‫اﻟﺮﲪﻦ اﻟﺮﺣﻴﻢ‬
‫ﺑﺴﻢ اﷲ‬
University of Khartoum
Faculty of Public and Environmental Health
Department of Food Hygiene and Safety
Microbiological Quality of Home –made Mayonnaise
Consumed at Ready to Eat Foods
Establishments in Khartoum Locality
A thesis submitted for partial fulfilment of the requirement of
Master Degree of Public and Environmental Health (MPEH) in
Food Hygiene and Safety
By :
Elrasheed Greeb Allah Mustafa Elzien
Supervisor:
Dr. Nazik Eltayeb Musa Mustafa
‫اﻵﻳـﺔ‬
‫ﻗﺎل ﺗﻌﺎﻟﻰ ‪-:‬‬
‫ﺑﺴﻢ اﷲ اﻟﺮﺣﻤﻦ اﻟﺮﺣﻴﻢ‬
‫)‪(25‬‬
‫ﺻ ﱠﺒ ًﺎ‬
‫ﺻ َﺒ ْﺒ َﻨﺎ اﻟ َﻤﺎ َء َ‬
‫ﻃ َﻌﺎ ِﻣ ِﻪ )‪َ (24‬أ ّﻧﺎ َ‬
‫ﺴﺎن ِإﻟﻰ َ‬
‫ﻈ ِﺮ ا ِﻹﻧ َ‬
‫) ًَﻓﻠﻴﻨ ًَُ‬
‫)‪(28‬‬
‫)‪(27‬‬
‫)‪(26‬‬
‫ﺷ ّﻘ ًﺎ‬
‫ض َ‬
‫ﺷ َﻘ َُﻘ َﻨﺎ ا َﻷ ْر َ‬
‫َ‬
‫ﻀﺒ ًﺎ‬
‫ﻋ َﻨﺒ ًﺎ َو َﻗ ْ‬
‫وِ‬
‫ﺣ ّﺒ ًﺎ‬
‫َﻓ َﺄ ْﻧ َﺒ ْﺘ َﻨﺎ ِﻓﻴ َﻬﺎ َ‬
‫ﻏ ْﻠﺒ ًﺎ )‪َ (30‬و َﻓﺎ ِآ َﻬ ًﺔ َوأ ّﺑ ًﺎ )‪َ (31‬ﻣ َﺘﺎﻋ ًﺎ‬
‫ﻖ ُ‬
‫ﺣ َﺪا ِﺋ َ‬
‫ﻼ )‪َ (29‬و َ‬
‫ﺨً‬
‫َو َز ْﻳ ُﺘﻮﻧ ًﺎ َو َﻧ ْ‬
‫)‪(32‬‬
‫ﱠﻟ ُﻜ ْﻢ َوﻷ ْﻧ َﻌ َﺎ ِﻣ ُﻜ ْﻢ‬
‫ﺻﺪق اﷲ اﻟﻌﻈﻴﻢ‬
‫ﺳﻮرة ﻋﺒﺲ اﻵﻳﺎت )‪(32 - 24‬‬
‫‪I‬‬
‫ُﺛ ﱠﻢ‬
Dedication
To the members of my family
To my teachers and colleagues
To my supervisor Dr. Nazik Eltayeb Musa
II
Acknowledgment
I would like to express my gratefulness and
indebtedness to my supervisor Dr. Nazik Eltayeb Musa for
her great assistance and research methodology . and also
would like to extend my gratefulness, to the staff of the
Faculty of Public and Environmental Health for their
beneficial courses, laboratory staff and Khartoum health
office staff.
III
CONTENTS
Subject
Page No.
Ayat from holly Koran
I
Dedication
II
Acknowledgment
III
Contents
IV - V
List of tables
VI
List of figure
VII
Abstract by English Language
VIII
Abstract by Arabic Language
IX
Chapter (1)
Introduction
1-2
Objectives
3
Chapter (2) Literature review
Composition and characteristics of Mayonnaise
Preparation of mayonnaise
4-5
5
Food poisoning and bacterial growth in mayonnaise
6 - 12
Chapter (3) Materials and Methods
Food handlers Questionnaire
13
Samples
13
Equipments
13 – 14
Culture Media
14 – 15
Chemicals and reagents
15
Oxidase test Materials
15
Motility medium
16
Methyl red and Voges – Proskauer medium
16
IV
Continue Chapter (3)
Simmons citrate medium
16
Indole test
16
O'meara reagent for Voges – Proskauer test
17
Oxidation Fermentation test.
17
Sterilization of Culture Media
17
Microbiological analysis
17
pH Measurement of Mayonnaise samples
17
Water content measurement of Mayonnaise samples
17 – 18
Aerobic Plate Counts (APCs) of Mayonnaise samples
18
Determination of the Most Probable Number (MPN) of
Coliforms.
Isolation and Identification of Enterobacteria
18 - 19
Enumeration
19 – 20
of
Staphylococcus
aureus
from
19
Mayonnaise samples
Gram Stain
20
Biochemical Tests
21 – 24
Chapter (4) Results
Results
25 - 41
Chapter (5)
Discussion
42 - 44
Recommendation
45
Conclusion
46
Chapter (6)
References
47 - 50
Appendix
1-3
V
List of Tables
Table title
Page No.
Recipes of home-made Mayonnaise samples.
Hygiene and sanitation conditions in food premises
27 – 28
29
where mayonnaise was prepared
Determination of the Most Probable Number (MPN) of
Coliforms:
30 - 31
Morphological and Biochemical tests showed the most
32 – 34
dominant types of bacteria in each samples
IMViC results carried on Gram negative bacilli
isolates
VI
35
List of Figures
Figure title
Showing percentage of the different
mayonnaise made from.
pH Measurement of Mayonnaise samples
Page No.
ingredient
36
36
Showing percentage by methods of using eggs.
37
Showing percentage by the source of eggs.
37
Showing percentage of the different isolate of Bacteria
from Mayonnaise.
Boiling potatoes for mayonnaise
38
peeling of potatoes for Mayonnaise
39
Materials and equipment of Mayonnaise preparation
40
Mixing of mayonnaise ingredients
40
homemade mayonnaise exposed for sale at room
41
temperature
VII
39
Abstract
The microbiological quantity of home-made mayonnaise samples,
prepared and consumed at fifty ready-to-eat food establishments in
Khartoum locality, North Administrative Unit were investigated for
hygienic status of premises. Laboratory work carried included
aerobic
viable count, coliform count and staphylococci species detection.
The mayonnaise samples showed viable counts between 5 × 10
5
and 2.4 × 107, with mean of 4.4 × 106. The mean of most probable
number for coliform bacteria was 3.4 × 103.
The samples showed an isolation and presumptive identification of
Staphylococcus species from 62 % of samples, Bacillus species from 32
% of samples. Escherichia coli from 20 % of samples and Salmonella
species from 6 % of the samples.
The high bacterial load of mayonnaise with hazardous bacteria was
attributed to bacterial contamination from ingredients and handling
conditions.
Although low PH was detected in samples due to vinegar, lemon juice and
citric acid and the presence of garlic appeared to be a significant safety
factors of hygienic situation of ready-to -eat establishment that should be
improved by handlers training and health education, and enforcement of
food safety laws.
