PROPOSAL FYP: EXTRACTION OF FLAVANOIDS FROM GARLIC AS ANTIMICROBIAL AGENT TO FOOD PRODUCT

UNIVERSITI KUALA LUMPUR MALAYSIANINSTITUTE OF

CHEMICAL & BIOENGINEERINGTECHNOLOGY (UNIKL MICET)

PROPOSAL

FINAL YEAR PROJECT (FYP)

Title of Project

The Extraction of Flavonoids inGarlic as Antimicrobial in Food

Product

Student’s Name

NurEzzati Binti Abd Aziz

Student’s ID Number

55104212122

Section/Department

Semester 5 in Diploma Chemical Engineering Technology Food

Project Supervisor’s Name

Miss Aida Safina Binti Aridi

Date of Submission

24 September 2014

CONTENT

No TABLE OF CONTENT PAGE1 INTRODUCTION2 OBJECTIVE

3 PROBLEM STATEMENT4 LITERATURE REVIEW5 METHODOLOGY

a. Preparation Sample: Extraction of

flavonoids from garlic

b. Microbiology Test: Determination of total

aerobic plate count

c. Fish balls formulation

d. Sensory Evaluation6 EXPECTED RESULT/POTENTIAL CONTRIBUTES7 RESEARCH PLAN8 BUDGET9 CONCLUSION10 REFERENCES

INTRODUCTION

People nowadays are very concern about safety issues that

involve in their everyday life consume food product especially

the food’s safety, quality and hygienic kind of standard.

Consumers tend to pay more attention towards these aspects of

characteristics because they value their life so much to have

a long life future ahead. Food is important as a substantial

choice in our lives. Our wellbeing depends on our nutritional

intakes primarily on our food intake and with several other

supplements.

According to Merriam Webster dictionary; medical

definition for microbiology is a branch of biology dealing

especially with microscopic forms of life like bacteria,

protozoans, viruses and fungi. Microbes can be anything in

microscopic view of forms with different kind of shapes and

sizes and can grow and exists in almost everywhere in this

world at any circumstances except to where places and things

are being sterilised.

There are so many factors influence the growth of

microorganisms. These factors can be divided into intrinsic

and extrinsic elements of parameter. The intrinsic factors are

shown by the food or the surface on which the microbes is

growing. The factors are pH, moisture content, nutrient

content, the biological structure of the food, redox potential

and its antimicrobial constituents of the food. All of these

factors have the ability to either encourage or discourage the

growth of the microorganisms. Some of these intrinsic

sometimes can be influenced by the extrinsic factor together

to stimulate the growth and retardness of microorganisms

growing. Extrinsic factors are the environment surrounding

factors around the food. This includes the temperature,

relative humidity, present of other microbes, the

concentration of gases and the chemical used like

preservatives and additives. (Hammer and Long, 1941).

In this study, garlic (Allium sativum) is chosen to be the

antimicrobial agent on food product. This study is to

determine the garlic extract effectiveness that act as one of

the additional ingredients to test one of the factor that

influence the microbial growth which is the antimicrobial

constituents to the food. The present of antimicrobial agent

to the food will provide some action against the growth of any

microbes that may live. Essential oil and spices will

influence the growth by acting as the antimicrobial agent.

Many medical plants have been found very effective in the cure

of bacterial disease which acts as the perfect antimicrobial

agent. Parts of plant like the flower, stem, leaves, root,

seeds and the whole plant can be used as one of the

ingredients in any food recipe which have a lot of benefits to

our health as well as functioning as the food additives. Some

of the antimicrobial components that play part as the

antimicrobial properties have been identified are eugenol from

cloves, cinnamon and sage, thymol from oregano, sage and

thyme, allicin from garlic, allylisothiocyanate from mustard,

vanillin from vanilla, etc. (Škrinjar and Nemet, 2009).

