Optimization and Preparation of Debittered Kinnow Beverage

The development of delayed bitterness in citrus products is a major problem to citrus producers and juice processors worldwide due to limonin and naringin, the bitter components, thereby affecting its consumer acceptability. The present study was carried out on fermentative production of debittered kinnow (Citrus reticulate Blanco) beverage using inducible enzymes naringinase, limonin dehydrogenase and α-L-rhamnosidase producing fermentative yeast Clavispora lusitaniae. The two process optimized are: bioprocess for low alcoholic naturally carbonated beverage and debittering of juice by partially purified enzymes. The physico-chemical characteristics of freshly prepared beverage were; TSS 13.00±0.20 °B, acidity 0.14±0.03%, pH 3.40±0.10, Brix acid ratio 92.85±0.00, limonin 6.90±0.10 ppm, naringin 443.00±10.00 ppm, total sugars 12.90±0.30%, reducing sugars 2.42±0.20 and ascorbic acid 27.80±1.00 mg/100 mL. The significant reduction of limonin and naringin content observed in T11 (beverage) during storage was 42.53% and 45.82%, respectively.

Citrus is the main fruit crop in the world, with a total production of 122 million tons in 2008. World production of citrus fruits in the period 2009-2010 was 123 million tonnes which includes grape fruit, lemons, limes, oranges, tangerines and other citrus fruits. India ranked 5 th in citrus production (5% of worlds production) with 6,286,000 tones. These fruits are well endowed with a variety of phytonutrients, vitamin C, folate, dietary fibres and minerals as well as many antioxidant phytophenolics, including flavonoids, amino acids, triterpenes, phenolic acids and carotenoids that can potentially protect health of the consumers. These are utilized primarily or juice recovery, where about half of the processed citrus including peels, segment membrane and seeds are generated as waste. These solid residues are referred to as citrus wastes. It is made up of pulp (juice sac residue), the peel (albedo and flavedo ), rag (membranes and cores), seeds(sugars (glucose, fructose, sucrose), lipids (linoleic, oleic, phyosterol, stearic acid, palmitic, glycerol and linolenic), acids (malic and citric acid and benzoic acid, succinic and tartaric acid), enzymes (pectinesterase, phosphatase, peroxidase), insoluble carbohydrates (pectin, cellulose), bitter principle (limonin, isolimonin), flavonoids (naringin, hesperidin), peel oil (D-limonene), vitamins (ascorbic acid, carotenoids, vitamin B complex), volatile constituents (esters, aldehydes, alcohols, hydrochloric acids, ketones), pigments (xanthophylls, carotenes), and minerals (potassium and calcium) (Bambidis and Robinson, 2006).
The processing of citrus fruits has faced many commercial restrictions due to bitterness caused by RESEARCH PAPER naringin, limonin and nomilin (Thammawat et al. 2008). The threshold have been established (Sahota et al. 2015) and excess level of these compounds causes intolerable bitterness in processed citrus products such as juice, wine and vinegar.
Many techniques were used to reduce bitterness by naringin such as adsorptive debittering (Fayoux et al. 2007), enzymatic hydrolysis (Puri and Kalra, 2005), poly-styrene divinyl benzene styrene resin treatment and β-cyclodextrin treatment (Mongkolkul et al. 2006). But these techniques alter the nutrient composition of juice and the nutrients, flavor and color.
To control the quality and improve the commercial value of the citrus juices, maintaining the health properties and increasing the acceptance by the consumer, the reduction of bitter component by enzymatic hydrolysis appears as one promising technique with industrial application due to its high specificity, efficiency and convenience on commercial scale.
The microbial enzymes are of paramount importance due to their cost efficient production, therefore replacing the chemical methods for debittering. Enzyme concentration, temperature, pH variation, and inducer concentration are important parameters responsible for stability and activity of an enzyme action. The yeast Clavispora lusitaniae (KF 633446) strain has the potential to produce fermentative debittering enzymes, naringinase, limonin dehydrogenase and α-L-rhamnosidase thereby, used for the debittering of citrus juices. The debittering enzymes can offer advantages such as cost effectiveness, single step hydrolysis, short incubation, preservation of flavour, retention of color, vitamins and organoleptic components of juice.
In the agro-industrial sector the use of enzymes led to a process optimization, reducing process-related energy costs, improving nutritional safety and quality of food, the development of new products. The enzyme naringinase is composed of α-L-rhamnosidase (EC 3.2.1.40) and β-D-glucosidase (EC 3.2.1.21). Naringin (4′,-5,7′-trihydroxyflavonone-7-rhamnoglucoside) is first hydrolyzed by α-L-rhamnosidase activity of naringinase to rhamnose and prunin (one third of the bitterness of naringin) which can be further hydrolyzed into glucose and naringenin by the β-Dglucosidase component of naringinase. Naringinase has potential biotechnological applications in citrus processing and bioprocess industries such as wine industries for aroma enhancement, glycolipids production, biotransformation of antibiotics, steroids. The enzyme limonin dehydrogenase eliminate the production of bitter component limonin by catalyzing the oxidation of Limonate A-Ring lactone (LARL), to the corresponding 17-dehydroxylimonate a nonbitter derivative. The enzyme rhamnosidase has many applications in industries as : debittering of citrus fruit juices (Busto et al. 2007), manufacture of prunin from naringin, aroma enhancement by enzymatic hydrolysis of terpenyl glycosides containing L-rhamnose, elimination of hesperidin crystals from orange juices, conversion of chloropolysporin B to chloropolysporin C (Feng et al. 2005).
In this work, optimization of minimal media for maximum enzyme production using fermentative yeast Clavispora lusitaniae was carried out by supplementation of kinnow, lemon and grapefruit peels for the low cost value aided substrate.

