THE USE OF HYDROGEN PEROXIDE FOR
PRESERVATION OF RAW MILK
By
Suzan Hamid Elamin
B.Sc. (Agric.)
University of Khartoum
(September 2001)
A dissertation Submitted to the University of Khartoum in
Partial Fulfillment of the Requirement for the
Degree of Master of Science in Food Science and Technology
Supervisor Dr. Nabila El Amir Yousif
Department of Food Science and Technology
Faculty of Agriculture
University of Khartoum
May 2005
DEDICATION
To my Family with Love
II
ACKNOWLEDGEMENT
I am very proud to have the opportunity to express my thanks in
the first to Allah who blessed me and gave me the power to complete
this work.
Also I would like to express my gratitude's to my Supervisor Dr.
Nabila El Amir Yousif for her advice and guidance throughout the
research process and for her help during preparation of this
dissertation.
Also I would like to express my gratitude to members of the Food
Science and Technology Department and my special thanks to all
friends who assisted me in any way.
III
ABSTRACT
This investigation was carried out to study the effect of hydrogen
peroxide on lengthening the shelf-life of raw milk. The milk samples
were obtained from the University of Khartoum Farm, Shambat
Farm and the Animal Production Research Centre Farm. The milk
samples
were
preserved
using
hydrogen
peroxide
(H2O2)
concentrations of 0.01 to 0.06% (30% w/v) . One sample was kept
without H2O2 and used as control sample, at room
temperature
20±7oC
The titratable acidity, fat content, protein content, beside ascorbic
acid, potassium iodide test and methylene blue test were measured
for all samples before preservation and then regularly at 6 hours
interval for 24 hours.
Results indicated an insignificant (P≤0.0.1) development in the
acidity (expressed as lactic acid percentage) of samples treated with
H2O2 at concentrations of 0.04 to 0.06%. After 6 hours of storage
the milk samples treated with 0.02 and 0.03% H2O2
showed a
gradual increase in lactic acid, while 0.01% H2O2 treated milk
samples gave result, similar to the control.
Hydrogen peroxide treatment has slightly affected the protein
content of milk. The results, however, showed the higher
concentrations of hydrogen peroxide (0.04%, 0.05% and 0.06%)
IV
significantly (P≤0.0.1) decreased the protein content compared to
the
other samples. Hydrogen peroxide treatment also has a destructive
effect on ascorbic acid.
The results indicated that fat of milk was not affected by hydrogen
peroxide treatment of milk. Also the results indicated that colour
reduction time of methylene blue of untreated milk samples were
shorter than H2O2 treated milk samples.
It can be concluded from these results that H2O2 added to milk at
a rate of 0.05% is a good preservative for raw milk and for up to 24
hour during transportation from production areas to consumption
areas without being subjected to deterioration under ambient
temperature prevailing in the sudan.
V
‫ﺑﺴﻢ ﺍﷲ ﺍﻟﺮﺣﻤﻦ ﺍﻟﺮﺣﻴﻢ‬
‫ﺨﻼﺼﺔ ﺍﻹﻁﺭﻭﺤﺔ‬
‫ﺃﺠﺭﻱ ﻫﺫﺍ ﺍﻟﺒﺤﺙ ﻟﺩﺭﺍﺴﺔ ﺃﺜﺭ ﻓﻭﻕ ﺃﻜﺴﻴﺩ ﺍﻟﻬﻴﺩﺭﻭﺠﻴﻥ‬
‫)‪ (H2O2‬ﻓﻲ ﺇﻁﺎﻟﺔ ﻤﺩﺓ ﺤﻔﻅ‬
‫ﻋﻴﻨﺎﺕ ﺍﻟﺤﻠﻴﺏ ﺍﻟﺨﺎﻡ ﻭﻗﺩ ﺃﺨﺫﺕ ﻋﻴﻨﺎﺕ ﺍﻟﺤﻠﻴﺏ ﺍﻟﺨﺎﻡ ﻤﻥ ﻤﺭﺯﻋﺔ ﺠﺎﻤﻌـﺔ ﺍﻟﺨﺭﻁـﻭﻡ ‪،‬‬
‫ﻤﺯﺭﻋﺔ ﺸﻤﺒﺎﺕ ﻭﻤﺯﺭﻋﺔ ﻤﺭﻜﺯ ﺃﺒﺤﺎﺙ ﺍﻹﻨﺘﺎﺝ ﺍﻟﺤﻴﻭﺍﻨﻲ‪.‬‬
‫ﺃﻀﻴﻔﺕ ﻤﺴﺘﻭﻴﺎﺕ ﻤﺨﺘﻠﻔﺔ ﻤﻥ ﻓﻭﻕ ﺃﻜﺴﻴﺩ ﺍﻟﻬﻴﺩﺭﻭﺠﻴﻥ ﻤﻥ ‪ %0.01‬ﺇﻟﻰ ‪ %0.06‬ﺇﻟﻰ‬
‫ﺍﻟﻌﻴﻨﺎﺕ ﺃﻋﻼﻩ ﻋﻠﻰ ﺩﺭﺠﺔ ﺤﺭﺍﺭﺓ ﺍﻟﻐﺭﻓﺔ ‪.20±7oC‬‬
‫ﻓﻲ ﻫﺫﻩ ﺍﻟﺩﺭﺍﺴﺔ ﺃﺠﺭﻴﺕ ﺇﺨﺘﺒﺎﺭﺍﺕ ﻟﻜل ﻤﻥ ﺍﻟﺤﻤﻭﻀﺔ ﺍﻟﻜﻠﻴﺔ ﻭﻨﺴﺒﺔ ﺍﻟـﺩﻫﻥ ﻭﻨـﺴﺒﺔ‬
‫ﺍﻟﺒﺭﻭﺘﻴﻥ ﺒﺎﻹﻀﺎﻓﺔ ﻟﻔﻴﺘﺎﻤﻴﻥ )ﺝ( ﻭﺇﺨﺘﺒﺎﺭ ﺍﻟﺒﻭﺘﺎﺴﻴﻭﻡ ﻭﺇﺨﺘﺒﺎﺭ ﺯﻤـﻥ ﺇﺨﺘـﺯﺍل ﺼـﺒﻐﺔ‬
‫ﺍﻟﻤﻴﺜﻠﻴﻥ ﺍﻷﺯﺭﻕ‪ .‬ﻭﻗﺩ ﺃﺠﺭﻴﺕ ﺍﻹﺨﺘﺒﺎﺭﺍﺕ ﻜل ﺴﺘﻪ ﺴﺎﻋﺎﺕ ﻟﻤﺩﺓ ‪ 24‬ﺴﺎﻋﺔ ﻟﻜل ﺍﻟﻌﻴﻨﺎﺕ‪.‬‬
‫ﺃﻅﻬﺭﺕ ﺍﻟﺩﺭﺍﺴﺔ ﻨﺘﺎﺌﺞ ﻤﻌﻨﻭﻴﺔ ﺒﺎﻟﻨﺴﺒﺔ ﻟﻠﺤﻤﻭﻀﺔ ﺤﻴﺙ ﻭﺠﺩ ﺃﻨﻪ ﻜﻠﻤﺎ ﺯﺍﺩ ﺘﺭﻜﻴﺯ ﻓﻭﻕ‬
‫ﺃﻜﺴﻴﺩ ﺍﻟﻬﻴﺩﺭﻭﺠﻴﻥ ﻗﻠﺕ ﺍﻟﺯﻴﺎﺩﺓ ﻓﻲ ﺍﻟﺤﻤﻭﻀﺔ ﻭﻗﺩ ﻜﺎﻥ ﺍﻷﺜﺭ ﻭﺍﻀـﺤﹰﺎ ﻓـﻲ ﺍﻟﻤـﺴﺘﻭﻴﺎﺕ‬
‫ﺍﻟﻌﺎﻟﻴﺔ )‪ (%0.04 ،%0.05 ،%0.06‬ﻭﺃﻤﺎ ﺍﻟﻤﺴﺘﻭﻴﺎﺕ ﺍﻟﻤﻨﺨﻔـﻀﺔ ‪ %0.02‬ﻭ ‪%0.03‬‬
‫ﻓﻘﺩ ﺃﻅﻬﺭﺕ ﺯﻴﺎﺩﺓ ﻤﺘﺩﺭﺠﺔ ﻓﻲ ﺍﻟﺤﻤﻭﻀﺔ ﺒﻌﺩ ‪ 6‬ﺴﺎﻋﺎﺕ ﻤﻥ ﺒﺩﺍﻴﺔ ﺍﻟﺤﻔﻅ‪ .‬ﺃﻤـﺎ ﺒﺎﻟﻨـﺴﺒﺔ‬
‫ﻟﻠﻌﻴﻨﺎﺕ ﺍﻷﻗل ﺘﺭﻜﻴﺯ ‪ %0.01‬ﻓﻘﺩ ﺃﻅﻬﺭﺕ ﻨﺘﺎﺌﺞ ﻤﺸﺎﺒﻬﺔ ﻟﻠﻌﻴﻨﺔ ﺍﻟﺘﻲ ﻟﻡ ﻴﻀﻑ ﻟﻬﺎ ﻓـﻭﻕ‬
‫ﺃﻜﺴﻴﺩ ﺍﻟﻬﻴﺩﺭﻭﺠﻴﻥ ) ﺍﻟﻌﻴﻨﺔ ﺍﻟﺸﺎﻫﺩ (‪.‬‬
‫ﺃﻅﻬﺭﺕ ﺍﻟﺩﺭﺍﺴﺔ ﺃﻥ ﻓﻭﻕ ﺃﻜﺴﻴﺩ ﺍﻟﻬﻴﺩﺭﻭﺠﻴﻥ ﻟﻪ ﺘﺄﺜﻴﺭ ﻤﻌﻨﻭﻱ ﻋﻠﻰ ﻨﺴﺒﺔ ﺍﻟﺒﺭﻭﺘﻴﻥ ﻓﻲ‬
‫ﺍﻟﺤﻠﻴﺏ ﻭﺃﺸﺎﺭﺕ ﺍﻟﻨﺘﺎﺌﺞ ﺃﻥ ﺍﻟﺘﺭﺍﻜﻴﺯ ﺍﻟﻌﺎﻟﻴﺔ ‪ ( %0.04 ، %0.05 ، %0.06‬ﻟﻬﺎ ﺘـﺄﺜﻴﺭ‬
‫ﻤﻌﻨﻭﻱ ﻓﻲ ﺘﻘﻠﻴل ﻨﺴﺒﺔ ﺍﻟﺒﺭﻭﺘﻴﻥ ﻓﻲ ﺍﻟﺤﻠﻴﺏ‪.‬‬
‫ﺇﻨﺨﻔﺽ ﻓﺘﻴﺎﻤﻴﻥ )ﺝ( ﺇﻨﺨﻔﺎﻀﹰﺎ ﻤﻌﻨﻭﻴﹰﺎ ﻓﻲ ﺍﻟﻌﻴﻨﺎﺕ ﺍﻟﻤﻌﺎﻤﻠﺔ ﺒﻔﻭﻕ ﺃﻜﺴﻴﺩ ﺍﻟﻬﻴـﺩﺭﻭﺠﻴﻥ‬
‫ﻭﺃﺸﺎﺭﺕ ﺍﻟﻨﺘﺎﺌﺞ ﺃﻥ ﻓﻭﻕ ﺃﻜﺴﻴﺩ ﺍﻟﻬﻴﺩﺭﻭﺠﻴﻥ ﻴﺤﻁﻡ ﻓﻴﺘﺎﻤﻴﻥ )ﺝ(‪.‬‬
‫‪VI‬‬
‫ﺃﻅﻬﺭﺕ ﺍﻟﺩﺭﺍﺴﺔ ﺃﻥ ﺩﻫﻥ ﺍﻟﺤﻠﻴﺏ ﻻ ﻴﺘﺄﺜﺭ ﺒﺈﻀﺎﻓﺔ ﻓﻭﻕ ﺃﻜﺴﻴﺩ ﺍﻟﻬﻴـﺩﺭﻭﺠﻴﻥ ﻭﻜـﺫﻟﻙ‬
‫ﺃﺸﺎﺭﺕ ﺍﻟﻨﺘﺎﺌﺞ ﺃﻥ ﺯﻤﻥ ﺇﺨﺘﺯﺍل ﺼﺒﻐﺔ ﺍﻟﻤﻴﺜﻠﻴﻥ ﺍﻷﺯﺭﻕ ﺒﺎﻟﻨﺴﺒﺔ ﻟﻠﻌﻴﻨﺎﺕ ﺍﻟﺨﺎﻟﻴﺔ ﻤﻥ ﻓـﻭﻕ‬
‫ﺃﻜﺴﻴﺩ ﺍﻟﻬﻴﺩﺭﻭﺠﻴﻥ ﺃﻗل ﻤﻥ ﺍﻟﻌﻴﻨﺎﺕ ﺍﻟﻤﻌﺎﻤﻠﺔ ﻴﻔﻭﻕ ﺃﻜﺴﻴﺩ ﺍﻟﻬﻴﺩﺭﻭﺠﻴﻥ‪.‬‬
‫ﺃﻭﺼﺕ ﺍﻟﺩﺭﺍﺴﺔ ﺒﺈﺴﺘﻌﻤﺎل ﺍﻟﺘﺭﻜﻴﺯﺍﺕ ‪ %0.05‬ﻓﻭﻕ ﺃﻜﺴﻴﺩ ﺍﻟﻬﻴﺩﺭﻭﺠﻴﻥ ﻟﺤﻔﻅ ﺍﻟﺤﻠﻴﺏ‬
‫ﻟﻤﺩﺓ ‪ 24‬ﺴﺎﻋﺔ ﺃﺜﻨﺎﺀ ﺘﺭﺤﻴل ﺍﻟﺤﻠﻴﺏ ﻤﻥ ﻤﻨﺎﻁﻕ ﺍﻹﻨﺘﺎﺝ ﺇﻟﻰ ﻤﻨـﺎﻁﻕ ﺍﻹﺴـﺘﻬﻼﻙ ﺘﺤـﺕ‬
‫ﺍﻟﻅﺭﻭﻑ ﺍﻟﻤﻨﺎﺨﻴﺔ ﺍﻟﺴﺎﺌﺩﺓ ﻓﻲ ﺍﻟﺴﻭﺩﺍﻥ ‪.‬‬
‫‪VII‬‬
CONTENTS
SUBJECT
Dedication ……………………………………………………………….....
Acknowledgement……………………………………………………….....
Abstract ……………………………………………………………….........
Arabic abstract……………………………………………………………...
Content………………………………………………………………...........
List of Table………………………………………………………………...
List of Figure……………………………………………………………….
CHAPTER ONE : INTRODUCTION…………………………………….
CHAPTER TWO : LITERATURE REVIEW………………………….......
2-1 Importance of Mik ………………………….......................................
2-2 Chemical Composition of Milk……………………………………......
2-3 Milk Production……………………………………………………….
2-3-1 Traditional Sector……………………………………………………
2-3-2 Semi-Traditional Sector……………………………………..............
2-3-3 The Urban Sector……………………………………………………
2-4 Sources of Milk Contamination……………………………………......
2-5 Types of Bacteria Found in Milk……………………………………....
2-6 Control of Contamination in Milk……………………………………...
2-7 Treatments and Methods of Milk Preservation ………………………..
2-7-1 Cooling………………………………………………………............