Figure 1: Garlic picture

The most common used spices together with shallots and

onion is garlic as one of the most crucial ingredients

especially in the Asian cuisine. Garlic also have been used

widely in the food supplements like vitamins to help

maintaining the human health or in the pharmaceutical used to

prevent and treat various illnesses. Its constituent value

added in food had become so familiar that people mistakenly do

not know its actual nutritional value. Garlic often well known

as one of the spice that can heal wounds by applying direct as

ground up or sliced up to prevent any spread of infections and

to slow down the symptoms of aging process. This is because it

of its antibacterial, antifungal, antioxidant and beneficial

effects on the cardiovascular and the human immune system. It

is the allicin, a sulphur containing compound found in garlic

that give the antimicrobial effects. (Guiñares, Belinario,

2014).

OBJECTIVES

To determine the percentage of garlic extraction yield

(%) by using water ethanol technique.

To do the microbial activity test on fishballs when mix

with different percentage of garlic extraction by using

total plate counting (TPC) method.

To study the effectiveness of garlic extract used in the fishballs.

PROBLEM STATEMENT

Several problems occur that involve in controlling the

microbial activity of the food processing especially in fish-

based food that requires attention. Food can become spoilt

depends on two factors; extrinsic factor and intrinsic factor.

For intrinsic factors; it depends on pH, moisture content,

nutrient’s content, biological structure of the food, redox

potential and its antimicrobial constituents of the food.

While for the extrinsic factors it depends on the surrounding

factors which are the temperature, relative humidity, present

of other microbes, the concentration of gases and the chemical

used as the ingredients. When one of these factors meet, the

microorganism can spread and grow and achieve certain level

for the food to go spoilt. This becomes so troublesome for

both the production and the consumer of food until many had

discovered the use of spices and extract oil as part of the

ingredients constituents to food. So, this final year project

is all about to have another alternative of using additives in

a much safer ways and to learn how to practically combine the

natural food additives into food production.

LITERATURE REVIEW

This new age, consumers are more demanding towards their

options of buying goods and foods at greater amount of quality

expectations. They expected everything to come up with higher

good quality in term of better taste, appearance and health

benefits. Thus this makes the food producer to come out with

new research to fill up the consumer’s level of expectation.

For health benefits and the food quality product, a new

approach must be done in order to achieve total maximum of

especially in controlling the microbial activity. Research

about spices and essential oil had become popular as another

organic antimicrobial constituent to the food product. This

includes garlic which has the both advantage for both health

benefits and act as antimicrobial agent.

To get the maximum benefits from garlic can be divided

into four states which is essential oil, dehydrated powder,

oil macerate and extract garlic. (Harunobu A, 2001). To get

the essential oil content, steam distillation can be made.

Steam distillation is used to extract and to manufacture the

essential oil by placing the botanical material and force the

steam over the material. Aromatic molecules from the plant

material (example: garlic) can be released by the hot steam

into volatile oils which then escape and evaporate into the

steam. The steam at a very high pressure that contains the

essential oil then will be condensed by passing it through a

cooling system. This separates the oil and water by forming a

liquid form after being condensed. The control of temperature

in this process is very important as if the temperature is too

hot, it can burn the plant material together with the

essential oil.

Figure 2: Steam Distillation Apparatus in Laboratory

METHADOLOGY

A. Preparation Sample: Extraction of flavonoids from Garlic

Material:

1. 750g of fresh garlic

2. 700mL of 95% Ethanol Food Grade

Apparatus:

1. 250mL and 1L beaker

2. 600mL conical flask

3. Aluminium foil

4. Analytical balance

5. Magnetic stirrer

6. Parafilm

7. Sterile drying oven

8. Vacuum filter

9. Waring Blender

10. Whatman no. 1 filter paper

Method:

1. 250g of fresh garlic is bought from the local market,

clean and wash properly.

2. Blend the garlic in the Waring Blender for 2 minute.

3. Add the 200g of the blended garlic into a 600mL conical

flask.

4. Add 400mL of 95% food based Ethanol in the conical flask.

5. A magnetic stirrer is used to homogenize the mixture for

3 more hours to increase the surface area.

6. Cover the conical flask with a parafilm and place it in a

dark surrounding like in the cupboard for 48 hours at

room temperature, 28°C

7. Filter the homogenized mixture by using vacuum filter and

uses a Whatman no.1 filter paper.

