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This Chapter present the different essential elements: The introduction which contains the
rationale, the literature of the study, and foundation, the general and specific problem, the scope
and delimitations that identifies the major variable the sub-variables and the indicators,
significance of the study which states the beneficiary of the further study and the corresponding
Introduction
Milk is the nutrient liquid secreted by the mammary glands of female mammals, which the
female’s ability to produce milk is one of the defining characteristics of mammals. The early
lactation milk is known as colostrum and carries the maternal antibodies to the baby. It can help
reduce the risk of various diseases in both the mother and baby that could benefit them in long
term. (USDA National Nutrient Data Base for standard references, 2005).
Exclusive Breastfeeding is recommended to mothers worldwide for the child's first six
months to achieve optimal growth, development and health. Thereafter, they should be given
nutritious complementary foods and continue breastfeeding up to the age of two years or beyond
(WHO, 2011). Although experts believe breast milk is the best nutritional choice for infants,
breastfeeding may not be possible for all women. For many, the decision to breastfeed or
formula feed is based on their comfort level, lifestyle, and specific medical situations which for
mothers who are unable to breastfeed or who decide not to, infant formula is a healthy alternative
that Formula Milk also provides babies with the nutrients they need to grow and thrive. (Ben-
which is based on a human mother’s milk nutrition. However, there are significant differences in
the nutrient of the Human Milk and the Infant Formula, but with that deficiencies, manufacturers
often innovate and add new ingredients to infant formulas in an attempt to mimic the
composition or performance of human milk (Wells, J.C.K., 1996). The popularity of Infant
Formula Milk increased because it has a longer shelf life than liquid milk and does not need to be
refrigerated due to its low moisture content and it can be accessible and convenient to mothers
Food spoilage, and food poisoning are caused by numerous types of microorganisms that
are influenced by many factors, that includes environment for which food was produced and
acquired, the microbiological quality of the food in its processed state, the sanitary conditions
under which the product was handled and processed and the adequacy of subsequent packing,
handling, and storage conditions in maintaining the microflora at low level (Jay, 1986).
Food spoilage can be defined as “any sensory change (tactile, visual, olfactory or flavor)”
which the consumer considers to be unacceptable. Spoilage may occur at any stage along food
chain. Spoilage may arise from insect damage, physical damage, indigenous enzyme activity in
the animal or plant tissue or by microbial infections. Most natural foods have a limited life.
Perishable foods such as fish, meat and bread have a short life span. Other food can be kept for a
considerably longer time but decomposes eventually. Enzymes can bring about destruction of
polymers in some foods while chemical reactions such as oxidation and rancidity decompose
others but the main single cause of food spoilage is invasion by microorganisms such as moulds,
yeast and bacteria. In case of mould spoilage a furry growth covers the food and it becomes soft
and often smells bad. Bacterial contamination is more dangerous because very often food does
not look bad even though severely infected, it may appear quite normal. The presence of highly
dangerous toxins and bacterial spores is often not detected until after an outbreak of food
poisoning, laboratory examination uncovers the infecting agent (Seema, R., 2015).
Chemical reactions that cause offensive sensory changes in foods are mediated by a
variety of microbes that use food as a carbon and energy source. These organisms include
prokaryotes (bacteria), single-celled organisms lacking defined nuclei and other organelles, and
eukaryotes, single-celled (yeasts) and multicellular (molds) organisms with nuclei and other
organelles. Some microbes are commonly found in many types of spoiled foods while others are
more selective in the foods they consume; multiple species are often identified in a single spoiled
food item but there may be one species (a specific spoilage organism, SSO) primarily
responsible for production of the compounds causing off odors and flavors. Within a spoiling
food, there is often a succession of different populations that rise and fall as different nutrients
become available or are exhausted. Some microbes, such as lactic acid bacteria and molds,
This study aims to identify the differences of the Microflora of different Spoiled Infant
Formula subjected to different PH Water Level. This study is to be conducted during the second
With regard to this, the researchers came up with following questions to be answered:
Specific:
I. What are the bacterial species can be found in Spoiled Infant Formula?
II. Are there any significant differences to the Microflora of Spoiled Infant Formula which are
a. Colony Shape
b. Colony Elevation
c. Colony Margin
d. Colony Size
e. Colony density?
