Transcript honey - Ning

HONEY
AS A NATURAL PRESERVATIVE
OF MILK
By ARCHANA.NATARAJAN
MSc.BT-09022
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HONEY
Introduction
Materials required
Methods
Result
Discussion
Limitations
MILK
• Milk and other dairy related products
comprise a major chunk of food products
for their nutritive values
• A good growth medium of many microorganisms like Klebsiella, Bacillus,
Pseudomonas and Staphylococcus
• Preservation of Milk – a major problem !
HONEY
• Natural food product produced by honey
bees
• Its usage since ancient times
• Major use in Ayurveda, Cosmetics and
Food products
• Composed of sugars – fructose, glucose
and with some amount of sucrose
HONEY
• Numerous studies on the anti-bacterial
activity of honey on clinically significant
bacteria have been reviewed
• The anti-bacterial activity of honey can be
grouped into peroxide component and
non-peroxide component
• The major anti-bacterial factor in honey is
HYDROGEN-PEROXIDE
MATERIALS
• CHEMICALS – Microbiological media and
individual components of agar and broths
of Luria-Bertani and Nutrient Agar were
from HiMedia laboratories,Mumbai,India
• Common chemicals salts and sugars were
also from HiMedia laboratories and Sisco
Research Lab,Mumbai
Luria-Bertani medium
Nutrient
agar
Milk and Honey
• Four commercially available pasteurized milk samples
were obtained and refrigerated at 4ºC until further use
• Samples stored from 1 to 4 days were serially diluted
and the isolated pure cultures were used for further
analyses
• Honey was obtained commercially and dilutions of honey
were made in sterile water with or without honey at
100ºC for 10 mins in water bath
• Artificial honey was prepared and used as control
• Sterile water was used as negative control for milk and
honey samples
Isolation of Bacterial Cultures
• 1ml of refrigerated milk samples were serially
diluted in sterile water
• From this,200µl of sample were plated onto
nutrient agar plates and incubated at 37ºC/24
hrs
• The randomly picked colonies were reinoculated in 15 ml LB broth and incubated for
14-16hrs at 37ºC
• Culture thus obtained was characterized by
various biochemical tests
DISC DIFFUSION ASSAY
• Sterile filter discs (10mm,diameter) were immersed in 5µl
diluted honey solutions and air dried
• The cells were harvested from the cultures grown to mid
log phase and the pallet was suspended in 3 ml fresh LB
medium
• From this culture,200µl corresponding to 1x107 CFU/ml
was plated on LB plates
• The discs containing honey of different concentrations
were placed on culture plates and incubated at 37ºC for
24 hrs
• The diameter of zones of inhibition were studied and
recorded.
Honey inhibits the growth of both Catalase
Positive and Negative Bacteria
• Cultures of bacteria isolated from milk
were grown with a density of 1x107
CFU/ml
• 5% inoculated into 1ml Nutrient Broth
• Each flask supplemented with honey of
different concentrations
• The change in absorbance at 550nm was
assessed using a photoelectric colorimeter
HONEY- as a Preservative
• Monitoring the bacterial growth in 500 ml of milk
samples that were stored with 100µl of
500mg/ml solution of honey added
• Stored at 4ºC for 3-6 days
• Comparison with milk sample without
broth(550nm) inoculated with 100µl milk sample
supplemented with honey and a similar volume
of the same milk sample without honey were
quantitated for its bacterial growth
RESULT
Spoilage of Milk during Shelf life
• Milk samples were evaluated for
contaminating bacterial species by serially
diluting the samples and plating them on
NA plates
• Sterile water used as negative control,did
not yield any bacterial colonies
Isolation and Characterization of
Bacteria
• Morphological characteristics, biochemical
and microbiological tests
• Most Common – Bacillus spp. ,
Staphylococcus spp. , Pseudomonas spp.
and Klebsiella spp.
• Broadly classified into Catalase positive
and catalase negative based on the
results of the catalase tests
Disc Diffusion Assay
• Zones of inhibition were measured
• Diameter of inhibition zones dependent more on
the bacterial species and the concentration of
honey
• Honey was found to inhibit catalase negative
cultures while catalase positive cultures were not
inhibited to the same extent
• Major role of Hydrogen Peroxide
• Artificial honey was used at various
concentrations and had no inhibitory effect on
the organisms
Honey inhibits growth of both
Catalase Positive and Negative
Bacteria
• The inhibition of growth increased in a
concentration dependent fashion at 24 hrs
incubation in catalase negative cultures
containing honey with a percent inhibition
of 50-60% in average
HONEY – As a Preservative
• Milk samples stored at 4ºC from 3-6 days
in the presence or absence of honey were
assayed for their bacterial content and
growth
• The addition of honey at a final
concentration of 50mg/ml had a
considerable inhibitory effect on bacterial
growth as compared to the samples
devoid of honey
DISCUSSION
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Preservation and storage of milk
The Lactoperoxidase system –
Its natural role in preservation of milk
Mechanism
Properties
Chemical preservation discouraged by
FAO/WHO
- Honey as a safe,natural product
LIMITATIONS
• Spores of C.botulinum have been found in
about 10% of honey by the Centre for
Disease Control and Preservation (CDC)
in the US.
• Known to cause Botulism in infants which
can cause serious illness in the first year
of life or can prove fatal
• Many people are found to be allergic to
Honey
REFERENCES
• Taornima PJ,Niemira B A & Beuchat P R ,
Inhibitory activity of honey against food borne
pathogens as infuenced by the presence of
hydrogen peroxide and the level of antioxidant
power, Intl J Food Microbiol,69(2001),217
• Roushdy A,Studies on the efficiency of hydrogen
peroxide and methods for its detection and
quantification. J Food Prot,59(1996),1223
• White J W, Honey Adv Food Sci,24 (1978),287