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beverages
Article
Shelf-Life Extension of Wood Apple Beverages
Maintaining Consumption-Safe Parameters and
Sensory Qualities
Md. Shakir Moazzem, Md. Belal Hossain Sikder * and Wahidu Zzaman
Department of Food Engineering and Tea Technology, Shahjalal University of Science and Technology,
Sylhet 3114, Bangladesh; shakir.sust@gmail.com (M.S.M.); wahidanft@yahoo.com (W.Z.)
*Correspondence: belalustc@yahoo.com; Tel.: +880-1911-212427
Received: 29 December 2018; Accepted: 1 March 2019; Published: 12 March 2019
Abstract:
An investigation was carried out to extend the shelf life of wood apple beverages by up to
50 days from its natural shelf life of 8–12 h. A wood apple beverage was prepared using freeze-dried
wood apple powder. Four samples were developed by pasteurizing the beverage at 85
◦
C for 10 min
and treatment with a combination of 50 ppm and 100 ppm of potassium metabisulphite, citric
acid, ascorbic acid, and sodium benzoate. Replications and controls were properly maintained.
The total soluble solids (
◦
Brix), pH, titrable acidity, ascorbic acid content, microbial growth and
sensory attributes of the prepared juice samples were evaluated at an interval of 10 days over a
storage period of 50 days. TSS was found to increase (16.30–18.25
◦
Brix) with storage period, while pH
(5.43–4.10), titratable acidity (0.67–0.08%), and ascorbic acid content (4.65–1.01 mg/100 mL) decreased
with time. The microbiological analysis showed little or no growth for samples treated with a
combination of 50 ppm potassium metabisulphite, citric acid, ascorbic acid and sodium benzoate up
to 50 days. Consumer acceptability of the beverage was found to be satisfactory. Thus, shelf life of
wood apple beverage was extended to 50 days satisfactorily, ensuring consumption-safe parameters
and satisfactory sensory qualities.
Keywords:
shelf-life extension; sensory qualities; pasteurization; product development;
consumption-safe parameters
1. Introduction
The wood apple (Aegle marmelos L. Correa) is an important indigenous fruit of the Indian
subcontinent, belonging to the family Rutaceae. It is native of the Indo-Malayan region and has
been known in India since prehistoric times [
1
]. It grows in the dry forests, hills, and plains of India,
Myanmar Bangladesh, and Pakistan [
2
]. The importance of wood apple lies in its medicinal and
curative properties [
3
]. It is one of the most useful medicinal fruits of the Indian subcontinent [
4
].
The wood apple is considered to be a natural source of anti-oxidants due to its potential radical
scavenging activity of various phytochemicals [
5
]. It also has hypoglycemic, antitumor, larvicidal,
antimicrobial, and hepatoprotective activity [
6
]. It has anti-diabetic and antioxidant potential in
terms of reducing levels of blood glucose and malondialdehyde [
7
]. Compounds purified from wood
apples have been proven to be biologically active against several major diseases including cancer and
diabetes. [
8
]. In Ayurveda, it is used to cure problems related to the heart, stomach, and intestine,
chronic constipation, dysentery, some forms of indigestion, typhoid, debility, fever, hemorrhoids,
hypochondria, melancholia, and heart palpitations [9].
The wood apple is one of the most nutritious fruits of the Indian subcontinent [
1
]. It contains
many vitamins, such as vitamin A, vitamin C, thiamine, riboflavin, and niacin, and minerals, such as
Beverages 2019,5, 25; doi:10.3390/beverages5010025 www.mdpi.com/journal/beverages
Beverages 2019,5, 25 2 of 14
calcium and phosphorus [
4
]. The edible pulp of 100 g of wood apple fruit contains 61.0 g of moisture,
1.6 g of protein, 0.2 g of fat, 1.9 g of minerals, 80.0 mg of calcium, 52.0 mg of phosphorus, 0.5 mg of
iron, 55
µ
g of carotene, 0.12 mg of thiamine, 1.19 mg of riboflavin, 1.0 mg of niacin, 8 mg of vitamin C,
610 mg of potassium, and 0.20 mg of copper [
10
]. Wood apple is very rich in vitamins, amino acids,
and minerals in comparison with other fruits [
11
] and can contribute significantly to the daily nutrient
needs of the individual. In addition, it can be used advantageously to supplement deficiencies of other
foods [
12
]. The ripe fruit of the wood apple is sweet, aromatic, nutritious, and very palatable and is
highly esteemed and eaten by all classes of people in India [
11
]. However, people prefer adding it to
other products to consuming it on its own [
2
]. People of the Indian subcontinent usually consume
wood apple as a beverage after blending it with other ingredients [13].
Fruit-based drinks are far superior to many synthetic preparations and are being replaced by
fruit beverages. Fruit beverages are easily digestible, highly refreshing, thirst quenching, appetizing,
and nutritionally far superior to many synthetic and aerated drinks [
14
]. They can help one to meet
the daily requirement of fruits and vegetables in one’s diet [
15
]. There are many pathways for the
deterioration of fruit beverages. However, many effective preservation methods can prevent spoilage.
The preservation of fruit beverages can be achieved through processing and the use of preservatives.
