WINE PREPARATION FROM BAEL JUICE EXTRACTED BY

ENZYMATIC TREATMENT

Dissertation Proposal
by
Karuna Kharel
B. Tech (Food), IV year

Department of Food Science and Technology

National College of Food Science and Technology
Institute of Science and Technology
Affiliated to Tribhuvan University, Nepal
2013

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 Contents

1. Introduction……………………………………………………………………. 1-5
1.1 Background…………………………………………………………………. 1-4
1.1.1 Fermentation………………………………………………………… 1
1.1.2 Bael (Aegle marmelos)………………………………………………. 2-4
1.2 Problem statement…………………………………………………………. 4
1.3 Justification………………………………………………………………… 4-5
1.4 Objectives of the work………………………………………………………. 5
1.4.1 General objective……………………………………………………… 5
1.4.2 Specific objectives………………………………………………………. 5

2. Methodology …………………………………………………………………… 6-11

2.1 Materials…………………………………………………………………….. 6-7
2.1.1 Raw materials…………………………………………………..…… 6
2.1.2 Equipments………………………………………………………… 6-7
2.1.3 Chemicals…………………………………………………………... 7
2.2 Methods………………………………………………………………..……. 8-11
2.2.1 Material collection…………………………………………………….. 8
2.2.2 Processing of Raw material…………………………………………… 8
2.2.3 Product Preparation……………………………………………………..8
2.2.4 Sensory Evaluation…………………………………………………… 8
2.2.5 Lab analysis……..…………………………………………………… 8-9
2.2.6 Data analysis….……………………………………………………. 9
2.2.7 Expected flowsheet……………………….…………………………. 10-11

3. Work Plan ………………………………………………………………… 12

4. Budget distribution………………………………………………………… 13
5. Expected outcome………………………………………………………….. 13
References……………….…………………………………………….... 14-15
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 Part 1
Introduction

1.1 Background
1.1.1 Fermentation
Fermentation is a term used for any process in which chemical changes are brought about by
the action of enzymes produced by living microbes in an organic substrate, whether
carbohydrate, fat, protein or other type of organic materials. Most alcoholic beverages are
manufactured by the metabolic activity of Saccharomyces cerevisiae or of closely related
yeasts through Embden Meyerhof Paranas (EMP) pathway. The yeasts ferment sugars in fruit
juices and grain mash, producing wine, beer and other beverages. The biochemical conversion
of grape juice occurs when it is inoculated by a yeast and incubated at 21-32⁰C for 3-5 days
under aerobic condition and followed by an anaerobic incuabation period, the yeast cells
enzymatically degrade the glucose and fructose in grape juice to acetaldehyde and then to
alcohol ( Aneja, 2009).

Wine is a product made by the alcoholic fermentation of grapes or grape juice by yeasts and
a subsequent ageing process. Wines, however, can be produced by the fermentation of juices
of fruits, berries, rhubarb, dandelions, honey etc (Frazier and Westhoff, 2009). It contains
vitamins, amino acids, esters, sugars, and tartaric acid and hundreds of chemical compounds
derived from grapes and yeast. In principle, any fruit having sufficient sugars would ferment to
make a wine. The common examples are cider (apple), perry (pears), cherry wine (cherry),
mead (honey), etc (Dhamija and Sangwan, 2006).

1.1.2 Bael ( Aegle marmelos)

Aegle marmelos (Rutaecae), commonly called as "Bael" is one of the indigenous wild fruits of
Nepal. It spreads throughout the country in the sub-tropical region covering the terai plain land
and the mid hills, as the non-timber forest products. Its religious and cultural values have been
very much recognized in Nepal in the traditional manner but its economic values have not been
exploited yet on a commercial basis ( Haq and Hughes, 2002).

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 Bael is a tropical seasonal fruit harvested during the months of May-June in many Asian
countries. The plant is 12-15 m tall and fruit is a globose, woody berry of 3-5" in diameter with
golden yellow rind (rich in β-carotene) when ripe. Due to the unorganized orchading of this
fruit tree in Asian countries, no country-wide data is available on area, production and
productivity of this fruit, albeit it is grown abundantly in Asian countries (Srivastava and
Singh, 2004).

