GOVERNMENT COLLEGE UNIVERSITY, FAISALABAD

Institute of Home and Food Sciences

Synopsis for MS. Food Science and Technology

Title: “Extraction and Application of Fruit By-product Phytochemicals for the

Development of Functional Fish Meat Products”
Name: Hassan Mazhar

Registration No.: 2012-GCUF-02299

ABSTRACT
The food industry generates large amount of wastes or by-products annually around the world
from a variety of sources. In that food wastes or by-products are an excellent source of
nutraceuticals, bioactive, inherently functional and possess many components that are good for
human health. Food wastes or by-products conversion to the functional food ingredients is the
healthy trends in the food industry. For this purpose, ultrasound technology assisted process will
be applied for the extraction of phytochemicals from fruit by-products (Papaya and Orange peel)
with distilled water, aqueous methanol and aqueous ethanol as the solvent. The ranges of
selected processing variables will be extraction temperature, sonication amplitude level,
water/waste ratio and sonication time conditions for maximum yield of phytochemicals from
fruit waste using Box-Behnken Design (BBD). Afterwards, the resultant extracts will be filtered
and subjected to drying process. The extracted phytochemical samples will be analyzed for
chemical composition (moisture, protein, fat, total phenols, cyanogenic glycosides and tannin
contents). Natural vegetable phytochemical extracts will be supplemented at different
concentration to fish meat for preparation of different meat products i.e. patties, balls/pops and
finger/sticks. The un-cooked and fried fish meat products will be tested for oxidative stability
(peroxide and free fatty acid values) and consumer acceptability at different intervals during 60-
days storage at refrigerator and −18°C. The data of phytochemical yield and product
development obtained for each treatment will be subjected to statistical analysis to determine the
level of significance by using the software package.

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 GOVERNMENT COLLEGE UNIVERSITY, FAISALABAD
Institute of Home and Food Sciences
Synopsis for Ms. Food Science and Technology

Title: “Extraction and Application of Fruit By-product Phytochemicals for the

Development of Functional Fish Meat Products”

1. Date of Admission: 1-09-2016

2. Date of Initiation (Research): 1-10-2017

3. Probable Duration (Research): 06 Months

Supervisory Committee:

1. Dr. Muhammad Imran (Chairman) ________________

2. Dr. Muhammad Kamran Khan (Member) ________________

3. Dr. Zarina Mushtaq (Member) ________________

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1. Introduction
Diet is an important factor affecting human health and well-being. Meat is an important
component of a healthy and well balanced diet due to its nutritional richness. Fish meat is a
valuable source of high biological value protein, omega fatty acids, vitamins and minerals. Lipid
and color oxidation are major causes of quality deterioration in meat products during storage.
These reactions lead to off-flavor, discoloration, and loss of nutritive value, ultimately
decreasing consumer confidence in the product and costing the industry over $700 million
annually. Intake of such food products with oxidized lipid constituents can modify DNA,
proteins, membrane structure and tumor initiation in biological system (Muik et al, 2005).

Fish meat is devoid of antioxidants and phytochemicals, and other helpful nutrients that have
shown to be protective against oxidation. Therefore one good possibility would be to generate
bioactive ingredients that could be applied to design stable fish foods. Due to increasing
production of plant food processing, by-products disposal represents a growing problem since
the plant material is usually prone to microbial spoilage, thus limiting further exploitation.
(Schieber et al., 2001; O'Shea et al., 2012; Jansen et al., 2013).

Undoubtedly, functional foods represent an important, innovative and rapidly growing part of the
overall food market. Epidemiological studies have pointed out that consumption of functional
foods imparts health benefits, e.g. reduced risk of coronary heart disease and stroke, as well as
certain types of cancer (Williamson, 2009).

Ultrasound is one of the emerging technologies that was developed to minimize processing,
maximize quality and ensure the safety of food products. Ultrasound is applied to impart positive
effects in food processing such as improvement in mass transfer, food preservation, assistance of
thermal treatments and manipulation of texture and food analysis (Knorr et al., 2011).

The action of ultrasound is due to cavitation, which generates high shear forces and micro
bubbles that enhances surface erosion, fragmentation and mass transfer resulting in high yield of
extracted materials and fast rate of extraction. The major advantages of ultrasound are minimum
effect on extractable materials, avoidance of organic solvents as its action also works in GRAS
solvents, reduction in extraction time, which can potentially enhance the extraction of heat

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sensitive bioactive and food components at lower processing temperatures and potentially in
large industrial scales (Awad et al., 2012).

