WATER DISPERSIBLE STEROL/STANOL ENRICHED POLYPHENOL RICH HERBAL TEAS IN AQUEOUS OR POWDERED FORMS TO REDUCE TOTAL AND LDL CHOLESTEROL LEVELS

Technical Field

The present invention relates to the field of compositions, either in aqueous or powdered forms, containing polyphenol rich herbal tea extracts and hydrophobic plant sterol s/stanols, and the methods for the production thereof. Unless otherwise indicated, the term "plant sterol", as used in this specification and claims, is intended to include both a plant sterol and a plant stanol. Specifically, the present invention relates to plant sterols containing tea drinks with excellent quality retention and stability in which plant sterol s/stanols remains stable during storage for a long period. The present invention also relates to plant sterols containing instant tea powders which easily dispersible in water. This invention further relates to the methods for producing such sterol/stanol containing polyphenol rich herbal tea products either in aqueous or powdered forms and their use in foods, beverages, pharmaceuticals and nutraceuticals, among others. Typical herbal teas include green, black, oolong, and white teas produced from Camellia sinensis. Other typical herbal teas include jasmine, linden, rooibos, rosehip, anise, ginseng, chamomile, rosemary, sage, mint, melissa, echinacea, guava leaves and their mixtures.

Background Art

It has been mentioned that dietary habits and changes in lifestyle lead to an extensive intake of cholesterol contained in food increased blood cholesterol, and thus is a factor in the onset of cardiovascular diseases such as hyperlipidemia, arteriosclerosis, arrhtymia, and myocardial infraction, among others, which currently tend to be increasing globally. Great efforts are focused on reducing the risk of coronary heart disease through dietary interventions. Plant sterols are found to interfere with the absorption of cholesterol into the body and lower its levels in serum.

Plant sterols have chemical structures resembling that of cholesterol is known to inhibit intestinal cholesterol absorption, thereby reducing the serum cholesterol level, as disclosed in U. S. Pat. No. 5,578,334. Plant sterols are only available to human through plant foods such as vegetable oils, nuts, seeds, cereals, legumes, fruits and vegetables. The current daily intake of plant sterols from conventional foods is estimated to be in the range of 160-400 mg among different populations. The daily doses, considered optimal for the purpose of lowering blood cholesterol levels, are 2-3 g of plant sterols.

However, plant sterols have the disadvantage of poor solubility in both water and oil, and therefore are not easily applicable to foods. Under such circumstances, plant sterol esters had been developed (US Patent No 3,751,569 and WO00061694), which shows an excellent solubility in an oil phase. The drawback to this process, is the use of non-food grade catalysts and reagents to carry out esterification. Another drawback, along with others, is the kinds of foods, into which plant sterol ester can be incorporated, are limited to specific fat based ones such as margarine, edible oils and mayonnaise. This would be quite inconvenient because of such a contradiction that the ingestion of such plant sterol leads to intake of large quantity of edible oil. For this reason, to develop a water based products which plant sterols easily dispersed or dissolved in is an urgent necessity. There have been several attempts to provide such a product with limited success. Known methods include grinding or milling the plant sterols to increase their solubility (US 3,881,005 and 4,195,084), however micronization sterol particles alone is not sufficient in order to achieve good incorporability in food matrix, these particles poorly wettable, aggregate readily and generally float on aqueous surface. Therefore, micronization of plant sterols combine with the additional use of emulsifiers. EP 0897671 Bl includes a method in which high melting point lipid, such as a plant sterol, melted at 150 °C is mixed with non-sterol emulsifier at a ratio of emulsifier to lipid less than 1 :2 (w:w), and melting this mixture by heating at 150 °C. Molten mixture was dispersed in the boiled water using high shear and temperature of the mixture is maintained at 95 °C. The phytosterols are reported to have particle size less than 15 μπι and preferably less than 10 μπι in aqueous dispersions.

In WO2003105611, Coca Cola incorporated plant sterol into fruit beverages without any added emulsifier or thickening agents to avoid the increase in viscosity and detracting from its organoleptic impression in case of using stabiliziers and gums or any formation of ring at the surface of juice or sink to the bottom.