The pulp is highly mucilaginous with 10-15 seeds and is rich in β-carotene. The fruit is
reported to contain important bioactive compounds such as carotenoids, phenolics, alkaloids,
pectins, tannins, coumarins, flavonoids and terpenoids (Maity et al., 2009). It is also reported
to contain a number of coumarins, alkaloids, sterols and essential oils such as palmitic, oleic
and linoleic acid (Sharma et al., 2007). The fruit has excellent aroma which is not destroyed
even during processing. Its' hard shell, mucilaginous texture and numerous seeds makes it
difficult to consume as a fresh fruit but in the excellent flavor, high nutritive and therapeutic
values of the Bael fruit lies an untapped potentiality for processing (Singh et al., 2012).

Bael fruits have medicinal uses like in the treatment of chronic diarrhea, dysentery, and
peptic ulcers, as a laxative while it also shows therapeutic effects viz. scavenging free radicals,
antioxidant, inhibition of lipid peroxidation, antibacterial, antiviral, anti-diarrheal,
gastroprotective, anti-ulcerative colitis, hepatoprotective, antidiabetic, cardioprotective and
radioprotective effects ( Baliga et al., 2011). Bael is considered to be one of the most nutritious
fruits as it is claimed to contain the highest riboflavin content among all the fruits and it is also
rich in vitamin C as the sweet orange (Haq and Hughes, 2002).

Bael fruits are of dietary use like the fruit pulp dressed with palm sugar is eaten as breakfast
(mostly in Indonesia), variety of products like bael sherbet, nectars, preserves, leather, squash,
juice, jam, jelly, marmalade, toffee are also present. Normally-harvested bael fruits can be
held for 2 weeks at 86º F (30º C) and 4 months at 48.2º F (9º C). Thereafter, mold is likely to
develop at the stem-end and any crack in the rind (Morton, 1987). So, fermentation of bael
juice into wine can aid in prolonging its shelf-life, increasing the nutritional content and act as
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a major tool for product diversification. However, there are very scarce literature on processing
of bael fruit pulp into fermented beverages such as wine. But there is ample scope to
bioprocess the fruits into value-added products such as wine to preserve the fruit nutrients,
aroma and taste to be available round the year (Panda et al., 2013).

Table 1.1 Nutritional value of Bael fruit (% or per 100g)

Components Value (%)

Water (moisture) 64.2
Protein 1.8
Fat 0.2
Mineral 1.5
Fibre 2.2
Carbohydrate 30.6
Calcium 0.09
Phosphorus 0.05
Potassium 0.6
Iron 0.3
Vitamin A (IU) 186
Vitamin B1 0.01
Nicotinic acid 0.9
Riboflavin 1.2
Vitamin C 0.01
Calorific value 129
(Source: Sharma et al., 2006)

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Table 1.2 Composition of Bael must and wine

Parameters Bael must Bael wine

TSS (⁰Brix) 20±0 2.9±0.0
Total Sugar(g/100ml) 12.5±0.004 2.03±0.12
Titrable acidity (g tartaric 0.08±0.03 0.15±0.07
acid/100ml)
pH 4.6±0.06 4.1±0.02
Phenol 1.02±0.41 0.93±0.03
Β-carotene (µg/100ml) 55±0.0 33±0.01
Ascorbic acid (mg/100ml) 86±0.0 80±0.01
Lactic acid (mg/100ml) 0.02±0.06 0.64±0.06
DPPH (%) 72.4±3 48±2.2
Ethanol (%) Not detected 7.87±0.31

(Source: Panda et al., 2013)

1.2 Problem Statement

Bael, despite being available countrywide, is still only significant in the traditional and
religious point of view as a sacred tree. But the nutritional aspect and therapeutic values it
carries as a food is scarcely exploited. Only a handful of cottage industries and medium scaled
industries are utilizing this valued product for preparation of products like jam, jellies and
juices in Nepal. As per Singh, et al., Its' hard shell and mucilagenous pulp also make it less
desirable as a fresh fruit among people. Large quantities of Bael rot away in the field itself
converting into compost because of the unapproached technologies, manpower and lack of
awareness of its' health benefits (Lama, 2011).

