Patent Application: US-58023309-A

Abstract:
the invention provides methods of purifying rebaudioside d from the stevia rebaudiana bertoni plant extract along with rebaudioside a . the methods are useful for producing high purity rebaudioside d and rebaudioside a . the high purity rebaudiosides are useful as non - caloric sweeteners in edible and chewable compositions such as any beverages , confectionaries , bakeries , cookies , chewing gums , and alike .

Description:
the present invention provides a process for isolation and purification of individual sweet glycosides from stevia rebaudiana bertoni plant extract , and more particularly for isolation and purification of rebaudioside d from stevia rebaudiana bertoni plant extract . advantages of the present invention will become more apparent from the detailed description given hereinafter . however , it should be understood that the detailed description and specific examples , while indicating preferred embodiments of the invention , are given by way of illustration only , since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description . among sweet glycosides existing in stevia only stevioside and rebaudioside a are available at moderate cost at & lt ; 80 % purity and at high cost at & gt ; 80 % purity . the highest purity of commercial product usually is more than 97 %. in the market there are no commercial quantities for rebaudioside b , rebaudioside d , and rebaudioside c . rebaudiosides e and f analytical standards in minor quantities are still unavailable . rebaudioside d is a high - potency diterpenoid glycoside sweetener having the chemical structure presented in fig2 . rebaudioside d is isolated and extracted , along with other steviol glycosides , from the stevia rebaudiana bertoni plant (“ stevia ”), which is commercially cultivated in japan , taiwan , malaysia , south korea , china , israel , india , brazil , australia , and paraguay . it is an ideal non - caloric sweetener with functional and sensory properties superior to those of many high - potency sweeteners . processed forms of stevia can be 30 to 400 times more potent than sugar . amongst the sweet diterpenoid glycosides of stevia , rebaudioside d is the least bitter , and with the least persistent aftertaste . at present there is no published commercial technology related to the isolation and purification of rebaudioside d , and certainly there is a need for efficient and economical method for comprehensive isolation and purification of individual sweet glycosides from stevia extract . the present invention provides a method for production of highly purified rebaudioside d from stevia extract . hereinafter , the term “ highly purified ” refers to a rebaudioside d composition that includes at least about 91 % to 100 % of the rebaudioside d on dry weight basis . exemplary embodiments of this invention are described in detail below and illustrated in fig3 - 6 . however , in the detailed description , only certain exemplary embodiments of the present invention are shown and described , by way of illustration . as those skilled in art will recognize , the invention can be embodied in many different forms and should not be construed as being limited to the embodiments set forth herein . diterpene glycosides , including sweet - tasting substances , are found in the stems , seeds and leaves of the s . rebaudiana bertoni plant , being present in the highest concentration in the leaves . the leaves , therefore , are the preferred starting material for recovery of sweet glycosides . rebaudioside d purification is developed starting from commercial stevia extract . the content of rebaudioside d in the extract can vary depending on stevia plant variety or technological scheme of the extract preparation . stevia extract containing stevioside — 25 . 40 %, rebaudioside a — 59 . 14 %, rebaudioside c — 9 . 71 %, rebaudioside d — 2 . 03 %, rebaudioside b — 0 . 56 %, rebaudioside e — 0 . 68 %, rebaudioside f — 1 . 02 %, steviolbioside — 0 . 11 %, and dulcoside a — 1 . 35 % was used as an exemplary starting material in illustrating the purification of rebaudioside d . now referring to fig3 , there is provided a one - stage purification of high purity rebaudioside a with relatively high rebaudioside d in accordance with one embodiment of the present invention . stevia extract was dissolved in a first ethanol - water solution at 50 - 70 ° c ., preferably 55 - 60 ° c ., for about 10 - 30 min , preferably 15 - 20 min , and then at 15 - 40 ° c ., preferably 20 - 22 ° c . for about 18 - 48 hours , preferably 20 - 24 hours with agitation . when the temperature reached to 22 ° c ., 1 - 2 % ( w / vol .) of highly purified rebaudioside a was added to the reaction mixture as a starter to initiate crystallization . the proportion of extract and the first ethanol - water solution depended on the content of minor glycosides and was between 1 . 