Indian goose berry fortified, anti-oxidant rich bael (Aegle marmelos) fermented beverage

Abstract

Bael (Aegle marmelos Correa), an important fruit of Indian subtropics, traditionally utilized in the preparation of preserve, candy, squash, RTS, etc. has immense therapeutic potential. An attempt was made to develop a bael based low alcoholic sweet fermented beverage ( may be called as bael cider), anti-oxidant fortified with 0.25 per cent bael leaf or 10 per cent Indian goose berry (Emblica officinalis Gaertn.) juice in separate batches. Unfortified bael pulp based drink was kept as control. The ameliorated pulp was fermented at 30 ± 2ºC using Saccharomyces cerevisiae. The gooseberry blended bael fermented beverage had higher anti-oxidant content in the form of phenolics (323 mg/100 ml) than leaf extract added fermented beverage (265 mg/100 ml) and control (266 mg/100 ml). Sensory evaluation of product revealed that gooseberry blended fermented beverage scored higher (8.2/10) than bael leaf blended fermented beverage (7.9/10) and control (7.0/10). Twelve months maturation study of beverage revealed increase in reducing sugars and decrease in phenolic content in all the treatments. Bael fermented beverage with gooseberry blend retained highest phenolic content (257 mg/100 ml) and sensory score 7.8/10. The study inferred that an acceptable quality fermented drink could be prepared from bael-gooseberry blend, which could be stored for one year with higher antioxidant value and minimum deterioration in the quality.

Introduction

Bael (Aegle marmelos Correa)is an indigenous fruit of Indian sub-continent. The fruit has high medicinal value and is highly useful in stomach related diseases like constipation, diarrhea, dysentery, peptic ulcer, etc. Bael is a rich source of carbohydrates, protein, fibre, phenolics and minerals. The chemical constituents of fruit include 61 per cent moisture, 36 ^obrix TSS, 22 mg/100 g vitamin-C, 0.42 per cent tannins, 3.6 per cent crude protein and 4.8 per cent fibre (Kaur and Kalia 2017). The mucilaginous pulp of bael fruit contains two therapeutically important compounds, psoralen and marmelosin. Psoralen is protective against sunlight and useful in skin diseases especially leucoderma. Marmelosin is beneficial in stomach related diseases. It is a good laxative as well as diuretic. Bael fruit is generally used in making non-fermented products like sherbet, squash, preserve and candy. There are few reports of bael wine (Garg et al. 2014; Panda et al. 2014).

Polyphenols are main contributors to the antioxidant activity of most of foods and beverages (Higdon and Frei 2003). Bael leaf is characterized by its bitter astringent taste and high medicinal properties. Asghar et al. (2018) reported aqueous extract of bael leaf contains alkaloids, flavonoids and phenolics. The extract is a good cardio-tonic and insulin promoter. Owing to rich amount of tannins, it may have ability to increase astringency of the product, a preferred sensory parameter for alcoholic drink. Similarly, Indian gooseberry juice is very rich in anti-oxidant compounds like vitamin-C and phenolics. Bhattacherjee et al. (2011) reported gallic acid, caffeic acid, catechin, epi-catechin, chlorogenic acid, p-coumaric acid and kaempferol as the prominent phenolic compounds in gooseberry juice. A number of research reports suggest that wine in moderation keeps heart healthy and might help losing weight and boost immunity (Guilford and Pezzuto 2011;Vidavalur et al. 2006). Cider is an alcoholic beverage made from the fermented juice of apples. Ciders are not as dry and astringent in taste as wines. Soft cider has alcohol content ranging from 1–5%, while hard cider has 6–7 percent (Joshi et al. 2011). The sweet cider has residual sugar from fermentation or is sweetened after fermentation. Ciders that were perceived to be comparatively sweet were accepted to a higher degree. Apple fermented beverage is the only available low alcoholic fermented beverage in the market. Garg and Goel (2006) developed gooseberry fermented beverage. In the present study, an attempt has been made to develop anti-oxidant rich low alcoholic bael fermented beverage having taste and aroma of bael, enriched with bael leaf extract and gooseberry juice.

Materials and Methods

Bael fruits were harvested from Institute’s orchard, brought to the laboratory and ripened at room temperature (35 ± 5 °C).The pulp was scooped out manually and mixed with equal amount of slight acidified (0.2% citric acid) warm water, homogenized and filtered through sieve to remove fibre and seeds. The pulp was further diluted and ameliorated with sugar and citric acid to maintain total soluble solids (TSS) to 20 ºBrix and acidity to 0.5 per cent. The must was treated with 200 ppm potassium metabisulphite (KMS) to kill any native microbial population in the pulp. The leaf extract was obtained by grinding the leaves with equal amount of water and filtering through fine muslin cloth manually by gentle squeezing. The gooseberry juice was extracted by first crushing the fruits using fruit mill and then pressed with hydraulic press to get the juice. Two separate batches of ameliorated bael pulp containing 0.25 per cent bael leaf extract and 10 per cent gooseberry juice were prepared for fermentation under same conditions.

