HONEY

1. HONEY AS A NATURAL PRESERVATIVE OF MILK By ARCHANA.NATARAJAN MSc.BT-09022

2. HONEY • Introduction • Materials required • Methods • Result • Discussion • Limitations

3. MILK • Milk and other dairy related products comprise a major chunk of food products for their nutritive values • A good growth medium of many micro-organisms like Klebsiella, Bacillus, Pseudomonas and Staphylococcus • Preservation of Milk – a major problem !

4. 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

5. 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

6. 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

7. 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

8. 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 re-inoculated in 15 ml LB broth and incubated for 14-16hrs at 37ºC • Culture thus obtained was characterized by various biochemical tests

9. 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.

10. 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

11. 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

12. RESULTSpoilage 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

13. 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

14. 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

15. 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

16. 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

17. DISCUSSION • 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

18. 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

19. 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