Microbiological & Analytical Techniques in Quality control of Beer
MICROBIOLOGICAL TECHNIQUES IN A BREWERY<br />PRESENTED BY<br />SINI P K<br />M.Sc.BIOTECHNOLOGY<br />St.BERCHMANS’COLLEGE<br />
DETAILS OF PROJECT WORK :<br />COMPANY NAME - SAB Miller Brewery<br /> chalakudy,kerala.<br />TOPIC - Microbiological & <br />Analytical techniques in<br /> BREWERY<br />Duration - May 21 , 2007 to June 15 , 2007<br />GUIDED BY - Mr. Nishil menon ,SAB Miller<br /> Mr.Ram kumar.s ,SAB Miller<br />
INTRODUCTION<br />Done the project work in the Quality control department of Brewery<br />Quality control department has Microbiology lab & Chemical analysis lab.<br />This department assures the quality of the product (Beer) by systematic monitering of raw material,product-in process and end product.<br />Uses standardized procedures and are guieded&monitered by theie central inistitution in Banglore.<br />
BREWERY MICROORGANISMS<br />Saccharomyces cerevisiae ( Bottom culture Yeast)<br /><ul><li>Growth in word cells are round, short oval or single and in pairs.
Spores will not normally be seen within 48 hours because a longer period of time is required for sporulation. If found they are round, 1-4 in the ascus and tightly packed.</li></ul>Saccharomyces cerevisiae ( Top culture Yeast)<br /><ul><li>Growth in wort; cells are round.
Short oval or single in pairs, short chains or groups.
They are round, 1-4 in ascus and fill the cell completely except for a wedge of cytoplasm separating them. This gives them a characterstic double walled appearance. </li></ul>saccharomyces carlsbergenesis<br /><ul><li>Mutated form of Saccharomyces cerevisiae
Developed by carlsberg brewery,Denmark</li></li></ul><li>Structure of Yeast cell<br />
OTHER MICRO ORGANISMS<br />A. BEER SPOILAGE BACTERIA<br />1 Lactic Acid bacteria<br /><ul><li>The only gram positive bacteria that cause series problems in the brewery environment
Two genera are commenly found, Lactobacillus pediococcus.
produces lactic acid, ethanol and CO2 from glucose; lactic acid acetic acid and mannitol from fructose. Alsoproduces acetic acid from glucose and also from fructose</li></ul>2. Acetic acid bacteria. <br /><ul><li>Acetic acid bacteria are acetifying organisms.
Gluconobactor oxidises alcohol to acetic acid, but it is further oxidised in to CO2 and H2O
It is gram negative. They are both aerobic. Some acectobacter are micro aerophilic. Grows and spoils beer in presence of O2</li></ul>3. Zymomonas<br /><ul><li> Heavy turbidty and production of acetaldehyde and H2S</li></ul>4. Pectinatus<br /><ul><li> Acid tolerent and produces organic sulphur components which wilproduce off flavor</li></li></ul><li>OTHER BREWERY MICRO ORGANISMS<br />B. NON BEER SPOILAGE BACTERIA.<br />1. Micrococcus<br />gram +ve cocci..<br />are hop sensitive and have never been shown to spoil beer.<br />aerobic<br />2. Clostridium<br />strictly anaerobic, gram +ve spore forming rods.<br />hop sensitive and cannot developed in hopped wort or beer. <br />rarely encountered in the brewery but one spices Clostridium bactyricum may develop in accumulations of wet spent mash grains.<br />3. Bacillus<br /> gram+vc, spore forming rods, aerobic or facultative<br /> hop sensitive and cannot develop in hopped wort or beer. <br />
OTHER BREWERY MICRO ORGANISMS<br />C WORT BACTERIA<br />Enterobacteriacea<br />capable of fermenting glucose to produce a mixture of acids and ethanol.<br /> Hafnia protea<br />able to survive the brewing process and may be carried from one fermentation to another in the picting yeast.<br />Enterobactor agylomerans<br />Coliform bacteria, able to ferment lactose <br /> also capable of surviving the brewing process and being carried forward in the picting yeast. <br />Acetic acid bacteria<br /> used commercially for producing Vinegar from wine, cider or unhoped malt beer <br />
