Project Proposal

Antibacterial Effects of Persimmon (Diospyros discolor) Extracts

on the Shelf Life of Canned Goods

A Research Proposal

by

Marionne G. Oliveros

Generose Christy M. Mateo

Maryam Amparo Concepcion R. Remoto

Submitted to the Faculty of the

Philippine Science High School – Main Campus

in partial fulfillment of the requirements for

Science and Technology Research 1

February 2011

1

ABSTRACT:

Antibacterial Effects of Persimmon (Diospyros discolor) Extracts on the Shelf Life of

Canned Goods

One of the main problems concerning our consumer society today is food spoilage.

Many consumers prefer to stock up, while most companies want to preserve their products –

however, they are unable to do so due to multiple factors which may lead to the shortening of

their shelf life. Food preservatives have offered some assistance, but most are chemical-

based, and, though effective, are not helpful towards the environment as a whole. Persimmon

leaves will be tested for their effectiveness in preventing food spoilage. They will serve as

natural alternatives rather than those available in the market.

Four types of canned goods will be tested (cocktail, corned beef, sardines, and meat

loaf). There will be three replicates of each sample. Persimmon leaves were crushed and

filtered thoroughly through a fine nylon mesh and then Rotavapped. Each replicate will have

have extracts 3.33% w/v. The samples would then be stored in an area of constant

temperature, and results will be checked after four days. Statistical data would be tested

through ANOVA, and significance in shelf life lengthening would determine the research's

success.

When proven that persimmon leaves may have a hand in food preservation, this will

encourage businesses to use natural alternatives rather than using chemicals which may harm

the surroundings. Persimmon is common in flora all around the world, including the

Philippines, providing manufacturers with a flourishing international supply of this organic

preservative

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TABLE OF CONTENTS

Introduction

4 Review of Related Literature

Persimmon

Mabolo (Diospyros discolor)

Flavonoids

Extraction of Flavonoids

Food Preservation Techniques

Shelf Life

Food-borne Bacteria

9 Proposed Methodology

Procurement and Preparation

of Persimmon Leaves (Diospyros discolor)

Procurement of Canned Goods

Extraction of Flavonoids from Persimmon Leaves

Addition of Persimmon Extract to Canned Goods

and Storage of Setups

Preparation of Canned Good Samples

after Four Days of Treatment

Gram Staining of Samples

Recording of Observation and Results

12 Bibliography

14 Task List

15 Network Chart

16 Gantt Chart

17 Proposed Budget

3

INTRODUCTION

PROPOSED TOPIC

Antibacterial Effects of Persimmon Leaves (Diospyros discolor) on the Shelf Life of

Canned Goods

BACKGROUND OF THE STUDY

Since prices of food had been high for the past years, consumers tend to by cheap and

long-lasting goods. Unfortunately, some preservatives utilized during the canning process are

harmful chemicals and inorganic materials that might lead to bodily dysfunctions and might

add to the factors of environmental crises.

Previous researches have shown that persimmon contain antibacterial properties.

These were utilized as an anti-allergenic dietary supplement. Chinese and Indians also use

persimmon fruit as an ingredient for “Holy teas” and a traditional remedy for hiccups.

Canning has been an extremely popular method for preserving foods. However, there

have been many incidents wherein improper canning has caused food spoilage (and in effect,

a dilemma for consumers worldwide). Plus, several canning techniques involve the addition

of chemicals and inorganic substances which, aside from being inefficient in terms of

resources, may harm the environment as well.

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STATEMENT OF THE PROBLEM

This study aims to identify whether Diospyros discolor, or the Mabolo will help in

prolonging the shelf life of canned goods since food spoilage has become a huge problem to

both contributors and consumers. This organic preservative may also be a cheaper and

healthier alternative since other preservatives nowadays contain factors that may ail the body,

causing illness or dysfunctions.

