KR20050067984A - Gardenia jasminoides ellis blue dye and preparation method thereof - Google Patents

Abstract

The present invention is a natural pigment having a variety of applications such as food additives, health food raw materials, dyes by enzymatic reaction of yellow pigment extract or geniposide of Gardenia jasminoides Ellis with various hydrolytic enzymes and amino acids It is about.

Description

Gardenia blue pigment and its preparation method {Gardenia jasminoides Ellis blue dye and preparation method

The present invention relates to a method for producing gardenia blue natural pigments from geniposide purified from Gardenia jasminoides Ellis yellow pigment extract or yellow pigment extract, and more specifically from gardenia blue pigment. It relates to a method for producing a blue natural pigment such as purple, blue, cyan, etc. by culturing the obtained geniside with an amino acid and a hydrolase, and improving the pigment productivity.

Pigments are currently used in a variety of additives or supplements in the food industry, cosmetics, pharmaceuticals and livestock feed. In particular, the color of the product plays a decisive role for consumers to finally distinguish and select the product, and plays an important role in determining the merchandise. Each natural food has its own color. However, these foods are inherently color-changed and nutritionally changed due to the interphysical and chemical action of various food ingredients, such as proteins, amino acids, sugars, fragrances and vitamins, during processing or storage. With the development of the food industry, the use of pigments is widely used as a means to increase the merchandise by masking the discoloration or fading of these colors, or to make the colors stand out more. Recently, there is a search for a method that can be applied to a variety of natural food pigments. Furthermore, these pigments are preferable because they exert a beneficial effect on health. For example, substances called anthocyanins are red, purple, and blue pigments that can be used as pigments in foods such as noodles, jams, beverages, yogurt, and ice cream. Dr. Izabela Konczak, director of plant product development at Food Science Australia, said, “The greatest benefits of ansocyanins are their antioxidant and other health-promoting effects. "We are working on lowering blood pressure, slowing blood clotting and protecting cells from a variety of harmful carcinogens." Ansocyanins are one of the polyphenolic compounds called 'vitamins of the 21st century' and they exert very beneficial medical and health benefits. Similarly, yellow pigment obtained from gardenia was used not only as an food coloring but also as a herbal medicine in the treatment of anti-inflammatory, soothing, fever, and bleeding according to Dongbogam treatment with various ingredients of gardenia. Carrot, watermelon, and tomato pigments are typical carotenoid pigments. Representative materials of carotenoid pigments are beta-carotene and lycopene, which not only give color but also remove harmful free radicals. In addition, a typical substance of the flavonoid pigment is a routine extracted from buckwheat, which is also called vitamin P, has the effect of strengthening capillaries. Therefore, there is a healing effect of sensitive skin that reddens the face easily. As described above, various natural pigments are generally excellent in safety and have physiologically active effects and effects, and thus, many studies for mass production have recently been conducted, and their useful value as additives for health foods has been proven.

Natural pigments are also used as dyes. Until the middle of the 19th century, dyes could only be obtained from natural sources such as plants, animals and minerals, but today people can afford to wear almost any color they want thanks to cheap synthetic dyes. Such synthetic dyes can exhibit vivid colors without stains regardless of the nature of the fibers. Despite these various advantages of synthetic dyes, interest in natural dyes has recently come to the fore. Dyeing with plant roots, bark, flowers, fruits, soil, etc. has attracted worldwide attention as it is more environmentally friendly and broader in application as a health product than chemical dyeing. Natural dyeing, which finds natural color, is a color that is composed of various ingredients unlike chemical dyes, so you can enjoy the unique color of natural dyeing while washing off the color little by little while washing. Can be. In addition, natural dyeing clothes are rarely worn. The side is poisonous and insect repellent that repels poisonous snakes and pests, so people in areas with a lot of poisons enjoy wearing natural dyed clothes when working outside. In the past, when skin diseases occurred, natural dyeing clothes were worn. In developed countries, natural dyeing clothes are developed and sold for athletes with athlete's foot and atopic dermatitis.