2-7-2 Boiling ………………………………………………………..........
2-7-3 Pasteurization ……………………………………………………….
2-7-4 Sterilization ……………………………………………………….....
2-7-5 Chemical Preservation………………………………………............
2-7-5-1 The Use of H2O2 in Milk Preservation…………………..............
2-7-5-2 Physical and Chemical Properties of H2O2………………….........
2-7-5-3 Concentration of H2O2……………………………………............
2-7-5-4 Effect of Hydrogen Peroxide on the Protein…………………….
2-7-5-5 Effect of Hydrogen Peroxide on the Ascorbic Acid……………...
2-7-5-6 Effect of Hydrogen Peroxide on the Milk Fat……………………
2-7-5-7 Effect of H2O2 on the Methylene Blue Reduction Test………….
CHAPTER THREE : MATERIALS AND METHODS
3-1 Source of Raw Milk…………………………………….......................
3-2 Methods ……………………………………………………………….
3-3 Chemicals and Reagents…………………………………….................
3-3-1 Hydrogen Peroxide…………………………………….....................
VIII
PAGE
I
II
III
V
VII
IX
XI
1
3
3
4
5
5
5
6
6
8
9
11
11
12
13
14
14
15
17
17
18
19
20
20
21
21
21
21
21
SUBJECT
3-3-2 Potassium Iodide…………………………………….........................
3-4 Hydrogen Peroxide Concentration…………………………………….
3-5 Chemical Tests…………………………………………………………
3-5-1 Acidity…………………………………………………….................
3-5-2 Protein Content……………………………………………………...
3-5-3 Fat Content……………………………………………………..........
3-5-4 Residual Hydrogen Peroxide Test …………………........................
3-5-5 Ascorbic acid content ………………………..................................
3-6 Biochemical Test………………………………………………………
3-6-1 The Methylene Blue Reduction Test………………………………..
3-7 Statistical Analysis…………………………………………………….
CHAPTER FOUR : RESULTS AND DISCUSSION
4-1 Acidity……………………………………………………....................
4-2 Protein Content…………………………………………………….....
4-3 Ascorbic Acid Content……………………………………………….
4-4 Fat Content……………………………………………………............
4-5 Methylene Blue Reduction Test……………………………………….
4-6 Residual H2O2 Test …………………………………………………..
Conclusion and Recommendation …………………………………………
References ………………………………………………………………..
PAGE
22
22
22
22
23
24
24
24
25
25
26
27
27
29
34
38
45
46
55
56
LIST OF TABLES
1.
TABLE
Composition of cow's milk …………………………………...
IX
PAGE
4
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
TABLE
PAGE
Compositional Analysis of Milk from different mamalls ……
4
Changes in protein content as affected by H2O2 treatment
during storage in Farm (1) ……………………………………
35
Changes in protein content as affected by H2O2 treatment
during storage in Farm (2) …………………………………...
36
Changes in progtein content as affected by H2O2 treatment
37
during storage in Farm (3) ……………………………………
39
Changes in ascorbic acid content as affected by H2Otreatment
during storage in Farm (1) ………………………...................
40
Changes in ascorbic acid content as affected by H2O2
treatment during storage in Farm (2)
…………………………………….
41
Changes in ascorbic acid content as affected by H2O2
treatment during storage in Farm
(3)……………………………………...
42
Changes in fat content as affected by H2O2 treatment during
storage in Farm (1)
……………………………………………..
43
Changes in fat content as affected by H2O2 treatment during
storage in Farm (2)
……………………………………………..
44
Changes in fat content as affected by H2O2 treatment during
storage in Farm
(3)……………………………………………...
Observation of average positive methylene blue reduction time
48
under various treatments of milk in Farm
(1)…………………
Observation of average positive methylene blue reduction time
49
under various treatments of milk in Farm (2)…………………
Observation of average positive methylene blue reduction time
50
under various treatments of milk in Farm (3)
…………………
52
Detection of residual H2O2 in treated milk in Farm
(1)………...
53
Detection of residual H2O2 in treated milk in Farm
(2)………..
X
TABLE
17. Detection of residual H2O2 in treated milk in Farm
(3)………..
PAGE
54
LIST OF FIGURES
1.
2.
3.
FIGURE
XI
Fig. (1) Changes in titratable acidity
of milk treated with
different levels of H2O2 in Khartoum University Farm
…….
Fig. (2) Changes in titratable acidity of milk treated with
different levels of H2O2 in Shambat Farm
……………………
Fig. (3) Changes in titratable acidity of milk treated with
different levels of H2O2 in Animal Production Research
Centre Farm
…………………………………………………..
XI
PAGE
30
31
32
INTRODUCTION
Sudan's milk production for the year 2001 is estimated (FAO, 2002)
at just over 7 million tons (MT) of which 37% was available for human
consumption, about 60% of production was suckled by the young stock
while about 3% went to waste, mainly because of high ambient
temperatures and inefficient handling Elsewhere, individual farmers
make their own arrangements for delivery and transport of their milk,
(FAO, 2002).
To transport milk, farmers use donkeys, donkey-tracted carts and
pick-up trucks depending upon availability, cost and the distances
involved.
Typically, donkeys are used for distances up to 5-7
kilometers (kms); donkey, carts for longer distances of 15 to 20 kms,
and pick-up trucks for longer distances. There are no refrigerated or
cooled transportation facilities which are necessary in a hot climate like
that of the Sudan. As a result, after several hours, the milk goes bad. It
is estimated that in remote areas milk losses due to poor transportation
and distribution facilities, could be as high as 10% (FAO, 2002). In
Sudan, apart from the Gezira Co-operative milk shed, there is no
organized system for milk collection and transportation.
Such problems are compounded by presence of bad roads and the
bacterial growth in raw milk resulting from absence of sanitary
methods of milk production and subsequent handling. Refrigeration,
suggested to overcome these problems (Luck, 1956), the first methods
are usually economically not feasible with chemical preservation being
the method of choice.
