KR102678647B1 - method of producing viniferin and pterostilbene from blueberry - Google Patents

Abstract

The present invention relates to a mass production process that can secure viniferine and pterostilbene, which are conventional stilbene-based functional materials, in large quantities through an easy and simple process, and solves problems in the process of producing conventional stilbene-based functional materials. And by overcoming the limitations of raw material resources, it is possible to induce and proliferate blueberry-derived callus that can produce about 18 to 89 times more viniferine and pterostilbene. In addition, by increasing the amount of viniferine and pterostilbene produced within the raw material resources, a high content of viniferine and pterostilbene can be secured from the same amount of raw materials, so it can be very important in related industries.

Description

Method for mass producing viniferin and pterostilbene from blueberries {method of producing viniferin and pterostilbene from blueberries}

The present invention relates to a mass production process that can secure large quantities of viniferine and pterostilbene, which are conventional stilbene-based functional substances, through an easy and simple process.

Recently, the importance of primary prevention through diet has increased due to the increase in metabolic syndromes such as lifestyle diseases such as diabetes, hyperlipidemia, cardiovascular disease, and obesity, and the development of functional foods and related materials to activate specific functions beyond simple nutritional intake. Research is actively underway. In particular, as the trend of food culture focuses on fresh local food, the health functional food industry is also focusing on research on phytochemicals, which are contained in large quantities in plant resources.

Stillbenes are a type of phytochemical mainly found in grapes and wine. They are naturally occurring phenolic compounds and exist in various forms ranging from monomers to polymers. Among these, resveratrol has been the most widely studied. Resveratrol is effective in preventing cardiovascular diseases and metabolic syndrome due to its strong antioxidant properties, and has various pharmacological effects to improve human health, such as cognitive dysfunction, inhibition of cancer cell growth, and cancer prevention effects in vitro and in vivo. It has been confirmed in vivo (Steiner, 2016). In addition, resveratrol is a phytoalexin that is produced to protect itself from physical, chemical, and biological stress of the external environment such as mold, bacteria, and UV irradiation, so it treats external stimuli such as UV to the plant resources themselves. Various studies have been attempted to improve antibacterial properties and increase the content of resveratrol by doing so.

However, in the case of stilbene-based substances other than resveratrol, studies have mainly explored the biological activity effects as pure substances without considering the raw materials, or simply manufactured extracts and then examined the biological activity effects of the extracts.

For example, there are glycoside isomers such as piceatannol, pterostilbene, astringin, and piceid, which are monomers of resveratrol, and 2 to 8 resveratrol subs. There are resveratrol oligomers composed of units.

Among them, pterostilbene has been studied to show that it has similar efficacy to resveratrol but has much greater bioavailability due to its relatively stable molecular structure. However, despite this effect, research on the industrialization of the production process to utilize it as a bioindustrial material is needed. is in poor condition. Furthermore, it is difficult to find basic data on the content of natural resources.

Among the resveratrol oligomers, viniferin, which is known to be mainly present in grapes, is a resveratrol dimer and is found in very small amounts even in wine, with a concentration of 0.1 to 4.3 mg/L. Viniferine is known to have excellent effects such as antioxidant, anti-inflammatory, anti-cancer and cardioprotective effects.

However, viniferine not only has a very low bioavailability among resveratrol oligomers, but its concentration in raw materials is very rare, and even if processed or extracted, there is a problem that it is difficult to produce viniferin at a sufficient concentration for its pharmacological properties.

In addition, in the prior art, there are examples of drying and pulverizing raw materials collected from cultivation areas, such as grapes that produce stilbene-based functional substances, and then producing a large amount of extract using organic solvents or ultrasonic extraction, but this still lacks extraction efficiency. and productivity is significantly low. In addition, a large amount of extract contains a large amount of impurities, which places an excessive burden on the subsequent separation and purification processes, so there is a problem that the content and quality of the stilbene-based functional material remain low.

In order to solve these problems, there are examples of introducing advanced separation and purification technology considering the purity and uniformity of the extract, but the production cost has increased excessively, making it difficult to secure competitiveness in terms of production cost, and the stilbene-based functional material Even if the content increases, viniferine still has a problem in that its concentration is very rare.

Patent Document 1. Republic of Korea Patent Publication No. 10-2015-0130006

The present invention was created to solve the above problems, and the purpose of the present invention is to provide a method for mass producing viniferin and pterostilbene from blueberries.

Another object of the present invention is to provide a culture composition for mass production of viniferin and pterostilbene from blueberry-derived callus.

In order to achieve the above object, the present invention

i) inducing blueberry callus by placing blueberry tissue on callus induction medium;

ii) subculturing the blueberry callus recovered from step i) 3 to 4 times at 3 to 6 week intervals to obtain callus;

iii) suspending and culturing the callus in a growth medium; and

iv) extracting the callus culture of step iii); A method for mass producing viniferin and pterostilbene derived from blueberries is provided.

The blueberry tissue in step i) may be blueberry skin or leaves.

The blueberry tissue in step i) was washed with 0.5-5% surfactant for 5 minutes, treated with water for 0.5-6 hours, then treated with 70% EtOH for 10-60 seconds, and then washed with 0.5-1.5% NaOCl 0.1-10. It may be a part of the tissue of a blueberry plant that has been subjected to a surface sterilization step of washing for several minutes.

The callus is transformed from the blueberry callus of step i) during the subculture process of step ii), and may be a red callus that appears red when visually observed.

The callus induction medium in step i) may be a solid medium containing auxin at a concentration of 0.1 to 1.0 mg/ml and cytokinin at a concentration of 0.1 to 0.2 mg/ml.

The growth medium in step iii) may be a liquid medium containing auxin at a concentration of 0.1 to 1.0 mg/ml.

The steps i), ii), and iii) may be cancer culture.

Step iv) may be performed by any one or more extraction methods selected from the group consisting of ultrasonic extraction, solvent extraction, and distribution extraction.

It may further include the step of freezing and drying the blueberry-derived callus extract obtained through step iv) above.

The viniferin may be delta (δ)-viniferin or epsilon (ε)-viniferin.

In order to achieve the above other object, the present invention provides viniferin and pterostilbene from blueberry-derived callus, which contains as an active ingredient any one growth regulator selected from the group consisting of auxin, cytokinin, and mixtures thereof. Provided is a medium composition for callus induction and subculture that induces transformation of mass-produced callus.

The auxin may be added at a concentration of 0.1 to 1.0 mg/ml, and the cytokinin may be added at a concentration of 0.1 to 0.2 mg/ml.

The present invention solves problems in the process of producing conventional stilbene-based functional materials and overcomes the limitations of raw material resources, thereby deriving blueberry-derived callus that can produce about 18 to 89 times more viniferine and pterostilbene. and can be multiplied. In addition, by increasing the amount of viniferine and pterostilbene produced within the raw material resources, high purity viniferine and pterostilbene can be secured from the same amount of raw materials, so it can be very important in related industries.

Figure 1 is a graph showing the analysis of stilbene content for each part of blueberry based on solid weight.
Figure 2 is a photograph of the callus induced by cutting the leaves, stems, and pericarp tissues of blueberry tissue culture seedlings grown under sterile conditions to a certain size to prepare tissue bodies (slices), and then pulverizing them in callus induction medium.
Figure 3 shows the cell culture results of tissues derived from blueberry leaves of the O'Neill variety treated under optimized surface sterilization conditions.
Figure 4 shows the results of cell culture of tissue derived from blueberry skin of the O'Neill variety treated under optimized surface sterilization conditions.
Figure 5 shows the cell culture results of tissues derived from the pericarp of O'Neill variety blueberry treated under surface sterilization conditions treated with 1% sodium hypochlorite for more than 10 minutes.
Figure 6 is a photograph of callus cultures prepared from groups 19, 20, and 21.

