WO2010021001A2

WO2010021001A2 - Process for preparing sweetener from stevia rebaudiana

Info

Publication number: WO2010021001A2
Application number: PCT/IN2009/000456
Authority: WO
Inventors: Ramakrishnan S. Kaushik; Antony Usha; Muthukumaran Geetha
Original assignee: Kaushik Ramakrishnan S; Antony Usha; Muthukumaran Geetha
Priority date: 2008-08-19
Filing date: 2009-08-17
Publication date: 2010-02-25
Also published as: WO2010021001A3

Abstract

The present invention provides a biological method for removing the color and odor causing components of stevia extracts and for producing a sweetener product which retains the beneficial anti-oxidant activity of a crude extract. Bacteria and fungi are capable of degrading plant pigments and polyphenols responsible for causing color in stevia extracts. Pleurotus ostreatus was employed as a test organism and reduced color by 70% and 50% in the yellow-brown (470 nm) and green (670 nm) region. Total polyphenol content was reduced by 40% but the free radical scavenging activity of the extract was maintained. Any bacteria or fungus capable of degrading plant pigments and polyphenols, but do not degrade stevioside, may be utilized in this method.

Description

COMPLETE SPECIFICATION

Process for preparing sweetener from Stevia rebaudiana

The present invention relates to the Process for preparing sweetener comprising of fermented extract of Stevia rebaudiana leaves, usable as a solid or liquid after stabilization in beverages or foods, and the fermentation process for producing the same. The invention seeks to retain the beneficial anti-oxidant property of the extracts of the leaf.

Description of prior art

Sugar (Sucrose) is very popular as a sweetener in beverages and foods. There is an increased demand for sugar substitutes because of the health problems associated with sugar. Synthetic alternatives to sugar include saccharin, aspartame and sucralose. More recently natural alternatives to sugar like glycyrrhizin, thaumatin and stevioside have gained popularity.

The plant Stevia rebaudiana produces Steviol glycosides. The plant is native to the highlands of Paraguay. These glycosides are intensely sweet and have sucrose equivalencies of between 50-300 times. They are highly heat, pH and microwave stable. They have been in use in Japan, Korea and Malaysia since many years. Their safety has also been investigated-extensively and FDA has approved the use of stevioside as a sweetener recently.

Aqueous and alcoholic extracts of stevioside are associated with a number of impurities, including plant pigments and phenolic which contribute to color, odor and taste not intrinsic to the molecule. Brown to yellow color is contributed by phenolic which also affect the stability of any formulation made out of a semi-pure extract, since phenolic compounds are easily oxidisable. Green and yellow color is contributed by chlorophylls and xanthophylls which impart a leafy odor and taste to the extract. Odor is contributed by volatile components which also give rise to herbal or leafy sensations. Taste is affected by the presence of polyphenols components. Low molecular weight phenolics are astringent and high molecular weight phenolics are bitter. Aqueous extracts of stevioside typically contain polymeric hydrolysable ^• polyphenols contributing to a bitter taste, which acts to amplify the inherent bitterness of the molecule. These factors make purification of the leaf extracts necessary.

One of the earlier patents for purification of stevioside (US Patent No: 3723410) involved defatting of coarsely ground leaves with chloroform and subsequent extraction with dioxane or water in the presence of an alkali. In this case the alkali served to precipitate out the phenolic acids by salting them out of the organic phase. Ion-exchange resins were already being used for purification of the molecule. The final step was crystallizing out the. molecule from cold methanol before recovery. Strong acidic and weak basic ion exchange resins have been used on a decoction of stevia leaves, followed by filtration to obtain pure products (Patent No.: 4892938). This was also one of the first patents to completely eliminate the use of organic solvents in the processing.

Chelating agents which perform the function of the ion-exchange resin in removing charged impurities (Patent No: 4599403) were also used.

A Japanese patent held by Tan (Patent No.: JP-A- 62-000496) involves the use of acetone along with methanol and further purification was achieved by adsorption;

US Patent No. 5112610 simply describes a method for using supercritical CO₂ to remove taste-impairing components without describing them. The gas is employed in leaves, crude extracts, purified extracts in solid form as well as in liquid extracts.

Cross linked starch and styrene gels have also been used on HPLC systems (Patent No.: 4171430) for purification of steviosides. HPLC has the disadvantage though of higher capital and operational costs.