Hydrogen peroxide is non-toxic preservative, and can easily be
removed without any residue. It is also cheap and it decomposes to
oxygen and water by the added catalase produced by milk normal
microflora (Dirar, 1975).
(FAO, 1957) recommended
the use of
hydrogen peroxide in low concentrations (0.01 – 0.08%), in warm
climates in less developed countries.
By using hydrogen peroxide as a preservative large quantities of
milk produced in the production areas can be transported to industrial
areas and thereby reducing the losses due to poor transportation.
Objectives :
This study is an attempt to check the possibility of using H2O2 for
preserving milk at ambient temperature prevailing in Sudan.
2
LITERATURE REVIEW
2.1 Importance of Milk :
Milk is a fluid secreted by female mammals to provide food for their
offspring from the time of birth until they are able to feed for
themselves. (Foley, et .al. 1974). Federal definition of U.S.A. " Milk
is the fresh, clean lacteal secretion obtained by the complete milking
of one or more healthy cows, properly fed and kept excluding that
obtained with fifteen days before and ten days after calving and
containing not less than 8.5% solids not fat and not less than 3.25%
of milk fat "(Rai and Phil 1980).
Milk is as ancient as mankind itself, as it is the substance created
to feed the mammalian infant. All species of mammals from man to
whales, produce milk for this purpose (Douglas, 1995). Milk and
milk products provide significant amount of protein and most
micronutrients, including calcium, B. group vitamin (particularly
riboflavin and B12 but also thiamin, niacin, B6), vitamin A, iodine,
Magnesium, phosphorus, potassium and zinc. (Anita, 2001). Milk
and dairy foods can increase the nutrient density of the diet and play
a pivotal role in ensuring that dietary intake are nutritionally
adequate. They can also help to improve bone and dental health and
possibly protect against hypertension and colon cancer (Anita, 2001).
Milk is the only food that provides a well balanced array of essential
nutrients including protein, fat, carbohydrates, vitamins and minerals
in the form which is palatables, digestible and sanitary.
3
Milk thus deserves recognition as whole meal and "perfect food".
Man consumes milk from different sources such as cows, goats,
sheeps, mares and reindeer (Kordylas, 1990).
2.2 Chemical Composition of Milk :
FAO (1997) reported that the composition of milk varies
considerably depending on species, bread, feeding, health status and
stage of laction. The average composition milk reported is shown
below (Table 1).
Table 1 : Average composition of cow's milk
MAIN CONSTITUENTS
Water
Total Solids
Fat
Lactose
Minerals
Protein
LIMIT VARIATION%
MEAN VALUES%
85.5 – 89.5
10.5 – 14.5
1.5 – 6.0
3.6 – 5.5
0.6 - 0.9
2.9 – 5.0
87.5
13.0
3.9
4.8
0.8
3.4
(FAO 1997).
The major constituents are water, fats, protein, lactose and mineral
matter. The milk of different mammals differs in composition as
reported by Foley, et. al. (1974).
Table 2 : Compositional analysis of milk fromDifferent
mammals
%
COW
Water
87.50
Fat
3.55
Protein
3.50
Sugar
4.80
Ash.
0.65
(Foley, et. al. 1974)
GOAT
RABIT
RAT
HUMAN
86.50
4.00
4.10
4.50
0.90
69.50
10.50
15.50
2.00
2.50
68.20
14.80
11.70
2.80
1.50
87.40
3.70
1.70
7.00
0.20
4
2.3 Milk Production :
In Africa milk is produced in most agricultural systems. It is either
sold fresh, consumed as fermented milk or manufactured into products
such as butter and cheese, (O'connor, 1995). In developing countries,
most milk is produced by a large number of small holders with small
quantities of milk (Barabas, 1994). Ali (1988) cited that dairy farming
is practiced on a limited scale in Sudan.
Few dairy farms are
established in the vicinity of big towns. Dirar (1975) reported that in
Sudan milk is produced on scattered farms usually in small quantities.
Sudan currently produces 7.1 million ton of milk per year, most milk
comes from indigenous cattle Zebu and up to 90% of milk animals are
found with in the range – based nomadic and transhumant areas (FAO,
2002).
The milk production is concentrated into three main sectors namely
the traditional sector (nomads), the semi traditional sector and the urban
sector (Hassan, 1985).
2.3.1 Traditional Sector :
Nomads own more than 90% of animals and their production is
characterized by fluctuation from higher yield to very low yield
depending upon availability of pasture (Babiker, 1987).
2.3.2 Semi-Traditional Sector :
In this sector the animal owners are found around towns to supply
people with cow's and goat's milk throught the year (Tag Eldin, 1985).
5
The Urban Sector :
This contains modern dairy plants which exist in the capital
Khartoum. They do exist close to the major population centers, but
farm from milk producing areas (Alla Gabo, 1986). Three broad cattle
production systems are operated in Khartoum namely :
1. the intensive production systems where farmers follow modern
dairy management practices.
2. the extensive range land-based system which is by far the largest
systems and contribute 60%.
3. the crop livestock integrated systems which contribute the rest
(around 30%) of milk production . (FAO, 2002).
2.4 Sources of milk contamination :
Milk is synthesized in specialized cells of the mammary gland and is
virtually sterile when secreted into alveoli of the udder (Tolle, 1980).
Beyond this stage of milk production, microbial contamination can
generally occur, from three main sources, from with in the udder, from
the exterior of the udder and from the surface of milk handling and
storage equipment (Bramley and Mckinnon, 1990). The milk secreted
into an uninfected cow's udder is sterile invariable it becomes
contaminated during milk, cooling and storage (FAO, 1989). The
health and hygiene of the cow, the environment in which the cow is
housed and milk and the procedures used in cleaning and sanitizing the
milking and storage equipment are all influencing the level of microbial
contamination of raw milk. Equally important are temperature and
length of time storage which allow microbial contaminant to multiply
6
and increase in numbers (Bramley and Mckinnon, 1990). Raw milk
as it leaves the udder of healthy cows normally contains very low
number of microorganisms and generally will contain less than 1000
total bacteria per ml (Kurweil and Busse, 1973). In healthy cows, the
teat cistern, teat canal and the teat apex may be colonized by a variety
of microorganisms though microbial contamination from with in the
udder of healthy animals is not considered to contribute significantly to
the total numbers of microorganisms in the bulk milk (Kurweil and
Busse, 1973). The detection of coli form bacteria and pathogens in
milk indicates a possible contamination of bacteria either from the
udder, milk utensils or water supply used (Bonfon, et. al. 2003). The
number of bacteria in aseptically drawn milk varies from animal to
animal and even from different quarters in the same animal.
On
average aspetically drawn milk from healthy udders contains between
500 and 1000 bacteria per ml. (O'connor, 1995) . Cow with mastitis
has the potential to shed large number of microorganism into the milk
supply (Bramley and Mckinnon, 1990). Mastitis organisms found to
most often influence the total bulk milk count Streptococcus spp., most
notably S. agalactiae and S. urberis (Jeffrey, and Wilson, 1987).
Contamination of mastitis milk with fresh clean milk may be one of the
reasons for high microbial load of bulk milk (Jeffery and Wilson,
1987). Teats and udders of cows inevitably become soiled while they
are laying in stalls or when allowed in muddy barnyard used bedding
has been shown to harbor large numbers of microorganisms. Total
counts often exceed 108 – 1010 per gram.
(Hogan, et al. 1989).
Organisms associated with bedding materials that contaminated the
surface of teats and udders include Streptococci,
7
staphylococci,
spore
formers
and
coliforms
(Bramley
and
Mckinnon, 1990). The degree of cleanliness of the milking system
probably influences the total bulk milk bacteria count (Olson and
Mocquat, 1980).
Milk residue left on equipment contact surfaces
supports the growth of a variety of microorganisms (Bramley and
Mckinnon 1990). Less efficient cleaning, using lower temperatures
and/or the absence of sanitizers tends to select for faster growing
principallhy gran-negative vs (Coliforms and Pseudomonoads) and
lactic Streptococci (Thoas, 1974). Fresh milk drawn from a healthy
cow normally contains a low microbial load (less than 1000/ml) but the
loads may increase up to 100 fold or more once it is stored at normal
temperature (Richter, et al. 1992). Keeping milk in clean containers at
refrigerated temperature immediately after milking process may delay
the increase of initial microbial load and prevent multiplication of
microorganism in milk between milking at the farm and transportation
processing plant (Adesium, 1994). Keeping fresh milk at an elevant
temperature together with unhygienic practices in the milking process
may result in microbiologically inferior quality (chye et , al 1994).
2.5 Types of bacteria found in milk :
Milk is a complex fluid containing a mixture of carbohydrates,
protein, fat and minerals in a different physio-chemical status and
forms. Its comprehensive nutritional properties and high moisture
content make it an excellent medium for supporting microbial growth
(FAO, 1997). Milk provides a favorable environment for the growth
of microorganisms (O'Connor, 1995). Microbes can enter milk via
the cow, air, feeds, milk handling equipment and milker. Bacteria
types
8
commonly associated with milk are Psuedomonas, Brucella,
Enterobacteriaceae,
Staphylococci,
Staphylococcus
aureus,
Streptococcus agalacteiae, S. thermophilus, lactococci, L. Laclis, L.
Lates – diacetylactis, L. cremoris, Leuconostoc lactis, Bacillus
cereus, Lactobacillus, L. Lactis, L. Bulgarious and Mycobacterium
tuberculosis (O'Connor, 1995).
Vasavada (1987) reported that many disease such as tuberculosis
brucellosis, diphtheria, scarlet fever and gastroenteritis are
transmissible through milk and milk products.
Pathogens that have involved in food borne outbreaks associated
with the consumption of milk include Listera, monocytogenes,
salmonella, campylobacter, Staphytococcus aureus, B. cereus and CI
botulimum (Ryser, 1998) Recently, E. Coli 0157:H7 has become a
serious threat to the dairy industry with several outbreaks reported in
developed countries ranging from milkd diarrhea to potentially fatal
hemolytic uremic syndrome, hemorrhagic colitis and thrombotic
thromboctyopaenic purpura. (Coia, et. al. 2001).
2.6 Control of Contamination in Milk :
The sources of entry of microorganisms in milk are interior of
udder, exterior of udder, atmosphere, utensils and milker. Therefore,
in order to obtain milk free from microbes, the hands of milkers and
the teats should be washed by antiseptic solutions. The utensils in
which milking is carried on should be sterilized before use, the
milking should be done in a clean atmosphere and the utensil should
be plugged with cotton wool after milking (Rai and Phil 1980). The
9
exterior of the udder can be a major source of bacteria
contamination to milk. Cleaning and removal of soil, bedding
material and manure from the udder and flanks of the cow before
milking is necessary to prevent the entry of many types of bacteria
into the milk (O'connor, 1995).