III. Which among the PH Water Level have the most bacterial growth?
Hypothesis
There are significant differences among the Microflora of Spoiled Infant Formula
a. Colony Shape
b. Colony Elevation
c. Colony Margin
d. Colony Size
e. Colony density
This study would primarily deal with the differences of the Effects of the PH Water Level
The selection of Infant formula would be based on the availability of the product in the
Philippine market, and the recommended age intake of Infant Formula from 6-12 months. The
brand of infant formula would be 'Lactum 6-12 months' . The infant formula would be prepared
in a controlled environment for the sake of limiting the effects of extraneous variables.
For the selection of Water sample, researchers would also base it on the availability of the
products in the Philippine Market. The Water sample would be Fresh Tap Water, Boiled Tap
Water, Distilled Water, and Sterilized Water. These Water would be tested with PH Meter to
For the culture media that would be used for isolation of bacterial species are Potato
Dextrose Agar, Standard Method Agar, Nutrient Agar, and Violet Red Bile salts Agar would be
acquired from Asiagel Corporation that are prepared under sterile and controlled conditions
following Laboratories Conda Spain procedures, standards, and protocols. Furthermore, these
microflora would be then subjected to purification to attain a more reliable and accurate bacterial
growth.
Identification of isolated bacteria would be crucial because this is also the part of gathering
of data. Several tests such as Gram Staining Technique, Motility Test, Coagulase Test, Catalase
Test, Oxidase Test, Indole Test, Voges-proskauer Test, Glucose-Gas Fermentation, Lactose
Fermentation, and Sucrose Fermentation. These tests would help the researchers identify the
This study aimed to prove if the use of different PH Water Level would have effects on the
Every parent heed about their health and feeding. The best source of babies feeding is
their mother’s milk thru breastfeeding. But in few cases the mother naturally fails to fulfill the
breast feeding requirement of baby due to disease factor or hormonal imbalance. But in some
cases the mother itself is not interested in breastfeeding, reasons manifested, the working females
can not provide proper feeding to their baby. In rare cases modern world females do not feed
their babies just to maintain their apparently beauty. So they follow the infant formula that is
available in market and suggested by their nutritionist or doctor. In this generation, the Infant
milk powders are generally considered as product of good microbiological quality with no risk of
spoilage, but several factors may contribute to change its physical and chemical properties which
reduce shelf-life and thus its commercial value (Cousins et al., 1987). Although the micro-
organisms in infant milk cannot grow due to its low moisture content, microbes can grow during
the preparation of the milk formula, which is then subjected to different external factors with
different effects such as temperature, the water used, time, and others.
The study would be beneficial to mothers in order to carefully assess the condition of
every formula milk that they would be giving to their baby. In this case they would be limiting
their exposure of the formula milk outside the environment and also lessening the exposure of
used as a core areas of researching in the discipline of food microbiology that could be reiterated
using distinctive variables that would result to an implicit research topic to their own study.
Furthermore, enrichment of the variables is recommended for them to have revolutionized results
to their study.