The extracted phytochemicals can be used in meat to further enhancing the meat
acceptability and improving the functional stability during storage. Therefore, it can be the most
viable approach to extract the phytochemicals from fruit and vegetable waste using the
innovative technology. Due to possible health hazards of some artificial antioxidants growing
attention has been paid to recognize natural antioxidants which are possibly more economically
feasible and effective antioxidants. One of Most significant category of natural antioxidant is
phenolic compound which is our concern, and much validation is extracted on the potency of
antioxidant and prevention of diseases (Scalbert et al., 2005; Fresco et al., 2006).

Objectives:

 Extraction of bioactive compounds from fruit and vegetable waste/by-products by using

ultrasound technology
 Develop a range of functional/healthier un-cooked and fried fish meat products using
extracted bioactive compounds
 Characterization of functional fish meat products for oxidative stability and consumer
acceptability at different storage intervals

2. Review of Literature

Norah O'Shea et al. (2012) producers are striving to create products which contain a
value added factor, such as dietary fiber or in more recent times, phytochemicals. The production
and addition of such nutrients can be quite costly for the producer. In the fruit and vegetable
industry, the preparation and processing procedures can lead to one third of the product being
discarded. This can be costly for the manufacturer and also may have a negative impact on the
environment. Research has shown that these by-products can have a high nutritional value. It has
also been suggested, that they could be used as a food ingredient due to their functional abilities
such as gelling and water binding. The focus of this review is on the nutritional and functional

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properties of the by-products of food processing and their potential applications as nutritional
new ingredients in foods.

Schieber et al. (2001) There is a rapidly growing body of literature covering the role of
plant secondary metabolites in food and their potential effects on human health. Furthermore,
consumers are increasingly aware of diet related health problems, therefore demanding natural
ingredients which are expected to be safe and health-promoting. By-products of plant food
processing represent a major disposal problem for the industry concerned, but they are also
promising sources of compounds which may be used because of their favourable technological
or nutritional properties. The purpose of this review is to highlight the potential of selected by-
products as a source of functional compounds.

Charanjit Kaur et al. (2002) The anti-oxidant activity of extracts from 36 vegetables was
evaluated by using a model system consisting of β-carotene and linoleic acid. The total phenolics
of the extracts was determined spectrophotometrically according to the Folin–Ciocalteau
procedure and ranged from 34 to 400 mg (100 g)−1 on a fresh weight basis. Mint, aonla, black
carrots, chenopodium, fenugreek, kachnar and ginger had high phenolic contents. The anti-
oxidant activity expressed as per percent inhibition of oxidation ranged from a high of 92% in
turmeric extracts to a low of 12.8% in long melon. Other vegetables found to have high anti-
oxidant activity (>70%) were kachnar, aonla, ginger, fenugreek, mint, beetroot, black carrots,
Brussels sprouts, broccoli, lotus stem, yam, coriander and tomato. Anti-oxidant activity
correlated significantly and positively with total phenolics (r2=0.6578, P < 0.05). The results
indicate that vegetables containing high phenolics may provide a source of dietary anti-oxidants.

Liu (2003) In general, fruits and plant by products are known to contain a wide variety of
phytochemicals, such as polyphenols, carotenoids and vitamins. More than 5000 individual
phytochemicals have been identified in fruits, vegetables and grains.

Wang and Weller (2006) Extraction is the first key step to isolate natural bioactive
compounds from plants and materials. Different extraction methods (i.e. solvent extraction,
expelling extraction process, supercritical fluid extraction (SFE) and microwave assisted
extraction) have been developed for the recovery of bioactive compounds and essential oils.

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 Samaram et al. (2013) However, several disadvantages like extra solvent amount in solvent
extraction, low yield in expelling process, massive investment in supercritical fluid extraction
(SFE) and the requirement for the aqueous phase in microwave assisted extraction indicates the
demand of comprehensive extraction method to recover different target compounds in economic
condition.

Samaram et al. (2015).Ultrasound-assisted extraction is a new simple technique for the

recovery of oil and bioactive compounds from different sources. The intensity of ultrasound
power creates extra vibration in sample molecules and facilitates the recovery of target
compounds from solid material to the liquid solvent phase. Therefore, high yield in short
extraction time beside the utilization of low solvent amount are remarkable advantages of
ultrasound-assisted extraction technique. Moreover, ultrasound- assisted extraction is adjustable
to be utilized with polar and non-polar solvents in various temperatures.

Nagendran Balasundram et al. (2006) Phenolic compounds, ubiquitous in plants are an

essential part of the human diet, and are of considerable interest due to their antioxidant
properties. These compounds posses an aromatic ring bearing one or more hydroxyl groups and
their structures may range from that of a simple phenolic molecule to that of a complex high-
molecular weight polymer. Flavonoids, which bear the C6–C3–C6 structure, account for more
than half of the over eight thousand different phenolic compounds. The antioxidant activity of
phenolic compounds depends on the structure, in particular the number and positions of the
hydroxyl groups and the nature of substitutions on the aromatic rings. Fruits, vegetables and
beverages are the major sources of phenolic compounds in the human diet. The food and
agricultural products processing industries generate substantial quantities of phenolics-rich by-
products, which could be valuable natural sources of antioxidants. Some of these by-products
have been the subject of investigations and have proven to be effective sources of phenolic
antioxidants. When tested in edible oils, and in fish, meat and poultry products, phenolic-rich
extracts have shown antioxidant activities comparable to that of synthetic antioxidants. Practical
aspects of extraction and production of sufficient amounts of natural antioxidants from most of
these sources remain to be elucidated.