Herbal tea extracts rich in polyphenol can be used to develop different food and beverage, pharmaceutical, and nutraceutical formulations due to their bioactive properties such as antimicrobial, immuno-stimulatory, anti-carcinogenic, and antiinflammatory activities; and also for its protective effect against metabolic syndrome, e.g., a clustering of several cardiometabolic risk factors including hyperglycaemia, dyslipidaemia and elevated blood pressure, where abdominal obesity and insulin resistance represent core parameters of this cluster. However, it is known that when tea extract rich in polypehnols is cooled down after brewing in hot water, the substances comprise polypnehol-protein and/or caffeine complexes (tea cream) starts to precitipate and this is increased in low pH (pH<5) values which beverages are mostly consumed due to sensory contribution of low acid foods to palatability. There have been many methods to obtain cold water soluble tea concentrates which have good colour and clarity including alkali treatment to increase the solubility of insoluble complexes and followed by centrifugation to remove any remaining cold-insoluble tea solids (U.S. 3,163,539) or the use of enzymes including tannase and cell wall degrading enzymes (U.S. 4,639,375). Employing alkali chemicals to increase the solubility of polypnehol -protein- caffeine complexes would degrade the polyphenols which is the main health related bioactive compounds of tea and applying centrifigation at high speed to increase the sedimented layer would also rise to lose these health related compounds in the final product. The use of enzymes would be expensive and might give tea liquors or infusions with very light colours. Therefore the incorporation of hydrophobic ingredients (such as sterol) into tea extracts require special treatment to ensure that these ingredients will not increase the system instability and final product will not separate due to density differences, and would have the health related bioactive compounds in its formula and without providing unpleasant effects on the texture of the final products.

The patent relates to the use of tea and sterol together, WO2006074752 (Al), relates to porous sachet comprising plant sterol, an emulsifier, and a particulate component such as tea. Sterol ester and emulsifier heated together around 60 or 80 °C and cooled to room temperature, mixed with tea leaves and put in a sachet, after that immersed in hot water to get final beverage. The inventors claim that presence of porous sachet would reduce the formation of plant sterol ester droplets on the tea-surface which makes a less attractive impression and, therefore, are preferably avoided or reduced.

Disclosure of Invention

The present invention was made in the light of the above circumstances. The stability of the system includes sterol and tea extract would be different any system containing sterol and fruit juice extract or any aqueous based foods due to the contribution of polyphenol-protein- caffeine complex in tea extract to the precipitation, even though the water solubility of sterol was imporoved before addition into the final product.

An object of the present invention is to provide foods and drinks containing aqueous polyphenol rich herbal tea extract fortified with substantially stable hydrophobic plant sterol, and a production method therefor. To achieve at least one of these or other advantages in accordance with the purpose of the invention, as emboided and broadly described, the inventors have found a process for producing a substantially stable dispersion consisting essentially of hydrohobic plant sterol and aqueous polyphenol rich herbal tea exract.

The process involves dispersing or otherwise suspending hydropobic plant sterols into a delivery medium of emulsifier containing aqueous polyphenol rich herbal tea exract, resulting mixture are exposed to impact forces by creating high shear. Shear can be generated in preparation of the dispersion of the invention by using, high shear mixer, a colloid mill, a ball mill, high pressure valve homogenizers or microfluidizer or other mechanical or sonic devices to produce micropaticles below 30 μιη preferably below 10 μιη (Figure 1) and will substantially follow bell curve distribution. When the particle size of mixture is decreased, the efficacy and stability of delivery medium is enhanced. The higher pressure applied not only reduced the particle size, but also would reduce the size of complexes produced by tea insoluble compounds. The smaller the particle size the better the dispersion, and less sandy feeling in the mouth is obtained.

If desired, for example if the final mixture is used to prepare beverage formulations, additional compounds such as buffers or agents for pH adjustments (e.g. citric acid, tartaric acid, phosphoric acid, acetic acid, salts of any of these acids, sodium hydroxide and sodium bicarbonate), colours, flavours, anti-oxidants (e.g. ascorbic acid, sodium bisulfite, sodium sulfite, butylated hydroxyanisole, butylated hydroxytoluene, alpha-tocopherol) can be included in the dispersion either before or after homogenization step, for example acidulants may be added before homogenization where as flavours and coloring agents might be added after this step.

In another embodiment, the aqueous medium comprise aqueous herbal tea extracts in combination with emulsifiers and or stabilizers. Emulsifiers/stabilizers can be any type of ionic and nonionic food grade emulsifiers or mixtures thereof, Hydrohilicity-Lipohicility Balance (HLB) is preferably high (more hydrophilic) about 15 to 18, and may be selected from sorbitan fatty acid esters, polyoxyethylene sorbitan fatty acid esters, lecithin, sucrose fatty acid esters, polyglycerol fatty acid esters, mono- and diglycerides of fatty acids and their acidic ester or any natural extract with emulsiying properties such as quillaia extract and polysaccharides such as gum arabic, gums, celluose, alginate. The emulsifiers will help to stabilize the system not only due to its sterol content but also providing higher solubility of polyphenols and reducing the amount of insoluble tea solids, and stability of the tea complex compounds can also be attained by the use of those stabilizers.

In its broader aspect, the solid content of aqueous medium such as solid content of herbal tea extract is from about 100 grams per liter of aqueous medium to 200 grams per liter of aqueous medium and it might be set depending on the aimed concentration in the final product formulation upon dilution.

