Microbiology in food processing presentation

Microbiology in Food Processing.

‘A study of detrimental and beneficial microbes in

Food Process Industries’

-Jaysing Parade

-Gargi Ramtirtha

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Contents:

� Micro-organisms-Definition ,types.

� Growth of microbes :

• Growth phases of microbes.• Factors affecting growth of microbes.

� Micro-organisms in-

• Milk Powder technology.• Beverages and viscous foods.• Vegetable oil technology.

� Designing a microbiologically safe plant.

� Fermentation Technology.

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Microbiology-Introduction

Definition: Micro-organisms are microscopic organisms, invisible to naked eye that may be unicellular or

multicellular.

Why do we study Micro-0rganisms in Food Processing?

Microbial Terminology:

• Cell

• Colony-CFU/ml or CFU/gm.

• Media

• Incubation

• Spores

SPORE STRUCTURE

COLONIES IN A MEDIA

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Microbiology-Introduction

Classification of Microbes:

Micro-organism

Prokaryotes

Bacteria

Eukaryotes

Fungi Protozoa Algae

Non-living

Viruses

Yeasts Protozoa-Amoeba Molds

Virus

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Growth Cycle of Micro-organisms:

All microbes undergo similar growth patterns.

Each ‘Growth Curve’ has 4 Phases:

� Lag phase: Cells don’t multiply as its still dormant and acclimatizing itself to media.

� Log phase: Microbes grow at an Exponential rate utilizing all nutrients in the media & producing toxic metabolic waste products.

� Stationary phase: Here Growth Rate = Death Rate, due to depletion of nutrients, increase in waste products.

� Death Phase: Death rate exceeds growth rate due to very low conc. of nutrients & high conc. of metabolic wastes.

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Growth of Micro-organisms- Factors affecting growth of microbes in Food

i Temperature:

� Psychrophiles: -5 to -14 °C

� Psychotrophs: -15 to +20 °C

� Mesophiles: +20 to +44 °C

� Thermophiles: +45 to +60 °C

� Thermoduric : +70°C

ii pH:

� Acidophiles (pH < 5.4),preferred by yeasts and molds.

� Neutrophiles (pH 5.4 - 8.5), preferred by bacteria

� Alkaliphiles (pH 7.0 - 11.5).

iii Oxygen requirements: Anaerobes ,aerobes ,facultative aerobes.

iv Osmotic pressure: Higher -Cell lysis.

v Presence or absence of moisture: Water activity 0.7 (Aw)

Food Product Aw

Skim milk Powder 0.3

Whole milk Powder 0.2

Fruit/Veg. Juices 0.97

Edible Oil 0.3

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Micro-organisms in food processing :

Commonly conducted tests in Food Plants :

� Total Count –It helps to enumerate total no. of MO’s in a media. The number of bacteria present can be

indicative of the sanitary condition of material. Mo’s tested Bacillus Cereus, S. Aureus , Salmonella

� Coliforms-Their presence is sign of pollution problems & therefore is particularly significant from a public

health standpoint. MO tested E. Coli.

� Yeast & Mold-They are indicative of poor CIP and sanitation, atmosphere etc. MO tested –Saccharomyces

sp. , mycelium sp.

TPC:Yeast & Mold:

COLIFORMS

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� Methods by which Microbes are tested:

� Pour Plate Technique

� Membrane Filtration

� Swab Method.

Membrane Filtration apparatus:

Pour Plate technique:

Swab Method

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Clean in place (CIP) system:

A system used in the preparation, distribution, delivery, and subsequent removal of cleaning solutions to soiled equipment and piping systems without dismantling them.

� CIP cycle: The executed recipe of rinses, washes, and air blows used to clean soiled equipment.

� CIP circuit: The sum of paths within a process unit operation that are cleaned as part of a single CIP cycle.

� Variables to be considered while designing CIP System:

(1) Time of exposure (contact time) to wash and rinse solutions

(2) CIP flow rate (3 - 5 ft./Sec)

(3) Temperature of wash and rinse solutions

(4) Chemical concentration of wash solutions

� Efficiency of CIP Design:

� Spray ball test – Riboflavin test.

� Cleanability – Checked using the full CIP protocol at the facility where the vessel is installed including cleansers and temperatures

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Micro-organisms and their role in-

• Milk Powder technology.

• Beverages and viscous foods.

• Vegetable oil technology.

– Microbial Profile

– Microbial Criteria

– Recommended CIP Cycle.

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Microbial Profile during Milk Powder Production:

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Microbial aspect of Milk Powder Production:

Milk has a pH closer to alkaline range i.e. 6.4-6.9 (favorable for microbial growth) and it is a rich source of nutrients like carbohydrates , proteins , water an excellent media for microbial growth ,hence its CIP is very critical.

Typical CIP Program in a Milk Processing plant:

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Microbial aspect of Milk Powder Production:

Microbiological limit for Milk Powder (Reference: Microbial standards specified by Codex Commission (1983),ICMSF-International Commission for Microbial Specifications for food (1986).

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Microbiology in Vegetable oil processing:

Vegetable oil processing involves very low use of water and veg oils have a low Aw hence microbial contamination is a low concern in these plants.

