US20200093166A1

US20200093166A1 - Process for manufacturing a sweetener and use thereof

Info

Publication number: US20200093166A1
Application number: US16/697,047
Authority: US
Inventors: Siddhartha PURKAYASTHA; Avetik Markosyan; Magomet MALSOGOV
Original assignee: PureCircle Sdn Bhd; PureCircle USA Inc
Current assignee: PureCircle Sdn Bhd
Priority date: 2005-10-11
Filing date: 2019-11-26
Publication date: 2020-03-26
Also published as: US10531683B2; US8790730B2; US20140242244A1; US20110195169A1

Abstract

Highly purified Stevioside, Rebaudioside A and a purified sweet steviol glycoside mixture were prepared from sweet glycoside extracts obtained from Stevia rebaudiana Bertoni leaves. The resulting sweeteners are suitable as non-calorie, non-cariogenic, non-bitter, non-lingering sweeteners, which may be advantageously applied in foods, beverages, and milk products.

Description

RELATED APPLICATIONS

This application is a continuation-in-part application of and claims the benefit of priority to U.S. patent application Ser. No. 12/720,888, filed Mar. 10, 2010, which is a continuation-in-part application of the following applications: U.S. patent application Ser. No. 11/246,066, filed Oct. 11, 2005, now U.S. Pat. No. 7,807,206; U.S. patent application Ser. No. 11/246,152, filed Oct. 11, 2005, now U.S. Pat. No. 7,862,845; U.S. provisional application Ser. No. 61/260,593, filed Nov. 12, 2009; U.S. provisional application Ser. No. 61/290,778, filed Dec. 29, 2009; U.S. patent application Ser. No. 12/684,129, filed Jan. 8, 2010; U.S. patent application Ser. No. 12/684,130, filed Jan. 8, 2010; and U.S. patent application Ser. No. 12/684,981, filed Jan. 11, 2010.

BACKGROUND OF THE INVENTION

Field Of The Invention

The present invention relates to a process for producing a highly purified sweet steviol glycoside mixture, Stevioside and Rebaudioside A from the extract of the Stevia rebaudiana Bertoni plant and use thereof in various food products and beverages.

Description Of The Related Art

In view of food sanitation, the use of artificial sweeteners such as dulcin, sodium cyclamate and saccharin has been restricted. However natural sweeteners have been receiving increasing demand. Stevia rebaudiana Bertoni is a plant that produces an alternative sweetener that has an added advantage of being a natural plant product. In addition, the sweet steviol glycosides have functional and sensory properties superior to those of many high potency sweeteners.
The extract of Stevia rebaudiana plant contains a mixture of different sweet diterpene glycosides, which have a single base—steviol and differ by the presence of carbohydrate residues at positions C13 and C19. These glycosides accumulate in Stevia leaves and compose approximately 10%-20% of the total dry weight. Typically, on a dry weight basis, the four major glycosides found in the leaves of Stevia are Dulcoside A (0.3%), Rebaudioside C (0.6%), Rebaudioside A (3.8%) and Stevioside (9.1%). Other glycosides identified in Stevia extract include Rebaudioside B, C, D, E, and F, Steviolbioside and Rubusoside.
The physical and sensory properties are well studied only for Stevioside and Rebaudioside A. They were tested for stability in carbonated beverages and found to be both heat and pH stable (Chang and Cook, 1983). The sweetness potency of Stevioside is around 210 times higher than sucrose, Rebaudioside A in between 200 and 400 times, and Rebaudioside C and Dulcoside A around 30 times (Phillips, 1989 and Tanaka, 1997).
However, apart from its high level of sweetness, they have also intrinsic properties of post-bitter taste and unpleasant and undesirable aftertaste. Some undesirable taste characteristics of glycosides can be as a result of contamination of other substances, presented in extract.
One of the main ways to improve the taste quality is the enzymatic glycosylation of mixture of semi-purified steviol glycosides. Another way to produce highly purified individual glycosides with standard characteristics and minimal content of accompanying compounds.
The invention related to the purification of two main glycosides—Stevioside and Rebaudioside A and use thereof.
A process for the recovery of diterpene glycosides, including Stevioside from the Stevia rebaudiana plant is described (U.S. Pat. No. 4,361,697). A variety of solvents, having different polarities, were used in a sequential treatment that concluded with a high performance liquid chromatographic (HPLC) separation procedure.
A method for the recovery of Rebaudioside A from the leaves of Stevia rebaudiana plants has been developed (U.S. Pat. No. 4,082,858). Again, final purification is achieved by liquid chromatography subsequent to an initial extraction with water and an alkanol having from 1 to 3 carbon carbons, preferably methanol. It is also known that water may be used as the initial solvent; their preferred solvent at this stage is a liquid haloalkane having from 1 to 4 carbon atoms. The preferred second solvent is an alkanol having from 1 to 3 carbon atoms, while the preferred third solvent is an alkanol having from 1 to 4 carbon atoms and optionally minor amounts of water.
Individual sweet glycosides can be obtained from the Stevia rebaudiana plant. A mixture of sweet glycosides extracted from the Stevia rebaudiana plant is processed to remove impurities by using two types of ion-exchangers. After removing the mixed sweet glycosides from the second column with methanol, the solution is dried. Upon refluxing the dried solids in a methanol solution and then cooling the solution, Stevioside precipitates out. The filtrate is further concentrated and cooled to precipitate out Rebaudioside A. This Rebaudioside A can be further purified as can the previously obtained Stevioside (U.S. Pat. No. 5,962,678). However, a large amount of toxic organic solvent, such as methanol is used.
However, all the above-mentioned methods allow the production of Stevioside and Rebaudioside A not in highly purified grade, which further possess a residual bitterness and aftertaste.
On the other hand, the unfavorable taste of the glycosides can be as a result of contamination of impurities, presented in extract. Highly purified Stevioside and Rebaudioside A possessing an improved taste profile and there is a need to provide an easy and commercially valuable process for the manufacturing the highly purified Stevioside and Rebaudioside A, and use thereof in various beverages and food products.