The extracts from Diospyros discolor should be able to prevent the multiplication or

existence of non-airborne bacteria that may cause food spoilage, such as species of

Pseudomonas, Micrococcus, and Proteus. The canned goods with Diospyros discolor extract

should surpass the quality of the control after four days of experimentation in terms of

presence of gram negative pathogenic bacteria.

SIGNIFICANCE

Canned goods have always been one of the most affordable products in the market,

appealing to all classes of society. Canning has helped in lengthening the time it took until

the foods inside them would expire. This preservation of foods is helpful for people who

want to stock up on food and for those who have limited budgets. Prolonging of the shelf life

of canned goods would be advantageous not only to the consumers, but also to the

manufacturers. There would be less wasted material and resources. A preservative offering

an all-organic alternative preservative is being proposed by the study. Instead of utilizing

chemicals which may affect the consumer's diet and health, Diospyros discolor extracts

provide a natural antibacterial food preservative.

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SCOPE AND LIMITATIONS

The use of one species of Persimmon, Diospyros discolor, which is native in the

Philippines, will be the focus of the study. Four types of canned goods were considered,

specifically meat loaf, corned beef, sardines, and cocktail. Replicates of these subjects will

involve the same brand, same amount, and same date of expiration prior to the addition of

Diospyros discolor. The subject of the research is only the preservation of canned goods

using the extracts from Diospyros discolor. The concentration of each extract will be

measured and maintained uniform all throughout the experiment. The only part of the tree

that will be utilized are the leaves, and no extracts from any other part will be taken. For

uniformity, leaves from only one specific tree will be gathered. One brand of each of the

canned good will also be used.

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REVIEW OF RELATED LITERATURE

PERSIMMON

The most common species of persimmon is Diospyros kaki, or the Oriental persimmon. It

originated in China, then spread to Korea, Japan, and other nearby countries. Its species also

cultivated in the United States of America when a United States Commander returned with a Japanese

variety to Washington, D.C. (usa.gov., 2006). Persimmons thrive in mild summers and moderate

winters, and due to this range, leaves do not flourish when temperatures reach 26ºF. High

temperatures may sunburn the bark as well (California Rare Fruit Growers, Inc., 1996).

The fruit of persimmons have been used as an ingredient for Chinese and Indian holy teas. It

has also been known as solution for hiccups. Persimmon cakes are known as a traditional remedy for

diarrhea, hemorrhoids, lung infections, and asthma (Kotani, et. al., 2000). Persimmon exhibits rapid

antibacterial effects against food-borne infections, such as Listeria monocytogenes and Vibro

parahacmolytic, ascribed to by the polyphenolic (or antioxidant) components of the fruit.

Persimmon leaves grow up to seven inches long and four inches wide, alternating with each

other in growth on branches. Their colors range from a yellowish green, a darker hue when maturing,

and reddish purple in the autumn season (California Rare Fruit Growers, Inc., 1996). Persimmon

leaves have been known to be a good source of important dietary antioxidants, such as vitamin A, C,

and flavonoids. They are also known to contain bioactive components, like tannins, phenolic

compounds, coumarins, organic acid, carotent, amino acids, and the aforementioned antioxidants (Li-

Lian J. et. al.) They are anti-allergenic and anti-puritic in composition, which makes them an allergy

or itching preventive (thewolfclinic.com). Further studies have shown that supplementation of

persimmon leaves improve lipid profiles and suppresses body weight gain in animals that were fed

high-fat diets.7

MABOLO

Mabolo (Diospyros discolor) is closely related to the

black persimmons and the Japanese persimmon (Diospyros kaki).

Mabolo, also known as the velvet apple, is native to the

Philippines and uncommon elsewhere. This species is known

locally as the kamagong (Darley, 2010). Its tree may grow to up

to 35 feet, while its leaves range from four to ten inches in

length. It bears fruit all throughout the year, therefore providing a

continuous food resource. Similar to its fruit, leaves (see Fig. 1) also thrive all year round. Mabolo's

main season, wherein the fruits are “... of a variety, rarer than the red … sweeter, and less astringent

…” (Wester, P.J.), is from June to September (Popenoe, W., 1920).

