NAME: NKOSINATHI MASHIANE

STUDENT NUMBER: 220000387

SUBJECT: FOOD MICROBIOLOGY 3 PRACTICAL

SUBJECT CODE: ABFMT3A

LECTURER: MS Z. MARRENGANE

PRACTICAL DONE AT: VUT F104 (SEPTEMBER 2022)

PROJECT THREE REPORT: THE EFFECT OF pH ON FOOD SPOILAGE.

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TABLE OF CONTENTS
INTRODUCTION……………………………………4,5
AIM……………………………………………………5
OBJECTIVES………………………………………..5
MATERIALS AND METHODS…………………….5,6
RESULTS……………………………………………5,6,7
DISCUSSION………………………………………..8,9
CONCLUSION………………………………………9
REFERENCES……………………………………...10

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INTRODUCTION
The pH value of a food is a direct function of the free hydrogen ions present in that
food. Acids present in foods release these hydrogen ions, which give acid foods their
distinct sour flavor. Thus, pH may be defined as a measure of free acidity. More
precisely, pH is defined as the negative log of the hydrogen ion concentration.
Therefore, if a food has a pH value of 3, then the concentration of hydrogen ions
present in that food is equal to 0.001 mol/l. If the pH value is 6, then the concentration
of hydrogen ions equals 0.000006 mol/l. These examples show the concentration of
decreases as the pH value of the food increases. This explains why a low-pH food is
a high-acid food and vice versa. The range of pH is commonly considered to extend
from zero to 14. A pH value of 7 is neutral, because pure water has a pH of exactly 7.
Values less than 7 are considered acidic, while those greater than 7 are considered
basic or alkaline (Anon, 1972).
It has been established that microorganisms grow best under conditions around
neutrality. Very few grow below pH 4. Bacteria tend to be the most fastidious in their
relationship to pH than yeast and moulds with pathogenic bacteria being the most
fastidious. Fruits, soft drinks, vinegar and wines all fall below the point at which
bacteria normally grow. Fruits generally undergo mould spoilage because moulds
have the capacity to grow at pH 3.5 or less. Meats and seafood have pH values around
5.6 making them prone to spoilage by bacteria, yeasts and moulds. Most vegetables
have higher pH’s than fruits and therefore be prone to bacterial than fungal spoilage.
Some foods are characterized by inherent acidity, others owe their acidity or pH to
action by certain microorganisms. Some foods are better equipped to resist pH
changes than others and are considered buffered.
Microorganisms, including yeasts, moulds and bacteria are sensitive to a food’s pH.
Very low or high pH value will prevent microbial growth. As a practical matter, no
unprocessed foods have a pH value high enough to offer much preservative value.
Many foods do have pH values low enough to offer some protection against microbial
growth. However, very few foods have pH low enough to completely inhibit the growth
of microorganisms, especially yeast and moulds which tolerate lower pH conditions
than most bacteria. Microorganisms that can survive in a lower pH are called
acidophiles and have the following properties: Extreme acidophilic archaea can grow
at pH 0.06, low pH destroys the cell membrane due to destruction of membrane
phospholipids but have unusual amino acids that sensitive to alkali but stable at low
pH and impermeable to hydrogen ion, at pH the cell membrane of acidophiles
disintegrate, killing the cell, example include Picrophilus. Neutrophiles are a type of
white blood cell that helps heal damaged tissues and resolve infections, lastly are
Alkaliphiles which are a class of extremophilic microbes that are capable of survival in
alkaline conditions (pH roughly 8.5 – 11) environments, growing optimally around a
pH of 10 (Anon, 1962).
When it comes to keeping food safe, food storage is a major issue. Food which is not
correctly stored can spoil or become contaminated, which can then lead to diseases.
Food storage refers to food which is kept in the fridge, freezer, and pantry or in some

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instances a warming receptacle. There are very specific rules regarding the
temperatures that food must be stored at, cooked to and if not followed, the risk of
becoming ill as a result of contamination increases (Newman et al, 2016).