The degree of cleanliness of the milking system probably
influence the total bulk milk bacteria count (Olson and Mocquat
1980). The key words in controlling bacteria in milk are cleanliness,
sanitation and cooling. Cleanliness applies to the cow, cow
environment, milking area, personnel involved in milk and the milk
storage area. Sanitation applies to the milking system and bulk tank.
Cooling refers to temperature of milk after it leaves the cow and how
quickly it is cooled (Bodman and Rice, 2004). To produce a clean
milk from healthy cows, O'connor, (1995) suggested an important
points to be followed and these are : (i) the udder should be washed
with clean water before milking, (ii) A strip cup should be used to
check for mastitis in each quarter before milking start, (iii) The body
of the cow should be free of soil, dirt and manure and contamination
of milk from external sources, (iv) milkers and milk handlers should
be in good health and their hands should be clean, (v) The milking
barn should have a good floor that is easy to clean and drain, there
should be good ventilation and lighting and (vi) Cooling milk is
essential to prevent an increase in bacterial numbers and spoilage of
milk.
10
Treatments and Methods of Milk Preservation
Milk is a medium very favorable for growth of microorganisms.
In order to keep it for a longer time, it needs careful handling
therefore, the most common methods of preservation of milk are
those which aim to reducing the number of microorganisms in milk
either by the action of heat or by the use of chemicals (Rai and Phil,
1980).
The most common methods of milk preservation that are used
today were described by Fraizeir and Westoff (1981), these include :
(i)
Use of heat such as pasteurization, ultra pasteurization
(sterilization), boiling and steam under pressure.
(ii)
Use of low temperatures such as refrigerated storage
(cooling) and freezing.
(iii) Drying.
(iv) Condensed products.
(v)
Use of irradiations.
(vi) Use of sound waves and electric currents.
(vii) Developed preservation (fermentation).
(viii) Use of chemicals preservative.
Milk preservation methods commonly used in dairy industry
include :2.7.1. Cooling :
To prevent growth of bacteria in milk and to maintain its quality
for domestic consumption or during transport to the processing plant,
11
it is essential to cool the fresh milk as quickly as possible (O'connor,
1995). Refrigeration does not reduce bacteria numbers it only slows
down their growth (O'connor, 1995). Milk is held at refrigeration
temperature during storage on the farm, in the truck or tank and
during transportation to plant. (Frazier and Westhoff, 1981).
Increase in total counts bacteria during prolonged storage of raw
milk (milk < 7oC) is caused by multiplication of its psychrotrophic
flora (Law, 1979). The storage and handling of milk at refrigeration
temperatures is selective for psychrotrophic bacteria which have
considerable spoilage potential (Law,1979).
The grade A
pasteurized milk ordinance of United States Public health service
stipulates that grade A raw milk for pasteurization shall be cooled to
10oC or less with in 2 hr after being drawn and kept that cold unlit
processed (Frazier and westhoff, 1981). Majewski (1975) reported
that milk cooled immediately and stored for 24 hr at 4oC had lower
mesophilic, Psychrotrophic, Caseolytic and lipolytic counts and
lower Coli-aerogens titres than non cooled. Ibrahim (1970) showed
that poor sanitation aggravated by high temperature can be expected
to result in high bacteria counts in milk.
2.7.2 Boiling :
Boiling of raw milk is usually practiced in developing countries.
The objectives of boiling is to destroy microorganisms, increase shelf
life of milk and better processing in areas where there is seasonal
surplus. The best methods for boiling milk with the minimum
nutrient loss is to bring it quickly to the boiling, stirring constantly
then immediately cooling it (Kin, 1972). A more sophisticated
alternative is to place heat proof jars of milk in deep vessels
12
containing water above the fill line of the milk. The water heated
until the milk temperature reaches 72oC, and held for 15 – 30 and
held for 15 – 30 minutes. Milk is then cooled quickly with ice or
cold water (Payne, 1990). Frazier and westoff (1981) reported that
boiling destroys all microorganisms except the spores of bacteria.
2.7.3 Pasteurization :
Practically, pasteurization, a heat treatment method, is sufficient
to kill almost pathogenic and most spoilage microorganisms of milk
(Herschoerfer, 1973). Pasteurization is heating of milk or other dairy
products to a temperature which destroys all the pathogenic
microorganisms without seriously affecting the composition of milk
or milk products to at least 62OC for 30 minutes or to at least 71oC
for 15 second (Rai and Phil 1980). Pasteurization make milk safe
with negligible effect on flavor and only very slight effect on
nutritive value (Payne, 1990). The efficiency of milk pasteurization
depends upon the temperature of pasteurization, the holding time, the
total numbers of bacteria and the proportion of the total microbial
load that are spore' farmers (Frazier and Westoff, 1981).
Pasteurization eliminates pathogens without causing significant
physicochemical change in milk (Havranek and Hadziosmanovic,
1966). O'connor (1995) reported that the process kills many
fermentative organisms as well as pathogens but putrefactive
microorganisms survive pasteurization. Although pasteurized milk
has storage stability of two to three days, subsequent deterioration is
caused by the putrefactive organism. Pasteurization has a little affect
on the nutritive value of milk as the major nutrients are not altered.
13
There is an insignificant loss of vitamin C and B group vitamins
(O'connor, 1995).
2.7.4 Sterilization :
Sterilization involves heating sealed bottles or metal containers of
homogenized milk for up to 120oC for 20 – 30 minutes usually using
saturated steam under pressure (Kon, 1972).
In sterilization, milk is subjected to severe heat treatment that
ensures almost complete destruction of the microbial population.
The product is then said to be commercially sterile . The milk is
subjected to above 100oC for 15 – 40 minutes. The product has a
much longer keeps for several months without refrigeration if un
opened (Payne, 1990).
2.7.5. Chemical Preservation :
One way of preserving food or lengthening its shelf-life is to use
certain chemical additives that can protect the food from microbial
spoilage by regarding growth of microbes, by destroying the cells, or
by doing both together (Collins, 1967).
Collins (1967) defined a potentially useful milk preservative as a
"compound that, in small quantities, inhibits undesirable organisms
but is utilized by the intended consumer, or at least, does not harm
him". The definition of a good chemical preservative for milk as
stated in the FAO (1957) report goes as follows, "An ideal
preservative for milk may be defined as any substance which when
added to milk enables the physical properties and chemical
composition of milk to remain unaffected by microbial or other
14
spoilage so that the milk retains its original wholesomeness and
nutritional value".
2.7.5.1 The Use of Hydrogen Peroxide (H2O2) in Milk
Preservation :
The use of H2O2 for preserving milk in warm countries has
gained the attention of the FAO and WHO of the United Nations. A
panel of experts sponsored by FAO (Report 1957) has recommended
the use of H2O2 under any one of certain circumstances. These
include :
1. where in a technically less developed country the production and
collection of milk is as yet insufficiently well organized.
2. where in a warm country milk is produced on scattered farms
usually in small quantities and has to be bulked and transported
over considerable distances before reaching a cooling or
pasteurizing center.
3. where in a warm country atmospheric temperatures are so high
at certain seasons of the year as to cause very rapid bacterial
multiplication and spoilage of milk.
4. where in a warm country roads and transportation are as such as
will not allow milk to reach the consuming area within a
sufficiently short time.
5. where, no refrigeration is available.
Some authors reported that among the various preservatives that
have been employed for increasing the keeping quality of milk
hydrogen peroxide has been found to offer the greatest possibilities
of
15
practical application under commercials conditions of handling milk
on account of its comparative in harmlessness on one hand and its
high bactericidal power on the other (Nambudripad, et. al. 1952).
Luck (1956) reported that a good milk preservative must have the
following properties :1. It must be easily destructible in the factory before using the
milk for human consumption or for industrial purpose.
2. It must not react with any of the constituent parts of the milk.
3. After destruction the remaining substance must be nonpoisonous, odorless and tasteless.
Hydrogen peroxide fulfill all these demands. Dirar (1975)
reported that tropical countries are faced with difficulty of keeping
raw sweet milk for a reasonable length of time. The quick souring of
milk is simply due to the high ambient temperatures, bad roads and
high bacterial counts of raw milk resulting from insanitary method of
milk production of the three means, namely refrigeration, double
pasteurization and chemical preservation suggested to combact this
problem, the first two method are usually economically unfeasible,
this leaves chemical choice, of all the many milk preservatives tested
hydrogen peroxide is the best one. The first person to suggest the
use of hydrogen peroxide for the preservation of raw milk in the
Sudan was Ibrahim (1970). Chu, et al. (1975) reported that the effect
of H2O2 on the bacteriological contents of milk has been reported by
several investigators namely : Luck (1956), Roundy (1958), Amin
and Olson (1967) and Nagib and Hussein (1972).
16
Physical and Chemical properties of H2O2 :
Have been studied by FAO/WHO 2004 :
Chemical name
Hydrogen peroxide
Empirical formula
H2O2
Molecula weight
4.02
Description
An odorless, or nearly odorless,
containing stabilizer appearing in
the residue on evaporation.
Powerful oxidizing agent.
2.7.5.2. Concentration of H2O2 :
Saha et al. (2003) found that keeping quality of milk treated with
H2O2 increased significantly when compared with untreated milk. It
was concluded that 0.04% to 0.05% is enough to preserve milk up to
24 hr. FAO (1957) reported that the use of any preservative in milk is
undesirable and should be adopted only in exceptional
circumstances. Countries which do not have a highly developed milk
handling system, recommended concentration of 0.01% to 0.08%
(w/v). Gregory, et. al. (1961) claimed that the 0.05% H2O2
treatment was effective in controlling acid development in milk
during 8 hr incubation at 24oC. Siegenhalar (1976) reported that in
tropical conditions it is possible to preserve milk for at least 25 hr
with addition of 0.06% to 0.08% of H2O2 in raw milk. Park and
Pack, (1977) found that growth of contaminating bacteria in raw
milk could
17
be checked for at least 8, 12 or 16 hours by treatment with 0.01, 0.02
or 0.03% H2O2 respectively. The bactericidal efficiency of H2O2
depends on its concentration and on the temperature and duration of
the treatment. (Gregory et. al. 1961). Dirar (1977), found that the
hydrogen peroxide treated milk (0.06%, 0.08%) were good fresh on
the other hand untreated milk developed high acidity and were,
therefore unacceptable.