FLAVONOIDS

Flavonoids are widely found in plants, which are non-nitrogenous biological pigments (also

known as biochromes). These are responsible for the red coloring of flower buds and purple-reds of

autumn leaves. Flavonoids are considered as an important part in contribution to the spread of plant

pollen due to the vivid colors it may give the plant, which in turn, attracts insects and other seed-

dispersing agents. (Norton, P., 1991). Apart from giving pigmentation to a plant, flavonoids have

bioactive properties that remain stable against heat, acid, and storage temperature. Research has

shown that hydroxylation of these bioactive properties may have effects on antibacterial activity.

Some flavonoids found in persimmon leaves are astraglin, isquercitrin, kaempferol, quercetin, rutin,

and kaempfetol 3-o-3-o-ß-D-glucopyronoside. (Li-Lian, J. et. al.)

EXTRACTION OF FLAVONOIDS

Optimized flavonoid extraction is possible through perfect conditions involving temperatures

at 85ºC, with a 1:05 material ratio and 75% ethanol as solvent, and four times of extraction within

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Fig. 1. Diospyros discolor leaves.

two hours (Sathishkumar, T., et. al., 2008). The leaves should be shredded and blended until it

becomes fine powder, and added a small volume of ethanol. The solution should be vacuum filtered

thoroughly and then washed with more ethanol (kay, 2008). Ethanol is because it, too, is polar,

therefore glycones in the flavonoids are soluble in it. Extracts should be stored in a cool dry place.

FOOD PRESERVATION TECHNIQUES

Food prepared using a certain procedure such as cooking, freezing, canning ,dehydrating,

milling, culturing with bacteria, or adding vitamins and minerals, has a lengthened shelf life than

other foods unprepared (Burdon, 1968). Foods gradually spoil or rot after hours or minutes after

harvesting, slaughtering or manufacturing. Some foods spoil rapidly, others are kept longer but for a

limited time only. The deterioration in some foods include biotic factors, such as bacteria (like yeast

and molds), and as well as abiotic factors, like temperature, moisture, oxygen, light and time, and

activities of enzyme (Singh, 2007).

The fundamental principle in food storage is to keep it cool, clean, and covered. High

temperature kills bacteria, while freezing preserves them in suspended animation (Singh, 2007).

SHELF LIFE

Canned goods that are unopened will store for 12 months or longer from the date of

processing. Canned fruit juices that are unopened will store for nine months

(FreezeDriedSurvivalFood.com). However, opened refrigerated canned foods which are well-covered

or sealed from the surrounding environment may last up to three or four days. However, its shelf life

may deteriorate depending on the surroundings, both abiotic and biotic factors, such as dampness,

which may corrode cans or metal lids and cause leakage so the food will spoil (QueryCAT, 2007).

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Table 1. Recommended Shelf Life of Canned Goods

Type of Canned Good Shelf Life (years)

Chicken, turkey, tuna, fish, seafood, meats 2-5

Baby Food 1

Tomato Sauce 1

Canned Fruits 2-5

Fruit Juice 1

Canned Vegetables 1-2

Baked Beans 1-2

Spaghetti Sauce 1

Soup 1

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FOOD-BORNE BACTERIA

Putrefactive types of bacteria are the main causes of food spoilage. Multiplication of bacteria

must be prevented to ensure the preservation of food. Once food is dried, or canned, the bacterial

growth will be inhibited due to lack of moisture (Burdon, et. al., 1968).

Examples of these bacteria are monerans. Monerans are simple-looking, unicellular

organisms. Most monerans do not make their own food, which means they have to rely on other

organisms to obtain it. These organisms have to break down or decompose other living things to

obtain energy (Daniel, et. al., 1994).