Fruit beverages tend to deteriorate and spoil easily if they do not undergo heat treatment and treatment
with preservatives. The most common method of inactivating microorganisms and enzymes for
increasing the shelf life of fruit juices is by thermal processing such as pasteurization. Chemical
preservatives are also used to prolong the shelf-life of fruit juice and beverages and inhibit microbial
growth [
16
]. The inhibitory action of preservatives is due to their interfering with the mechanism of
cell division, the permeability of cell membranes, and the activity of enzymes. Chemical preservatives
such as potassium metabisulphite, sodium benzoate, citric acid, ascorbic acid, and their mixtures
have been used widely in fruit beverages and are generally recognized as safe (GRAS) [
17
]. However,
excessive consumption of these preservatives may be hazardous to human health [
18
]. In order to
ensure the safety of fruit beverages for human consumption, chemical preservatives have to be used
within acceptable limits. The improper use of preservatives exceeding the acceptable limit can be
a potential risk for human health. In the last decades, food safety has become very important for
governments, producers of food products, as well as consumers [
19
]. According to the 2018 Food
Safety (Food Hygiene) Regulations of the Bangladesh Food Safety Authority (BFSA), the maximum
limits of potassium metabisulphite and sodium benzoate in the industrial production of fruit beverages
are 1000 and 600 ppm, respectively, whereas no limit has been set for using citric acid and ascorbic acid
in fruit beverages because of their abundance in citrus fruits [
20
]. The 2011 Food Safety and Standards
(Food Product Standards and Food Additives) Regulation of the Food Safety and Standards Authority
of India (FSSA) specifies that the maximum limits of potassium metabisulphite and sodium benzoate
for the industrial production of fruit juices and beverages are 700 and 600 ppm, respectively [
21
].
It should be noted that potassium metabisulphite, sodium benzoate, citric acid, and ascorbic acid are
considered GRAS by the FAO [22] and the FDA [23].
The influence of chemical preservatives on the quality attributes of orange juice was investigated
by Stephen et al. It was possible to store orange juice effectively for three weeks using 0.03%
sodium benzoate, sodium metabisulphite, potassium sorbate, or its combinations of preservatives [
24
].
The effect of chemical preservatives on strawberry juice was investigated by Ayub et al. Pasteurization
and treatment with 20% sucrose, 0.1% sodium benzoate, and 0.1% potassium sorbate prolonged the
shelf-life of strawberry juice by up to three months [
25
]. The clarification, preservation, and shelf life
evaluation of cashew apple juice were investigated by Talasila et al. It was possible to prolong its
shelf life by up to 90 days by treatment with 0.1 g/L citric acid and benzoic acid [
26
]. Various factors
impacting wood apple beverage production were investigated by Minh [
27
]. The comparative effect of
crude and commercial enzymes on the juice recovery from wood apple (Aegle marmelos Correa) using
principal component analysis was investigated by Singh et al. The effects of incubation time, incubation
temperature, and crude enzyme concentration on the yield, viscosity, and clarity of the juice obtained
Beverages 2019,5, 25 3 of 14
from wood apple fruit pulp were observed. It was concluded that wood apple juice yield, viscosity, and
clarity are functions of enzymatic hydrolysis conditions [
28
]. The effect of thermal processing on the
preparation of ready-to-serve wood apple beverage blended with aonla was studied by Rathod et al.
It was observed that pasteurization by thermal processing (90
◦
C for 25 s) was effective for inactivating
microbial flora [
14
]. The effects of the processing quality and storage stability of functional beverages
prepared from aloe vera blended with wood apple fruit were studied by Sasikumar. It was concluded
that a formulation of functional beverages can satisfy consumers’ acceptance [
2
]. It was also indicated
by Tiwari and Deen that wood apple and aloe vera can be utilized for a valuable RTS (ready-to-serve)
beverage and is acceptable to consumers in terms of taste, color, flavor, and medicinal properties [
29
].
The processing and storage stability of wood apple squash for nutritional security was investigated by
Mandal et al. It was concluded that it may play an important role in food and nutritional security [
3
].
The wild wood apple fruit was studied by Kenghe et al. in terms of value addition. Squash was
prepared from this fruit by adjusting the TSS (total soluble solids) of the pulp [
30
]. The preparation
and shelf-life of mixed juice based on wood apple and papaya were investigated by Chowdhury
et al. It was concluded that potassium metabisulphate (KMS) was effective for the prevention of
spoilage against microbial growth in bottled mixed juices [
31
]. Jam and fruit bars with wood apple
were developed by Vidhya and Narain and were safe and fit for consumption for up to 90 days [
32
].
The effect of different pre-treatments on the physicochemical properties, organoleptic quality, and
shelf life of wood apple candy was investigated by Kumar et al. for up to 4 months [
1
]. Candy, jam,
murabba, and chutney using wood apple by incorporation of various herbs (ginger, cardamom, and
rose extract in wood apple) were developed by Srivastava et al., and the acceptability and nutritive
value of the products were assessed [33].
Currently, processed products based on wood apple are becoming popular because of its rich
nutrient profile [
27
]. Wood apple has an excellent aroma that is not removed during processing.
Therefore, there is untapped potential for processing wood apple into various products [
34
]. It also
has great potential for commercial processing and can be processed into various products such as
beverages, preserves, candy, squash, toffee, slabs, pulp powder, and nectar [35].
