INTRODUCTION

Lycopene is an open chain pigment of unsaturated caro- tenoid which is responsible for red colour found in toma- toes, grapes, watermelon, and other bright colored fruits and vegetables. Its function in plants is to absorb light in photosynthesis and to protect plants from the excess of

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Received 20th May, 2014

Accepted after revision 30th June, 2014 BBRC Print ISSN: 0974-6455 Online ISSN: 2321-4007

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light (Amin et al., 2010). Lycopene is an acyclic isomer of ß-carotene with 11 conjugated double bonds, normally in the all-trans configuration. Because its double bonds are conjugated, lycopene absorbs radiation at long wave- length of the visible spectrum (Maheswari et al., 2012).

Lycopene has been characterized as a strong antioxi- dant, implicated in reduced incidence of cardiovascular

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Meena Vankudre, Vina D. L. Putra and Vaibhav Zamare

disease and reduced frequency of several types of can- cers, most notably prostate (Giovannucci et al., 2002). Lycopene can prevent carcinogenesis by protecting vital bio molecules including DNA, proteins, enzymes and lipids (Breemen et al., 2008 and Salvadori, 2007). It has been shown to have the highest antioxidant activ- ity among the carotenoids in cell protection against free radicals (Salvadori et al., 2007). Supplementation with tomato based products also increase lycopene level in human serum and protect against UV-light induced erythema (Aust et al., 2003).

Lycopene content in tomatoes can vary signifi- cantly, depending on the type of tomato and ripening. In the reddest strain, lycopene levels are close to 50mg kg-1, compared with only 5mg kg-1 in the yellow strain (Veazie et al., 2006). The colour was generally an accurate indicator of lycopene content, with a yellow cultivar containing less lycopene than red cultivars, and the red cultivars containing more than an orange culti- var. However, black cultivars as a group did not contain more lycopene than red (Cox et al., 2003).

Processing of food helps to release the lycopene from food matrix, thus improving accessibility of the lipophilic compound for the formation of lipid micelles together with dietary lipid and bile acid. Lycopene uptake is higher after ingestion of processed tomatoes (e.g tomato paste, tomato juice) (Stahl and Sies, 2006).

The antioxidant and anticancer properties of Lyco- pene make it an ideal component for daily food supple- ments. It is one of the popular pigments highly accepted by food industry as food additives and also for its health benefits (Rao and Agarwal, 1999). At the sixty-seventh meeting of The Joint FAO/WHO Expert Committee on Food Additives (JECFA), it was agreed that both syn- thetic lycopene and lycopene extracted from any organ- isms were acceptable as food colours and established a group ADI of 0-0.5 mg/kg body weight/day for both preparations (FAO/WHO 2006).

Nowadays, Lycopene supplements are available in the form of tablets or gel beads which are relatively high of cost. For this reason, improved form of Lycopene sup- plement is developed in the form of widely consumed cookies. This provides the consumer the easiest way to include Lycopene in the diet by consuming one package, to fulfil the daily requirement of lycopene.

MATERIALS AND METHODS

EXTRACTION AND PURIFICATION OF LYCOPENE

Tomatoes, obtained from the local market, were ground with water in a mortar and pestle to obtain a paste. The

paste was mixed with acetone and stirred for 3-4 min- utes (Aghel et al., 2011). The extra liquid was removed and the residue is collected by squeezing in between fil- ter paper. The red dehydrated paste was the mixed with mixture of n-hexane and ethyl acetate (1:1). Anhydrous MgSO4 was added to remove the extra water content. The solution was filtered by using filter paper to sepa- rate chunks of tomato paste. The final solution was sub- jected into column chromatography packed with silica gel by using solvent mixture of n-hexane and acetone (1:1) to elute ß-carotene and pure Lycopene (Lehman, 2009). Lycopene extract from tomato was a dark-red viscous liquid. It was freely soluble in ethyl acetate and n-hexane, partially soluble in ethanol and acetone, and insoluble in water.

EVAPORATION OF SOLVENT

The collected compound was subjected to rotary evapo- rator to evaporate and recollect the solvent. The water bath was maintained at temperature 80o . The solvent, such as ethyl acetate and n-hexane were recollected and reused for another round of extraction.

COLOR TEST FOR CAROTENOID

To the small amount of extracted sample, concentrated sulphuric acid was added drop by drop. Blue colour was obtained, indicating the presence of carotenoid (Maheswari et al., 2012).

