest, and it was confirmed that the NO concentration of the experimental group tended to decrease as the concentration of the experimental substance increased.

In fact, the NO concentration decreased most significantly at a concentration of 1,000 μg/ml in each of the samples according to the embodiment of the present invention and the samples according to Comparative Examples 1 to 3 (BN-1 to 4), and in particular, the sample according to the embodiment of the present invention (BN-4) was confirmed to have the lowest concentration of 6.0±0.7 μM. Next, the sample according to Comparative Example 3 (BN-3), the sample according to Comparative Example 2 (BN-2), and the sample according to Comparative Example 1 (BN-1) were analyzed to be 6.6±0.1 μM, 7.9±0.3 μM, and 8.3±0.3 μM, respectively.

<Experimental Example 4> Antibacterial activity evaluation

In order to evaluate the antibacterial activity of the samples obtained in Example 1 and Comparative Examples 1 to 3, a death test was performed on a total of five types of bacteria, including bacteria causing dental caries ( Streptococcus sobrinus , Streptococcus mutans ), bacteria mainly found in root canals ( Enterococcus faecalis ), and bacteria mainly found in periodontal disease ( Porphyromonas gingivalis , Prevotella intermedia ), using the minimum inhibitory concentration method.

The strains were obtained from the Korea Research Institute of Bioscience and Biotechnology (KCTC) and used. Each strain was cultured anaerobically ( _N2 85%, _H2 10%, _CO2 5%) at 37°C in Brain Heart Infusion (BHI) liquid medium and Tryptic soy broth (TSB) solid medium.

The experimental group was diluted to various concentrations using a liquid medium by the 2-fold dilution method. After that, 2 mL of samples of various concentrations were taken and injected into 20 mL of TSB medium, and then 100 μL of each oral bacteria were injected and cultured in an incubator at 37°C for 48 hours.

After culture, the concentration at which each fungus did not grow when observed with the naked eye was determined as the MIC, and the results are shown in Table 1 below.

Strains MIC(μg/mL) BN-1 BN-2 BN-3 BN-4 Streptococcus sobrinus 500 500 62.5 62.5 Streptococcus mutans 500 250 62.5 62.5 Enterococcus faecalis 500 500 62.5 62.5 Porphyromonas gingivalis 250 250 62.5 62.5 Prevotella intermedia 250 250 62.5 62.5

As shown in Table 1 above, the test substances with the lowest minimum inhibitory concentrations were the sample (BN-4) according to the example of the present invention and the sample (BN-3) according to comparative example 3, and the minimum inhibitory concentration was 62.5 μg/mL in all strains.

Additionally, the sample (BN-1) according to Comparative Example 1 and the sample (BN-2) according to Comparative Example 2 were confirmed to have a minimum inhibitory concentration of 250 to 500 μg/mL in all strains.

Through the above antibacterial activity evaluation, it was confirmed that the fermentation process increased the antibacterial activity of the bacopa mixed extract against oral bacteria, but the steaming process did not significantly affect the antibacterial activity.

<Experimental Example 5> Cytotoxicity assessment

Cytotoxicity was evaluated using RAW 246.7 cells by varying the concentration of the sample obtained in Example 1 and the sample obtained in Comparative Examples 1 to 3. The results are shown in Fig. 4. It was confirmed whether the NO concentration was reduced.

Specifically, the MTT assay, a cytotoxicity test, was used as a safety test for samples. RAW 264.7 macrophages were dispensed into a 96-well plate at a concentration of 1Х10 ⁵ cells/well, and 24 hours after dispensing, 100 μL of the test substance diluted to a concentration of 100 to 1,000 μg/mL was added and cultured for 48 hours.

Then, 20 μL of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) solution was added, incubated for 4 hours to form formazan, and the supernatant was removed.

Then, 150 μL of DMSO was added to dissolve Formazan, and the absorbance was measured at 570 nm using an ELISA reader (Epoch ^TM 2, BioTek, USA), and the cell viability was calculated using the following equation.

[formula]

Cell viability (%) = [(Exp. - Blank)/Control] Х 100

(Exp: Absorbance of the extract containing cells, Blank: Absorbance of the extract not containing cells, Control: Absorbance of distilled water containing cells)

As shown in Figure 4 below, the cytotoxicity of the bacopa mixed extracts (BN-1 to 4) of 100 μg/mL, 250 μg/mL, 500 μg/mL, 750 μg/mL, and 1,000 μg/mL was confirmed based on the cytotoxicity (cell viability: 100%) of the untreated group. As a result, the cytotoxicity of the sample according to Comparative Example 1 (BN-1), the sample according to Comparative Example 2 (BN-2), the sample according to Comparative Example 3 (BN-3), and the sample according to Example 1 (BN-4) was not observed at any concentration. Therefore, the safety of the bacopa mixed extracts (BN-1 to 4) was confirmed in RAW 264.7 cells.

In conclusion, when the steamed dried product using Bacopa monnieri and nutmeg is inoculated with a fermentation strain and the powder of the steamed fermented extract or the extract is included as an active ingredient, the obtained composition has excellent ABTS and DPPH free radical scavenging activity and total polyphenol content and total flavonoid content, and thus has an excellent antioxidant effect, and exhibits anti-inflammation, antibacterial activity and cytotoxicity effects, and thus is suitable for applications requiring the above effects.

Claims (6)

A composition for preventing and improving periodontal disease, comprising as an effective ingredient a powder of a steamed fermented extract obtained by inoculating a fermented strain into a solid steamed dried product using Bacopa ( Bacopa monnieri ) and nutmeg , or an extract thereof,
The above fermentation strain is at least one selected from Bifidobacterium longum , Lactobacillus acidophilus , and Leuconostoc mesenteroides .
A composition for preventing and improving periodontal disease, characterized in that the steamed fermented extract is a fermented extract of a steamed product steamed at 80 to 110°C for 1 to 48 hours, dried at 35 to 75°C for 1 to 24 hours, and having a moisture content of 10% or less. In the first paragraph,
A composition for preventing and improving periodontal disease, characterized in that the above bacopa ( Bacopa monnieri ) and nutmeg (Nutmeg) satisfy a weight ratio of Bacopa (a) : Nutmeg (b) of 1:0.25 to 2. delete delete In the first paragraph,
A composition for preventing and improving periodontal disease, characterized in that the above steamed fermented extract is extracted using a reflux extraction solvent at 70 to 100° C. for 2 to 5 hours under reflux. In paragraph 5,
A composition for preventing and improving periodontal disease, characterized in that the reflux extraction solvent comprises at least one selected from chloroform, ethanol, methanol, water, ethyl acetate, hexane, and diethyl ether in an amount of 5 to 20 times the weight of the fermented product.