The yeast culture, Saccharomyces cerevisiae was obtained from culture collection of microbiology laboratory at CISH, Lucknow. The ameliorated pulp was inoculated with actively growing 24 h old culture (cells in log phase of growth) of Saccharomyces cerevisiae at 5% inoculum level having 10⁶ cells/ml. The inoculated must was fermented at 30 ± 2ºC under anaerobic condition till ethanol content reached to 4 per cent level. The fermented beverage was siphoned out and bottled in 200 ml capacity pre-sterilized glass bottles. The bottles were pasteurized at 55 ºC for 45 min and matured for one year at room temperature. Samples were withdrawn at 4 months intervals and analyzed for various parameter.

Biochemical analysis of stored bael fermented beverage was carried out at regular intervals of 4 months up to one year. Microbiological quality of the wine was carried out as per the method described by Speck (1985). TSS was recorded by using hand refractometer (Erma, Japan). Titratable acidity, vitamin-C, total phenolics, ascorbic acid, and reducing sugars were determined as per the methods described by Ranganna (2000). The concentration of ethanol in beverage was measured spectrophotometrically as per the method of Caputi et al.(1968). The phenolic, marmelosin & psoralen contents of bael fermented beverage were analyzed by applying modified protocol of Basha et al. (2004) using Shimadzu high performance liquid chromatograph with C-18 column and acetonitrile: water and methanol -water as mobile phases.

The sensory evaluation of fermented beverage was carried out by a panel of semi-skilled judges on hedonic scale on the basis of colour, clarity, aroma and taste (Amerine et al. 1965).

The experiment was laid in two factor CRD design using values in triplicate and data was subjected to statistical analysis applying Web Agri Stat Package (WASP 2.0) developed by Jangam and Wadekar (2010), ICAR-Central Coastal Agricultural Research Institute, Ela, Old Goa.

Results and discussion

Fermentation temperature is critical in the production of wine, because temperature above35 or below 5^OC affect the growth of yeast. Lower temperature of 12–16 °C is recommended for white wine, while 20–32 °C is recommended for red wine (Reynolds et al. 2001). Girard et al. (1997) reported that 30 °C fermentations produced more intense wine color and flavor than 20 °C fermentations and resulted into increased extraction of anthocyanins in wines. Hence, the fermentation was carried out at 30 ± 2ºC.

The final ethanol level in each treatment of prepared fermented beverage was estimated to be around 4 per cent at zero time, which remained the same throughout the maturation and aging period. Generally, the pasteurization temperature is 60–65 °C. However, the bottles were pasteurized at lower temperature of 55 °C for longer period (45 min) to preserve the bael aroma.

The microbial analysis indicated that there was no microbial growth in any of the samples at any stage of storage. Compared to apple cider where the starting TSS is kept up to 14^oB, comparatively higher TSS (20^oB) was kept in bael must. Since bael, in addition to fermentable sugar, is also a rich source of non fermentable polysaccharides, vitamins, antioxidants, organic acids, proteins and minerals (Kaur and Kalia, 2017), theoretically visible reduction in TSS was not observed. In case of apple cider, 6–7 percent ethanol is obtained from 14–15 percent sugars and the final TSS level is less than 1^oB. However, in bael fermented drink after the completion of fermentation, a TSS of 16.8–16.9^oB was observed which reached to a marginal increased level of 17.2^oB in all the samples after 12 months of storage (Table 1). The increase in TSS might be due to breakdown of complex polysaccharides in to simpler water soluble mono or polymers (Yahia et al. 2019).

Table 1.

Changes in total soluble solids (TSS) and titratable acidity of bael cider during storage

Treatment	TSS (0 Brix)				Acidity (%)				Reducing sugars (%)
	0 day	4 month	8 month	12 month	0 day	4 month	8 month	12 month	0 day	4 month	8 month	12 month
Control	16.9	16.7	16.8	17.2	0.68	0.67	0.66	0.66	1.45	1.56	1.75	1.92
0.25% bael leaf extract	16.8	16.7	16.8	17.2	0.68	0.67	0.67	0.66	1.58	1.59	1.75	1.89
10% aonla Juice	16.8	16.8	16.8	17.2	0.70	0.70	0.70	0.68	3.77	3.86	4.31	4.69
C.D at 5%	Treatment (T): 0.071 Period (P): 0.082 T X P: 0.141				Treatment (T): 0.010 Period (P): 0.012 T X P: 0.020				Treatment (T): 0.057 Period (P): 0.066 T X P: 0.115

The titratable acidity of bael fermented beverages at zero day were 0.68–0.7 per cent. It decreased slightly during storage to final values of 0.66–0.68 per cent at the end of storage (Table 1). The storage data on TSS and acidity indicated stability of the product in terms of sugar-acid blend.

