WO2001040495A1

WO2001040495A1 - A method for preparing dioxin decomposers from stevia, a dioxin decomposer prepared by the method and a method for decomposing dioxins using it

Info

Publication number: WO2001040495A1
Application number: PCT/KR2000/001381
Authority: WO
Inventors: Young-Gug Kim
Original assignee: Stevia Bio Tech Co., Ltd.
Priority date: 1999-12-01
Filing date: 2000-11-30
Publication date: 2001-06-07
Also published as: KR100304314B1; KR20010068166A

Abstract

Disclosed is a dioxin decomposer derived from Stevia. Also, a method is provided for preparing the dioxin decomposer. After being crushed, dried leaves and stems of Stevia rebaudiana Bertoni are separately steeped in water to give extracts which are then filtered. Each extract filtrate is concentrated in a multi-stage process to control its saccharinity. Stevia yeast (zymogen), which has been cultured in the sludge remaining after the filtration, are added to each of the concentrates which are then forcibly aged using ultrasonic waves, followed by fermenting the forcibly matured solutions for a long period of time. The fermented leaf solution is mixed at an appropriate ratio with the fermented stem solution and, using ultrasonic waves, the mixture is refermented to give the dioxin decomposer. To materials, which produce dioxin upon being incinerated, a dilution of the dioxin decomposer is applied at room temperature at an appropriate ratio, so as to prevent dioxin from being discharged into the air during the incineration of the materials.

Description

A METHOD FOR PREPARING DIOXIN DECOMPOSERS FROM STEVIA, A DIOXIN DECOMPOSER PREPARED BY THE METHOD AND A METHOD FOR DECOMPOSING DIOXINS USING IT

TECHNICAL FIELD

The present invention relates to a method for preparing dioxin decomposer from Stevia, a dioxin decomposer prepared by that method and a method for decomposing dioxins by using the dioxin decomposer.

More particularly, the present invention relates to the effective decomposition of dioxins by use of a dioxin decomposer prepared by fermenting extracts from Stevia leaves and ste s with Stevia yeast.

PRIOR ART

Stevia is a perennial Compositae plant native to Paraguay and Brazil, scientifically named St evi a reba udi ana Bertoni, and thus far, 154 species of Stevia have been found in total.

Stevia is so highly resistant to damages by blight and harmful insects as not to require pesticides or chemical fertilizers for its growth. Extracts of this perennial plant are readily dissolved in water and alcohol, but show such high thermal resistance that they do not begin to decompose before they are heated to at least 196 °C . In addition, extracts of Stevia can be stored for a long time period because they hardly undergo any decomposition after processing owing to their high resistance to acid or salts. Besides, extracts of Stevia have been found to be non-carcinogenic and non-addictive.

The Stevia species is cultivated in mass scales in China to supply a sweetener material, which is 300 times greater in saccharinity and 90 times lower in calories than the sugar extracted from sugar canes.

One of prior arts concerning Stevia can be found in Japanese Pat. Appl'n No. Hei . 3-193301, which discloses the antimicrobial activity of an extract from leaves and stems of Stevia against pathogenic enteric bacteria, such as 0-157, and Salmonella. In particular, the extract neutralizes the verotoxin, produced by 0-157. Also, the patent describes that the extract from Stevia can significantly decompose nicotine. With such data, therefore, extracts of Stevia can be interpreted as non-carcinogenic, non- additive and safe materials.

Dioxin, a very toxic environmental hormone, is one of the most problematic pollutants in the environmental field. 95 % of the total quantity of the dioxin existing in nature was produced when wastes comprising organic chlorine compounds, such as PCB, were incinerated. Additionally, dioxin is released during the preparation of herbicides, sterilizing agents, antibacterial agents, pulp, and paper, and then adsorbed in the natural ecosystem.

Dioxin, a deadly poison, is now treated by four known methods: complete incineration method, pyrolysis method, photolysis method, and microbial decomposition method.

In the complete incineration method, chlorine gas generated upon incineration in a burning chamber remains in the chamber as a high temperature atmosphere to retard the generation of dioxin. To be completely ignited, the gas is required to stay in the burning chamber for such a long period of time as to form a uniform gas distribution in the chamber. In addition, the burning temperature of 1073 K or higher makes the complete incineration method difficult to apply to an industrial purpose.