SUMMARY OF INVENTION

An object of the present invention is to provide a commercially valuable process for producing a highly purified sweetener from the extract of Stevia rebaudiana Bertoni plant and use thereof in various food products and beverages, which overcomes the disadvantages of the related art.
The invention, in part, pertains to the dried and powdered leaves being subjected to water extraction and the resulted extracts is purified using treatment with a base such as calcium hydroxide and then iron chloride. The filtrate was deionized on, e.g., Amberlite FPC23H, Amberlite FPA51, and Amberlite FPA98C1. The filtrate is concentrated under vacuum and spray dried. The isolation and purification of Stevioside and Rebaudioside A were developed using alcoholic precipitation and ultrafiltration. The highly purified Stevioside and Rebaudioside A were obtained. Any type of existing Stevia extract with various ratios of sweet steviol glycosides are feasible.
The highly purified glycosides were applied in various foods and beverages as sweetener.
The invention, in part, pertains to a purified sweet glycosides extract produced from the Stevia rebaudiana plant, wherein the main sweet glycosides are Rebaudioside A and Stevioside, obtained by a process including drying Stevia rebaudiana leaves, treating the leaves to extract an aqueous liquid solution containing mixed sweet steviol glycosides, extracting the Stevia rebaudiana leaves, obtaining an extract, filtering the extract, obtaining a filtrate, treating the filtrate with a base such as calcium hydroxide, treating the extract with trivalent iron chloride, desalting, decolorizing, and evaporating the filtrate to dryness.
In the invention, purified Rebaudioside A and Stevioside can be obtained by dissolving sweet steviol glycosides in methanol at ambient temperatures to precipitate Stevioside, filtering the solution to recover a precipitate of Stevioside, purifying, recovering a high purity Stevioside, concentrating the remaining solution and evaporating to dryness, suspending the powder in ethanol, heating and then cooling the solution to precipitate Rebaudioside A. Suspending the crystalline Rebaudioside A obtained in ethanol-water solution at cool conditions (10-12° C.) prepares a high purity of Rebaudioside A. Stevioside or Rebaudioside A has a purity of at least 98%. Applications are found in various foods such as chocolate, ice cream, beverage, dairy products, as a sweetener in a tablet form.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory and are intended to provide further explanation of the invention as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings are included to provide a further understanding of the invention. The drawings illustrate embodiments of the invention and together with the description serve to explain the principles of the embodiments of the invention.

FIG. 1 shows a sensory evaluation of raw Stevia extract, Stevioside, and Rebaudioside A;

FIG. 2 shows a sensory evaluation of Stevioside with a different grade of purity; and

FIG. 3 shows a sensory evaluation of Rebaudioside A with a different grade of purity;

DETAILED DESCRIPTION

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.
The dried leaves of Stevia rebaudiana Bertoni were extracted by 10 volumes of water. The proportion of extraction water preferably was about 5 liters to about 15 liters (pH 6.0 to 7.0) to one kilogram of leaves. Greater volumes of solvent can be used, however, it was not preferable from the practical standpoint. The duration of extraction may be from 0.5 hours to 24 hours, with a period of from about 1 hours to about 6 hours preferred.
The extraction temperature can be in the limits of 25-90° C., however the temperatures between 45-75° C. are more preferable.
The plant material was separated from the solution by filtration, and the pH of the filtrate was adjusted to about 10 with calcium hydroxide and heated between 40-60° C., preferably from 50° C. to 55° C., for about 0.5-1.0 hours, cooled to ambient temperature with slow agitation, and neutralized by FeCl₃. After mixing for 10-15 minutes, the precipitate was removed by filtration; the filtrate was passed through the Celite, deionized, and decolorized by Amberlite FPC23H, Amberlite FPA51, and Amberlite FPA98Cl by conventional manner. The solution was concentrated and spray dried.
The resulting sweet steviol glycoside mixture was a yellow powder and had a content of 3.4% Dulcoside, 64.6% Stevioside, 6.7% Rebaudioside C and 25.3% Rebaudioside A.
An HPLC analysis of the obtained product was carried out using an Agilent Technologies 1100 Series (USA) equipped with Zorbax-NH₂column using acetonitrile-water gradient from 80:20, v/v (2 minutes) to 50:50, v/v during 70 minutes and UV detector at 210 nm.
The obtained powder was dissolved in methanol and maintained at a temperatures 20-50° C., preferably at 20-25° C., for 0.5-6.0 hours, preferably 0.5-1.0 hours with agitation. The proportion of extract and methanol was between 1:2-1:7, w/v, preferably 1:5. During this time the precipitate was formed, which was filtered and dried. According to the HPLC analysis, the powder contents were around 90-91% of Stevioside. A second treatment by methanol was not efficient to prepare high purity Stevioside.
For the further purification, the powder was mixed with two volumes of 90% of ethanol and at 10-12° C. and maintained for about 30 minutes with slow agitation. The precipitate was separated by filtration and dried under vacuum. The Stevioside with about 98.5-99.4% purity was obtained.
The filtrates were combined and used for recovery of Rebaudioside A. For this purpose the remaining solution was evaporated to remove the methanol, the syrup obtained diluted with water and passed through polysulfone based ultrafiltration membranes (with a filtering discrimination of 2.5 kDa) (Liumar Technologies, Ottawa, Canada) with diafiltration. The filtrate was concentrated and spray dried. The obtained powder was mixed with 96.2% ethanol and maintained at 45-50° C. for about 30 minutes with agitation. The proportion of syrup and ethanol was between 1:2-1:7, w/v, preferably 1:5. During this time the precipitate was formed, which was filtered and dried. Rebaudioside A with 88-90% purity was obtained. For the further purification the powder was mixed with two volumes of 92% ethanol and maintained at 10-12° C. for about 60 minutes with slow agitation. The crystals were filtered and dried. Rebaudioside A with 98.9% purity was obtained.
Based on the results of preliminary test on the sweetening power of the sweeteners, aqueous solutions of commercial Stevia extract (0.05%) commercialized by Ganzhou Julong High-Tech Food Industry Co., Ltd (China), Stevioside (0.07%), and Rebaudioside A (0.028%) were prepared.
The organoleptic test was carried out with 30 previously trained panel members. It was observed that Rebaudioside A has the highest sweetness level (5.96), followed by Stevioside with a mean score of 4.62, and commercial Stevia extract had the lowest mean score of 2.96. Rebaudioside A had the lowest score for bitterness (1.76), and commercial Stevia extract was the most bitter compared to the other samples. For overall acceptability, Rebaudioside A had the highest score of 4.05 followed by Stevioside (3.81) and raw extract (3.16) (FIG. 1).
The taste profile of Stevioside with 99.3% of purity was more preferable as compared with 90.2 and 95.4% (FIG. 2). The similar feature was obtained for Rebaudioside A with various grades of purity (FIG. 3).
The highly purified sweeteners can be favorably used for seasoning various food products (for instance, soy sauce, soy sauce powder, soy paste, soy paste powder, dressings, mayonnaise, vinegar, powdered vinegar, bakery products and confectioneries, frozen-desserts, meat products, fish-meat products, potato salad, bottled and canned foods, fruit and vegetables) in intact or mixed forms with other sweeteners, such as corn syrup, glucose, maltose, sucrose, lactose, aspartame, saccharin, sugar alcohols, organic and amino acids, flavors and/or coloring agents.
The products are favorably usable as a low-cariogenic and low-calorie sweetener because it is less fermentable by oral dental-caries causative microorganisms. Exemplary applications include low-cariogenic food products such as confectioneries including chewing gum, chocolate, biscuits, cookies, toffee and candy. Additionally applications include soft drinks such as coffee, cocoa, juice, carbonated drinks, sour milk beverage, yogurt drinks and alcoholic drinks, such as brandy, whisky, vodka and wine. In addition to the above-described uses, the sweeteners are usable for sweetening drugs and cosmetics.
The following examples illustrate preferred embodiments of the invention.

