SURNAME; SCOTCH

INITIAL; K
ID; 201901195
DATE; 5\5\2023

BIO 420 MINI PROJECT

THE MICROBIOLOGICAL ASSESSMENT OF POST HARVEST IN TOMATOES AND
ITS SHELF LIFE

INTRODUCTION

Tomato (Solanum lycopersicum) fruit, naturally exists as a wild plant in the Americas to the tens
of thousands of varieties farmed now, tomatoes have developed into one of the most well-liked
food crops in the world. Today's tomatoes have a variety of sizes and shapes, ranging from small
and spherical to enormous and irregularly shaped (Jaksuma Pongsetkul, and Soottawat Benjakul,
2021). They were once wild plants in the Andes that now flourish in Bolivia, Chile, Colombia,
Ecuador, and Peru. An elongated fruit shape that normally grows 180 cm (6 ft.) or more above
the ground if supported—although upright bush types have been created, typically 100 cm (3 ft.
3 in) tall or shorter is a common physical trait separating many cultivated varieties from
undomesticated accessions. The "tender" perennials indeterminate kinds eventually die since
they came originating from tropical highlands, indeterminate varieties are "tender" perennials
that perish every year in temperate climates. Although in certain situations they can survive up to
three years in a greenhouse. Lycopene, a red pigment and antioxidant found mostly in tomatoes,
has been linked to a number of health advantages, including a decreased risk of cancer and heart
disease. Although technically a fruit, they are typically consumed and served as vegetables and
are an excellent source of vitamin C, potassium, foliate, and vitamin K., (Xiao Su. et al., 2021).
They also contain about 95% water and 5% that is primarily made up of carbs and fiber. They
can also be used to make fruit spreads like marmalades, jams, and jellies. Although they can be
kept chilled (below 40 degrees Fahrenheit) for a short while, raw tomatoes and raw tomato
products will eventually go bad because of bacteria, yeasts, and molds.

Tomatoes (Solanum lycopersicum), which are farmed and consumed worldwide, come in a
variety of fresh market and processed varieties. Up to 50% of the harvest might be lost due to
post-harvest losses, which are a significant contributor in crop production declines.
Characterizing fruit quality and assessing tomato fruits' post-harvest qualities are crucial steps in
the selection and breeding of outstanding tomato varieties. This involves examining a fruit's shelf
life (the amount of time it can be stored without losing quality), color, and sensitivity to
pathogens. The pace of fruit softening, which occurs along with fruit ripening and exacerbates
damage during shipping and handling, affects tomato shelf life (Jaksuma Pongsetkul, and
Soottawat Benjakul, 2021). Additionally, fruit ripening is sometimes connected to tomatoes'
susceptibility to fruit diseases, particularly for neurotropic fungi like Botrytis cinerea, also
known as gray mold.

Due to contamination by foodborne pathogens, fresh fruit supply has been connected to
multistate epidemics. Acidified sodium benzoate (NaB) has been shown to be beneficial in
decreasing pathogens inoculated on cherry tomatoes and preventing cross-contamination.
Organic acids like benzoic acid are efficient antimicrobials. The simplest aromatic carboxylic
acid and a type of organic acidifier is benzoic acid (C7H6O2, BA), a colorless crystalline solid.
Because it can prevent the growth of harmful microbes, benzoic acid is used as a preservative in
the food and feed industries (Del Olmo A., 2015). Additionally, it might promote health and
growth while preventing the diarrhea brought on by an E. coli challenge. The use of benzoic acid
as a food additive to enhance health is possible. Benzoic acid is an organic acid that was initially
utilized in foods about a century ago. According to Halas D., et al., 2009, the acid is naturally
found in prunes, cinnamon, and cloves. Because the free acid form is poorly soluble in water, the
sodium salt (sodium benzoate) is frequently used. The antibacterial activity of benzoic acid is
predominantly against yeasts and molds.

