This is a part of my project in Quality control department of SAB-MILLER brewery.It includes the usual test they conduct to ensure quality of Beer.

1. MICROBIOLOGICAL TECHNIQUES IN A BREWERY PRESENTED BY SINI P K M.Sc.BIOTECHNOLOGY St.BERCHMANS’COLLEGE

2. DETAILS OF PROJECT WORK : COMPANY NAME - SAB Miller Brewery chalakudy,kerala. TOPIC - Microbiological & Analytical techniques in BREWERY Duration - May 21 , 2007 to June 15 , 2007 GUIDED BY - Mr. Nishil menon ,SAB Miller Mr.Ram kumar.s ,SAB Miller

3. INTRODUCTION Done the project work in the Quality control department of Brewery Quality control department has Microbiology lab & Chemical analysis lab. This department assures the quality of the product (Beer) by systematic monitering of raw material,product-in process and end product. Uses standardized procedures and are guieded&monitered by theie central inistitution in Banglore.

4. Brewery micro organisms Culture media used in lab Checking yeast count Checking yeast consistancy Checking yeast viability Membrane filtration method Spread- plate method Anaerobic incubation method Yeast propagation method CONTENTS

7. Short oval or single in pairs, short chains or groups.

8. . Spores usually detectable after 48 hours.

12. Two genera are commenly found, Lactobacillus pediococcus.

14. Includes gluconobacter and acetobactor.

15. Gluconobactor oxidises alcohol to acetic acid, but it is further oxidised in to CO2 and H2O

17. OTHER BREWERY MICRO ORGANISMS C WORT BACTERIA Enterobacteriacea capable of fermenting glucose to produce a mixture of acids and ethanol. Hafnia protea able to survive the brewing process and may be carried from one fermentation to another in the picting yeast. Enterobactor agylomerans Coliform bacteria, able to ferment lactose also capable of surviving the brewing process and being carried forward in the picting yeast. Acetic acid bacteria used commercially for producing Vinegar from wine, cider or unhoped malt beer

18. CULTURE MEDIA USED IN BREWERY

19. Media used are : 1 Mac Conkey Agar (HIMEDIA) For detection of coliforms For preparing Mac Conkey suspend 51.5g. in 1000ml distilled water. Boil with gentle swirling to dissolve the medium completely. Sterilise the medium by autoclaving. 2. Plate Count Agar / PCA(HIMEDIA) All forms of bacteria For preparing PCA, suspend 23.5g. in 1000ml distilled water. Boil to dissolve the medium completely. Sterilise by autoclaving. 3.Walerstein Nutrient Medium / WLN (HIMEDIA) All forms of bacteria For preparing WLN, suspend 80.25g. in 1000ml distilled water.Heat to boil to dissolve the medium completely. Sterilise by autoclaving

20. Media used are : 4.Schwarz Differential Medium / SDM (HIMEDIA) Only for wild yeast. For preparing SDM suspend 44.5g. in 1000ml distilled water. Boil with constant stirring for 15 minutes. Donot autoclave 5. Lysine Medium (HIMEDIA) Only for wild yeast For preparation of lysine medium base, suspend 6.6g. in 100ml of distilled water containing 1ml potassium lactate (50%). Heat to boiling to dissolve the medium completely. Donot autoclave 6.Raka Ray Medium (HIMEDIA) Anaerobic Medium To prepare Raka Ray Medium, 3ml of Tween 80 is added to 100ml of distilled water. To this 29g. of Lactic acid broth (selective media) is added and mixed well. To this 6g. of agar is added and is made up to 300ml using distlled water. After dissolving the medium sterilize by autoclave for 15 minutes. After cooling it to room temperature add lactose supplement to media before it sets.

21. CHECKING YEAST COUNT

23. Fill chamber in hemocytometer by setting pipette tip on edge of chamber. Avoid over fill.

25. YEAST COUNT CALCULATION Yeast count = No. of bacteria X 25 squres X 102 X104 RESULT Yeast count = 5 X 25 X 102 X 104 = 125 X 106 = 125 million cells.

26. YEAST CONSISTANCY

27. CHECKING YEAST CONSISTANCY AIM To determime the consistancy of yeast slurry and thus to find out the pitching rate PREPARING SAMPLE Yeast sample are taken directly from fermentation tanks.

28. CHECKING YEAST CONSISTANCY METHOD The empty weight of the centrifuge tubes are taken by using a weighing machine. The samples are then filled in two certifuge tubes and the initial weight of the samples are taken. The tubes are then placed for centrifugation for 15 minutes at 3000 pm After 15 minutes the tubes are taken and the supernatent is drained off. The final weight of the yeast cells are taken.