It is evident that wood apple beverages are highly nutritious, with genuine medicinal value, and
they hold great potential in the developing market of beverages to become a popular, high-quality
beverage of commercial interest. Some attempts have been made very recently to develop value-added
products such as jam, fruit bars, squash, murabba, chutney, and candy. However, no serious effort has
been made to extend the shelf-life of wood apple beverages to the best of our knowledge. Therefore,
it would be of great significance to extend the shelf-life of such highly nutritious beverages to make
them available for longer periods of time. The present investigation was carried out to extend the
shelf-life of a wood apple beverage by up to 50 days from its natural shelf-life of 8–12 h while
maintaining physicochemical, microbial, and sensory attributes. It was also the aim of this study to
establish a set of formulae for the commercial production of shelf-stable wood apple beverages on an
industrial scale.
2. Materials and Methods
2.1. Materials and Chemical Reagents
Healthy, fresh, and fully ripe wood apple fruits, with a rich sweet aroma and without any visual
defects, were purchased from the Swapno super shop of Subidbazar, Sylhet, Bangladesh. All the
chemicals used in this experiment were purchased from Hi Media
®
, India, and were of analytical
grade. The Laboratory of Dept. of Food Engineering and Tea Technology provided this investigation
with potassium metabisulphite, sodium benzoate, ascorbic acid, citric acid, plate counting agar for
total viable bacterial count, potato dextrose agar for total fungal count, McConkey agar for coliform
counts, refined sugar, distilled water, and other chemicals and reagents. Experiments mentioned in
Beverages 2019,5, 25 4 of 14
this study were conducted between June and August of 2018 in Sylhet, Bangladesh. Room temperature
is usually around 28–32 ◦C.
2.2. Preparation of Freeze-Dried Wood Apple Powder from Raw Wood Apple Pulp
Wood apple fruits were taken to the laboratory of Dept. of Food Engineering and Tea Technology
and washed in tap water to wash out adhering dirt and dust particles. Fruits were cut in slices by a
wood cutter. Bark and seeds were removed, and the pulp was scooped out with the help of a stainless
steel spoon. The extracted pulp was then kneaded and heated at 75
◦
C for 2 min. A No. 20 mesh
stainless steel sieve (0.841 mm/841 microns) was used to sieve the pulp and remove the seeds and
fibers. A TelStar LyoQuest freeze drier (Telstar Technologies, Barcelona, Spain) was used to make
freeze-dried wood apple powder from the pulp.
2.3. Preparation of Beverage
A total of 2 kg of freeze-dried powder was obtained from 6 kg of ripe wood apple fruits. A total
of 1.5 L of wood apple beverage was produced in 3 batches by mixing a total of 150 g of freeze-dried
wood apple powder and a total of 150 g of sugar with a total of 1.5 L of distilled water. In each batch,
500 mL of wood apple beverage was made by mixing 50 g of freeze-dried wood apple powder and
50 g of sugar with 500 mL of distilled water. Pasteurization of the prepared wood apple beverage
was done by heating at 85
◦
C for 10 min. It was then cooled down at a room temperature of 32
◦
C
for 15 min. The main beverage sample was then taken into 12 different glass bottles and treated with
a combination of 50 ppm and 100 ppm of potassium metabisulfite (KMS), citric acid (CA), ascorbic
acid (AA), and sodium benzoate (SB). The glass bottles were then corked tightly and wrapped with
aluminum foil in an aseptic condition. Flow chart showing procedure for preparing wood apple
beverage samples is shown in Figure 1.
2.4. Labeling of Samples
The labelling of samples is given in Table 1. Control samples, i.e., samples not treated with
any preservatives, were labeled as Sample A. The samples treated with 100 ppm KMS (potassium
metabisulphite) + 100 ppm CA (citric acid) were labeled as Sample B. The samples treated with 100 ppm
of AA (ascorbic acid) + 100 ppm SB (sodium benzoate) were labeled as Sample C. The samples treated
with 50 ppm KMS (potassium metabisulphite) + 50 ppm CA (citric acid) + 50 ppm of AA (ascorbic
acid) + 50 ppm SB (sodium benzoate) were labeled as Sample D. The beverage samples treated with
these preservatives were stored in a room temperature of 28–32 ◦C for further evaluation.
Table 1. Labeling of samples showing doseage of heat treatment and concentrations of preservatives.
Sample Labels Heat Treatment,
i.e., Pasteurization
Potassium
Metabisulphite
(KMS)
Sodium
Benzoate
(SB)
Citric Acid
(CA)
Ascorbic Acid
(AA)
Sample A
(Control) 85 ◦C for 10 min - - - -
Sample B 85 ◦C for 10 min 100 ppm - 100 ppm -
Sample C 85 ◦C for 10 min - 100 ppm - 100 ppm
Sample D 85 ◦C for 10 min 50 ppm 50 ppm 50 ppm 50 ppm
- designated preservative has not been used in any form or concentration.
Beverages 2019,5, 25 5 of 14
Beverages 2018, 4, x FOR PEER REVIEW 5 of 14
Figure 1. Flow chart showing procedure for preparing wood apple beverage samples.
2.5. Determination of Physicochemical Properties
Figure 1. Flow chart showing procedure for preparing wood apple beverage samples.