In the past, colorants, which are artificially added to products to express colors, or pigments, were mainly made of natural materials extracted from materials that exist in nature, but have been used in a limited way due to difficulty in extraction and limitation of materials and uses. Since then, with the development of chemical industry in the 1900s, chemical synthetic pigments have been developed around the United States. The classification of pigments varies according to the purpose of use and the purpose of use, but the main classifications are classified into classifications based on raw materials, classifications based on dissolution properties, classifications based on permits, and classifications based on natural and artificial synthesis. Natural pigments include animal pigments, anthocyanin-based vegetable pigments, and pigments using biotechnology. Representative natural pigments include cochineal extract pigment, lactose pigment, safflower yellow pigment, gardenia yellow pigment, gardenia blue pigment, beet red pigment, red cabbage pigment, grape skin extract pigment, berry pigment, turmeric pigment, high quantity pigment, Monascus pigment, Paprika extract pigment, and Anato pigment. Synthetic pigments include tar-based and natural pigment composites. Among these, synthetic pigments such as tar dyes are decreasing in number due to the hazard problem, and natural pigments are very important commercially because many consumers are interested in health-oriented products.

The domestic food coloring market is estimated to be around 11 billion won per year. Among them, the synthetic pigment market including tar pigment forms about 1 billion won, and the natural pigment market forms about 10 billion won. Table 1 shows the change in the size of the pigment market (www.Foodi.com) (Unit: KRW 10,000).

year Market size 1994 6.9 billion won 1995 $ 1,198 million 1996's 5.55 billion won 1997 9.95 billion won 1998 11 billion 1999 12 billion

As the demand for natural pigments is on the rise, the market size will gradually increase, but as the amount used is small compared to the price, the market expansion in terms of volume will not happen rapidly. However, through the development of natural dye production process technology for more economical natural pigment production, it is necessary to lower the unit price and expand the scope of application.

There are about 260 domestic pigment manufacturing companies in Korea, and all of these companies import raw materials or finished products from abroad, except for caramel pigments and some natural pigments. In Korea, synthetic coloring is gradually regulated, and the number of natural coloring is expected to increase gradually. The reason for this is that natural pigments have higher safety and reliability than synthetic pigments. However, chemical synthetic products have been used for a long time, and thorough research is being conducted more than natural products, and synthetic pigments are still used a lot in terms of economics and stability. The reason why the use of natural pigments does not spread rapidly is that the price of natural pigments is several times to several tens of times higher than chemical synthetic products, and the color is changed or decolorized depending on the nature of the raw material, so that the stability is lowered, and more than conventional synthetic pigments are used. This is because the color retention period is short and the industry is reluctant to use it. Also, in order to produce pigments in Korea, research on existing pigments should be carried out a lot. Recently, with the claim of synthetic pigments, the replacement with natural pigments is being made. Since the harmful theory of synthetic pigments has been around for a long time, research on realistic alternatives and development of safer pigments should be conducted. Therefore, if you look at the current problems of natural pigments by selecting enzymes for improving productivity as in the present invention, the need for cheaper natural pigments by optimizing the culture conditions.

In natural pigments, natural yellow pigments such as safflower, red chrysanthemum, turmeric, and gardenia are obtained from cochineal, safflower, red chrysanthemum, and natural blue pigments are commercially obtained from gardenia and algae. However, in the case of natural blue pigment, phycocyanin obtained from seaweed is not available because it is very unstable in acidic beverages containing alcohol, while the glycosides contained in the extract of Gardenia fruit are not colored by themselves but It is known that stable water-soluble blue pigment can be obtained by decomposition to remove glucose and react with primary amine.

The blue and red pigments of Gardenia jasminoides were isolated from the mixture of Iridoid glycosides and proteolytic products obtained from the fruit of Iridoid-based marshes and Gardenia augusta MERRILL Var. Grandflora HORT., Gardenia jasminoides ELLIS. It is a pigment obtained by making. This pigment does not change color tone even with pH change and is relatively stable against heat and light. Research on the production of gardenia blue pigment has been reported, and Fujikawa et al. Can continuously produce blue pigment by using microbial growth cells instead of using microorganisms or attached enzymes to hydrolyze the geniposide of gardenia. Geniposide has been reported to be beneficial for the growth of microorganisms as well as for the production of blue pigments (Fujikawa, S., et al . J. Ferment. Technol ., 65: 419-424, 1987). Even when used, stable blue pigments can be obtained, and six main components have been reported by HPLC (US Patent No. 4,878,921). In Korea, organic solvent extracts from gardenia fruit are adsorbed on activated carbon to separate the geniposide, cellulase is added and hydrolyzed to obtain genipine, and then reacted with amino acids, primary amines or peptides to produce gardenia blue pigment. Patented (Korean Patent Application 10-2000-0020213).

