Walailak Procedia 2019; 2019(1): IC4IR.

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Development of anthocyanin-rich jelly by Thai mulberry (Morus alba) fruit powder

Jutamas TONGMAI, Chaowanee CHUPEERUCH, Uthaiwan SUTTISANSANEE,
Rungrat CHAMCHAN, Chanakan KHEMTHONG and Nattira ON-NOM*
Institute of Nutrition, Mahidol University, Nakhon Pathom 73170, Thailand

(*Corresponding author’s e-mail: [email protected])

Abstract
Mulberry fruit is well known as a good source of anthocyanins with many biological activities. This study was
investigated the physico-chemical properties of Thai mulberry (Morus alba) fruits powder (MFP) and use as an ingredient to
developing an anthocyanin-rich jelly. The physical and chemical properties and sensory evaluation of anthocyanin-rich jelly by
varying four levels of MFP at 0, 13, 20 and 26% (w/w) were determined. The results showed that MFP contains carbohydrate
(73.46%), protein (10.24%), fat (1.74%), and dietary fiber (11.29%). The acidity, total soluble solid and pH were 6.43%,
13.72% and 4.28, respectively. MPF exhibited the antioxidant activities by 1-diphenyl-2-picrylhydrazyl (DPPH) radical
scavenging, ferric reducing antioxidant power (FRAP) and oxygen radical absorbance capacity (ORAC) assay of 1.20 µmol
trolox equivalent (TE)/100g dry weight (DW), 166.16 µmol TE/g DW and 595.11 µmol TE/g DW, respectively. The result of
total phenolic contents (TPCs) using Folin-Ciocalteu method was 26.88 mg GAE/g DW and total anthocyanin was 6.13 mg
cyanidin-3-glucoside equivalents/g DW. As the results of anthocyanin-rich jelly, the increase of MFP resulted in the increasing
of total soluble solid, acidity and hardness. For color measurement, lightness (L*) was decreased but redness (a*) and
yellowness (b*) were increased. The sensory results showed anthocyanin-rich jelly from 13% MFP obtained the highest overall
liking score of 7.5 (like moderately to like very much). The antioxidant activities, TPCs and total anthocyanin content of the
developed jelly with MFP were significantly higher (p<0.05) than the jelly without MFP. Therefore, anthocyanin-rich jelly
could be developed as functional food with consumer acceptability.
Keywords: Jelly, mulberry fruit, anthocyanin, antioxidant activity, total phenolic

Introduction
Mulberry belongs to the genus Morus of the family Moraceae and it is grown wild or cultivated in all regions such as
Asia, Europe, North and South America and Africa. In general, there are three types of mulberry, including white (Morus
alba), black (Morus nigra) and red (Morus rubra). However, the color of mulberry fruits cannot be used to identify the
mulberry species (Ozguven & Ozcelik, 2013; Hamid & Joshi, 2016). Mulberry tree has been cultivated traditionally for their
leaves (food for silkworms (Bombyx mori L.) and animal fodder) and as ornamental trees (Vijayan, 2010); however, due to its
nutritive value, the mulberry fruit is nowadays consumed in both fresh and processed forms, such as juice, jams, syrups,
beverages, natural dyes, or dried fruits (Ercisli & Orhan, 2007; Gundogdu et al. 2011; Sánchez, 2000). In previous studies,
mulberry fruits have been reported to exhibit a variety of biological activities, such as anti-thrombotic (Yamamoto et al. 2006),
antioxidant (Kim et al. 1999; Naderi, Asgary et al. 2004), antimicrobial (Takasugi et al. 1979), anti-inflammation (Kim & Park,
2006) and neuroprotective effects (Kanget al. 2006). These activities are generated by anthocyanins, which are a group of
naturally occurring phenolic compounds that are responsible for the color of mulberries. Cyanidin-3-glucoside and cyanidin-3-
rutinoside are the major anthocyanins (Liu et al. 2004).
Processing can alter and often damage anthocyanin and its antioxidant properties in fruit and vegetable. Maceration,
heating, and various separation steps can result in oxidation, thermal degradation, leaching, and other events that can lead to
lowering the levels of antioxidants in processed foods compared with the fresh ones (Kalt, 2005). Nevertheless, in contrast,
there are also recent studies which have shown that the antioxidant levels of processed food products, derived from e.g. tomato
(Capanoglu et al. 2008), and sour cherry (Toydemir et al. 2013) are maintained by the high recovery of the compounds during
processing or may even enhanced by the appearance of new compounds which have a higher antioxidant capacity (Nicoli,
Anese & Parpinel, 1999). Therefore, this study aimed to investigate nutritive values, physical properties and total phenolic
contents, total anthocyanin content and antioxidant activities by various assays including ORAC, DPPH and FRAP from Thai
mulberry (Morus alba). In addition, anthocyanin-rich jelly was developed using sensory evaluation and determined its physico-
chemical properties.