Beverages 2019,5, 25 6 of 14
2.5. Determination of Physicochemical Properties
TSS (
◦
Brix) was determined by the method of AOAC (2012) by using an Abbe Hand Refractometer
(Erma, Tokyo, Japan) [
36
]. A sample replaceable prism was inserted in the refractometer. It was then
held against a light source. The reading was regarded as the total soluble solids of the samples in
◦
Brix. pH was determined by the method of AOAC (2012) [
36
]. pH values of the beverage samples
were measured at room temperature with a digital glass electrode pH meter (Model 744, Metrohm,
Herisau, Switzerland), which was calibrated prior to sample pH measurement using standard buffer
solutions of pH 4 and 7. About 10 mL of sample was measured into a beaker, and the pH meter was
dipped into the beaker to measure the pH of the sample. The pH values were then recorded. Titratable
acidity (as citric acid) was estimated by the method described by Ranganna [
37
]. Titratable acidity
was determined by dissolving a known weight of sample in distilled water and titration against 0.1 N
NaOH using phenopthalein as an indicator. Ascorbic acid content (as vitamin C) was estimated by a
method described by Ranganna [
37
]. Ascorbic acid content was determined by a direct colorimetric
method using 2,6-dichlorophenol indophenol as a decolorizing agent by ascorbic acid in the sample
extract and in a standard ascorbic acid solution.
2.6. Microbial Examination
Samples were analyzed for total bacterial count, total fungal count, and coliform count according
to the procedure described by APHA [
38
]. The serial dilution and plating method was followed
to enumerate the microbial load of wood apple beverage samples. Enumeration of total bacterial
count, total fungal count, and coliform count was done by using plate counting agar, potato dextrose
agar, and McConkey agar, respectively. One milliliter of juice from each sample was taken into a test
tube containing 9 mL of sterile water and homogenized by shaking to make the 10
−1
dilution. Serial
dilutions of 10
−2
, 10
−3
, 10
−4
, 10
−5
, and 10
−6
were then prepared from it. The plate counting agar plates
(with 10
−6
dilutions) and potato dextrose agar plates (with 10
−4
) were incubated at 32
◦
C for 48 h for
total bacterial count and total fungal count, respectively. McConkey agar plates (with 10
−2
dilutions)
were incubated at 37
◦
C for 24 h for coliform count. The colony enumeration was done using a digital
colony counter, and values were expressed as colony forming units/mL (cfu/mL) of the sample.
The following formula was used to calculate the number of colony forming units per mL (cfu/mL)
after the incubation time:
cfu =
Number of colony
volume of sample added ×dilution factor .
2.7. Sensory Evaluation
Data on sensory evaluation of this study was obtained according to the recommendations made by
the Society of Sensory Professionals [
39
]. The consumers’ acceptance of the juice was evaluated by 150
consumers (naïve panelists). The sensory panel comprised of randomly selected people from Sylhet,
Bangladesh with ages ranging from 8 to 67 years. Samples were presented in 200 mL glass bottles, and
the panelists were asked to evaluate the samples for appearance, aroma, taste, and overall acceptability
using a nine-point hedonic scale, varying from “dislike extremely” (Score 1) to “like extremely” (Score
9). Sensory booths were set up in the laboratory of Food Engineering and Tea Technology with proper
ventilation, a neutral background, proper lighting, good internet connectivity, minimal traffic, and no
distractions, noise, or odors. Samples were prescreened to ensure that they represent the important
parameters being tested and did not contribute to unnecessary bias. The serving bottles were masked
with aluminum foil with no suggestive information so that respondents could not identify samples
unintentionally. Brand new palate cleaners and glass bottles were used. Bias was minimized by
serving the samples to the panelists in masked 200 mL glass bottles at a room temperature of 28–32
◦
C.
All samples were held for at least 3 min for proper sensation so that the panelists could distinguish
Beverages 2019,5, 25 7 of 14
between nuanced differences. Five minutes of break time was maintained after each evaluation to
eliminate fatigue and exhaustion.
2.8. Interval at Evaluation Samples
Physicochemical properties (TSS, pH, titratable acidity, and ascorbic acid content), microbial
growth (total bacterial count, total fungal count, and coliform count), and sensory attributes
(color, flavor, taste, and overall acceptability) were evaluated at an interval of 10 days over a period of
50 days.
2.9. Statistical Analysis
Data were analyzed using SPSS software (SPSS Inc., Chicago, IL, USA), version 25 for Windows.
Results of TSS (
◦
Brix), pH, titratable acidity (%) and ascorbic acid content (mg/100 mL) were reported
as mean
±
standard deviation (SD) for three (3) replicates, n= 3. Results of sensory evaluation for
appearance, aroma, taste, and overall acceptability were reported as mean
±
standard deviation
(SD) of 9-point hedonic scale ratings given by 150 consumers, n= 150. One-way analysis of variance
(ANOVA) followed by Duncan’s multiple range test (DMRT) (multiple comparison post-hoc test)
was used to analyze the statistical difference. Differences with p-values < 0.05 were considered
statistically significant.