The pyrolysis method is characterized by heating dioxins in a low oxygen atmosphere to dechlorinate and hydrogenate dioxins. However, this method suffers from the disadvantage of regenerating dioxins when they are not reacted in low oxygen atmospheres.

In the photolysis method, dioxins are dechlorinated by exposure to solar light or ultraviolet light. When tetrachlorobenzoparadioxin, the most toxic compound, is exposed to ultraviolet light and solar light, it takes long periods of time, for example, 8 hours and 24 hours, respectively, to completely decompose the compound. Furthermore, since the decomposition and fixation is generally conducted in an open system, there may be caused a burden to the environment.

Finally, the microbial decomposition method is to dissolve dioxin-containing materials in a solvent such as ethyl acetate with the aim of promoting the metabolic activity of dioxin-decomposing microbes to neutralize dioxins. However, it takes a longer period of time for the microbial decomposition method to remove dioxins than for the photolysis method to do it. Additionally, since the decomposition and fixation is conducted in an open system, reaction parameters, including temperature and pH, must be controlled to conditions suitable to maintain the optimal activity of the microbes. Further, the performance of the microbes to decompose dioxins is increasingly deteriorated with an increase of the number of chlorine substituents .

DISCLOSURE OF THE INVENTION

It is, therefore, an object of the present invention to overcome the problems encountered in prior arts and to provide a method for preparing such a dioxin decomposer from non-toxic and safe Stevia.

It is another object of the present invention to provide a dioxin decomposer capable of effectively reducing the toxicity of dioxins.

It is a further object of the present invention to provide a method for effectively decomposing dioxins even at room temperature by use of such a dioxin decomposer.

In accordance with a first aspect of the present invention, there is provide a method for preparing a dioxin decomposer from Stevia, comprising the steps of: extracting useful ingredients by steeping crushed dry leaves and stems of Stevia reba udi ana Bertoni, separately, in water at an amount of 5-10 liters of water per kg of the crushed plant, said leaves and stems having a water content of 6 % or less; filtering the leaf extract and the stem extract and concentrating the supernatants in a multi-stage manner at a temperature varying in the range from about 80 °C to 150 °C, to give a leaf concentrate with a saccharinity of 20-30 ° and a stem concentrate with a saccharinity of 15-25 °; culturing Stevia yeast

(zymogen) in sludge, said sludge being obtained after the extracting step; forcibly fermenting the leaf concentrate and the stem concentrate, separately, using ultrasonic waves in the presence of about 0.1- 2 % by volume of the cultured Stevia yeast (zymogen); concentrating the forcibly fermented leaf solution and stem solution by further fermenting them for 3-6 months; and mixing the fermented leaf concentrate at a volume ratio of about 2:8 to 4:6 with the fermented stem concentrate and re-fermenting the mixture using ultrasonic waves. In accordance with a second aspect of the present invention, there is provided a dioxin decomposer prepared by the method.

In accordance with a third aspect of the present invention, there is provided a method for decomposing dioxins, comprising the steps of: diluting the dioxin decomposer with about 10-100 volumes of water; and immediately applying the dilute dioxin decomposer to a subject to be treated, at an amount of about 2-20 cc per g of the subject at room temperature.

BEST MODES FOR CARRYING OUT THE INVENTION

The present invention relates to the effective decomposition of dioxins by use of a dioxin decomposer prepared by fermenting extracts from Stevia leaves and stems with Stevia yeast.

Used in the present invention is St evi a re_bauc.ia.r_a Bertoni the extract from whose leaves are highly sweet, but low in calories. The plant's seeds are cultured for a certain period of time. After being vegetatively reproduced through cuttage or division, the saplings are then transplanted in during April to May. During October to November, the transplants have grown to maturity, from which leaves can be gathered.

Leaves and stems of the Stevia species are both used as source materials in the present invention. The stems, although being lower in saccharinity than the leaves, are useful because they are richer in xylem vessel and fibrous material so that their adsorption effect is large enough to remove organic chlorine.

After being gathered, Stevia' s leaves and stems are dried separately. Natural and artificial drying processes are both useful. It is preferred that leaves and stems are dried to a water content of 6 % or less. Because the water content affects the concentration of the final extract, lower water contents are better. If the water content exceeds 6 %, the content of sweet materials in the dried Stevia plant is too large to effectively perform a subsequent crushing step.

