KR100753982B1 - Method of recovering pinitol in high yield from carob syrup - Google Patents

Abstract

The present invention relates to a method for separating high yield of finitol from carob syrup, comprising the steps of: (a) diluting carob syrup to an appropriate concentration to remove common sugar components through cultivation of microorganisms; (b) a pretreatment step of removing the cells produced by the microbial culture through centrifugation or filtration; (c) removing distilled ethanol produced when the microorganism is yeast; (d) efficiently recovering finitol using the activated carbon column from the sample; And (e) concentrating the sample and then adding ethanol to form pinitol crystals. The method of the present invention increases the content of pinitol in carob syrup and effectively removes other sugars contained in the raw material. There is a very good effect of separating the finitol in high yield.

Kerop, Finitol, Cairoinositol, Separation

Description

Method of recovering pinitol in high yield from carob syrup

1 is a schematic process diagram illustrating a process of separating finitol and chiroinositol from soybean by-product using an activated carbon column.

The present invention relates to a method of separating pinitol from carob syrup in high yield, and more specifically, to treating microorganisms and activated carbon from carob syrup, which is a liquid concentrate of a large amount of sugar components in a pod of carob fruit. A process for economically separating finitol in high yield using a column.

Chiro-inositol and its methylether type pinitol, a natural hypoglycemic component contained in natural products such as soybean and pine needles, have recently attracted much attention from scientists. Cairo inositol and finitol are well-known substances, and chiranoinositol is an isomer of myo-inositol as its structural isomer. In addition, finitol is a structure in which a methyl group is linked to an ether bond with carbon 3 of the chiranoinositol.

The hypoglycemic effect of Cairoinositol has been reported since the early 1990s (Ortmeyer et al., Endocrinol. 132: 646-651, 1993; Huang et al., Endocrinol . 132: 652-657, 1993; Farese et al., Proc. Natl. Acad. Sci. USA , 91: 11040-11044, 1994; Fonteles et al., Diabetologia 39: 731-734, 1996). In addition, Cairoinositol has no side effects such as gastrointestinal disorders, liver damage, and hypoglycemia when overused by existing oral hypoglycemic agents, and it is included in natural foods, so it can be used safely when developing it as a health supplement or pharmaceutical material. Your chances of success are high. In addition, Cairoinositol is known to be effective in the treatment of obesity, polycystic ovary, etc. (Nestler JE et al., New Eng. J. Med., 340: 1314-1320, 1999). In addition, it was found that pinitol, which is a predominant chiroinositol component of soybean, has an equivalent hypoglycemic effect on its own (US Pat. No. 5,827,896; Narayanan et al., Current Science, 56 (3): 139-141, 1987).

Until now, in the method of preparing chiroinositol, a method of hydrolyzing pinitol (methyl ether of di-chiroinositol) extracted from plant leaves (Anderson et al., Ind. Eng. Chem. , 45: 593-596, 1953), A method of converting myo-inositol to chiroinositol by an organic chemical method (Shen et al., Tetrahedron Letters , 131: 1105-1108, 1990) has been disclosed in the literature, but such known manufacturing processes have been time consuming. It takes a long time and has low yield and is not economical, and the method of synthesizing from kasugamycin (U.S. Patent No. 5,091,596) also has a low yield and is considered to be a considerably high production price. In order to develop a production method, a method of using edible resources, especially soybeans, soybean processed products and pine needles, containing chiroinositol, as a material for lowering blood sugar And a method for separating and purifying chiroinositol components using acid hydrolysis from this edible resource (Korean Patent Application No. 10-2000-12881, Korean Patent Application No. 10-2000-12882) As an improved process, a method using an activated carbon column was developed to use a conventional zeolite (US Pat. No. 4,482,761), a method using a cation exchange resin (US Pat. No. 5,096,594), a method using an anion exchange resin ( It has been patented for a method of separating finitol and chiroinositol more economically than US Pat. No. 5,482,631) and the like (Korean Patent Application No. 10-2001-001611). In order to solve the disadvantage of not recovering the phosphorus compounds, glycosides or phosphorylation using microorganisms such as bacteria, yeast and mold Such as developed by giving way to more effectively recover pinitol and Cairo inositol was converted to the present composition to a different form to the pinitol and Cairo inositol bar was filed patent (Republic of Korea Patent Application No. 10-2001-44677).