There are three basic types of food spoilage which are appearance textural changes
and changes in colour and taste. These types of food spoilage on ground beef are
caused by Gram negative aerobic rods (Pseudomonas, Acinetobacter and Moraxella),
Bacillus and clostridia. Bacillus are gram positive spore that form rods, aerobic when
to anaerobic clostridia and have two pathogens B. anthacis and B. cerecis.
Acinetobacter are organisms with gram negative rods with affinity to Neisserriacea,
they differ from Moraxellae in being oxidase negative, strict aerobes that reduce
nitrate, when the culture is young, they are rod shaped and when they get old, they
become cocci shaped. Clostridia organisms are gram positive spore forming rods, they
grow in mesotrophic, psychotropic and thermotropic conditions (Jay, Loessener, &
Golden, 2005).

To isolate the type of microorganism in the sample a series of dilutions were

performed; the purpose of this is to decrease the concentration of the microorganism
to yield single colonies between 30 and 300. The first dilution contained 1 gram of the
sample and 9 millilitres of diluent, the rest of the dilutions contained 1 millilitres of
previous dilution and 9 millilitres of diluent (Wiley, et al., 2008).

The serial dilutions were plated in spread plate methods. In the spread plate
method=the dilutions are transferred on to the centre of the agar plate and is then
evenly spread over the surface of the agar with a sterile bent-rod. The numbers of
colonies that grow on the plate equal the number of viable organisms in the sample
(Wiley, et al., 2008).
There were two types of agars used in this experiment; the Plate Count Agar and the
Potato Dextrose Agar. Plate Count Agar (PCA) is free from selective supplements and
relatively rich in nutrients, making it ideal for the enumeration of viable organisms. It is
used for the enumeration of bacteria in water, wastewater, food and dairy products in
a laboratory setting (Safety, n.d.).
Potato Dextrose Agar (PDA) is a general-purpose basal medium for the identification,
cultivation and enumeration of yeast and moulds in foods and dairy products. PDA
contains dextrose as a carbohydrate source which serves as a growth stimulant, and
potato infusion that provides a nutrient base for luxuriant growth of most fungi. Agar
is added as the solidifying agent. A specified amount of sterile tartaric acid (10%) may
be incorporated to lower the pH of the medium to 3.5, so that bacterial growth is
inhibited (Rijal , 2015).

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AIM
The effect of pH on food spoilage
OBJECTIVES
1.To take pH reading of sample
2.To perform a serial dilution of sample
3.To perform a spread plate technique on Plate Count Agar and Potato Dextrose Agar
4.To perform a plate count
MATERIAL
• Pineapple juice
• Peach Chutney
• Mustard
• Lemon juice
• pH meter
• Saline
• Plate Count Agar
• Potato Dextrose Agar
PROCEDURE
The sample of the above food was brought to class and a pH meter was used to
measure the pH of the different products. Also, the appearance (texture, smell and
texture) of the product was recorded. A 1:10 dilution series of all sample was made
and 1 ml was plated from each dilution on the Plate Count Agar and Potato Dextrose
Agar. After plates were incubated at 37°C for 24 hours, they were checked for growth.
The food sample were stored at room temperature and were used in week 2.
Week 2
The pH of each of the products was measured and results were recorded. The
appearance, smell and texture of all samples was recorded. Another 1:10 dilution
series was prepared and 1 ml was plated from each dilution on the Plate Count Agar
and Potato Dextrose Agar.
RESULTS
Week 1
Table1: Ph values and morphology of some food products for week 1

Sample Appearance Smell Colour pH

Apple juice Sweet and 4.0
Liquid fresh Brownish

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 Tomato sauce Grainy smooth Fresh Brick red 3.0
like texture
Pineapple juice Not Sweet and
Cloudy fresh Yellowish 3.3
Lemon juice Fresh and
Liquid strong Lime 2.6

Week two
Table 2: pH values and morphology for food products week 2

Appearance/
Sample texture Smell Colour pH
Apple juice Cloudy Smells sour Brownish 3.7
Tomato sauce Grainy, Bit sour Brick red 4.1
smooth and
liquid like
texture
Pineapple There was Smells sweet Brownish
juice growth 3.9
Lemon juice Thick Smells sweet Lime milky 2.2

Table 3: morphology on some food products found on PCA and PDA plates

PLATE COUNT AGAR POTATO DEXTROSE AGAR

Shape Texture Elevation Colour Number Shape and Texture Elevation Colour Number
and size of size of
colonies colonies
(CFU/ml) (CFU/ml)
APPLE JUICE