Raw material
Fresh, fully ripened kinnow (Citrus reticulata Blanco) fruits were procured from the Department of Fruit Science, Punjab Agricultural University, Ludhiana. The selected fruits were washed in the chlorinated water and then used for the extraction of juice. The juice was extracted aseptically under hygienic conditions with a juice extractor.

Preparation of sugar solution
The sugar solution was prepared by boiling (500 g) granulated sucrose in one litre of water for 10 min and then, allowed to cool at room temperature and stored aseptically in sterilized glass bottles.

Inoculum preparation
The yeast inoculum was prepared in diluted juice with Brix adjusted to 13°B. A loopful culture of 24 h old yeast (Clavispora lusitaniae KF633446) was inoculated in 100 mL diluted kinnow juice in 250 mL Erlenmeyer flask and incubated at 30±5 °C for 24 h to achieve concentration of 10 5 -10 6 cells mL -1 .

Preparation of debittered citrus beverage
A debittered beverage was prepared under optimized conditions of inoculum concentration-(0.75% v/v), TSS (13 °B), incubation temperature (30±5 °C) and incubation time (48 h). The diluted juice was inoculated @ 0.75% v/v with freshly prepared inoculum and incubated at 30±5°C for 48 h in batch scale glass digester.

Fermentation
The physico-chemical analysis (pH, % acidity, TSS, Brix acid ratio, naringin, limonin and juice yield) of fresh kinnow juice was performed. Juice was diluted in the ratio 1:1.5 with water. Diluted juice was pasteurized at 82 °C for 15 Sec, cooled and Brix adjusted to 13 °B by adding sugar solution followed by inoculation of yeast i.e. 0.75% (v/v).

Bottling and storage
It was incubated for 48 h at 30±5 °C. The beverage was refrigerated for 24 h, siphoned, bottled and stored in refrigerated conditions.

Enzymatic debittering of citrus juice
Technology for the debittering of kinnow juice under optimized fermentation conditions were developed by yeast Clavispora lusitaniae (Kaur 2017). The concentration and incubation time of purified debittering enzymes for debittering of kinnow juice has been optimized. The purified enzymes Naringinase (0.6 mL), limonin dehydrogenase (0.8mL) and α-L-rhamnosidase (0.6mL) was added the diluted kinnow juice (13 °B) and kept for 10 h at room temperature.
The samples were drawn after regular interval of two hours for estimation of naringin and limonin.