The product was found to have 3.0 mg/100 ml vitamin-C in control (Fig. 1). Leaf extract addition did not significantly enhanced vitamin-C content of the product (3.1 mg/100 ml). Addition of gooseberry fermented beverage resulted into more than 14 per cent increase in vitamin-C of bael fermented beverage (3.5 mg/100 ml). After 12 months of storage, vitamin-C content decreased to only 1.0 mg/100 ml in gooseberry juice added sample and further below in other two treatments. Loss in vitamin-C content may be attributed to gradual oxidation of ascorbic acid during storage (Sapei and Hwa, 2014).

Fig.1.

Changes in total phenolics and vitamin-C contents of bael cider during storage Vit- C: CD at 5%: Treatment (T): 0.074; Period (P): 0.085; T X P: 0.148 Total phenolics: CD at 5%: Treatment (T): 0.074; Period (P): 0.085; T X P: 0.148

The total phenolics content of pure bael fermented beverage was 266 mg/100 ml, which was at par to leaf extract sample with 265 mg/100 ml (Fig. 1). Addition of gooseberry hiked the phenolics level to 323 mg/100 ml. The main phenolics identified in pure bael fermented beverage through HPLC were catechin (166 mg/100 ml), followed by kaempferol (102 mg/100 ml), gallic acid (27 mg/100 ml) and caffeic acid (14 mg/100 ml) while in aonla juice fortified treatment these were 180, 112, 42 and 15 mg/100 ml, respectively. Bael leaf supplemented treatment did not show much variation from control. The marmelosin and psoralen contents were found to be 23, 20 and 18 µg/100 ml and 38, 34 & 33 µg/100 ml, in pure bael, bael leaf extract and aonla juice fortified treatment, respectively. Gradual but regular decline in total phenolics was observed in all samples. However, after twelve months of storage, gooseberry enriched sample still had maximum phenolic content (257 mg/100 ml), followed by leaf extract sample (209 mg/100 ml) and least in control (203 mg/100 ml). It might be due to the fact that contents of most of the phenolics of fermented drinks diminished with the time (Kallithraka and Salacha, 2009).

Reducing sugar contents of control, leaf extract sample and gooseberry enriched samples were found to be 1.45, 1.58 and 3.77 per cent, respectively at zero day. Reducing sugars increased gradually with the time, reaching to 1.92, 1.89 and 4.69 per cent in control, leaf extract sample and gooseberry enriched samples, respectively (Table 1). The increase in reducing sugars content may be attributed to break-down of sucrose into glucose and fructose units with the storage period (Kalra and Tandon 1985).

Sensory evaluation of bael fermented beverage on the basis of parameters like colour, clarity, aroma and taste, revealed that addition of gooseberry juice enhanced the organoleptic qualities of the product. It obtained 8.2 score out of 10 at zero day, followed by 7.9 of leaf extract sample and 7.0 of control (Fig. 2). The scores reached peaks at 4 months which might be due to increased clarity of cider as a result of settling of suspended viscous bael particles during aging. After that slight decrease in score was observed this might be due to reduction in vitamin C and phenolic content (Techakanon and Sirimuangmoon, 2020) or difference in scoring by sensory experts, over a period of time, while agreeing on a wine’s overall quality (Grohmann et al. 2018). After 12 months, gooseberry enriched sample was still most preferred (7.8), followed by leaf extract sample (7.4) and then control (7.0).

Fig. 2.

Changes in sensory scores of bael cider during storage

Conclusion

The study revealed that a good quality of fermented beverage could be prepared from bael pulp. The product possesses a good blend of pleasant aroma and taste of bael fruit along with refreshing astringency of alcohol. Fortification with gooseberry juice resulted in better quality bael fermented beverage in terms of nutritional properties and sensory acceptability.

Authors' contributions

NG conceived the presented idea. SK and PY carried out the experiments. All authors discussed the results and contributed to the final manuscript.

Funding

The author(s) received no financial support for the reported research work.Consent to participate and publication: Authorship of the paper is accurately represented. All authors participated in the actual authorship of the work and have given consent for publication.

Data availability

The research work is original and has not been previously published and has been submitted only to the journal. The data is real and available with authors.

Declarations

Conflicts of interest

The authors declare no real or apparent conflicting or competing interest.

Ethics approval

This article does not contain any studies with animal subjects. The sensory evaluation of fermented beverage was carried out by a panel of semi-skilled judges who were fully informed about the study. Participants expressing an interest were further made to undergo judging of the fermented drink prepared as per standard protocol.