Materials and methods

Raw materials
Thai mulberry fruit (Morus alba) powder (MFP) from fully mature fruits by Freeze-drying were obtained from
Kamnulchul Farming Co., Ltd., Thailand and kept at -20°C until studies. Other ingredients used for jelly included carrageenan,
locust bean gum, and citric acid (Chemipan Corporation Co., Ltd., Thailand) and sugar (Mitr Phol Sugar Corporation., Ltd.,
Thailand).
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International Conference on 4 Industrial Revolution and Its Impacts, 27-30 March 2019, Walailak University, Thailand
Development of anthocyanin-rich jelly by Thai mulberry (Morus alba) fruit powder
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Jelly processing
The jelly formulation consisted of 19 g of sugar, 0.2 g of citric acid, 0.85 g of carrageenan, 0.25 g of locust bean gum,
and 3-7 g of mulberry fruit powder. Distill water was added to mulberry fruit powder. Sugar previously dissolved in boiling
water with jelly agents was warmed up to 80°C for 20 min or until clear solution was obtained. Then, MFP dispersion, citric
acid and flavor were added into the clear solution. The dispersion was then heated again for approximately 3 min at 80°C. In
order to obtain a desirable gel consistency, the end point was judged as the point at which the total soluble solids of the viscous
dispersion reached 72ºBrix, as determined by a hand refractometer (ATAGO Co. Ltd., Tokyo, Japan). The dispersion was then
poured into a round mold and cooled at 4°C for 24 h to produce a jelly. The produced jelly was then freeze-dried, subsequently
ground into a fine powder using a grinder to a size of 60 messes, and stored at −20°C in an aluminum foil container for
analysis.

Jelly agents + sugar

MFP Mixing with distilled water

Water Heating and stirring
(80°C, 20 min)

Citric acid
Heating and stirring
(80°C, 3 min)

Cooling
(4°C, 24 h)

Jelly

Figure 1 Flow diagrams for production of mulberry fruit powder jelly.