VIII
‫ﺍﻟﻤﺴﺘﺨﻠﺹ‬
‫ﻨﻭﺍﺤﻰ ﺍﻟﺠﻭﺩﺓ ﺍﻟﻤﻴﻜﺭﻭﺒﻴﻭﻟﻭﺠﻴﺔ ﻟﻠﻤﺎﻴﻭﻨﻴﺯ ﺍﻟﻤﺼﻨﻊ ﻤﺤﻠﻴﹰﺎ ﺘﻡ ﺘﻘﺼﻴﻬﺎ ﻓﻰ‬
‫ﻤﺤﻼﺕ ﺍﻟﻭﺠﺒﺎﺕ ﺍﻟﺴﺭﻴﻌﺔ ﻭﺍﻻﻏﺫﻴﺔ ﺍﻟﺠﺎﻫﺯﺓ‪ ،‬ﺤﻴﺙ ﺘﻡ ﺘﻘﺼﻰ ﻫﺫﻩ ﺍﻟﺨﻭﺍﺹ ﻟﻌﺩﺩ‬
‫ﺨﻤﺴﻭﻥ ﻋﻴﻨﺔ ﻤﻥ ﺍﻟﻤﺎﺩﺓ ﺍﻟﻐﺫﺍﺌﻴﺔ ﻤﺎﺨﻭﺫﺓ ﻤﻥ ﻋﺩﺩ ﺨﻤﺴﻭﻥ ﻤﻨﺸﺄﺓ ﻟﺒﻴﻊ ﺍﻟﻁﻌﺎﻡ‬
‫ﺍﻟﺠﺎﻫﺯ ﻟﻺﺴﺘﻬﻼﻙ ﺒﻭﺤﺩﺓ ﺍﻟﺨﺭﻁﻭﻡ ﺸﻤﺎل ﺍﻻﺩﺍﺭﻴﺔ ﺍﻟﺘﺎﺒﻌﺔ ﻟﻤﺤﻠﻴﺔ ﺍﻟﺨﺭﻁﻭﻡ‪.‬‬
‫ﺍﻟﺘﺤﻠﻴل ﺍﻟﻤﻌﻤﻠﻰ ﺍﻟﺫﻯ ﺍﺠﺭﻯ ﺸﻤل‪-:‬‬
‫ﻋﺩ ﺍﻟﺒﻜﺘﺭﻴﺎ ﺍﻟﻬﻭﺍﺌﻴﺔ ﺍﻟﺤﻴﺔ ﻭﺍﻟﻘﺎﺒﻠﺔ ﻟﻠﻨﻤﻭ‪ ،‬ﻋﺩ ﺒﻜﺘﺭﻴﺎ ﺍﻟﻜﻭﻟﻴﻔﻭﺭﻡ‪ ،‬ﺍﻟﺘﻌﺭﻑ ﻋﻠﻰ‬
‫ﺍﻟﺒﻜﺘﺭﻴﺎ ﻤﻥ ﻨﻭﻉ ﺍﻟﻤﻜﻭﺭﺍﺕ ﺍﻟﻌﻨﻘﻭﺩﻴﺔ‪.‬‬
‫ﻭﻗﺩ ﻜﺎﻥ ﻋﺩ ﺍﻟﺒﻜﺘﺭﻴﺎ ﺍﻟﺤﻴﺔ ﻭﺍﻟﻘﺎﺒﻠﺔ ﻟﻠﻨﻤﻭ ﺒﻴﻥ ‪ 510 x 5‬ﻭ ‪10 x 2.4‬‬
‫ﺒﻤﺘﻭﺴﻁ‬
‫‪7‬‬
‫‪ 610 x 4.4‬ﻭﻤﺘﻭﺴﻁ ﻋﺩ ﺒﻜﺘﺭﻴﺎ ﺍﻟﻜﻭﻟﻴﻔﻭﺭﻡ ‪. 310 x 3.4‬‬
‫ﻭﻗﺩ ﺘﻡ ﻋﺯل ﺍﻟﺒﻜﺘﺭﻴﺎ ﻤﻥ ﻨﻭﻉ ﺍﻟﻤﻜﻭﺭﺍﺕ ﺍﻟﻌﻨﻘﻭﺩﻴﺔ ﻤﻥ ‪ %62‬ﻤﻥ ﺍﻟﻌﻴﻨﺎﺕ‪،‬‬
‫ﺍﻟﺒﺎﻜﺘﺭﻴﺎ ﺍﻟﻌﺼﻭﻴﺔ ﻤﻥ ﻨﻭﻉ ﺍﻟﺒﺎﺴﻠﺱ ﻤﻥ ‪ %32‬ﻤﻥ ﺍﻟﻌﻴﻨﺎﺕ‪ ،‬ﺒﺎﻜﺘﺭﻴﺎ ﺍﻻﺸﺭﻴﻜﻴﺔ‬
‫ﺍﻟﻘﻭﻟﻭﻨﻴﺔ ﻤﻥ ‪ %20‬ﻤﻥ ﺍﻟﻌﻴﻨﺎﺕ ﻭﺍﻟﺴﺎﻟﻤﻭﻨﻴﻼ ﻤﻥ ‪ %6‬ﻤﻥ ﺍﻟﻌﻴﻨﺎﺕ‪.‬‬
‫ﺍﻟﻌﺩ ﺍﻟﺒﻜﺘﺭﻴﻰ ﺍﻟﻌﺎﻟﻰ ﻟﻌﻴﻨﺎﺕ ﺍﻟﻤﺎﻴﻭﻨﻴﺯ ﺍﻟﻤﻌﺩ ﻤﺤﻠﻴﺎﹰ‪ ،‬ﻭﻭﺠﻭﺩ ﺍﻨﻭﺍﻉ ﻤﻥ‬
‫ﺍﻟﺒﻜﺘﺭﻴﺎ ﺍﻟﻤﺴﺒﺒﺔ ﻟﻸﻤﺭﺍﺽ‪ ،‬ﻴﻌﺯﻯ ﻟﺘﻠﻭﺙ ﺍﻟﻤﺎﻴﻭﻨﻴﺯ ﺍﻟﻤﻌﺩ ﻤﺤﻠﻴﹰﺎ ﻤﻥ ﺍﻟﻤﻜﻭﻨﺎﺕ‬
‫ﺍﻟﻤﺘﻌﺩﺩﺓ ﻭﺍﻷﺩﻭﺍﺕ ﻭﺍﻻﺸﺨﺎﺹ ﺍﻟﻌﺎﻤﻠﻴﻥ ﻓﻰ ﺘﺠﻬﻴﺯ ﻫﺫﺍ ﺍﻟﻁﻌﺎﻡ ﻭﺒﻴﻌﻪ ﺒﺎﻟﻤﺤﻼﺕ‪.‬‬
‫ﺒﺎﻟﺭﻏﻡ ﻤﻥ ﺍﻟﻜﺸﻑ ﻋﻥ ﺭﻗﻡ ﺁﻴﺩﺭﻭﺠﻴﻨﻰ ﻤﻨﺨﻔﺽ ﻟﻬﺫﻩ ﺍﻟﻌﻴﻨﺎﺕ ﻨﺴﺒ ﹰﺔ‬
‫ﻻﺴﺘﺨﺩﺍﻡ ﺍﻟﺨل ﻭﻋﺼﻴﺭ ﺍﻟﻠﻴﻤﻭﻥ ﻭﺤﺎﻤﺽ ﺍﻟﺴﺘﺭﻴﻙ ﻤﻊ ﻭﺠﻭﺩ ﺍﻟﺜﻭﻡ ﻭﺍﻟﺘﻰ ﺘﻌﻤل‬
‫ﻜﻌﻭﺍﻤل ﺴﻼﻤﺔ ﺫﺍﺕ ﺍﻫﻤﻴﺔ‪ ،‬ﻨﺠﺩ ﺍﻥ ﺍﻟﻭﻀﻊ ﺍﻟﺼﺤﻰ ﻟﻤﺤﻼﺕ ﺒﻴﻊ ﺍﻻﻁﻌﻤﺔ ﺍﻟﺠﺎﻫﺯﺓ‬
‫ﻟﻺﺴﺘﻬﻼﻙ ﻴﺠﺏ ﺍﻥ ﻴﺭﻗﻰ ﺒﺘﺩﺭﻴﺏ ﺍﻟﻌﺎﻤﻠﻴﻥ ﻭﺍﻟﺘﺜﻘﻴﻑ ﺍﻟﺼﺤﻰ ﻭﺘﻨﻔﻴﺫ ﻗﻭﺍﻨﻴﻥ ﺍﻟﺭﻗﺎﺒﺔ‬
‫ﻋﻠﻰ ﺍﻷﻁﻌﻤﺔ ﻭﺘﺤﺴﻴﻥ ﺍﻨﺘﺎﺝ ﺍﻟﻤﺎﻴﻭﻨﻴﺯ ﺍﻟﻤﻌﺩ ﻤﺤﻠﻴﹰﺎ ﻭﺭﻓﻊ ﺩﺭﺠﺔ ﺍﻷﻤﺎﻥ ﺒﻪ‪.‬‬
‫‪IX‬‬
Introduction
Eating out home increasingly becoming an important daily habit of
individuals inhabit in cites, and in many cases is driven by necessity due
to the increase in workers and students population. Adolescents and
young adults are eating out more frequently than ever as seen in many
countries.
This situation and changing in eating habits bring to the local Sudanese
food market new kinds of foods, which are recognized, know
internationally such as burgers and sausages sandwiches, which were not
part of Sudanese diets. Changes in Sudanese diet profile may bring food
safety hazards to consumers especially in absence of good hygiene
practices and poor personal hygiene situations.
Although Mayonnaise is of good nutrient content, good taste and flavour,
it is composed of different groups of foods, which would combine their
microbial contents; the final product may furnish a good culture medium
for microorganisms that previously had little chance to grow (Frazier and
Westhoff, 1978).
The water activity commonly found in this product is about 0.95 (Snyder,
2002). Approximate minimum aw value for the most spoilage bacteria 0.9,
most spoilage yeasts 0.88 and most spoilage moulds 0.80 (Jay, 1986).