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3. Materials and Methods
3.1. Procurement of raw materials

Fruit by-products (Papaya and Orange peel) will be collected from local fruits and
processing industries in Faisalabad, Pakistan. The selected raw materials will be cleaned to
remove the adhered dirt, dust and other foreign debris.

3.2. Extraction of phytochemicals from fruit by-products

Ultrasound (model VCX 750, Sonics & Materials, Inc., USA) technology assisted
process will be applied for the extraction of phytochemicals from fruit by-products with distilled
water, aqueous methanol and aqueous ethanol as the solvent. Response surface methodology will
be used to establish the optimum conditions for the sonication assisted extraction of
phytochemicals. A quadratic polynomial regression model describing extraction efficiency as a
function of four independent variables such as extraction temperature, sonication amplitude
level, water/waste ratio and sonication time will be established based on a Box-Behnken
experimental design. Afterwards, the resultant extracts will be filtered and subjected to drying
process.

3.3. Analysis of fruit phytochemical extracts

The phytochemical extracts will be analyzed for chemical composition.

3.3.1. Moisture, protein and fat contents

Moisture content will be analyzed by using air forced draft oven (Model: DO-1-30/02,
PCSIR, Pakistan). Protein will be calculated by the Method No. 990.03 (AOAC 2006). Crude
fat will be estimated by AACC (2000) Method No. 30-10.

3.3.2. Total polyphenols

Total polyphenols will be measured by using Folin-Ciocalteu method following the

protocol of Singleton et al. (1999). For the intention, 50 µL extract will be added to 250 µL of
Folin-Ciocalteu reagent with 750 µL of 20% sodium carbonate solution and the volume will be

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made upto 5 mL with distilled water. After 2 hours, absorbance will be recorded at 765 nm with
UV/Visible Spectrophotometer against control.

3.3.3. Determination of cyanogenic glycosides

The cyanogenic contents in extract samples will be estimated by alkaline titration

according to the method outlined in AOAC (1990) Method No. 26.115. 20 g of extract will
be taken in kjeldhal flask following the addition of 200 mL distilled water and will slowly be
mixed the sample. The sealed flask will be given rest (3 h) to get proper hydrolysis process
of the mixture. Then, distillation process will be carried out by connecting the flask to a
vapor distilling apparatus and distillate will be collected in a flask containing 20 mL of 2.5%
NaOH solution until a marked volume. 8 mL of 6 M NH 4OH and 2 mL of 5% KI solution
will be transferred into the distillate solution before titration against 0.02 M AgNO 3 by
microburette. The used volume of AgNO 3 will be noted for each individual extract sample.

3.3.4. Estimation of tannin

For tannins, 0.5 g of the sample extract and 100 mL of distilled water will be added in
a conical flask, gently boiling for 1 hour and will be filtered using Whatman No. 44 filter
paper. The filtrate will be diluted to 100 mL and then be cooled. For the greenish blue color
development, 50 mL aliquot will be put into each flask. This will be followed by 5 mL
Folin–Dennis reagent, 10 mL of saturated sodium carbonate solution and will be diluted to
100 mL with distilled water. After thorough mixing, the flasks will be allowed to stand in a
water bath at 250 °C for 20 min and the optical density will be measured at 700 nm through
spectrophotometer. Distilled water will be used as blank regarding the calibration curve.
Standard tannic acid solution will be prepared from which a standard curve wil l be drawn
(absorbance versus concentration in mg/cm 3). From this curve, the concentrations for each
sample will be used for the tannin content calculation (Nwinuka et al., 2005).

3.4. Functional meat product development and analysis

Natural fruit phytochemical extracts will be supplemented at different concentration to

fish meat for preparation of different meat products i.e. patties, balls/pops and finger/sticks. The
partial/parfrying of the products will be carried out to determine the lipid stability. Un-cooked

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and fried meat products will be vacuum sealed in plastic bags and then will be stored at
refrigerator and −18 °C in a freezer for a storage period of 60-days. The oxidative stability of oils
extracted from fish meat products will be assessed by measuring peroxide value (AOCS 1998
Method No. Cd 8‒53) and free fatty acid value (AOCS 1998 Method No. Ca 5a‒40),
respectively.