Below, we will look at various aspects and various implementation examples of the present invention in more detail.

One aspect of the present invention provides a method for mass producing viniferin and pterostilbene derived from blueberries, comprising the following steps.

i) inducing blueberry callus by placing blueberry tissue on callus induction medium;

ii) subculturing the blueberry callus recovered from step i) 3 to 4 times at 3 to 6 week intervals to obtain callus;

iii) suspending and culturing the callus in a growth medium; and

iv) Separating cells from the callus culture of step iii) above and extracting them with a solvent.

The blueberry tissue in step i) may be blueberry stems, leaves, and pericarp, preferably blueberry pericarp or leaves, and more preferably blueberry leaves. The leaves may be leaves obtained from blueberry tissue culture seedlings. As confirmed in the experimental examples described below, it can be seen that various stilbenes are present in blueberry skin and leaves (Figure 1 and Table 2). Furthermore, it can be seen that it is desirable to use leaves that are not damaged or contaminated after surface treatment, have a high survival rate close to 100%, and have the shortest incubation time required for callus induction.

First, the blueberry tissue in step i) was washed with 0.5-5% surfactant for 5 minutes, treated with water for 0.5-6 hours, then treated with 70% EtOH for 10-60 seconds, and then washed with 0.5-1.5% NaOCl 0.1- It may be a part of the tissue of a blueberry plant that has gone through a surface sterilization step of washing for 10 minutes. However, surface sterilization treatment involves washing with 0.2-2% surfactant for 5 minutes, treating with water for 1-6 hours, followed by treatment with 70% EtOH for 10-30 seconds, and washing with 0.5-1% NaOCl for 0.5-5 minutes. It is preferable to use part of the tissue of a blueberry plant that has gone through several stages, as it can achieve a survival rate of 80-100% or more.

If the blueberry tissue does not undergo the surface sterilization process as described above, or is manufactured using a plant that has undergone the surface sterilization process under different conditions, as in the experimental example described later, the tissue is damaged or contaminated, resulting in callus. There is a problem where it is not formed properly.

It was confirmed that the survival rate of blueberry tissue produced through the above-described surface sterilization process can reach more than 50%, and when performed under the above preferred conditions, it reaches 80-100%.

Above i) Induce blueberry callus by placing the blueberry tissue on callus induction medium. At this time, it is preferable that step i) is cancer culture.

Afterwards, ii) the blueberry callus recovered from step i) can be subcultured 3 to 4 times at 3 to 6 week intervals to obtain red callus. Specifically, in step ii), when the cells of the tissue undergo cell division and callus, a mass of cells, is formed, the callus is separated from the callus induction medium and subcultured in a medium under the same conditions to proliferate. In this case, subculture Cultivation should be done at intervals of 3 to 6 weeks, and it is desirable to subculture more than three times. Most preferably, it may be subcultured 3 to 4 times.

The medium used in step ii) is preferably the same as the callus induction medium in step i). Therefore, the medium used in step ii) and the callus induction medium used in step i) are referred to as 'callus induction medium' unless a separate distinction is required or otherwise stated.

The callus is transformed from the blueberry callus of step i) during the subculture process of step ii), and is characterized by being red when confirmed with the naked eye.

In order to obtain red callus, both callus induction medium and subculture conditions must be satisfied. For example, it is preferable that both steps i and ii are dark cultured. If both steps ⅰ and ⅱ above satisfy the cancer culture, it is desirable to culture at intervals of 4 to 6 weeks and subculture 3 to 4 times. If subculture is performed less than 3 times or more than 4 times, it is transformed into red callus. Since this does not occur and remains as yellow callus, there is a problem in that red callus cannot be obtained. Therefore, most preferably, callus induction culture and subculture of blueberry callus are performed under dark culture conditions, and subculture may be performed 3 to 4 times.

If the subculture step is omitted, a problem may arise in which red callus cannot be obtained. The present invention can mass-produce up to 89 times the content of viniferine and pterostilbene by culturing red callus in suspension and obtaining extracts from the cultured cells. In other words, if any one of steps i and ii is omitted (particularly step ii) or the culture conditions are different, there is a problem in that the above-described purpose of the present invention cannot be achieved.

Next, iii) the callus is cultured in suspension in a growth medium.

Step iii) may be a step of suspension culture to ensure sufficient growth and proliferation of callus, especially red callus, obtained through step ii). For example, the callus obtained in step ii) above is transferred to a liquid medium and cultured in liquid suspension, and cultured in the liquid medium for 2 to 15 weeks, preferably 4 to 8 weeks, to produce a red callus culture grown from red callus. You can get it.

After the primary suspension culture is completed, if necessary, repeated subculture can be performed by re-inoculating the callus culture in the same medium and under the same conditions and continuously culturing, but this is not particularly limited. However, considering the production of unexpected by-products during the culture period and the maintenance period of red callus, it is preferable to perform only the first suspension culture from a cost and effectiveness perspective.

As described above, the method of producing cells by inducing callus from blueberry tissue, sub-culturing it, and culturing it in suspension is a suitable method of callus-derived cells (especially plant cells) well known to those skilled in the art of the present invention. Any manufacturing method can be used and is not particularly limited.

It is preferable that step iii) is also a dark culture. If any one of the above processes is performed in light culture, red callus may not be formed or may be transformed back into the original yellow callus, so only step iii) Rather, it is most desirable to perform both steps i and ii in dark culture.

In the present invention, step iii) is a suspension culture, in which red callus derived from blueberries is dispersed and cultured in a growth medium, which is a liquid nutrient medium, and can be derived from a sufficiently grown callus culture. Step iii) is cultured for 2 to 15 weeks, preferably 4 to 8 weeks, and after the culture period has passed, the cultured red callus culture can be recovered. If necessary, additional subculture can be performed repeatedly by inoculating the cultured red callus culture into the same medium under the same conditions.

The basic essential components of the medium used in the suspension culture are similar to those used for callus induction, but the mixing amount of growth regulators contained is different, and unlike the callus induction medium, it is a liquid medium, so it may not contain a solidifying agent. there is.

The callus induction medium and growth medium are medium to which a growth regulator that induces callus culture and proliferation is added, and are not particularly limited thereto, but may include a conventional solid medium or liquid medium. For example, MS (Murashige and Skoog, 1962) medium, B5 (Gamborg et al., 1968) medium, SH (Schenk and Hildebrandt, 1972) medium, LS (Linsmaier and Skoog, 1965) medium, and WPM (Lloyd and McCown, 1980) may be any one selected from the group consisting of media.

The most widely used medium is MS (Murashige and Skoog, 1962) medium, which is designed for tissue culture of tobacco. MS medium is characterized by high concentrations of ammonium, nitrate, and potassium. LS (Linsmaier and Skoog, 1965) medium is similar to MS medium, but has differences in the composition of some organic substances. White medium (White, 1963) is characterized by low salt concentrations and was designed for culturing tomato roots. B5 medium (Gamborg et al., 1968) was designed to cultivate most callus and is characterized by a higher content ratio of nitrate ion than ammonium ion. SH medium (Schenk and Hildebrandt, 1972) was developed to cultivate callus of dicotyledonous and monocotyledonous plants. NN (Nitsch and Nitsch, 1969) medium was developed for weak culture and has a lower salt concentration than MS medium but higher than White medium.

When cultivating a new species, it is advantageous to select a medium by referring to the previously reported culture results of highly related plant species. When inducing only callus from plant tissue, there is no need to test various media to set a special media. Rather, selecting growth regulators with different concentrations and combinations in the medium is often more effective.

Growth regulators are essential elements involved in the differentiation of stems and roots and the growth of cells, but in plant tissue culture, the type, concentration, and combination of growth regulators are the most important requirements for successful culture. Or, depending on the combination, callus with completely different properties may be induced. Therefore, in order to obtain blueberry-derived callus or callus extract that simultaneously mass-produces bipiperine and pterostilbene as in the present invention, 'callus induction medium' and 'proliferation medium' limited to the optimized conditions of the growth regulator through the experimental examples described later. It is most preferable to use .