 The present inventors have found that in the course of continuously studying raw materials and technologies capable of producing finitol most economically, carob contains the largest amount of finitol than conventionally known natural products such as soybean and pine needles. Carob is a legume plant that lives primarily in Mediterranean countries and has been used as a raw material for locust bean gum. Carrot's fruit peel (bean pods) contains a large amount of sugar, which is collected and concentrated by carob syrup. Carob syrup is as shown in Table 1 of Example 1 most of the sugar, fructose, glucose and the like is composed of a general sugar, the unusual thing is that about 10% of the sugar component is finitol. This is a very high amount in the case of soybean, pine needles and the like, the content of finitol in the total solid content is only 0.5 to 1.0%, indicating that carob syrup is a very good raw material for the production of finitol. The present inventors have applied some of the techniques for recovering finitol from soybean by-products, which have been invented, to carp syrup, resulting in more economical production of finitol. More specifically, research and testing a process that can further increase the economic efficiency in recovering the finitol component from the carob syrup, the results of the microbial treatment to convert the pinitol derivatives in the carp syrup to finitol to increase the concentration of pinitol and to remove all common sugar components After removal, the cells are removed by filtration or centrifugation, and the solution is concentrated and dried to produce a low purity finitol product with 40% to 50% finitol, or by further performing activated carbon treatment and crystallization on the solution. High purity finitol products with a content of 90% or more can be obtained to complete the present invention.

Accordingly, it is an object of the present invention to provide a method for producing a low purity finitol product by effectively enhancing the content of pinitol in carob syrup using microbial bioconversion and at the same time removing common sugars.

It is yet another object of the present invention to provide a process for producing high purity finitol products from carob syrup using microbial treatment, activated carbon adsorption and crystallization methods.

Hereinafter, the configuration of the present invention will be described in detail. Other objects and advantages of the invention will be more clearly understood by the following examples.

The present invention cultivates microorganisms such as bacteria, yeast, mold, and the like in carp syrup to increase the content of pinitol and remove other sugars, while selectively adsorbing and eluting using an activated carbon column to fractionate pinitol at a high concentration. Provides a high-cost, economical way to produce finitol from carob syrup. The method of the present invention, which has a technical configuration different from that of the conventional method, can effectively remove other sugar components in the carob syrup, thereby making it possible to configure the subsequent treatment process very efficiently. In the present specification, about 10% of the total solid is present in the form of finitol, and the remaining 80% is a general sugar such as sugar, fructose, and glucose, and the remaining 10% is composed of protein, fat, and unknown components. It is.

The inventors of the present invention (Korean Patent Application No. 10-2001-44677) show that when treating natural products containing pinitol with microorganisms, the content of pinitol is increased by decomposing the pinitol to increase the content of pinitol and at the same time remove common sugars. I found the facts. In the present invention, this principle is equally maximized in the recovery rate of finitol even when applied to the carob syrup, and effectively removes other sugars contained in the carob syrup, thereby remarkably reducing the processing burden in the subsequent separation and purification process. Could.

In a microorganism used in the present invention is the invention of the telegraph a saccharide that had shown the best effect my process Carlsbad Bergen sheath (Saccharomyces carlsbergensis) and relative to a saccharide that had shown an excellent effect my process serenity non-zero (Saccharomyces cerevisae) and Aspergillus Aspergilus niger was selected. Among them, Saccharomyces Carlsbergensis was evaluated as the most desirable because of the ability to produce pinitol and its ability to remove sugar components. Aspergillus niger is excellent in sugar availability, but at the end of culture when other nutrients are depleted, finitol is also used as a nutrient, which is high risk for practical use.