Circular Smooth Convex Cream Too 10- Circular, Smooth Raised Cream 72 with 1
white, numerous 1 irregular Yeasts, huge
yellowish to count form in the rough mould
middle moulds
Circular Smooth Convex Yellow, Too -
10 Irregular Smooth Convex Cream 15 yeasts
cream numerous 2 shape all yeasts, white colonies
white to count over the rough and many
plate with moulds moulds
few
circular
TOMATO SAUCE

Circular Smooth Most of them Creamy 157 10- Circular Smooth Convex Creamy 77
and Yeast were white 1 and Yeast and white
colonies spherical colonies

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spherical convex(raised) some
shape and few flat were flat
Circular Smooth Most of them Creamy 49 10- Circular Smooth Convex Creamy Too few
and Yeast were convex white 2 and Yeast and flat white to count
spherical colonies and few were spherical colonies
shape flat
PINEAPPLE JUICE

Mostly Smooth Convex White, Too 10- Irregular Cottony Flat Cream Too
circular, creamy numerous 1 shape colonies (mould) white numerous
3 had an white to count Spread (mould) Convex to count
irregular throughout Smooth (yeast)
shape most of yeast
the colonies
surface
Mostly Smooth Convex White Too 10- Circular Cottony Convex White 25 mould
circular, and numerous 2 (mould), colonies colonies
a few cream to count small (mould) 50 yeast
had an white Circular Smooth colonies
irregular and yeast
shape Irregular colonies
(yeast),
big

LEMON JUICE

Circular Smooth Flat White 6 10- Circular Smooth Convex Creamy Too
1 yeast white numerous
colonies to count
Irregular Rough Flat Creamy Too 10- Irregular Smooth Convex Cream 41
white numerous 2 and few Yeast white
to count circular colonies

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DISCUSSION
In the 1st week, the apple juice had a sweet and fresh smell, it’s appearance or texture
was liquid like with a brownish colour and a pH of 4.0 but in the 2nd week the apple
juice smelled sour, it’s appearance and retained its brownish colour however the pH
had decreased to 3.7 making the apple juice more acidic. The apple juice was serially
diluted and plated on the PCA and PDA plates. The growth observed in the first week
on the PCA plates was only for the 10-1 and 10-2 dilutions. For the 10-1 plate on PCA,
the colonies were too numerous to count, cream white yellowish in colour, circular
shape, smooth texture and convex elevated. for PCA 10-2 plate, the colonies were too
many to count, yellow and cream white colour circular shape, smooth texture and
convex elevated. The decrease in pH of the apple juice and the lemon juice from week
1 to week 2 is as a result of samples deemed unfit. Both a high lactic acid content and
a low pH indicated that the sample was spoiled
For PDA 10-1 plate, the number of colonies were 72 with one huge mould, cream
colour, circular and irregular form of size in the middle, smooth yeasts and rough
moulds in texture with a raised elevation and for PDA 10-2 plate, number of colonies
were 15 yeast colonies and many mould, cream white colour, Irregular shape all over
the plate with few circular colonies, smooth yeasts and rough moulds in texture and
colonies were convex elevated.
The tomato sauce had a fresh smell, grainy smooth texture like appearance, with a
brick red colour and a pH of 3.0 for the 1st week but for the 2nd week the smell had
changed to be a bit sour, the appearance and colour still remained the same as in
week one. However, the pH has increased to 4.1. The tomato sauce PCA 10-1 plate,
the number of colonies were 157 in cream white colour, circular and spherical in shape,
smooth yeast colonies in texture and most of the colonies were raised in elevation with
few flat and for the PCA 10-2, the microbial load had decreased to 49, shape, size,
smoothness and texture remained the same as in 10-1 plate. For PDA 10-1 plate, the
number of colonies were 77 and for the 10-2 plate the colonies were too numerous to
count. However, both plates PDA plates for 10-1 and 10-2, shape was circular and
spherical with colonies in cream white colour, smooth yeast colonies in texture and in
a convex and flat elevation.
The pineapple juice had a sweet and fresh smell, not cloudy appearance, yellowish
colour, and a pH of 3.3 for the 1st week but for the 2nd week pineapple juice smelled
sweet, appearance showed no growth, colour had changed to brown and the pH had
slightly increased to 3.9. The pineapple juice PCA 10-1 and 10-2 plates, for both plates
the number of colonies were too many to count, both in white and creamy white colour,
both plates were convex elevated, same smooth texture, the only difference was she
shape as PCA 10-1 had mostly circular with only 3 colonies in irregular shape and for
10-2 plate most colonies in circular and few colonies in irregular shape. For PDA 10 -1
plate, colonies were too numerous to count in cream white colour, colonies were in
irregular shape and were spread throughout most of the surface, texture was cottony
colonies (mould) smooth yeast colonies and the mould flat elevated, yeast convex
elevated and for the 10-2 plate, we had 25 mould colonies and 50 yeast colonies,