In particular, in the present invention, red callus modified from yellow callus must be derived, rather than the yellow callus generally obtained from blueberry tissue, and also, excellent efficacy is achieved without changing the properties of the red callus recovered through the previous step. It must be possible to promote proliferation and growth while maintaining the same, and it can be confirmed through the experimental examples described below that this can be achieved through the culture steps and culture conditions proposed in the present invention.

That is, the content of the growth regulator contained in the callus induction medium of step i) and the growth medium of step iii) may be the same or different from each other. Specifically, both auxin and cytokinin were added to the callus induction medium in step i), preferably auxin at a concentration of 0.1 to 1.0 mg/ml, and auxin at a concentration of 0.1 to 0.2 mg/ml. It may be a solid medium containing cytokinin.

The growth medium in step iii) may be one to which auxin or cytokinin or both are added, preferably a liquid medium to which only auxin is added as a growth regulator, more preferably 0.1 It may be a liquid medium containing only auxin at a concentration of from 1.0 mg/ml as a growth regulator.

The auxin may be any one selected from the group consisting of 2,4-D (2,4-dichlorophenoxyacetic acid), NAA (naphthalatene acetic acid), IBA (indolebutric acid), and IAA (indole-acetic acid), but is preferred. It may be 2,4-D. The cytokinin may be any one selected from the group consisting of kinetin (6-furfuryl-amino purin), BA (banzyladenine), and zeatin {6-(4-hydroxy-3-methyltrans-2-butenylamino) purine}. There is, preferably BA.

Specifically, the callus induction medium in step i) may be MS (Murashige & Skoog) medium containing 2,4-D at a concentration of 0.1 to 1.0 mg/ml and BA at a concentration of 0.1 to 0.2 mg/ml, and 0.5 mg/ml. MS (Murashige & Skoog) medium containing 2,4-D at a concentration of /mL and BA at a concentration of 0.2 mg/mL is most preferred.

The growth medium in step iii) may be MS (Murashige & Skoog) medium containing 2,4-D at a concentration of 0.1 to 1.0 mg/ml, most preferably 2,4-D at a concentration of 0.2 mg/ml. It may be an MS (Murashige & Skoog) badge containing .

Specifically, the growth temperature suitable for inducing and proliferating the callus culture is not particularly limited, and may be, for example, 20 to 30°C, and preferably 22 to 27°C. The appropriate pH range for inducing and proliferating the callus culture is not particularly limited, and for example, the preferred pH may be 5 to 7, and more preferably pH 5 to pH 6. The culture time appropriate for the induction and proliferation of the callus culture is not particularly limited, and may be, for example, 2 to 15 weeks, preferably 4 to 8 weeks. In order to induce the callus, it can be cultured under light conditions where the time and light intensity are controlled through an artificial light source, and under dark conditions where no light is provided, and preferably, it can be cultured under dark conditions.

It was confirmed that the blueberry-derived red callus culture induced and propagated by the above method contained 0.01 to 15% by dry weight of blueberry-derived red callus.

Finally, iv) the red callus culture from step iii) is extracted to prepare a callus extract.

After going through all the above-mentioned processes, red callus derived from blueberry tissue is produced, and the culture is recovered by suspension culture. Through the extraction process, the productivity of viniferine and pterostilbene is higher than that of blueberry leaf extract. Both can produce and obtain significantly better callus extracts (18 times and 89 times more, respectively).

The red callus culture is the culture medium itself, the suspension culture cells (plant cells) grown and proliferated from blueberry-derived red callus separated by filtration or centrifugation of the culture medium (pellet excluding the centrifuged supernatant) ), etc.

In order to separate the suspension cultured cells, any suitable method widely known to those skilled in the art of the present invention can be used without particular limitation, for example, separating solids through centrifugation, distillation under reduced pressure, freeze drying, or It may be powdered through a process such as spray drying.

In step iv), the red callus culture recovered in step iii) can be extracted through various methods, and is not particularly limited as long as it is an appropriate method widely known to those skilled in the art of the present invention. For example, one or more extraction methods selected from the group consisting of ultrasonic extraction, solvent extraction, and distribution extraction can be used.

Specifically, ultrasonic extraction is obtained by immersing the red callus culture in an extraction solvent or an acid-added extraction solvent and treating it with ultrasonic waves, and solvent extraction is obtained by immersing the red callus culture in an extraction solvent for extraction, and distribution extraction After adding water and the same amount of organic solvent to the red callus culture, partitioning, each layer separated into two phases is separated and extracted. Distribution extraction can be repeated as needed by adding an equal amount of water and an organic solvent to one of the separated layers obtained through the above process, distributing the layer, and separating the layer into two phases.

The extraction solvent may be any one or more selected from the group consisting of water, lower alcohols having 1 to 5 carbon atoms, chloroform, dichloromethane, benzene, acetone, hexane, ethyl acetate, and mixtures thereof. The organic solvent may be any one or more selected from the group consisting of butanol, chloroform, dichloromethane, benzene, hexane, and ethyl acetate.

Specifically, the extraction process is as follows. Adding one or more extraction solvents selected from the group consisting of water, lower alcohols having 1 to 5 carbon atoms, chloroform, dichloromethane, benzene, acetone, hexane, ethyl acetate, and mixtures thereof to the red callus culture recovered in step iii) above. It may be an ultrasonic extract in which the active ingredients contained in the callus are effectively extracted by ultrasonic treatment under specific conditions after addition. At this time, the extraction solvent used in the ultrasonic extraction may further include an acid solution.

Alternatively, it is a solvent extract obtained by adding an extraction solvent to the recovered red callus culture, or extracted by a partition extraction method using one or more organic solvents selected from the group consisting of butanol, chloroform, dichloromethane, benzene, hexane, and ethyl acetate. It may be a distributed extract.

Most preferably, the extraction step iv) is performed by adding a mixed solvent of extraction solvent and acid to the red callus culture recovered from step iii) and then treating it with ultrasound under specific conditions to effectively remove the active ingredients contained in the red callus. It may be an extracted ultrasonic extract, and the ultrasonic extract extracted through this extraction process may be distributed and extracted using one or more organic solvents selected from the group consisting of butanol, chloroform, dichloromethane, benzene, hexane, and ethyl acetate. .

Therefore, the extract prepared through the method of the present invention is a material with various functionalities and can be usefully used in various industrial fields such as medicine, food, and cosmetics. In particular, since blueberries are edible as natural medicinal plants, the composition of the present invention containing extracts derived from blueberries as an active ingredient has the advantage of being safe even for long-term use.

Summarizing the above, the final form of the method for mass producing blueberry-derived viniferin and pterostilbene of the present invention may include the following steps and conditions.

First, i) Surface sterilization by washing with 0.5-5% surfactant for 5 minutes, treating with water for 0.5-6 hours, then treating with 70% EtOH for 10-60 seconds, and washing with 0.5-1.5% NaOCl for 0.1-10 minutes. A portion of the tissue of the blueberry plant that has undergone the processing step is separated to prepare a blueberry tissue, and auxin at a concentration of 0.1 to 1.0 mg/ml and cytokinin at a concentration of 0.1 to 0.2 mg/ml are added to the prepared blueberry tissue. Blueberry callus is induced by planting on MS medium (callus induction medium).

ii) Subculture the blueberry callus recovered from step i) 3 to 4 times at 3 to 6 week intervals on the same medium as step i) to obtain red callus.

iii) The red callus is cultured in suspension in dark culture conditions in MS medium (proliferation medium) supplemented with auxin at a concentration of 0.1 to 1.0 mg/ml for 2 to 15 weeks, preferably 4 to 8 weeks.

iv) Cells are separated from the red callus culture in step iii) above and extracted with a solvent to prepare a callus extract.