3. Results and Discussion
3.1. Changes in Physicochemical Properties
TSS (
◦
Brix) of the samples gradually increased from the first day (16.30
◦
Brix to 16.50
◦
Brix)
to the end of storage (16.78
◦
Brix to 18.25
◦
) throughout the storage period (Table A1). Sample D
showed better retention of TSS in comparison with other samples. The changes in TSS (
◦
Brix) of
the beverages are shown in Figure 2. The increase in TSS during storage may be attributed to a
conversion of polysaccharides into simple sugars and a degradation of pectic substances in soluble
solids [
40
]. This result was also found in reports of Singh et al. [
40
], Minh [
27
], Tiwari and Deen [
29
],
Mandal et al. [3]
, Rathod et al. [
14
], and Chowdhury et al. [
31
]. Similar observations were also reported
by Kumar et al. [1] for wood apple candy.
pH of the samples gradually decreased from the first day (5.43–4.88) to the end of storage
(4.97–1.30) throughout the storage period (Table A1). The higher retention of pH by Sample D in
comparison with other samples is depicted in Figure 3. A decline in pH during storage was observed,
which may be due to the action of citric and ascorbic acid on the sugar and protein component of the
product [
10
]. This result is in accordance with those of Rathod et al. [
14
] and of Minh [
27
]. Such a
decrease in pH over the storage period was also reported by Yang et al. [
18
] for orange juice and by
Krishnakumar et al. [
41
] and Khare et al. [
42
] for sugarcane juice. However, a better retention of pH
could be obtained in this study, which indicates the substance is safe for human consumption.
Titratable acidity (%) of the samples gradually decreased from the first day (0.69–0.43%) to the end
of storage (0.43–0.08%) throughout the storage period (Table A1). The decrease in acidity was rapid in
Sample A, which was not treated with any preservatives. A decrease in acidity during storage might
be due to chemical interactions between organic constituents and enzymatic reactions [
40
]. It might
also be due to the conversion of acids into salt sand sugars by enzymes, particularly, Invertase [
9
]. This
result is in agreement with those of Chowdhury et al. [
31
], Singh et al. [
40
], and Verma and Gehlot [
43
].
Similar observations were reported by Dhaka et al. for kinnow juice [
44
] and Paul and Ghosh for
pomegranate juice [
45
]. The higher retention of titratable acidity (%) by Sample D in comparison with
other samples is shown in Figure 4.
Ascorbic acid content of wood apple beverage samples gradually decreased from the first day
(4.65–4.43 mg/100 mL) to the end of storage (3.01–1.01 mg/100 mL) throughout the storage period
(Table A1). This reduction may be due to the oxidation of ascorbic acid in to dehydrate ascorbic acid by
Beverages 2019,5, 25 8 of 14
oxygen [
3
]. Ascorbic acid is sensitive to oxygen, light, and heat and is easily oxidized in the presence
of oxygen by both enzymatic and non-enzymatic catalysts [
14
]. This finding was also found in the
reports of Tiwari and Deen [
29
], Rathod et al. [
14
], Mandal et al. [
3
], and Chowdhury et al. [
31
]. Similar
observations were reported by Bhardwaj and Mukherjee [
46
] on kinnow juice blends, Talasila et al. [
26
]
on cashew apple juice, and Khare et al. [
42
] on sugarcane juice. The higher retention of ascorbic acid
content (mg/100 mL) by Sample D in comparison with other samples is depicted in Figure 5.
Beverages 2018, 4, x FOR PEER REVIEW 8 of 14
ascorbic acid content (mg/100 mL) by Sample D in comparison with other samples is depicted in
Figure 5.
Figure 2. TSS (°Brix) evaluation of samples.
Figure 3. pH evaluation of samples.
Figure 2. TSS (◦Brix) evaluation of samples.
Beverages 2018, 4, x FOR PEER REVIEW 8 of 14
ascorbic acid content (mg/100 mL) by Sample D in comparison with other samples is depicted in
Figure 5.
Figure 2. TSS (°Brix) evaluation of samples.
Figure 3. pH evaluation of samples.
Figure 3. pH evaluation of samples.
Beverages 2019,5, 25 9 of 14
Beverages 2018, 4, x FOR PEER REVIEW 9 of 14
Figure 4. Acidity (%) evaluation of samples.
Figure 5. Ascorbic acid evaluation of samples.
3.2. Changes in Microbial Load
The microbial evaluation of wood apple beverage is given in Table 2. The bacterial load of
Sample A (control samples) increased considerably from the day of preparation (1.09 × 107 cfu/mL at
Day 0) to the end of storage (2.43 × 107 cfu/mL at Day 50). This might be due to the fact that Sample
A was not treated with any preservatives. As for treated juice samples, bacterial load was observed
to be less than 10 cfu/mL at the day of preparation (Day 0). Heat and the action of preservatives
Figure 4. Acidity (%) evaluation of samples.
Beverages 2018, 4, x FOR PEER REVIEW 9 of 14
Figure 4. Acidity (%) evaluation of samples.
Figure 5. Ascorbic acid evaluation of samples.
3.2. Changes in Microbial Load
The microbial evaluation of wood apple beverage is given in Table 2. The bacterial load of
Sample A (control samples) increased considerably from the day of preparation (1.09 × 107 cfu/mL at
Day 0) to the end of storage (2.43 × 107 cfu/mL at Day 50). This might be due to the fact that Sample
A was not treated with any preservatives. As for treated juice samples, bacterial load was observed
to be less than 10 cfu/mL at the day of preparation (Day 0). Heat and the action of preservatives
Figure 5. Ascorbic acid evaluation of samples.