Physiochemical and microbiological analysis of kinnow juice and beverage
The total soluble solids and pH of kinnow juice and beverage were determined by using Erma hand refractometer of 0-32 °B (Erma, Tokyo, Japan) and pH meter (ECIL, Hyderabad, type 101; Electronic Corporation of India Ltd., Hyderabad, India). Total acidity expressed as citric acid was estimated following the procedure of AOAC (1999). Brix-acid ratio was calculated through dividing TSS value by total acidity of the juice and carbonated beverage. Total sugars were estimated by phenol sulphuric acid method (Dubois et al. 1956). Reducing sugars were estimated by the method of Miller (1959). The titration method using 2, 6-dichlorophenol indophenol dye was used to estimate ascorbic acid (AOVC 1996). The total phenolic content (TPC) was determined by spectrophotometry, using gallic acid as a standard, according the method described by Singleton and Rossi (1965). Limonin content was estimated by colorimetric method (Vaks and Lifshitz, 1981) and naringin content was estimated by Davis method (1947). Carbon dioxide volumes in beverage bottles were determined by Zahm and Nagel piercing device (CO 2 tester, Zahm and Nagel Co., Inc., Holland, New York, USA) and percent alcohol (v/v) was calculated by spectrophotometric determination method of ethanol (Caputi et al. 1968). Total yeast count was enumerated on GYE agar by serial plate dilution method.

Sensory evaluation
The organoleptic evaluation of kinnow beverages was done on the basis of appearance, taste, color, aroma, bouquet, body, flavor, astringency and overall acceptability by a panel of judges. Consumer acceptance for the products was evaluated on a nine point "Hedonic scale" (Amerine et al. 1965).

Statistical analysis
Statistical analysis was done by using CPCS1 software. Standard errors were calculated for all mean values. Differences were considered significant at the p ≤ 0.05 level.

Shelf-life studies of kinnow beverage
Shelf-life of fermented debittered kinnow beverage stored at refrigerated temperature (4 °C) and room temperature was studied and evaluated fortnightly for physico-chemical, microbiological and organoleptic qualities.
The results showed a significant decrease in Brix from 13±0.2 °B to 11.2±0.3 °B at refrigerated temperature. The brix acid ratio also decreased from 92.85±0.0 to 19.31±0.0. Similar results have been reported in lime juice (Sarolia and Mukherjee 2002.The increase in TSS content of juice during storage might be due to hydrolysis of polysaccharides into monosaccharide and oligosaccharides. The pH decreased from 3.4±0.1 to 3.0±0.1 while acidity increased from 0.14±0.03% to 0.56±0.01% during storage. pH is inversely proportional to the acidity of any medium. This decrease in pH and increase in acidity was attributed due to formation of acidic compounds by degradation of reducing sugars (Zia 1987;Akhtar et al. 2010). Our results are in accordance with Saleem (1980) and Ahmed (2008).
The total sugars decreased from 12.9±0.3% to 9.5±0.4%. The reducing sugars also decreased from 2.42±0.2% to 1.32±0.1%. The sugars in citrus are mainly glucose, laevulose and sucrose. Similar results were also observed by Jairath (2012) for preparation of amla beverage. The increase in total sugar content of juice during storage might be due to hydrolysis of polysaccharides into monosaccharide and oligosaccharides.
Ascorbic acid (vitamin C) content reduced from 27.8±1 mg/100 mL to 6.48±0.4 mg/100 mL. The decrease of ascorbic acid (vitamin C) in beverage during storage results from oxidation of ascorbic acid by ascorbic acid oxidase due to a combined effect of oxygen and light (Bhardwaj and Mukherjee 2011). But the decrease was significantly less under refrigerated storage condition which may be attributed to low temperature and high relative humidity in storage, which inhibited the conversion of acid in sugars and decreased rate of ascorbic acid oxidation.
The mean polyphenol contents of kinnow beverage decreased significantly from 53.48±3 mg GAE/100 mL to 40.8±2 mg GAE/100 mL. According to Ritter et al. (1992) and Karadeniz and Eksi (2001) reports, clarification also decreased the polyphenolic contents of commercial fruit juices. Polyphenol contents decreased constantly with the progress of the ripening, while in red coloured varieties it increased during the last ripening stage due to the maximal accumulation of anthocyanidines and flavonols (Marinova et al 2005).The decrease of limonin from 6.9±0.1 to 3.52±0.1 ppm might be due to production of CO 2 during storage. Similar results were reported by Singh et al. (2015). Sahota et al. (2015) reported that limonin content decreased from 7.6 ppm to 4.0 ppm in kinnow beverage. Naringin content was decreased from 443±10 to 143.7±4 ppm .The decrease in naringin content occurreddue to hydrolysis of naringin into rhamnose and prunin by α-L-rhamnosidase activity of yeast Clavispora lusitaniae.
The alcohol production started after 10 days and gradually increased from 0.11±0.01% to 0.89±0.05% after 90 days The CO 2 pressure 0.65±0.05 bar started building after 10 days and reached up to 1.46±0.06 bar after 90 days.
Total yeast count was increased from 58×10 5 ±10 to 58x10 8 ±20 cfu mL -1 at the end of 90 days at refrigerated temperature.