EXAMPLE 1

Extraction of Sweet Steviol Glycosides

The leaves of Stevia rebaudiana are dried at 55° C. for three hours in a vacuum oven and powdered (30 mesh). One kg of the obtained material was mixed with 10 liters of water (pH 6.5) and heated to 55° C. with slow agitation for 10 hours. The plant material was separated from the solution by filtration and the pH of the filtrate was adjusted to 10 with about 24 grams of calcium hydroxide and heated to 50° C. for 0.5 hours. The obtained mixture was cooled to ambient temperature and the pH was adjusted to about 7.0 by about 53 grams of FeCl₃. After mixing for 15 minutes the precipitate was removed by filtration.
The slightly yellow filtrate was passed through the Celite, deionized, and decolorized by conventional manner on Amberlite FPC23H, Amberlite FPA51, and Amberlite FPA98Cl commercialized by ROHM & HAAS Co., Germany. The solution was concentrated and spray dried. The yield was 122 grams of powder with a content of sweet glycosides of about 91%. The sweet steviol glycoside mixture contains 3.4% Dulcoside, 64.6% Stevioside, 6.7% Rebaudioside C and 25.3% Rebaudioside A. In another embodiment, a sweet steviol glycoside mixture with a purity of at least about 95% was obtained. In a further embodiment, a sweet steviol glycoside mixture with a purity of at least about 98% was obtained.

EXAMPLE 2

Preparation of Stevioside

100 grams (on the base of dry material) of the sweet steviol glycoside powder obtained by the process of EXAMPLE 1 was mixed with 0.5 liters of methanol and maintained at 25° C. for 45 minutes with slow agitation. The precipitate Stevioside was filtered and dried. 61.2 grams of Stevioside with 90.6% purity was obtained.
For the further purification the powder was mixed with two parts of 90% of ethanol, and maintained at 10-12° C. for about 30 minutes with slow agitation. The precipitate was separated by filtration and dried under vacuum. The product weighed 58.8 grams and contained 99.3% Stevioside. In another embodiment, Stevioside with a purity of at least about 98% was obtained.

EXAMPLE 3

Preparation of Rebaudioside A

The remaining solutions after separation of Stevioside (EXAMPLE 2) were combined, and methanol was removed by evaporation. The syrup was diluted with water and passed through polysulfone based ultrafiltration membranes (with a filtering discrimination of 2.5 kDa) (Liumar Technologies, Ottawa, Canada) with diafiltration. The filtrate was concentrated and spray dried. 40.8 grams of powder with content of Rebaudioside A of around 60% were obtained. The powder was mixed with five volumes (w/v) of 96.2% ethanol and maintained at 50° C. for 30 minutes with slow agitation. The precipitate was filtered and dried. Rebaudioside A with 89.8% purity was obtained. The powder was mixed with two volumes of 92% of ethanol and maintained at 12° C. for 60 minutes with slow agitation. The crystals were filtered and dried. 23.6 grams of Rebaudioside A of 98.9% purity was obtained. In another embodiment, Rebaudioside A with a purity of at least about 98% was obtained.