Media used are :<br />1 Mac Conkey Agar (HIMEDIA)<br />For detection of coliforms<br />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.<br />2. Plate Count Agar / PCA(HIMEDIA)<br />All forms of bacteria<br />For preparing PCA, suspend 23.5g. in 1000ml distilled water. Boil to dissolve the medium completely. Sterilise by autoclaving.<br />3.Walerstein Nutrient Medium / WLN (HIMEDIA)<br />All forms of bacteria<br />For preparing WLN, suspend 80.25g. in 1000ml distilled water.Heat to boil to dissolve the medium completely. Sterilise by autoclaving<br />
Media used are :<br />4.Schwarz Differential Medium / SDM (HIMEDIA)<br />Only for wild yeast.<br />For preparing SDM suspend 44.5g. in 1000ml distilled water. Boil with constant stirring for 15 minutes. Donot autoclave<br />5. Lysine Medium (HIMEDIA)<br />Only for wild yeast<br />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<br />6.Raka Ray Medium (HIMEDIA)<br />Anaerobic Medium<br />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.<br />
CHECKING YEAST COUNT<br />AIM<br /><ul><li>To determine the number of yeast cells in the yeast slurry</li></ul> PREPARING SAMPLE<br /><ul><li>. If the number of cells is very high sample can be diluted by mixing a drop of sample in100 ml distilled water. In this case the dilution factor (102) must be multiplied.</li></li></ul><li>FIILING THE HEMOCYTOMETER CHAMBER<br /><ul><li>Take a portion of your sample by placing the glass pipette tip in to the sample mixture and letting it fill Via capillary action.
Fill chamber in hemocytometer by setting pipette tip on edge of chamber. Avoid over fill.
Carefully place hemocytometer on microscope stage. Focous the objective at 40X.</li></li></ul><li>YEAST COUNT <br />CELL COUNTING<br />will be counting squares within the 1 mm2 ruled area centrally located on the chamber.<br /> It is helpful to establish a counting protocol for all cell counts<br />. For eg: cells touching or lying on the boundary lines are not counted where as cells touching or lying inside the squares can be counted. Yeast cellbuds emerging from mother cells are counted as a separate cell if the bud is at least one half the size of the mother cell.<br />
YEAST COUNT<br />CALCULATION <br />Yeast count = No. of bacteria X 25 squres X 102 X104<br />RESULT<br />Yeast count = 5 X 25 X 102 X 104<br /> = 125 X 106<br /> = 125 million cells. <br />
CHECKING YEAST CONSISTANCY<br />AIM <br />To determime the consistancy of yeast slurry and thus to find out the pitching rate<br />PREPARING SAMPLE<br />Yeast sample are taken directly from fermentation tanks.<br />
CHECKING YEAST CONSISTANCY<br />METHOD<br />The empty weight of the centrifuge tubes are taken by using a weighing machine.<br />The samples are then filled in two certifuge tubes and the initial weight of the samples are taken.<br />The tubes are then placed for centrifugation for 15 minutes at 3000 pm<br />After 15 minutes the tubes are taken and the supernatent is drained off. <br />The final weight of the yeast cells are taken.<br />
CHECKING YEAST VIABILITY<br />AIM<br /><ul><li>To determine the viability of yeast cells</li></ul> METHOD<br /><ul><li>Mix the sample and transfer enough quantity of the sample to test tube containing
Transfer two drops of above yeast suspension and add two drops of methylene blue. Mix well and allow to stand for I minute.
Take a drop of this suspension and transfer on to the heamocytometer. Examine under 40 X bright field objective,.