Whereas pH does not affect the elution of the glycosides in the first stage of water extraction, lower pH reduced the amount of colored impurities. This was the basis of a patent (Patent No.: 5972120) which also utilized ultra and nano filtration processes for complete purification.

Electro coagulation for removing the pigments provides an interesting method of reducing solvent usage to remove chlorophylls. This process needs further study before it can be used industrially.

Summary of the invention:

An objective of the current invention is to prevent the use of chemicals in processing extracts of stevia leaves, to remove color and odor. Thus the process describes the use. of micro-organisms capable of degrading plant pigments and polyphenols to process the extracts of stevia leaves.

Furthermore, it is beneficial to retain the anti-oxidant activity of crude extracts of the leaf, which is not possible by any other method of processing. The product thereof becomes uniquely endowed with acceptable organoleptic properties while being colorless and beneficial for health.

Description of the invention:

The sweetener described in the present invention involves extracts of Stevia rebaudiana leaves. The extracts may be used as sweeteners provided polyphenols and plant pigments which contribute to color and odor are removed.

The polyphenols in leaf of Stevia rebaudiana are hydrolysable tannins, which are polymers of gallic and ellagic acid. These polymers may be broken down into the monomers, Gallic or ellagic acid, and glucose. Once they are broken down, the monomers are colorless.

Plant pigments like chlorophyll, xanthophylls and carotenoids contribute to color and a green leafy odor. These pigments are complex organic molecules which can be utilized to provide energy to the degrading organism.

The process of degrading plant components is a naturally occurring cycle, like composting, which constantly renews the sequestered carbon in plant and animal bodies and makes it available to the growing plants and animals. Many microorganisms in nature are .capable of degrading such plant and animal components.

In the current invention, an aqueous extract of stevia leaves is made by a decoction process. The leaves are extracted in boiling water and filtered to remove the particulate matter. This extract now contains steviol glycosides and the impurities that are to be removed in subsequent processing steps. It is to be noted that there are many processes described in prior art that reduce the amount of impurities being eluted out in the extraction step.. These may be used in the current process without any deleterious effects, in fact, reducing the amount of impurities in the first step may be beneficial.

To this decoction of stevia leaves, glucose is added at the rate of 1 gram per liter. The glucose serves as an inducer for the growth of the preferred micro-organism. It may be noted that any simple sugar, like glucose, which is preferred by. the micro-organism may be added to the extract. The amount of carbon source added may be increased or decreased depending on how much is actually utilized by the micro-organism during the fermentation. Some molecules like Di-methyl sulphoxide that influence the secretion of enzymes in micro-organisms may also be used as required. There is no restriction on the use of any inducer as long as it is complementary to the process.

To this extract, inoculums of the micro-organism of choice are added. The microorganism is chosen by an earlier screening step, keeping in mind the following points:

1. It should be able to degrade color causing components.

2. It should not degrade the steviol glycosides.

3. It should be considered safe for use in food products.

Based on such a screening process, any micro-organism from the class of fungi or bacteria may be chosen. The fungus Pleurotus ostreatus was chosen for the current invention since it is considered a GRAS (generally regarded as safe classification by USFDA), a food fungus and was able to remove color very efficiently in the screeninci step. It may be noted that any other micro-organism based on a screening experiment would suit just as well for the preferred decolorisation step.

The inoculated extract is now allowed to ferment for 48-100 hours. Preferably 48- 72 hours with 60 hours being the optimum. Temperature may be maintained within 27°C to 37°C, optimum being 30⁰C.

The decolorisation is followed at characteristic wavelengths 470 nm (yellow-brown region) and 670 nm (green region) for reduction of color. Once the reduction is seen to be stabilized, the fermentation may be stopped and the extract taken up for further processing as required.

The ferment is then filtered through a 0.2 μm filter to remove all suspended spores and microbial cells. In case of requirement of stabilization of the ferment as liquid, it may be processed as such. In the current invention, it was enough to add glycerol at the rate of 3% v/v and storage at 4°C for stabilization of the liquid for up to a week. This step may be adapted as per methods known in prior art for stabilizing liquid foods.

For requirement of powder, the ferment may be spray dried, lyophilized or vacuum evaporated as prescribed by prior art. The choice of drying method will not influence the stability of the molecule or the powder.