3.5. Sensory evaluation of meat products

Experienced and untrained assessors will carried out the sensory analysis of meat product
samples according to the instructions given by Meilgaard et al. (2007). Each judge will give the
written informed consent after explanation of risks and benefits of participation prior to the
study. The panelists will be provided informative instructions and brief definitions of attributes
such as color, odor, flavor, off-flavor and overall acceptability. Samples will be presented to
participants in sensory booths under white lighting. The order of presentation will be balanced to
avoid carry-over effects. Each panelist will receive the samples assigned with random three‒digit
code numbers. Each panelist will be asked to list their preference on a 9‒cm comparison line (1 =
dislike extremely to 9 = like extremely). Consumers will also rate their expected liking and
intention to purchase. The sensory analysis will be performed at different storage intervals for
experimental treatments.

3.6. Statistical analysis

The behavior of the Box-Behnken Model will be explained by the following the quadratic
equation. The data of phytochemical yield obtained for each treatment will be subjected to
statistical analysis to determine the level of significance by using the software package
(MATLAB) according to the method described (Montgomery, 2008). The average of the three
runs will be reported as the measured value with standard deviation. The Duncan’s multiple
range (DMR) test will be used to estimate the level of significance that existed between the mean
values for product data analysis. The sample analysis for storage stability and consumer
acceptability will be carried out in triplicate and the significant differences will be calculated
among means at a probability level of 5%.

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4. References

AOAC. (2006). Official Methods of Analysis, 18th Ed. Association of Official Analytical
Chemists, AOAC Press, Arlington, VA, USA.

Awad,T. S., Moharram, H. A., Shaltout, O. E., Asker, D. and Youssef, M. M. (2012).
Applications of ultrasound in analysis, processing and quality control of food: A review.
Food Research International, 48: 410–427.

Charanjit Kaur, Harish C Kapoor (2002) Anti‐oxidant activity and total phenolic content of some
Asian vegetables. International Journal of Food Science & Technology. 37(2) 153-161.

Jansen RJ, Robinson DP, Stolzenberg-Solomon RZ, Bamlet WR, de Andrade M, Oberg AL,
Rabe KG, Anderson KE, Olson JE, Sinha R and Petersen GM. 2013. Nutrients from fruit
and vegetable consumption reduce the risk of pancreatic cancer. Journal of Gastrointest
Cancer. 44(2):152-161.

Knorr, D., Froehling, A., Jaeger, H., Reineke, K., Schlueter, O. and Schoessler, K. (2011).
Emerging technologies in food processing. Annual Review of Food Science and
Technology, 2: 203–235.

LijunWang Curtis L.Weller 2006. Recent advances in extraction of nutraceuticals from plants.
Trends in Food Science & Technology 17(6) 300-312.

Liu RH (2003) Health benefits of fruits and vegetables are from additive and synergistic
combination of phytochemicals. Am J Clin Nutr 78:517S–520S.

Meilgaard MM, Civille GV and Carr T. 2007. Overall Difference Tests: Does a Sensory
Difference Exist Between Samples? In: Sensory Evaluation Techniques (4th edn). CRC
Press, New York, USA. pp. 63–104.

Muik, B., Lendl B, Molina-Díaz A and Ayora-Cañada MJ. 2005. Direct monitoring of lipid
oxidation in edible oils by Fourier transform Raman spectroscopy. Chemistry and Physics
of Lipids. 134(2):173–182.

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NagendranBalasundram KalyanaSundram SamirSamman. 2006. Phenolic compounds in plants
and agri-industrial by-products: Antioxidant activity, occurrence, and potential uses. Food
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Nwinuka NM, Ibeh GO and Ekeke GI. 2005. Proximate composition and levels of some
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O'Shea N, Arendt EK and Gallagher E. 2012. Dietary fibre and phytochemical characteristics of
fruit and vegetable by-products and their recent applications as novel ingredients in food
products. Innovative Food Science and Emerging Technologies. 16:1–10.

P. Fresco, F. Borges, C. Diniz, M.P.M. Marques 2006. New insights on the anticancer properties
of dietary polyphenols. Medicinal Research Reviews. 26(6):747–766

Schieber A, Stintzing FC and Carle R. 2001. By-products of plant food processing as a source of
functional compounds – recent developments. Trends in Food Science and Technology.
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Schieber A, Stintzing FC and Carle R. 2001. By-products of plant food processing as a source of
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Shadi Samaram , Hamed Mirhosseini , Chin Ping Tan and Hasanah Mohd Ghazali.
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AlirezaSerjouie. 2015. Optimisation of ultrasound-assisted extraction of oil from papaya
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Singleton VL, Orthofer R and Lamuela-Raventos RM. 1999. Analysis of total phenols and other
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Vilkhu, K., Mawson, R., Simons, L. and Bates, D. (2008). Applications and opportunities for
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