The blueberry-derived callus extract obtained through the above-described series of processes contains viniferine and pterostilbene contents 18 and 89 times higher, respectively, compared to natural blueberry leaf extract, and the manufacturing process is simple culture to produce red callus. Since it can be recovered sufficiently through a simple extraction process, it has the advantage of being able to obtain a large amount of blueberry-derived callus extract showing excellent function. In addition, since most processes use minimal solvents and are therefore very environmentally friendly, the blueberry-derived red callus and its extract prepared according to the present invention are therapeutic agents or functional food materials that have the effect of treating liver toxicity, protecting and improving liver function. It can be used as.

In addition, in the present invention, in order to recover a high-concentration product, the extract can be concentrated by a method such as reduced pressure concentration or purified to high purity using an additional purification method. Since it is possible to produce a blueberry-derived callus extract containing more than viniferin and 89 times more pterostilbene, it can be said to be very cost-effective compared to the conventional process for producing viniferin or pterostilbene.

Additionally, the extract may be prepared in powder form by additional processes such as reduced pressure distillation, freeze drying, or spray drying.

Another aspect of the present invention is to mass-produce viniferin and pterostilbene from blueberry-derived callus, containing as an active ingredient any one growth regulator selected from the group consisting of auxin, cytokinin, and mixtures thereof. It relates to a medium composition for callus induction and subculture that induces callus transformation. The medium composition for callus induction and subculture is one in which both auxin and cytokinin are added, preferably auxin at a concentration of 0.1 to 1.0 mg/ml, and auxin at a concentration of 0.1 to 0.2 mg/ml. It may be a solid medium containing cytokinin.

The auxin may be any one selected from the group consisting of 2,4-D (2,4-dichlorophenoxyacetic acid), NAA (naphthalatene acetic acid), IBA (indolebutric acid), and IAA (indole-acetic acid), but is preferred. It may be 2,4-D. The cytokinin may be any one selected from the group consisting of kinetin (6-furfuryl-amino purin), BA (banzyladenine), and zeatin {6-(4-hydroxy-3-methyltrans-2-butenylamino) purine}. There is, preferably BA.

Specifically, the callus induction medium in step i) may be MS (Murashige & Skoog) medium containing 2,4-D at a concentration of 0.1 to 1.0 mg/ml and BA at a concentration of 0.1 to 0.2 mg/ml, and 0.5 mg/ml. MS (Murashige & Skoog) medium containing 2,4-D at a concentration of /mL and BA at a concentration of 0.2 mg/mL is most preferred.

Through the above-described medium composition for callus induction and subculture, it was confirmed that when tissue isolated from blueberries was seeded and subcultured, red callus was formed when confirmed with the naked eye, and the red callus was formed by viniferin. It was confirmed through the experiment described later that the productivity of pterostilbene was very significant. In particular, in the case of the extract obtained by suspension culturing and extraction of the red callus, it was confirmed that a callus extract that was significantly superior to the blueberry leaf extract (18 times and 89 times, respectively) could be produced and obtained.

According to one aspect of the present invention, subculture is performed at intervals of 3 to 6 weeks, and subculture is preferably performed three or more times. Most preferably, it may be subcultured 3 to 4 times.

As described above, the callus extract can be used as a food composition or cosmetic composition.

Ingredients included in the cosmetic composition of the present invention may include ingredients commonly used in cosmetic compositions in addition to the callus extract as an active ingredient, for example, stabilizers, solubilizers, vitamins, pigments, and fragrances. Includes adjuvants and carriers.

The formulation of the cosmetic composition of the present invention is not particularly limited and can be appropriately selected depending on the purpose. For example, the cosmetic composition of the present invention can be manufactured in formulations such as skin ointment, lotion, softening lotion, nourishing lotion, nutritional cream, massage cream, essence, pack, emulsion, oil gel, etc.

The cosmetic composition of the present invention contains the callus extract in an amount of 0.00001 to 50% by weight, preferably, 0.0001 to 20% by weight, and more preferably 0.1 to 10% by weight, based on the total weight of the composition. However, the composition as described above is not necessarily limited to this and may change depending on the patient's condition and progress.

Meanwhile, in the above 'food composition', there is no particular limitation on the type of food. Examples of foods to which the extract can be added include drinks, meat, sausages, bread, biscuits, rice cakes, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, It includes beverages, alcoholic beverages, vitamin complexes, dairy products and milk-processed products, and includes all health functional foods or health supplements in the conventional sense.

In the food composition of the present invention, the callus extract can be added as is to food or used together with other foods or food ingredients, and can be used appropriately according to conventional methods. The amount of callus extract added in the food composition of the present invention can be appropriately determined depending on the purpose of use (prevention or improvement). It is preferable to include 0.1 to 50% by weight of the callus extract based on the total weight of the food composition of the present invention, but it is not limited thereto. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount may be below the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range.

When manufacturing a beverage, there are no particular restrictions on other ingredients other than containing the callus extract, and various flavoring agents or natural carbohydrates can be contained as additional ingredients like regular drinks. The natural carbohydrates include monosaccharides, such as glucose and fructose; Disaccharides such as maltose, sucrose, etc.; and polysaccharides, such as common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As the flavoring agent, natural flavoring agents (thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The natural carbohydrate The ratio is about 1 to 20% by weight, preferably about 5 to 12% by weight, based on the total weight of the food composition of the present invention.

In addition to the above, the food composition of the present invention contains various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavors, colorants and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and It may contain its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the food composition of the present invention may contain pulp for the production of natural fruit juice, fruit juice beverages, and vegetable beverages.

These ingredients can be used independently or in combination. The proportion of these additives is not that important, but is generally selected in the range of 0.1 to about 20% by weight based on the total weight of the food composition of the present invention.

Hereinafter, the present invention will be described in more detail through examples, etc., but the scope and content of the present invention should not be construed as being reduced or limited by the examples below. In addition, based on the disclosure of the present invention including the following examples, it is clear that a person skilled in the art can easily carry out the present invention for which no specific experimental results are presented, and the patent to which such variations and modifications are attached It is natural that it falls within the scope of the claims.

In addition, the experimental results presented below describe only the representative experimental results of the examples and comparative examples, and the effects of each embodiment of the present invention that are not explicitly presented below will be described in detail in the corresponding section.

Experiment example One. Blueberries By part styrene content analysis

1) Experimental materials and reagents

Among the blueberries produced domestically, the O'Neal variety, an early maturing variety, harvested in the Seongnam region in June 2016 was collected. The collected blueberries were divided into skin, leaves, and stems, and each part was freeze-dried to analyze the stilbene content.

All reagents used in the experiment were HPLC grade and were purchased from JT Baker (Phillipsburg, NJ, USA) and Sigma-Aldrich (St Louis, MO, USA). Resveratrol (t-resveratrol, R5010), pterostilbene (P1499), and pinosylvin (56297) used as standard substances were obtained from Sigma-Aldrich (St Louis, MO, USA) and viniferine (ε- Viniferin (FV30429) was purchased from Carbosynth (Berkshire, UK).

By analyzing the stilbene content of each tissue part of blueberry, we attempted to select tissues to be used for tissue culture.

2) Blueberries By part stilben content analysis

In order to analyze the stilbene content in each part (pericarp, leaf, stem) of the blueberry prepared in step 1) above, stilbene was extracted using the method below, and then the stilbene content was analyzed by HPLC.