However, unlike soybean by-products such as tofu sprouts, in the case of carp syrup, nutritional ingredients necessary for the growth of microorganisms such as nitrogen sources and minerals, in addition to carbon sources, must be supplemented with nutrients lacking from the outside. When the culture is completed using Saccharomyces Carlsbergensis microbial cells are removed by centrifugation or filtration. The total solid content in the culture medium from which the cells were removed was 40-50% of the initial level, of which 20% is ethanol produced during the culture. Concentration of this liquid and spray drying or lyophilization can produce a low purity finitol product with a content of about 40-50%. Alternatively, the ethanol is removed by vacuum distillation or the like, and the mixture is added to a column filled with activated carbon and separated from other impurities. Thus, a liquid having a finitol content of 70-80% can be obtained. Concentrate this solution, add twice the volume of ethanol, and leave it at low temperature to obtain pinitol crystals with a purity of 90% or more.

In addition, Saccharomyces Carlsbergensis is able to grow under both anaerobic and anaerobic conditions, so carob syrup can be treated under anaerobic conditions. As a result, the treatment time is about 50% longer than under aerobic conditions, but the treatment effect is almost the same. It was a level. If the same purpose can be achieved even in anaerobic conditions, there is a lot of economic advantages because the high speed stirring and air supply necessary to maintain the aerobic condition is unnecessary.

The process of the present invention for producing a low purity finitol product and a high purity finitol product from carob syrup will be described in more detail with reference to the separation and purification process shown in FIG. 1.

Low Purity Finitol Product Production Process

First, dilute carob syrup having a total solid content of 65-70% by 4-5 times with water to make a solution of 15-20% of total solid content, and then additionally add yeast extract as needed for the smooth growth of microorganisms. In general, the amount of suitable yeast extract added is 2-8% of total solids. Subsequent microbial incubation for 48 hours removes most of the common sugars, leaving only finitol and some non-fermentable nutrients. The produced microbial cells are removed by centrifugation or filtration.

In the above process, the microbial culture was completed and the cells were removed, and the content of finitol in the total solids was 40-50%. In some cases, it is concentrated in this state and then powder dried or lyophilized to obtain a low purity finitol product containing 40-50% of finitol. In order to improve the chromaticity of the product, 20-30% of powdered activated carbon is added to the total solids before condensation, stirred for 1-2 hours, decolorized, concentrated and dried to obtain a light yellow low purity finitol product. Can be.

High Purity Finitol Product Production Process

First, a carob syrup having a total solid content of 65-70% is diluted 4-5 times with water to make a solution having a total solid content of 15-20%, and additionally, the yeast extract is added as much as necessary for the smooth growth of microorganisms. In general, the most appropriate amount of yeast extract is 2-8% of total solids. Subsequent microbial incubation for 48 hours removes most of the common sugars, leaving only finitol and some non-fermentable nutrients. The produced microbial cells are removed by centrifugation or filtration. When using yeast as a microorganism, about 20% of the total solids are converted to ethanol during the cultivation process. If ethanol is present during the adsorption of activated carbon, it inhibits the adsorption of other substances. Therefore, ethanol must be removed by vacuum distillation. do. First, in order to increase the adsorption capacity of the activated carbon, pH is adjusted to 6 to 8 using caustic soda. Samples of 1.5 to 3 times the volume of activated carbon packed column can be processed at a time based on the culture completion liquid with 10% total solids concentration. The proper flow rate is 1 to 2 packed volume per hour (Bed Volume). Display).