EXAMPLE 4

Low-Calorie Orange Juice Drink
Orange concentrate (35%), citric acid (0.38%), ascorbic acid (0.05%), sodium benzoate (0.02%), orange red color (0.01%), orange flavor (0.20%), and sweetener (0.06%) containing 90.2, 95.4 or 99.3% of Stevioside, or 80, 90, or 98.9% of Rebaudioside A were blended and dissolved completely in the water (up to 100%) and pasteurized. The sensory evaluation of the samples is summarized in the TABLE 1. The data shows that best results were obtained for highly purified Rebaudioside A and Stevioside.

	TABLE 1

	Comments

Sample	Flavor	Aftertaste	Mouth feel

Stevioside -	Sweet and balanced flavor	Slight bitterness in	Acceptable
90.2%		aftertaste
Stevioside -	Sweet and balanced flavor	Slight bitterness in	Acceptable
95.4%		aftertaste
Stevioside -	Sweet, pleasant, balanced flavor	Clean, no bitterness	Quite full
99.3%
Rebaudioside	Sweet, rounded and balanced	Almost no any	Acceptable
A - 80.0%	Flavor	bitterness
Rebaudioside	Sweet, rounded and balanced	Almost no any	Full
A - 90.0%	Flavor	bitterness
Rebaudioside	High quality of sweetness,	Clean, no unpleasant	Quite full
A - 98.9%	pleasant, taste similar to	aftertaste
	sucrose, balanced flavor

By the same way can be prepared juices from other fruits, such as apple, lemon, apricot, cherry, pineapple, etc.

EXAMPLE 5

Low-Calorie Carbonated Lemon-Flavored Beverage

The formula for the beverage was as below:


	Ingredients	Quantity, kg

Sugar	30.0
Sweetener	0.4
Citric acid	2.5
Green tea extract	25.0
Salt	0.3
Lemon tincture	10.0	L
Juniper tincture	8.0	L
Sodium benzoate	0.17
Carbonated water	up to 1000	L

Sensory and physicochemical characteristics of the drink are presented in the TABLE 2.
The drinks with highly purified Rebaudioside A and Stevioside were superior with an excellent flavor and taste.

	TABLE 2

	Characteristics

		Stevioside -	Rebaudioside	Rebaudioside
Item	Stevioside - 90.2%	99.3%	A - 90.0%	A - 98.9%

Appearance	Transparent liquid,	Transparent	Transparent	Transparent
	free of sediment	liquid, free of	liquid, free of	liquid, free of
	and strange	sediment and	sediment and	sediment and
	impurities. A light	strange	strange	strange
	opalescence, caused	impurities. A	impurities. A	impurities. A
	by features of used	light	light	light
	raw materials is	opalescence,	opalescence,	opalescence,
	possible.	caused by	caused by	caused by
		features of	features of	features of
		used raw	used raw	used raw
		materials is	materials is	materials is
		possible.	possible.	possible.
Color	From light yellow up to	From light	From light	From light
	Yellow	yellow up to	yellow up to	yellow up to
		yellow	yellow	yellow
Taste	Sour-sweet, some	Sour-sweet,	Sour-sweet,	Sour-sweet,
	bitterness in	expression of	almost no any	expression of
	aftertaste	sweetness is	bitterness,	sweetness is
		rapid. The	expression of	rapid.
		taste is	sweetness is
		satisfactory.	rapid.

EXAMPLE 6

Low-Calorie Carbonated Drink

The formula for the beverage was as below:


	Ingredients	Quantity, %

	Cola flavor	0.340
	Phosphoric acid (85%)	0.100
	Sodium citrate	0.310
	Sodium benzoate	0.018
	Citric acid	0.018
	Sweetener	0.030
	Carbonated water	to 100

The beverages prepared with different sweeteners were given to 10 judges for comparison.
TABLE 3 shows the results.

	TABLE 3

	Number of panelists

Comparison	Stevioside-	Stevioside-	Rebaudioside A	Rebaudioside A
Point	90.2%	99.3%	90.0%	98.9%

Bitter taste	6	2	3	0
Astringent taste	6	2	3	0
Aftertaste	6	2	3	0
Quality of	Sweet,	Clean (7 of the	Sweet, some	Clean (10 of the
sweet taste	bitterness in	10 judges)	bitterness in	10 judges)
	aftertaste (6 of		aftertaste (5 of
	the 10 judges)		the 10 judges)
Overall	Satisfactory (5	Satisfactory (8	Satisfactory (8	Satisfactory (10
evaluation	of the 10	of the 10	of the 10	of the 10
	judges)	judges)	judges)	judges)

The above results show that the beverages prepared using highly purified Stevioside and Rebaudioside A possessing good organoleptic characteristics.

EXAMPLE 7

Chocolate

A composition containing 30 kg of cacao liquor, 11.5 kg of cacao butter, 14 kg of milk powder, 44 kg of sorbitol, 0.1 kg of salt, and 0.1 kg of sweetener prepared according to the EXAMPLES 2 or 3, was kneaded sufficiently, and the mixture was then placed in a refiner to reduce its particle size for 24 hours. Thereafter, the content was transferred into a conche, 300 grams of lecithin was added, and the composition was kneaded at 50° C. for 48 hours. Then, the content was placed in a shaping apparatus, and solidified.
The products are low-cariogenic and low-calorie chocolate with excellent texture. Also, the organoleptic test carried out with 20 panelists revealed no lingering after-taste. The most desirable ones were the products with Rebaudioside-98.9% (19 members) and Stevioside 99.3% (16 members).

EXAMPLE 8

Ice-Cream

1.50 kg of whole milk were 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 sweetener prepared according to the EXAMPLES 2 or 3, were added into the milk and was stirred until the ingredients completely dissolved. The mixture then was pasteurized at a temperature of 80° C. for 25 seconds. The homogenization of the obtained mixture was carried out at a pressure of 800 bars and 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 ice creams produced 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. Organoleptic test carried out with 20 panelists. The most desirable ones were the products with 98.9% Rebaudioside A (18 members) and 99.3% Stevioside (14 members).