In yet another embodiment, the aqueous medium where hydropobic plant sterols suspended into it at varying concentrations, an amount from about 10 gram to about 200 grams per liter of aqueous medium, is exposed to high shear created by either with an high shear mixer, colloidal mill or high pressure valve homogenizers or microfluidizer to produce micropaticles.

In one embodiment, the dispersion before homogenizing may not require heating, however heating is desirable for pasteurization to prevent microbial spoilage. Thus the process may need one or more heating steps and this step may be applied before or after homogenization, or heating both.

In one embodiment, the dispersion before homogenization is optionally heated to temperature of from 20 °C to about 65 or 80 °C for a period of time of from about 0.1 minute to 60 minutes. In another embodiment, the dispersion is optionally heated to 100 °C for a period of time 1 to about 20 seconds before homogenization. In yet another embodiment, heated dispersion is cooled to about 25 °C.

In further embodiment, homogenization of the dispersion is conducted in a homogenizer such as using microfluidizer or high pressure homogenizer. The dispersion may be treated with high shear mixer previously. The homogenization may be operated preferably between 4.000 and 20.000 psi. The invention also relates to conducting homogenization at different pressures in single or multiple stages, increasing the number of passes through the chamber further decreases particle size.

In another embodiment, the aqueous polyphenol rich herbal tea extract may be produced by solvent (generally hot water) extraction of tea leaves with various equipment such as fixed bed reactors, continuous reactors with co-current or counter-current flow or just brewing tea. By using hot water (about 80-85 °C) and continuous reactors with counter -current flow, more concentrated liquid extract may be produced than in normally brewed tea. The tea extracts produced by continuous reactors with counter -current flow and just brewing tea have tea solids concentration in the region of about 9 to 25 °Brix. The solvent could be acidic buffer which would facilitate separation of insoluble parts and preservation of polyphenols. Insoluble parts in the extract may be separated by centrifugation. The solids concentration or pH of clear centrifugate or filtrate may be adjusted after or before separation process.

In a further embodiment, this aqeuous homogenized dispersion compirimising mainly plant sterol and herbal tea extract is either spray dried, or freeze dried, or vacuum dried to get water dispersible sterol fortified powder tea in the presence of excipients such as, but not limited to polysaccharides, maltodextrin, gum arabic, starch and starch derivatives, modified starches, agar, alginic acid, gums, cellulose, mycrocystalline cellulose or proteins such as skimmed milk powder, whey proteins. The content of excipients in the dried product is in the range of 2 to 40%, preferably about 5 to 20% by weight. The powder may contain from about 10% to about 60%) of plant sterols, preferably about 30%> to 40% by weight. The rest of the composition may composed of the dry matter of aqueous herbal tea extracts and excipients, stabilizers. The fluidity of the powder can be improved through the use of anti-caking agents, such as tricalcium phosphate, talc, stearic acid and its salts, polyethylene glycol or fumed silicon dioxide.

In a further aspect of the invention, this plant sterol enriched herbal tea extract and its dried form can be used in the preparation of pharmaceutical or nutritional products and beverages and the medical applications of such products. The food formulations according to the present invention comprises further constituents, such tea polyphenols which act independently, simultaneously and/or in synergy with the plant sterols, thus increasing the cholesterol lowering capacity of the final products prepared according to this invention. The consumption of plant sterol enriched herbal tea extract (e.g. black tea) and its dried form in for example beverage formulation would reduce blood total and LD and reduce the oxidative stress of subjects, who were consuming sterol enriched tea bevarage regularly, due to possibly its antioxidative polyphenol content. LDL-cholesterol levels significantly reduced (Figure 2a- c) in comparison to the consumption of same beverage without sterol or without sterol and tea extract in its composition. Total antioxidant status and oxidative stress of subjects would improved upon the consumption of sterol enriched black tea product (Table 1) Table 1 Change of total antioxidant status, total oxidant status and oxidative stress index of consumers after 4 weeks consumption period of ice tea products

Control Ice tea Functional (sterol enriched) ice tea

(n=33) (n=34) (n=32)

Before After P Before After P Before After P

TAS (mmol/L) 1.02 ± 0.10 0.77 ± 0.34 0.000* 0.92 ± 0.18 1.02 ± 0.24 0.014* 0.70 ± 0.13 0.75 ± 0.11 0.009*

TOS (μιηοΙ H ₂ 0 ₂ Eq/L) 9.7 ± 3.8 9.6 ± 5.3 0.929 12.8 (7.5-22.1) 11.8 (9.9-16.3) 0.224 7.7 (5.0-9.0) 6.1 (2.3-9.6) 0.090

OSI (TOS/TAS) 9.37 ± 3.20 13.33 ± 7.49 0.011 * 20.40 16.21 15.73±11.28 0.054 12.52 ± 8.47 8.41 ± 5.30 0.037*

TAS : Total antioxidant status

TOS : Total oxidant status

OSI: Oxidative stress index (TOS/TAS)