The drying is carried out under conventional conditions, for example, at 60-90 °C for 16-36 hours. Almost all of the leaves can be dried to a water content of 6 % or less under these conditions. Because stems are more difficult to dry, they may be cut into pieces 5 cm long or less, followed by repeating the drying step if the water content is not reduced to less than 6 % . Before being steeped in water, the dried Stevia leaves and stems are separately crushed. In this regard, they are preferably crushed to a size of about 100 meshes or less to extract effective materials therefrom. A crusher suitable for use in this purpose is made of SUS 304 or 306 or PVC materials because those materials do not allow the contamination with impurities, but the present invention is not limited to these materials.

As an example suitable for the extraction of effective materials from Stevia, but not by way of limitations, there is suggested a method comprising steeping the crushed leaves and stems in water and then heating the mixture to boiling water. It is preferred that water is used at an amount of 5-10 liters per kg of the crushed plant material. If the amount of water used is outside this range, the extract is too concentrated or too dilute to bring about an excellent decomposition effect of dioxin.

The extraction time period is preferably between about 30 min and 2 hours for leaves and between about 1 hour and 3 hours for stems. For example, if the heating is performed for a shorter time period, the resulting extract cannot be obtained in a maximum quantity. On the other hand, even if the heating time period is longer than the upper limits, no additional quantities of the extract are obtained.

Thereafter, the extracts from the leaves and the stems are separately filtered and each extract filtrate is concentrated in a multi-stage process. A suitable concentrator is exemplified by a stainless steel or PVC machine which is operated in 2-4 stages. The filtration may be achieved with the supernatant obtained after cooling the extract to room temperature .

When account is taken of the fact that Stevia' s extracts are decomposed at 196 °C, the concentration of each extract filtrate is carried out by heating in the temperature range from 80 to 150 °C under reduced pressure conditions in multiple stages. Through this multi-stage concentration, the extracts of the leaves and the stems are controlled in saccharinity. The reduced pressure condition is determined according to the temperature. For example, the temperature of the concentration step may start at 80 °C under 150atm, increase to 120 °C under 120atm and terminate at 150 °C under lOOatm in two-stage concentrator.

Preferably, the leaf concentrate is controlled to range in saccharinity from 20 to 30 ° while the stem concentrate's saccharinity is in the range from 15 to 25 °. The term "saccharinity" as used herein means an amount of a concentrated extract necessary for deposition. In the present invention, saccharinity ranges limited to the leaves and stems are the maximums that can be obtained by the concentration process. The time period taken to achieve the concentration is about 2 hours on average, although differing from the stems to leaves.

After the filtration of the extracts, the remaining sludge is allowed to stand for about 6 months at room temperature to culture Stevia yeast

(Zymogen) . The term "zymogen" as used herein means the Stevia yeast cultured in such sludge, composed mainly of Candi a kyusei , Tri chaspai on pencll a t um, beneficial fungus or microbes, and other fermenting microbes .

The zymogen cultured is added to each of the leaves and stems concentrates, followed by forcibly fermenting the concentrates by use of ultrasonic waves. The addition amount of the zymogen is preferably between about 0.1 and 2% by volume and more preferably between about 0.5 and 1% by volume. For example, if the zymogen is added at an amount less than 0.1 % by volume, the fermentation period of time is doubled. On the other hand, more than 2 % by volume of the zymogen is economically unfavorable.

For the forcible fermentation of the concentrates, ultrasonic waves are utilized as follows. An ultrasonic generator is divided largely into an oscillator and a vibrating device. When pulses generated from the oscillator are applied to the vibrating device, it is vibrated according to vibrating cycle. In the present invention, ultrasonic waves are at a frequency of about 100,000 cycles/sec, based on a 3-vibration technique per sec. The 3- vibration technique per sec is established on the astronomical term "degree day number". According to the degree day number, 3.5 vibrations per sec are set for the equator, 2.5 vibrations per sec for the north and south poles, and 3 vibrations per sec for the subtropical and the temperate zone. The forcible fermentation of the concentrates is preferably carried out at room temperature for example, 15-35°C for about 24 hours, but the present invention is not limited to these conditions.