EXAMPLE 9

Yogurt

In 5 kg of defatted milk, 4.0 grams of sweetener, prepared according to EXAMPLES 2 and 3, were dissolved. After pasteurizing at 82° C. for 20 minutes, the milk was cooled to 40° C. A starter in amount of 150 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.

EXAMPLE 10

Table Top Tablet

A mixture, consisting of 58.5% lactose, 10% calcium silicate, 5% cross-carmellose, 5% L-leucine, 1% aerosol 200, 0.5% magnesium stearate, and 20% of a sweetener, obtained according to the EXAMPLE 2 or 3, was kneaded sufficiently. Then the mixture was shaped with the use of a tabletting machine, equipped with punchers of 6.2 mm diameter, into tablets of 70 mg each, 3.0 mm thick, and 10±1 kg hardness
The tablets can be easily administrated due to their appropriate sweetness. However, the formulations using low grade of Stevioside and Rebaudioside A were somewhat sticky with solubility about 3-4 minutes in water at 25° C. The tablets, prepared with highly purified Rebaudioside A show the best characteristics with the solubility around 20-30 seconds.

EXAMPLE 11

Tooth Paste

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 A or Stevioside, obtained according to the EXAMPLE 2 or 3, 0.2%; and water to 100%, by usual way. The product possesses good foaming and cleaning abilities with appropriate sweetness.

EXAMPLE 12

Soy sauce

0.8 g of Rebaudioside A/Stevioside mixture (1:1, w/w) obtained according to the invention was added to 1000 mL of soy sauce and mixed homogenously. The products had an excellent taste and texture.

EXAMPLE 13

Bread

1 kg of wheat 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 A or Stevioside, obtained according to the EXAMPLE 2 or 3 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 smooth texture.

EXAMPLE 14

Diet Cookies

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 A or Stevioside (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.

EXAMPLE 15

Cake

123 g of hen eggs, 45 g of sugar, 345 g of sorbitol liquid, 2.0 g of sucrose fatty acid ester, 0.35 g of Rebaudioside A or Stevioside was mixed with 100 g of wheat flour and 200 g of water in order to prepare a cake according to a conventional method. The product had an excellent taste with an optimal sweet flavor.
The following examples describe a process to make a sucrose-based sweetener with stevia-based sweeteners having improved taste, mouthfeel and flavor properties. Because sucrose and stevia-based sweeteners have very different melting characteristics and solubility, conventional co-crystallization techniques would not result in a suitable product. For example, while sugar is about 66% soluble in water, Rebaudioside A is only about 1% soluble, and Stevia 95 (containing steviol glycosides at 95% purity) is only about 34% soluble in water. Sugar has a melting point of about 186° C., while Rebaudioside A has a melting point of about 240° C., and Stevia 95 is mostly amorphous.
Because of these and other disparities, conventional processes would not lead to a suitable product.
Instead, the following process was developed to overcome the difficulties associated with working with sweetening components having such different properties. The resulting low-calorie sweetener can be used in any food, beverage or consumer healthcare product. Other ingredients can be incorporated into the low-calorie sweetener using this process, including but not limited to sugar, salt, vitamins, minerals, supplements, homeopathic agents, preservatives, citric acid, juniper tincture, ascorbic acid, sodium benzoate, colorings, flavorings, and a combination thereof.
Although the processes described in the following Examples make references to the sweeteners made in Examples 1-3, it is to be understood that these processes can be used with any stevia-based sweeteners, including but not limited to Rebaudioside A, Stevioside, and purified sweet steviol glycoside mixtures, made by using any process, including the enzymatic transglycosylation processes described in co-pending U.S. Patent Application Ser. Nos. 11/246,066, 12/684,129 and 12/684,130, the entire contents of which are incorporated by reference herein. The processes described in these co-pending applications involve an enzymatic transglycosylation process using CGTases (cyclodextrin glycosyltransferase) produced by cultures of Bacillus stearothermophilus to produce stevia-based sweeteners, including, but not limited to, Steviosides, Rebaudioside A and purified sweet steviol glycoside mixtures.

EXAMPLE 16

Sugar (Sucrose) based Sweetener with Stevioside

1488 g of granulated sugar with moisture content adjusted to 0.80% was distributed to form a layer with thickness of 30 mm on a vibrating tray. 12.05 g stevioside obtained according to EXAMPLE 2, was dissolved in 18.07 g of solvent mixture containing 4 volumes of water per 1 volume of ethyl alcohol to make stevioside 40% (w/w) solution. The solution was heated up to 40° C. for prevention of crystal formation and was dispersed on the granulated sugar by means of an air-powered pneumatic method with pressure of compressed air at 0.1 MPa over a period of 100 seconds while maintaining the intensity of vibration at 1200 vibrations per minute (vpm). The granulated sugar was dried over a period of 10 minutes by means of a convective method in a drum-type drying apparatus set at a temperature of 65° C. until its moisture content was 0.09%.
The sweetener produced has a homogenous and intact structure of crystal sugar and has a taste profile identical to sucrose, with sweetness power about or at least about 3 times higher than sugar.

EXAMPLE 17

Sugar (Sucrose) based Sweetener with Rebaudioside A

1490 g of granulated sugar with moisture content adjusted to 0.90% was distributed to form a layer with thickness of 30 mm on a vibrating tray. 10.03 g rebaudioside A obtained according to EXAMPLE 3, was dissolved in 15.04 g of solvent mixture containing 4 volumes of water per 1 volume of ethyl alcohol to make rebaudioside A 40% (w/w) solution. The solution was heated up to 40° C. for prevention of crystal formation and was dispersed on the granulated sugar by means of an air-powered pneumatic method with pressure of compressed air at 0.1 MPa over a period of 100 seconds while maintaining the intensity of vibration at 1200 vpm. The granulated sugar was dried over a period of 10 minutes by means of a convective method in a drum-type drying apparatus set at a temperature of 65° C. until its moisture content was 0.08%.
The sweetener produced has a homogenous and intact structure of crystalline sugar and has a taste profile identical to sucrose, with sweetness power about or at least about 3 times higher than sugar.