After the activated carbon adsorption of the culture complete solution is completed, washing with 1 to 2 BV of distilled water to remove non-adsorbed components remaining in the packed column. Suitable washing rates are 1 to 2 BV per hour. Subsequently, an eluent containing 1 to 2 BV of an organic solvent having a concentration of 5 to 15% (v / v) is supplied to elute only the finitol adsorbed on the activated carbon. Methanol, ethanol, isopropanol, or acetone may be used as the organic solvent. However, ethanol has been found to be the most suitable solvent in consideration of efficiency, safety, and economic efficiency. The pH of the eluent is suitably 3 to 4, in which the equilibrium constant of the chiroinositol component is lowered, and an appropriate feed rate is 0.5 to 2 BV per hour. Subsequently, a regeneration solution 1 BV containing the same solvent at a concentration of 40 to 80% is passed through to remove components strongly adsorbed to activated carbon such as oligosaccharides. The appropriate feed rate for this step is also 0.5 to 2 BV per hour. The eluate is fractionated into appropriate volume units, analyzed using high performance liquid chromatography (HPLC), and the fractions containing finitol are collected together. When the experience of the elution is accumulated, only the concentration of the solvent containing high finitol component can be collected without a separate fractionation by measuring only the concentration of the solvent in the eluate using a refractometer. In the order of weak adsorption to activated carbon under eluting conditions, the finitol fraction comes out first from 0.6 to 2.2 BV after the start of elution, and most impurities including other sugars come from 2.6 to 4.0 BV. The finitol fraction contains 70 to 80% of finitol and the impurity fraction contains 3 to 7% of finitol. After passing through the regeneration solution, the solvent component remaining in the activated carbon is sufficiently removed by washing with 3 to 6 BV of distilled water, and the activated carbon can be reused for the next adsorption operation. The eluate fractions are distilled under reduced pressure at a temperature below 60 ° C. to recover the solvents in the fractions and to carry out concentration for the subsequent steps. Concentration is carried out until the solids content is at least 10%. The recovered solvent can be reused for the next operation after adjusting the concentration.

The pinitol solution obtained by the above method was concentrated to a solid content of 60 to 70% and 1 to 2 times the volume of ethanol solution was added and left for 12 hours at a low temperature of 10 ° C. or lower to precipitate pinitol. Recovering by centrifugation or vacuum filtration and then depressurizing yields a high purity finitol product with a purity of 90% or more.

The advantage of the process of the present invention as compared to known methods for recovering finitol by chromatographic methods using ion exchange resins is that most other sugars are removed by microbial culture. It is possible to significantly reduce the size of the column.

In addition, the advantages of the present invention as compared to the technology of separating the finitol from the soybean by-product of the present invention (Korean Patent Application No. 10-2001-44677) are the same size of activated carbon column because of the high content of finitol in the case of carob syrup. The amount of liquid that can be treated with is not only 3 to 5 times higher, but also the contents of components irreversibly adsorbed to activated carbon such as proteins and fats increase the lifespan of the activated carbon column, and the content of finitol after microorganism culture is 40-50%. It can be commercialized even if it is recovered immediately in this state.

Unlike soybean by-products of telegrams in microbial culture, carob syrup lacks the necessary nitrogen sources and other minerals and vitamins necessary for microbial growth, so these nutrients must be supplemented externally. As a result of intensive research, the present inventors were the best yeast extract (yeast extract) and the amount was 2 to 8% of the total solids present in the solution.

Finitol prepared by the method of the present invention can be used in pharmaceuticals or foods to prevent or treat its complications, such as diabetes, obesity, cataracts, etc., which is used in the form of a general pharmaceutical composition or functionally added finitol as an active ingredient. It can also be used in the form of a drink or food.

Hereinafter, the configuration and working effects of the present invention will be described in more detail with reference to Examples. However, the following examples are only for the purpose of illustrating the present invention, and the scope of the present invention is not limited by these examples according to the gist of the present invention.

Example 1 Component Analysis of Carob Syrup

The results of the component analysis of two different types of carob syrup obtained by the inventors of different origin are shown in Table 1 below. Both types of carob syrup contained 6-8% of total weight and 9-12% of total solids.

Table 1

Example 2 Treatment of Carob Syrup by Saccharomyces Carlsbergensis

100 mL of a 15% total solid carob syrup dilution is placed in a 500 mL Erlenmeyer flask with baffle, 0.6 g of yeast extract corresponding to 4% of total solids is added, and Saccharomyces carlsbergensis is added. Inoculation was incubated for 48 hours in a shaker maintained at 30 ℃, 150 rpm to obtain the results shown in Table 2. After 48 hours of incubation, finitol increased by 16.1% from 15.5 g / L to 18.0 g / L and 90.7% of total sugar was removed.

[Table 2]

Example 3 Carob Syrup Treatment with Saccharomyces cerevises

100 ml of 15% total solids of carob syrup was added to a 500 ml Erlenmeyer flask with baffle, and 0.6 g of yeast extract corresponding to 4% of total solids was added and inoculated with Saccharomyces cerevisae . Incubated for 48 hours in a shaker maintained at 30 ℃, 150 rpm to obtain the results shown in Table 3. After 48 hours of incubation, finitol increased by 10.3% from 15.5 g / L to 17.1 g / L, and 70.5% of total sugar was removed.