Tomatoes should typically be stored around 12-14oC (53-57oF) with 85% relative humidity. The
fruit can be properly preserved in this condition for 2 to 3 weeks. Tomatoes can also be canned,
dried, frozen, or pickled to extend their shelf life. Tomatoes have a short shelf life due to an
enzyme that digests their cell wall and softens the fruit. Pectin is found in tomato cell walls and
is destroyed by polygalacturonase. Polygalacturonase is a pectinase (a pectin-degrading enzyme).

The most recent reference genome, published in 2021, was 799 MB in size and encoded 34,384
(predicted) proteins scattered across 12 chromosomes (Xiao Su. et al., 2021).

The purpose of this study was to compare the quality changes and shelf-life of fresh tomato fruit
treated with 0.1% sodium benzoate (SB) and stored at room temperature (25-28 °C) for two
weeks to samples without preservatives
MATERIALS AND METHODS

For the experiment, tomato fruits (Solanum lycopersicum), were bought from Botswana Building
Society (BBS) mall in March 2023. The experiment was conducted at the University of
Botswana science laboratories and took a duration of two weeks until its termination time. The
fruits bought were sorted or packaged on the basis of uniformity in size, color, ripeness and
absence of visible injury and then divided into 3; the control, and treatment 1 and for treatment 2.
Fruits of treatment 1 and for treatment 2 were dipped for 5 minutes in solution of sodium
benzoate of concentrations1%. The control fruits were dipped in water only. The experiment
comprised of two treatments with one replications in each treatment. The fruits from every
replication of each treatment and control were packed in transparent plastics (perforated) and
kept at 4ºC and 25ºC for 14 days. For study of storage behavior, fruit samples were analyzed
after 1, 5, 10 and 14 days of storage for various physicochemical characteristics.

Preparation of 1% sodium benzoate

1.42g of sodium benzoate powder was weighed using a weighing balance. It was then poured
into a 1 liter bottle and stirred until dissolved. The solution was kept to be used for the treatment
of fresh tomato fruits.

Tomato swabbing, serial dilutions and plaiting

The surface of the tomato fruit was swabbed and the tip was vortexes for about 45-60 minutes. 1
ml of the starting sample was added to 9 ml of Tube 1. This is then followed by the same
procedure, where 1 ml from Tube 1 is added to 9 ml of Tube 2, 1 ml from Tube 2 is added to 9
ml from Tube 3, and so on making some serial dilutions. 0.5ml of the solutions from tube 1 and
from tube 3 was pipetted onto the plates with media. A rod sterilized by flame was used to
spread the inoculum plated around the plate. The plates were stored at a temperature of 37ºC for
24 hours for growth to occur and then moved to 4ºC to inhibit microbial growth and cross
contamination of the of the colonies. The colonies were counted and recorded. Cells from a
previous culture was transferred into fresh growth medium by using a loop and streaking into a
fresh growth media to get pure colonies from the culture which was then used for biochemical
tests, DNA extraction and gram staining.
Gram staining

A clean slide was used. And smeared with the colony sample using a sterilized loop .the wet
smear was air dried and heat fixed. Crystal violet was poured and kept for about 30 seconds to 1
minutes and rinse with water. The smear was then flooded with iodine for 1 minute and wash
with water. Then, washed with 95% alcohol for about 45 seconds and rinse with water, safranin
was added for about 1 minute and washed with water. The slide was then air dried and observed
under the microscope and results were recorded.

Preparations of media

a. Nutrient agar (NA)

56g of nutrient agar powder was weighed using a weighing boat and weighing balance into a 2L
distilled water bottle. The mixture was mixed using a magnetic stirrer for 10-15 minutes until it
had completely dissolved. It was then boiled for 30 minutes and then autoclaved at 121°C for 15
minutes. It was then left to cool and poured into the petri dished and wait for the media to
solidify and then refrigerated at 4°C.

b. Potato Dextrose Agar (PDA)

Add 79g of Potato Dextrose Agar (PDA) to 2 liter of distilled water. Mix the mixture using a
magnetic stirrer for 10-15 minutes until it had completely dissolved. It was then boiled for 30
minutes and then autoclaved at 121°C for 15 minutes. It was then left to cool and poured into the
petri dished and wait for the media to solidify and then refrigerated at 4°C.

c. Sabouraud Dextrose Agar (SDA)

Weigh 84g of Sabouraud Dextrose Agar (SDA). Mix the mixture using a magnetic stirrer for 10-
15 minutes until it had completely dissolved. It was then boiled for 30 minutes and then
autoclaved at 121°C for 15 minutes. It was then left to cool and poured into the petri dished and
wait for the media to solidify and then refrigerated at 4°C.