0 : 2 . 5 - 1 . 0 : 10 . 0 , w / v , preferably 1 . 0 : 3 . 0 - 5 . 0 , w / v . during this time a first precipitate was formed , which was separated by filtration or centrifugation . the concentration of ethanol in the first ethanol - water solution is between 75 - 99 %, preferably 82 - 88 %. the content of rebaudioside a and rebaudioside d in the first precipitate ranges between 79 - 99 % and 0 . 8 - 4 . 0 % respectively . the purity and yield of rebaudioside a depended on the ration of extract to ethanol - water solution and concentration of ethanol . the data at various concentrations of ethanol is summarized in the table 3 . the extract : methanol ratio was 1 : 3 . 0 , w / v . the purification level and output of rebaudioside a at various volumes of 88 % ethanol solution is summarized in the table 4 . the rebaudioside d content increases with the increase of the concentration of ethanol up to 88 - 90 % and the decrease of the ration of ethanol - water solution to extract . at the same time the purity of rebaudioside a increased with more diluted ethanol solutions and higher ratios of ethanol - water solution to extract . the yield of the product at this stage for stevia extracts with various contents of rebaudioside a , after treatment with 1 : 3 ( w / vol .) ratios of 88 % ethanol is summarized in the table 5 . as it could be expected the yield of the product increases with increase of the content of rebaudioside a in the initial extract . the precipitate was separated by filtration or centrifugation , washed with about two volumes of absolute ethanol and dried . any type of equipment which allows separation of precipitate from liquid , such as various types of centrifuges or filtration systems can be used in this stage . different type dryers , such as rotary vacuum dryer , fluid bed dryer , rotary tunnel dryer or plate dryer , are suitable to produce purified steviol glycosides in powder form . in case if initial extract contains high amount of rebaudioside b and rebaudioside d , for rebaudioside a and later rebaudioside d purification lower concentrations of ethanol and higher ratio of ethanol - water solution to the extract are preferred to use ( table 6 ; table 7 ). in this series of experiments the rebaudioside a content in the initial extract was 48 . 7 %. the yield of the product with high content of rebaudioside a and rebaudioside d can be increased by using ethanol for after - precipitation . for that purpose at the end of crystallization , 0 . 5 - 1 . 0 , v / w , preferably 0 . 5 - 0 . 8 , v / w , of absolute ethanol to the initial solid , was added to the mixture and the process was continued for another 2 - 3 hours . the yield and purity of the product from extract with 48 . 7 % of rebaudioside a content are summarized in table 8 . to produce high purity rebaudioside a the process can be carried out at 30 - 50 ° c . without cooling stage . although the purity of rebaudioside a was higher it resulted in lower yield of the product . the quality of the product increased at higher washing temperatures . the results obtained using 3 . 5 volumes of 85 % ethanol to one part of extract after 24 hours and with and without after - precipitation stage are summarized in table 9 . when the content of rebaudioside a in the final product was less than 97 % mainly due to high content of rebaudioside b and / or rebaudioside d , the product was additionally washed with aqueous solution of ethanol . for that the rebaudioside a obtained after the precipitation was suspended in the ethanol - water mixture at room temperature for 30 - 40 min . after homogeneous suspension was obtained the temperature was increased up to 35 - 50 ° c . preferably 38 - 42 ° c . and agitated for about 10 - 20 hours , preferably 12 - 15 hours , and then at 10 - 25 ° c ., preferably 20 - 22 ° c . for about 3 - 20 hours , preferably 5 - 10 hours . the proportion of rebaudioside a and ethanol was 1 . 0 : 2 . 0 - 1 . 0 : 5 . 0 , w / v , preferably 1 . 0 : 2 . 5 - 4 . 