8. The mixture is rinsed by using 300mL 95% food based

Ethanol.

9. Evaporate the excess ethanol by using the drying oven for

24 hours at 60°C.

10. Weight and record the remaining desired solid by

using analytical balance.

11. Store the extract in the universal bottle at 4°C

covered with aluminium foil.

12. Step 1 to 11 is repeated for the next 2 replicates.

13. Calculate the extract yield using these formula:

GarlicExtractYield (% )= TotalWeightExtract(g)TotalWeightofShallot(g)

×10

B. Formulation of Fishballs

Materials:

Table 1: Material needed to make fishballs

Ingredients Formulations (%)Control 2 3 3

Surimi(white fish

meat)

500 g 500 g 500 g 500 g

Wheat flour 25 25 25 25Salt 15 15 15 15

White Pepper 2.5 2.5 2.5 2.5Ice water 175ml 175ml 175ml 175ml

Extraction of

flavonoids

0 5 10 15

Apparatus:

1. Mixer

Method:

1. Surimi (white fish meat) is soften up by defrosting.

2. The white fish meat is mix with salt for 5 minutes by

using a mixer.

3. All the other ingredients will be mix together and make

sure to be stir uniformly.

4. Ice water is added slowly until the mixture is well

blended.

5. The garlic extract is added accordingly by using the

formulation table. Mixing is continue for another 30

minutes.

6. The mixture is then will be shaped manually into round

same shape by using hands.

7. Set the fishballs in warm water 40°C for 30 minutes.

8. Immerse the fish ball into hot water at 95°C for another

30 minutes.

9. Immerse the fish ball in cold water for 30 minutes and

the fish ball is ready to be stored at 4°C.

C. Preparation of Peptone Water

Method:

1. Add 20g-1L of distilled water. Mix well and distribute

into final containers.

2. Sterilize by autoclaving at 121°C for 15 minutes.

D. Preparation of Nutrient Agar

Materials:

1. 10g agar plate count agar (PCA)

2. 500mL distilled water.

Apparatus:

1. Analytical balance

2. Hot plate

3. Stirrer

4. Screw cap bottle

5. Oven

Method:

1. Weight 10g agar plate count agar (PCA) and dilute with

500mL of distilled water.

2. Use beaker to heat the liquid with a hot plate. Stir by

using stirrer until mixture is clear.

3. Transfer into screw cap bottle and autoclave for 15

minutes with 121.1°C for 2 hours.

4. Place the solution into oven with 60°C-70°C.

E. Microbiology Test

Materials:

1. Nutrient agar.

2. Peptone water

3. Sample

Apparatus:

1. Colony counter

2. Incubator

3. Petri dish

4. Pipette

5. Universal bottle

Method:

1. Take 3 fishballs form each sample (1,2,3) and store it at4°C for the period of 0, 2, 6 and 12 days.

2. Microbiological test will be conducted as the period of

days reached. Sample will be taken out to be observed and

recorded.

3. Weight 10g of samples and added into 90mL 0.1% of peptone

water.

4. The mixture is homogenised using stomacher for 3 minutes.

1. Serial dilution is prepare 10-2, 10-3,10-4,10-5,10-6

respectively.

2. Pipette 0.1 ml of the garlic extraction onto the centre

of an agar plate.

3. The L-shaped glass rod was dip into a beaker of ethanol

and then the rod was tap on the side of the beaker to

remove any excess ethanol.

4. Ethanol-soaked spreader must be passed through the flame

to burn off the alcohol to sterilise the spreader first

before spreading the fishballs.

5. The homogenized fishballs with extract is spread evenly

with the sterilise spreader making sure all the plate is

covered.

6. The spreader then must be immersed in the ethanol, tapped

on the side of the beaker to remove any excess ethanol

and reflamed.

7. The procedure is repeated to inoculate the remaining

three plates.

8. 3 replicates must be done for this experiment with

different percentage of flavonoid extraction and one

control sample.

9. Incubation: undisturbed in upright position at 30-33°C

for 1 month. The microbial activity and growth is

observed one week after another.