3.2. Changes in Microbial Load
The microbial evaluation of wood apple beverage is given in Table 2. The bacterial load of Sample
A (control samples) increased considerably from the day of preparation (1.09
×
10
7
cfu/mL at Day 0)
to the end of storage (2.43
×
10
7
cfu/mL at Day 50). This might be due to the fact that Sample A was
not treated with any preservatives. As for treated juice samples, bacterial load was observed to be less
than 10 cfu/mL at the day of preparation (Day 0). Heat and the action of preservatives destroyed most
Beverages 2019,5, 25 10 of 14
of the microbes. The presence of bacterial load was noticed at the end period of storage, notably at
Day 40 (under 3
×
10
5
cfu/mL) and Day 50 (under 10
×
10
5
cfu/mL) in Sample C. Sample B developed
bacterial load at Day 50 (under 2
×
10
5
cfu/mL). Sample D developed no bacterial colony during the
storage of 50 days, but the presence of bacterial load was noticed at Day 60 (3.78
×
10
5
cfu/mL). Total
fungal count (yeast and mold count) of control samples (Sample A) increased considerably from the
day of preparation (no growth at Day 0) to the end of storage period (7.0
×
10
5
cfu/mL at Day 50).
As for treated juice samples, no fungal load was observed at Day 0. The presence of a few fungal loads
was noticed at the end period of storage in Sample B (under 4
×
10
4
cfu/mL at Day 50) and Sample C
(under 2
×
10
4
cfu/mL at Day 50). Sample D developed no fungal growth over the storage period
of 50 days, but fungal growth was observed at Day 60 (2.83
×
10
4
cfu/mL). No growth of indicative
organisms such as coliforms was observed in the samples over the storage period. Growth of coliforms
was inhibited by heat treatment and the inhibitory action of preservatives.
Thus, pasteurized wood apple beverage samples treated with a combination of 50 ppm and
100 ppm of preservatives were safe to drink for up to 50 days. The results of microbial evaluation
found in this study is in accordance with the reports of Sasikumar [
2
] and Chowdhury et al. [
31
] and
are in agreement with the reports of Akinola et al. [
24
], Ayub et al. [
25
], Bhardwaj and Mukherjee [
36
],
Khare et al. [42], Dhaka et al. [44] and Chatterjee et al. [47].
Table 2. Evaluation of total viable count of samples over storage period.
Type Sample
No.
Time of Storage (Days)
Day 0
(cfu/mL)
Day 10
(cfu/mL)
Day 20
(cfu/mL)
Day 30
(cfu/mL)
Day 40
(cfu/mL)
Day 50
(cfu/mL)
Day 60
(cfu/mL)
Total
bacterial
count
Sample A 1.09 ×1071.52 ×1071.71 ×1071.88 ×1072.00 ×1072.43 ×1073.53 ×107
Sample B Nil Nil Nil Nil Nil ≤2.00 ×1053.26 ×106
Sample C Nil Nil Nil Nil ≤
3.00
×
10
5≤1.00 ×1062.63 ×106
Sample D Nil Nil Nil Nil Nil Nil 3.78 ×105
Total yeast
and Mold
count
Sample A Nil 3.30 ×1054.00 ×1054.90 ×1056.00 ×1057.00 ×1051.50 ×106
Sample B Nil Nil Nil Nil Nil ≤4.00 ×1049.00 ×104
Sample C Nil Nil Nil Nil Nil ≤2.00 ×1047.00 ×104
Sample D Nil Nil Nil Nil Nil Nil 2.83 ×104
Total
coliform
count
Sample A Nil Nil Nil Nil Nil Nil Nil
Sample B Nil Nil Nil Nil Nil Nil Nil
Sample C Nil Nil Nil Nil Nil Nil Nil
Sample D Nil Nil Nil Nil Nil Nil Nil
Nil = No microbial growth.
3.3. Changes in Sensory Attributes
Results on sensory evaluation are presented in Table 3. The DMRT test that followed ANOVA
revealed that there was a significant difference (with significance level of 0.05) among samples in their
sensory attributes. Sample D was significantly “better” than other samples, and a higher retention of
sensory attributes was observed in Sample D in comparison with other samples. The sensory attributes
of the samples reduced significantly (with a significance level of 0.05) over the storage period. Sample
D scored the highest rating in terms of appearance (8.92
±
0.07 at Day 0 and
7.60 ±0.03
at Day 50),
aroma (8.81
±
0.02 at Day 0 and 7.50
±
0.02 at Day 50), taste (8.92
±
0.07 at Day 0 and
7±0.03
at
Day 50), and overall acceptability (8.90
±
0.05 at Day 0 and 7.51
±
0.04 at Day 50). The Hedonic
scale rating of wood apple beverages for appearance ranged from 8.92
±
0.07 to 7.90
±
0.03 (like
very much/like moderately) at the day of preparation (Day 0) and gradually decreased to the range
of 7.60
±
0.03 to 6.70
±
0.02 (like moderately/like slightly) at the final day of preparation (Day 50).
Aroma of the beverage samples was in the range of 8.81
±
0.02 to 7.90
±
0.02 (like very much/like
moderately) at Day 0 following a gradual reduction in rating towards the end of storage. The score for
aroma was in the range of 7.50 ±0.02 to 6.50 ±0.03 (like moderately/like slightly) at the final day of
storage (Day 50). Taste of the samples were within the range of 8.92
±
0.07 to
8.00 ±0.02
(liked very
much) at the preparation day (Day 0) and decreased to be in the range of 7.00
±
0.03 to
5.50 ±0.02
Beverages 2019,5, 25 11 of 14
(like moderately—neither like or dislike) at the end of storage (Day 50). Overall acceptability of
samples ranged from 8.90
±
0.05 to 7.80
±
0.03 (like very much/like moderately) at the day of
preparation (Day 0) and followed a decreasing trend towards the end of storage. The scores for overall
acceptability at the final day of storage (Day 50) were within the range of 7.51
±
0.04 to 6.50
±
0.02
(like moderately/like slightly). The results found in this study on the sensory attributes of wood apple
beverage are in accordance with those of Sasikumar [
2
], Tiwari and Deen [
29
],
Rathod et al. [14]
, and
Chowdhury et al. [31].