1.3 Justification

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According to Haq, 2002, Bael is one of the most nutritious fruits with medicinal values for
curing different diseases like cholera and diarrhea when used at the half ripe stage and is also
considered as a tonic for heart and brain. It is believed to show some antibacterial, anti-viral
activity against virus diseases and anti-helminthes. It also prevents leucoderma, acts as
antioxidant, antidiabetic and cardioprotective agent (Baliga et al., 2011).

Also, Bael is a wild fruit grown abundantly throughout the country but it is still one of the
underutlised crops of Nepal which, if would be converted to a value added product, could
generate employment and income at the farm and village levels with upliftment of economy
from domestic to national level at large. This fruit is unknown in internal and international
market thus development of such fruits into a reasonable product can attract both (Singh et al.,
2012).

Extraction of bael juice by enzymatic process will increase juice yield from 69.1% (when
untreated) to 72.5-86.6% (Singh et al., 2012). Apart from this fact it will also degrade the high
amount of pectin present in the pulp which could otherwise be a source for unwanted product
like methanol on the course of fermentation. So, commercialization of a value added product
from Bael (ex: as a medicinal wine) is a need of time.

1.4 Objectives of the work

1.4.1 General Objective

The general objective of this work is to prepare wine from enzymatically extracted bael juice
and evaluate its' quality.

1.4.2 Specific Objectives

1. To extract juice from the Bael pulp by pectinase treatment.
2. To evaluate physicochemical properties (Moisture, TSS, total sugar, titrable acidity, pH, β-
carotene, ascorbic acid, methanol, ester, aldehyde, ethanol, phenol, tannin and ash content)
of bael pulp.
3. To run the fermentation.
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4. To determine the biochemical changes during the fermentation.
5. To evaluate the physicochemical properties of the prepared bael wine.
6. To carry out the sensory evaluation of the products.

Part II
Methodology

2.1 Materials
2.1.1 Raw materials
1. Ripe Bael fruit
2. Pulverised sugar
3. Water
4. Pectinase
5. Yeast Nutrient
6. Wine yeast
7. KMS
8. Nitrogen Source

2.1.2 Equipments
1. Hammer
2. Spoon
3. Muslin Cloth
4. Fermenter Vessel
5. Siphoning tube
6. Glass bottles
7. Refractometer
8. pH meter
9. Burette
10. Pipette
11. Test tubes

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12. Thermometer
13. Pycnometer
14. Beaker
15. Volumetric flask
16. Test tube stand
17. Filter paper
18. Incubator
19. Funnel
20. Burette stand
21. Porcelain basin
22. Hot air oven
23. Electronic balance
24. Filtration arrangement
25. Conical flask
26. Bunsen burner
27. Silica crucible
28. Measuring cylinder
29. Muffle furnace

2.1.3 Chemicals
1. Potassium Ferricyanide
2. Zinc acetate
3. Glacial acetic acid
4. Fehling's solution
5. Methylene Blue Indicator
6. Standard Dextrose
7. Conc. H2SO4
8. Sodium Hydroxide
9. Phenolphthalein
10. 2,6-dichlorophenol indophenols dye
11. Metaphosphoric acid
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12. Standard Ascorbic acid
13. Sodium Carbonate

2.2 Methods
2.2.1 Material Collection
Raw material (Bael) will be collected from Alternative Herbal Products (P) Ltd, Balkhu
Kathmandu.

2.2.2 Processing of Raw materials

Bael fruit will be washed, split into two halves by hammer and pulp will be scooped out with a
spoon. Seeds and fibres will be separated out and then mixed with equal amount of water as
pulp i.e, in 1:1 ratio. In it KMS and pectinase will be added and left overnight. The juice will
then be filtered through muslin cloth. Amelioration will be done to required ⁰Bx and then juice
will be heated to about 80⁰C for 2 minutes.