The generated ultrasonic waves render the microbes aged, so that the useful microbes can be maximally proliferated, fermenting the concentrates.

In the presence of the Stevia extract containing the cultured useful microbe, dioxins are decomposed to lose chlorine.

In addition, the forcible fermentation by use of ultrasonic waves has the advantage of rapidly removing characteristic offensive odor and toxicity from the Stevia extract. Therefore, the dioxin decomposer prepared from the Stevia extract which is deodorized and detoxified in such a manner can be used as a food material or a food additive.

After being forcibly fermented, the leaf and stem extract concentrates are further fermented for 3-6 months in respective storage vessels. During this fermentation period, all microbes except for the useful microbe which have been maximally proliferated by the forcible fermentation die. Next, the resulting fermented leaf concentrate is mixed at a volume ratio of 2:8~4:6 with the resulting fermented stem concentrate, after which the mixture is subjected to re-fermentation for 15-20 days under ultrasonic waves to give a dioxin decomposer. The oscillation with ultrasonic waves is carried out in the same manner as above for the same reasons as above.

As for the volume ratio between the leaf concentrate and the stem concentrate, it is suitably controlled according to the target to be treated, e.g., dioxin-containing burn-outs or foods. For instance, the volume ratio of the leaf concentrate to the stem concentrate is about 2:8 for the decomposition of the dioxins contained in incinerates. For use in the decomposition of pure dioxins contained in foods, feedstuffs, fertilizers or cosmetics, the leaf concentrate is preferably mixed at a volume ratio of 4:6 with the stem concentrate. The re-fermentation under the ultrasonic waves, although its period is as short as 15-20 days, can bring about the effect similar to that obtained when the fermentation is carried out for 6 months in the absence of the ultrasonic waves.

For practical use, the dioxin decomposer prepared from Stevia is diluted with 10-100 volumes fold of water. When the dilute dioxin decomposer is applied to burn-outs, the useful microbe effective to decompose dioxins serve as mediators for dioxin decomposition in such a way that chlorine molecules of dioxins are adsorbed to pores which are formed as a result of the secession of sweet materials from xylem vessels and sieve tubes during the concentration. Finally, dioxins are decomposed.

Experimental data shows that the dilute dioxin decomposer can reduce the dioxin quantity of burnouts by at least 46%.

In addition, the 10-1,000 fold diluted dioxin decomposer is used in the quantity of about 2-20 cc per g of burn-outs. Such quantities of the dilute dioxin decomposer ensure to the reduction of the dioxin content to 2-3 nm per g of burn-outs.

The dioxin decomposer of the present invention should not be stored in dilute forms because the useful microbe effective to decompose dioxins die at room temperature. After a period of time has passed, the dilute dioxin decomposer is unsuitable for the decomposition of highly toxic dioxins, such as 2,3,7,8-TCDD and 2, 3 , 7 , 8-TCDF. Therefore, it is preferred that the dioxin decomposer of the present invention is diluted with water just before application to dioxin-containing materials or dioxins themselves. The application is achieved by simply mixing the dilute dioxin decomposer with the materials of interest at room temperature with stirring . Depending of the subject to be treated, the dioxin decomposer shows different decomposition capacities. For instance, when the dioxin decomposer of the present invention is mixed with burn-outs immediately after being diluted with 10 volumes fold of water, the dioxin content of the burn-outs are decreased by at least 46 % after 10 days of stirring. Of course, the decomposition effect is increased with the stirring time.

As explained above, the dioxin decomposer of the present invention is very effective for decomposing dioxins or chlorine-containing, toxic materials like dioxins, at room temperature without being processed additionally. Further, because steviosides, an extract of Stevia, are used as sweeteners for foods, the dioxin decomposer of the present invention is applied to foods, with the aim of bringing about the effects of sweetening the foods as well as decomposing the dioxins contained in the foods. Moreover, the dioxin decomposer can find applications in a broad spectrums of subjects, including feedstuffs, fertilizers, and cosmetics.

A better understanding of the present invention may be obtained in light of the following examples which are set forth to illustrate, but are not to be construed to limit the present invention.

EXAMPLE 1 In this example, the characteristic odor and toxicity of the Stevia extract were removed by culturing microbes in abundance therein.