First, each freeze-dried part (pericarp, leaf, stem) prepared in step 1) above was pulverized using a mortar and pestle. At this time, the freeze-dried stems of blueberries were pulverized using a grinder (Cyclotec 1093, Foss, Hoganas, Sweden). 25 ml of methanol solution mixed with 0.1% HCl was added to 3 g of the pulverized sample, suspended, and treated with ultrasonic waves at 27°C for 30 minutes to prepare an extract. The extract was centrifuged at 12,000 Filtered through 4 and the residue was collected. The residue was re-extracted using the extraction process described above and filtered. The re-extracted liquid was mixed with the filtrate, concentrated under reduced pressure at 40°C (rotary evaporator, EYELAN-1110, EYELA, Tokyo, Japan) to remove the solvent, and then 20 mL of distilled water and an equal amount of ethyl acetate were sequentially added. It was left at 4°C for 10 minutes. The ethyl acetate fraction was transferred to a new tube, and the remaining distilled water layer was separated. The separated residual distilled water layer was separated into fractions two more times using ethyl acetate. All ethyl acetate fractions were mixed and then concentrated under reduced pressure at 40°C (rotary evaporator, EYELA N-1110, EYELA, Tokyo, Japan) to prepare a concentrate. The concentrate was dissolved in 1 ml of methanol, filtered through a 0.22 μm syringe filter, and then injected into HPLC to analyze the stilbene content.

3) HPLC analyze

To analyze the stilbene content of blueberries, the conventional HPLC analysis method (Lyons et al. 2003) was used with some modifications. Specifically, the HPLC system (Agilent 1100 series) consists of a pump, injector, UV/VIS detector, integrator (Chemstation software), and column (Agilent eclipse XDB-C18: 4.6 mm x 250 mm, 5 μm, ZORBAX). The analysis conditions are shown in Table 1 below. Water with 0.1% formic acid (A) and acetonitrile (B) with 0.1% formic acid were used as solvents at a flow rate of 1.0 ml/min under gradient conditions. It was operated for 45 minutes. At this time, the sample injection volume was 20 μl, and the solvent gradient conditions started with an A/B ratio of 90:10, maintained at a 20:80 ratio until 30 minutes, and then adjusted to 90:10 until 35 minutes. The ratio was maintained at 90:10 for 10 minutes. The detection wavelength was measured at 300 nm, and the oven temperature of the analysis column was kept constant at 25°C. Meanwhile, as standard substances, resveratrol, pterostilbene, and pinosylvin were purchased from Sigma-Aldrich (St Louis, MO, USA), and viniferine was used from Carbosynth (Berkshire, UK). Each experimental value was expressed as the average of 5 repeated measurements.

As a result of the analysis, the stilbene standard solution was separated without interference peaks in the order of resveratrol, viniferine, and pterostilbene, and the RT values were 14.02 minutes for resveratrol, 20.19 minutes for pinosylvin, and 24.69 minutes for pterostilbene, respectively. It was confirmed that all species were separated within 30 minutes, and this was used as a calibration curve.

parameters condition Column Agilent eclipse XDB-C18 (4.6 x 250mm, 5m, ZORBAX) Column temperature 25℃ flow rate 1.0ml/min injection volume 20 μl menstruum A: 0.1% formic acid in water,
B: 0.1% formic acid in acetonitrile gradient time(min) % A %B 0 90 10 30 20 80 35 90 10 45 90 10 wavelength 300 nm

4) Statistical processing

For stilbene content, analysis of variance (ANOVA) was performed using SAS 9.2 version for Windows (SAS Institute Inc., Cary, NC, USA), and significance was tested using Duncan's multiple range test at the p<0.05 level.

5) Conclusion

Figure 1 is a graph showing the analysis of stilbene content for each part of blueberry based on solid weight.

Blueberry part Stilbenoid concentration
(Stilbeoid content)(㎍/g dw) Resveratrol
(Resveratrol) -Viniferine
(-Viniferin) pinosylvin
(Pinosylvin) pterostilbene
(Pterostilbene) pericarp
(Skin) 1.45±0.24 2.88±1.50 0.03±0.03 0.14±0.02 stem
Stem 1.38±0.16 7.19±1.43 0.00±0.00 0.03±0.01 leaf
Leaf 2.86±0.64 1.00±0.12 0.09±0.03 0.10±0.02

Looking at Figure 1 and Table 2, it was confirmed that resveratrol was most abundantly contained in blueberry leaves at 2.86 ㎍/g dw, and that the skin and stem of blueberries contained 1.45 and 1.38 ㎍/g dw, respectively. It was confirmed that among the four types of stilbenes, viniferine content varies significantly depending on the part of the blueberry. In particular, it was found to be contained most highly in the stems and skin of blueberries.

However, in the case of pinosylvin, it was confirmed that there was a significant difference in each part of the blueberry, and it was confirmed to contain the lowest content overall, but in particular, it was observed that the stem of the blueberry contained almost no pinosylvin.

The content of pterostilbene was highest in the pericarp at 0.14 μg/g dw, and a significant difference was found in leaves and stems at 0.10 and 0.03 μg/g dw, respectively.

In summary, blueberry skin, stems, and leaves contain different stilbenoids in various amounts and ratios depending on the type, so any stilbenoid can be used. However, it is the leaves and skin of blueberries that contain various stilbenes. As confirmed, it can be seen that it is preferable to use the leaves and skin of blueberries.

Experiment example 2. Blueberries origin callus Check inducibility

1) Plant preparation

Before using adult blueberry tissues, we first sought to obtain uncontaminated blueberry tissues from blueberry tissue culture plants under sterile conditions and then use them to determine whether callus formation was possible.

The leaves and stem tissues of blueberry tissue culture seedlings were cut into sections of a certain size and placed on callus induction medium. Callus was generated about 2-3 weeks after rooting, and at about 6-8 weeks, callus was formed throughout the tissue to the extent that it could be used for subculture. At this time, callus derived from leaves of blueberry tissue culture seedlings was cut out under aseptic conditions and subcultured. Transferred to a plate medium for culture. Callus is a shapeless mass of cells that dedifferentiates, but it is by no means uniform, so only the part containing the division center (outer layer of the callus mass), that is, the pale and shiny fresh part, is picked out with tweezers and a scalpel and transferred to new medium for subculture. The callus was stabilized by performing . It was confirmed that callus was formed throughout the tissue enough to enable subculture, and in the present invention, considering the degree of proliferation, culture period, and cost, blueberry tissue culture seedlings were continuously transplanted into new media at about 4-6 week cycles. Leaf-derived callus cell lines were obtained. Therefore, it was confirmed that callus formation was possible from blueberry tissue under appropriate sterile culture conditions.

The callus induction medium used at this time was MS (Murashige & Skoog) medium including B5 vitamins (M0231) containing plant hormones (auxin 0.2 mg/ml), sucrose 30 g/l, and gelite 4 g/l (pH 5.8). medium was used. The culture conditions were 1500 Lux light culture (16/8 h day/night cycles) and dark culture conditions in a growth chamber with a temperature of 26°C and a humidity of 60%.

2) Conclusion

Figure 2 is a photograph of the callus induced by cutting the leaves, stems, and pericarp tissues of blueberry tissue culture seedlings grown under aseptic conditions to a certain size to prepare tissue bodies (slices), and then pulverizing them in callus induction medium.

Specifically, it was confirmed that callus was generated about 2-3 weeks after the tooth was wound (left side of Figure 2), and at about 6-8 weeks, callus was formed throughout the tissue to the extent that it could be used for subculture (right side of Figure 2). At this time, the callus is cut under aseptic conditions and transferred to a plate medium for subculture. Callus is a shapeless mass of cells that dedifferentiates, but it is by no means uniform, so only the part containing the division center (outer layer of the callus mass), that is, the pale and shiny fresh part, is picked out with tweezers and a scalpel and transferred to new medium for subculture. The callus was stabilized by performing .

As shown in Figure 2, it was confirmed that callus was formed throughout the entire tissue to enable subculture in both leaves and stems, and the leaf-derived callus was continuously transplanted to new media at about 4-6 week cycles to produce blueberry tissue culture seedlings. Leaf-derived callus cell lines were obtained.