2.2.3 Product Formulation

For wine preparation, two formulations will be used. Both formulation's TSS will be
maintained as 20⁰Bx but one will be with pure cane sugar while the other will be by a sugar
syrup of 20⁰Bx.

2.2.4 Product Preparation

Then, starter culture, nitrogen source and raw materials will be added in calculated amount and
it is left for fermentation for 7 days and racked subsequently at regular intervals of 20days for
3 times. The product is then clarified, bottled, corked and pasteurized.

2.2.5 Sensory evaluation

Fermented product will be evaluated by 9-point Hedonic rating test as described by
Ranganna,2009 by semi trained panelist (students and teachers) of National College of Food
Science and Technology, Khusibu.

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2.2.6 Lab analysis
2.2.6.1 Moisture
Moisture content will be determined by specific gravity method as per described by Kirk and
Sawyer, 1999.
2.2.6.2 TSS
Total Soluble Solids will be determined using refractometer.
2.2.6.3 Reducing Sugar
The reducing sugar will be determined as per Lane and Enyon method described in AOAC,
2005.

2.2.6.4 pH
pH will be determined using calibrated pH meter.
2.2.6.5 β-carotene
β-carotene will be determined as per the methods of AOAC, 2005.
2.2.6.6 Ascorbic acid
Vitamin C content will be determined by methods as per Ranganna, 2009.
2.2.6.7 Methanol
Methanol content of samples will be determined by methods as per Kirk and Sawyer, 1999.
2.2.6.8 Alcohol content
Alcoholic content of the samples will be measured by pycnometer as per the method described
by Kirk and Sawyer, 1999.
2.2.6.9 Ester
Ester content of samples will be determined as per methods described by Kirk and
Sawyer,1999.
2.2.6.10 Aldeyde content
Aldehyde content of samples will be determined as per method described by Kirk and Sawyer,
1999.

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2.2.6.11 Ash content
Ash content of samples will be determined by methods described by Kirk and Sawyer,1999.
2.2.6.12 Phenolic content
Total Phenolic content of samples will be determined as per the method described in Current
protocols in Food Analytical Chemistry, 2002.
2.2.6.13 Tannin content
Tannin content of samples will be determined by methods as per Ranganna, 2009.

2.2.7 Data analysis

The sensory data will be analyzed by Analysis of Variance (ANOVA) using genstat
programming.

2.2.8 Expected Flowsheet

Ripened Bael fruit

Weighing and Splitting into two halves by thrusting with hammer

Scooping out the pulp with spoon

Separating the seeds and fibres from the fruits

Mixing pulp with water in 1:1 ratio

Addition of KMS (100µg/ml)

Addition of pectinase (0.02%) and leaving it for overnight

Straining
Addition of bentonite

Filtration of bael juice by muslin cloth

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Amelioration of must (adjustment of TSS to about 20⁰Bx with i) sugar and ii) 20⁰Bx Sugar
syrup)

Heating of the juice for about 80⁰C for 2 minutes

Cooling to room temperature

Inoculation with 5-10% starter culture along with nitrogen source

Fermentation at ambient temperature for 7 days

Racking
(First racking is carried out at 2-3⁰Bx,
Racking is repeated for three times in 20days interval)

Clarification with 0.04% bentonite before final racking

Bottling and corking

Pasteurisation at 70⁰C for 5 minutes

Ageing/Maturation

Bael wine

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 Part III
Work Plan

The research is supposed to take place within a time limit of May, 2014-Oct, 2014

S.N. Activities Dec May Jun Jul Aug Sept Oct

1. Topic and
proposal

2. Literature
review work

3. Lab work

4. Data Processing

5. ReportWriting

6. Report
Submission

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 Part IV
Budget distribution

i. Raw materials
ii. Chemicals/Apparatus
iii. References and stationary
iv. Report writing and binding
v. Research station overhead
vi. Miscellaneous

Part V
Expected Outcome

A beverage rich in anti-oxidants with an alcoholic concentration of about 7-8% (v/v) is

expected as a result. An appreciable yield of juice and thereby a good quality wine is expected
as a result of enzymatic treatment during juice extraction than done by hot and cold methods.