Preparation of 0.8% agarose gel

Preparation for 25ml 50X TAE buffer, 6.05g of Tris-HCL, 0.4625ml of EDTA and 1.4275ml of
glacial acetic acid were mixed and dissolved. The 1ml EDTA was prepared whereby 0.1861g of
EDTA was mixed with 1ml of distilled water. 1X TAE buffer of 1L was prepared by adding
20ml of 50X TAE buffer and 980ml of distilled water. For the gel electrophoresis(0.8%), 1.6g of
gel agarose, 200ml of TAE buffer, 1X(gel preparations(2x trays), 700ml of TAE buffer 1x (gel
running), ethidium bromide 3µl\ 100ml, loading dye of 2µl and the molecular ladder of 3µl

Preparations for biochemical tests

I. Catalase test
A drop of Hydrogen peroxide was poured on top of a clean slide and the colony sample
of bacteria was mixed with it. If bubbles appear (due to the production of oxygen gas) the
bacteria are catalase positive. If no bubbles appear, the bacteria are catalase negative.

II. TSI (500g)

Take 5ml of the autoclaved TSI media in a sterile test tube and slant was made by tilting
the media till it solidified. A flamed inoculating loop was used to pick a colony from the
bacterial culture and then streak on the whole slant surface of the medium. Incubate at
37°C for 18-24 hours. The color change in the slant and butt was observed, gas
production and H2S production.

III. Oxidase test

In a sterile petri plate, place a strip/disc of Whatman no. 1 filter paper. Soak the filter
paper with 1% Kovacs’ oxidase reagent and let it dry. Using a sterile inoculating loop,
pick up a well-isolated colony of test bacteria from a fresh (18 to 24 hours old) culture
and make a smear on the reagent-soaked filter paper piece. Observe for color change and
note the time required for change in color for up to 60 seconds

IV. Tryptone broth (500ml)

To test for indole production, inoculate a 4-ml tryptone broth tube with one loopful of
culture. After 24 to 48 hours of incubation, add to the culture 1.0 ml of either ether or
xylene. Mix well and then allow organic solvent to rise to the top of the medium
DNA quantification and gel electrophoresis

For optimal performance, add beta-mercaptoethanol (user supplied) to the Genomic Lysis Buffer
to a final dilution of 0.5 % (v/v) i.e., 500µl per 100 ml. Add 50 – 100 mg (wet weight) fungal or
bacterial cells1 that have been suspended in up to 200 µl of water or isotonic buffer (e.g., PBS)
to a ZR Bashing Bead™ Lysis Tube (0.1 mm & 0.5 mm). Add 750 µl Bashing Bead™ Buffer to
the tube2. Secure in a bead beater fitted with a 2 ml tube holder assembly and process at
maximum speed for ≥ 5 minutes. (Required processing time will vary depending on the device
and application and therefore should be evaluated on a case by case basis). Centrifuge the ZR
Bashing Bead™ Lysis Tube (0.1 & 0.5 mm) in a micro centrifuge at 10,000 x g for 1 minute.
Transfer up to 400 µl supernatant to a Zymo-Spin™ III-F Filter in a Collection Tube and
centrifuge at 8,000 x g for 1 minute. Add 1,200 µl of Genomic Lysis Buffer to the filtrate in the
Collection Tube from Step 4. Transfer 800 µl of the mixture from Step 5 to a Zymo-Spin™ IICR
Column3 in a Collection Tube and centrifuge at 10,000 x g for 1 minute. Discard the flow
through from the Collection Tube and repeat Step 6. Add 200 µl DNA Pre-Wash Buffer to the
Zymo-Spin™ IICR Column in a new Collection Tube and centrifuge at 10,000 x g for 1 minute.
Add 500 µl g-DNA Wash Buffer to the Zymo-Spin™ IICR Column and centrifuge at 10,000 x g
for 1 minute. Transfer the Zymo-Spin™ IICR Column to a clean 1.5 ml micro centrifuge tube
and add 100 µl (35 µl minimum) DNA Elution Buffer directly to the column matrix. Centrifuge
at 10,000 x g for 30seconds to elute the DNA. Ultra-pure DNA is now ready for use in the
experiments.
RESULTS