Experiment example 3. Blueberries origin callus Surface sterilization method of plants for cell culture Choi red

1) Plant preparation

First, we optimized the surface disinfection method of plants for cell culture of blueberry-derived callus. The plants used were fruits and leaves of the O'Neal variety, an early maturing variety of blueberries produced in Korea, harvested in the Seongnam region in June 2016.

When the plant was a fruit, the fruit was first sterilized as a whole to make it sterile, and then the pericarp tissue with the pulp removed was used.

After washing the plant several times with water to completely remove contaminants and contaminants, the type of washing solution (detergent, hypochlorous acid, etc.), concentration and treatment time, treatment with 70% ethanol, etc. were set as variables, and the specific washing order was performed. and conditions are as shown in Table 3 (washing proceeds in the order 1->2->3->4). When changing the washing solution, the product was washed with sterilized water three times for at least 30 seconds, washed in a shaking incubator at 25°C and 100 rpm, and then the washing solution to be changed was applied.

For the reproducibility and reliability of the experiment, 10 to 11 samples under the same conditions were made in each experiment and cultured under dark culture conditions in a growth chamber at a temperature of 25 ± 1 ℃ and humidity of 60%, and the survival rate and contamination rate were analyzed. To eliminate cross-contamination factors in each experiment, one tissue was tested for each sample.

Washing order cleaning solution density(%) Washing time One Detergent 0.2, 2 5 minutes 2 tap water - 1, 3, 6 hours 3 Ethanol 70 0, 30 seconds 4 NaOCl 0.5, 1 30 seconds, 1, 5 minutes

2) Conclusion

Figure 3 shows the cell culture results of tissues derived from blueberry leaves (old leaf) of the O'Neill variety treated under optimized surface sterilization conditions, and Figure 4 shows the cell culture results of the pericarp tissue derived from blueberry fruit of the O'Neill variety treated under optimized surface sterilization conditions. This is the result of cell culture.

In the case of leaves of the O'Neill variety, separating and culturing the tissue from the plant washed with 2% detergent for 5 minutes → tap water for 1 hour → 70% EtOH for 30 seconds → 1% NaOCl for 5 minutes prevents damage and contamination and increases the survival rate ( 100%) was the highest (Figure 3).

In the case of fruit from O'Neill variety blueberries, prepare the plant by washing with 2% detergent for 5 minutes → tap water for 1 hour → 70% EtOH for 30 seconds → 0.5% NaOCl for 30 seconds, and use the tissue obtained from this to clean the tissue cell surface. It was confirmed that surface sterilization conditions enabled callus cell culture by minimizing damage (Figure 4) (survival rate 80%).

In particular, when inducing callus by separating the pericarp tissue from sterilized fruit, the optimal conditions for surface sterilization were different depending on the variety of blueberry. Specifically, in the case of Darrow, which has larger and harder flesh than O'Neill, 1 in the final fourth step. It was confirmed that washing with % NaOCl for 1-5 minutes was preferable.

In addition, when treated with bleach (1% sodium hypochlorite) for more than 10 minutes, the contamination rate was low, but regardless of the order, not only were the tissues damaged or discolored, but the survival rate also decreased (Figure 5).

Experiment example 4. Blueberries leaf origin callus Judo Badge conditions optimization

1) Experimental materials

Leaves of tissue culture seedlings of the O'Neal variety grown under sterile conditions were used, and in the case of adult blueberries, O'Neal (O'Neal), an early maturing variety harvested in June 2016 in the Seongnam region among blueberries produced in Korea, was used. ) The leaves of the variety were surface disinfected under the conditions of Experimental Example 3 and then used as a sterile material. MS (Murashige & Skoog) medium used in the medium including B5 vitamins (M0231), sucrose (S0809), gelite (G1101), 2,4-Dichlorophenoxy acetic acid (2,4-D) (D0911), BA (6) -Benzylaminopurine, 6-BAP) (B0904) was purchased from Duchefa Biochemie (Haarlem, Netherland).

2) Blueberries leaf origin of callus Judo

In the present invention, callus was derived from O'Neill, a representative blueberry variety. Blueberry leaves of the O'Neill variety were collected, washed with running water, and then surface sterilized by washing with 2% detergent for 5 minutes → tap water for 1 hour → 70% EtOH for 30 seconds → 1% NaOCl for 5 minutes. Blueberry leaf tissues that had completed surface sterilization were cut into pieces of 5 x 5 mm, and each tissue was placed on a culture medium. The callus induction medium used at this time was MS (Murashige & Skoog) medium including B5 vitamins (M0231) containing plant hormones (auxin or cytokinin), 30 g/l sucrose, and 4 g/l gelite (pH 5.8). used. The culture conditions were 1500 Lux light culture (16/8 h day/night cycles) and dark culture conditions in a growth chamber with a temperature of 25 ± 1 ℃ and 60% humidity. For each group, 10-11 dishes were prepared to place tissues, and this experimental set was repeated 4 times.

The first subculture period was optimized by observing the growth rate on the growth medium, and the growth rate was determined by analyzing the increase rate (area) 3 weeks after the callus tissue was seeded (transplanted).

Afterwards, from the second subculture, the same cycle was conducted uniformly, and subculture was performed using a solid medium of the same composition.

3) 1st subculture Timing analysis results

When plants (blueberries) were treated under the optimal sterile conditions of Experimental Example 3 and blueberry leaf tissues were cultured under various medium conditions, we attempted to analyze whether callus formation was induced and the proliferation rate. All results are shown in Table 4.

Table 4 shows the results of induction and proliferation of callus derived from blueberry leaves in various media conditions.

division organization Hormone conditions added to the medium Degree of callus induction and proliferation Auxin (2, 4-D) ^a concentration (mg/ml) Cytokinin ^a
Concentration (mg/ml) degree of proliferation
(score) ^b Proliferation rate (%) ^c 1st army leaf
(cancer culture)
young leaf 0.2 0 - 15 2nd army 0.5 0 - 25 3rd army 1.0 0 - 42 4th Army 0.2 0.05 + 64 Fifth Army 0.5 0.05 + 78 6th Army 1.0 0.05 + 74 7th Army 0.2 0.1 + 69 8th Army 0.5 0.1 + 61 9th Army 1.0 0.1 + 72 10th Army 0.2 0.2 ++ 67 11th Army 0.5 0.2 +++ 66 12th Army 1.0 0.2 ++ 66 13th Army leaf
(Photoculture)
young leaf 0.2 0.2 +++ 70 14th Army 0.5 0.2 +++ 67 15th Army 1.0 0.2 ++ 68 16th Army leaf
(cancer culture)
old leaf 0.2 0.2 ++ 65 17th Army 0.5 0.2 ++ 63 18th Army 1.0 0.2 ++ 52

^a 2, 4-D: 2,4-dichlorophenoxyacetic acid), BA: 6-benzylaminopurine.

^b Score for blueberry explants: - Weak callus initiation and poor growth, + Good induction of callus but poor growth, ++ Good initiation and moderate growth, +++ Best induction and vigorous growth of callus.

^c Induction coefficient(%) = (total number of induced calli/number of cultured explants) x 100%.

Table 4 evaluates callus induction and proliferation in each group using the change in increase rate (area) of callus derived from blueberry leaves after 3 weeks. Through this, callus was induced after about 3-6 weeks, and the medium condition with the highest induction of callus from blueberry tissues (leaves) under appropriate sterile culture conditions was when auxin (2, 4-D) and cytokinin (BA) were added to the medium. It was confirmed that it was preferable that the medium was added simultaneously. (auxin 0.5 mg/ml and cytokinin 0.2 mg/ml)

In addition, even when using the same medium, light culture was superior to dark culture, and tissue culture seedling leaves (young leaf) were superior to blueberry (adult) leaves (old leaf), and the conditions described above (medium conditions, It was confirmed that if all conditions (light culture/dark culture, young leaf) are satisfied, the time required for callus induction can be shortened by about 2-4 weeks.