10. Plate count is recorded like:

For colonies more than 300, report as TNTC(too

numerous to count)

For colonies less than 30, report as TNTS (too small

to count)

For plate which appears no growth, express as NC(no

colony)

If half of the plate is cover with spreader, report

as SPR(spreader)

F. Sensory Evaluation

Materials:

1. Boiling water.

Apparatus:

1. 1L pot

2. Serving bowl

3. Stove

Method:

1. Scoring test will be conducted to 50 trained panellists

to determine the most preferable amount of flavonoid

extraction to be used as ingredients in the fishcakes.

2. Boil the fishballs at 100°C for 2 minutes and served at

room temperature.

3. Trained panellists aimed to be diploma and degree food

students who already taken their sensory evaluation

subject and teachers of food sections. They will be

provided questions based rating score like:-

Rating

score

1 2 3 4 5

Outstandi

ng

Above

expectati

on

Meet

expectati

on

Below

expectati

ons

Poor

Table 3: Rating score

EXPECTED RESULT

A. Result: Extraction Yield (%)

Percentage of Garlic Extraction (%) ¿ TotalWeightExtract (g)TotalWeightGarlic(g)

×100

Replicate Replicate Replicate 3

1 2Total Weight

Garlic (g)Total Weight

Extract (g)Percentage of

Garlic Extraction

(%)

B. Result: Microbiology Test on Fishballs

Incubation day: 0

Table 1: 10% of flavonoids extraction of garlic

MicroorganismsDilutio

n

Factor

No. of colonies10-1 10-2 10-3 10-4 10-5 10-6

Plate 1Plate 2Average



n

Factor

No. of colonies10-1 10-2 10-3 10-4 10-5 10-6




n

No. of colonies10-1 10-2 10-3 10-4 10-5 10-6

FactorPlate 1Plate 2AverageIncubation day: 2



n

Factor

No. of colonies10-1 10-2 10-3 10-4 10-5 10-6




n

Factor

No. of colonies10-1 10-2 10-3 10-4 10-5 10-6



Microorganisms

Dilutio

n

Factor

No. of colonies10-1 10-2 10-3 10-4 10-5 10-6


Incubation day: 6



n

Factor

No. of colonies10-1 10-2 10-3 10-4 10-5 10-6




n

Factor

No. of colonies10-1 10-2 10-3 10-4 10-5 10-6




n

Factor

No. of colonies10-1 10-2 10-3 10-4 10-5 10-6


Incubation day: 12



n

Factor

No. of colonies10-1 10-2 10-3 10-4 10-5 10-6




n

Factor

No. of colonies10-1 10-2 10-3 10-4 10-5 10-6




n

Factor

No. of colonies10-1 10-2 10-3 10-4 10-5 10-6


C. Result: Sensory Test

Sensory evaluation form

Name:

Semester/section:

Matrix card:

1. Food code:_____

Ratin

g

1 2 3 4 5 6 7

Scale

d

likin

g

Dislik

e

extrem

ely

Dislike

moderat

ely

Dislik

e

slight

ly

Neithe

r like

nor

dislik

e

Like

slight

ly

Like

moderat

ely

Like

extrem

ely

2. Food code:_____

Ratin

g

1 2 3 4 5 6 7

Scale

d

likin

g

Dislik

e

extrem

ely

Dislike

moderat

ely

Dislik

e

slight

ly

Neithe

r like

nor

dislik

e

Like

slight

ly

Like

moderat

ely

Like

extrem

ely

3. Food code:_____

Ratin

g

1 2 3 4 5 6 7

Scale

d

likin

Dislik

e

extrem

Dislike

moderat

Dislik

e

slight

Neithe

r like

nor

Like

slight

Like

moderat

Like

extrem

g ely ely ly dislik

e

ly ely ely

RESEARCH PLAN

No ACTIVITY WEEK1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

1 Title released

2 FYP

implementation&Job

Safety Analysis(JSA)

briefing

Submit a title form

3 Title released

Meeting with SV

about the flow of

the project4 Submit first draft

5 Submission of JSA

form to Madam

NorainiBurok

Proposal submit6 Chapter

1:Introduction7 Chapter

2:Literature review8 Chapter

3:Methadology9 Chapter

4:Result&Discussion10 Chapter

5:Conclusion&Recomm

endations11 Acknowledgement,

Appendix and

References12 Table of content

Cover page13 Final draft

(Complete)14 Prepare a slide

15 Presentation about

projectBUDGET

NO Item Quantity Price (RM)Total

(RM)