CHAPTER II
Food spoilage is defined as any change that makes a product unacceptable for human
consumption. It can result from different causes involving physical-chemical and biochemical
changes and also microbial growth and activity (Huis in’t Veld, 1996). Spoilage microbes are
often common inhabitants of soil, water, or the intestinal tracts of animals and may be dispersed
through the air and water and by the activities of small animals, particularly insects. It should be
noted that with the development of new molecular typing methods, the scientific names of some
spoilage organisms, particularly the bacteria, have changed in recent years and some older names
are no longer in use. Many insects and small mammals also cause deterioration of food but these
Yeasts
Yeasts are a subset of a large group of organisms called fungi that also includes molds
and mushrooms. They are generally single-celled organisms that are adapted for life in
specialized, usually liquid, environments and, unlike some molds and mushrooms, do not
produce toxic secondary metabolites. Yeasts can grow with or without oxygen and are well
known for their beneficial fermentations that produce bread and alcoholic drinks. They often
colonize foods with a high sugar or salt content and contribute to spoilage of maple syrup,
pickles, and sauerkraut. Fruits and juices with a low pH are another target, and there are some
tolerate high sugar and high salt concentrations and are the usual spoilage organisms in foods
such as honey, dried fruit, jams and soy sauce. They usually grow slowly, producing off-odors
and flavors and carbon dioxide that may cause food containers to swell and burst. Debaryomyces
hansenii can grow at salt concentrations as high as 24%, accounting for its frequent isolation
from salt brines used for cured meats, cheeses, and olives. This group also includes the most
important spoilage organisms in salad dressings (Mandrell, R.E., et. Al. 2006). Saccharomyces
spp. are best known for their role in production of bread and wine but some strains also spoil
wines and other alcoholic beverages by producing gassiness, turbidity and off- flavors associated
with hydrogen sulfide and acetic acid. Some species grow on fruits, including yogurt containing
fruit, and some are resistant to heat processing (Elez-Martinez, P., et. Al., 2004)
Candida and related genera are a heterogeneous group of yeasts, some of which also
cause human infections. They are involved in spoilage of fruits, some vegetables and dairy
products (Casey, G.D., 2003). Dekkera and Brettanomyces are principally involved in spoilage of
fermented foods, including alcoholic beverages and some dairy products. They can produce
volatile phenolic compounds responsible for off-flavors (Couto, J.A., et. Al., 2005)
Molds
Molds are filamentous fungi that do not produce large fruiting bodies like mushrooms.
Molds are very important for recycling dead plant and animal remains in nature but also attack a
wide variety of foods and other materials useful to humans. They are well adapted for growth on
and through solid substrates, generally produce airborne spores, and require oxygen for their
metabolic processes.
Most molds grow at a pH range of 3 to 8 and some can grow at very low water activity levels
(0.7–0.8) on dried foods. Spores can tolerate harsh environmental conditions but most are
12 minutes at 90 degrees centigrade . Different mold species have different optimal growth
temperatures, with some able to grow in refrigerators. They have a diverse secondary
metabolism producing a number of toxic and carcinogenic mycotoxins. Some spoilage molds are
Spoilage molds can be categorized into four main groups: Zygomycetes are considered
relatively primitive fungi but are widespread in nature, growing rapidly on simple carbon sources
in soil and plant debris, and their spores are commonly present in indoor air. Generally they
require high water activities for growth and are notorious for causing rots in a variety of stored
fruits and vegetables, including strawberries and sweet potatoes. Some common bread molds
also are zygomycetes. Some zygomycetes are also utilized for production of fermented soy
products, enzymes, and organic chemicals. The most common spoilage species are Mucor and
Rhizopus. Zygomycetes are not known for producing mycotoxins but there are some reports of
Penicillium and related genera are present in soils and plant debris from both tropical and
Antarctic conditions but tend to dominate spoilage in temperate regions. They are distinguished
by their reproductive structures that produce chains of conidia. Although they can be useful to
humans in producing antibiotics and blue cheese, many species are important spoilage organisms,
and some produce potent mycotoxins such as patulin, ochratoxin, citreoviridin, penitrem.
Penicillium spp. cause visible rots on citrus, pear, and apple fruits and cause enormous losses in
these crops. They also spoil other fruits and vegetables, including cereals. Some species can
attack refrigerated and processed foods such as jams and margarine. A related genus,
Byssochlamys, is the most important organism causing spoilage of pasteurized juices because of
Aspergillus and related molds generally grow faster and are more resistant to high
temperatures and low water activity than Penicillium spp. and tend to dominate spoilage in
cyclopiazonic acid. Aspergilli spoil a wide variety of food and non-food items (paper, leather,
etc.) but are probably best known for spoilage of grains, dried beans, peanuts, tree nuts, and
some spices.
Other molds, belonging to several genera, have been isolated from spoiled food. These
generally are not major causes of spoilage but can be a problem for some foods. Fusarium spp.
cause plant diseases and produce several important mycotoxins but are not important spoilage
organisms. However, their mycotoxins may be present in harvested grains and pose a health risk.
Bacteria
Spore-forming bacteria are usually associated with spoilage of heat-treated foods because
their spores can survive high processing temperatures. These Gram-positive bacteria may be
strict anaerobes or facultative which is capable of growth with or without oxygen. Some spore-
formers are thermophilic, preferring growth at extreme temperatures with as high as 55 degrees
machines.
