Isolation of Bacteria Playing a Role in Food Fermentation

Isolation of Bacteria Playing a Role in Food Fermentation Basic theory : Microorganisms that are abundant in nature apparently have a role varied, can be destructive but there are also many benefits, especially inside fermented products. Lactic acid bacteria (LAB) are a group of gram bacteria positive in the form of cocci or rods, does not form spores, optimum temperature ± 40oC, generally non-motile, anaerobic, catalase negative and oxidase positive, with lactic acid as the main product of carbohydrate fermentation. LAB consists of 4 genera, namely: Lactobacillus, Leuconostoc, Streptococcus and Pediococcus. But as identification techniques develop, new characteristics emerge has 16 genera. Lactic acid bacteria are bacteria that are often used as a starter culture for fermented milk, has potential as an anti-cholesterol thought to be due to the presence of exopolysaccharides (EPS). Homofermentative bacteria are fermenting only lactic acid bacteria produces lactic acid as the end product. Homofermentative bacteria are common used in food preservation, due to the production of lactic acid in large quantities High levels in food can inhibit the growth of other bacteria causes food spoilage. In this group of bacteria pyruvic acid is formed from the glycolysis pathway (EMP) acts as a hydrogen acceptor, where Reduction of pyruvic acid by NADH2 produces lactic acid. Lactic acid bacteria can be divided into 2 groups, namely: 1. Homofermentative bacteria; Glucose is fermented to produce lactic acid as the only product. Examples: Streptococcus, Pediococcus, and several Lactobacillus. 2. Heterofermentative bacteria: glucose is fermented in addition to producing acid Lactate also produces other compounds, namely ethanol, acetic acid. Example:Leuconostoc, and several species of Lactobacillus. Instructional Objectives 1. Able to know the bacteria that play a role in fermentation food and able to isolate bacteria that play a role in food fermentation B. Materials and tools Ingredients:     Salted vegetables Starter yoghurt Yoghurt Kefir Tool:     Bunsen Petri dish OR Goblet Erlenmeyer Media :            Micropipette MRS Agar Lactose Broth (with durham tube) MR –VP Alphanaftol KOH H2O2 solution Diluent Solution Spirit Alcohol Diluent solution C. Work procedures: Effect of Different Temperatures on Lactic Acid Bacteria 1. Prepare a diluent solution (physiological NaCl), also prepare MRS Agar media 2. Take 1 cc of the material to be tested and dilute it with 9 ml diluent solution. Meanwhile, for foodstuffs that are not liquid, weigh 1 gram of the solution in 9 ml diluent solution 3. Dilute to 101 4. Turn on MRS Agar media 5. Incubate for 2 days, at temperatures of 25oC and 37oC, observe 6. Do it aseptically 7. Perform a quadrant scratching technique on the sample Carbohydrate breakdown (testing with lactose broth) 1. From the sample, inoculate 1cc into Lactose broth) 2. Incubate at 37oC 3. Observe growth (marked by turbidity) and gas formation in the Durham tube. The emergence of gas indicates a heterofermentative reaction Catalase Testing 1. Take one lop of bacteria from the agar (can be from an agar slant or a petri dish) 2. Then drip with hydrogen peroxide 3. If gas bubbles are released then catalase is positive, if there are no gas bubbles then catalase is negative 4. Do this on several bacteria and record the results in the table Methyl Red Testing First day 1. Mark the MR-VP broth tube with the bacterial culture used 2. Inoculate MR-VP broth with bacterial culture 3. Incubate at 37oC for 5x24 hours Day Two 1. Add 5 drops of methyl red reagent into the MR VP tube 2. Report results. The test is positive if the broth is red after addition methyl red reagent. The test is negative if the MR-VP broth turns yellow or orange after adding reagents Voges Proskauer Test First day 1. Label the MR-VP broth with the bacterial culture used 2. Inoculate MR-VP broth with bacterial culture 3. Incubate at 37oC for 24-48 hours Day Two 1. Add 10 drops of 40% KOH solution and 15 drops of alphanaphthol solution in the broth MR-VP 2. Shake the jar until the broth looks frothy. Shake well increasing aeration resulting in increased oxidation of 2-3 butanediol to acetoin and clarify the reaction results of this test. The reaction results can be seen at the latest after 30 minutes 3. The test is positive if the broth is red within 30 minutes after addition of reagents. The test is negative if MR-VP broth does not show color change after adding reagents Observation with a Microscope 1. Make observations with a microscope with staining 2. Describe what you see (photo if necessary)