Mayonnaise contains oil, which may become oxidized or hydrolyzed.
The acidity of mayonnaise is affected by a pH 3 to pH 4 which is
favoured by yeasts, moulds and acid tolerant bacteria (Frazier and
Westhoff , 1978 ).
1
Mayonnaise in Sudanese Market is either imported or home-made in huge
amounts. Food safety authorities in Khartoum State destroyed bulk
shipments of mayonnaise and ketchup from cold stores during 2007 due
to violation of expiry date and/or spoilage signs.
2
Objectives
The role of the microbial load in evaluating the quality of this product is
not fully discussed. This study is designed to study the following
objectives:1. To
observe
the
pH
for
each
mayonnaise
sample
prior
microbiological analysis.
2. To count aerobic bacterial viable count.
3. To evaluate contamination of mayonnaise with coliform bacteria as
an indicator for faecal contamination of product and its ingredients.
4. To detect Staphylococcal species presence in the samples.
5. To know about the ingredient of home-made mayonnaise.
3
Literature Review
Composition and characteristics of Mayonnaise
Mayonnaise, as described and defined by the FDA standard of identity, is
an emulsified, semisolid food prepared from vegetable oils, one or more
acidifying ingredients (vinegar, lemon and / or lime juice), egg yolk- containing ingredients, and one or more optional ingredients that include
salt, nutritive carbohydrate, sweeteners, spice, monosodium glutamate,
sequesterants, and crystallization inhibitors. Mayonnaise, in its original
recipe contains not less than 65%
content
of
egg
are
protein,
oil (Snyder, 2002). The nutrient
fat
(saturated,
monounsaturated,
polyunsaturated), carbohydrate, calcium, vitamin A, B vitamins, B12, and
vegetable oils containing, monoglycerides and diglycerides, free fatty
acids, phospholipids, sterols, tocopherols, carotenoids, chlorophyll,
vitamins, E, A and D; while spices are rich in minerals like calcium, iron,
phosphorus, manganese and zinc. Fresh spices contain significant
amounts of β-carotene and vitamin C. Also mayonnaise contains salt and
water (Mann and Truswell, 2002). Mayonnaise must contain mustard
flour in some proportion in order to give it a characteristic flavour, it is
from different seeds, and contains two kinds of oils, fixed or non- volatile
oil and aromatic oil (Lowell and Kilgore 2006). In 1996 study published
by the New England Journal of Medicine found a significant reduction in
risk of heart disease among women who ate the most vitamin E-rich
foods, such as mayonnaise and creamy salad dressings (Gibson. 2006).
The form of water in oil emulsions of mayonnaise and salad dressing,
particularly the chemical composition of the water phase, plays a key role
in their microbiological stability. The pH range is 3.2 to 4.0 due to acetic
acid; the aqueous phase salt content, 0.9 to 1.1% and the sugar content is
4
7 to 10%. This composition provides an aw of 0.925 (Smittle,
2000;Anon., 2001).
Preparation of mayonnaise
Mayonnaise is made by slowly adding oil to an egg yolk, mustard,
vinegar, and salt. Mustard helps to keep the emulsion stable while
whisking vigorously to disperse the oil into the liquid. Egg yolk contains
lecithin, which acts as emulsifier. All ingredients are added at the
beginning of the process to prevent speckles. Home made mayonnaise
can approach 85% fat before the emulsion breaks down; commercial
mayonnaise are more typically 70-80% fat. Low fat mayonnaise products
contain starches, cellulose gel, or other ingredients to simulate the texture
of real mayonnaise.
Homemade mayonnaise can also be made using raw egg whites, with no
yolks at all, at least if it is done at high speed in a food processor. The
resulting texture appears the same, and – if properly seasoned with salt,
pepper, mustard, lemon juice, vinegar a little paprika – it tastes similar to
traditional mayonnaise made with egg yolks.
If mayonnaise is made during wet, humid weather, high humidity and
heat will weight it down and yield a greasy result (Filippone, 2006).
The Moisture holding power of mustard is most suitable for mayonnaise
and salad dressing. It contain materials, which absorb and hold a
considerable amount of water, such mustard flour could hold at least
twice its weight of water and form a heavy paste (Kilgore, 2006).
5
Food poisoning and bacterial growth in mayonnaise
Bacterial food poisoning is a common illness which could be caused by
Staphylococcus aureus, Bacillus species and Salmonella just to name a
few.
An early report associating food poisoning with Bacillus spp. was made
in 1906 when Lubenau described an outbreak in a sanatorium where 300
inmates and staff developed symptoms of profuse diarrhoea, stomach
cramps and vomiting .A spore forming bacillus was isolated from meat
balls incriminated meal, Although Lubenau named the organism Bacillus
peptonificans, the properties he described resemble those of Bacillus
cereus(Adams and Moss, 2000).
The first description of food poisoning caused by staphylococci is thought
to be that of Vaughan and Sternberg who investigated a large outbreak of
illness in Michigan. Salmonellas are now established as one of the most
important causes of food borne illness worldwide. In Europe in 1989 the
annual incidence of salmonellas was around 50 per 100.000 inhabitants in
most countries(Adams and Moss, 2000).
Escherichia coli was first isolated in 1885, the food borne illness types
are not very common causes of food borne illness in developed countries
, but an important cause of childhood diarrhoea in less developed
countries (Adams and Moss, 2000).
According to the Centre for Disease Control food borne illness
cases carry a high price tag, each year in the United States about 76
million people become ill, 325,000 are hospitalized and 5000 die due to
food borne illness (Garden-Robinson, 2005). In the United State, the
largest outbreak of food poisoning recorded occurred in 1994, when 3.4
6
million people in forty different states ate ice-cream that was
contaminated with Salmonella from unpasteurrized egg (Trenev, 1998).
There was concern that mayonnaise, salad dressings, and some egg based
sauces such as hollandaise sauce and béarnaise sauce could be potentially
hazardous because of their link to food borne illness outbreaks (Snyder,
2002).
USDA food microbiologists and public health authorities have named
home-made mayonnaise, Caesar dressing, and hollandaise and béarnaise
sauces as items that pose risks of a hazard if prepared from raw shell
eggs. Possible contamination of intact shell egg by Salmonella enteritidis
was recognized in Europe and the United States during the latter 1980s
when it become established that this pathogen could be transferred from
the infected ovaries of laying hens to the egg yolk before the shell was
formed. Hollandaise and béarnaise sauces are examples of hot butter
sauces, in many ways, those products can be considered as hot
mayonnaise and include more than enough acid, however, the amount of
acid in these foods is not recognized as a critical hazard control (Snyder,
2002).
Food poisoning cases in New south Wales restaurant by Salmonella
species Potsdam strain were attributed to shell egg-based Caesar salad
dressing mayonnaise, a swab of a cap from a mayonnaise bottle collected
and tested positive (Unicomb et. al. 2003)
Due to its ingredients mayonnaise regarded as source of many bacterial
contaminants especially Salmonella spp.
In microbiological survival experiments carried by Hwang and Tamplin
(2005) Listeria monocytogenes was inactivated in mayonnaise at pH 4.0
7
(4.0 log 10 cfu/g reduction in 4 days) and 4.5 (4.0 log 10 cfu/g reduction
in 10 - 15 days). At both storage temperatures, no significant level of
inactivation or growth was observed at pH 5.0.
Meldrum et al. (2006) found in microbiological survey of ready to eat
foods carried in Wales that egg mayonnaise sandwiches were the heavily
contaminated foods which contain 8.8% of unsatisfactory level as set by
the study levels.
Mckellar et.al.(2002) studied the probabilities of Escherichia coli
0157:H7 growth in mayonnaise and specify the factors enhancing growth
and suggest that pH, salt concentrations and temperatures are the main
factors affecting growth.
Lock and Board (1995) suggested that the larger the infection of the egg
with salmonella, the longer the viable organisms persisted in the
mayonnaise prepared from them. They also found that in mayonnaise
prepared from yolks of eggs artificially contaminated with Salmonella
enteritidis and with acetic acid as the acidulant, the growth of the
organisms
was
both
dose-
and
temperature-dependent.
Several
Mayonnaise spoilage bacteria have been investigated by Kurtzman et.al.
(1971) during their study of antibacterial agents which could be added to
delay Mayonnaise spoilage.
In a research study about commercial mayonnaise, the test mayonnaise
was inoculated with salmonella and mixed with chicken and ham and
because of its acidity, the growth of bacteria was decreased in a reduced–
calorie mayonnaise which contains more water and less oil and vinegar.
the lower calorie version killed fewer bacteria. However, other
researchers, have shown that certain strains of bacteria can survive the
acidic environment of mayonnaise (Garden-Robinson, 2005). There are
8
ingredients in mayonnaise vinegar that kills many types of bacteria but
they do not make mayonnaise completely safe, especially when other
food ingredients start to be mixed in with the mayonnaise, for example
potato salad can contain quite a bit of mayonnaise, but once combined
with potatoes, onions, eggs, etc it can form a health hazard depending on
the degree and type of biological contamination.