Spore-forming bacteria represent the most important group of canned food-spoiling

microorganisms because of their heat-resistant nature. In addition, there are other microorganisms

which are mesophilic, but through the leakage of the container during cooling, may spoil the food.

This would classify food-borne bacteria into two categories: biological spoilage by thermophilic

bacteria, and by mesophilic microorganisms (studentsguide.in)

Most foods have water in them, and that may cause the growth of bacteria. Gram-

negative bacteria are known to be pathogenic especially when found in food (Masschalk, B.,

et. al., 2003). Pathogenic bacteria are usually colorless or odorless (such as Salmonella or E.

coli), usually invisible to the naked eye (Alfaro, D., 2010).

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MATERIALS AND METHODOLOGY

Process Flowchart

Procurement and Preparation of Persimmon Leaves (Diospyros discolor)

In the preparing the persimmon extracts, persimmon (Diospyros discolor) leaves will

be obtained, washed and then shredded into pieces. The shredded leaves will be made into

powder with the use of a common blender.

Procurement of Canned Goods

In this set up, there will be four experimental setups from each of the canned goods,

thus, 12 experimental canned goods to be tested, with the other four canned goods set as

control. The canned goods considered in this experiment are corned beef, cocktail, meat loaf,

and sardines, the samples in each group having the same expiration date as indicated on their

labels.

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Extraction of Flavonoids from Persimmon Leaves

In the resulting leaf powder, 96% ethanol was added as a solvent. In order to obtain the

flavonoids, the solution will filtered four times through a fine nylon wire mesh. To remove the

solvent, the solution will go to RotaVaporation for ten minutes. The resulting solution will then

stored in the refrigerator in a clean beaker, capped with foil.

Addition of Persimmon Extract to Canned Goods and Storage of Setups

Each of the three replicates of canned good will have persimmon extracts 3.33% of

their weight. Upon adding the extract, the canned good should be covered with parafilm or

foil. The canned goods, including the control, will be placed on a shelf at room temperature

and avoiding direct sunlight.

Preparation of Canned Good Samples after Four Days of Treatment

Finally, after four days after the expiration of the canned goods, a loopful of sample

will be obtained from each of the canned goods for observation. Each sample will be placed

on corresponding slides and will be prepared for gram staining.

Gram Staining of Samples

In gram staining, each of the sample slides will be heat fixed by quickly passing the

slide over the fire of the alcohol lamp. Crystal violet stain will then be added to the heat fixed

sample for one minute. Next will be washing with distilled water. Iodine will then be added

for three minutes. Then will be washed with distilled water. Afterwards, it will be

decolorized with 95% ethanol for 20 seconds and then will be washed again with water.

Lastly, the sample will be counter-stained with safranin for one to two minutes and then

washed for the last time and blot dried with tissue.

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Recording of Observation and Results

The sample slides will then be observed under the microscope and will be classified

as gram positive or gram negative. Gram negative bacteria will be regarded as pathogenic

food-borne bacteria. Results and observations will then be recorded, and statistical data will

be determined by ANOVA.

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BIBLIOGRAPHY

Baumann, L.S. (2006). Persimmon. Retrieved from: http://www.skinandallergynews.com/views/cosmeceutical-critique-by-leslie-s-baumann/fadf7d9135.html

Burdon, K.L., Williams, R.P. (1968). Microbiology. Toronto, Ontario, Canada: The Macmillan Company.

Canned Food Tips and Hints. Canned Food Cooking Tips and Hints. (2010). Retrieved from: http://www.homecooking.about.com/od/specificfood/a/cannedfoodtips.htm

Caton G. Using Preservatives. Retrieved from: http://www.gregcaton.com/lumen/preserve_ecart.htm

City of Dipolog: Dipolog Fruits. Retrieved from: http://dipologcity.com/Fruits.htm

Daniel, L., Ortleb, E.P., Biggs, A. (1994). Kingdom Monera. Merrill Life Science. United States of America: Glencoe Division of Macmillan: McGraw-Hill School Publishing Company.