As such, it is known that beta glucosidase and cellulase from iripoid glycosides of gardenia berry are changed to blue color by culturing microorganisms, but the method of preparing blue pigment from gardenia fruit is a yield problem. It is difficult to put into practical use, and it is limited to some enzymes in producing gardenia blue pigment.

An object of the present invention is to provide a method for mass production of gardenia blue pigment by reacting a hydrolase with an amino acid from Gardenia yellow pigment extract or geniposide. Instead of the conventional complex multi-step reaction, it improves the dye production rate through only one step reaction and enables mass production.

The present inventors select Isolase from Trichoderma sp., And select glycine, alanine, tyrosine, and the like which are the most suitable amino acids for pigment production using the enzyme. It is characterized in that the pigment is produced in high yield by optimizing the enzyme reaction conditions. It is also characterized by producing a pigment that is stable to a wide range of temperature and pH. According to the present invention, blue pigments such as violet, blue and cyan were prepared by enzymatic reaction from nipposide which is a component of gardenia jasminoides and solutions containing respective amino acids.

The present invention isolase from the genus Trichoderma (Isolase from Trichoderma sp.), Beta glucosidase from the genus Summer (β-glucosidase from Thermus sp.), Hemicellulase from Aspergillus (Hemicellulase from Aspergillus sp .), Beta-amylase from barley and β-glucanse from Bacillus sp. The present invention relates to a method for producing a gardenia blue pigment.

The hydrolytic enzymes can be prepared and used by genetic recombination in addition to the natural microorganisms.

In addition, the present invention is characterized by using the geniposide in the gardenia yellow pigment extract.

In addition, the present invention is characterized in that the enzyme reaction incubation or shaking culture for 10-72 hours under the conditions of 40-80 ℃, pH 4-7.

In addition, the present invention is characterized by reacting with an enzyme amount of 0.04-2g (160-8000IsoU) per 1g of Gardenia yellow cow extract.

In addition, the present invention is isolated from trichoderma genus (Isolase from Trichoderma sp.), Beta glucosidase from the genus Summers (β-glucosidase from Thermus sp.), Hemicellulase from the genus Aspergillus (Hemicellulase) from Aspergillus sp.), beta amylase from barley (β-amylase from barley), and beta glucanase (β-glucanse from Bacillus sp.) from the group of Bacillus spp. It relates to a gardenia blue pigment obtained by reacting.

In addition, the present invention relates to a gardenia blue pigment, characterized by using the geniposide in the extract of Gardenia yellow.

In addition, the present invention is characterized in that the pigment can be used as health supplements, food additives, adjuvants or dyes.

In the method for preparing a blue pigment according to the present invention, an enzyme of 0.04-2 g / g based on the weight of the geniposide is added to an enzyme reaction product containing a substrate concentration of 0.4 mM-10 mM and a concentration of 0.4 mM-10 mM of an amino acid. Reaction is carried out, and the blue pigment powder obtained by stopping or shaking the culture for 10-72 hours under the reaction temperature according to the characteristics of the enzyme, that is, the reaction temperature of 40-80 ℃, pH 4.0-8.0.

Specific details of the present invention have been described through examples. However, the scope of the present invention is not limited only to the Examples.

<Example 1: Selection of an enzyme excellent in blue pigment-producing ability together with alanine from nipposide>

In this example, in order to select a hydrolase that represents a blue pigment, alanine, an amino acid, and geniposide extracted from gardenia were reacted. A total of 10 hydrolase enzymes, including beta and alpha-binding hydrolase, were tested for dye production capacity according to the reaction conditions of Table 2. Table 2 shows the reaction pH and reaction temperature of each enzyme. Enzymes with reaction conditions between 45-55 ° C. were reacted at 50 ° C., and recombinant enzymes were reacted at 70 ° C.