Sample Biochemical Test

TSI catalase oxidase Tryptose
broth
Slant Butt
Sample 5 red (+) yellow(+) - + -
Sample 6 red(+) Yellow (+) + - -
Table 1. Biochemical test results for bacteria found on tomato fruit surface

KEY; -Reflects positive results + Reflects negative results

The results shows positive results for TSI with a color change from red to yellow, for catalase
test, a positive test for sample 6 as bubbles appeared indicating production of oxygen gas and
negative for sample 5 as no bubbled formed. The oxidase test results showed positive results for
sample 5 as the litmus paper color changed to blue and negative for sample 6 as they was no
color change For the iodole test they was no red color formed, indicating negative results for
both sample 5 and 6.

Table 2. Gram stain results

sample Gram stain

Cell morphology Gram status
Sample 5 rods shape, aggregates, purple positive
Sample 6 cocci, clustered cells, purple positive

Samples were positive for the gram stain and showed positive gram stains and sample 5 with rod,
aggregates and sample 6 with cocci, clustered cellular morphology

Microbial growth from tomato fruit surface

70
66

60
54.2

50

43.4
log CFUs/ml tomatoes

39.8
40
control
treat. 1
32
treat 2
30
24.8

20

11.8
10 7.2 8

0.040.010.12 0.390.410.48 0 0.380.47

0
1 7 14

Days

KEY; each day has results for 0.1 Dilution and 0.001 dilutions respectively

The result shows that sodium benzoate had an effect in prolonging the shelf life of the tomato
fruit in terms of microbial growth and fruit firmness as they are lower rates of microbial growth
indicated by log CFUs. The graph also shows that they are a decreased number of microbial
growths in 0.001 dilutions as compared to 0.1 dilutions which shows to have containing more
microorganisms.

NB; they was no results for the gel electrophoresis, which may have been to errors during
pipettes, wrong gel dilutions and bacterial extractions.

DISCUSSION

Benzoic acid is frequently used in foods and feeds as an antibacterial and antifungal preservative.
Its antibacterial action is highest at pH 2.5-4.0 (Andersen F. A., 2001). Benzoic acid inhibits the
growth of bacteria and yeasts, which is a primary cause of food spoilage. Although the addition
of benzoic acid might extend the shelf life of drinks and avoid nutritional losses, excessive
benzoic acid consumption can cause diarrhea, abdominal pain, and other symptoms, as well as
interfere with the body's intermediary metabolic processes. According to Andersen F. A., 2001,
Benzoic acid has long been used as an antibacterial ingredient in foods, usually in the form of its
sodium salt, sodium benzoate. Carbonated and still beverages, syrups, fruit salads, icings, jams,
jellies, preserves, salted margarine, mincemeat, pickles and relishes, pie, pastry fillings, prepared
salads, fruit cocktail, soy sauce, and caviar are all made with it. The level of utilization ranges
from 0.05 to 0.1%. Sodium benzoate is an organic acid with strong antibacterial action at low
pH. The impact is caused by the unionized form's increased permeability to microbes.

The result indicates that samples treated with 0.1% sodium benzoate exhibited slower rate of
quality changes throughout storage, including microorganisms, change in the surface texture,
moisture quantity observed in the fruit as well as its size. These samples can store at room
temperature for at least 2 weeks without any spoilage. However samples without preservatives,
revealed a higher rate of the changes in quality characteristics, underwent spoilage during
2weeks storage at different times depending on the temperature. The shelf-life of treatment 1 was
1 and a half week and that of treatment 2 was 2 weeks as indicated by the excess of total
microorganisms (>1.00 × 104 CFU/g sample). A higher number of CFUS might have been to
contamination when free handling the tomatoes when swabbing. Overall, the results indicated
that using sodium benzoate at the level of 0.1% can effectively extend the shelf-life of fresh
tomato fruits for more than 2 weeks.