Darley, J.J. (2010). Casimiroa, Black Persimmons, and Mabolo: Know and Enjoy Tropical Fruit. Retrieved from: http://www.proscitech.com.au/trop/details.htm

Drummond, K.E. (1996). The Dictionary of Nutrition and Dictetics. United States of Amercia: International Thompson Publishing, Inc.

Kay (2008). How to Extract Flavonoids? [Message 1]. Message posted to http://answers.yahoo.com/question/index?qid=20080410143206AAp5PlZ

Lerner, R. (2000). Persimmon – Leaf. Retrieved from: http://www.hort.purdue.edu/ext/senior/fruits/Persimmon3.html

Li-Lian Ji, et. al., Natural Medicine: Antibacterial Effect of Extracts from Persimmon Leaves. Institute of Functional Biomolecules, Nanjing University, China.

Microbes Associated With Food Spoilage. Retrieved from: http://www.microbiologyproecedure.com/food-microbiology-microbes-associated- with-

food-spoilage.htm

Microbial Spoilage. Causes of the Spoilage of Food. Retrieved from: http://home.pacific.net/hk/~ppleung/Chem/spoilage.htm

Mika, H., Ryuji, S., Takeshi, W., Tokuya, W., Hiroshi, K., Atsushi, N. (2006). Antibacterial and Deodorizing Effect of Persimmon Tannin (Pancil). Japanese Journal of Conservative Dentistry (Vol. 48 No.2). Japan Science and Technology Agency.

Norton, P., et. al., (1991). Flavonoids. Encyclopedia Britannica, Fifteenth Edition. Encyclopaedia Britannica Inc., USA.

Pirie, N.W. (1978). Leaf Protein and its By-Products in Human and Animal Nutrition. Oakleigh, Melbourne, Australia: Cambridge University Press.

15

Satishkumar, T., et. al. (2008). Optimization of Flavonoids Extraction from the Leaves of Tabernaemontana heyneana Wall. Using L16 Orthogonal Design . Nature and Science, 6(3),

2008, ISSN: 1545-0740

Singh, M. (2007). Encyclopedia & Food Chemistry. New Delhi, India: Anmol Publications, PVT. LTD.

What is Food Spoilage? (2010). Culinary Arts. Retrieved from: http://culinaryarts.com/od/safetysanitation/a/bacteria-2.htm

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TASK LIST

Activity

Code

Activity Description Estimate

Duration

(days)

Observable Indicators of

AccomplishmentsImmediate

Preceding

Activity

A Procurement of Persimmon Leaves

7 100 grams (approximately

100 leaves)

N/A

B Procurement of Extracting Tools 5 0.75 mm wire mesh, 1 -

1000 mL beaker, 2 - 400

mL beaker, 1 – 250 mL

beaker

N/A

C Procurement of Canned Goods 1 Same brand, same

expiration date

N/A

D Extraction of Flavonoids from Persimmon Leaves

1 96 % Ethanol, RotaVap A and B

E Application of Persimmon Extracts

23 3.33% of Canned Good Net

Weight

C and D

17

NETWORK CHART

18

GANTT CHART

Legend:

Activity 1. Procurement of Persimmon Leaves

Activity 2. Procurement of Extracting Tools

Activity 3. Procurement of Canned Goods

Activity 4. Extraction of Flavonoids from Persimmon Leaves

Activity 5. Application of Persimmon Extracts

19

PROPOSED BUDGET

A. List of Materials

Materials Description and

Quantity

Source Estimated Cost

4 - Corned Beef Robinsons Market

4 – Cocktail Robinsons Market

4 – Meat Loaf Robinsons Market

4 – Sardines Robinsons Market

Total Budget for Materials

B. Rentals and Services

Not Applicable

C. Other Projected Expenses

Not Applicable

TOTAL BUDGET:

20