Title of Invention	"MEDIUM FOR DETERMINATION OF TOTAL PLATE COUNT IN FOOD"
Abstract	The present invention relates to a medium for the determination of total plate count in food comprising 0.4 - 0.6 % of peptone or tryptone; 0.2 - 0.3 % of beef extract or meat extract; 0.2 - 0.3 % of sugar or sugar alcohol; 0.001 - 0.003 % by weight of phenol red and 1 - 2 % by weight of agar and process of preparation thereof.

Full Text

FIELD OF INVENTION The present invention relates to a medium for the determination of total plate count in food and a process for preparation thereof. BACKGROUND OF INVENTION In food microbiology the microbial load is assessed by counting the total number of micro-organisms in a specified quantity of food. A number of bacteriological media are recommended from time to time for this purpose. One of the media described in the art for total plate count is Dextrose Tryptone Agar (DTA). This.media is.particularly recommended by National Canners Association in canned foods for the determination of thermophilic "flat sour" causing bacteria. Standard Methods for the Examination of Dairy Products recommended this media for the total plate count of mesophilic and or thermophilic aerobes. One of the disadvantages of this media is that it requires 36 to 48 hrs of incubation at 37°C or at 55°C. Another disadvantage of the said media is poor readability of colony as the color change of bromocresol purple is slow and pH less than 5.2. Another media described in the art for total count is Beef Infusion Lactose Agar. This media is particularly recommended for the total plate count of butter and butter products by American Dairy Science association. The major disadvantage of this medium is that ft fs particularly useful for the enumeration of lipolytic bacteria. Other media recommended by Standard Methods for the Examination of Dairy Products is the Tryptone Glucose Extract Agar. One of the major disadvantages of this media is that it does not differentiate between acid producing bacteria and non-acid producing bacteria from glucose. Another disadvantage of this medium is that it requires 48 hours of incubation at 37°C. OBJECTIVE OF THE INVENTION The principal object of the present invention is to develop a medium for the determination of total plate count which gives results within 24 hours of incubation at37°C and 55°C Another object of the present is to develop a medium for the determination of total plate count to have clear readability of the colony developed during incubation by creating a contrasting background Still another object of the present invention is to develop a medium for the determination of total plate count of thermophilic and mesophilic bacteria. Yet another object of the present is to develop a medium for the determination of total plate count that can be used for the study of aerobic and anaerobic bacteria. Still another object of the present is to develop a medium for the determination of total plate count that can be used in all types of foods produced in the food industry. Yet another object of the present invention is to develop a media for the determination of total plate count to differentiate between acid producing bacteria and non-acid producing bacteria even at low levels of acid production. SUMMARY OF THE INVENTION The present invention relates to a medium for the determination of total plate count in food comprising 0.4 - 0.6 % of peptone or tryptone; 0.2 - 0.3 % of beef extract or meat extract; 0.2 - 0.3 % of sugar or sugar alcohol; 0.001 - 0.003 % by weight of phenol red and 1 - 2 % by weight of agar The present invention further relates to a process for the preparation of the medium for the determination of total plate count in food comprising the steps of dissolving 0.4 - 0.6 % of peptone or tryptone, 0.2 - 0.3 % of beef extract or meat extract, 0.2 - 0.3 % of glucose, 0.001 - 0.003 % of phenol red in one-fourth volume of deionized water at pH of 7.0 to 7.6; melting 1 - 2 % of agar by heating in three-fourth volume with deionised water; mixing the molten agar with the solution of step (i) by si'toclaving at 118-123°C for 10 to 20 minutes. DESCRIPTION OF INVENTION According to the present invention a medium is developed which is used for the determination of total plate count for routine food analysis in food industry. The medium is simple and easy to prepare. It gives faster growth thereby the results are obtainable within 24 hours of incubation. The said medium also differentiates between acid producing bacteria and non-acid producing bacteria even at low levels of acid production. The readability of the inoculated culture plate is very high due to contrasting background. On comparison with other total plate count media the growth rate of microorganism is high and the number of colony developed in the said media is also more. The medium contains enriched nutrients like peptone or tryptone at 0.1 to 1 percent. Enzymes, amino acids and other vitamins are supplemented in the form of meat extract or beef extract preferably in the concentration of 0.2 to 0.5 percent. Sugar for fermentation by acid producers is supplemented as lactose, dextrose or dulcitol or the like at a concentration of 0.1 to 0.5 per cent. The background colour of the medium is given by dyes, or indicators like phenol red or methylene blue which are non-inhibitory to bacteria. pH of the medium is adjusted to alkaline in reaction preferably between-pH 7.0 to 7.6, most suitably at 7.25. The ingredients other than agar are dissolved in one-fourth volume required in distilled or deionized water and pH is adjusted to 7.6. The agar for solidification at 1.5% concentration1 is melted by heating in