0 , w / v . the ethanol concentration was between 85 - 93 % preferably 88 - 90 %. in case if purity of rebaudioside a was lower than 97 % due to high content of stevioside , the product was washed with absolute ethanol by the same way as it was described above for rebaudioside b and rebaudioside d contaminated product . the proportion of rebaudioside a and ethanol was 1 . 0 : 2 . 0 - 1 . 0 : 5 . 0 , w / v , preferably 1 . 0 : 2 . 5 - 4 . 0 , w / v . now referring to fig5 , there is provided a functional flowchart for purification of rebaudioside a and rebaudioside d in accordance with one embodiment of the present invention . purification of rebaudioside d from the crystals / precipitates with rebaudioside a and rebaudioside d content around 75 - 80 % and 2 . 0 - 3 . 5 % respectively , was developed as follows . it is to be noted that the crystals with rebaudioside a and rebaudioside d can be obtained from the process as described in connection with fig3 . the precipitate with high content of rebaudioside a and rebaudioside d was mixed with a second ethanol - water solution and incubated at 45 - 65 ° c . preferably 50 - 55 ° c . for 2 - 6 hours preferably 3 - 4 hours with agitation . then , the mixture was cooled down to room temperatures for 1 - 3 hour preferably 0 . 5 - 1 . 0 hour . the precipitate was separated by filtration . the preferable ratio of solids to aqueous ethanol solution was 1 to 5 , w / v , and the optimum concentration of ethanol was 78 %. however ethanol concentration can be in the limits 70 - 80 % and ratio 1 : 2 . 5 - 1 . 7 , w / v . to facilitate the filtration of high rebaudioside d precipitate , activated carbon in amount of 0 . 5 - 3 . 0 vol . % preferably 1 . 0 - 1 . 5 vol . % was added to the mixture before filtration . the precipitate was then mixed with 3 - 5 volumes of 30 - 50 % of methanol . the suspension was maintained with agitation at 45 - 65 ° c . preferably 57 - 62 ° c . for 1 - 5 hours preferably 2 - 3 hours and subjected to filtration . elution of adsorbed on activated carbon glycosides was carried out with methanol . both precipitates obtained without and with carbon application contain 19 - 22 . 1 % of rebaudioside d at the optimal conditions ( table 10 ). in principle the higher the applied volume of methanol the faster can be elution process . the process can be completed in shorter time period when aqueous solution of methanol was used . when the initial material containing 95 . 6 % of rebaudioside a and 3 . 5 % rebaudioside d ( fig4 a ) was mixed with 3 . 5 volumes of 78 . 0 % of ethanol , the mixture was boiled for 10 - 15 min and undissolved material was separated by hot filtration , the output of precipitate was in the limits of 6 - 7 . 0 % with 52 - 53 . 0 % and 43 - 45 . 0 % of rebaudioside a and rebaudioside d ( fig4 b ) contents , respectively . for the further purification the precipitate was suspended in 50 % ethanol at the ratio of 1 : 2 , w / v and at 30 - 40 ° c . preferably 33 - 37 ° c ., and maintained for 2 - 15 hours preferably 10 - 12 hours with agitation . the suspension was filtered and dried . the yield of precipitate with content of about 15 - 17 . 0 % rebaudioside a and 80 - 82 % rebaudioside d was in the range of 42 - 46 . 0 %. in principle up to five volumes of aqueous ethanol can be applied at this stage . the concentration of ethanol can be in the limits of 10 - 80 % preferably 45 - 52 %. the precipitate was subjected to similar treatment . the precipitate was separated by filtration , washed with about two volumes of anhydrous methanol and dried . any type of equipment , which allows separating precipitate from liquid , such as various type centrifuges or filtration systems , can be used in this stage . different type of dryers , such as rotary vacuum dryer , fluid bed dryer , rotary tunnel dryer or plate dryer are suitable to produce purified rebaudioside d in powder form . the purity of rebaudioside d was around 95 - 99 % content ( fig4 c ). the yield of the product was around 58 - 60 %. the remaining combined solution after isolation of rebaudioside d was mixed with small amount of rebaudioside a as starter and left for crystallization at 20 - 22 ° c . for 20 - 24 hours . rebaudioside a content in the crystals ranged 97 . 7 - 99 . 4 %. the remaining solution from the first precipitation can be used for isolation of rebaudioside a or highly purified mixture of steviol glycosides . high purity rebaudioside d obtained in this invention has 1129 . 