The findings of this study suggest that wood apple beverage preserved with chemical
preservatives retained maximum sensory attributes during storage because the 9-point Hedonic
scale rating of Sample D (50 ppm KMS + 50 ppm SB + 50 ppm CA + 50 ppm AA) for sensory attributes
ranged from 8.92
±
0.07 to 7.00
±
0.03 (Table 3) over the storage period of 50 days. It means that Sample
D was either “liked very much” or “liked moderately” by the panelists over the entire storage period.
This result is in accordance with that of Ayub et al. who reported that strawberry juice preserved with
chemical preservatives retained maximum sensory attributes during storage [
25
]. It was also revealed
that the use of preservatives in wood apple beverage did not have a negative influence in the opinion
of consumers. This finding is supported by the reports of Akinola et al. and Sasikumar who reported
that fruit beverages preserved using chemical preservatives can be acceptable to consumers [24].
Table 3. Sensory evaluation of samples over the storage period.
Sensory
Attributes
Sample
No.
Storage Time (Days)
Day 0 Day 10 Day 20 Day 30 Day 40 Day 50
Appearance
Sample A 7.90 ±0.03 aF 7.70 ±0.02 aE 7.50 ±0.03 aD
7.20
±
0.03
aC 7.00 ±0.02 aB 6.70 ±0.02 aA
Sample B 8.39 ±0.02 bF
8.10
±
0.01
bE 8.00 ±0.03 bD
7.70
±
0.02
bC
7.40
±
0.02
bB 7.00 ±0.03 bA
Sample C 8.71 ±0.02 cF 8.50 ±0.04 cE 8.30 ±0.03 cD 8.00 ±0.02 cC 7.70 ±0.01 cB 7.40 ±0.04 cA
Sample D
8.92
±
0.07
dF
8.80
±
0.02
dE 8.50 ±0.03 dD
8.10
±
0.02
dC
7.90
±
0.02
dB 7.60 ±0.03 dA
Aroma
Sample A 7.90 ±0.02 aF 7.70 ±0.02 aE 7.30 ±0.04 aD
7.00
±
0.03
aC 6.70 ±0.02 aB 6.50 ±0.03 aA
Sample B 8.10 ±0.02 bF
7.80
±
0.03
bE 7.50 ±0.03 bD
7.30
±
0.03
bC
7.00
±
0.02
bB 6.70 ±0.03 bA
Sample C 8.50 ±0.02 cF 8.10 ±0.02 cE 7.91 ±0.02 cD 7.50 ±0.04 cC 7.20 ±0.03 cB 7.01 ±0.02 cA
Sample D
8.81
±
0.02
dF
8.60
±
0.03
dE 8.30 ±0.02 dD
8.00
±
0.02
dC
7.80
±
0.03
dB 7.50 ±0.02 dA
Taste
Sample A 8.00 ±0.02 aF 7.70 ±0.02 aE 7.10 ±0.03 aD
6.50
±
0.03
aC 6.00 ±0.02 aB 5.50 ±0.02 aA
Sample B 8.50 ±0.01 bF
8.10
±
0.02
bE 7.90 ±0.02 bD
7.51
±
0.02
bC
6.99
±
0.02
bB 6.51 ±0.02 bA
Sample C 8.80 ±0.03 cF 8.50 ±0.02 cE 8.10 ±0.02 cD 7.80 ±0.02 cC 7.30 ±0.03 cB 6.90 ±0.03 cA
Sample D
8.92
±
0.07
dF
8.70
±
0.02
dE 8.31 ±0.02 dD
8.01
±
0.02
dC
7.51
±
0.03
dB 7.00 ±0.03 dA
Overall
Acceptability
Sample A 7.80 ±0.03 aF 7.50 ±0.02 aE 7.20 ±0.01 aD
7.01
±
0.03
aC 6.70 ±0.03 aB 6.50 ±0.02 aA
Sample B 8.20 ±0.03 bF
8.00
±
0.03
bE 7.60 ±0.03 bD
7.40
±
0.03
bC
7.00
±
0.02
bB 6.80 ±0.03 bA
Sample C 8.60 ±0.02 cF 8.40 ±0.03 cE 8.10 ±0.03 cD 7.80 ±0.02 cC 7.50 ±0.04 cB 7.10 ±0.03 cA
Sample D
8.90
±
0.05
dF
8.70
±
0.03
dE 8.40 ±0.03 dD
8.10
±
0.02
dC
7.81
±
0.02
dB 7.51 ±0.04 dA
Values represent the mean
±
standard deviation of 9-point hedonic scale rating given by 150 consumers
(
n= 150
);
abcdef
column—means within the different superscript letter were significantly different (p< 0.05);
ABCDEF row—means within the different superscript letter were significantly different (p< 0.05).