Enzymes Microbial origin pH & Temperature β-glucosidase Thermus caldophilus (recombinant enzyme) 7.0 & 80 ℃ α-glucosidase Thermus caldophilus (recombinant enzyme) 7.0 & 80 ℃ Amylomaltase Thermus caldophilus (recombinant enzyme) 7.0 & 80 ℃ α-amylase Aspergillus sp. 4.5-6.0 & 55 ℃ β-glucanase Bacillus sp. 4.2-7.5 & 55 ℃ Cellulase t Trichoderma viride 4.5 & 45 ℃ Cellulase a Aspergillus niger 4.5 & 55 ℃ Hemicellulase Aspergillus niger 4.5 & 50 ℃ Barly-β-amylase Barley - Isolase Trichoderma longibrachiatum 4.0-8.0 & 40 ℃

Enzymes Absorbance (590 nm) 1 hours 3 hours 5 hours 19 hours β-glucosidase 1.0 1.4 1.4 1.3 α-glucosidase - - - - Amylomaltase - - - 0.1 Hemicellulase 0.2 0.3 0.4 0.6 Cellulase a - 0.1 0.1 0.1 Cellulase t 0.7 1.4 1.4 1.5 Isolase 0.7 1.5 1.7 1.7 α-amylase 0.1 0.4 0.8 1.1 β-glucanase - - - 0.1 Barly-β-amylase 0.2 0.4 0.9 1.2

Absorbance at 590 nm of the absorption wavelength of the blue pigment using a spectrophotometer that can analyze the characteristics of the pigment to determine the properties of the pigment over time as the 10 enzymes are incubated with the reaction product containing the geniposide and amino acids Was measured and presented in Table 3. That is, in the early stage of the enzyme reaction, there is a difference in pigment production for each enzyme at 590 nm, which is a characteristic of the blue pigment, but as time passes, the difference in pigment production for each enzyme is clearly shown, and the final yield was also confirmed. Among them, the enzyme with the highest pigment production capacity was isolated from Trichoderma sp., Followed by Cellulase from Trichoderma sp. Β-glucosidase from Thermus sp. Showed a relatively good pigment production. Isolase from Trichoderma sp., Cellulase from Trichoderma sp., Β-glucosidase from Thermus sp., Beta-binding substances such as hemicellulase from Aspergillus sp., Beta-amylase from barley, and beta-glucanse from Bacillus sp. The hydrolytic enzymes were slightly different in their yield, but showed blue pigments, and the remaining alpha-binding hydrolase was almost absent (Table 3).

In addition, as a result of investigating the characteristics of the spectrum of the pigment produced from the mixed solution of the geniposide and alanine by the enzyme from Trichoderma sp. As time passed, the absorbance was increased at 590 nm, which is a characteristic of the blue pigment.

Example 2 Amino Acid Selection for Pigment Production from Geniposide

Hydrolyzed enzyme from Trichoderma sp.Isolase from Trichoderma sp. Was reacted with 0.4mM of geniposide and 4mM of various 20 amino acids, respectively, at 50 ° C. There was a difference in color, and it can be seen that there was a slight difference in the yield over time. The specific absorption wavelength is measured by using a spectrophotometer that can recover the reaction solution over time and analyze the characteristics of color to examine the color and the amount produced according to each amino acid. The measurements are given in Table 4. The range of absorption wavelengths in which the color produced as described above shows the maximum absorbance was 560-580 nm for violet, 580-600 nm for blue, and 600-620 nm for cyan. In addition, glycine (Glycine) having the lowest molecular weight among these 20 amino acids showed a maximum of 2.5 at an absorbing wavelength of 570 nm, which is purple. 2 is a result of observing the difference between the amino acid molecular weight and the produced color. As the molecular weight of the amino acids increases, the color produced also shifted from purple to blue and cyan. Therefore, it was confirmed that various pigments could be prepared from violet to cyan according to the amino acids by molecular weight. Table 4 shows the results obtained by measuring the specific absorption wavelength values and the absorbances of the dyes obtained by enzymatic reaction between nipposide and isolase according to various amino acids.