EXAMPLE 18

Sugar (Sucrose) based Sweetener with Reb A and Steviol Glycoside Mixtures

A mixture of 50 kg of sugar with 192 gm of Reb A obtained according to EXAMPLE 3 was thoroughly blended in a ribbon blender and then compacted into sheets of product using a roll compaction unit (IR 520 roll compactor or similar unit) applying roll speeds ranging between about 12 to 16 rpm under a roll pressure of about 14 to 17 bar, which was then milled and classified to get optimum sized granules of the sugar-Reb A blend.
The sugar-Reb A granules had sweetness profile like sugar with about two times the sweetness potency of sugar
A mixture of 65 kg of sugar with 188 gm of Steviol Glycoside obtained according to EXAMPLE 1 was thoroughly blended in a ribbon blender and then compacted into sheets of product using a roll compaction unit (IR 520 roll compactor or similar unit) applying roll speeds ranging between about 12 to 16 rpm under a roll pressure of about 14 to 17 bar, which was then milled and classified to get optimum sized granules of the sugar-Steviol Glycoside blend.
The sugar-Steviol Glycoside granules had a sweetness profile like sugar with at least 50% higher sweetness than sugar alone.

EXAMPLE 19

Sugar based Sweetener with Reb A

A sugar solution of 65 kg of refined sugar in 35 kg of warm water was made and mixed thoroughly with 138 gm of Reb A obtained according to EXAMPLE 3 . The solution mixture was passed through multiple-effect evaporators, and then to the vacuum pans to reach the desired level of super-saturation, when the sugar liquor is “seeded” with invert sugar to initiate formation of sugar crystals. The sugar liquor with developing crystals is then discharged in a mixer to complete the crystallization and then to a centrifugal for separation of crystals from mother liquor. The separated crystals are then dried in a fluidized bed dryer/cooler.
The co-crystallized sugar-Reb A had sweetness profile like sugar with at least 50% higher sweetness potency than sugar alone.

EXAMPLE 20

Sugar (Sucrose) based Sweetener with a Purified Sweet Steviol Glycoside Mixture

1488 g of granulated sugar with moisture content adjusted to 0.80% was distributed to form a layer with thickness of 30 mm on a vibrating tray. 11.58 g of the purified sweet steviol glycoside mixture obtained according to EXAMPLE 1 was dissolved in 17.37 g of solvent mixture containing 4 volumes of water per 1 volume of ethyl alcohol to make 40% (w/w) solution. The solution was heated up to 40° C. for prevention of crystal formation and was dispersed on the granulated sugar by means of an air-powered pneumatic method with pressure of compressed air at 0.1 MPa over a period of 100 seconds while maintaining the intensity of vibration at 1200 vpm. The granulated sugar was dried over a period of 10 minutes by means of a convective method in a drum-type drying apparatus set at a temperature of 65° C. until its moisture content was 0.05%.
The sweetener produced has a homogenous and intact structure of crystalline sugar and has taste profile identical to sucrose with sweetness power 3 times higher than sugar.
In the following examples, a low intensity sweetener, such as sucrose, is combined with a crystallization inhibitor in water. The ratio of the low intensity sweetener to the crystallization inhibitor is between about 1:1 to about 100:1 (w/w), or preferably between about 50:1 to about 80:1 (w/w).
In Examples 21 and 22, the resulting solution is then purged with nitrogen at a rate of about 1 to about 10 liters per minute per liter of solution, at a temperature of about 50° C. to about 80° C., preferably between about 65° C. to about 75° C. The purging takes place for a period of about 0.5 hours to about 10 hours, preferably about 1 hour to about 2 hours.
The solution is then combined with a solution of a high intensity sweetener, and the combined solution is evaporated to obtain a concentrated sweetener composition with about 90% to about 97% (w/w) total solids. The evaporation can be carried out using any suitable device, at a temperature of about 100° C. to about 150° C., preferably about 120° C. to about 140° C., under a vacuum of about 300 mbar to about 800 mbar, preferably about 600 mbar to about 800 mbar, for a period of about 0.5 minute to about 2 minutes, preferably for about 0.7 minute to about 1.3 minutes.
The concentrated sweetener composition is then fed to an impact beating apparatus, such as, but not limited to, a shear mixer, turbulizer, aerator, extrusion device or any other device capable of introducing shear mechanical impact or agitation to the concentrated sweetener composition. The mechanical impact duration on the concentrated sweetener composition is about 0.01 second to about 300 seconds, preferably about 0.5 second to 100 seconds, and more preferably about 10 seconds to about 60 seconds.
In one embodiment, the impact beating apparatus is provided with a water vapor removal system to remove water vapor formed by the fast release of the heat of crystallization of the concentrated sweetener composition as granules are formed. The water vapor removal system can be, but is not limited to, a vacuum condenser, a dry gas supply, and the like. If dry gas is used, examples of suitable gas are air and nitrogen.
The resulting sweetening composition granules are then dried using any apparatus suitable for drying granules or powder, such as, but not limited to, a vacuum dryer, belt dryer, fluid bed dryer, and the like. The drying can take place at a temperature of about 50° C. to about 80° C., preferably about 60° C. to about 70° C.
The granules are then sifted to obtain fractions of various particle sizes. In one embodiment, the particle sizes ranged from about 0.5 mm to about 2 mm.