To form the salad the over-all pH of the mayo-salad is increased from
that of mayonnaise alone, so, if disease causing bacteria are present the
pH is high enough and the temperature is in the danger zone (40-140 F ) ,
then the right conditions for a food borne disease outbreak will occur
(Wolf and Hall, 2000). No microbiological safety or spoilage differences
were observed between the salads prepared with real mayonnaise or
reduced calorie mayonnaise dressings (Erickson et. al. 1993).
Saccharomyces bacilii was isolated from two third of the spoiled
mayonnaise samples examined, most of the rest were spoiled by
Lactobacillus fructivor, samples contained large number of both
Saccharomyces bacilii and Lactobacillus plantarum. Two of samples
contained small numbers of bacilli; Bacillus subtilis, Bacillus pumilis,
and Bacillus. licheniformis were also present in one unspoiled sample
(Kurtzman, Roger, and Hazeltine, 1971).
Storage at law temperature protected the salmonellas against the effect of
the acid. These principles did not apply to mayonnaise prepared from
artificially contaminated eggs with lemon juice as the acidulant, in this
case the fate of Salmonella enteritidis
appeared to be largely
independent, of temperature, but dependent upon inoculum size (Lock
and Board ,1995).
9
The survival of Salmonella Spp., Clostridium perfringens and
Staphylococcus aureus in mayonnaise is influenced by the pH of the
mayonnaise and the choice of the acidulant used in preparation (Radford
and Board, 1993).
Although mayonnaise is often blamed for food poisoning, it seldom
causes food poisoning or spoilage, commercial mayonnaise is mostly
vinegar (acid) and oil emulsified by a small amount of egg yolk, the high
acid level means that bacteria do not grow in mayonnaise, mixing spoons
can transfer bacteria in to the mayonnaise, usually the bacteria will live
only a short time because of the acid. But if the mayonnaise is soon
mixed with other food, this may be long enough to contaminate those
foods, the acidity of mayonnaise will not make unsafe food safe to eat
(Roberts and Hertzler 2001). One general statement is possible, in the
majority of mayonnaise Salmonella enteritidis was protected to a limited
extent by storage at 4 °C (Lock and Board, 2002).
Spoilage in mayonnaise and salad dressings result from a variety of
causes including separation of the emulsion, oxidation and hydrolysis of
the oils by chemical or biological action, and growth of microorganisms
that produce gas or off-flavour, microbial spoilage of these products is
generally caused by yeasts and bacteria (Kurtzman et. al. 1971). Salad
dressings contain oil, which may become oxidized or hydrolyzed, and
enough moisture to permit microbial growth, for the most part, however,
their acidity (about PH 3 to 4) is too great for most bacteria but
favourable for yeasts or moulds. Egg or egg products, pickles, relish,
pimientos, sugar, starch, gums, gelatin, spices, and other ingredients may
add microorganisms, sometimes in appreciable numbers, and may make
the dressings better media for microbial growth. The three types of
spoilage of mayonnaise and similar dressing are (1) separation of the oil
10
or water from the emulsion (2) oxidation and hydrolysis of the oils by
chemical or biological action, and (3) growth of microorganisms to
produce gas, off-flavours, or other defects, darkening often takes place.
The decomposition can be caused by bacteria, yeasts, or moulds Acetic
acid (from vinegar) in the commercial salad dressings and mayonnaise
has the major preservative effect due to its bactericidal effect on
vegetative
cells
of
pathogenic
microorganisms, especially food
pathogens. Salt and sugar have minor effects, (Kurtzman, et. al. 1971).
A total of 30 samples of commercial mayonnaise deliberately
inoculated with Salmonella enteritidis PT4 have been studied. The fastest
death rate of the test organism was obtained with a fat –free mayonnaise
PH 2.6. there was a trend indicating that the antimicrobial attributes of
mayonnaise were enhanced when the pH become more acid, especially
with acetic acid as an acidulant. The looseness of the correlation of death
rates with pH in the range 4-5 may well reflect the contribution of the
diverse range of ingredients other than acids to the antimicrobial
properties of this commodity (Lock and Board, 1995).
Food products compounds combined their microbial contents, water and
flavouring materials are potential sources of contamination, spices and
other condiments are important sources of microorganisms although
spices maybe treated with propylene oxide gas. The shells of eggs
become contaminated by faecal matter from the hen, by the lining of the
nest, by wash water if the eggs are washed, by hand ling and perhaps by
the material in which the eggs are packed, and also microorganisms can
be added from pickles and salt (halophilic) (Frazier and westhoff, 1978).
11
Using pasteurized eggs yolks at 65.6 oC has been proved by snyder
(2006) to be effective in eliminating bacterial contaminants and maintain
mayonnaise characteristics.
12
Materials and Methods
Food handlers Questionnaire
A questionnaire aiming to access the hygiene and sanitation conditions of
food handlers was prepared and distributed among food premises where
mayonnaise samples were taken from May 2007 to ٍSeptember 2007.
Samples:Fifty samples of home -made mayonnaise were collected randomly from
fifty food services establishments (restaurants and cafeterias) in
Khartoum Locality. Information regarding recipes was collected along
with each sample.
Observation of preparation methods of mayonnaise and sanitary
conditions was also performed.
Around 100 grams of sample was collected aseptically from each food
establishment in sterile glass bottle and transferred in ice box to the
laboratory of the department of food Hygiene and Safety at the University
of Khartoum in not more than two hours.
Materials
Equipments
1- Autoclave.
2- Colony counter.
3- Cotton wool.
4- Durham's tubes.
5- Filter papers.
6- Graduated conical flask.
13
7- Hockey stick.
8- Hot air oven.
9- Incubator.
10- Loops.
11- Marker pens.
12- Oil- lens Microscope (Olympus)
13- Petri dishes.
14- pH meter (Hanna instruments).
15- Pipettes.
16- Racks.
17- Sensitive balance.
18- Slides.
19- Sterile universal bottles.
20- Test tubes.
21- Volumetric flasks.
22- Water bath.
Culture Media:
1. MacConkey's broth(SD-Fine
– Chem. India) composition :bile salts,
5.0 g/L ; lactose, 10.0 g/L ; neutral red, 0.075 g/L ; peptone, 20.0 g/L ; ,sodium
chloride, 5.0 g/L ; final pH 7.2±0.2 (37 °C).
2. MacConkey's agar
(SD-Fine – Chem. India)composition: agar, 12.0 g/L
;bile salts, 5.0 g/L ; lactose, 10.0 g/L ; neutral red, 0.075 g/L ; peptone, 20.0 g/L
; sodium chloride, 5.0 g/L ; final pH 7.4±0.2 (25 °C)
3. Mannitol salt agar(Scharlu,
Spain) composition peptone, 10 g/L ; D-
mannitol, 10 g/L ; meat extract, 1 g/L ; phenol red, 0.025 g/L ;sodium chloride,
75 g; final pH 7.4±0.2.
4. Nutrient agar
(Scharlu, Spain); composition agar, 15 g/L ;meat extract,
1 g/L ; peptone, 5 g/L ; sodium chloride, 5 g/L ; yeast extract, 2 g/L ; final pH
7.1±0.2 (37 °C).
14
5.
Peptone water
(Biomarks Laboratories) ;composition peptone, 10 g/L ;
sodium chloride, 5 g/L ; final pH 7.2±0.2 (37 °C).
6. Plate count agar
(Biomarks Laboratories);composition agar, 9.0 g/L ;
dextrose, 1.0 g/L ; tryptone, 5.0 g/L ; yeast extract, 2.5 g/L ; final pH 7.0±0.2
(37 °C).
Chemicals and reagents
1. Bromothymol blue.
2. Creatine powder.
3. Crystal Violet.
4. Dipotassium hydrogen phosphate.
5. Ethanol.
6. Glucose.
7. Gram's iodine.
8. Hydrogen peroxide (H2O2).
9. Kovac's Oxidase Reagent.
10. Methyl red.
11. Potassium hydroxide.
12. Rabbit Plasma
13. Safranine.
14. Sodium chloride.
Materials for biochemical reactions
Oxidase test Materials:
1. Filter paper discs 10 cm in diameter
2. 1%
aqueous
N,
N,
N’,
N’-tetra-methyl-p-phenylenediaminem
dihydrochloride Hcl (Kovac's oxidase reagent)
3. Sterile applicator sticks.
15
Motility medium
Semi-solid agar in prepared semi-solid agar media, motile bacteria swarm
and gave a diffused spread growth, that was easily seen and recognized
by the naked eye.