The product obtained by the above mentioned process is now sufficiently decolorized, deodorized and has excellent organoleptic properties. Since Gallic and Ellagic acids are still present in the extract, they contribute to the anti-oxidant activity of the product which is synergistic with any food that it is added to.

Extract of Stevia rebaudϊana leaves

Preparation of optimum fermentation condition ( Addition of inducers, optimising temperature and time )

Addition of inoculum { micro-organism )

Follow decolorϊsation in the ferment by absorption measurements at visible wavelengths and stop when absorbance becomes stable

Remove the ferment and process according to product requirement

BRIEF OUTLINE OF PROCESS FOR PRODUCTION OF SWEETENER FROM STEVIA REBAUDIANA PLANT MATERIAL

Example:

100 grams of Stevia leaves were extracted with 1 liter of boiling water for 10 minutes. The extract was filtered and 1 gram of glucose added to it. The fungus, Pleurotus ostreatus, was added to the extract under sterile conditions and the flask allowed sitting in an incubator at 3O⁰C for 60 hours. Upon completion of 60 hours, the ferment was taken out and analyzed for the presence of color at 470 nm and 670 nm. Fig 1 shows the Decolorisation of the stevia extracts by the fungus Pleurotus ostreatus as seen in a full visible region wavelength scan. Profile 1 is the crude extract and profile 2 is the fermented extract after 60 hours. Fig 2 shows the HPLC profile of the extract before and after fermentation. There is no change in the stevioside concentration even after fermentation. The anti-oxidant activity was followed using the DPPH ( expand) assay. Fig 3 shows that the free radical scavenging activity is maximum at the 60 th hour, the extract was then added to coffee and tea and the synergistic anti-oxidant activity was found to be good. After spray drying, a pale yellow powder was obtained, which upon addition to coffee or tea did not cause any contribution to color, odor or taste except contributing to the sweet taste.

Brief description of the diagrams

Fig No 1:

Decolorisation of the stevia extracts by the fungus Pleurotus ostreatus as shown in a full visible region wavelength scan. Profile 1 is the crude extract and profile 2 is the fermented extract after 60 hours.

Fig No 2:

HPLC profile of the crude and fermented extract of stevia leaves. There is no change in the stevioside concentration even after fermentation. Profile 1 is the crude extract and profile 2 is the fermented extract after 60 hours.

Fig No 3:

The free radical scavenging activity of the fermented extract as followed by the DPPH assay. The free radical scavenging activity is maximum at the 60th hour.

Claims

We claim:

1. A process for preparing the sweetener; comprising the step of fermenting the extract of the Stevia rebaυdiana plant material with a micro-organism, bacteria or fungus, so as to remove the color and odor causing impurities

2. The process for preparing sweetener, according to claim 1 may utilize any micro-organism, bacteria or fungus, capable of degrading the color and odor causing impurities without degrading the steviol glycosides of interest in the extract.

3. The process for preparing sweetener according to claim 1, may utilize aqueous or organic solvent extracts of Stevia rebaυdiana leaves for fermentation.

4. The process for preparing sweetener according to claim 1, may preferably be conducted with the addition of additional carbon sources, inducers or any other fermentation condition variables for increasing the efficiency of decolorisation.

5. The process for preparing sweetener according to claim 1, may include further downstream processing of the fermented extract, like ion-exchange, column chromatography, solvent fractionation, or other methods for increasing the purity of the end product.

6. The said process resulting in a sweetener product containing steviol glycosides and other sweet components of Stevia rebaudiana leaves, but with considerably reduced color and odor causing impurities. The sweetener additionally retains the anti-oxidant activity inherent in the crude extracts of the leaf.

7. The sweetener according to claim 6, may be a stabilized liquid of the ferment produced by process.

8. The sweetener according to claim 6, may include dehydration of the ferment . produced, by processes such as spray drying or lyophilisation, or any other method for use as solid powder, as a table top sweetener or to be added to other formulations.

9. A process for producing a sweetener with substantially reduced color and odor causing impurities while retaining the beneficial anti-oxidant activity of the crude plant material extract, by the use of bacterial or fungal fermentation for degradation of the said impurities without affecting the steviol glycoside content of the Stevia rebaudiana plant material extracts.