QUALITATIVE AND QUANTITATIVE ANALYSIS OF LYCOPENE

The pure lycopene sample was analyzed by thin layer chromatography by using Lycopene standard. Other qualitative analysis was done by using Double beam UV-vis spectrophotometer at wavelength range 400- 500nm. The concentration was also determined by this method and by comparing with standard. A lycopene solution in n-hexane showed an absorption maximum at approximately 472 nm (Alda et al., 2009).

COOKIES AND LYCOPENE EMBEDDED CREAM PRODUCTION

The cookies are made by following the simple butter cookies recipe (Lamke et al., 2011). The whole batter pro- duces 24 cookies. The cookies are coated with lycopene embedded chocolate cream. 1 cookie weighs about 27.8 grams and contains 1 mg of lycopene. The product is formulated in such a way that they will provide about 3 mg lycopene per serving.

SENSORY EVALUATION OF THE COOKIES

10-20 panellists were employed from Sinhgad College of Science to evaluate the cookies quality on the basis of appear- ance, size, odour, texture, colour, taste, overall acceptabil- ity. The cookies were rated on a 9 point hedonic scale (1 = dislike extremely, 9 = like extremely) (David Peryam, 1998) and an overall quality rating on a 10 point scale (Peryam et al., 1998; Azizah et al., 2012; Kilcast, 2010). The panel- lists were given the original cookies (without Lycopene) and Lyco-cookies containing 1mg lycopene per cookie.

Meena Vankudre, Vina D. L. Putra and Vaibhav Zamare

RESULTS AND DISCUSSION

The method of lycopene extraction by solvent-solvent extraction (fig. 1) and purification by column chroma- tography (Fig. 2) was successfully yielding maximum amount of lycopene (40mg kg-1 tomato paste), and 95% of solvent recovery was achieved by rotary evapora- tor. The method is considered to be cost-effective for lycopene extraction. Characterization of lycopene was done by spectrophotometric analysis in which lycopene showed maximum absorbance at 472 nm (fig. 3).

FIGURE 1: Extraction of Lycopene by solvent-

solvent extraction method

FIGURE 2: Purification of lycopene by column

chromatography

Meena Vankudre, Vina D. L. Putra and Vaibhav Zamare

In the presence investigation, lyco-cookies were suit- able in any parameters for public consumption and mar- ket trials, as a commercially valuable cookie product. There is no significant difference between the rates of parameters listed in table 1, of lyco-cookies and other cookies products. The relationship was calculated using following equation:

Pair T-test:

Where X1 and X2 are the data of sample 1 and 2 respectively, S is the standard deviation of data of sam- ple 1 and 2, and n is the number of data. α = 5% = 0.05 and degree of freedom is 10. The value of t is 2.282603. The value of p is 0.071306. The result is not significant at p ≤ 0.05.

The additional lycopene content in the cookies pro- vides an improved nutritional value, which is more pre- ferred by many people than simple consumption of large amount of tomatoes and other forms of lycopene sup- plements such as tablets, syrups, and gels. Apart from its consumption by wide range of consumers, the production of lyco-cookies also targets the people undergoing treat- ment of skin problem, skin cancer, and other free radical associated diseases. The modified form of this lycopene supplement also is believed to contribute the adequate lycopene intake by people, especially for those in tropical regions where UV radiation has become a health issue.

Agricultural sector is also benefited during the present study, as it provides an efficient way for tomato waste management. As tomato is a fast ripening fruit, there is a need to distribute the fruit for efficient utilization. The surplus of tomatoes during the overproduction period can be directed for lycopene extraction and lycopene enriched food products.

ACKNOWLEDGEMENTS

The authors are thankful to the Principal, Sinhgad Col- lege of Science, Ambegaon, Pune and Sinhgad College of Pharmacy, Narhe, Pune for providing facility, coop- eration and help during the course of the present work.

Meena Vankudre, Vina D. L. Putra and Vaibhav Zamare

FAO/WHO (2006). Evaluation of certain food additives (Sixty-seventh report of the Joint FAO/WHO Expert Committee on Food Additives). Geneva, World Health Organization (WHO) Technical Report Series, No. 940.

John W. Lehman, (2009). Operational organic chemistry: 4th Edition, Pearson, 63-68.