4. Conclusions
Pasteurization at 85
◦
C for 10 min and a combined treatment with potassium metabisulphite,
sodium benzoate, citric acid, and ascorbic acid could effectively extend the shelf life of wood apple
beverages by up to 50 days. Consumption-safe parameters of wood apple beverage were properly
maintained over the storage period with satisfactory consumer acceptability. Pasteurization (85
◦
C for
10 min) and treatment with 50 ppm potassium metabisulphite + 50 ppm sodium benzoate + 50 ppm
citric acid + 50 ppm ascorbic acid was found to be more efficient than other treatments in retaining
the physicochemical properties, microbial growth, and sensory attributes of wood apple beverages.
Findings of this study will help interested parties to produce safe-to-consume and shelf-stable wood
apple beverages on an industrial scale and promote this highly nutritious beverage commercially in
the developing market of beverages.
Beverages 2019,5, 25 12 of 14
Author Contributions:
M.S.M. conceived, designed and carried out the experiments, handled and collected
raw data, conducted statistical analysis, analyzed and interpreted the data and prepared the manuscript; W.Z.
participated in manuscript preparation, facilitated data collection and reviewed the manuscript before submitting;
M.B.H.S. conceptualized the project, acted as corresponding author and supervised the whole research work.
Funding: This research received no external funding.
Acknowledgments:
The authors express their gratitude to the Department of Food Engineering and Tea
Technology, Shahjalal University of Science and Technology, Sylhet, for continuous material support and
technical assistance.
Conflicts of Interest: The authors declare no conflicts of interest.
Appendix A
Table A1.
Evaluation of physicochemical parameters of treated wood apple beverage samples
during storage.
Para
Meters
Sample
No.
Storage Time (Days)
Day 0 Day 10 Day 20 Day 30 Day 40 Day 50
TSS
(◦Brix)
Sample A 16.50 ±0.03 dA 16.82 ±0.02 cB 16.98 ±0.03 cC 17.40 ±0.01 cD 17.85 ±0.03 dE 18.25 ±0.05 cF
Sample B 16.41 ±0.02 cA 16.51 ±0.02 bB 16.61 ±0.02 bC 16.73 ±0.02 bD 16.88 ±0.03 cE
16.91
±
0.03
bE
Sample C 16.36 ±0.03 bA 16.48 ±0.02 bB 16.57 ±0.04 bC 16.62 ±0.07 aC 16.81 ±0.03 bD
16.88
±
0.02
bE
Sample D 16.30 ±0.02 aA 16.40 ±0.01 aB 16.48 ±0.02 aC 16.59 ±0.01 aD 16.69 ±0.02 aE
16.78
±
0.02
aF
pH
Sample A 5.09 ±0.10 aF 4.91 ±0.04 aE 4.70 ±0.02 aD 4.44 ±0.05 aC 4.30 ±0.05 aB 4.10 ±0.02 aA
Sample B 5.20 ±0.02 bF 5.15 ±0.02 bE 5.09 ±0.02 bD 5.00 ±0.02 bC 4.95 ±0.02 bB 4.73 ±0.02 bA
Sample C 5.30 ±0.02 bcF 5.22 ±0.02 cE 5.15 ±0.02 cD 5.08 ±0.02 cC 4.98 ±0.02 bcB 4.78 ±0.02 cA
Sample D 5.43 ±0.01 cF 5.34 ±0.02 dE 5.28 ±0.02 dD 5.15 ±0.01 dC 5.08 ±0.02 cB 4.97 ±0.03 dA
Acidity
(%)
Sample A 0.43 ±0.02 aF 0.37 ±0.01 aE 0.26 ±0.02 aD 0.17 ±0.02 aC 0.12 ±0.01 aB 0.08 ±0.01 aA
Sample B 0.57 ±0.02 bE 0.52 ±0.02 bD 0.47 ±0.01 bC 0.41 ±0.01 bB 0.39 ±0.01 bB 0.34 ±0.01 bA
Sample C 0.60 ±0.02 cF 0.56 ±0.01 cE 0.53 ±0.01 cD 0.49 ±0.01 cC 0.45 ±0.006 cB 0.36 ±0.01 cA
Sample D 0.69 ±0.01 dF 0.63 ±0.006 dE 0.58 ±0.006 dD 0.53 ±0.01 dC 0.49 ±0.01 dB 0.43 ±0.01 dA
Ascorbic
Acid
Content
(mg/100mL)
Sample A 4.23 ±0.02 aF 3.41 ±0.02 aE 2.74 ±0.04 aD 2.11 ±0.04 aC 1.44 ±0.05 aB 1.01 ±0.07 aA
Sample B 4.40 ±0.02 bF 4.10 ±0.02 bE 3.80 ±0.01 bD 3.31 ±0.01 bC 2.99 ±0.02 bB 2.77 ±0.01 bA
Sample C 4.50 ±0.02 cF 4.17 ±0.01 cE 3.89 ±0.01 cD 3.49 ±0.01 cC 3.10 ±0.02 cB 2.84 ±0.01 cA
Sample D 4.65 ±0.02 dF 4.25 ±0.01 dE 4.00 ±0.02 dD 3.70 ±0.01 dC 3.39 ±0.02 dB 3.01 ±0.02 dA
Values represent the mean
±
standard deviation of 3 replicates (n= 3);
abcdef
column—means within the different
superscript letter were significantly different (p< 0.05);
ABCDEF
row—means within the different superscript letter
were significantly different (p< 0.05).
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