Typical CIP in Vegetable oil Plant: Only Deodorizer and Alfa Laval VHE Economizer (High steam sparging heat exchanger) is CIP’ed with Water pre-rinse+caustic soln of 8-10%+Hot water final rinse at temp 70 to 80 degree C ,each cycle being of 5-10 minutes.

Frequency-Once in a year.

Microbiological limit for Fats & oil (Reference: Microbial standards specified by Codex (1983),ICMSF(1986):

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Microbiology in Beverages & Viscous foods:

Pasteurization: Partial sterilization of foods at a temperature that destroys harmful microorganisms without major

changes in the chemistry of the food.

Sterilization: Total destruction of all microorganisms (whether or not pathogenic) and their spores

Destruction of all micro-organisms and spores in viscous foods, involves sterilization, usually at temperatures between 110°C and 115°C for 60-90 seconds.

Alfa Laval supplies sterilizer (Steritherm) or pasteurizer units (SS PHE) for viscous fruits or vegetable pulps or

juices .It is mostly aseptic process (Asepto) with zero chance of contamination due to no contact with air or water.

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Microbiology in Beverages & Viscous foods:

� Fruit and vegetable juices or pulps are low acid foods, mainly harboring acidophiles (pH < 5.4)

such as Clostridium botulinum, which are completely destroyed in sterilization

� CIP in Beverage plant: CIP Cycle involves Hot water pre-rinse+caustic soln of 2-5 % + a final rinse at temp 70 to 80 degree C. Frequency-After each production batch.

� Microbial testing - Total plate count post sterilization and cooling: Standard- Not detectable or 5-10cfu/100gm.

� Fruit Juice Pasteurizer:

� Pasteurization of juice not only destroys spoilage organisms, but also inactivates the pectic enzymes, which cause chemical changes. Lactic acid and acetic acid bacteria do not grow in juice at temperatures above 54°C, and temperatures above 71°C are sufficient to destroy most other spoilage organisms including most fermentation organisms.

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Designing a Microbiologically safe Equipment & Plant :

A.) Drainablity:

� Equipment like tanks , vessels, hoppers, with discharge openings must be fully drainable .For good drainablity & cleanability sharp corners must be avoided. Horizontal surfaces must have a slope of > 3 °

� Designing properly sloping floors to drains (¼ inch per foot)

� Design floor drains to prevent the accumulation of water in or around the drains and making drains accessible for cleaning with drain covers to avoid pest entry.

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B.)Top Rims and equipment covers:

� Top rims of tanks, hoppers etc. must avoid ledges or crevices where product can lodge & are difficult to clean.

� Covers should be completely detachable for cleaning, if hinged covers are used, the hinge must be designed

such that hinge is easily cleanable & accumulation of dust/dirt is avoided.

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C.)Smooth food-contact surfaces:

� There should not be any unreachable crevices which can be formed by unsealed joints, tack welds &threaded fittings. Ideally product contact surfaces should have surface finish of 0.8 µm Ra or better.

� All food contact surfaces be smoothly bonded (e.g., free of pits, folds, cracks, crevices, open seams, exposed threads, and piano hinges) to avoid harboring pathogens.

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D.)Installation:

� Elevating food-contact surfaces sufficiently above the floor or away from wall, ideally 300 mm for cleaning & inspection.

� Un-clad framework should be constructed with hollow square or round section members.

� Supports for piping or equipment must be fabricated & installed such that no water or soils can remain on surface or within the supports.

� Insulation must be clad with Stainless steel which must be fully welded.

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F.)Piping:

• Dead leg : An area of entrapment in a vessel or piping that could lead to contamination.

Dead legs will be measured by the term L/D, where L is the leg extension from the I.D. wall perpendicular to the flow pattern or direction, and D is the I .D. of the extension or leg of a tubing fitting or the nominal dimension of a valve or instrument.

As a standard currently in Hygienic process industry L/D of 2:1 is preferred.

� Cross contamination of product streams shall be physically prevented by:(a) removable spool piece(b) U-bend transfer panel(c) double block-and-bleed valve system –Mix proof valve cluster

� Prevent use of by-pass as much as possible.

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� Orifice plates, shall be installed in a drainable position.

� Eccentric reducers shall be used in horizontal piping to eliminate pockets in the system.

� Ball valves are not recommended in fluid hygienic piping systems.

� Piping and tubing design should have routing and location priority over process and mechanical support

systems.

� Product hold-up volume should be minimized.

Dead leg in Ball ValvesDead leg at equipment attachment points

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E.) Layout:

• Designing the plant for one direction of personnel traffic, product, and air flow

• Locating catwalks with open grating so they do not pass over areas of food-contact

surfaces.

� References:

• EHEDG Standards for Hygienic design for food processing equipment.

• ASME-BPE for Bioprocessing Equipment.

• 3-A Sanitary Standards and 3-A Accepted Practices.

• Essentials of Food Science - 3rd Ed Springer 2007

• European Union (EU) Directives and Statutory Commission of England & Wales.

• Giese J. Ultra pasteurized liquid systems earn 1994 IFT Food Technology Industrial Achievement

Award. Food Technology 1994; 48(9): 94–96.