15 molecular weight , c 50 h 80 o 28 molecular formula and structure presented in the fig2 , and is in the form of white and odorless powder . the compound is about 180 - 200 times sweeter than sugar when compared to 10 % sucrose solution . the infrared absorption spectrum is shown in the fig6 . rebaudioside d exhibits a characteristic absorption maximum at around 1730 cm − 1 . other properties of the pure rebaudioside d are as follows : specific rotation : [ α ] d 25 − 29 . 5 ° in 50 % ethanol ( c = 1 . 0 ). solubility in water is around 0 . 2 % which is increasing with increase in temperature . it precipitates again upon cooling the solution . highly soluble during chromatographic separation stage and before crystallizing . rebaudioside d obtained according to this invention may be incorporated as a high intensity natural sweetener in foodstuffs , beverages , pharmaceutical compositions , cosmetics , chewing gums , table top products , cereals , dairy products , toothpastes and other oral cavity compositions , etc . the examples which follow show representative proportions which may be employed . in addition , rebaudioside d can be used as a sweetener not only for drinks , foodstuffs , and other products dedicated for human consumption , but also in animal feed and fodder with improved characteristics . during the manufacturing of foodstuffs , drinks , pharmaceuticals , cosmetics , table top products , chewing gum the conventional methods such as mixing , kneading , dissolution , pickling , permeation , percolation , sprinkling , atomizing , infusing and other methods can be used . the sweetener obtained in this invention can be used in dry or liquid forms . it can be added before or after heat treatment of food products . the amount of the sweetener depends on the purpose of usage . it can be added alone or in the combination with other compounds . the following examples illustrate preferred embodiments of the invention for the isolation and purification of rebaudioside d and related compounds and the use thereof in foodstuffs and pharmaceuticals . it will be understood that the invention is not limited to the materials , proportions , conditions and procedures set forth in the examples , which are only illustrative . one kg of stevia extract containing stevia extract containing stevioside — 25 . 40 %, rebaudioside a — 59 . 14 %, rebaudioside c — 9 . 71 %, rebaudioside d — 2 . 03 %, rebaudioside b — 0 . 56 %, rebaudioside e — 0 . 68 %, rebaudioside f — 1 . 02 %, steviolbioside — 0 . 11 %, and dulcoside a — 1 . 35 % was dissolved in 3000 ml of 95 % ethyl alcohol and maintained at 80 ° c . for 35 min , and then at 15 ° c . for 12 hours with agitation . when temperature reached 22 ° c ., 1 . 0 % of highly purified rebaudioside a was added to the reaction mixture as starter to initiate crystallization . precipitate was separated by filtration and washed with about two volumes of 99 . 5 % ethanol . the yield of crystalline material was 47 . 1 % with content of stevioside ( 8 . 8 %), rebaudioside a ( 81 . 7 %), rebaudioside c ( 5 . 1 %), rebaudioside d ( 3 . 3 %), rebaudioside b ( 0 . 1 %), rebaudioside e ( 0 . 3 %), rebaudioside f ( 0 . 4 %), and dulcoside a ( 0 . 3 %). the remaining solution contains stevioside ( 40 . 2 %), rebaudioside a ( 39 . 1 %), rebaudioside c ( 13 . 8 %), rebaudioside d ( 0 . 9 %), rebaudioside b ( 1 . 0 %), rebaudioside e ( 1 . 0 %), rebaudioside f ( 1 . 6 %), steviolbioside ( 0 . 2 %), and dulcoside a ( 2 . 3 %), and can be used for the isolation of rebaudioside a or highly purified mixture of steviol glycosides . the precipitate was mixed with 3 . 5 volumes of 77 . 7 % ethanol and incubated at 50 ° c . for 3 hours with agitation . then , the mixture was cooled down to room temperature and the precipitate was separated by filtration . the output of crystals was around 14 % and 65 . 9 g of product was obtained with content of stevioside ( 1 . 4 %), rebaudioside a ( 72 . 8 %), rebaudioside c ( 1 . 5 %), rebaudioside d ( 21 . 4 %), rebaudioside b ( 0 . 1 %), rebaudioside e ( 2 . 1 %), and rebaudioside f ( 0 . 7 %). the content of various glycosides in the filtrate was as follows : stevioside ( 10 . 0 %), rebaudioside a ( 83 . 15 %), rebaudioside c ( 5 . 69 %), rebaudioside d ( 0 . 35 %), rebaudioside b ( 0 . 1 %), rebaudioside e ( 0 . 01 %), rebaudioside f ( 0 . 35 %), and dulcoside a ( 0 . 35 %). for further purification of rebaudioside d the precipitate was suspended in 50 % ethanol at 1 : 2 w / v ratio and maintained for 12 hours at 35 ° c . with agitation . the suspension was filtered and precipitate was dried . the yield of precipitate was around 23 % and it contains stevioside ( 0 . 8 %), rebaudioside a ( 16 . 2 %), rebaudioside c ( 0 . 7 %), rebaudioside d ( 81 . 6 %), rebaudioside e ( 0 . 