*statistically significant at a value of p<0.05

⁽¹⁾ Every group consumed 250 ml of product twice in a day. The product consumed by control group neither includes tea or sterol, but other ingredients in ice- tea; the product consumed by ice-tea group does not include sterol, but includes tea and other ingredients in ice-tea; and the product consumed by functional ice-tea group indues both sterol (lg/250ml), tea and other ingredients in ice-tea formulation

Description of Drawings

Figure 1 (a) Particle size of unprocessed plant sterol having a total plant sterol content of 99%, and composed of β-sitosterol, campasterol, stigmasterol, brassicasterol, campastanol, β- sitostanol, the mixture having a melting point of about 138 °C to about 170 °C, derived from vegetable oils and tall oils, and supplied by VitaeNaturals, Vitae Caps, Spain

Figure 1 (b) Particle size of processed plant sterol and tea extract mixture

Figure 2 (a) Total Cholesterol (mg/dL) levels of 3 groups of consumers after 4 weeks consumption period of ice tea products; blue and red bars represent the total cholesterol levels belongs to the before and after consumption period.

Figure 2 (b) LDL-Cholesterol (mg/dL) levels of 3 groups of consumers after 4 weeks consumption period of ice tea products; blue and red bars represent the total cholesterol levels belongs to the before and after consumption period.

Figure 2 (c) % of reduction in blood lipid parameters of 3 groups of consumers after 4 weeks consumption period of ice tea products

EXAMPLES

The invention will now be illustrated by, but is not intended to be limited to, the following examples. In these examples, parts and percentages are by weight, unless noted otherwise.

Preparation of ice tea beverage from black tea extract fortified with plant sterols

Example 1

Black tea leaves were extracted by continuous extractor with counter-current flow (Niro Atomizer). Hot water (80-85 °C) was used as extraction solvent with, tea: water ratio of 1 :5 (w:w) and black tea feed spent 15 minutes inside the extractor. After that, tea extract was centrifuged to reduce tea cream and haze to a spesicif level (with around 14°Bx value, soluble solids content), and centrifuged black tea extract was mixed with citric acid-sodium citrate buffer to make pH around 3.25. Later on, emulsifiers (Polyoxyethylene (20) sorbitan monooleate and Sucrose fatty acid ester (HLB 16); 0.2 and 0.1% w/v) was dissolved in this mixture and hydrophobic plant sterol (20%) was added into, and the mixture heated to 65 to 80 °C, and it was blended using a high shear mixer (Ultra-Turrax), 1000 rpm for about 5 min. After that, it was homogenized by using high pressure microfludizer (Microfluidics) at 12000 psi and 5000 psi to get particle size lower than 10 μιη.

This base mixture was, then diluted with water, and flavoured with sugar or artifical/natural sweeteners and aroma compounds (e.g., peach, lemon, mango) to make sterol content 4 g per liter of ice tea beverage at around having pH of 3.45 (citric acid-sodium citrate, 1.6-0.6 g per liter) and, where its final serving size was aimed to have 1 g per 250 ml of beverage. Finally, the beverage was pasteurized at 85-100 °C for 30-60 seconds (or other viable combination of time and temperature), cooled and bottled aseptically and followed by cooling to 20°C.

Example 2

Black tea extract in example 1 could be replaced with green tea extract or any other herbal tea extract. The highest plant sterol added to base mixture to be able to work sufficiently with microfluidizer was around 15-25%; dilution ratio would be around 1 to 40-60 (w:w) to get a product 1 g of plant sterol in 1 serving size of beverage (250 ml), which tea extract would be diluted to having around 3-2.25 g tea per liter when the °Brix ratio in the beginning was around 14. The final solid content of beverage could it can be set different values according to sensorial acceptance and °Brix value of herbal tea extract, sterol content of base mixture

Example 3

Preparation of sterol enriched powdered tea

The base mixture in Example 1, 2 mixed with gum arabic or gum arabic:maltodextrin (1 :4, w:w) at 5% in total weight, can be spray dried with an inlet temperature of 140 °C and temperature at the outlet was 90 °C . The final sterol content of powder produced the mixture from Example 1 was around 50%. Tea extract can also be agglomerated by using fluidized bed reactor at following conditions: air flow rate: 105-80 m ³ /h, air temperature: 85 °C, exhaust air temperature: 45-50 °C, product temperature: 50-55 °C, spraying pressure: 2.5 bar, pump position: 3-8. Final powder can be mixed with sugar or sweeteners as an ice tea beverage that is ready to drink after redispersion in water.

Powdered tea fortified with plant sterol can be used in any product such as, but not limited to, beverage formulations, nutraceutical preparations, ice-cream, and baked goods among others.