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Honey as a Natural Preservative in Milk


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Honey as a Natural Preservative in Milk

  2. 2. HONEY <ul><li>Introduction </li></ul><ul><li>Materials required </li></ul><ul><li>Methods </li></ul><ul><li>Result </li></ul><ul><li>Discussion </li></ul><ul><li>Disadvantages </li></ul>
  3. 3. MILK <ul><li>Milk and other dairy related products comprise a major chunk of food products for their nutritive values </li></ul><ul><li>A good growth medium of many micro-organisms like Klebsiella, Bacillus, Pseudomonas and Staphylococcus </li></ul><ul><li>Preservation of Milk – a major problem ! </li></ul>
  4. 4. HONEY <ul><li>Natural food product produced by honey bees </li></ul><ul><li>Its usage since ancient times </li></ul><ul><li>Major use in Ayurveda, Cosmetics and Food products </li></ul><ul><li>Composed of sugars – fructose, glucose and with some amount of sucrose </li></ul>
  5. 5. HONEY <ul><li>Numerous studies on the anti-bacterial activity of honey on clinically significant bacteria have been reviewed </li></ul><ul><li>The anti-bacterial activity of honey can be grouped into peroxide component and non-peroxide component </li></ul><ul><li>The major anti-bacterial factor in honey is HYDROGEN-PEROXIDE </li></ul>
  6. 6. MATERIALS <ul><li>CHEMICALS – Microbiological media and individual components of agar and broths of Luria-Bertani and Nutrient Agar were from HiMedia laboratories,Mumbai,India </li></ul><ul><li>Common chemicals salts and sugars were also from HiMedia laboratories and Sisco Research Lab,Mumbai </li></ul>Luria-Bertani medium Nutrient agar
  7. 7. Milk and Honey <ul><li>Four commercially available pasteurized milk samples were obtained and refrigerated at 4 º C until further use </li></ul><ul><li>Samples stored from 1 to 4 days were serially diluted and the isolated pure cultures were used for further analyses </li></ul><ul><li>Honey was obtained commercially and dilutions of honey were made in sterile water with or without honey at 100 º C for 10 mins in water bath </li></ul><ul><li>Artificial honey was prepared and used as control </li></ul><ul><li>Sterile water was used as negative control for milk and honey samples </li></ul>
  8. 8. Isolation of Bacterial Cultures <ul><li>1ml of refrigerated milk samples were serially diluted in sterile water </li></ul><ul><li>From this,200 µl of sample were plated onto nutrient agar plates and incubated at 37ºC/24 hrs </li></ul><ul><li>The randomly picked colonies were re-inoculated in 15 ml LB broth and incubated for 14-16hrs at 37ºC </li></ul><ul><li>Culture thus obtained was characterized by various biochemical tests </li></ul>
  9. 9. DISC DIFFUSION ASSAY <ul><li>Sterile filter discs (10mm,diameter) were immersed in 5 µl diluted honey solutions and air dried </li></ul><ul><li>The cells were harvested from the cultures grown to mid log phase and the pallet was suspended in 3 ml fresh LB medium </li></ul><ul><li>From this culture,200µl corresponding to 1x10 7 CFU/ml was plated on LB plates </li></ul><ul><li>The discs containing honey of different concentrations were placed on culture plates and incubated at 37ºC for 24 hrs </li></ul><ul><li>The diameter of zones of inhibition were studied and recorded. </li></ul>
  10. 10. Honey inhibits the growth of both Catalase Positive and Negative Bacteria <ul><li>Cultures of bacteria isolated from milk were grown with a density of 1x10 7 CFU/ml </li></ul><ul><li>5% inoculated into 1ml Nutrient Broth </li></ul><ul><li>Each flask supplemented with honey of different concentrations </li></ul><ul><li>The change in absorbance at 550nm was assessed using a photoelectric colorimeter </li></ul>
  11. 11. HONEY- as a Preservative <ul><li>Monitoring the bacterial growth in 500 ml of milk samples that were stored with 100 µl of 500mg/ml solution of honey added </li></ul><ul><li>Stored at 4ºC for 3-6 days </li></ul><ul><li>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 </li></ul>
  12. 12. RESULT Spoilage of Milk during Shelf life <ul><li>Milk samples were evaluated for contaminating bacterial species by serially diluting the samples and plating them on NA plates </li></ul><ul><li>Sterile water used as negative control,did not yield any bacterial colonies </li></ul>
  13. 13. Isolation and Characterization of Bacteria <ul><li>Morphological characteristics, biochemical and microbiological tests </li></ul><ul><li>Most Common – Bacillus spp. , Staphylococcus spp. , Pseudomonas spp. and Klebsiella spp. </li></ul><ul><li>Broadly classified into Catalase positive and catalase negative based on the results of the catalase tests </li></ul>
  14. 14. Disc Diffusion Assay <ul><li>Zones of inhibition were measured </li></ul><ul><li>Diameter of inhibition zones dependent more on the bacterial species and the concentration of honey </li></ul><ul><li>Honey was found to inhibit catalase negative cultures while catalase positive cultures were not inhibited to the same extent </li></ul><ul><li>Major role of Hydrogen Peroxide </li></ul><ul><li>Artificial honey was used at various concentrations and had no inhibitory effect on the organisms </li></ul>
  15. 15. Honey inhibits growth of both Catalase Positive and Negative Bacteria <ul><li>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 </li></ul>
  16. 16. HONEY – As a Preservative <ul><li>Milk samples stored at 4 ºC from 3-6 days in the presence or absence of honey were assayed for their bacterial content and growth </li></ul><ul><li>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 </li></ul>
  17. 17. DISCUSSION <ul><li>Preservation and storage of milk </li></ul><ul><li>The Lactoperoxidase system – </li></ul><ul><li>Its natural role in preservation of milk </li></ul><ul><li>Mechanism </li></ul><ul><li>Properties </li></ul><ul><li>Chemical preservation discouraged by FAO/WHO </li></ul><ul><li>Honey as a safe,natural product </li></ul>
  18. 18. DISADVANTAGES <ul><li>Spores of C.botulinum have been found in about 10% of honey by the Centre for Disease Control and Preservation (CDC) in the US. </li></ul><ul><li>Known to cause Botulism in infants which can cause serious illness in the first year of life or can prove fatal </li></ul><ul><li>Many people are found to be allergic to Honey </li></ul>
  19. 19. REFERENCES <ul><li>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 </li></ul><ul><li>Roushdy A,Studies on the efficiency of hydrogen peroxide and methods for its detection and quantification. J Food Prot,59(1996),1223 </li></ul><ul><li>White J W, Honey Adv Food Sci,24 (1978),287 etc </li></ul>