Effect of enzymatic treatments on limonin content in kinnow juice
The yeast Clavispora lusitaniae has the efficiency to degrade limonin content in juice. The limonin concentration of freshly drawn juice was 7.5 ppm. During 30 days storage at ambient temperature, the limonin content was recorded, the decreasing trend of limonin concentration was observed in treatments containing debittering enzymes viz. T2 (0.8ml  The limonin content in T2 decreased from 7.5 ppm to 5.18 by 17.6%, 26% and 30% and in T4 limonin concentration reduced from 7.5 ppm to 4.36 ppm by 29%, 35.6%, and 41.9% over the period of 10, 20 and 30 days respectively (Fig. 1).
The moderate reduction was also observed in T10 by 7.73% on 10 th of storage and with the increase in storage time, limonin reduced by 12.4%, 18.53% on 20 and 30 days period.
In T10 (beverage), the limonin content was decreased to 18.53% due to efficiency of C. lusitaniae to metabolize and eliminate the bitter component by the production of limonin dehydrogenase. In T11, the limonin content reduced by 42.53% during the storage period of 30 days due to synergistic effect of yeast strain with the combination of debittering enzymes. The limonin content decreased significantly in T4, T7, T11 whereas limonin content in T6, T9 were found to be non-significant.
These results are in agreement with Singh et al. (2015) who showed decrease in limonin content from 6.90 ppm to 3.53 ppm in kinnow beverage. Puri et al. (2002) also purified limonin dehydrogenase from the cell culture of Rhodococcus fascians which degrades limonin by 66% kinnow juice. Hasegawa et al. (1982) showed 70% reduction in limonin content in naval orange juice serum with Arthrobacter globiformis cells in acrylamide gel.
The addition of 0.8 mL/100mL concentration of partially purified limonin dehydrogenase enzyme is an optimized concentration in debittering of citrus juices as maximum reduction in limonin content was recorded in T7 (45.33%). This reduction in limonin content is due to the combination of debittering enzymes and their stability during period of 30 days.
The results are in accordance with Prakash et al. (2002) who showed that naringin content reduced to 75% in Indian grapefruit juice. Yalim et al. (2004) reported the naringin elimination upto 30-40% in various citrus products. Patil et al. (2014) showed that maximum decrease of naringin (74%) was obtained at naringinase concentration of 100U/100mL with incubation at 40°C for 4hrs.
It is concluded from the study that the optimized concentration of naringinase enzyme is 0.6mL/100mL that is highly able to reduce the naringin content in citrus juices. The low alcoholic naturally carbonated beverage obtained was found to be fresh, safe, stable, more natural, minimally processed free from additives, contaminants, adulterants, and harmful pathogenic bacteria. It offers more nutrients, effervescence, longer shelf life and other physiological benefits. CO 2 act as a critical solvent which forms carbonic acid and hence responsible for enhancing shelf life of the beverage. The acceptability and higher sensory score of beverage is dependent upon its physiochemical characteristics and loss of some compounds that imparts flavor and aroma of the beverages during storage (Sahota et al. 2009). The presence of alcohol with <1% (v/v) is also responsible for enhancing the shelf life of the beverage.

CONCLUSION
On the basis of results it can be concluded that the strain Clavispora lusitaniae is capable of producing debittered kinnow beverage using the optimized process parameters. The beverage can be stored for a period of 3 months at refrigeration temperature with minimum changes of all physico-chemical characters.