International Federation Total Count of Potential IFU Methed No. 2 of Fruit Juice Producers Spoilaging Microorganisms of April 1996 (IFU) Fruits and Related Products Page 1 of 5 A. PREFACE It is known that no single plating medium can be perfect in all respects in determining the Total Count of microorganisms present in all products. The purpose of a sultable culture medium is to enable the growth of all the microorganisms having the potential to spoil finished products, the so-called “potential spoilaging microorganisms*. Due largely to the low pH of fruit products, growth of most microorganisms is inhibited and limited mostly to the ACIDURIC TYPES mentioned below: LACTIC ACID BACTERIA, predominantly the genera Lactobacillus and Leuconostoc, can be isolated from low Brix level products, such as juices, and during the earliast stages of the juice concentration process. YEASTS species mostly exhibit osmoduric characteristics and may be encountered as the main potential spoilaging microorganisms of products with Brix level of more than 32° Bx and up to 65° Bx. MOULDS should be regarded mainly as secondary contaminants, due either to rotten raw material or improper storage and re-infection during processing. Some other ACIDURIC TYPES, such as Acetic Acid Bacteria and or Gluconobacter, may be part of the spoilage flora, especially in soft drinks. B. PRINCIPLE There are numerous growth media in use for examination of fruit products. The recommended procedure described below is being successfully used by many research and industrial laboratories. This test-method provides a very informative picture of microbial stability and processing (1), (2), (3). The medium used in testing allows the growth of most spoilaging microorganisms: > Ordinary Yeasts + only + Lactic Acid Bacteria without any further differential typing + Moulds + without any differentiation possibility - Other Aciduric Bacteria without any further differential typingInternational Federation Total Count of Potential IFU Method No, 2 of Fruit Juice Producers Spollaging Microorganisms of April 1996 (FU) Frults and Related Products Page 2 of 5 Cc. METHOD 1. Pour Plate Method using ordinary 90 mm petri dishes and testing at a1:10 fold dilution rate are recommended. A sample of 20 g weighed into 180 ml phosphate buffered dilution water should be used for routine work (pH =7.1 40.1). Phosphate-Buffered Dilution Water (4) a) Stock solution KH2PO4 34.0 g Water 500 mi Adjust pH to 7.1 0.1 with 1 N NaOH, Bring volume to 1 litre with water. Sterilizatior 15 min. at 121° C Store in refrigerator. b) Working solution Take 1.25 ml of above stock solution and bring volume to 1 litre with water. Sterilization: 15 min, at 121° Remark: When working with either sensitive bacteria or with microaerophilic types, the following phosphate butfered peptone water should be used (5): Phosphate - Buffered - Peptone - Water KH2P04 15 og NagHPO4.2H20 90 g NaCl 50 g Peptone 10.0 g Water 100 smi Adjust pH to 7.1 + 0.1 by use of 0.1 N NaOH. Sterilization: 15 min, at 121° C It should be kept in mind that this method is reliable only for general information concerning potential spoilaging microorganisms. For further and more intensive testing the following groups might be considered: + Yeasts: + Osmoduric and Osmophilic types - Osmotolerants - Preservative Resistant TypesInternational Federation Total Count of Potential IFU Method No. 2 of Fruit Juice Producers Spoilaging Microorganisms of April 1996 (FU) Fruits and Related Products Page 3 of 5 + Lactic Acid Bacteria (Lactobacillaceae): - Homofermentative types - Heterofermentative types + Moulds: = Mycotoxinogenic types - Heat Resistant (thermoduric) types. ~ Xerop! types * — Other Aciduric Bacteria: + Acetobacter + Gluconobacter 2. Growth Medium OSA - Orange Serum Agar Medium Formulation Of OSA: Tryptone 10.0 g Yeast Extract 3.0 g Glucose (anhydrous) 40 9 K2HPO4 25 9 ‘Agar (powder) 170 9 Orange Serum* 200 mi Water 800 mi Final pH = 5.5 40.1 ** Sterilization: 15 min. at 121° C * The orange serum is prepared by using fresh orange juice or reconstituted orange juice at 11° Brix. Note that if orange juice concentrate is used for dilution, it should be free of preservatives. ‘While being stirred, the juice (11° Bx) is heated to 93° C. The heated juice is filtered by use of a cotton layer. The filtered fluid, which constitutes the serum, should be added to the already dissolved ingredients before autoclaving. Commercially available media must be in accordance with the above and should be tested for their efficacy. ** Measure the pH of the medium after sterilization on a sterile sample at 45° C, So that it is at the required pH. The adjustment of the pH (4.0.1 unit), if necessary, should be done by use of either sodium hydroxide (1N), or hydrochloric acid (1N).International Federation Totat Count of Potential 1FU Method No. 2 of Fruit Juice Producers + Spollaging Microorganisms of April 1996, (IFU) Fruits and Related Products Page 4 of 5 3. Incubation: Time / Temperature Because of the versatility of the various systematic microbial groups, a median temperature is recommended, Incubation: Temperature: 30° + 1° C Time: 2-3 days First counting: 48 hours Mino growth: Final counting: 72 hours 4, Expression of Results 1. Total Count of Microorganisms {tis highly recommended to mention the number of Clu per 1g sample and the incubation time, 2. Additional informative data by Differential Counting a. Colonies of Yeasts - identified by colony and cell morphology. The final analysis should be based on the size of the yeasts cells as determined by a professiohal analyst. b. Colonies of Acidurle Bacteria identified morphologically; no interpretation should be made without microscopic examination after Gram-staining. Lactic Acld Bacteria are Gram-positive stained, while Acetobacter andGluconobacter are Gram-negative stained. If necessary, further biochemical tests could be carried out for identification. c. Colonies of Moulds - typical colonies identified by either mycelium spread or spore formation. Remark: The identification of any pin-point or questionable colonies should be examined microscopically.International Federation Total Count of Potential IFU Method No. 2 of Fruit Juice Producers Spoilaging Microorganisms of April 1996 (IFU) Fruits and Related Products Page 5 of S REFERENCES: (1) Mislivec, P.B., Beuchat L.K. and Cousin M.A. (1992) "Yeasts and Moulds" ‘Compendium of Methods for the Microbiological Examination of Foods. ‘American Public Health Ass. APHA. (2) Weissman, Sh. (1992) “Microbiology of Citrus Products" - 1969-1992. Inst. for Food Microbiology, !srael. (3) Schildmann, J.A. (1993) “Peter Eckes" KG. Private Communication. (4) FDA- USA (1995) “Media and Reagents" FDA - Bacteriological Analytical Manual, 8th Edition. (5) Besling, J.R. (1993) Private Communication.