Physico-chemical analysis
The physico-chemical properties of mulberry fruit powder (MFP) and jelly samples were analyzed at the Laboratory of
Food Science and Food Chemical unit, Institute of Nutrition, Mahidol University.
Proximate analysis: the moisture content, ash, protein, fat, carbohydrate, dietary fiber and sugar were determined using
standard method of AOAC (2016). The calorie content was calculated based on the contents of protein, fat and carbohydrate.
Soluble solids, pH, total acidity and color: the soluble solids were evaluated through a digital pocket refractometer
(ATAGO Co. Ltd., Tokyo, Japan) and expressed in °Brix. The pH was measured using a digital pH meter (METTLER
TOLEDO, USA). Five g of MFP was dispersed with 25 mL deionized water at 25°C after pH meter calibration with standard
buffers pH 4 and 7 (Kha et al., 2010). In case of jelly, pH meter was introduced electrode directly into the jelly. Total acidity
was measured by titration 10% solution of product with 0.1 N NaOH up to pH 8.1 and recorded as mg citric acid/100 mL
(Mutlu et al., 2018). The color of samples were analyzed by a HunterLab ColorFlex EZ spectrophotometer using C.I.E. LAB
(L*, a*, b*) system, whereas L* is lightness, a* is redness/greenness, and b* is yellowness/blueness.
Texture: texture properties of jelly were evaluated using Texture Analyzer (TA.XT plus, Stable Micro System Ltd., YL,
UK). The method was modified from Lee et al. (2010). Hardness, springiness, cohesiveness, gumminess and chewiness of
mulberry jelly were determined using radiuses cylinder probe P/0.5. Ten mulberry jellies with 15×15×10 mm3 were
individually compressed using the penetration mode under conditions of pre-test speed of 1.5 mm/s, a test speed 1.5 mm/s,
post-test speed 10 mm/s, 50% strain.
Sensory evaluation: Nine point hedonic test (1=dislike extremely, 9=like extremely) was followed for conducting the
sensory evaluation of mulberry jelly. The panels of thirty judges evaluated the product for sensory parameters including
appearance, color, odor, flavor, texture and overall acceptability. Ten gram of mulberry jelly was served in a capped
transparent plastic cup which was coded with 3-digit numbers. The panelists were asked to rinse their mouth with water in
between the sample (Amerine et al. 1965).

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Total phenolic contents and antioxidant activities: Approximately 50 mg of mulberry fruit powder was mixed with 1 mL
distilled water. However, 40% ethanol was used to extract jelly samples. After thoroughly mixing (1min) with Vortex mixer,
the mixture was heat in the water bath for 2 h at 50°C. The residue was extracted after it was re-mixed and centrifuge at 4600
rpm for 15 min at 4°C. The clear supernatant was separated for the analysis of total phenolic compounds using the Folin-
Ciocalteu colorimetric method (Ainsworth & Gillespie, 2007) and the results were expressed in gallic acid equivalents per
gram dry weight (mg GAE/g DW) of sample. Antioxidant activity were determined using the DPPH-radical scavenging assay
(Fukumoto & Mazza, 2000) , ORAC (oxygen radical absorbance capacity) assay (Ou et al. 2001) and FRAP assays (Benzie &
Strain, 1996) and the results were expressed as Trolox equivalents per gram dry weight (TE/g DW).
Total anthocyanin: Total anthocyanins were determined by pH differential method (Giusti & Wrolstad, 2005) using a
spectrophotometer. The pigments were extracted by combined 1 g of each sample with 12 mL HCl (1%). After incubation for
48 h at 3 to 5°C, the tubes were centrifuged at 4000 rpm. Samples were filtered and stored at –20° C until analysis. Absorbance
was measured at 530 nm and 657 nm in buffers at pH 1.01 and 4.5 using A = [(A530 – A657) pH 1.0 - (A530 – A657) pH 4.5]
with a molar extinction coefficient of 29.600. Results were expressed as mg of cyanidin-3-glucoside equivalents (CGE) in g
dry weight (mg cy-3-glue/ 100 g-1 DW).

Statistical analysis
The data were expressed as mean ± standard deviation. Data were analyzed using an analysis of variance and t-test.
Duncan multiple range test was used to determine significant difference among the various samples in triplicate. Data was
analyzed using the software, Statistical Package for Social Science (SPSS) version 19 SPSS Inc., Chicago, II, USA at the 0.05
level.