Chu et al. (1975) found that the use of
at 35oC for 30
0.01%, 0.02%, 0.03%, 0.04% and 0.05% H2O2
minutes reduced the total bacterial count by 63.49%, 88.73%,
94.76%, 95.23% and 99.92% respectively. VonRuden, et. al. (1967)
reported that effectiveness of H2O2 in treating milk depends on
concentrations of treatment and resistance of different bacterial
species present.
The FAO experts (Report, 1957) have
recommended the use of hydrogen peroxide concentration of
between 0.01% and 0.08% for preserving raw milk.
2.7.5.3. Effect of Hydrogen Peroxide on The protein :
Gregory et. al. (l961) found that treatment of milk with 0.05%
(w/v) H2O2 for 8 hour at 24oC had no significant effect on the B
complex vitamins or on the fat – soluble vitamins and only slightly
reduced the nutritive value of the protein.
al.(1961)
Also Gregory et.
reported that the prolonged treatment and high
concentrations of H2O2 bring about changes in the milk protein.
Fish and Michelsen (1967) found that
K
– casein and
B
–
Lactoglobulin did not make a complex when treated with hydrogen
18
peroxide prior to heating, but it did not completely prevent the
interaction.
Individual milk protein solutions treated with 1%
hydrogen peroxide either slowed down its electrophoretic mobility,
migrated faster or remained unchanged (Grindrod and Nickorson,
1966). Also they found that treatment with 1% H2O2 of individual
whey protein solutions slowed down the electrophoretic mobility of
B-Lactoglabulins, speeded up that of bovine serum albumine and
did not affect that of
- Laclalbumin. Gaafar (1992) found that
hydrogen peroxide treated milk at 0.05%, 0.1 and 0.2% for
24 hours, significantly decreased the methionine content of milk
protein. At 0.1% for 24 hours hydrogen peroxide markedly
lowered the modified protein efficiency ratio and biological value
of milk protein. Mohieldin (1995) found that a slight reduction in
the nutritive value of the protein was obtained in the high hydrogen
peroxide concentrations (0.07 and 0.08%).
2.7.5.5. Effect of Hydrogen Peroxide on Ascorbic Acid :
The ascorbic acid oxidation varies inversely with the quantity of
H2O2 added milk (Luck, 1956). Gregory et. al. (l961) found that
(0.05% w/v) H2O2 treatment during 8 hours incubation at 24oC had
no significant effect on the levels of biotin, nicotinic acid,
riboflavin, thiamine but had destructive effect on ascorbic acid in
milk. The vitamins of milk are damaged very little by treatment
with H2O2 only ascorbic acid is seriously influenced (Luck, 1956).
19
2.7.5.6. Effect of Hydrogen Peroxide on the Milk fat :
Raj and Singhal (1990) found that addition of 0.1%, 0.2%, 0.3%
or 0.4% H2O2 to milk produced an initial decrease of 0.05 – 0.1%
in fat determined by Gerber method, but levels returned to those of
control as the hydrogen peroxide was decomposed. Cock (1963)
reported that pH, lactose and fat of milk are not affected by
hydrogen peroxide treatment of milk.
2.7.5.7. Effect of H2O2 on the Methylene Blue Reduction Test
:
The length of time milk take sto decolorise methylene blue is a
good measure of its bacterial content and hence of its hygienic
quality (O'connor, 1995). Saha, el at (2003) reported that
methylene blue reduction test was conducted to get an idea about
the bacterial population in milk. Saha, et al. (2003) found that
colour reduction time of methylene blue test was 8, 10, 11, 12, 12,
14 and 15 hours for untreated, 001, 0.02, 0.03, 0.04, 0.05 and
0.06% H2O2 treated milk respectively. Also he found that color
reduction time of untreated milk was lower than H2O2 treated milk.
Hossain et al (1989) reported that color reduction time for untreated
milk samples were less than H2O2 treated milk samples.
20
CHAPTER THREE
MATERIALS AND METHODS
3.1 Source of Raw Milk :
Milk samples were collected from three different sources ; namely
University of Khartoum Farm (Farm 1), Shambat Farm (Farm 2) and
Animal Production Research Centre Farm (Farm 3).
Fresh milk samples were collected from university of Khartoum
and Shambat Farm one hour after-milking. However, milk samples
obtained from Animal Production Research Centre were collected 3
hours after milking. Samples were collected in sterile plastic
containers and were packed in an ice box and brought to the
laboratory for analysis.
3.2 Methods :
In the laboratory the collected milk samples, after thorough
mixing were divided into seven equal parts. Out of the seven parts,
one part was kept as untreated milk (control) and six portions were
treated with 0.01, 0.02, 0.03, 0.04, 0.05 and 0.06% of hydrogen
peroxide (30% w/v) at room temperature 20±7oC.
3.3 Chemicals and Reagents :
3.3.1 Hydrogen Peroxide :
Hydrogen peroxide (30% w/v) was obtained from Merck 30% E
Merck, D-6100 Darmstadt F-R, Germany.
21
3.3.2 Potassium Iodide :
Forty percent (40%) solution was prepared for the residual
hydrogen peroxide test.
3.4 Hydrogen Peroxide Concentration :
The required concentration of hydrogen peroxide was prepared as
described by Dirar (1967). The required concentration of hydrogen
peroxide were all prepared by dilution on the basis that the
commercial preparation contained 30% H2O2 for example, if 0.02
final hydrogen peroxide concentration was needed the following
procedure was used – one ml of the 30% commercial solution was
added to 9 ml. of distilled water to give a 3% solution, one ml of
latter was added to 2 ml of distilled water to give a 1% H2O2
solution of this solution, 2 ml solution was taken, using a sterile
pipette and added to 100 ml. of milk under aseptic conditions to give
the required final concentration of 0.02 H2O2.
3.5 Chemical Tests :
3.5.1 Acidity :
The acidity of fresh and treated with H2O2 milk samples was
determined according to commercial testing and product control in
the Dairy Industry (1974). Ten milliliters of milk were measured
into a white porcelain dish and five drops of phenolphthalein
indicator were then added. This was titrated against N/9 sodium
hydroxide until a faint pink colour lasting for not less than 30
seconds was obtained. The titration figure was divided by 10 to give
the acidity of the sample expressed as percent lactic acid.
22
3.5.2 Protein Content :
The protein content of milk was determined by the Kjedahl
method according to the AOAC method (1990). In a Kjedahl flask
10 ml milk sample were placed followed by addition of two Kjedahl
tablets (each tablets contain l gm Na2So4 and the equivalent of 0.1
mg Hg). Twenty five milliliters of concentrated sulfuric acid
(density 1.815 gm/ml at 20oC) were added to the flask and mixture
was then digested on a heater until a clean solution was obtained (3
hours), the flasks were removed and left to cool. The digested
sample was poured in a volumetric flask (100 ml) and diluted to 100
ml with distilled water. Five milliliters were taken and neutralized
using 10 ml of 40% NaOH. The distillate was received in a conical
flask containing 25 ml of 2% boric acid and 3 drops of indicator
(bromocrsol green + methyl red). The distillation was continued
until the volume in the flask was 75 ml. The flask was then removed
from the distillator, and the distillate was titrated against 0.lN HCl
unitl the end point was obtained (pink color). Protein content was
calculated as follows :
Nitrogen (%) = T X 0.01 X 20 X 0.014
Weight of Sample
Protein (%) = Nitrogen (%) X 6.38
Where :
T
= Titration figure
0.1
= Normality of Hcl
0.014 = Atomic weight of nitrogen/1000
20
= Dilution factor
6.38 = Conversion factor of N to protein.
23
3.5.3 Fat Content :
Fat content was determined by Gerber method according to the
AOAC method (1990) : Ten milliliters sulfuric acid (density 1.815
gm/ml at 20oC) were poured into clean dry Gerber tubes, then 10.94
ml milk were added followed by addition of 1 ml amyl alcohol, and
distilled water at 20oC. The contents of the tube were thoroughly
mixed till no white particle were seen. The tubes were then
centrifuged at 1100 revolutions per minutes (rpm) for 5 minutes.
The tubes were transferred to a water bath at 65oC for 3 minutes after
which the fat content was immediately read.
3.5.4 Residual Hydrogen Peroxide Test :
A test for residual hydrogen peroxide was carried out by adding
0.5 milliliters of freshly prepared 40% potassium lodide solution to
5 ml of treated milk samples. A positive result was indicated by
development of yellow color. This test was adopted from method
described by loane (1969).
3.5.5 Ascorbic Acid :
Ascorbic acid content of milk was determined by using 2,6 –
dichlorophenol – indophenol titration method of Ruck (1963). Thirty
ml of clear filtrate of milk sample was first blended with a reasonable
amount of 0.4% oxalic acid for 2 minutes and then filtered. The
volume of filtrate was made up to 250 ml using 0.4% oxalic acid.
An amount of 20 ml of filtrate was titrated with 2,6 – dichlorophenol
– indophenol dye to a faint pink colour. The ascorbic acid content
was calculated according to the following formula :
24
Pure ascorbic acid (0.05 grams) was dissolved in 60 ml of 20%
metaphosphoric acid and diluted to 250 ml. Ten milliliters of
ascorbic acid solution were titrated against 2,6 – dichlorophenol –
indophenol solution until faint pink color persisted for 15 seconds.
The result was expressed as mg/100 ml.
Ascorbic acid
(mg/100 ml)
= titration figure X dye strength X 100
Factor
Factor
= Sample wt. (30 ml) X sample vol. for titration
Total vol. of sample (250 ml)
Dye Strength =
1
Titre
2.6 Biochemicals Test :
3.6.1. The Methylene Blue Reduction Test :
The methylene blue reduction test was determined according to
Rural Dairy Technology (1995) method.
Apparatus :
• A water bath set at 37 – 38 C.
• Test tubes graduated at the 10 ml mark. The tubes were
plugged with cotton and sterilized before use.
• A supply of 1 ml pipettes were also plugged with cotton and
sterilized.
• The rubber stoppers for closing the tubes were sterilized
before use by immersing in boiling water for minutes.
25
• A methylene blue solution was made up from standard
methylene blue milk testing tablets.
Procedure :
The milk was mixed thoroughly. The sample was taken with a
clean sterile dipper and the test tube was filled to the 10 ml mark.
The test tube was stoppered with a sterile rubber stopper. The test
tube was placed in the test rack. One ml of the methylene blue
solution was added to each tube. The rubber stopper was replaced
with aseptic precautions. Each tube was inverted twice so as to mix
the milk and solution thoroughly, and the tubes were placed in the
water bath at a temperature of 37 – 38oC.A note of the time at which
the tubes were put into the water bath was made. The samples were
examined after 30 mintes. Decolourised samples were removed from
the water bath. Partly decolourised samples were not disturbed. At
half-hourly intervals decolourised samples were examined again and
were repeated as above.