The biochemical tests for TSI was positive, oxidase and catalase test had both the positive and
negative results whereas the tryptone broth had tested negative for both of the samples 5 and 6.
In TSI, an acid/acid (yellow slant/yellow butt) reaction, It indicates the fermentation of dextrose,
lactose and/or sucrose. An alkaline/alkaline (red slant, red butt) reaction shows the absence of
carbohydrate fermentation results. Microorganisms are oxidase positive when the color changes
to dark purple and they are oxidase negative if the color does not change or it takes longer than 2
minutes. The chemicals in Kovac's reagent react with the indole to produce a red color. In a
positive test, a red layer will float at the surface of the broth. Lastly catalase is an enzyme that
converts hydrogen peroxide to water and oxygen gas. Bacteria are simply mixed with H2O2. If
bubbles appear (due to the production of oxygen gas) the bacteria are catalase positive. If no
bubbles appear, the bacteria are catalase negative.

Form the gram staining results it can be identified that some of the microorganisms that
contributes to the spoilage of tomatoes is Botrytis cinerea, Tricophyton mentagrophytes,
Monascus purpureus fungi, Rhodotorula mucilaginosa yeast.

When consumed in tomatoes, sodium benzoate salt can have a positive impact on humans
because benzoic acid administration can improve gut functions such as digestion, absorption, and
barrier, which is an important reason why benzoic acid can improve growth and health in animal
studies (del Olmo.et al., 2015). This is because it has the ability to affect enzyme activity, redox
status, immunity, and microbiota. We can further ensure that benzoic acid may be employed as a
type of gut health product in humans and animals by analyzing the outcomes of these
investigations. It also has the potential to be utilized as a dietary supplement to improve health,
particularly for some convalescent patients, (Sim G. A., et al., 1955). Should be supplemented at
a rate of 0.2- 0.5% in feeds. However, Excessive BA consumption, on the other hand, causes
acute or chronic toxicity symptoms that adversely damage human and animal health and growth
(Andersen F. A., 2001). Recent studies using piglets as the model have also revealed that
excessive dietary BA supplementation causes liver, spleen, and lung malfunction and damage, as
well as impairing the mucosal morphology of the duodenum, jejunum, and ileum Excess BA
administration decreased the growth of IPEC-1 cells in an in vitro experiment, which could be
connected to the impairment of redox state mediated by the Nrf2 pathway.

Boron has a positive influence on the level of cracking and shelf life of the tomato fruit. Any
cracks on the fruit shoulder increases water loss and susceptibility to pathogen attack and can be
also used for better preservation of tomatoes and increase their shelf life, their safety and quality
for consumption.

CONCLUSION

The study indicated that postharvest treatment with 0.1% sodium benzoate was most successful
in preserving the physicochemical properties and enzymatic activity of tomato fruits stored at
low temperatures. It also greatly reduced postharvest deterioration and increased the storage life
of the fruits together with its safety and quality.

REFERENCES

Andersen F. A., 2001, Final report on the safety assessment of Benzyl Alcohol, Benzoic Acid
and Sodium Benzoate. International Journal of Toxicology.;20(3):23–50. doi:
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del Olmo A., Calzada J., Nuñez M., 2015, Benzoic acid and its derivatives as naturally occurring
compounds in foods and as additives: Uses, exposure, and controversy, Critical Reviews in Food
Science and Nutrition.;57 (14):3084–3103. doi: 10.1080/10408398.2015.1087964.

Halas D., Hansen C. F., Hampson D. J., Mullan B. P., Wilson R. H., Pluske J. R., 2009, Effect of
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Torrallardona D., Badiola I., Broz J, 2007, Effects of benzoic acid on performance and ecology
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Xiao Su, Baoan Wang, Xiaolin Geng, Yuefan Du, Qinqin Yang, Bin Liang, Ge Meng, Qiang
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APPENDIX
Gram staining results ad the oxidase test results

Bacterial gel electrophoresis results and biochemical results