Other thermophiles (Bacillus and Geobacillus spp.) cause a flat sour spoilage of high or
low pH canned foods with little or no gas production, and one species causes ropiness in bread
held at high ambient temperatures (Boor, K., 2006). Mesophilic anaerobes, growing at ambient
canned products, early blowing of cheeses, and butyric acid production in canned vegetables and
fruits (Clostridium spp.); and "medicinal" flavors in canned low-acid foods (Alicyclobacillus)
(Chang & Kang, 2003). Psychrotolerant sporeformers produce gas and sickly odors in chilled
meats and brine-cured hams (Clostridium spp.) while others produce off-odors and gas in
Lactic acid bacteria (LAB) are a group of Gram-positive bacteria, including species of
producing fermented foods such as yogurt and pickles. However, under low oxygen, low
temperature, and acidic conditions, these bacteria become the predominant spoilage organisms
on a variety of foods. Undesirable changes caused by LAB include greening of meat and gas
formation in cheeses (blowing), pickles (bloater damage), and canned or packaged meat and
vegetables. Off-flavors described as mousy, cheesy, malty, acidic, buttery or liver-like may be
detected in wine, meats, milk, or juices spoiled by these bacteria. LAB may also produce large
amounts of an exopolysaccharide that causes slime on meats and ropy spoilage in some
beverages.
Pseudomonas and related genera are aerobic, gram-negative soil bacteria, some of which
can degrade a wide variety of unusual compounds. They generally require a high water activity
for growth (0.95 or higher) and are inhibited by pH values less than 5.4. Some species grow at
refrigeration temperatures (psychrophilic) while other are adapted for growth at warmer, ambient
viridiflava), Shewanella putrefaciens, and Xanthomonas campestris are the main food spoilage
organisms in this group. Soft rots of plant-derived foods occur when pectins that hold adjacent
plant cells together are degraded by pectic lyase enzymes secreted by X. campestris, P.
fluorescens and P. viridiflava. These two species of Pseudomonas comprise up to 40% of the
naturally occurring bacteria on the surface of fruits and vegetables and cause nearly half of post-
harvest rot of fresh produce stored at cold temperatures. P. fluorescens, P. fragi, P. lundensis, and
S. putrefaciens cause spoilage of animal-derived foods (meat, fish, milk) by secreting lipases and
proteases that cause formation of sulfides and trimethylamine (off-odors) and by forming
biofilms (slime) on surfaces (55;73). Some strains are adapted for growth at cold temperatures
number of human pathogens (Salmonella, E. coli, Shigella, Yersinia) and also a large number of
spoilage organisms. These bacteria are widespread in nature in soil, on plant surfaces and in
digestive tracts of animals and are therefore present in many foods. Erwinia carotovora is one of
the most important bacteria causing soft rot of vegetables in the field or stored at ambient
temperatures.
Biogenic amines are produced in meat and fish by several members of this group while
others produce off-odors or colors in beer (Obesumbacterium), bacon and other cured meats
(Proteus, Serratia), cheeses (several genera), cole slaw (Klebsiella), and shell eggs (Proteus,
Enterobacter, Serratia). Temperature, salt concentration, and pH are the most important factors
acyl homoserine lactones (AHLs) to regulate the expression of certain genes, such as virulence
factors, as a function of cell density. These AHL quorum-sensing signals may regulate
proteolytic enzyme production and iron chelation during spoilage of some foods (Rasch, M., et.
Al., 2005)
Other bacteria are associated with spoilage of chilled, high protein foods such as meat,
fish, and dairy products. They may not be the predominant spoilage organisms but contribute to
the breakdown of food components and may produce off-odors.Most species are aerobic
although some grow at low oxygen levels and may survive vacuum packaging,and one
Psychrobacter, which are predominant bacteria on poultry carcasses on the processing line and
have been isolated from a variety of spoiled meat and fish. Acinetobacter grows at a pH as low
as 3.3 and has been detected in spoiled soft drinks. These two genera do not produce extracellular
lipases, hydrogen sulfide, or trimethylamine (fishy odor) and so are considered to have a low
spoilage potential.