Results and discussion

Physico-chemical properties of mulberry fruits powder (MFP)
The proximate composition of mulberry fruits powder is shown in Table 1. Similar results regarding the chemical
compositions of dried mulberry were previously reported by Sadia et al. (2013). The principal component of Mulberry fruits
(Morus alba) powder var. Chiang Mai was carbohydrates (73.46%), particularly total sugars (31.98%). Ozguven & Ozcelik
(2013) found that the ripen mulberry contains the highest amount of total sugar which is related to the sensory acceptability.
Moreover, MFP contained dietary fiber (11.29%) and protein (10.24%), similar to some staple foods; this implies that MFP
may also be a source of important nutrients (Marconi et al. 1997). The several studied reported that dried mulberry (Morus
alba) contains higher amount of protein than strawberry (Ozcan & Hacıseferogulları, 2007) and sun berry (Patel et al. 2011).
The dietary fiber consisted of soluble dietary fiber (1.27%) and insoluble dietary fiber (10.03%). The relevant health
organizations recommend ingesting 18-32 g of dietary fiber per day (Spiller, 1986), and food products formulated to contain
abundant dietary fiber are currently being developed (Grigelmo et al. 1999). Therefore, the MFP may be considered to be an
alternative source of dietary fiber or a specialty food ingredient for use in the production of functional food products. The
moisture content of MFP was 9.99% which was in line with the quality standard of Thai community product standard (Thai
industrial standards institute, 2016).
The MFP pH value was 3.29 which indicate that the products are from a food technology point of view, within a safe
range in terms of microbial development. According to Azeredo (2012), food with pH lower than 4.0 are classified as very acid
foods. Total acidity was related to pH and reported at 6.34%. Moreover, the soluble solid of MFP was 13.72°Brix. The color
of MFP was 21.78 of brightness, 10.36 of redness and 3.30 of yellowness. This could be explained that the color of the
powders influenced by the maturity of fruit ripening (Lee & Hwang, 2017).

Table 1 Physico-chemical properties of mulberry fruits powder (MFP)1.

Physico-chemical properties MFP

Energy (kcal/100g) 350.43±11.92
Moisture (%) 9.99±3.07
Protein (g/100g) 10.24±0.40
Total fat (g/100g) 1.74±0.05
Total carbohydrate (g/100g) 73.46±2.68
Ash (g/100g) 4.58±0.04
Dietary fiber (g/100g) 11.29±1.55
Soluble dietary fiber (g/100g) 1.27±0.18
Insoluble dietary fiber (g/100g) 10.03±1.37
Total sugars (g/100g) 31.98±8.82
pH 3.29±1.82
Total acidity (mg/100ml) 6.34±0.32
Soluble solids (°Brix) 13.72±0.90
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Physico-chemical properties MFP

Color
L* 21.78±4.92
a* 10.36±4.45
b* 3.30±1.61
1
the data are displayed as having a mean±SD.

Total phenolic contents, antioxidant activities and total anthocyanins of mulberry fruits powder (MFP)
Table 2 shows total phenolic contents, antioxidant activities and total anthocyanin of mulberry fruits powder (MFP).
MPF exhibited the antioxidant activities by 1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging, ferric reducing
antioxidant power (FRAP) and oxygen radical absorbance capacity (ORAC) assay of 1.20 µmol trolox equivalent (TE)/100g
dry weight (DW), 166.16 µmol TE/g DW and 595.11 µmol TE/g DW, respectively. The result of total phenolic contents
(TPCs) using Folin-Ciocalteu method was 26.88 mg GAE/g DW and total anthocyanin was 6.13 mg cy-3-glue/ 100 g-1 DW.
Equally to our results, Lee & Hwang (2017) found similar level of total anthocyanins in Korean Morus alba L. However,
Aljane & Sdiri (2016) presented the lower TPCs and total anthocyanins in mulberry fruit (Morus alba).

Table 2 Total phenolic contents, antioxidant activities and total anthocyanins of mulberry fruits powder (MFP)1.