3.7 Statistical Analysis :
Data were analysed according to the standard statistical procedure
(Gomez and Gomez 1984). Analysis of variance was carried out for
each parameter as for a completely randomized design. The means
were separated using the Least Significant Difference (LSD).
26
CHAPTER FOUR
RESULTS AND DISCUSSION
4.1. Titratable acidity :
Fig. (1) shows development in acidity of samples collected from Farm
(1) and stored at room temperature 20±7oC. Fig. (1) samples collected
from Farm (1) shows the acidity against time- the results indicated that
the acidity positively (P ≤ 0.01) corrected with time. Milk samples
preserved with 0.06% treated samples showed no mentionable change
in titratable acidity, while a slight increase in the acidity of the 0.05%
treated samples obtained after 24 hours of storage. The acidity of the
samples treated with 0.04% H2O2 increased slightly after 18 hours of
storage maintained
a maximum titratable acidity after 24 hours of storage. After 6 hours of
incubation the samples treated with 0.03% and 0.02% H2O2 showed a
gradual increase in lactic, while the control and 0.01% concentration
maintained the highest value of titratable acidity expresses as lactic
percent.
Fig. (2) shows changes in titratable acidity of samples from farm
(2) the results indicated that after 24 hours of storage the 0.06% H2O2
treated samples showed no mentionable change in titratable acidity,
while a slight increase in the acidity of the 0.05% H2O2 treated
samples obtained after 24 hours of storage. Milk samples treated with
0.04% H2O2 increased markedly after 18 hours on incubation. After 6
hours of storage the samples treated with 0.03% and 0.02% H2O2
27
showed a gradual increase in lactic acid, while the control and 0.01%
H2O2 concentration showed the highest value of titratable acidity.
Fig. (3) shows changes in titratable acidity of samples from Farm
(3) results show that the acidity was positively correlated with time.
Milk samples preserved with 0.06% H2O2 resulted in a slight increase
in titratable acidity however, after 24 hours of storage they show lactic
acid comparable to the original raw milk. After six hours of storage
0.02% H2O2 treated samples recorded a slight increase and 0.01%
treated samples and control recorded high titratable acidity levels.
Significant difference (P≤ 0.01) was found with the acidity of
different types of milk samples. The results of acidity test are in
agreement with several research workers. Siegnhalar (1976) reported
that, in tropical condition it is possible to preserve milk for at least 24
hours with addition of 0.06% to 0.08% of H2O2 in raw milk. From
another experiment park and pack (1977) reported that growth of
contaminating bacteria in raw milk could be checked for at least 8, 12
or 16 hours by treatment with 0.01, 0.02 or 0.03% H2O2 respectively.
The initial acidity levels of the raw milk used in this study were
0.17, 0.17 and 0.16% for farms 1, 2, and 3 respectively. These value
however, are considered similar to values reported by Mohi Eldien
(1996) who reported 0.17, 0.15, 0.16 and 0.19% but a slighty higher
than those reported by Radhakrishnan and Srinivason (1951) who
reported 0.013 and 0.15%. Lubis (1983) observed that when hydrogen
peroxide was added at 0.05% the titratable acidity in milk after 24
hours was 0.14%.
In general the present results showed that lactic acid percentage
decrease with the increase of hydrogen peroxide concentration, which
is in agreement with observation of Mohi Eldien, (1995) Dirar (1975)
28
and Lubis (1983). Gregory et. al. (1981) reported that the H2O2
treatment was effective in controlling lactic acid development in milk.
It is well known that the acidity in milk is developed due to the
break down of milk sugar lactose into lactic acid by fermentative effect
of acid producing bacteria, H2O2 prevent bacterial fermentation by the
inhibiting the growth of acid producing bacteria or destroying the cells
or by doing both together and this is in agreement with the results of
Dirar (1975), Dirar (1967) and Gregory et. al. (1981).
4.2. Protein content:
result in Table 3 (farm 1) indicated that the use of 0.06%, 0.05%,
0.04% and 0.03% as milk preservation resulted in a significant (P≤
0.01) decrease in protein levels 2.90, 2.92, 2.94 and 2.96 when
compared with other H2O2 concentrations. Protein content for
concentration of 0.02% and 0.01% were statistically similar, however,
the higher protein content revealed with the lower H2O2 concentration
(0.02% and 0.01). The mean protein value of the control was
significantly (P≤0.01) higher compared to the other concentrations after
24 hr of storage.
The data presented in table 4 (farm 2) showed that the use of
0.06%, 0.05% and 0.04% as milk preservatives resulted in a
significant (P≤0.01) decrease in protein levels (2.98, 3.00 and 3.01%)
when compared with other H2O2 concentrations. Milk samples
preserved with 0.03% resulted in a slight decrease in protein content.
The higher protein content revealed with lower (0.01% and 0.02%)
H2O2 concentrations while the control was significantly (P≤0.01)
higher as compared to the other concentrations after 24 hours of
storage.
29
XXXI
XXXII
Fig. (3) Changes in Titratable acidity of milk treated with different
levels of H2O2 in Animal Production Research Centre Farm
Storage time (Hours)
Results in Table 5 (Farm 3) indicated that the use of 0.06%,
0.05% and 0.04% preservatives resulted in a significant (P≤0.01)
decrease in protein content.
A slight decrease in the protein
content was obtained the 0.03% and 0.02% H2O2 concentrations,
however, the higher protein content revealed with lower (0.01%)
H2O2 concentration. The mean protein content of the control was
significantly (P≤0.01) higher as compared to the other
concentration after 24 hours of storage.
The results showed that a slight reduction in the protein
content of milk was obtained using the high hydrogen peroxide
concentrations (0.04%, 0.05%, 0.06%).
This results is in an
agreement with Gaafar (1992), who found that hydrogen peroxide
treated milk at 0.05%, 0.1% and 0.2% for 24 hours, significantly
decreased the methionine content of milk protein. Mohi Eldien
(1995) reported that a slight reduction in the nutritive value of the
protein was obtained in the high H2O2 concentration (0.07% and
0.08% H2O2). Dirar (1967) reported that 0.01, 0.03 and 0.05%
H2O2 treated samples showed either insignificant effect of the
hydrogen peroxide treatment on the protein value or aslight
decrease in the protein value. Gregory, et. al. (1961) found that
treatment of milk with 0.05% (w/v) H2O2 for 8hr at 24°C had no
significant effect on the B-complex vitamin or on the fat-soluble
vitamins and only a slight reduction in nutritive value of the
protein. Also found that prolonged treatment and high
concentrations of H2O2 bring about changes in the milk protein.
Fish and Michelson (1967) reported that
K-casien
and
B-
Lactoglobulin did not make a complex when treated with
hydrogen peroxide prior to heating – but it did not completely
prevent the interaction.
XXXIII
4.3. Ascorbic Acid Content:
results in Table 6 (Farm 1) indicated that the use of H2O2 as
preservative resulted in a significant (P≤0.01) decrease in ascorbic
acid content when compared with the control. The average
ascorbic acid content of untreated, as well as milk samples treated
with 0.01, 0.02, 0.03, 0.04 0.05 and 0.06%H2O2 were 1.17, 0.53,
0.46, 0.43, 0.38, 0.37 and 0.33 mg/100 ml respectively after 24 hr
of storage. The higher ascorbic acid content revealed with lower
(0.01%) H2O2 concentration. The mean ascorbic acid content of
the control was significantly (P≤0.01) higher as compared to the
other concentration.
Results in Table 7 (Farm 2)
showed that the average
ascorbic acid of untreated as well as milk samples treated with
0.01, 0.02, 0.03, 0.04 0.05 and 0.06%H2O2 were 1.00, 0.41, 0.31,
0.25, 0.22, 0.21 and 0.20 mg/100 ml respectively after 24 hours of
storage. The higher ascorbic acid content revealed with the lower
(0.01%) H2O2 concentration as compared with the control.
The data presented in Table 8 (Farm 3) showed that highly
significant (P≤0.01) difference was obtained in the treated milk
samples as compared to the control. The average ascorbic acid
content of untreated as well as milk samples treated with 0.01,
0.02, 0.03, 0.04 0.05 and 0.06%H2O2 were 0.83, 0.68, 0.60, 0.56,
0.50, 0.46 and 0.43 mg/100ml respectively. The results showed
that the control had the highest mean ascorbic acid content while
0.06% H2O2 had the lowest mean after 24 hours of storage.