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References

Aneja,K.R. (2009). Experiments in Microbiology Plant Pathology and Biotechnology. In:

"Food, Dairy, Industrial and Agricultural Microbiology" (4th Ed.). pp. 383-384. New age
International (P) Ltd., New Delhi.

AOAC. (2005). "Official Methods of Analysis" (18th ed.). Arlington, VA, USA. Association of
Official Analytical Chemists, Washington DC.

Baliga,M.S., Bhat, H.P., Joseph, N. and Fazal, F. (2011). Phytochemistry and medicinal uses
of the bael fruit (Aegle marmelos Correa): A concise review. Food Research
International 44, 1768-1775.

Dhamija, S.S. and Sangwan, S. (2006). Industrial Production of Alcohol in India, Food and
Industrial Microbiology.

Frazier,W.C. and Westhoff,D.C. (2009). Food Microbiology. In: "Food fermentations" (4th
Ed.). p. 339. Tata McGraw-Hill Publishing Company Limited, New Delhi.

Haq, N. and Hughes, A. (2002). Fruits for the future in Asia. In: "Status report of processing
and marketing of underutilized fruits in Nepal". p.174, ICUC, University of
Southampton.

Kirk.R.S. and Sawyer, R. (1999). "Pearson's Composition and Analysis of Foods" (9th ed.).
Longman publishing group, London, England.

Lama, T.N. (2011). Cool operator The Himalayan Times.com Retrieved from
http://www.thehimalayantimes.com/perspectives/fullnews.php?
headline=Cool+operator+&newsid=MjA0MA== [Accessed on 19th Dec, 2013]
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Maity, P., Hansda,D., Bandyopadhyay,U. and Mishra,D.K. (2009). Biological Activity of
crude extracts and chemical constituents of bael, Aegle marmelos (L.) Corr. Indian
Journal of experimental Biology, 47, 849-861. [Cited in Panda, S.K., Sahu, U.C.,
Behera S.K. and Ray, R.C. (2013). Bio-processing of bael [Aegle marmelos] fruits into
wine with antioxidants. Food Bioscience. 5, 34-41].

Morton, J. 1987. Bael Fruit. In: Fruits of warm climates. p. 187–190. Julia F. Morton, Miami,
FL.

Panda, S.K., Sahu, U.C., Behera S.K. and Ray, R.C. (2013). Bio-processing of bael [Aegle
marmelos] fruits into wine with antioxidants. Food Bioscience. 5, 34-41.

Ranganna. S. (2008). "Handbook of Analysis and quality control for Fruits and Vegetable
Products" (2nd ed.). Tata McGraw-Hill Publishing Co., New Delhi.

Sharma, P.C., Bhatia, V., Bansal, N. and Sharma, A.(2007). A review on bael tree. Natural
Product Radiance, 6(2), 171-178. [Cited in Panda, S.K., Sahu, U.C., Behera S.K. and
Ray, R.C. (2013). Bio-processing of bael [Aegle marmelos] fruits into wine with
antioxidants. Food Bioscience. 5, 34-41].

Singh, A., Kumar, S. and Sharma, H.K. (2012). Effect of enzymatic hydrolysis on the juice
yield from bael fruit (Aegle marmelos Correa) pulp. American. J. of Food Technol.
7(2), 62-72.

Srivastava, K.K. and Singh, H.K. (2004). Physico-chemical quality of bael (Aegle marmelos
Correa.). Agricultural Science Digest, 24(1), 65-66. [Cited in Panda, S.K., Sahu, U.C.,
Behera S.K. and Ray, R.C. (2013). Bio-processing of bael [Aegle marmelos] fruits into
wine with antioxidants. Food Bioscience. 5, 34-41].

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