Semi-solid agar was prepared by dissolving 3.25 gram of nutrient broth
powder in 250 ml distilled water to form 0.4 percent of agar solution.
The final medium was quite clear and transparent. Ten ml amounts were
dispensed in test tubes, and sterilized in to the autoclave at 121 °C for 15
minutes. The tubes were left in vertical position until became semi-solid
Methyl red and Voges – Proskauer medium
compositionPeptone
0.5 g/L; Glucose 0.5 g/L ;Dipotassuim hydrogen phosphate 0.5 gram :Distilled water
100 ml .
The peptone and di potassium hydrogen phosphate are dissolved.
Dispensed in 5 ml amounts and sterilized in the autoclave at 1210C for 15
minutes, glucose was sterilized in the autoclave at 1000C for 30 minutes,
and 0-25 ml to each tube was added, by sterile pipette.
Methyl red indicator solution: Methyl red 0-019; Ethanol;
30ml distilled
Water.
Simmons citrate medium( Micr Master laboratories, India); composition:
agar, 15 g/L ; ammonium dihydrogen phosphate, 0.2 g/L ; bromothymol blue,
0.08 g/L ; disodium ammonium phosphate, 0.8 g/L ; magnesium sulfate
heptahydrate, 0.2 g/L ; sodium chloride, 5 g/L ; trisodium citrate, 2 g/L ; final
pH 7.0 Dispensed into tubes and sterilized into the autoclave at 121 0C
for 15 minutes then Allowed to set as slopes.
Indole test: Medium composition: ,Peptone ( containing tryptophen ) 5
g /L; Sodium chloride . NaCl, 1.25 g/L; Distilled water 250 ml.
16
O'meara reagent for Voges – Proskauer test: The same media made
for methyl red test. O'meara reagent containing ten grams of potassium
hydroxide and 0.075 gram of creatine powder were mixed in 25 ml
distilled water .
Oxidation Fermentation test
using Hugh and Leifson’s
medium ;composition :agar, 2.5 g/L ;bromothymol blue, 0.08 g/L;
2 g/L
;
dipotassium hydrogen phosphate, 0.3 g/L
;
OF
peptone,
sodium chloride, 5 g/L ;final
pH 7.1±0.2 (37 °C).
Sterilization of Culture Media:
All Culture media were sterilized by autoclave at 121oC under steam
pressure of 15 pound per square inch, for 15 minutes, except glucose
which was sterilized in the autoclave by steaming at 100oC for 30
minutes.
Microbiological analysis
All microbiological analysis and tests were carried aseptically near gas
burner flame placed in a laminar flow cabinet.
pH Measurement of Mayonnaise samples
pH values were measured at room temperature (approximately 25 -30 °C)
using a Hanna pH meter (HANNA Instruments) by inserting the
calibrated electrode of the pH meter in small portion of the sample.
Water content measurement of Mayonnaise samples
Water content of Mayonnaise samples was measured by oven drying. In
this method; Five grams of sample were spread by a spatula on an
aluminium foil, the edges of the foil were raised to form a weighting boat.
17
The sample was weighted using a sensitive balance then heated in hot air
oven first at 50 °C for one hour then at 90 °C until the weight became
stable. The deference between the initial weight and the last weight was
calculated as water content percentage (Isengard, 1995)
Aerobic Plate Counts (APCs) of Mayonnaise samples:
Twenty-five grams of each Mayonnaise sample were mixed with 225 ml
of sterile distilled water and homogenised by gentle shaking in sterile
conical flask for several minutes, followed by serial dilutions in the same
diluent. The serial dilutions were prepared up to 10 -6.
From suitable
dilution tubes; 0.1 ml was spread by means of a flamed glass spreader
onto the entire surface of Plate Count Agar plate. This process was done
in duplicate for each selected dilution (Andrews, 1992).
Inoculated plates were inverted and incubated at 37 °C for 24 and 48
hours. The number of colony forming units per ml of Mayonnaise was
calculated by multiplying the number of bacteria by the dilution factor.
Results were reported as means of cfu/ mL of Mayonnaise samples
analyzed
Determination of the Most Probable Number (MPN) of
Coliforms:
Determination of the Most Probable Number (MPN) of coliform bacteria
was carried out according to 8th edition of Bacteriological Analytical
Manual (Feng, Weagant, and Grant, 1998; Andrews, 1992.) using nine
tubes series containing MacConkey's broth (Blood and Curtis, 1995).
For each Mayonnaise samples examined, three tubes containing sterile
double strength MacConkey's broth and inverted Durham's tubes were
prepared.
Another six tubes containing single strength MacConkey's
18
broth and inverted Durham's tubes were prepared. The tubes containing
the medium were sterilized by autoclaving.
From the same mayonnaise homogenate prepared for viable plate count,
from each sample 10 ml portions were added to each of the three tubes
containing double strength MacConkey's broth. One ml portions of the
sample homogenate were added to each of three tubes containing single
strength MacConkey's broth. Other 0.1 ml portions of each homogenized
sample were added to each of the remaining three tubes containing single
strength MacConkey's broth.
The tubes were incubated at 37 °C for 24 hours , After the incubation
period the tubes were examined for the presence of gas in the Durham
tubes. Those showed gas bubbles were considered positive.
Those showed negative results were incubated for another 24 hours
period and examined again for gas production. The results were recorded
and interpreted from the Most Probable Numbers tables (Andrews, 1992).
Isolation and Identification of Enterobacteria:
From the mayonnaise homogenate 0.1 ml aliquot of selected dilution
levels were spread on MacConkey's agar plates and incubated at 37 °C
for 24 hours. Developed colonies morphology was examined and they
were subjected to Gram stain reaction. Sugar fermentation tests and
IMViC reactions were carried to identify isolates.
Enumeration of Staphylococcus aureus from Mayonnaise
samples:
From the Mayonnaise homogenate prepared as described above for APC,
0.1 ml portion of various dilution levels were spread-plated on Mannitol
Salt Agar (MSA) and incubated at 37 °C for 24 hours (Baird & Lee,
19
1995). Colonies with typical Staphylococcus morphology (i.e., circular,
yellow , convex and surrounded by yellow zone due the fermentation of
mannitol
were picked, subjected to Gram staining, examined
microscopically to confirm spherical cells morphology then tested for
catalase and coagulase reactions.
Methods used in Identification of Isolates:
Gram Stain:
1- One drop of distilled water was dropped on the sterile slides, and a
smear was made.
2- The smear was air-dried and heat fixed.
3- Crystal violet was applied for two minutes.
4- The excess stain was washed by water.
5- The slide was flooded with gram's iodine for one minute .
6- The slide was rinsed with water then The excess water was removed
by gentle shacking off, but not to dryness.
7- The slide was decolorized by 90% alcohol for 10 second and rinsed
with water.
8- The slide was flooded with safranine for one minute.
9- The excess stain was drown from the slide and thoroughly washed,
and air dried.
10- The slide was examined under the microscope; gram reaction,
morphology and cell arrangement of microorganism were noted.
Those organisms retain the complex purple or blue are gram positive,
while those losing it (pink or red) are considered gram- negative.
20
Biochemical Tests:All
Biochemical Tests were carried out according to 3rd Edition of
Cowan and Steel's manual for the identification of medical Bacteria
(Barrow and Felthman, 2003).
Catalase production:
1-0.5 ml of 2%H2O2 was placed in a sterile tube.
2- Small amount of the growth from the pure culture was taken by sterile
applicator stick and inserted in the tube containing H2O2.
3- The presence of gas bubbles means that the organism produces
catalase and the test then considered positive, other wise negative.
Oxidase production:
Oxidase test distinguishes major subgroups of chemohetrotrophs by
revealing the absence or presence of cytochrome oxidase enzyme.
Oxidase positive bacteria possess cytochrome oxidase or indophenol
oxidase (an iron containing haemoprotein). These both catalyse the
transport of electrons from donor compounds (NADH) to electron
acceptors (usually oxygen).
1. Two drops from Kovac's Oxidase reagent were dropped on filter paper
disc.
2. Immediately heavy mass of cell were used to cover the moist area, by
using sterile applicator stick.
3. After few minutes a change in colour of the cells along the streak to a
deep purple or black considered as an oxidase positive reaction. If no
21
apparent change in colour from that of the reagent occurred the
organism was considered oxidase negative.
Detection of bacterial motility
The motility medium tubes were inoculated by straight wire; a single stab
down the centre of the tubes to about half the depth of the medium was
made. The tubes were incubated at 37°C for 2-4 days. Those showed
diffuse, hazy growths that spread throughout the medium and rendered it
slightly opaque, were considered motile bacteria. Those gave growths
that were confined to the stab- line, had sharply defined margins and left
the surrounded medium clearly transparent, were considered non-motile.