5 %), and rebaudioside f ( 0 . 2 %). around 15 . 2 g of dry material was obtained at this stage . the content of various glycosides in the resulted filtrate was as follows : stevioside ( 1 . 6 %), rebaudioside a ( 89 . 7 %), rebaudioside c ( 1 . 7 %), rebaudioside d ( 3 . 4 %), rebaudioside b ( 0 . 1 %), rebaudioside e ( 2 . 6 %), and rebaudioside f ( 0 . 8 %). it was combined with the filtrate from previous stage . the precipitate was subjected to similar treatment with 50 % ethanol solution to get a product with content of 3 . 8 % rebaudioside a and 95 . 7 % rebaudioside d . the product also contains stevioside , rebaudioside c and rebaudioside f 0 . 1 % each as well as 0 . 2 % rebaudioside e . the yield of this product was around 75 % and around 11 . 4 g of crystals were obtained . the quantity of filtrate at this stage was around 3 . 8 g with 39 . 3 % and 53 . 4 % rebaudioside d and rebaudioside a respectively . the obtained rebaudioside d was dissolved in 2 volumes of 30 % methanol and treated with 0 . 3 % of activated carbon at 60 ° c . for 30 min then subjected to hot filtration . rebaudioside d spontaneously precipitated after filtration . the crystals were separated by filtration and dried at 80 ° c . for 12 hours . the yield of precipitate was around 8 . 8 g and it contains 98 . 4 % rebaudioside d on dry base . the combined filtrate from second and third stage of precipitation was 455 . 8 g and contains stevioside ( 9 . 1 %), rebaudioside a ( 83 . 9 %), rebaudioside c ( 5 . 2 %), rebaudioside d ( 0 . 7 %), rebaudioside b ( 0 . 1 %), rebaudioside e ( 0 . 3 %), rebaudioside f ( 0 . 4 %), and dulcoside a ( 0 . 3 %). it was mixed with 1 % rebaudioside a as starter and left for crystallization at 22 ° c . for 12 hours . the crystals were separated by filtration and washed with about two volumes of ethanol . rebaudioside a content in the crystals was 98 . 8 % on dry base . the quantity was 273 . 5 g after drying . the purity of the rebaudioside d was determined using hplc which was performed using a zorbax nh 2 column ( 150 × 4 . 6 mm , 5 μm ) at a temperature of 30 ° c . the mobile phase comprised a solution of 20 % buffer ( 0 . 0125 % acetic acid and 0 . 0125 % ammonium acetate ) and 80 % acetonitrile at a flow rate of 1 . 0 ml / min . 12 μl of each sample was injected in duplicate and the sample was analyzed using a uv detector at 210 nm ( 4 nm bandwidth ) with a reference of 260 nm ( 100 nm bandwidth ). the analysis required a run time ranging from 40 to 60 min . a buffer solution of 0 . 0125 % acetic acid and 0 . 0125 % ammonium acetate was prepared by dissolving 0 . 125 g ammonium acetate and 125 μl glacial acetic acid in one liter of water . the retention time of rebaudioside b was adjusted by varying the ratio of ammonium acetate to acetic acid while maintaining a total of 0 . 025 % of both combined . increasing the amount of acetic acid decreased the retention time of rebaudioside b . a diluent solution was prepared by mixing 500 ml of ethyl alcohol and 500 ml of the buffer solution . rebaudioside d standards were prepared by diluting 10 . 0 ± 0 . 5 mg ( recorded to the nearest 0 . 1 mg ) of the rebaudioside d standard with 4 ml of the diluent solution to make a standard solution of approximately 2500 mg / l . the rebaudioside d standard solution was injected at 10 . 8 , 11 . 4 , 12 . 6 and 13 . 2 μl . the moisture content was measured by karl fischer analysis every time a standard was prepared and corrections were made based on the solvent purity according to the certificate of analysis . stevioside standards were prepared by diluting 12 . 5 ± 0 . 5 mg ( recorded to the nearest 0 . 1 mg ) of the stevioside standard with 5 ml of the diluent solution to make a standard solution of approximately 2500 mg / l standard ( stock a ) ( correcting for moisture and purity ). the stevioside standard was then diluted using one ml of stock a to ten ml of diluent to produce a 250 mg / l standard ( stock b ), and stock standards were diluted to final concentrations ranging from 2 . 5 to 50 mg / l . samples of the rebaudioside d compositions were prepared by diluting 125 ± 2 mg ( recorded to the nearest 0 . 1 mg ) of the rebaudioside d composition with 50 ml of the diluent solution to make a sample solution of approximately 2500 mg / l ( correcting for moisture ). individually prepared duplicate samples were injected at 12 μl . if the samples were not analyzed immediately , they were stored without headspace , under nitrogen , and desiccated . the table 11 provides a guideline for retention times for rebaudioside d and other steviol glycosides . however , those of ordinary skill in the art should appreciate that the retention times may be modified as needed . 