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colonies white in colour, circular shaped (mould), small circular and shaped big yeast,
convex elevation and texture was cottony colonies (mould) smooth yeast colonies.
Lemon juice had strong and fresh smell, liquid appearance, lime colour and a pH of
2.6 for the 1st week but for the 2nd week, the lemon juice had a sweet smell, its
appearance had turned thick, lime milky colour and pH of lemon juice had slightly
decreased to 2.2. The PCA for 10-1 plate, there were 6 number of colonies which were
white in colour, circular in shape, smooth texture and colonies were flat elevated. For
10-2 plate, the colonies were too many to count in cream white colour, irregular shape,
rough texture and flat elevated colonies For PDA plates, 10-1 and 10-2 had the same
convex elevation, both plates colonies were cream white in colour, their texture was
smooth yeast colonies. 10-1 plate colonies were too numerous to count and were
circular shaped, 10-2 plate had 41 colonies and were irregular with fewer colonies
circular shaped.
The decrease in pH of the apple juice and the lemon juice from week 1 to week 2 is
as a result of samples deemed unfit. Both a high lactic acid content and a low pH
indicated that the sample was spoiled and the increase in pH of the tomato sauce and
pineapples is as a result of both samples being very much susceptible to microbial
spoilage. pH in the context of preserving food is important because it determines the
time and temperature to which we must submit a food product to effectively eliminate
the microbiological and enzymatic activity, ensuring the stability of the final food
product.
Potato Dextrose Agar plates pH 5.6 and pH 3.5 are solid media plates recommended
for use in qualitative procedures for the identification, cultivation, and enumeration of
yeast and moulds as we saw with the 10-1 and 10-2 plate for the four different types of
product samples.
Plate count agar (PCA) is a bacteriological substrate used for the determination of the
total number of live, aerobic bacteria in a sample (Ronald and James, 2014). It is not
a selective medium as we observed in the 10-1 and 10-2 plates in different product
samples. PCA is used for the enumeration of bacteria in food, water and other
materials of sanitary importance and it is also suitable for enumerating bacterial count
of sterile rooms as observed with the counting of the colonies in the 10-1 and 10-2
plates under PCA for the four product samples. PCA is a general-purpose medium
and between the 10-1 and 10-2 plates and may not support the growth of fastidious
organisms.
CONCLUSION
The pH of foods can affect them in a way that when the product or food is not stored
properly its microbial load will increase thus making the food to be too acidic as the
pH decreases hence the storage conditions of food and the pH correlate. Low
temperature prevents the microbes from growing. Low temperature prevents the
microbes from breathing and also reducing the pH of the food helps to reduce food
spoilage as the low pH kills the microbes.

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REFERENCES

1. Anon, 1962. pH value of food products. Food Eng. 34(3):98-99. ‘’Acidified

Foods’’ Title 21, Part 114 (21CFR114) in Code of Federal Regulations,
Government Printing Office, Washington D.C. 1999.
2. Jay, J. M., Loessener, M. J. & Golden, D. A., 2005. Mordern Food Microbiology.
New York: Springer Science+ Business Media, Inc.
3. Newman DJ, Cragg GM (2016) National Research Council of ground
meat1981- 2014
4. Rijal , N., 2015. Potato Dextrose Agar (PDA): principle, composition and
colony characteristics, s.l.: microbeonline. available at:
https://microbeonline.com/potato-dextrose-agar-pda-principle-composition-
colony- characteristics/>. Accessed: 15/10/2021
5. Ronald M. Atlas and James W. Snyder (2014). Handbook of media for clinical
and public health microbiology. CRC Press. Taylor & Francis Group, LLC.
6. Last updated: August 10, 2022 by Sagar Aryal.
https://microbiologyinfo.com/potato-dextrose-agar-pda-principle-uses-
composition-procedure-and-colony-characteristics/
7. Willey, J. Sherwood. L. Woolverton. C. (2008). Prescott’s, Marley, and Klein's
Microbiology, 7th edition. New York: The McGraw-Hill Companies.

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