In other words, it was confirmed that the first subculture period was preferably 4 to 6 weeks, and in this experimental example, it was performed uniformly at the most desirable interval of 4 weeks, considering the stilbene content in callus and the expression level of related genes.

In addition, when using the pericarp, callus induction was delayed by 2 to 4 weeks, so it was preferable to use the leaves.

As described above, among the callus induced by primary culture, calli from groups 11 and 14 were subcultured 3rd to 5th time. As a result, it was confirmed with the naked eye that not only the proliferation rate but also the physical properties (hardness, color, etc.) of the callus showed differences depending on the culture conditions and subculture order. Specifically, regarding the color of callus, in the case of callus derived from blueberry (tissue cultured seedlings) leaves, in light culture, overall yellow callus was obtained up to the 4th subculture, but in dark culture, some callus appeared in the 3rd subculture. It was confirmed that red callus, which was red on the surface, was induced. This can be clearly confirmed with the naked eye, and only the generated red callus was separated and used.

In the case of callus derived from blueberry skin, it was confirmed that red callus could be induced and obtained in the 4th subculture only under dark culture conditions. However, in the case of callus derived from the pericarp, the subculture period is 2 to 4 weeks longer than callus derived from the leaves, so mass production is difficult.

Experiment example 5. Blueberries ( tissue culture seedlings ) leaf origin callus Suspension culture conditions optimization

One) Blueberries ( tissue culture seedlings ) leaf origin of callus Suspension culture

In this experiment, yellow callus (DY) obtained by subculturing blueberry leaf-derived callus derived from group 11 in Experimental Example 4 on a growth medium to the fourth time, and callus derived from blueberry leaves derived from group 14 in Experimental Example 4. Suspension culture was performed using yellow callus (LY) obtained by subculturing callus from blueberry leaves derived from group 11 to the fourth time and red callus (DR) obtained by subculturing callus derived from group 11 to the fourth time.

Since callus is a group of heterogeneous cells, the proliferation rate is slow, so uniform cell proliferation and mass production can be induced by transferring it to a liquid medium and culturing it in suspension. Therefore, the yellow callus (LY, DY) and red callus (DR) were cultured in suspension under various conditions to confirm the optimal conditions.

At this time, it is desirable for the callus mass to be uniformly dispersed to form a cell group. To this end, in the experimental example of the present invention, the callus mass was finely chopped using a sterilized scalpel under aseptic conditions, and then cultured so that it was homogeneously released and proliferated in a liquid medium. ('Early suspension culture' in Figure 6).

Suspension culture contains plant hormones (mix of auxin (2,4-D) 0.2 mg/ml and cytokinin (BA) 0.0, 0.2, 4 mg/ml), sucrose 30 g/l, and CaCl2 30 mM (pH 5.7). MS (Murashige & Skoog) medium including B5 vitamins (M0231) liquid medium was used. The liquid medium was sterilized under high pressure and then used by dispensing 40 ml into each 100 ml Erlenmeyer flask. 400 mg of blueberry-derived callus (yellow callus (LY, DY) and red callus (DR)) was placed in 40 ml of liquid medium and cultured in suspension using a shaking incubator at 25°C, 100 rpm, and dark culture conditions for 7 weeks. The callus culture was then harvested.

The cell proliferation rate was analyzed by measuring the fresh and dry weights before and after of callus cultures cultured in three types of suspension culture media, and are shown in Figure 6 and Table 5.

Figure 6 is a photograph of callus cultures prepared from groups 19, 20, and 21.

division callus Hormone conditions added to liquid medium for suspension culture degree of proliferation Auxin (2, 4-D) ^a concentration (mg/ml) Cytokinin ^a
Concentration (mg/ml) degree of proliferation
(score) ^b Proliferation rate (%) ^c 19th Army Yellow callus (Group 11)_DY 0.0 0.0 - -
(fungal contamination or cell necrosis) 20th Army 0.2 0.0~0.2 +++ 52 21st Army 0.2 4 - -
(cell necrosis) 22nd Army Yellow callus (Group 14)_LY 0.0 0.0 - -
(fungal contamination or cell necrosis) 23rd Army 0.2 0.0~0.2 +++ 68 24th Army 0.2 4 - -
(cell necrosis) 25th Army Red callus (11th Army_4th)_DR 0.0 0.0 - -
(fungal contamination or cell necrosis) 26th Army 0.2 0.0~0.2 ++ 54 27th Army 0.2 4 - -
(cell necrosis)

As shown in Figure 6 and Table 5, when the suspension culture medium contained 4 mg/mL of Cytokinin (BA), it was confirmed that the cells were completely necrotic after about 3 weeks (Group 21).

Additionally, in the case of group 19, which used a suspension culture medium that did not contain both Auxin (2, 4-D) and Cytokinin (BA), it was confirmed that the yellow callus gradually lost its yellow color and then became necrotic or died due to mold contamination.

Referring to this, if Cytokinin (BA) is contained in excess of 1 mg/ml in the suspension medium, it may act toxic to callus and cause necrosis, and Auxin (2, 4-D) and Cytokinin (BA) ) are all 0 mg/ml, the problem of insufficient proliferation occurring due to the absence of ingredients essential for callus suspension culture and vulnerability to mold contamination may occur.

Meanwhile, in the case of group 20, where only Cytokinin (BA) was adjusted to 0.0, 0.1, and 0.2 mg/ml, the growth rate of the callus culture was almost similar, and through this, Auxin (2, 4-D) 0.2 mg/ml , it was confirmed that it is preferable to use a suspension medium containing 0.0 to 0.2 mg/ml of Cytokinin (BA). In this case, it was confirmed that the callus culture had grown sufficiently to be harvested after 6 to 7 weeks. Subculture is also possible if necessary.

Experimental Example 6. Production of callus derived from blueberry leaves, isolation and content analysis of viniferin and pterostilbene (maximum enrichment culture of pterostilbene)

One) Blueberries leaf origin callus Culture Recovery

In this experiment, yellow callus (DY) obtained by subculturing blueberry leaf-derived callus derived from group 11 in Experimental Example 4 on a growth medium to the fourth time, and callus derived from blueberry leaves derived from group 14 in Experimental Example 4. Suspension culture was performed using yellow callus (LY) obtained by subculturing callus from blueberry leaves derived from group 11 to the fourth time and red callus (DR) obtained by subculturing callus derived from group 11 to the fourth time.

Suspension culture medium optimized in Experimental Example 5 (MS (Murashige & Skoog) medium containing a mixture of auxin 0.2 mg/ml and cytokinin 0.0-0.2 mg/ml, sucrose 30 g/l, CaCl2 30 mM (pH 5.7) B5 vitamins (M0231)) were used, and their composition is shown in Table 6 below. The liquid medium was sterilized under high pressure and then used by dispensing 40 ml into each 100 ml Erlenmeyer flask. The selected callus (LY, DY, DR) was finely chopped using a sterilized scalpel under aseptic conditions, and then 400 mg of callus was placed in 40 ml of liquid medium and incubated at 25°C, 100 rpm, using a shaking incubator. They were harvested after being cultured in suspension for 7 weeks under dark culture conditions.

The dry weight of callus extracts cultured in three types of suspension culture media was measured and shown in Table 6.

The callus used in Table 6 is indicated by LY, DY, and DR, and the numbers after them indicate the contents of auxin (2,4-D) and cytokinin (BA) in the suspension culture medium. Specifically, 0.2/0.0 uses a mixed medium of auxin 0.2 mg/ml and cytokinin 0.0 mg/ml, 0.2/0.1 uses a mixed medium of auxin 0.2 mg/ml and cytokinin 0.1 mg/ml, and 0.2/0.2 used a medium containing 0.2 mg/ml of auxin and 0.2 mg/ml of cytokinin.