Two tubes were made from each pure culture tested for motility.
Sugar Oxidation and Fermentation test (OF):
This test has been carried according to Barrow and Feltham (2003).
Tubes containing semi sold Hugh and Leifson’s OF medium were
inoculated using straight wire and one of them covered by sterile paraffin
oil. The tubes incubated for up to five days and examined daily for signs
of fermentation and oxidation.
Carbohydrates attack tests
The breakdown of carbohydrates was tested by phenol red – glucose
broth:
Each tube inoculated with the growth from the surface of the pure culture
media and incubated at 37 °C for 48 hours .Then observed for gas
production and colour change to yellow.
22
Coagulase test:
The enzyme coagulase, produced by a few of the Staphylococcus species,
is a key feature of pathogenic Staphylococcus species. Two drops of
rabbit plasma were placed on sterile slide. One drop of water was placed
on the slide near the plasma. Small amount of the growth from the pure
culture was taken by a wood applicator and placed on the drop of water
and emulsified and then mixed with plasma. The slide were observed for
visible clumping of cells within 10 to 15 seconds Those showed
coagulation or clumping considered positive for coagulase test .
Methyl red test:
The liquid medium was inoculated from the 24 hours pure culture and
incubated at 37 0C for 48 hour. Five drops of the methyl red reagent was
added, mixed. Those tests showed bright red were considered positive
tests, and those showed yellow colour were considered negative.
Voges – Proskauer test:
The liquid medium was inoculated from the young pure culture and
incubated at 370C for 48 hour. 0.5 ml of the O'meara reagent was added
to each tube. The tubes were placed in 37 oC water bath for 4 hours and
aerated by shaking at intervals. Those showed eosin –pink colour were
considered positive tests, otherwise negative.
Indole test
The medium was dispending into the tubes and sterilized into the
autoclave at 121 0C for minutes. The medium was inoculated with the
pure culture and incubated for 95 hours at 37 0C. 0.5ml of kovac's
23
reagent was, added to each tube, and shacked gently. Those tubes showed
red colour in the alcohol layer were considered positive.
Citrate utilization test:
The medium was inoculated with the pure culture incubated for 96 hours
at 37 0C. Those tubes showed blue colour and streak of growth were
considered positive and those showed the original green colour were
considered negative.
24
Results
Samples
The information collected by questionnaire and observations showed that
the fifty samples of home-made mayonnaise were prepared according to
various recipes. All of them contained vinegar, oil while 98% contained
whole eggs or eggs yolks and the majority of the samples (82%)
contained garlic and 62% salt (Table 1). Sixty percent of samples
contained smashed potatoes where 38% contain lemon juice and limited
numbers of samples contain ketchup, pepper, yogurt, carrots, sugar or
bread crumbs. (Figures 1, 2 and 3).
Observations showed that the sanitary conditions of places and utensils
used for mayonnaise preparation were not satisfactory from the point of
public health view and that mayonnaise was not handled according to
good hygienic practices (Table 2).
Water content measurement of Mayonnaise samples
Water content of samples was between 26% - 52 % with mean water
content of 38.8%. Table 1.
The pH measurements of the samples were within 2.9 to 5.8 and the mean
pH of the samples was 3.96 (Figure 2).
Aerobic Plate Counts (APCs) of Mayonnaise samples:
The mean of the Aerobic pale count APC of microorganisms was 4.4 x106,
the mean of the Most Probable Number for coliform baeteria was 3.4 x103
per 100 ml. Table (3) shows these results.
25
Enumeration of Staphylococcus aureus from of Mayonnaise samples:
Isolation and identification of bacterial species from samples reveal the
following microorganisms (Table 4):
1. Staphylococcus spp. in 62% of the samples .
2. Bacillus spp. in 32% of the samples .
1. Enterobacteriaceae in 26% of the samples .
Isolation and Identification of Enterobacteria:
Thirteen samples (26%) showed the presence of Gram negative bacteria
belonging to Enterobacteriaceae ;
77% of them
were presumptively
identified as Escherichia coli while 23% of these isolates were
presumptively identified as Salmonella spp. (Table 5).
26
Table (1) Recipes of home-made Mayonnaise samples
Recipe
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
Whole eggs (raw)
Egg yolk
9
9
9
9
9
9
X
X
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
X
X
X
X
X
X
X
9
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
9
9
Vinegar Garlic
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
X
9
Vegetable oil Potato Ketch-up
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
9
X
X
9
9
X
9
9
9
X
9
X
X
X
9
9
9
9
9
9
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Bread criemb
Yohgurt
X
X
X
X
9
X
X
X
X
X
X
9
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
9
9
9
9
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
Table Key: S & G : Sugar & Gum
27
Lemon Sodium chloride
X
X
X
X
X
X
X
9
X
9
X
X
X
X
X
X
X
X
X
X
X
X
X
9
9
X
X
X
9
9
X
X
X
X
9
9
X
9
X
X
X
9
X
X
X
9
9
9
9
9
Carrot
Pepper
Water
X
9
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
9
9
X
X
X
X
X
9
X
X
9
9
X
X
X
X
X
X
X
X
X
X
X
X
X
9
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
9
9
Mustard S&g P H
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
9
9
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
9
9
3
3
3
3
4
4
3
3
4
4
3
3
3
3
3
3
3
3
4
4
4
3
4
3
4
.
.
.
.
.
.
.
.
9
7
9
8
1
1
9
8
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
4
8
9
7
9
8
6
8
1
0
0
8
1
9
1
Water content
4 2 %
3 9 %
4 3 %
4 8 %
5 0 %
4 4 %
5 2 %
4 1 %
4 5 %
3 5 %
3.4%
3 7 %
4 0 %
3 7 %
4 2 %
3 4 %
2 6 %
3 2 %
3 2 %
3 1 %
3 4 %
3 2 %
4 2 %
4 2 %
4 5 %
Table (1) continue
Recipe Whole eggs (raw) Egg yolk Vinegar Garlic
9
9
9
9
26.
9 X
9
9
27.
9
9
9 X
28.
9 X
9
9
29.
9 X
9
9
30.
9 X
9
9
31.
9 9
9
9
32.
9 X
9
9
33.
9 X
9
9
34.
9 X
9
35.
X
9 X
9
9
36.
9 X
9
9
37.
9 X
9
9
38.
9
9
9 X
39.
9 X
9
9
40.
9 X
9
9
41.
X
9 X
9
42.
X
9 X
9
43.
X
9
9 X
44.
9 X
9
9
45.
X
9 X
9
46.
X
9 X
9
47.
X
9 X
9
48.
X
9 X
9
49.
X
9
9
9
50.