Count total number of cells and number of blue stained dead cells. Viable cells are viewed colourless and dead cells appeared blue in colour because the cell wall of dead cells absorb the methylene blue stain.</li></li></ul><li>CHECKING YEAST VIABILITY<br />CALCULATION<br />% Viable cells = Number of viable cells x 10<br /> Total number of cells<br />RESULT<br />% Viable cells = No. of Viable cells x 100 <br /> Total number of cells<br />No. of Viable cells / colourless cells = 25<br />Total Number of cells = 26 <br />% Viable cells = 25 X 100<br /> 26 <br /> = 96.15 % <br />
MEMBRANE FILTRATION METHOD<br />INTRODUCTION<br /><ul><li>This method is also used to trap microorganisms in purification analysis of various liquids ;like water, beer etc.</li></ul>PRINCIPLE<br />This method is used when we want to separate micro organisms which are scanty in the fluids<br />In the method membrane filters are fitted in the apparatus.<br /> This apparatus is connected to a vaccum pump.<br /> The vaccum pump creates a negative pressure and the fluid is sweked through the filter pad. <br />The pore size of the membrane filter should be less than that of the size of micro organism to be trapped. Thus the micro organisms get trapped in the membrane filter<br />
MEMBRANE FILTRATION METHOD<br />INSTRUMENTATION<br /><ul><li>Membrane filtration apparatus consists of a manifold, membrane filter of a particular pore size.
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.
A Vaccum pump is connected to another opening of the side arm flask by means of Vaccum tubing.
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
. These filters consists of pores. A wide variety of membrane filter with different pore sizes ranging from 0.01 – 10 micro meter are available.
Here in brewery membrane filter of 0.45micrometer pore size is used.
Membrane filters are also called molecular filter / Milli pore filters / poly pore filters. These membranes are held in special holders. Thickness of the membrane filter is 0.1mm.</li></li></ul><li>MEMBRANE FILTRATION METHOD<br />PROCEDURE<br />The filtration apparatus must be sterilized .first. For this the apparatus is wrapped in aluminium foil and sterilized in an autoclave.<br />Dry the apparatus in hot air over.<br />sterilise the forceps by dipping them in the alcohol for atleast one minute, flash flaming and allow to cool for a few seconds. .<br />Unwrap the filtration apparatus after placing it in Laminar air flow unit.<br />Disassemble the filtration cup and using the sterile forceps place a sterile membrane filter on the apparatus in such a way that the ruled side of filter is up, flat on the membrane support grid.<br />Re assemble the apparatus, connect it to the opening of a sterile side arm flask, which inturn is connected toa Vaccum pump.<br />Pour the solution to be analysed (here beer) through the top of the filter cups and switch on the Vaccum pump and open the valves of the manifold.<br />Continue the suction until all the solution has been drawn through the membrane into the side arm flask.<br />Close the manifold valves and switch off the Vaccum pump.<br />Detach the funnel from the filter support suction.<br />Using sterile forceps asceptically remove the membrane from the grid and place it (ruled side up) on the surface of agar plate. Ensure that there is immediate contact between agar surface and the membrane.<br />Incubate the agar plate at 250c for 48 hrs. <br />Only colonies that have formed on the membrane must be counted. Count the number of colonies that have developed on the membrane surface and record.<br />
RESULT<br />Absence of colonies on membrane filter after incubation. The beer sample is pure<br />
SPREAD – PLATE METHOD<br />Spread plate method is an indirect plate court or viable count technique.<br /> Method is used to detect the presence of viable microorganisms in a sample. <br /> 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.<br />
SPREAD – PLATE METHOD<br />PRINCIPLE<br /> The peculiarity of viable cells is that they can grow, reproduce and thus produces colones.<br /> 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.<br /> Usually the count is made more accurate by use of special colony counter.<br />REQUIREMENTS<br />Sterile plate with sterile solid media (eg:- Mac conkey agar, Plate count Agar, WLN, Raka Ray etc.)<br />Sample which is to be analysed (eg: beer, wort, gas dissolved in saline etc.)<br />Sterile, graduated 1ml pipette<br />L- rod.<br />
SPREAD – PLATE METHOD<br />PROCEDURE<br />If the sample is thickly populated with microorganism. It should be diluted.<br />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.<br />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.<br />Hold the petridish in the hand and lift the lid of it slightly. Expel the sample on to the surface of the agar.<br />Sterlise the L-rod by flash flaming after dipping in alcohol cool the L-rod.<br />On the edge of the agar spread the sample over the surface. While rotating the plate to ensure even distribution of sample.<br />Replace the lid of the petridish<br />Incubate the petridishes ar appropriate temperature and check for colonies.<br />RESULT<br />No colonies obtained. Beer is of GOOD QUALITY<br />