Antioxidant activities
Total phenolic Total anthocyanins
DPPH radical FRAP assay ORAC assay
Sample contents (mg cy-3-glue/ 100 g-1
scavenging assay (umol TE/g DW) (umol TE/g DW)
(mg GAE/g DW) DW)
(umol TE/100g DW)
MFP 26.88± 1.76 1.20 ± 0.06 166.16±16.39 595.11±63.59 6.13±0.59
1
the data are displayed as having a mean±SD.

Effect of MFP on physico-chemical properties of jelly

Data pertaining to physico-chemical charateristics of different mulberry fruit jelly formulas was presented in Table 3.
The control jelly showed the lowest pH (2.91) and total acidity (0.17g/100g) which due to no MFP was added in the formula as
compared to other formulas. The hardness of the jellies increased significantly with increase in the quantities of MFP added to
the jellies. Considering the fact that the other conditions known to affect the formation of jelly, such as cooking time,
temperature, and concentration of sugar were fixed in this study, the amount of MFP added profoundly influenced the hardness
of the gels. Chandra & Shamasundar (2014) were also reported that the hardness of jelly could be increased as the pH
decreased. Moreover, the jellies produced in this study exhibited hardness values lower than the commercial fruit jelly (848 g),
probably as the result of gel additives, like pectin. For the color measurement, lightness (L*) was decreased but redness (a*)
and yellowness (b*) were increased in the mulberry jelly when compared with the control formula.

Table 3 Physico-chemical properties of mulberry fruits jelly (MFJ)1,2.

Physico-chemical Jelly Jelly with mulberry fruits powder (%)

properties (control) 13 20 26
pH 2.91±0.02c 3.74±0.03 b
4.02±0.03 a
4.06±0.02a
Total acidity (g/100g) 0.17±0.02d 0.32±0.02 c
0.40±0.03 b
0.62±0.02a
Hardness (g) -1.69±1.00d 524.98±6.84c 592.39±13.83b 604.05±18.44a
Adhesiveness (g.mm) -2.94±0.88a -15.95±2.78 c
-27.19±1.19 d
-10.74±1.03b
Springiness (mm) 0.57±0.06d 0.85±0.02b 0.90±0.003a 0.82±0.02c
Chewiness (g.mm) -0.23±0.10c 224.96±9.11 a
224.22±2.39 a
164.64±7.84b
Color
L* 16.99±0.04a 4.28±0.03b 4.08±0.04c 4.11±0.01c
a* -0.66±0.03d 11.96±0.05a 9.55±0.05b 10.64±0.04c
b* -0.09±0.02d 2.88±0.02a 1.96±0.05c 2.40±0.04b
1 2
the data are displayed as having a mean±SD. Different letters in same row indicate significant difference at p<0.05 using one
way ANOVA followed by Duncan multiple range test.

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Development of anthocyanin-rich jelly by Thai mulberry (Morus alba) fruit powder
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Total phenolic contents, antioxidant activities and total anthocyanins of mulberry fruit jelly
Phenolic compounds are plant constituents that possess antioxidant activity and prevent the decomposition of
hydroperoxides into free radicals (Pokorny, Yanishlieva & Gordon, 2001). The total phenolic contents, antioxidant activities
and total anthocyanins of mulberry fruit jelly are shown in Table 4. The mulberry jellies contained higher amount of total
phenolic contents than the control formula. This was due to the MFP addition. Moreover, it was found that the amount of
phenolic is higher than the calculation which might because of the heat processing. The heat treatment might induce the
changes in extractabilities of the phenolics and flavonoids due to the disruption of the plant cell wall, and consequently, the
enhanced release of the bound polyphenolic and flavonoid compounds (Choi et al. 2006). A similar result was noted in the
study of Khatun et al. (2006), reporting that the phenolic contents of spices increased by a factor of approximately 2-4 after
cooking. Moreover, the antioxidant activities of mulberry jellies were determined by DPPH radical scavenging, FRAP and
ORAC assay. The results showed that antioxidant activities increased with increases in the MFP concentration in the jellies, a
trend which was similar to those of TPCs of the jellies. Therefore, the antioxidant activities of mulberry jellies were attributed
to the contributions of TPCs. The total anthocyanins of the jelly with 13%MFP estimated to be 4.51 mg cy-3-glue/ 100 g-1
DW was found to increase to 5.25 and 5.54 mg cy-3-glue/ 100 g-1 DW in jelly with 20 and 26% MFP, respectively. In
addition, it was found the antioxidant activities and total anthocyanins remained after jelly production.

