Microbiological & Analytical Techniques in Quality control of Beer

MICROBIOLOGICAL TECHNIQUES IN A

BREWERY

PRESENTED BYSINI P K

M.Sc.BIOTECHNOLOGYSt.BERCHMANS’COLLE

GE

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

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.

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

BREWERY MICROORGANISMSSaccharomyces cerevisiae ( Bottom culture

Yeast)

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.

Saccharomyces cerevisiae ( Top culture Yeast)

Growth in wort; cells are round. Short oval or single in pairs, short chains or groups. . Spores usually detectable after 48 hours. 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.

saccharomyces carlsbergenesis

Mutated form of Saccharomyces cerevisiae Developed by carlsberg brewery,Denmark

Structure of Yeast cell

OTHER MICRO ORGANISMSA. BEER SPOILAGE BACTERIA

1 Lactic Acid bacteria 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 2. Acetic acid bacteria. Acetic acid bacteria are acetifying organisms. Includes gluconobacter and acetobactor. 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

3. Zymomonas Heavy turbidty and production of acetaldehyde and H2S

4. Pectinatus Acid tolerent and produces organic sulphur components which wilproduce off flavor

OTHER BREWERY MICRO ORGANISMS

B. NON BEER SPOILAGE BACTERIA.

1. Micrococcus gram +ve cocci.. are hop sensitive and have never been shown to spoil beer. aerobic 2. Clostridium strictly anaerobic, gram +ve spore forming rods. hop sensitive and cannot developed in hopped wort or beer. rarely encountered in the brewery but one spices Clostridium bactyricum may

develop in accumulations of wet spent mash grains. 3. Bacillus gram+vc, spore forming rods, aerobic or facultative hop sensitive and cannot develop in hopped wort or beer.

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

CULTURE MEDIA USED IN BREWERY

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

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.

CHECKING YEAST COUNT

CHECKING YEAST COUNTAIM To determine the number of yeast cells in the yeast slurry

PREPARING SAMPLE . 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.

FIILING THE HEMOCYTOMETER CHAMBER 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.

YEAST COUNT CELL COUNTING

will be counting squares within the 1 mm2 ruled area centrally located on the chamber.

It is helpful to establish a counting protocol for all cell counts

. 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.

YEAST COUNTCALCULATION

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.

YEAST CONSISTANCY

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.

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.

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

RESULTInitial weight = 132.1508Final weight = 112.0493X = 132.1508 - 69.9135 = 62.2373Y = 112.0493 - 69.9135 = 42.1358Consistancy = Yx100 = 42.1358x100 x 62.2373

= 67.70 %

YEAST VIABILITY

CHECKING YEAST VIABILITY

AIM To determine the viability of yeast cells METHOD Mix the sample and transfer enough quantity of the sample to test tube containing distilled water and mix well. 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.

CHECKING YEAST VIABILITY

CALCULATION% Viable cells = Number of viable cells x 10 Total number of cells

RESULT% Viable cells = No. of Viable cells x 100 Total number of cellsNo. of Viable cells / colourless cells = 25Total Number of cells = 26 % Viable cells = 25 X 100 26 = 96.15 %

MEMBRANE FILTRATION METHOD


INTRODUCTION This method is also used to trap microorganisms in purification analysis of

various liquids ;like water, beer etc.

PRINCIPLE This method is used when we want to separate micro organisms which are

scanty in the fluids In the method membrane filters are fitted in the apparatus. This apparatus is connected to a vaccum pump. The vaccum pump creates a negative pressure and the fluid is sweked

through the filter pad. 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


INSTRUMENTATION Membrane filtration apparatus consists of a manifold, membrane filter of a

particular pore size. Manifold includes the filtration cups. 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.


PROCEDURE The filtration apparatus must be sterilized .first. For this the apparatus is wrapped in

aluminium foil and sterilized in an autoclave. Dry the apparatus in hot air over. sterilise the forceps by dipping them in the alcohol for atleast one minute, flash flaming and

allow to cool for a few seconds. . Unwrap the filtration apparatus after placing it in Laminar air flow unit. 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.

Re assemble the apparatus, connect it to the opening of a sterile side arm flask, which inturn is connected toa Vaccum pump.

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.

Continue the suction until all the solution has been drawn through the membrane into the side arm flask.

Close the manifold valves and switch off the Vaccum pump. Detach the funnel from the filter support suction. 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.

Incubate the agar plate at 250c for 48 hrs. 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.

RESULT Absence of colonies on membrane filter after

incubation. The beer sample is pure

SPREAD – PLATE METHOD

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.

SPREAD – PLATE METHODPRINCIPLE

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.

SPREAD – PLATE METHODPROCEDURE 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

ANAEROBIC INCUBATION

METHOD

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.


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.


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.


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. .

YEAST PROPAGATION METHOD

YEAST PROPAGATION Brewing utilizes strains of Saccharomyces

calsbergensis a bottom yeast and Saccharomyces cerevisiae both 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.

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.

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

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.

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

YEAST PITCHING

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..

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.

“THANK YOU”

Microbiological & Analytical Techniques in Quality control of Beer

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