2) callus Extract Preparation

In order to analyze the contents of pterostilbene, viniferin, and resveratrol in callus cultures cultured in the three types of liquid suspension culture media, extracts were obtained.

Specifically, suspension culture cells were separated from callus cultures cultured in the above three types of suspension culture media, freeze-dried, and pulverized using a mortar and pestle.

25 ml of methanol/HCl 0.1% solution was added to 3 g of the powder sample to suspend it, followed by ultrasonic extraction at 27°C for 30 minutes. The extract was centrifuged at 12,000ㅧg for 10 minutes using a centrifuge (Centrikon T-324, Kontron, Milano, Italy) and filtered using whatman filter paper No. It was filtered through 4, and the residue was collected and re-extracted in the same way. The filtrate was mixed and concentrated under reduced pressure at 40°C (rotary evaporator, EYELA N-1110, EYELA, Tokyo, Japan) to remove the solvent. 20 ml distilled water and an equal amount of ethyl acetate were sequentially added and left at 4°C for 10 minutes. The ethyl acetate layer was transferred to a new tube, and the remaining distilled water layer was treated twice in the same manner as above. All ethyl acetate layers were mixed and concentrated under reduced pressure at 40°C (rotary evaporator, EYELA N-1110, EYELA, Tokyo, Japan). The concentrate was dissolved in 1 ml of methanol, filtered through a 0.22 μm syringe filter, and then injected into HPLC. .

Specifically, the blueberry leaf control was obtained by performing the same extraction process as above, except that leaves of blueberry tissue culture seedlings were used.

3) HPLC analyze

For content analysis of resveratrol, pterostilbene, and viniferine, the conventional HPLC analysis method (Lyons et al. 2003) was used with some modifications. Specifically, the HPLC system (Agilent 1100 series) consists of a pump, injector, UV/VIS detector, integrator (Chemstation software), and column (Agilent eclipse XDB-C18: 4.6 mm x 250 mm, 5 μm, ZORBAX). The analysis conditions are shown in Table 1 below. Water with 0.1% formic acid (A) and acetonitrile (B) with 0.1% formic acid were used as solvents at a flow rate of 1.0 ml/min under gradient conditions. It was operated for 45 minutes. At this time, the sample injection volume was 20 μl, and the solvent gradient conditions started with an A/B ratio of 90:10, maintained at a 20:80 ratio until 30 minutes, and then adjusted to 90:10 until 35 minutes. The ratio was maintained at 90:10 for 10 minutes. The detection wavelength was measured at 300 nm, and the oven temperature of the analysis column was kept constant at 25°C. Meanwhile, as standard substances, resveratrol, pterostilbene, and pinosylvin were purchased from Sigma-Aldrich (St Louis, MO, USA), and viniferine was used from Carbosynth (Berkshire, UK). Each experimental value was expressed as the average of 5 repeated measurements.

5) Conclusion

To explore the optimal culture conditions for callus, stilbene was extracted from blueberry leaf extract (control) and callus extract, and the content of four stilbene target substances (resveratrol, ε-viniferin, pinosylvin, and pterostilbene) in the extract was quantified by HPLC. It was analyzed, and the results are shown in Table 6 below.

division ε-viniferine Resveratrol pterostilbene pinosylvin blueberry leaves
(Control group) 1.00±0.12 2.86±0.64 0.10±0.02 0.09±0.03 Experimental group 1
(LY_0.2/0.0) 4.40±0.05
[4.4] 0.06±0.03
[0] 0.56±0.02
[15.6] 0.29±0.00
[3.2] Experimental group 2
(DY_0.2/0.0) 9.38±0.15
[9.4] 0.33±0.01
[0] 0.37±0.03
[13.7] 0.57±0.01
[6.3] Experimental group 3
(DR_0.2/0.0) 17.98±2.89
[18] 0.00±0.00
[0] 8.91±1.30
[89.1] 0.60±0.02
[6.7] Experimental group 4
(LY_0.2/0.1) 5.72±0.22
[5.7] 0.07±0.01
[0] 0.93±0.04
[9.3] 0.36±0.05
[4] Experimental group 5
(DY_0.2/0.1) 4.72±0.31
[4.7] 0.20±0.02
[0] 0.16±0.04
[1.6] 0.69±0.01
[7.7] Experimental group 6
(DR_0.2/0.1) 4.18±0.53
[4.2] 0.10±0.04
[0] 0.95±0.03
[9.5] 1.14±0.07
[12.7] Experimental group 7
(LY_0.2/0.2) 9.95±0.12
[10] 0.50±0.02
[0] 0.50±0.13
[5] 0.00±0.00
[0] Experimental group 8
(DY_0.2/0.2) 7.06±0.33
[7.6] 0.17±0.02
[0] 0.23±0.02
[2.3] 0.84±0.03
[9.3] Experimental group 9
(DR_0.2/0.2) 1.58±0.39
[1.6] 0.10±0.03
[0] 0.72±0.06
[7.2] 1.49±0.04
[16.6]

- Resveratrol, ε-viniferin, and pterostilbene content (μg/g DW) in blueberry leaf (control) extract and callus extract and amplification rate compared to the control

As shown in Table 6, the contents of ε-viniferin, resveratrol, pterostilbene, and pinosylvin were analyzed in the callus extract. As a result, it was confirmed that the callus extract of experimental group 3 showed the highest viniferine and pterostilbene content.

In the case of suspension culture of blueberry-derived red callus obtained through the cancer culture process described above and the extract obtained from it (experimental group 3), the content of resveratrol was hardly confirmed, but the content of pterostilbene was 89 times higher on average, ε -It was confirmed that ε-viniferin was contained 18 times more. This is more than 6 times higher than the extract of yellow callus culture cultured with suspension culture medium under the same conditions. In addition, it can be seen that the content is more than 12 times higher than that of the extract of the culture medium using the same red callus but cultured in suspension culture medium under different conditions. In addition, it was confirmed that the ε-viniferin content was more than two times higher.

Claims (14)

1) After separating a part of the tissue from the blueberry leaf, it was placed on callus induction medium containing auxin at a concentration of 0.5 mg/ml and cytokinin at a concentration of 0.2 mg/ml to grow blueberry callus under dark culture conditions. inducing step; and
2) subculturing the blueberry callus recovered from step 1) 3 to 4 times at 3 to 6 week intervals to recover only red callus; viniferin and pterostilbene (containing pterostilbene) Blueberry callus culture method for mass production. delete i) After separating a part of the tissue from the blueberry leaf, it was placed on callus induction medium containing auxin at a concentration of 0.5 mg/ml and cytokinin at a concentration of 0.2 mg/ml to grow blueberry callus under dark culture conditions. inducing step;
ii) subculturing the blueberry callus recovered from step i) 3 to 4 times at 3 to 6 week intervals to recover only red callus;
iii) suspend-culturing the callus in a growth medium containing auxin at a concentration of 0.2 mg/ml; and
iv) extracting the callus culture of step iii); Method for mass producing blueberry-derived viniferin and pterostilbene. delete According to paragraph 3,
The blueberry tissue in step i) was washed with 0.5-5% surfactant for 5 minutes, treated with water for 0.5-6 hours, then treated with 70% EtOH for 10-60 seconds, and then washed with 0.5-1.5% NaOCl 0.1-10. A method characterized in that part of the tissue of a blueberry plant that has been subjected to a surface sterilization step of washing for several minutes is separated. delete delete delete delete According to paragraph 3,
Wherein step iv) is performed by one or more extraction methods selected from the group consisting of ultrasonic extraction, solvent extraction, and distribution extraction. According to paragraph 3,
The method further comprising the step of freezing and drying the blueberry-derived callus extract obtained through step iv). According to paragraph 3,
A method wherein the viniferin is delta (δ)-viniferin or epsilon (ε)-viniferin. delete delete