60 g of concentrated orange juice were mixed with 1 . 1 g of citric acid , 0 . 24 g of vitamin c , 1 . 0 g of orange essence , 0 . 76 g of rebaudioside d and water , to create a homogeneously dissolved mixture of 1000 ml total amount . then , the mixture was pasteurized for a period of 20 seconds at about 95 ° c . in order to prepare an orange juice similar to one made by conventional method . the product had excellent taste profile . juices from other fruits , such as apple , lemon , apricot , cherry , pineapple , etc . can be prepared using the same approach . 1 . 50 kg of whole milk was heated to 45 ° c ., and 300 grams of milk cream , 100 grams of tagatose , 90 grams of sorbitol , 6 grams of carrageenan as a stabilizer , 3 grams of polysorbate - 80 as an emulsifier , and 1 . 0 gram of rebaudioside d as in example 10 , were added into the milk and stirred until the ingredients completely dissolved . the mixture then was pasteurized at a temperature of 80 ° c . for 25 seconds . after homogenization the samples were kept at a temperature of 4 ° c . for 24 hours to complete the aging process . vanilla flavor ( 1 . 0 % of the mixture weight ) and coloring ( 0 . 025 % of the mixture weight ) are added into the mixture after aging . the mixture was then transferred to ice cream maker to produce ice cream automatically . samples of produced ice creams were transferred to seal containers and were kept in the freezer at a temperature of − 18 ° c . the application of sweeteners does not affect the physicochemical properties of ice cream , as well as the overall attributes of color , smoothness , surface texture , air cell , vanilla aroma intensity , vanilla taste , chalkiness , iciness and melting rate . in 1 kg of defatted milk , 0 . 8 grams of high purity rebaudioside d , prepared according to the invention was dissolved . after pasteurizing at 82 ° c . for 20 minutes , the milk was cooled to 40 ° c . a starter in amount of 30 grams was added and the mixture was incubated at 37 ° c . for 6 hours . then , the fermented mass was maintained at 10 - 15 ° c . for 12 hours . the product is a low - calorie and low - cariogenic yoghurt , without foreign taste and odor . all ingredients were blended and dissolved in the water , and pasteurized . the product possesses excellent taste and flavor . 1 kg of flour , 37 . 38 grams of fructooligosaccharide syrup , 80 grams of margarine , 20 grams of salt , 20 grams of yeasts , and 0 . 25 grams of high purity rebaudioside d , obtained according to the invention were placed into the blender and mixed well . 600 ml of water was poured into the mixture and kneaded sufficiently . at the completion of the kneading process , the dough was shaped and raised for 30 to 45 minutes . the ready dough was placed in oven and baked for 45 minutes . bread samples had creamy white color , and a smooth texture . flour , 50 . 0 %; margarine , 30 . 0 %; fructose , 10 . 0 %; maltitol , 8 . 0 %; whole milk , 1 . 0 %; salt , 0 . 2 %; baking powder , 0 . 15 %; vanillin , 0 . 1 %; rebaudioside d , 0 . 55 %; obtained according to this invention were kneaded well in dough - mixing machine . after molding of the dough the cookies were baked at 200 ° c . for 15 minutes . the product is a low - calorie diet cookie with excellent taste and appropriate sweetness . 0 . 8 g of rebaudioside d was added to 1000 ml of soy sauce and mixed homogenously . the product had an excellent taste and texture . a tooth paste was prepared by kneading a composition comprising of calcium phosphate , 45 . 0 %; carboxymethylcellulose , 1 . 5 %; carrageenan , 0 . 5 %; glycerol , 18 . 0 %; polyoxyethylene sorbitan mono - ester , 2 . 0 %; beta - cyclodextrin , 1 . 5 %; sodium laurylsarcosinate , 0 . 2 %; flavoring , 1 . 0 %; preservative , 0 . 1 %; rebaudioside d , obtained according to this invention , 0 . 2 %; and water to 100 %, by usual way . the product possesses good foaming and cleaning abilities with appropriate sweetness . it is to be understood that the foregoing descriptions and specific embodiments shown herein are merely illustrative of the best mode of the invention and the principles thereof , and that modifications and additions may be easily made by those skilled in the art without departing for the spirit and scope of the invention , which 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