Seeds of the St evi a reba udi ana Bertoni species, which exhibit a high sweetening effect, but are low in calories, were bred to saplings. They were vegetatively reproduced through division and then, transplanted in during April to May. The transplants grew to maturity, from which leaves could be gathered, during October to November.

300 g of the Stevia leaves and 700 g of the Stevia stems were separately dried at 90 °C for 26 hours. The dried Stevia leaves were crushed to 100 meshes in an SUS 304 crusher. As for the stems, they were cut to pieces 5 cm long and re-dried to a water content of 6 % or less, followed by crushing the dried stems to pieces with a size of 100 meshes in the same crusher.

Water was poured at an amount of 8 liters per kg of the plant material to the water content-controlled leaves and stems, separately. Thereafter, the leaves were immersed in water and boiled for 1.5 hours and the stems were immersed in water and boiled for 2 hours, followed by extracting the supernatants .

After being cooled to room temperature, the extracts were filtered, and the supernatants were concentrated about for 2 hours in 2-stage SUS 306 concentrator, e.g., at 80 °C under 150atm and then at 150 °C under lOOatm. The leaf and the stem concentrates were measured to have saccharinitys of 25 ° and 18 °, respectively. Separately, the sludges remaining after the filtration were mixed and fermented at room temperature for 6 months to culture zymogen.

To each of the leaf concentrate and the stem concentrate, 2% by volume of the zymogen was added and then forcibly fermented at 30 °C for 24 hours under ultrasonic waves. The fermented concentrates were tested for toxicity according to toxicity measurement standards prescribed by food safety regulations. Also, an examination was made of their odors with the senses of smell and taste. Stevia concentrates were initially bitter to the sense of taste and gave off a bad smell, but the bad smell was significantly alleviated after the ultrasonicatio . As for the toxicity, it was found to be below base toxicity values after the ultrasonication and disappear naturally with the lapse of time.

As apparent from the experiment, the characteristic offensive odor of Stevia, which was not removed even by long-term fermentation, was significantly alleviated within a relatively short time by the ultrasonication. Also, the ultrasonication reduced its toxicity to nil.

EXAMPLE 2 In this example, the dioxin decomposer prepared according to the present invention was tested for dioxin decomposition.

The fermented leaf solution and the fermented stem solution obtained in Example 1 were separately fermented at room temperature for 4 months in vessels while being exposed to sunlight.

The resulting fermented leaf concentrate was mixed at a volume ratio of 4:6 with the fermented stem concentrate and the mixture was subjected to re- fermentation in an ultrasonic mixer to produce 500 g of a dioxin decomposer. The dioxin decomposer was found to be not offensive to the sense of smell as measured in the same manner as in Example 1.

After the dioxin decomposer was diluted with two volumetric folds of water, 100 ml of the dilute dioxin decomposer was mixed with 1 g of burn-outs (heterogeneous mixture) at room temperature by stirring. Dioxin concentrations of the burn-outs before and after the treatment with the dioxin decomposer were measured to determine the decomposition effect of the decomposer.

Separately, the dioxin decomposer was diluted with 10 volumetric folds of water, after which 100 ml of the dilute dioxin decomposer was mixed with 1 g of burn-outs (homogeneous mixture). After two days of stirring at room temperature, the dioxin concentration was measured .

The dioxin concentrations measured are given in Table 1, below.

The dioxin concentrations were measured by an EPA method with the aid of High-Performance Gas Chromatography (HRGC) and High-Performance Mass Analyzer (HRMS, resolution 10,000 or higher). The data was obtained by the Research Institute of Industrial Science & Technology, Korea, using an EPA method.

TABLE 1

As apparent from the data, dioxin concentrations are reduced by at least 46 % simply by mixing the dioxin decomposer of the present invention with dioxins or dioxin-containing materials at room temperature. Also, the dioxin decomposer of the present invention is found to have excellent decomposition effects on 2,3,7,8-TCDD and 2,3,7,8- TCDF, known as the most toxic among dioxin species.

Because the data is obtained by the treatment of dioxins with the dioxin decomposer for 2-10 days, it is expected that a longer time period of the treatment could bring about higher improvement in the decomposition of dioxins.