Alcaligenes is a potential contaminant of dairy products and meat and has been isolated
from rancid butter and milk with an off-odor. These bacteria occur naturally in the digestive tract
of some animals and also in soil and water. Flavobacterium is found widely in the environment
and in chilled foods, particularly dairy products, fish, and meat. It uses both lipases and proteases
to produce disagreeable odors in butter, margarine, cheese, cream, and other products with dairy
ingredients. Moraxella and Photobacterium are important constituents of the microflora on the
surface of fish. Photobacterium can grow and produce trimethylamine in ice-stored, vacuum-
packaged fish. Brochothrix has been isolated from meat, fish, dairy products and frozen
vegetables. During spoilage, it produces odors described as sour, musty, and sweaty (Russo, F.,
Milk is an excellent medium for growth for a variety of bacteria (Deeth, H.C., 2008).
Spoilage bacteria may originate on the farm from the environment or milking equipment or in
processing plants from equipment, employees, or the air. LAB are usually the predominant
microbes in raw milk and proliferate if milk is not cooled adequately. When populations reach
about 106 cfu/ml, off-flavors develop in milk due to production of lactic acid and other
compounds. Refrigeration suppresses growth of LAB and within one day psychrophilic bacteria
(Pseudomonas, Enterobacter, Alcaligenes and some spore-formers) grow and can eventually
produce rancid odors through the action of lipases and bitter peptides from protease action
Pasteurization kills the psychrophiles and mesophilic bacteria (LAB), but heat-tolerant
species (Alcaligenes, Microbacterium, and the sporeformers Bacillus and Clostridium) survive
and may later cause spoilage in milk or other dairy products. Immediately following
does occur (Stevenson, R.G., et. Al., 2003). Spoilage problems in cheese can sometimes be traced
to low quality milk but may also result from unhygienic conditions in the processing plant. Hard
and semi-hard cheeses have a low moisture content (<50%) and a pH ~5.0, which limits the
growth of some microbes. Some coliforms and Clostridium spp. that cause late gas blowing can
grow under these conditions as can several species of molds. Other psychrotrophs produce
biogenic amines, particularly tyramine, during storage of cheese (Novicka et al., 2003). Soft
cheeses with a higher pH of 5.0–6.5 and a moisture content of 50–80% may be spoiled by
spoiled processed cheese, where it produces gas holes and off-flavors (Meyer, J.D., et. Al.,
2003).Yeasts and molds are the main spoilage organisms found in cultured milks (yogurt, sour
cream and buttermilk) because the higher acidity in these products inhibits many bacteria
(Mayoral, M.B., et. Al., 2005). Pseudomonas, yeasts and molds can spoil butter and “light”
butters. Since the light butters have higher moisture content than butter, they can support more
microbial growth. Cream may become rancid when populations of Pseudomonas and
Enterobacter proliferate.
CHAPTER III
Methodology
This chapter details out the research methodology for the present study. It explains the
research objectives and a suitable methodology to achieve those objectives. The objective of this
study is to identify the Microflora of Spoiled Infant Formula subjected to different PH Water
Level. This chapter discusses the kind of research, the research design that researchers would use,
Kind of research
The researchers will use an experimental research to identify the Microflora of Spoiled
Infant Formula subjected to different PH Water Level. This refers to a research in which one
variable is manipulated by the researcher to determine any impact on other variable. The element
being manipulated by the researchers is known as the independent variable, whereas the change
resulting from implementation of the independent variable is the dependent variable (Tierrey, S.,
2008).
Researchers identified the PH Water Level as independent variable which could have an
impact to overall result of the dependent variable . The Spoiled Infant Formula is the dependent
After identifying the variables in the problem situation, the next step is to design the
research in a way that the requisite data can be gathered and analyzed to arrive at a result. A
proper planned research design is important in order to ensure the accuracy, and confidence of
the study.
This experimental research will use the Randomized Block Experimental Design
specifically Within-Subject Design. In this design the PH Water Level would be classified into
subgroups called blocks. These blocks would homogenized the results to have an accurate and
unbiased data.
Legend:
Culture Media
products, and also from clinical samples. The coliform group consists of several
of the prepared petri dish is reddish purple and slightly opalescent gel. Numerous
Standard Method Agar or Plate Count Agar is recommended for the determination of
plate counts of microorganisms in food, dairy, water, waste water and also from
clinical samples. The appearance of the prepared petri dish is slightly light yellow.