Table 3

Example 4 Carob Syrup Treatment by Aspergillus Niger

100 ml of 15% total solids of carob syrup was added to a 500 ml Erlenmeyer flask with baffle, and 0.6 g of yeast extract corresponding to 4% of total solids was added and inoculated with Aspergilus niger . Three days incubation in a shaker maintained at 30 ℃, 150 rpm to obtain the results shown in Table 4. After two days of incubation, the pinotitol peaked at 16.8 g / L from the initial 15.5 g / L and then decreased back thereafter, almost disappearing after three days, and 93% of the total sugar was removed.

Table 4

Example 5 Nutrient Supplementation Effect on Microbial Treatment

Distilled water was added to Australian carob syrup having the composition as shown in Table 1 of Example 1 to prepare a 500 mL solution adjusted to a total solid content of 20%. The solutions were divided into 5 portions, each of 100 mL divided into a 500 mL Erlenmeyer flask. To this, yeast extract was added so as to be 0, 1, 2, 4, 8% of the total solids in the solution, respectively. After adjusting the pH of each flask to 7.0, it sterilized at 121 degreeC for 15 minutes, and cooled to room temperature. 5 mL of Saccharomyces Carlsbergensis culture medium prepared in advance were added in aseptic state and then incubated for 48 hours in a shaker maintained at 30 ° C. and 150 rpm. As a result, the higher the amount of yeast extract, the faster the treatment rate, but the total solid removal rate after 48 hours was similar (Table 5). However, when no yeast extract was added, the solids removal efficiency was significantly lowered.

Table 5

Example 6 Pretreatment of Carob Syrup by Anaerobic Microbial Treatment

100 ml of 15% total solids of carob syrup is added to a 200 ml Erlenmeyer flask with baffle, and then 0.6 g of yeast extract corresponding to 4% of total solids is added and Saccharomyces carlsbergensis is added. Inoculation was incubated for 72 hours in a state of natural standing at 30 ℃ as shown in Table 6. After 72 hours of culture, finitol increased by 14.8% from 15.5 g / L to 17.8 g / L and 87.6% of total sugar was removed.

TABLE 6

Example 7 Production of Low Purity Finitol Products by Microbial Treatment

300 mL of distilled water was added to 100 mL of Australian carob syrup composed of the components shown in Table 1 of Example 1 to make 400 mL of a solution of Brix 20, and 3.5 g of yeast extract was added thereto to dissolve well. The pH of this solution was adjusted to 7.0, then placed in a 2 L Erlenmeyer flask and sterilized at 121 ° C. for 15 minutes, and 20 mL of Saccharomyces Carlsbergensis culture solution previously incubated therein was inoculated under aseptic conditions. After incubation for 48 hours in a constant temperature incubator maintained at 30 ℃ was obtained 396 mL of the supernatant from which the cells were removed by centrifugation. The constituents of this supernatant were measured and Brix 10.2, total solids content after alcohol distillation was 63.8 g / L, and pinitol content 26.8 g / L. The solution was concentrated to Brix 30 and then lyophilized to obtain 65.6 g of a light brown dried product. As a result of analyzing the dried matter, the content of finitol was 40.9%.

Example 8 Production of High Purity Finitol Products

First step: microbial culture

300 mL of distilled water was added to 100 mL of Australian carob syrup composed of the components shown in Table 1 of Example 1 to make 400 mL of a solution of Brix 20, and 3.5 g of yeast extract was added thereto to dissolve well. The pH of this solution was adjusted to 7.0, then placed in a 2 L Erlenmeyer flask and sterilized at 121 ° C. for 15 minutes, and 20 mL of Saccharomyces Carlsbergensis culture solution previously incubated therein was inoculated under aseptic conditions. After incubation for 48 hours in a constant temperature incubator maintained at 30 ℃ was obtained 388 mL of the supernatant from which the cells were removed by centrifugation. The constituents of this supernatant were measured and Brix 9.8, total solids content after ethanol removal was 65.3 g / L, and pinitol content was 27.5 g / L.