XXXIV
Table 3 : Changes in protein content as affected by H2O2
treatment
during storage in Farm (1)
STORAGE
LEVELS OF H2O2 IN MILK
TIME
HOUR
LSD
LEVEL OF
SIGNIFICANCE
Control 0.01% 0.02% 0.03% 0.04% 0.05% 0.06%
0
3.269
3.269
3.257
3.269
3.269
3.257
3.257
6
3.269
3.257
3.257
3.251
3.255
3.251
3.245
1
+0.0185
2
+0.024
12
3.253
3.198
3.186
3.156
3.132
3.090
3.043
18
3.253
3.197
3.182
2.572
2.543
2.519
2.495
24
3.262
3.197
3.181
2.571
2.507
2.501
2.459
Mean
3.261
3.224
3.213
2.964
2.938
2.922
2.900
± SD
± 0.072
±
±
±
±
±
±
0.110
0.118
0.454
0.353
0.354
0.367
1
+ LSD 0.05
2
+ LSD 0.01
XXXV
**
Table 4 : Changes in protein content as affected by H2O2
treatment
during storage in Farm (2)
LEVEL
STOR
LEVELS OF H2O2 IN MILK
LSD
OF
AGE
SIGNIFIC
TIME
ANCE
HOUR
0
6
12
18
24
Mean
± SD
Cont 0.01 0.02 0.03 0.04 0.05 0.06
rol
%
3.40
3.41 3.41 3.41 3.41 3.41 3.41
8
8
3.40
3.40 3.37 3.36 3.34 3.34 3.34
1
7
6
198
3.38
3.22 3.16 3.13 3.09 3.07 3.04
2
7
7
26
3.39
3.22 3.16 2.66 2.61 2.59 2.54
0
6
3.38
3.22 3.16 2.66 2.59 2.54 2.52
6
4
3.39
3.30 3.25 3.04 3.01 3.00 2.97
5
0
6
7
3
0
6
±
±
±
±
±
±
±
0.08
0.11 0.12 0.48 0.36 0.38 0.39
0
8
1
+ LSD 0.05
2
+ LSD 0.01
%
6
0
8
6
3
2
%
4
4
2
2
2
1
%
6
9
0
4
6
0
XXXVI
%
8
7
3
0
9
2
%
6
6
3
9
5
8
+0.0
+0.0
**
Table 5 : Changes in protein content as affected by H2O2
treatment
during storage in Farm (3)
LEVEL
STOR
LEVELS OF H2O2 IN MILK
LSD
OF
AGE
SIGNIFIC
TIME
ANCE
hour
0
6
12
18
24
Me
an
±
SD
Cont 0.01 0.02 0.03 0.04 0.05 0.06
rol
%
3.51
3.51 3.51 3.51 3.51 3.51 3.51
9
3
3.51
3.49 3.47 3.46 3.44 3.41 3.39
1
9
5
145
3.51
3.32 3.27 3.25 3.23 3.22 3.18
2
9
3
193
3.51
3.32 3.27 3.10 3.08 3.06 3.03
7
0
3.51
3.32 3.26 3.10 3.04 3.03 3.00
7
0
3.51
3.39 3.36 3.28 3.26 3.25 3.22
9
4
0
9
4
0
4
±
±
±
±
±
±
±
0.10
0.11 0.10 0.19 0.19 0.19 0.21
6
2
1
+ LSD 0.05
2
+ LSD 0.01
%
4
2
5
2
9
6
%
1
6
5
8
6
5
%
3
2
3
4
9
3
XXXVII
%
1
2
2
7
7
3
%
3
4
0
1
1
2
+0.0
+0.0
**
Results showed that the H2O2 had a destructive effect on
ascorbic acid. Also results indicated that the use of H2O2 as a
preservatives resulted in a significant (P≤ 0.01) decrease in ascorbic
acid content which agrees with the findings of Luck (1956) who
found that the ascorbic acid oxidation varies inversely with the
quantity of H2O2 added to milk. Gregory et. al. (1961) found that
0.05% (w/v) H2O2 treatment during 8 hours incubation at 24°C had
no significant effect on the levels of biotin, nicotinic acid, riboflavin,
thiamine or pyridoxal but had destructive effect on ascorbic acid in
milk. Weinstein and Trout (1951) reported that 0.028 ml. of 30%
hydrogen peroxide solution oxidized the naturally occurring vitamin
C in milk in 30 minutes. Luck (1956) reported that the vitamins of
milk are damaged very little by treatment with H2O2 only ascorbic
acid is seriously influenced. Krukowsky (1949) found that ascorbic
acid was oxidized rapidly and completely by hydrogen peroxide in
milk heated to 61.1°C . It was also found that the peroxidase of milk
and also copper ions helped in this reaction which converted
ascorbic acid into dehydro- ascorbic acid.
4.4. Fat Content :
Table 9 shows the effect of H2O2 on fat content of milk.
Results in Table 9 (Farm 1) indicated that no significant difference
(P≤0.01) was observed in the treated milk samples as compared to
the control. The average fat content of untreated, as well as treated
0.01, 0.02, 0.03, 0.04 0.05 and 0.06% H2O2 milk samples were
3.46, 3.48, 3.48, 3.43, 3.40, 3.50 and 3.48% respectively after 24
hours of storage.
XXXVIII
Table 6 : Changes in ascorbic acid content as affected by
H2O2 treatment
during storage in Farm (1)
STORAGE
TIME
(HOUR)
LEVELS OF H2O2 IN M ILK
LSD
Control
0.01%
0.02%
0.03%
0.04%
0.05%
0.06%
mg/100ml
mg/100ml
mg/100ml
mg/100ml
mg/100ml
mg/100ml
mg/100ml
0
1.873
0.701
0.666
0.619
0.670
0.571
0.524
6
1.760
0.683
0.570
0.570
0.520
0.473
0.463
1
+0.030
12
1.111
0.619
0.540
0.524
0.476
0.460
0.397
2
+0.022
18
0.571
0.317
0.302
0.238
0.222
0.190
0.190
24
0.540
0.302
0.238
0.206
0.127
0.111
0.095
Mean
1.171
0.525
0.463
0.431
0.383
0.365
0.334
± SD
± 0.587
± 0.185
± 0.171
± 0.181
± 0.182
± 0.188
± 0.171
Hour
1
2
* LSD 0.01
* LSD 0.05
XXXIX
LEVEL OF
SIGNIFICANCE
**
Table 7 : Changes in ascorbic acid content as affected by
H2O2 treatment
during storage in Farm (2)
STORAGE
TIME
(HOUR)
LEVELS OF H2O2 IN M ILK
LSD
Control
0.01%
0.02%
0.03%
0.04%
0.05%
0.06%
mg/100ml
mg/100ml
mg/100ml
mg/100ml
mg/100ml
mg/100ml
mg/100ml
0
1.987
0.688
0.466
0.400
0.377
0.344
0.333
6
1.167
0.388
0.333
0.278
0.222
0.211
0.200
12
0.877
0.355
0.322
0.255
0.200
0.167
0.167
18
0.500
0.322
0.300
0.167
0.167
0.167
0.167
1
24
0.477
0.289
0.244
0.167
0.155
0.144
0.133
2
Mean
1.002
0.408
0.311
0.253
0.224
0.206
0.200
± SD
± 0.575
± 0.151
± 0.098
± 0.089
± 0.085
± 0.075
± 0.072
1
* LSD 0.05
2
* LSD 0.01
XL
LEVEL OF
SIGNIFICANCE
**
+0.020
+0.027
Table 8 : Changes in ascorbic acid content as affected by
H2O2 treatment
during storage in Farm (3)
STORAGE
TIME
(HOUR)
LEVELS OF H2O2 IN M ILK
LSD
Control
0.01%
0.02%
0.03%
0.04%
0.05%
0.06%
mg/100ml
mg/100ml
mg/100ml
mg/100ml
mg/100ml
mg/100ml
mg/100ml
0
1.334
1.260
0.195
1.158
1.093
0.992
0.862
6
0.862
0.639
0.600
0.492
0.417
0.412
0.389
12
0.714
0.612
0.537
0.426
0.352
0.334
0.334
18
0.648
0.556
0.473
0.398
0.325
0.306
0.278
1
+0.0156
24
0.575
0.334
0.334
0.315
0.306
0.278
0.278
2
+0.021
Mean
0.827
0.680
0.608
0.558
0.499
0.455
0.428
± SD
± 0.281
± 0.320
± 0.312
± 0.317
± 0.310
± 0.281
± 0.229
1
2
* LSD 0.05
* LSD 0.01
XLI
LEVEL OF
SIGNIFICANCE
**
Table 9 : Changes in fat content as affected by H2O2
treatment
during storage in Farm (1)
STORAGE
TIME
(HOUR)
LEVELS OF H2O2 IN M ILK
lsd
Control
0.01%
0.02%
0.03%
0.04%
0.05%
0.06%
0
3.467
3.467
3.500
3.433
3.433
3.500
3.467
6
3.433
3.433
3.500
3.500
3.500
3.533
3.467
12
3.467
3.500
3.433
3.400
3.433
3.467
3.400
18
3.500
3.500
3.500
3.433
2.500
3.467
3.467
24
3.433
3.433
3.433
3.400
3.400
3.433
3.467
Mean
3.460
3.467
3.473
3.433
3.400
3.500
3.467
± SD
±
0.112
±
0.072
±
0.110
±
0.118
±
0.100
* NS : Non Significant
XLII
±
0.000
±
0.057
LEVEL OF
SIGNIFICANCE
NS
Table 10 : Changes in fat content as affected by H2O2
treatment
during storage in Farm (2)
STORAGE
TIME
(HOUR)
LEVELS OF H2O2 IN M ILK
LSD
Control
0.01%
0.02%
0.03%
0.04%
0.05%
0.06%
0
3.733
3.800
3.800
3.700
3.667
3.633
3.633
6
3.776
3.633
3.633
3.700
3.700
3.567
3.633
12
3.733
3.567
3.600
3.600
3.600
3.533
3.633
18
3.767
3.767
3.667
3.667
3.667
3.700
3.633
24
3.733
3.700
3.667
3.700
3.700
3.667
3.733
Mean
3.747
3.693
3.673
3.673
3.640
3.620
3.653
± SD
± 0.064
±
0.122
±
0.080
±
0.122
±
0.063
• NS : Non Significant
XLIII
±
0.094
±
0.0916
LEVEL OF
SIGNIFICANCE
NS
Table 11 : Changes in fat content as affected by treatment
during storage in Farm (3)
STORAGE
TIME
(HOUR)
LEVELS OF H2O2 IN M ILK
Control
0.01
%
0.02
%
0.03
%
LSD
0.04
%
0.05
%
0.06
%
0
4.067
4.233
4.200
4.100
4.033
4.000
4.100
6
4.067
4.167
4.100
4.100
4.000
4.167
4.033
12
4.167
4.000
4.067
4.000
4.133
4.100
4.067
18
4.100
4.167
4.067
4.200
4.033
4.167
4.233
24
4.100
4.000
4.187
4.067
4.100
4.100
4.167
Mean
4.100
4.113
4.120
4.093
4.060
4.107
4.120
± SD
± 0.065
±
0.106
±
0.094
±
0.088
XLIV
±
0.063
LEVEL
OF
SIGNIFI
CANCE
±
0.103
±
0.108
NS
The data presented in Table 10 (Farm 2) shows that the
hydrogen peroxide treated milk samples were statistically similar as
compared to the control. The average Fat content of untreated as
well as milk samples treated with 0.01, 0.02, 0.03, 0.04, 0.05 and
0.06% H2O2 were 3.75, 3.69, 3.67, 3.64, 3.62 and 3.65%
respectively after 24 hours of storage.
Results in Table 11 (Farm 3) shows that no significant
difference was obtained in the treated milk samples as compared to
the control in the fat content after 24 hours of incubation. Results
indicated that fat of milk are not affected by hydrogen peroxide
treatment of milk. The results of this experiment are in agreement
with the findings of Cook (1963) who found that pH, lactose and fat
of milk are not affected by hydrogen peroxide treatment of milk. Raj
and Singhal (1990) reported that addition of 0.1, 0.2, 0.3, or 0.4%
H2O2 to milk produce an initial decrease of 0.05 – 0.1% in fat
determined by Gerber method, but levels returned to those of the
control as the H2O2 was decomposed.
4.5 Methylene Blue Reduction Test:
The results of methylene blue reduction test are shown in
Tables 12, 13 and 14 for Farms 1, 2 and 3 respectively. From table
12 it is evident that color reduction time of methylene blue test 3
hours, 10 hours and fifteen minutes, 11 hours and fifteen minutes, 12
hours and 30 minutes, 13 hours and fifteen minutes, 14 hours and 15
minutes and 15 hours and 25 minutes for untreated, 0.01, 0.02, 0.03,
0.04 0.05 and 0.06%H2O2 treated milk respectively for Farm (1).