Example 3 Optimal Temperature and pH of Enzyme Reaction

In order to optimize the reaction pH and reaction temperature, the enzyme was reacted by varying the temperature and pH by adding isolase from Trichoderma sp. To the mixed solution of nipposide and glycine. As a result, as shown in Figure 3a, 3b for the maximum pigment production reaction temperature is 40-70 ℃ more preferably 55 ℃ was suitable, the reaction pH is 4.5-6 is most appropriate and in the alkaline environment of the blue pigment Little production was observed.

Example 4 Suitable Mixing Ratio of Zeniposide and Amino Acid for Maximum Production of Blue Pigment

In order to produce the maximum blue pigment, the concentration of nipposide and glycine was adjusted to 0: 10-10: 0 based on a total of 10 mM, and the enzyme reaction was performed at 55 ° C. and pH 4.5 while the result is shown in FIG. 4. . As shown in FIG. 4, the ratio of 2: 8 to 8: 2, more precisely, 5: 5-6: 4 was found to be most suitable for pigment production.

Example 5 Investigation of Suitable Amount of Enzyme for Maximum Production of Blue Pigment

Based on the reaction conditions according to Examples 1 to 4, the amount of enzyme addition suitable for the maximum pigment yield was measured when the reaction was performed for a long time. The amount of enzyme with 4 IsoU units per mg of enzyme activity was investigated by increasing the number of g-sides of the geniposide as a substrate. As shown in FIG. 5, the pigment yield increased with the increase of enzyme until 10 hours, but was 0.04 g after 22 hours. Except for the amount of enzyme / g substrate (160IsoU) showed similar final yield, 0.2g / g substrate (800IsoU) enzyme amount is the most suitable addition of the enzyme if the reaction operation is continued for more than 20 hours. In addition, as a result of more detailed investigation between 0.2g / g substrate enzyme amount and 0.04g / g substrate enzyme amount, 0.1g / g substrate (400IsoU) enzyme amount was found to be most suitable when operated for 20 hours or more. Therefore, the amount of enzyme can be flexibly controlled in the range of 0.04 g / g substrate enzyme amount to 2 g / g substrate enzyme amount depending on the reaction operation time and economy.

Example 6: Preparation of Blue Pigment Powder

Glycine of 25mM and 20 amino acids obtained from Gardenia jasminoides was prepared at a concentration of 25mM. The bio-reactor was used in a bio-reactor with 600ml of phenylposide and 600ml of glycine and 10mM sodium acetate buffer (pH 4.5). After addition and mixing, the solution was sterilized at 121 ° C. for 20 minutes, cooled to 55 ° C., and a solution prepared by dissolving 6 g enzyme in 300 ml of sterile water was added to a bioreactor to initiate a reaction, followed by reaction with stirring at 10-20 rpm. After sufficient reaction for up to 40 hours, the reaction solution is sterilized, the protein is denatured to stop the enzyme reaction, and the denatured protein is removed by centrifugation (3,000 g, 20 min). The purple pigment solution from which the protein was removed was concentrated to 300 ml by the vacuum concentrator, and the concentrate was lyophilized to obtain 12.7 g of the purple pigment.

In addition, yellow pigment extracted from Gardenia jasminoides contained in the extraction process was not separated during the extraction process to achieve the substrate replacement effect to replace the yellow Gardenia extract yellow pigment with 12g and 1g glycine and 1g enzyme as a reactant It was operated under the same reaction conditions as above, recovered, concentrated and lyophilized to obtain a blue pigment.

Example 7: Stability of the Prepared Pigment

In the present Example, the degree of fading of the color was measured by observing the absorbance variation rate of the dye prepared in Example 6 at the characteristic absorption wavelength of the dye according to pH and temperature. 50 mM buffer, pH 4-12 range (pH4-5 is sodium acetate buffer, pH6-8 is potassium phosphate buffer, pH9-12 is sodium borate / sodium hydroxide buffer (sodium borate) / NaOH buffer)) was stopped at 55 ℃ was observed over time. As a result of standing for more than 100 hours, the residual ratio of 80% or more was shown in the pH 5-12 range (Fig. 6a). In addition, temperature stability was measured by adjusting to 25-75 degreeC in a constant temperature water bath. In addition, at 25 ° C., the residual percentage was 100%, and even at 30-55 ° C., the residual percentage was 80% or more, but at 75 ° C., the residual percentage was 70% or more at 140 hours (FIG. 6b).