1. Surimi 2.5kg RM 9.50/kg RM 23.75

2. Cap Lili Wheat Flour 1kg RM 1.35/kg RM 1.35

3. Tesco Fine Salt 450g/1packRM

0.65/pack

RM 0.65

4. Ice Cube 3 pack RM RM 9.00

3.00/pack

5. Shallot 3kg RM 6.00/kg RM 18.00

TOTAL RM 52.75

CONCLUSION

In conclusion, garlic extract can be used as antimicrobial

constituent to food as it discourage the growth of

microorganism in the food. Food had been tested and can

approach the consumer level of expect. Besides having the best

quality reason from the health benefit, garlic can be also

used as an additive in food to replace the chemical additive

used by the market. There are certain people who can accept

slightly garlicky taste in the food while others cannot stand

the pungent taste of it. But overall people can face this

approach of new constituent of food.

REFERENCES

1. Ibtisam Mohammed Ababutain, Antimicrobial Activity of

Ehtanolic Extracts From Some Medical Plant, Australian

Journal of Basic and Applied Sciences, 2011.

2. Investigation of Antioxidant and Antimicrobial Properties

of Garlic Peel Extract (Allium sativum) and Its Use as

Natural Food Additive in Cooked Beef, Journal of

Scientific Research & Reports, Department of Food Science

and Technology, Federal University of Technology Akure,

Nigeria, 2014

3. Hiba Ali Hassan, Ayad Mohammed RasheedRaauf,

BasamaMonjdAbdRazik and Bassam Abdul Rasool Hassan,

Chemical Composition and Antimicrobial Activity of the

Crude Extracts Isolated from ZingiberOfficinale by Different

Solvents, PharmaceuticaAnalyticaActa, 2012.

4. R. C. Guinares, M. C. E. B. Agbon, A. S. B. Campeon, M.

F. M. T. Belinario, Effects of Garlic Allium sativumExtract

on Lipid Oxidation, Microbiological, and Organoleptic

Qualities in Hot-smoked Sardines, Sardinellalongiceps During

Frozen Storage, International Journal of Fisheries and

Aquatic Studies, 2014

5. Lillian, How to Make Your Own Fishballs, (2014). [online]

Available at: http://www.bashaar.org.il/files/5389.pdf

[Accessed 24 Sep. 2014].

6. Serge Ankri, David Mirelman, Antimicrobial properties of

allicinfrom garlic, Department of Biological Chemistry,

Weizmann Institute of Science, Rehovot, Microbes and

Infection, 2, 1999

7. Settharaksa, S., 1Jongjareonrak, A.,Hmadhlu,

P.,Chansuwan, W. and ,Siripongvutikorn, S. Flavonoid,

phenolic contents and antioxidant properties of Thai hot

curry paste extract and its ingredients as affected of

pH, solvent types and high temperature, Nutraceutical and

Functional Food Research and Development Center,

Department of Food Technology, Faculty of Agro-Industry,

Prince of Songkla University, 2012.

8. B. W. Hammer and H. F. Long, Factors Influence the

Bacterial Growth in Butter, Dec 1941

9. Marija M. Škrinjar and Nevena T. Nemet. Antimicrobial

Effects of Spices and Herbs Essential Oils, 15 October

2009

10. Oxoid.com, (2014). CM0009, Peptone Water | Oxoid - Product Detail. [online] Available at: http://www.oxoid.com/UK/blue/prod_detail/prod_detail.asp?pr=CM0009&c=UK&lang=EN [Accessed 24 Sep. 2014].

PROPOSAL FYP: EXTRACTION OF FLAVANOIDS FROM GARLIC AS ANTIMICROBIAL AGENT TO FOOD PRODUCT

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