Vegetable oil Potato Ketch-up Bread criemb
X
X
9
9
X
X
9
X
X
X
9
9
X
X
9
9
X
X
X
9
X
X
X
9
X
X
9
9
X
X
9
9
X
X
9
9
X
X
9
9
X
X
9
9
X
X
9
9
X
X
9
9
X
X
9
9
X
X
9
X
X
X
9
9
X
X
9
9
X
X
X
9
X
X
X
9
X
X
X
9
X
X
X
9
X
X
9
9
X
X
9
9
X
X
9
9
X
X
9
X
Yohgurt
X
X
X
X
X
X
X
9
X
X
X
X
9
X
X
X
X
X
X
X
X
X
X
X
X
28
Lemon Sodium chloride
9
9
X
9
X
9
X
X
9
X
X
X
X
X
X
X
9
X
X
X
9
9
X
X
X
X
9
9
9
9
9
9
9
9
9
9
9
9
X
X
9
9
9
9
9
9
9
X
X
9
Carrot
Pepper
Water
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
9
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
9
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
9
9
X
9
9
X
X
X
Mustard S&g P H
X
X
3.8
X
X
3.9
X
X
3.8
X
X
3.2
X
X
3.8
X
X
4.2
X
X
3.4
X
X
3.9
X
X
3.4
X
X
3.6
X
X
3.9
X
X
2.9
X
X
3.2
X
X
3.8
X
X
3.9
X
X
5.3
X
X
5.2
9
9 4.3
9
9 4.2
X
X
5.8
9
9 4.3
9
9 3.7
X
X
4.6
X
X
4.5
X
X
4.4
Water content
4 9 %
3 4 %
3.7%
3 4 %
3 9 %
3 9 %
3 8 %
4 9 %
3 9 %
3 5 %
3 9 %
3 1 %
4 0 %
3 6 %
3 9 %
2 7 %
3 8 %
3 9 %
4 1 %
3 9 %
4 0 %
4 3 %
3 8 %
3 7 %
3 9 %
Table (2) Hygiene and sanitation conditions in food premises
where mayonnaise was prepared
Hygiene situation
Workers medical examination Cards:
Valid
Not Valid
No of cases
Percent
319
48
87
13
308
59
84
16
3
364
0.8
99.2
Present
Absent
31
336
8.4
91.6
Present
Absent
6
44
23
12
88
46
Uniforms:
Worn
Not worn
Wearing glove during preparation of Mayonnaise:
Worn
Not worn
Hair Cover:
Tab water:
Presence of latrines
2
Table (3)
Determination of the Most Probable Number (MPN) of
Coliforms:
Sample number
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
Viable count per gram
9.8 × 106
5.2 × 106
4.6 × 106
1.2 × 106
2.3 × 107
1.4 × 107
6.9 × 106
5.5 × 106
1.1 × 107
5.1 × 106
9.3 × 106
9.7 × 107
1.2 × 107
5.2 × 106
4.2 × 106
3.7 × 106
2.6 × 106
2× 106
3.2 × 106
5 × 105
1.3 × 106
8.2 × 106
9.4 × 106
1.8 × 106
3 × 106
6.6 × 106
1.1 × 107
5.7 × 106
2.8 × 106
7.4 × 106
3
MPN per 100 ml
2.3×103
7.5 ×103
2.1×103
2.1×104
4.6×104
2.8×103
2.3×103
9.3×103
6.4×103
3.9×103
2.8×103
2.3×103
2.4×103
2.3×103
7.5×103
1.2×104
2.8×103
7.× 102
2.8×103
3× 102
7×102
2.8×103
1.5×104
1.4×103
2×103
1.2×104
2.8×103
3.9×103
2.3×103
Table (3) continue
Sample number
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
Viable per gram
1.1 × 107
8.2 × 106
1.8 × 106
5 × 106
8.7 × 106
1.1 × 107
1.3 × 106
5.4 × 106
7.5 × 106
3.8 × 106
2.2 × 107
2.4 × 107
3.2 × 106
4.2 × 106
2.3 × 107
4.4 × 106
3.8 × 106
9.5 × 106
7.6 × 106
3.4 × 106
4
MPN
2.1× 104
2.8× 103
1.1× 103
2.3× 103
2.8× 103
3.9× 103
1.5× 103
2.8× 103
4.8× 103
6.4× 103
1.5× 104
2.1× 104
2× 103
2.8× 103
2.4× 104
2× 103
3.9× 103
6.4× 103
7.5× 103
2.3× 103
5
Table (4)Morphological and Biochemical tests showed the most dominant types of bacteria in each samples:
Sample number
Gram stain
Spores
Motility
Growth in air
1
2
S
R
S
-
+
-
+
+
+
3
4
5
6
7
S
R
R
S
R
S
+
-
+
+
D
-
8
9
R
S
R
-
10
11
12
13
14
S
S
S
R
R
S
Growth
anaerobically
+
Oxidase
Glucose
Carbohydrates
Plasma
-
+
+
+
F
+
F
-ve
+
+
Catalase
production
+
+
+
+
+
+
+
+
+
+
+
+
+
D
+
+
+
+
+
+
+
d
-
+
+
+
+
D
+
F
+
+
F
F /0
F
+
+
+
+
+
+
+
+
+
+
+
-
+
+
+
+
F
+
+
+
D
+
+
+
+
+
+
+
+
+
D
+
+
+
+
+
d
+
+
+
+
D
F
F
F
+
F/0
Coa
-ve
-ve
?
Staphylococcus spp.
Staphylococcus spp.
Staphylococcus spp.
Enterobacteriaceae
Bacillus spp
-
-
+
+
+
-
+
F
- ve
Staphylococcus spp.
-ve
-ve
-ve
?
- ve
-ve
Genus or Family
Staphylococcus spp.
Enterobacteriaceae
Staphylococcus spp.
Staphylococcus spp.
Enterobacteriaceae
Enterobacteriaceae
Staphylococcus spp.
Bacillus spp
Staphylococcus spp.
Enterobacteriaceae
Staphylococcus
Enterobacteriaceae
Table Key:
S: sphere, R: Rod shape, D: different reactions in the duplicated test, Coa: Coagulation, ?: not known, F: fermentation, O: Oxidation
32
Gramstain
Spores
Mortility
Growth in air
Growth anaerobically
Catalaes
Oxidase
Glucose
Carbohydrates
Plasma
S
R
-
+
+
+
+
+
+
+
-
F
+
-ve
R
S
R
R
R
R
S
R
S
R
R
S
S
S
S
R
S
R
+
+
+
+
+
+
+
D
D
D
D
+
D
+
+
D
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
D
D
D
D
+
+
+
D
+
+
+
+
+
+
+
D
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
d
d
d
d
d
d
+
+ no
gas
+
+
D
D
D
D
+
+
+
D
+
+
+
+
+
+
+
D
Sample number
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
+
F
F/0
F/0
F/0
F/0
F
+
F
F/0
+
F
F
F
F
+
F
F/0
-ve
?
?
?
?
-ve
-ve
?
Coa
-ve
-ve
-ve
- ve
?
Genus or Family
Staphylococcus spp.
Enterobacteriaceae
Enterobacteriaceae
Staphylococcus spp.
Bacillus spp.
Bacillus spp.
Bacillus spp.
Bacillus spp.
Staphylococcus spp.
Enterobacteriaceae
Staphylococcus spp.
Bacillus spp.
Enterobacteriaceae
Staphylococcus spp.
Staphylococcus spp.
Staphylococcus spp.
Staphylococcus spp.
Enterobacteriaceae
Staphylococcus spp.
Bacillus spp.
Table Key:
S: sphere, R: Rod shape, D: different reactions in the duplicated test, Coa: Coagulation, ?: not known, F: fermentation, O: Oxidation
33
Sample number
Gramstain
Spores
Mortility
Growth in air
Growth anaerobically
Catalaes
Oxidase
Glucose
Carbohydrates
Plasma
33
34
35
S
S
S
-
-
+
+
+
+
+
+
+
+
+
-
+
+
+
F
F
F
Coa
- ve
- ve
Staphylococcus spp.
Staphylococcus spp.
Staphylococcus spp.
36
37
38
R
R
S
R
+
+
+
D
D
D
+
+
+
+
D
D
+
D
+
+
+
+
d
d
d
D
D
+
D
F/0
F/0
F
F/0
?
-ve
?
Bacillus spp.
Bacillus spp.
Staphylococcus spp.
Bacillus spp.
39
40
S
R
R
S
+
+
-
D
D
-
+
+
+
+
+
D
D
+
+
+
+
+
d
d
-
+
D
D
+
F
F/0
F/0
F
- ve
?
?
-ve
Staphylococcus spp.
Bacillus spp.
Bacillus spp.
Staphylococcus spp.
41
R
+
D
+
D
+
d
D
F/0
?
+
+
-
+
+
D
D
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
D
+
+
D
+
D
+
+
+
+
+
+
+
+
+
+
d
d
-
+
+
+
+
D
+
+
D
+
d
F
+
F
+
F/0
F
F
F/0
F
F
-ve
42
43
44
45
46
47
48
49
50
S
R
S
R
R
S
S
R
S
S
-ve
?
-ve
-ve
?
-ve
-ve
Genus or Family
Bacillus spp.
Staphylococcus spp.
Enterobacteriaceae
Staphylococcus spp.
Enterobacteriaceae
Bacillus spp.
Staphylococcus spp.
Staphylococcus spp.
Bacillus spp.
Staphylococcus spp.
Staphylococcus spp.
Table Key:
S: sphere, R: Rod shape, D: different reactions in the duplicated test, Coa: Coagulation, ?: not known, F: fermentation, O: Oxidation
34
Table (5)
IMViC results carried on Gram negative bacilli isolates
Sampl Indo Meth Voges Citra Member of Enterobacteriace
numb
Red proskau
2.
+
+
-
- Escherichia coli
4.
+
+
-
- Escherichia coli
5.
+
+
-
- Escherichia coli
8.
+
+
-
- Escherichia coli
13.
+
+
-
- Escherichia coli
16.
+
+
-
- Escherichia coli
23.
+
+
-
- Escherichia coli
26.
+
+
-
- Escherichia coli
31.
+
+
-
- Escherichia coli
44.
+
+
-
- Escherichia coli
9.
-
+
-
+ Salmonella spp.
15.
-
+
-
+ Salmonella spp.
42.
-
+
-
+ Salmonella spp.