Example 8: Cloth Dyeing

After making the dye prepared in the dyeing solution and put the cloth in the dyeing container in the condition of adjusting the fire so that the temperature of the dyeing solution is 20-100 ℃ and then slowly boil while stirring slowly for 30-60 minutes on low heat. Take out fire in dyeing container and dehydrate after standing. Wash in hot water, dehydrate second, and dry to complete the product. It is preferable to perform dyeing by immersing for 30 to 60 minutes at 60-100 degreeC.

As a result of dyeing the material, it has a good affinity for protein fibers (dogs, hairs), but it is not good for cellulose fibers. In the case of protein fibers, dyeing is possible at about pH 4 and at 60 ° C for 30 minutes.

As can be seen from the above, the present invention provides a method of simply preparing a gardenia blue pigment in high yield through excellent enzymatic reaction with amino acids from the geniposide of gardenia. According to the present invention, the hydrolase decomposing the beta-binding material among the various hydrolase was confirmed to form a pigment together with the amino acid by digesting the geniposide.

In addition, the present invention can produce a variety of blue-based pigments of violet, blue and cyan with a variety of amino acids in a single step at a relatively moderate temperature. That is, in the present invention, the enzyme is hydrolyzed for 1-3 days by the enzymatic reaction, which is a conventional production method of blue pigment, to form phenyl, and the obtained phenyl is reacted with primary amines at a high temperature of 70 ° C. or higher. On the contrary, it was possible to produce the pigment through only one step without requiring high temperature and other separation process at relatively medium temperature.

In addition, the present invention can achieve a reaction optimization for the maximum pigment production, simplify the pigment production process will significantly shorten the production cost and production time will be able to increase the industrial utilization.

In addition, the present invention can have bioactive effects, effects by containing the intrinsic agonist of gardenia can be used not only in food additives, but also in health supplements. In addition, due to the excellent stability of the pigment produced by the present invention can be used as a raw material for dyeing the fibers as a natural dye.

1 is a result of investigating the characteristics of the spectrum of the pigment produced from the mixed solution of nipposide and alanine.

Figure 2 is a result of measuring the color of the dye obtained by the enzymatic reaction of amino acids having different molecular weights, nipposide and isolase.

Figure 3a is the result of measuring the dye production amount according to the reaction temperature.

Figure 3b is the result of measuring the dye production amount according to the reaction pH.

Figure 4 is the result of measuring the pigment production amount according to the mixing ratio of nipposide and amino acid.

5 is a result of measuring the dye production amount according to the amount of hydrolase added according to the reaction time.

Figure 6a is the result of measuring the stability according to the reaction pH of the dye prepared in the present invention.

Figure 6b is the result of measuring the stability by reaction temperature of the dye prepared in the present invention.

Claims (7)

Isolase from Trichoderma sp., Beta glucosidase from Thermus sp., Hemicellulase from Aspergillus sp., It is characterized by enzymatically reacting one of the group consisting of beta amylase from barley (β-amylase from barley) and beta glucanase from Bacillus sp. A method of producing a gardenia blue pigment. The method for producing a gardenia blue pigment according to claim 1, wherein the genome yellow pigment extract contains geniposide. The method of claim 1 or 2, wherein the enzyme reaction is incubated for 10-72 hours under a condition of 40-80 ° C. and pH 4-7. The method for producing a gardenia blue pigment according to claim 1 or 2, which is reacted with an amount of 0.04-2 g (160-8000 IsoU) enzyme per 1 g of Gardenia yellow pigment extract. Isolase from Trichoderma sp., Beta glucosidase from Thermus sp., Hemicellulase from Aspergillus sp., Gardenia blue system obtained by enzymatic reaction of one kind from the group consisting of beta amylase from barley and beta glucanase from Bacillus sp. Pigment. According to claim 5, Gardenia blue pigment, characterized in that using the geniposide in the gardenia yellow pigment extract. The gardenia blue pigment according to claim 5 or 6, wherein the pigment is usable as a dietary supplement, a food additive, an adjuvant or a dye.