ANAEROBIC INCUBATION METHOD<br />Anaerobic microorganisms are used in a brewery for the production of beer, It includes the saccharomyces strains of yeast.<br /> In addition to this various unwanted anaerobic microorganisms affects the beer quality. <br />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.<br /> Here anaerobic cultivation is done by using anaerobic Jar. <br />
ANAEROBIC INCUBATION METHOD<br />PRINCIPLE<br />usingthe anaerobic Jar an anaerobic environment is created. <br />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. <br />Indicator tablets inside the Jar become pink Colour after complete anaerobisis is achieved. <br />
ANAEROBIC INCUBATION METHOD<br />INSTRUMENTATION<br />Apparatus is called Gas Pack Jar or anaerobic Jar with a lid. Lid consists of screws and a pressure gague.<br /> 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. <br />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.<br />
ANAEROBIC INCUBATION METHOD<br />PROCEDURE<br />The sample to be analysed ( beer, air dissolved in water) is inoculated on an medium<br />Anaerobic medium (eg. – Raka Ray medium) was used<br />The Petri dish is placed inside the anaerobic jar. <br />The outer plastic cover of the gas pack is removed and placed inside the anaerobic jar. Indicator tablets are also placed inside the jar. <br />Immediately close the lid of the anaerobic jar and tightly close the screws. Check the pressure gague.<br />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.<br />After some hours indicator tablets become pink in colour shows the complete removal of O2 inside the jar. <br />Incubate for one week check the pressure.<br />After one week open lid and take and the Petri plates.<br />Check for the presence of colonies. .<br />
YEAST PROPAGATION<br />Brewing utilizes strains of Saccharomyces calsbergensis a bottom yeast and Saccharomyces cerevisiaeboth bottom and top yeast strains.<br /> 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.<br />
YEAST PROPAGATION<br />In a multi plant situation yeast pure culture is provided from the main laboratory to other regional laboratories of the brewery. <br /> 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:<br />As Slant cultures<br />As pure culture in 10 – 50ml flask<br />As pressed pure yeast cake (25-40% dry weight)<br />As yeast slurry in 10-20litre stainless steel flasks.<br />Transportation is usually in an insulated cooler box or packaged in dry ice or both. Transportation time should not exceed 24 hours.<br />The yeast seed lots received by the regional laboratory should propagate it. So that a satisfactory yeast count is obtained for online pitching.<br />
YEAST PROPAGATION<br />PROCEDURE<br />Yeast propagation is a multistep process which needs atleast two weeks.<br />Wort at a low temperature (usually 200c or below) is used as a medium for yeast propagation oxygenation is achieved mainly by agitation <br />
YEAST PROPAGATION<br />Step 1 : Recovering yeast cells from agar slants.<br />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<br />Step 2: I ml to 15ml stage<br />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.<br />Step 3: 15ml to 200ml stage<br />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.<br />Step 4: 200ml to 10 litre stage<br />After peak count is obtained 200ml yeast culture is transferred to 10 litre wort in a special laboratory Vessel called Cornelius Vessel.<br />
YEAST PROPAGATION<br />Step 5 : 10 litre to 2hl stage<br />Step 6: 2hl to 20hl stage<br />.<br />Step 7: 20hl stage to Fermentation tank<br />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”<br />Step 8 : Combination of pitched wort with several brews<br />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 <br />
YEAST PITCHING<br />Defined as the process of inoculating or direct injection of yeast into the cold wort before it enters the fermentor tank.<br /> 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.<br /> This pitched, aerated wort is then transferred to fermentation vessal (600hl tank) which already contains 300 hl aerated wort. <br />Thus a total of 500hl wort is formented in one tank..<br />
CONCLUSION<br />Control of the quality of the manufactured product and quality assurance is strictly followed in SAB Miller.<br />Samples from raw material,product in- process,final packaged products are tested<br />The results of these tests are analysed and check whether the results are within specification.<br />Microbiologist should also comment on reasons of results being out of specification.<br />Standardization of the procedures are done once in an year under the guidence of central laboratory of SAB Miller.<br />Products of each unit of SAB Miller are analysed and ranked by the central laboratory once in a month.<br />