29. YEAST CONSISTANCY CHECKING CALCULATION Note the values of empty weight , initial weight and final weight. Initial weight - Empty weight = X Final weight - Empty weight = Y Yeast consistancy = Y x 100 X RESULT Initial weight = 132.1508 Final weight = 112.0493 X = 132.1508 - 69.9135 = 62.2373 Y = 112.0493 - 69.9135 = 42.1358 Consistancy = Yx100 = 42.1358x100 x 62.2373 = 67.70 %

30. YEAST VIABILITY

32. distilled water and mix well.

33. Transfer two drops of above yeast suspension and add two drops of methylene blue. Mix well and allow to stand for I minute.

34. Take a drop of this suspension and transfer on to the heamocytometer. Examine under 40 X bright field objective,.

36. MEMBRANE FILTRATION METHOD

39. Manifold includes the filtration cups.

40. The filtration cups are connected to a common hollow, stainless steel pipe. This pipe is inturn connected to one opening of a side arm flask.

41. A Vaccum pump is connected to another opening of the side arm flask by means of Vaccum tubing.

42. The major part of a filtration apparatus is the membrane filter. These are circular filter made of cellulose acetate, cellulose nitrate, poly carbonate, poly vinyl chloride, or other synthetic materials

43. . These filters consists of pores. A wide variety of membrane filter with different pore sizes ranging from 0.01 – 10 micro meter are available.

44. Here in brewery membrane filter of 0.45micrometer pore size is used.

46. RESULT Absence of colonies on membrane filter after incubation. The beer sample is pure

52. SPREAD – PLATE METHOD

53. SPREAD – PLATE METHOD Spread plate method is an indirect plate court or viable count technique. Method is used to detect the presence of viable microorganisms in a sample. This method is also useful to study the colony characteristics and biochemical properties of the colonies which leads to the identification of the particular microorganism.

54. SPREAD – PLATE METHOD PRINCIPLE The peculiarity of viable cells is that they can grow, reproduce and thus produces colones. Each viable cell develops a distinct colony. The original number of viable micro organisms in the sample is proportional to the number of colonies in the agar plate after incubation. Usually the count is made more accurate by use of special colony counter. REQUIREMENTS Sterile plate with sterile solid media (eg:- Mac conkey agar, Plate count Agar, WLN, Raka Ray etc.) Sample which is to be analysed (eg: beer, wort, gas dissolved in saline etc.) Sterile, graduated 1ml pipette L- rod.

55. SPREAD – PLATE METHOD PROCEDURE If the sample is thickly populated with microorganism. It should be diluted. L –rod is taken and its bend portion is dipped in alcohol and dlame heated for sterilization. The L-rod is allowed to cool for 10-15 seconds. Mix the contents of the sample in bottle or test tube. Remove the lid or cap, flame the mouth and withdraw 0.5ml sample into a sterile pipette. Replace the lid of the bottle. Hold the petridish in the hand and lift the lid of it slightly. Expel the sample on to the surface of the agar. Sterlise the L-rod by flash flaming after dipping in alcohol cool the L-rod. On the edge of the agar spread the sample over the surface. While rotating the plate to ensure even distribution of sample. Replace the lid of the petridish Incubate the petridishes ar appropriate temperature and check for colonies. RESULT No colonies obtained. Beer is of GOOD QUALITY

57. ANAEROBIC INCUBATION METHOD

58. ANAEROBIC INCUBATION METHOD Anaerobic microorganisms are used in a brewery for the production of beer, It includes the saccharomyces strains of yeast. In addition to this various unwanted anaerobic microorganisms affects the beer quality. The final Packaged beer must be free of all these microorganisms. So for the purification analysis of beer, O2, Co2 Anaerobic cultivation methods are used. Here anaerobic cultivation is done by using anaerobic Jar.

59. ANAEROBIC INCUBATION METHOD PRINCIPLE usingthe anaerobic Jar an anaerobic environment is created. This is achieved by using a Gas pack inside the anaerobic Jar Gas Pack consists of hydrogen, CO2 and palladium as catalyst. H2 and CO2 inside the Gas Pack diffuses into the Jar H2 combines with O2 inside the Jar to form H2O in presence of palladium catalyst and thus O2 is removed. The CO2 also creates an anaerobic environment. Indicator tablets inside the Jar become pink Colour after complete anaerobisis is achieved.

60. ANAEROBIC INCUBATION METHOD INSTRUMENTATION Apparatus is called Gas Pack Jar or anaerobic Jar with a lid. Lid consists of screws and a pressure gague. In addition to the anaerobic jar a gas pack is also present gas pack is commercially available in a disposable envelope . It consists of chemicals that generate H2, CO2 and palladium. An indicator tablet is also available along with the gas pack. It is also placed inside the jar. If the environment is completely anaerobic the tablet becomes pink in colour and in presence of O2 it becomes lilac colour.