35
FIGURE (1) SHOWING PERCENTAGE OF
INGREDIENT MAYONNAISES MADE FROM
THE
DIFFERENT
M
O
100%
90%
80%
70%
60%
50%
40%
30%
20%
10%
0%
A
B
C
D
E
F
G
H
I
J
K
L
N
A. Vegetable oils 100%
I. Water 12%
B. Vinegar 100%
J. Sugar and Gum 12%
C. Whole raw eggs or eggs yolks 98%
K. Mustard 12%
D. Garlic 82%
L. Pepper 10%
E. Sodium chloride 62%
M. Carrot 8%
F. Potato 60%
N. Bread crumb and other starches 4%
G. Lemon 24%
O. Ketchup 02%
H. Yoghurt 12%
Figure (2) pH Measurement of Mayonnaise samples
pH of Mayonnaise samples
35
30
Number of samples
25
20
Series1
15
10
5
0
2.9-3.9
4-4.9
5-5.9
pH
36
FIGURE (3) SHOWING PERCENTAGE BY METHODS OF USING
EGGS
80%
70%
60%
50%
40%
30%
20%
10%
0%
ii
iii
i
iv
ii. Whole raw eggs
80%
iii. Whole raw eggs + eggs yolks 12%
i. eggs yolk
06%
iv. Unused
02%
FIGURE (4) SHOWING PERCENTAGE BY THE SOURCE OF EGGS
80%
70%
60%
50%
40%
30%
20%
10%
0%
A
B
C
A. Farms
80%
B. Companies 18%
C. Unused
02%
37
FIGURE (5) SHOWING PERCENTAGE OF
ISOLATE OF BACTERIA FROM MAYONNAISE
THE
DIFFERENT
70%
60%
50%
40%
30%
20%
10%
0%
A
B
A. Staphylococcus
B. Bacillus
C. Escherichia coli
62%
32%
20%
D. Salmonella
06%
C
38
D
Figure (6) Boiling potatoes for mayonaise
Figure (7) peeling of potatoes for Mayonnaise
39
Figure (8) Materials and equipment of Mayonnaise prepration
Figure (9) Mixing of mayonnaise ingredients
40
Figure (10) homemade mayonnaise exposed for sale at room
temperature
41
Discussion
This study showed high bacterial load of mayonnaise with hazardous
bacteria. This bacterial contamination of mayonnaise may come from the
ingredients used in making mayonnaise like yoghurt, carrot, pepper, water, eggs,
potato, and breadcrumbs.
The eggs could be contaminated from the infected hens or their shells became
contaminated from faecal matters from the hen, the lining of the nest or by
washing water.
Food handlers or food processor during cleaning and breaking of the egg
shells, peeling of potatoes or garlic,carrot, or during mixing of yoghurt,
vegetable oil , ketchup could be a major source of mayonnaise contamination
especially in the recognized bad handling habits and low personal hygiene and
sanitation conditions observed in food establishments from where samples were
taken.
Knifes, mixing spoons, food utensils and surfaces which were used for holding
different foods e.g meat
and chicken could also act as sources of cross
contamination as observed during mayonnaise preparation in dusty crowded
environments. (Frazier and westhoff, 1978).
This food is of higher moisure content, the water activity commonly found
in this products is about 0.95(Snyder, 2006), but the detection methods used in
this study did not help in releasing all the bound water. This food has a various
degrees of complexity. They were rich in nitrogenous materials in addition to
their sufficient fermentable carbohydrates. All of these materials could act as
necessary growth factors for microorganisms. The mean pH of four is near the
minimum growth pH favoured by microorganism as explained by Jay, Loessner
and Golden (2005).The mayonnaise samples were considered within the safe pH
range(4.10 or 4.00) as mentioned by Xiong, Xie and Edmondson (2000).
Microorganisms can come in contact with other types of ready food rich in
42
water content and have more optimum pH as they usually consumed through
topping of sandwiches and dishes
Acidic food some times could be associated with food borne outbreak such as
apple juice and fermented sausages as stated by Adams and Moss(2000).
Previous laboratory investigations carried on mayonnaise showed high potential
of bacterial survival. Adams and Moss(2000) showed that Escherichia coli
survived for prolonged periods at pH values that do not permit growth,
particularly is refrigerated foods. Smittle, R.B. (2000) also stated that,
Salmonella is tolerant of acids. Meldruma et. al. (2006) indicated the presence
of Escherichia coli, Staphylococcus aureus and Listeria monocytogenes in egg
mayonnaise sandwiches in Wales, UK.
Salmonella species although associated with eggs they were not isolated from
many samples in this study, this may be due to the methods used for isolation of
bacterial contaminants, which did not take into account the injury of cells
caused by acetic acid in vinegar, it expected that the prevalence of Salmonella
species is much higher than the five percent detected.
In the absence of heat treatment, using much vinegar appear the main safety
factor as it contributes in lowering pH of the products especially most of
mayonnaise samples were prepared from whole eggs which contain the alkaline
egg white.
Garlic (Allium sativum) also can lower bacterial content (Ross et. al. 2001).
Lemon due to citric acid can play a good role in minimizing Salmonella species
in mayonnaise products as suggested by Xiong et. al ( 2002)
Type of vegetable oil used could also affect survival of Salmonella species in
mayonnaise as shown by Lock and Board (1996).
43
Staphylococci species isolated are a result of excessive handling during
preparation of home made mayonnaise and indicate the poor personal hygiene
of food handlers
Enterobacterial load was high as indicated by MPN count. Their presence
suggests poor raw materials quality and poor hygiene preparation conditions.
The presence of Bacillus species and the high viable counts demonstrate clearly
the poor hygiene of mayonnaise preparation.
The study did not examine the samples for moulds but they expected to be high
due to vegetables used as ingredients.
Mayonnaise samples that showed more than one isolate are suggested to be
more hazardous.
44
Recommendation
1- Raw eggs used in homemade mayonnaise must be forbidden in the food
control legislation, because the shells of eggs from the farms are
contaminated by faecal matter from the hens and by the lining of the nest.
2- Hygienic situation of ready - to – eat - food establishment should be
improved by food handler training and health education and enforcement of
food safety laws, by ministry of health and public health inspectors in the
localities.
3- The food control health inspector must insure the certain signs of good
hygienic standards, such as clean toilets, clean cutlery or crockery, clean
walls and floors, clean uniforms and fingernails, short or covered hair, valid
medical card.
4- Wash hand basins and soap must be available in the room of food processing
to help food handler to clean their hands.
5- Good sanitation in Cafeterias must be enforced by the public health
inspector of the locality, because insects are vectors and transmitter of
microorganisms.
6- Vegetables and spices must be cleaned with clean water.
7- Eggs used in homemade mayonnaise must be pasteurized as mentioned in
the literature review.
45
Conclusions
Homemade mayonnaise appeared to be widely used in ready to eat foods
establishments in Khartoum locality. High bacterial loads were detected in
mayonnaise including hazardous bacteria inspite of a high acidity of the product.
This high light the importance of improving production situations and hygienic
status in ready to eat foods establishments in Khartoum locality.
46
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50
IN THE NAME OF ALLA THE MOST GRACIOS AND MOST MERCIFUL
University of Khartoum
Faculty of Public and Environmental Health
Department of Food Hygiene and Safety
Questionnaire about Assessment of Microbiological Quality
of Mayonnaise Consumed at Ready to Eat Foods
Establishments in Khartoum Locality
(1) Ingredients :
(A) Whole eggs
(
)
(B) Eggs yolks
(
)
(C) Vinegar
(
)
(D) Garlic
(
)
(E) Vegetable oils.
(
)
(F) Potato
(
)
(G) Ketchup
(
)
(H) Bread crumb
(
)
(I) Yohgurt
(
)
(J) Lemon
(
)
(K) Sodium Chloride
(
)
(L) Carrot
(
)
(M) Pepper
(
)
(N) Water
(
)
(O) Mustard
(
)
(p) Sugar and Gum
(
)
(Q) Others
(
1
) specify
2. Source of eggs
(A) Farm
( )
(B) Company
(
)
(C) Not using
(
)
(A) Whole raw eggs
(
)
(B) Eggs Yolks
(
)
(C) Whole eggs plus eggs yolks
(
)
3. Methods of using Eggs :
(D) Not using
(
)
(A) By hands
(
)
(B) Other
(
)
(A) Home made
(
)
(B) factories
(
)
(A) Home made
(
)
(B) Factories
(
)
(A) Valid
(
)
(B) Invalid
(
)
(
)
4. Methods of eggs yolks isolation :
5. The source of yoghurt :
6. The source of ketchup :
7. Validity of Medical card :
8. Wearing of overalls:
(A) Wearing
2
specify
(B) Not wearing
(
)
9. Wearing gloves during preparation of Mayonnaise:
(A) Wearing
(
)
(B) Not wearing
(
)
10. The presence of latrine:
(A) Present
(B) Absent
(
)
(
)
3