remaining three-fourths volume with distilled or deionised water and mixed before sterilization by autoclaving at 118-123°C and preferably at 121°C for 15 minutes. The inocula are prepared at suitable dilutions and plates are prepared as poured plate for total count. For determining anaerobic growth, the broth without agar is over layered with sterile liquid paraffin. After incubation for 24 hrs the bacterial colonies on the plates are counted for total plate count of the sample. The colour of the colony changes from pinkish red to yellow due to fermentation of glucose. To determine the presence of aerobic and anaerobic organism liquid broth is used without agar where the colour changes from pink to yellow is noted. Food samples received from production centre for routine microbiological analysis were utilized. Fbocf samples received were pre-incubated at 37°C and at 55°C before analysis. Sample macerated in quarter strength of Ringer Solution were appropriately diluted and plate count was carried out. The results were compared with that of the one used conventionally such as Dextrose Tryptone Agar (DTA) obtained from HiMedia Laboratories, Mumbai. The new media was prepared as explained above. Total Plate count was carried out by poured plate method and recorded as colony forming units (CFU) per ml. Plates were incubated at 37°C and reading was taken after 24 ± 2 hours of incubation. Incubation of conventional media was prolonged up to 48 hours and reading was taken at 24 and 48 hrs. Liquid broth was used to determine presence or absence of aerobic and anaerobic organisms where the colour change to yellow is noted. For anaerobic growth liquid media is over layered with sterile liquid paraffin. Conventional liquid broth used was Dextrose Tryptone Broth (DTB) OBSERVATIONS Table I: Retort Processed Spicy Foods (Table Removed) CFU = Colony forming Unit, DTA = Dextrose Tryptone Agar, MTGEA Extract Agar Modified Tryptone Glucose Table II: Retort Processed Sweet Foods (Table Removed) Table III: Liquid Foods / Drinks (Incubation at 37°C) (Table Removed) CFU = Colony forming Unit, DTA = Dextrose Tryptone Agar, MTGEA = Modified Tryptone Glucose Extract Agar Table IVA: Other foods (Reading taken after 24 a 48 hrs of incubation at 37°C) Aerobic Growth in Liquid broth (Table Removed) DTB = Dextrose Tryptone Broth, MTGEB = Modified Tryptone Glucose Extract Broth Table IVB: Other foods (Reading taken after 24 & 48 hrs of incubation at 37°C) Anaerobic Growth in Liquid broth (Table Removed) DTB = Dextrose Tryptone Broth, MTGEB = Modified Tryptone Glucose Extract Broth Table IVC: Other foods (Reading taken after 24 & 48 hrs of incubation at 37°C) Aerobic Growth in Solid media (Table Removed) CFU = Colony forming Unit, DTA = Dextrose Tryptone Agar, MTGEA = Modified Tryptone Glucose Extract Agar, NR = Not readable due food particles and hazy background APPENDIX Table 1: Comparison of two media for total plate count (TPC) within 24 hrs (Table Removed) Table 2: Growth at different temperatures (Table Removed) Table 3: Performance Evaluation of MTGEA at Different pHs (Table Removed) Table 4: Standardization of glucose requirements at pH 7.25 (Table Removed) Note: Original concentration TGE -0.1 %; DTA- -0.5% EXAMPLE The amount of the ingredients used in the media are as follows (Table Removed) 5.0g The pH was adjusted to 7.25. 5g of peptone, 3g of beef extract, 3g of glucose and phenol red were dissolved in one-fourth volume of deionised water at pH of 7.25. 15g of agar for solidification is melted by heating in remaining three-fourth volume with distilled water and mixed before sterilization by autoclaving at 121 °C for 15 mins We Claim, 1. A media for the determination of total plate count in food comprising : i. 0.4 - 0.6 % of peptone or tryptone; ii. 0.2 - 0.4 % of beef extract or meat extract; iii. 0.2 - 0.4 % of sugar or sugar alcohol; iv. 0.001 - 0.003 % by weight of phenol red; v. 1 - 2 % by weight of agar 2. The media for the determination of total plate count as claimed in claim 1, wherein the sugar is glucose. 3. The media for the determination of total plate count as claimed in claim 1, wherein the sugar alcohol is dulcitol. 4. A process for the preparation of the media for the determination of total plate count in food comprising the steps of : i. dissolving 0.4 - 0.6 % of peptone or tryptone, 0.2 - 0.4 % of beef extract or meat extract, 0.2 - 0.4 % of glucose, 0.001 -0.003 % of phenol red in one-fourth volume of deionized water at pH of 7.0 to 7.6; ii. melting 1 - 2 % of agar by heating in three-fourth volume with deionised water; Hi. mixing the molten agar with the solution of step (i) by autoclaving at 118-123° C for 10 to 20 minutes; 5. The process for the preparation of the media for the determination of total plate count in food as claimed in claim 4, wherein the molten agar is mixed with the solution of step (i) preferably at 121 °C. 6. A media for the determination of total plate count in food as herein described with reference to foregoing examples. 7. The process for the preparation of the media for the determination of total plate count in food as claimed in claim 4, wherein the pH is preferably 7.25.

Documents:

2617-del-2008-Abstract-(03-01-2014).pdf

2617-del-2008-abstract.pdf

2617-del-2008-Claims-(18-07-2014).pdf

2617-del-2008-claims.pdf

2617-del-2008-Correspondence Others-(03-01-2014).pdf

2617-del-2008-Correspondence Others-(15-07-2014).pdf

2617-del-2008-Correspondence Others-(18-07-2014).pdf

2617-del-2008-correspondence-others.pdf

2617-del-2008-description (complete).pdf

2617-del-2008-form-1.pdf

2617-del-2008-form-2.pdf

2617-del-2008-form-3.pdf

2617-del-2008-GPA-(03-01-2014).pdf