Table 4 Total phenolic contents, antioxidant activities and total anthocyanins of mulberry fruit jelly1,2.

Antioxidant activities
Total phenolic Total anthocyanins
Sample contents DPPH radical (mg cy-3-glue/ 100 g-1
(mg GAE/g DW) FRAP assay ORAC assay DW)
scavenging assay
(umol TE/g DW) (umol TE/g DW)
(umol TE/100g DW)
Control 2.93±0.12c 0.17±0.02c 4.11±0.39d ND ND
b b
13% MFP 21.66±1.13 3.43±0.11 117.13±5.23c 500.03±23.78b 4.51±0.26c
20% MFP 22.50±1.75b 3.55±0.25b 124.09±4.92b 532.21±52.95b 5.25±0.20b
a a a a
26% MFP 25.64±0.95 4.12±0.30 140.84±5.05 600.60±24.34 5.54±0.24a
1
the data are displayed as having a mean±SD. 2Different letters in same column indicate significant difference at p<0.05 using
one way ANOVA followed by Duncan multiple range test. ND=not detected

Sensory evaluation of mulberry fruit jelly

Sensory evaluation of jellies with different levels of MFP were performed on 6 different attributes such as appearance,
color, odor, taste, texture and overall liking using 9-point hedonic scale (Table 5). There was no significant difference in all
sensory attributes between jellies with different levels of MFP. However, the results showed the tendency of adding more MFP
could result in decreased in all the attributes. Therefore, 13% MFP was recommended to use in the production of anthocyanin-
rich jelly because it obtained the highest overall liking score of 7.5 (like moderately to like very much).

Table 5 Sensory evaluation of mulberry fruits jelly1,2,3.

Jelly Jelly with mulberry fruits powder (%)

Sensory attributes
(control) 13 20 26
Appearance 7.33±1.30 a
7.47 ±0.94 a
7.27±1.11 a
7.07±1.20a
Before test
Color 7.30±1.26ab 7.70±0.84a 7.40±1.04ab 7.00±1.49b
Odor 7.00±1.20a 7.23±1.01a 7.20±0.85a 6.83±1.21a
Taste 6.77±1.57a 7.53±1.20a 7.37±0.89a 7.17±0.95a
After test
Texture 7.27±1.08a 7.33±0.71a 7.00±1.11a 6.77±1.55a
Overall liking 6.93±1.23a 7.47±0.78a 7.30±0.88a 7.30±1.09a
1
All data were represented as mean±SD. 2Different letters in same row indicate significant difference at p<0.05 using one way
ANOVA followed by Duncan multiple range test. 39-points hedonic scale (1=Dislike extremely, 5=Neither Like nor Dislike,
9=Like Extremely.

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Conclusions
Mulberry fruit powder could be use as ingredient to the production of delightful, nutritious and delicious jellies.
Anthocyanin-rich jelly with 13% MFP was found to be accepted by the consumer. Moreover, the developed jelly was
contained higher total phenolic contents, antioxidant activities and total anthocyanins than the control formula. This
development will not only improve the nutrients of the product but it helps to increase the values of the ingredient and the
product. Therefore, it might be concluded that mulberry fruit powder is one of functional ingredient which can use in food
products in order to increase applications of such value-added food ingredients.

Acknowledgements
This work was supported by Institute nutrition Mahidol University and funded by Capacity Building Program for New
Researcher 2018 from National Research Council of Thailand (NRCT).

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