Although the data of Table 2, as described in Example 2, was obtained by the Research Institute of Industrial Science & Technology, Korea, using 100 ml of the 2-fold diluted dioxin decomposer per g of a subject, a smaller amount of the dioxin decomposer, e g., 2-20 cc of 10-100 fold diluted dioxin decomposer was also highly effective in decomposing dioxins.

INDUSTRIAL APPLICABILITY As described hereinbefore, the dioxin decomposer is prepared by forcibly fermenting extracts of Stevia leaves and stems with the aid of ultrasonic waves in the presence of the zymogen cultured in the Stevia sludge remaining after the extraction and thus comprises rich microbe effective to decompose dioxins. The microbe serve as mediators for decomposing dioxins in such a way as to adsorb chlorine molecules of dioxins to the pores formed as a result of the secession of sweet materials from xylem vessels and sieve tubes of Stevia during the concentration of the Stevia extracts. Therefore, the dioxin decomposer of the present invention can effectively decompose dioxins at room temperature without requiring high reaction temperatures, complex reaction controlled processes, nor causing the regeneration of dioxins during reaction.

Besides, the dioxin decomposer of the present invention is free of the characteristic offensive odor and toxicity of Stevia, finding applications in various fields, including foods, cosmetics, and other dioxin-containing products.

The present invention has been described in an illustrative manner, and it is to be understood that the terminology used is intended to be in the nature of description rather than of limitation. Many modifications and variations of the present invention are possible in light of the above teachings.

Therefore, it is to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described.

Claims

1. A method for preparing a dioxin decomposer from Stevia, comprising the steps of: extracting useful ingredients by steeping crushed dry leaves and stems of Stevia reba udiana Bertoni, separately, in water at an amount of 5-10 liters of water per kg of the crushed plant, said leaves and stems having a water content of 6 % or less; filtering the leaf extract and the stem extract and concentrating the supernatants in a multi-stage manner at a temperature varying in the range from about 80 °C to 150 °C, to give a leaf concentrate with a saccharinity of 20-30 ° and a stem concentrate with a Saccharinity of 15-25 °; culturing Stevia yeast (zymogen) in sludge, said sludge being obtained after the extracting step; forcibly fermenting the leaf concentrate and the stem concentrate, separately, using ultrasonic waves in the presence of about 0.1-2 % by volume of the cultured Stevia yeast (zymogen); concentrating the forcibly fermented leaf solution and stem solution by further fermenting them for 3-6 months; and mixing the fermented leaf concentrate at a volume ratio of about 2:8 to 4:6 with the fermented stem concentrate and re-fermenting the mixture using ultrasonic waves.

2. The method as set forth in claim 1, wherein said extracting step is carried out for about 0.5-2 hours for the leaves and for about 1-3 hours for the stems .

3. The method as set forth in claim 1, wherein said Stevia yeast (zymogen) is composed of Candi a kyusei , Tri chaspai on pencll a tum, useful fungus and microbes, and other fermenting microbe.

4. The method as set forth in any one of claims 1 or 3, wherein said forcible fermentation step is carried out in the presence of about 0.5-1 % by volume of the Stevia yeast (zymogen).

5. The method as set forth in claim 1, wherein said forcible fermentation step is carried out at 15- 35 °C using ultrasonic waves with a frequency of 100,000 cycles/sec or higher.

6. The method as set forth in claim 1, wherein said re-fermenting step is carried out for 15-20 days

7. A dioxin decomposer prepared by the method of any of claim 1 to 6.

8. The dioxin decomposer as set forth in claim 7, wherein the dioxin decomposer comprises the fermented leaf concentrate and the fermented stem concentrate at a volume ratio of about 4:6 and is suitable for use in decomposing dioxins of foods, feedstuffs, fertilizers and cosmetics.

9. The dioxin decomposer as set forth in claim 7, wherein the dioxin decomposer comprises the fermented leaf concentrate and the fermented stem concentrate at a volume ratio of about 2:8 and is suitable for use in decomposing dioxins incurred in incineration.

10. A method for decomposing dioxins, comprising the steps of: diluting the dioxin decomposer of claim 7 with about 10-100 volumes of water; and immediately applying the dilute dioxin decomposer to a subject to be treated, at an amount of about 2-20 cc per g of the subject.