EXAMPLE 21

Sugar (Sucrose) based Sweetener with a Purified Sweet Steviol Glycoside Mixture

50 kg of granulated sugar and 1 kg of invert sugar were dissolved in 30 liters of water at 80° C. The obtained solution was maintained at 70° C. and purged with nitrogen through a nozzle installed at the bottom of the tank during 1 hour. The nitrogen was delivered at a rate of about 1 liter per minute per liter of solution. 175 g of purified sweet steviol glycoside mixture obtained according to EXAMPLE 1 was dissolved in 0.3 liter of water and added to the sugar solution. The solution was evaporated in vacuum evaporator at 800 mbar vacuum and 125° C. until 95° brix was achieved. The feed rate to evaporator was adjusted to achieve solution residency time of 0.5 to 1 min. The evaporated solution was fed to an impact beating machine (Bepex Turbulizer type) operating at 50 rpm. The shear mixing action of the impact beater initiated intense crystallization and residual water evaporation due to the fast release of heat of crystallization. This transformed the sugar solution into free flowing granules approx 1-2 mm in diameter. The impact beating device was connected to a vacuum condenser to facilitate quick removal of water vapors from crystallization zone and preventing “caking” of obtained granules. The residual moisture of obtained granulated powder was 2.5%. The granules were transferred to a drum-type drying apparatus set at a temperature of 65° C. and dried until its moisture content was 0.06%.
The produced sweetener appeared as white granular powder and had taste profile identical to sucrose with a sweetness power 2 times higher than sugar.

EXAMPLE 22

Sugar (Sucrose) based Sweetener with Rebaudioside A

50 kg of granulated sugar and 1 kg of invert sugar were dissolved in 30 liters of water at 80° C. The obtained solution was maintained at 70° C. and purged with nitrogen through a nozzle installed at the bottom of the tank during 1 hour. The nitrogen was delivered at a rate 1 liter per minute per liter of solution. 150 g of Rebaudioside A obtained according to EXAMPLE 3 was dissolved in 0.3 liter of water and added to the sugar solution. The solution was evaporated in vacuum evaporator at 800 mbar vacuum and 125° C. until 95° brix was achieved. The feed rate to evaporator was adjusted to achieve solution residency time of 0.5 to 1 min. The evaporated solution was fed to an impact beating machine (Bepex Turbulizer type) operating at 50 rpm. The shear mixing action of the impact beater initiated intense crystallization and residual water evaporation due to the fast release of heat of crystallization. This transformed the sugar solution into free flowing granules approx 1-2 mm in diameter. The impact beating device was connected to a vacuum condenser to facilitate quick removal of water vapors from crystallization zone and preventing “caking” of obtained granules. The residual moisture of the obtained granulated powder was 3%. The granules were transferred to a drum-type drying apparatus set at a temperature of 65° C. and dried until its moisture content was 0.08%.
The produced sweetener appeared as white granular powder and had taste profile identical to sucrose with sweetness power 2 times higher than sugar.

EXAMPLE 23

Sugar (Sucrose) based Sweetener with Rebaudioside A

50 kg of granulated sugar and 1 kg of invert sugar were dissolved in 30 liters of water at 80° C. 150 g of Rebaudioside A obtained according to EXAMPLE 3 was dissolved in 0.3 liter of water and added to the sugar solution. The solution was evaporated in a vacuum evaporator at 800 mbar vacuum and 125° C. until 95° brix was achieved. The feed rate to the evaporator was adjusted to achieve solution residency time of 0.5 to 1 min. The evaporated solution was fed to an impact beating machine (Bepex Turbulizer type) operating at 50 rpm. The shear mixing action of the impact beater initiated intense crystallization and residual water evaporation due to fast release of heat of crystallization. This transformed the sugar solution into free flowing granules approx 1-2 mm in diameter. The impact beating device was connected to a vacuum condenser to facilitate quick removal of water vapors from crystallization zone and preventing “caking” of obtained granules. The residual moisture of obtained granulated powder was 3%. The granules were transferred to drum-type drying apparatus set at a temperature of 65° C. and dried until its moisture content was 0.08%.
The produced sweetener appeared as yellowish granular powder and had taste profile close to sucrose with sweetness power 2 times higher than sugar.

EXAMPLE 24

Control Solutions of Sweetener Compositions

Sucrose, invert sugar, Rebaudioside A obtained according to EXAMPLE 3, and purified sweet steviol glycoside mixture obtained according to EXAMPLE 1 were dissolved in the water to prepare the control samples for sweetener compositions. The concentration of components is indicated in Table 4.

TABLE 4

Control samples

Concentration, mg/L

		Invert	Steviol
Control samples	Sucrose	sugar	glycosides	RebA

Control sucrose	50,000	0	0	0
Control Steviol glycosides	24,420	488.5	91.5	0
Control RebA	24,440	488.8	0	73.3

EXAMPLE 25

Taste Evaluation of Sugar based Sweeteners

A trained panel of 15 sensory analysts evaluated the control samples and the sweeteners compositions prepared according to Examples 21, 22, 23. The sweetener compositions were dissolved in water at 2.5% (wt/vol) concentrations. The evaluation results are summarized in Table 5.

TABLE 5

Evaluation of sweetener compositions

Evaluation results

			Sweetness	Licorice
Samples	Sweetness	Bitterness	lingering	aftertaste

Control sucrose	++++	−	−	−
Control Steviol	++++	++++	++++	++++
glycosides
Control RebA	++++	+++	+++	+++
Steviol glycoside	++++	+	+	−
composition (EXAMPLE
21)
Reb A composition	++++	−	+	−
(EXAMPLE 22)
Reb A composition	++++	+++	++	++
(EXAMPLE 23)

The samples prepared according to Examples 21, 22, 23 demonstrated significant improvements in key taste attributes in comparison with respective control samples. The samples prepared by process of Examples 21 and 22 were significantly superior to sample prepared by process of Example 23. Therefore it can be concluded that the deoxygenation of solution by nitrogen purge reduces the color formation and undesirable taste characteristics of final compositions.
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 is therefore understood to be limited only by the scope of the appended claims.