63. ANAEROBIC INCUBATION METHOD PROCEDURE The sample to be analysed ( beer, air dissolved in water) is inoculated on an medium Anaerobic medium (eg. – Raka Ray medium) was used The Petri dish is placed inside the anaerobic jar. The outer plastic cover of the gas pack is removed and placed inside the anaerobic jar. Indicator tablets are also placed inside the jar. Immediately close the lid of the anaerobic jar and tightly close the screws. Check the pressure gague. Indicator tables is now lilac in colour because of presence of O2 inside the jar The H2 and Co2 diffuses from gas pack. H2 combine with O2 inside the jar to form water in presence of catalyst. After some hours indicator tablets become pink in colour shows the complete removal of O2 inside the jar. Incubate for one week check the pressure. After one week open lid and take and the Petri plates. Check for the presence of colonies. .

64. YEAST PROPAGATION METHOD

65. YEAST PROPAGATION Brewing utilizes strains of Saccharomyces calsbergensis a bottom yeast and Saccharomyces cerevisiaeboth bottom and top yeast strains. Yeast propagation is a multi step process that involves a gradual increase in number of yeast cells from a single colony to many hecto liters.

66. YEAST PROPAGATION In a multi plant situation yeast pure culture is provided from the main laboratory to other regional laboratories of the brewery. This is to ensure that the same yeast strain is used by all the plants of the company otherwise it would affect the quality of the final product. The main laboratory supplies yeast in many ways: As Slant cultures As pure culture in 10 – 50ml flask As pressed pure yeast cake (25-40% dry weight) As yeast slurry in 10-20litre stainless steel flasks. Transportation is usually in an insulated cooler box or packaged in dry ice or both. Transportation time should not exceed 24 hours. The yeast seed lots received by the regional laboratory should propagate it. So that a satisfactory yeast count is obtained for online pitching.

67. YEAST PROPAGATION PROCEDURE Yeast propagation is a multistep process which needs atleast two weeks. Wort at a low temperature (usually 200c or below) is used as a medium for yeast propagation oxygenation is achieved mainly by agitation

68. YEAST PROPAGATION Step 1 : Recovering yeast cells from agar slants. For recovering the cells from the agar slants add 10ml of saline solution into the slant shake well. Inoculum for further propagation is obtained from this Step 2: I ml to 15ml stage 1 ml of inoculum is taken from the slant using a micro pipette and is inoculated to 4 sets of 15ml wort (at 200c) in concial flasks. 1 ml of inoculum is added to each set . The flasks are then cotton plugged and allowed to shake (to supply air /O2) in an orbital shaker. Incubation is at 250c until a peak count of yeast cell is obtained. Incubation period is approximately 22-24 hours. Peak count is nearly 23 million cells/ml. Step 3: 15ml to 200ml stage After peak count is obtained 4ml of inoculum is taken from 15ml each and transferred to 4 sets of 200ml wort in conical flask. Placed in orbital shaker and incubate at 250c until peak count is obtained. Incubation period is approximately 22 hours. Step 4: 200ml to 10 litre stage After peak count is obtained 200ml yeast culture is transferred to 10 litre wort in a special laboratory Vessel called Cornelius Vessel.

69. YEAST PROPAGATION Step 5 : 10 litre to 2hl stage Step 6: 2hl to 20hl stage . Step 7: 20hl stage to Fermentation tank After desires peak count is obtained 20hl yeast is online pitched with 200hl aerated wort. The temperature of wort is 10 to 110c. This process is called “online Pitching” Step 8 : Combination of pitched wort with several brews The 200hl pitched wort is then transferred to 300 hl aerated wort (temperature 10-110c) in fermentation Vessel with capacity 600hl. Rapid yeast cell multiplication occurs after 40-60hours of pitching. Yeast cell at this stage is “Zero” generation yeast. Yeast cells have high metabolic activity that evolves heat and the temperature rises to 12 – 130c which is the optimum temperature for fermentation

70. YEAST PITCHING

71. YEAST PITCHING Defined as the process of inoculating or direct injection of yeast into the cold wort before it enters the fermentor tank. Usually in breweries the 20 hl yeast culture is incoulted into 200hl wort in pipes leading to fermentation vessel. This pitching is called “online pitching” which facilities proper mixing of yeast cells with the wort. This pitched, aerated wort is then transferred to fermentation vessal (600hl tank) which already contains 300 hl aerated wort. Thus a total of 500hl wort is formented in one tank..

72. CONCLUSION Control of the quality of the manufactured product and quality assurance is strictly followed in SAB Miller. Samples from raw material,product in- process,final packaged products are tested The results of these tests are analysed and check whether the results are within specification. Microbiologist should also comment on reasons of results being out of specification. Standardization of the procedures are done once in an year under the guidence of central laboratory of SAB Miller. Products of each unit of SAB Miller are analysed and ranked by the central laboratory once in a month.

73. “THANK YOU”