Second Process: Activated Carbon Treatment

Ethanol in the supernatant was completely removed by adding the same volume of water and concentrating 3-4 times in a vacuum concentrator, and then water was added to obtain 240 mL of activated carbon feed liquid of Brix 10. The pH of this solution was adjusted to 8.0 and the sample was injected into a glass column filled with 200 mL of activated carbon (inner diameter 3.5 cm x length 30 cm) at a flow rate of 200 mL per hour to allow the finitol to be adsorbed onto the activated carbon. Activated carbon was used granular activated carbon of 30 to 80 mesh size. After the adsorption of the sample was completed, 200 mL of distilled water was passed to wash out the unadsorbed impurities. Then, 200 mL of 10% (v / v) ethanol solution and 50% (v / v) ethanol solution were each flowed at a flow rate of 200 mL per hour to elute the components adsorbed on the activated carbon. Subsequently, the resultant was washed with 2 L of distilled water to remove ethanol remaining in activated carbon and reused in the next adsorption operation. 40 mL (0.2 BV) of the eluted sample was collected from the start of the elution with ethanol solution until the end of the wash. Finitol and other sugars (sucrose, fructose, glucose and myo-inositol) in each fraction were analyzed using HPLC. The analytical column was Dionex Carbopak MA-1 (Dionex U.S.A) and confirmed by a pulsed electrochemical detector. The buffer used was 60 mM NaOH and eluted for 90 minutes at a flow rate of 0.4 mL per minute. The concentration of ethanol in each fraction was analyzed using gas chromatography. Finitol was eluted in the 0.6-2.0 BV fraction and peaked at 1.4 BV. Other impurities were eluted in the 1.8-3.6 BV fraction and peaked at 2.8 BV. All the fractions of 0.6-2.0 BV containing the peak of finitol were collected and analyzed to obtain 320 mL of the finitol fraction containing 31.3 g / L of finitol. The content of finitol in the dry weight of the finitol fraction was 80.1%.

Third Step: Crystallization and Drying

The solution obtained in the second step was concentrated about 8 times to 40 mL, and 80 mL of 95% ethanol was added thereto and left at 4 ° C. for 12 hours while slowly stirring. The precipitate produced at this time was recovered by vacuum filtration and vacuum dried at 40 ° C. for 12 hours to obtain 23.5 g of a high purity pinitol white powder product having a content of 94.7%.

As described above, the method of the present invention effectively removes 60% of the other sugars in the carob syrup through microbial treatment, which greatly reduces the burden in the subsequent recovery process and converts the derivative of finitol to pinitol. This is the most economical way to produce finitol because it has a very excellent effect of maximizing the recovery rate of finitol.

Claims (24)

delete delete delete delete delete delete delete delete delete Finitol contained in the carob syrup is adsorbed to activated carbon, and the finitol adsorbed on the activated carbon is eluted with an organic solvent solution selected from the group consisting of ethanol, isopropanol, methanol, and acetone, wherein the concentration of the organic solvent solution is initially 5 to 15. A method of producing high purity finitol, comprising eluting finitol adsorbed on activated carbon by stepwise increase from% (v / v) to 40 to 80% (v / v). delete delete delete delete delete delete delete The method of claim 10, Method wherein the carob syrup is any of the following: (Iii) Sugars extracted from carob fruit (Ii) Sugar solution extracted from parts other than the fruit of the carob tree (Iii) the concentrate of (iii) or (ii) above; (Iii) the diluent of (iii) or (ii) above. The method of claim 10, Prior to the isolation of finitol, microorganisms selected from the group consisting of Saccharomyces carlsbergensis , Saccharomyces cerevisiae and Aspergillus niger were added to the carob syrup. Thereby increasing the pinitol content in the carob syrup. The method of claim 19, Culturing the microorganisms under aerobic conditions. The method of claim 19, A method for culturing microorganisms under anaerobic conditions. The method of claim 19, A method for replenishing nitrogen sources to promote microbial culture. The method of claim 22, The nitrogen source is a yeast extract characterized in that the method. The method of claim 19, After culturing, the method comprising the step of removing the microbial cells and ethanol.

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