Claims

What is claimed is:

1. A process for producing a low-calorie sweetener composition, comprising:

a) providing a low intensity sweetener;

b) providing a crystallization inhibitor;

c) dissolving the sucrose and crystallization inhibitor in water to make a solution with 60% to 80% (w/w) total solids content;

d) purging the solution (c) with nitrogen to obtain a purged sugar solution;

e) providing a high intensity sweetener;

f) dissolving the high intensity sweetener in water to obtain a high intensity sweetener solution with about 10% to about 70% (w/w) solids;

g) adding the high intensity sweetener solution to the purged sugar solution to obtain a sweetener composition solution containing about 50% to about 80% (w/w) total solids;

h) evaporating the sweetener composition solution to obtain a concentrated sweetener composition solution with about 90% to about 97% (w/w) solids content;

i) feeding the concentrated sweetener composition solution to an impact beating apparatus to induce intensive crystallization and residual water evaporation, and to produce free flowing granules of the sweetener composition;

j) drying the granules to form the low-calorie sweetener composition.

2. The process of claim 1, wherein the high intensity sweetener is made by a process comprising the steps of: i) drying Stevia rebaudiana leaves; ii) treating the dried Stevia rebaudiana leaves with water to extract sweet glycosides, wherein a leaves-water mixture is formed; iii) filtering the leaves-water mixture to obtain an aqueous filtrate; iv) directly treating the aqueous filtrate with calcium hydroxide or calcium oxide to raise the pH of the aqueous filtrate to about 10; v) directly neutralizing the treated aqueous filtrate with trivalent iron chloride; and vi) filtrating the neutralized aqueous filtrate to obtain a filtrated aqueous filtrate, and evaporating the filtrated aqueous filtrate to dryness to obtain purified sweet glycosides.

3. The process of claim 2, further comprising: vii) dissolving the purified sweet glycosides of claim 2 in methanol at temperatures 20-50.degree. C. in an amount of 1:2-1:7 (w/v) to precipitate Stevioside from solution; viii) filtering the solution from step (i) to recover the precipitate of Stevioside; ix) suspending the precipitate of Stevioside obtained in step (ii) in an alcohol-water solution to further purify the Stevioside; and x) recovering a higher purity of Stevioside with a purity of at least 98%.

4. The process of claim 3, which further comprises: combining remaining alcohol solutions; removing the alcohol; ultrafiltering to obtain a filtrate; and recovering Rebaudioside A with a purity of at least 98%.

5. The process of claim 1, wherein in step (d) the solution is purged with nitrogen at a rate of about 1 to 10 liters per minute per liter of solution at a temperature ranging from about 50-80° C., for about 0.5-10 hours.

6. The process of claim 1, wherein in step (h) evaporation is carried out with any equipment suitable for evaporating water from aqueous solution to obtain concentrated sweetener composition with about 90% to 97% (w/w) total solids;

7. The process of claim 6 wherein the evaporation is carried out at about 100-150° C., under about a 300-800 mbar vacuum, for about 0.5-2 minutes.

8. The process of claim 1 wherein step (i) the impact beating apparatus is a device capable of introducing shear mechanical impact or agitation to the concentrated sweetener composition solution.

9. The process of claim 8 wherein the mechanical impact duration on the concentrated sweetener composition solution is between about 0.01-300 seconds.

10. The process of claim 8 wherein the impact beating apparatus is provided with a water vapor removal system.

11. The process of claim 1 wherein step (j) the granules are dried by means of any apparatus suitable for drying powder.

12. The process of claim 11 wherein the drying is conducted at about 50-80° C.

13. The process of claim 1 further comprising sifting the obtained granular low calorie sweetener composition to obtain fractions with specific particle size.

14. The process of claim 13 wherein the particle size in the range of 0.5-2 mm.

15. A low-calorie sweetener made by the process of claim 1, comprising a high intensity sweetener and a low intensity sweetener.

16. The low-calorie sweetener of claim 15, wherein the low intensity sweetener is selected from the group consisting of sucrose, glucose, maltose, fructose, lactose, tagatose, palatinose, sugar alcohols, and combinations thereof.

17. The low-calorie sweetener of claim 15, wherein the high intensity sweetener is selected from the group consisting of: steviol glycosides including a purified sweet steviol glycoside mixture, stevioside, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside D, rebaudioside E, rebaudioside F, dulcoside A, dulcoside B, rubusoside, glycosylated steviol glycosides, glucosylated steviol glycosides and stevia; siamenoside; mogroside IV; mogroside V; Luo Han Guo sweetener; monatin and its salts (monatin SS, RR, RS, SR); glycyrrhizic acid and its salts; curculin; thaumatin; monellin; mabinlin; brazzein; hernandulcin; phyllodulcin; glycyphyllin; phloridzin; trilobatin; baiyunoside; osladin; polypodoside A; pterocaryoside A; pterocaryoside B; mukurozioside; phlomisoside I; periandrin I; abrusoside A; cyclocarioside I; or a combination thereof.

18. The low-calorie sweetener of claim 15, wherein the crystallization inhibitor is selected from the group comprising, invert sugar, glucose, fructose, fructo-oligosaccharides, inulo-oligosaccharides, fructose-terminated oligosaccharides, lactulose or combinations thereof.

19. The low-calorie sweetener of claim 15, wherein the low intensity sweetener to high intensity sweetener dry weight ratio is between about 1:50 to about 1:400 (w/w).

20. The low-calorie sweetener of claim 15, wherein the low intensity sweetener to crystallization inhibitor ratio is between about 1:1 to about 100:1 (w/w).