Numerous bacteria can be cultured in this petri dish such as Bacillus subtilis,
aureus.
Potato Dextrose Agar is recommended for the isolation and enumeration of yeasts
and molds from dairy and other food products. The appearance of the prepared petri
dish is light amber. Numerous bacteria can be cultured in this petri dish such as
Saccharomyces cerevisiae.
appearance of the prepared petri dish is light yellow. Numerous bacteria can be
A total of 4 samples would be collected from the Spoiled Infant Formula which would be
I. Primary Isolation
All samples would be cultured in different agar plates with the use of sterile standard
Examination of all cultures on solid media was performed for detection of growth,
pigmentation, colonial morphology as well as changes in the media. Plates which showed visible
growth were subjected to further bacteriological tests while those which did not show visible
growth were incubated for further 48 hours and then discarded if no growth was detected.
The primary isolates would be subcultured in Nutrient Agar in order to attain pure
Gram Stain Technique is the most important step in identification of bacteria. This
differentiate bacteria into two types which are the Gram Positive and the Gram Negative. In
examining the bacteria, Gram Positive bacteria would appear violet, while the Gram Negative
Motility Test
This test is done to help differentiate species of bacteria that are motile from non-motile.
Basically means that Motile bacterias have the ability to propel themselves through liquids by
means of flagella. Motility media contains tryptose, sodium chloride, agar, and color indicator.
Coagulase Test
Staphylococci S. aureus which produces two form of coagulase, the bound and free. Bound
coagulase or clumping factor, is bound to the bacterial cell wall and reacts directly with
fibrinogen. When a bacterial suspension is mixed with plasma, this enzyme causes alteration in
fibrinogen of the plasma to precipitate on the staphylococcal cells, causing the cells to clump.
Free coagulase however, produced extra-cellularly by the bacteria that causes the formation of a
clot when S. aureus colonies are incubated with plasma (Pharm, M., Kavitha, R., 2016)
Catalase Test
This test demonstrate the presence of enzyme catalase in the organism. The enzyme
catalase mediates the breakdown of hydrogen peroxide in to oxygen and water. The presence of
the enzyme in a bacterial isolate is evident when a small inoculum is introduced into hydrogen
peroxide, and the rapid effervescence of oxygen bubbles occur (Pharm, M., Kavitha, R., 2016).
Oxidase Test
Oxidase test is used to determine the presence of bacterial cytochrome oxidase enzyme
Indole Test
Indole Test is performed to determine the ability of the organism to split triptophan
molecule into indole. Indole is one of the metabolic degradation product of the amino acid
tryptophan. Bacteria that possess the enzyme tryptophanase are capable of hydrolyzing and
deamination tryptophan with the production of indole, pyruvic acid, and ammonia. This test help
to differentiate species of the family Enterobacteriaceae (Pharm, M., Kavitha, R., 2016).
Voges-proskauer Test
Voges-proskauer test helps to determine the ability of the organisms to produce neutral
trsl end product acetyl-methyl-carbinol or acetoin from glucose fermentation. Positive test would
appear pinkish red color, and for the Negative test, it would appear Yellowish in color (Al-
Lactose Fermentation
Lactose fermentation identifies the ability of the bacteria to ferment lactose as a carbon
source which also helps to differentiate species of the family Enterobacteriaceae. A Positive test
would indicate a yellowish color, and for the Negative test, there would be no change in color or
Sucrose Fermentation
Enterobacteriaceae. This tests the bacteria's ability to ferment sucrose and the production of acid
end-product. A Positive result would appear yellowish color, and for the Negative result, there
would be no change in color or between the tone of reddish to none (Pharm, M., Kavitha, R.,
2016).
Glucose-Gas Fermentation
which tests the ability of the bacteria to ferment glucose and produce gas and or an acid end-
product. A Positive test would indicate a yellow result, and for the Negative test, there would be
no color change or little to reddish color (Pharm, M., Kavitha, R., 2016).
Statistical Treatment
One-way ANOVA would be used to determine the significant differences among the
samples, which would be backed up with the p-value to rationalize the significance of the results.
The researchers will compute for the mean of the number of samples that inhibits growth of