XLV
Results in Table 13 (Farm 2) showed that color reduction time of
methylene blue test was 7 hours and 30 minutes, 11 hours and 40
minutes, 12 hours and 55 minutes 14 hours, 14 hours and 50 minutes, 15
hours and 50 minutes, 16 hours and 10 minutes for untreated, 0.01, 0.02,
0.03, 0.04, 0.05 and 0.06% H2O2 treated milk samples respectively.
In Table 14 (Farm 3), it is evident that color reduction time of
methylene blue test was 5 hours and 45 minutes, 9 hours and 30 minutes,
10 hours and 35 minutes, 11 hours and 30 minutes, 12 hours and 30
minutes, 13 hours and 40 minutes, and 14 hours and 40 minutes for
untreated, 0.01, 0.02, 0.03, 0.04, 0.05 and 0.06% H2O2 treated milk
samples respectively.
Results indicated that color reduction time of methylene blue for
untreated milk samples were shorter than H2O2 treated milk samples. The
results of this experiment are in agreement with the findings of Saha, et.
al. (2003) who found that color reduction time of methylene blue test
was 8, 10, 11, 12, 12, 14 and 14 hours for untreated, 0.01, 0.02, 0.03,
0.04 0.05 and 0.06% H2O2 treated milk respectively. Hossein, et al
(1989) found that colour reduction time of metylene blue for H2O2
treated milk samples were more than untreated milk samples.
4.6 Residual H2O2 Test :
The data on K1 test were given in Table 15, 16 and 17 for Farms 1,
2 and 3 respectively. The results indicated that at zero time of storage,
hydrogen peroxide could not be detected in the control and 0.01% H2O2
treated samples, whereby in the other treated samples hydrogen peroxide
was present. After 6 hours of storage the
XLVI
results showed that hydrogen peroxide disappeared from the 0.02% H2O2
treated milk samples for Farms 1, 2 and 3, while hydrogen peroxide
disappeared from 0.03% H2O2 treated samples after 12 hours for farms 1,
2 and 3. Hydrogen peroxide disappeared from the 0.04% and 0.05%
H2O2 treated milk samples after 12 and 18 hours respectively for farms 1,
2 and 3. Hydrogen peroxide disappeared from the 0.06% H2O2 treated
milk samples after 24 hours for farms 1, 2 and 3.
The FAO (1957) experts and some authorities in the field (Cook,
1963, Patil, 1980) agreed to the requirement that hydrogen peroxide must
be removed from the treated dairy product before the product reaches
consumer. Patil (1980) showed that the reduction in the residual
hydrogen peroxide content was greatest when the milk was boiled after
the addition of hydrogen peroxide. Luck (1956) reported that H2O2 is one
of the ideal preservatives because it can be destroyed easily, quickly and
completely through addition of catalase (the enzyme which split H2O2)
and there is no residual toxic effect after a destruction of hydrogen
peroxide. Ibrahim (1970) reported that hydrogen peroxide is added to
fresh milk to reduce the bacterial load, excess of hydrogen peroxide can
later be removed from milk by the addition of the enzyme catalase.
Dirar. (1975) reported that hydrogen peroxide can easily be decomposed
to water and oxygen by the action of catalase added or by the catalase
naturally found in raw milk.
Santha and Ganguli (1975) reported that different types of milk appear
to decompose hydrogen peroxide at different rates, cows milk seemed to
decompose hydrogen peroxide faster than buffalo milk and this could be
attributed to the higher levels of catalase enzymes in cows milk than
buffalo milk. Dirar (1967) reported that many factors are involved in the
XLVII
decomposition of hydrogen peroxide such as temperature, pH and
enzymes.
Table 12 : Observation on average positive methylene blue
reduction time under various treatments of milk
in Farm (1)
OBSERVATION TIME
8.15 am
11.15
6.30 am
7.30am
8.45pm
9.30pm
10.30pm
11.40pm
H2O2
Control
-
+
+
+
+
+
+
+
0.01%
-
-
+
+
+
+
+
+
0.02%
-
-
-
+
+
+
+
+
0.03%
-
-
-
-
+
+
+
+
0.04%
-
-
-
-
-
+
+
+
0.05%
-
-
-
-
-
-
+
+
0.06%
-
-
-
-
-
-
-
+
LEVELS
XLVIII
-Negative (-) sign denotes blue Color of methylene blue appearance
in untreated and H2O2 treated milk samples .
- Positive (+) Sign denotes Reduction Color of methylene blue from
untreated and H2O2 treated milk samples.
Table 13 : Observation on average positive methylene blue
reduction time under various treatments of milk
in Farm (2)
H2O2
OBSERVATION TIME
LEVELS
8.40am 4.10pm 8.20pm 9.35pm 10.40pm 11.30pm 12.30pm 12.50pm
Control
-
+
+
+
+
+
+
+
0.01%
-
-
+
+
+
+
+
+
0.02%
-
-
-
+
+
+
+
+
0.03%
-
-
-
-
+
+
+
+
0.04%
-
-
-
-
-
+
+
+
0.05%
-
-
-
-
-
-
+
+
0.06%
-
-
-
-
-
-
-
+
- Negative (-) sign denotes blue Color of methylene blue appearance
in untreated and H2O2 treated milk samples .
XLIX
- Positive (+) Sign denotes Reduction Color of methylene blue from
untreated and H2O2 treated milk samples.
L
Table 14 : Observation on average positive methylene blue
reduction time under various treatments of milk
in Farm (3)
H2O2
LEVELS
OBSERVATION TIME
8.00am
1.45am
5.30pm
6.35pm
7.30pm
8.30pm
9.40pm
10.40pm
Control
-
+
+
+
+
+
+
+
0.01%
-
-
+
+
+
+
+
+
0.02%
-
-
-
+
+
+
+
+
0.03%
-
-
-
-
+
+
+
+
0.04%
-
-
-
-
-
+
+
+
0.05%
-
-
-
-
-
-
+
+
0.06%
-
-
-
-
-
-
-
+
- Negative (-) sign denotes blue Color of methylene blue appearance
in untreated and H2O2 treated milk samples .
Positive (+) Sign denotes Reduction Color of methylene blue from
untreated and H2O2 treated milk samples.
LI
Hydrogen peroxide concentration 0.05% was chosen in this study
as being suitable to preserve milk samples up to 24 hours during
transportation from production area to consumption area under ambient
temperature (20±7oC) prevailing in the Sudan. This level preserve milk
up to 24 hours without the need to add catalase to remove the residual
hydrogen peroxide. These results agreed with the findings of Mohi
Eldein (1995) who reported that hydrogen peroxide concentrations
0.03% to 0.05% are suitable for controlling the bacterial flora of milk
during transportation from farm to consumption area. Gregory et. al.
(1961) recommended the level of 0.05% H2O2 for milk preservation
during transportation in hot climate. The FAO (1957) experts report had
recommended the use of hydrogen peroxide concentration between
0.01% to 0.08% (w/v) for preserving raw milk. Dirar (1975) report that
hydrogen peroxide is an effective preservative of raw milk and that milk
preserved with hydrogen peroxide compared well with fresh milk, the
concentration of 0.06% H2O2 is recommended for preserving raw milk
for 9 hours. Saha ea al. (2003) reported that the keeping quality of milk
treated with H2O2 increased significantly when compared with untreated
milk. It was concluded that 0.04% to 0.05% H2O2 is enough to preserve
milk up to 24 hours.
From the results of the above mentioned parameters it is now clear
that H2O2 is effective to inhibit the growth of bacteria in raw milk and
can be used as a preservative of milk under climatic condition of Sudan.
Addition of 0.05% H2O2 to raw milk is enough to preserve it for up to 24
hours.
LII
Table 15 : Detection of residual H2O2 in treated milk
in Farm (1)
STORAGE
TIME
(HOUR)
Control
0.01%
0.02%
0.03%
0.04%
0.05%
0.06%
0
-
-
+
+
+
+
+
6
-
-
-
**
+
+
+
12
-
-
-
-
-
**
+
18
-
-
-
-
-
-
+
20
-
-
-
-
-
-
-
LEVELS OF H2O2 IN MILK
- Positive (+) sign denotes the appearance of H2O2 from treated milk
samples.
- Negative (-) sign denotes the absence of H2O2 from treated milk
samples.
LIII
Table 16 : Detection of residual H2O2 in treated milk
in Farm (2)
STORAGE
TIME
(HOUR)
Control
0.01%
0.02%
0.03%
0.04%
0.05%
0.06%
0
-
-
+
+
+
+
+
6
-
-
-
**
+
+
+
12
-
-
-
-
-
**
+
18
-
-
-
-
-
-
**
20
-
-
-
-
-
-
-
LEVELS OF H2O2 IN MILK
- Positive (+) sign denotes the appearance of H2O2 from treated milk
samples.
- Negative (-) sign denotes the absence of H2O2 from treated milk
samples.
LIV
Table 17 : Detection of residual H2O2 in treated milk
in Farm (3)
-
STORAGE
TIME
(HOUR)
Control
0.01%
0.02%
0.03%
0.04%
0.05%
0.06%
0
-
-
+
+
+
+
+
6
-
-
-
**
+
+
+
12
-
-
-
-
-
**
+
18
-
-
-
-
-
-
+
20
-
-
-
-
-
-
-
LEVELS OF H2O2 IN MILK
Positive (+) sign denotes the appearance of H2O2 from treated milk
samples.
-
Negative (-) sign denotes the absence of H2O2 from treated milk
samples.
LV
CONCLUSION AND RECOMMENDATIONS
• The present investigation was conducted to assess the properties
of hydrogen peroxide as a milk preservative.
• The acidity, ascorbic acid, protein content, fat content potassium
iodide test and methylene blue reduction test were used as
evaluation criteria.
• The results indicated a significant reduction on the development
of the acidity of milk samples treated with H2O2 at certain
concentrations.
• Hydrogen peroxide has a destructive effect on ascorbic acid of
milk.
• Hydrogen peroxide has no effect on the milk protein level.
• Milk fat is not affected by hydrogen peroxide treatment and
color reduction time of methylene blue of untreated milk is
shorter than H2O2 treated milk.
• It appears from the results that H2O2 is an effective preservative
in controlling the acid development in raw milk and that milk
preserved with H2O2 compare well with fresh milk.
• The effectiveness of hydrogen peroxide is dependent upon
increased its concentration.
• Residual H2O2 was removed by the naturally catalase found in
raw milk.
• Hydrogen peroxide at 0.05% level extend the shelf-life of raw
milk for at least 24 hr.
LVI
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