• Department of Food Science, North Carolina State University, Raleigh, NC.

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FERMENTATION PROCESS

What is Fermentation?

• Fermenation is the process in which complexorganic substance (molecules) is converted in tothe simpler organic substance (molecules) by theaction of enzymes secreated by the micro-organisms.

–Micro-organism+substrate= more microbial cells+ metabolic products

Two types:- -Aerobic fermentation

�Production of Acetic Acid, citric acid

- Anaerobic fermentation

�Alcohol,acetone,butanol,lactic acid


a. Rapid growth in suitable organic substrates with easy cultivation in large quantities.

b. Ability to maintain physiological costancy under fermentation condition and to produce necessary enzymes readialy in order to bring about desired chemical changes.

c. Ability to carry out the transformations under simple and workable modifications of enviornmental conditions.

Characterstics of M.O


Selection of Micor-organism

-Carry out desired reaction efficienty.

-Stock cultures

-Individual organism may be isolated from natural sources


Industrially important micro –organisms may be classified under yeast,bacteria & molds

Micro-organism Uses

YEAST BACTERIA MOLDS

Ethyl Alcohol Lactic Acid -lactobacillus

Penicillin

Backers Yeast Acetone &Butanol-Clastidium

Streptomycin

Beer Vinegar&-Acetobactor

Whisky Dextran-Leconostoc

Wine (Saccharomycesellipsoidens)

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Industrially important yeasts

© Alfa Laval Slide 29

YEAST PRODUCT

Distiller’s Yeast:-Saccharomyces cerevisiae

Ethyl Alchol, potable liquors, backers yeast, pharmaceutical yeast, Invertase, Flavors enhancing agents

Schizosaccharomyces pombe Ethyl Alcohol (Alfa Laval Fermetation)

Saccharomyces cerevisiae & Saccharomyces carlsbergensis

Beer_Brewers Yeast

Saccharomyces cerevisiaevar.ellipsoideus

Wine

Candida species Food & fooder yeast

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YEAST

• They are generally defined as fungi, which in astage of their life cycle occurs as a single cells,reproducing commonly by budding or lessfrequently by fission

• They are >1500 strains of yeast, 500 species & 50genera.

• Asexual reproduction of yeast- doubling 1 to 2hours

• Occurrence:- nature soil, marine, many forms oforg-matter-particulearly with carbohydrates.

• Isolation:- Soil of wine yards, fruits etc© Alfa Laval Slide 30

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Yeast• Shapes :- Ovidal, elipsoid,

Spherical

• Size :Depends upon

– age/culture conditions etc.

– Width 1 to 9 mic,length 2-10 mic

– Much larger than bacteria.

– Budding:-

– Fission:-


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Yeast structure & function of cellular components-Cell wall:- Responsible for the shape

Bud scares are formedCan produce avg.24 buds

Schizosaccharomyces – reproduce by fission,exibit scare rings at the point where crsoo-wall divide after septum fomation.

Cytoplasmic membrance:- betn cell wall layers & cytoplasm

Nucleus :- Generic & Heriditarycontrol.

Vacuoles :- Contains amino acids

Endoplasmic Reticulum:-

Mitorchondria:- Power house of the cell.

Lipid globules:-


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Growth requirements of yeast

• Other organic carbon sources:-

– Nitrogen

– Aminoacids

– Other organic sources

– Vitamins

– Minerals

Carbohydrates (carbon &

Engergy sources)

Monosaccharides:- All yeast are

able to metabolise

D-glucose,D-fructose & D-mannose

Di,tri & polysaccharides are first hydrolysed to their respective hexose & then metaboliszed,Hydrolysis is carried out only if the species can utilise the hexose & then metabolised,

Glucose,fructose,Cellulose and melezitose

Glucose,fructose,mannose, sucrose,galactose, maltose,raffinose,mellibiose,lactose & trehalose are most commonly utilised sugars© Alfa Laval de 33

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Fundamentals of yeast growth(Facultative-Vegetative re-production, td=2hrs.)

Aerobic-Respiration

• EMP-TCA-Oxidative Phosphorylation(Mito.)

• O2 is final e acceptor

• Complete degradation

• 36ATP/glucose

• Cell membrane with UFA & sterols (Nicotinic acid)

Anaerobic- Fermentation

• Glycolytic(EMP) Pathway (cytoplasm)

• EtOh is final elec.acceptor

• Partial gedradation

• 2ATP/glucose

• Weak cell membrance


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Aerobic & Anaerobic growth

Aerobic(respiration) Anaerobic (fermentation


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Metabolism of yeast

Glycolytic Pathway(EMP)


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Metabolism of yeast


SUCROSE

INVERTASE

GLUCOSE+

FRUCTOSE

EMP

C2H5OH+CO2

• How Yeast is working

• - Sucrose (Invertase)

• - Glucose

• EMP pathway

• C2H5OH+Co2+Heat

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Basic Chemistry of Ethanol

C12H22O11+ H20 → C6H12O6+ C6H12O6 → 2(C2H5OH) + 2(CO2)

Sucrose Water Glucose Ethanol Carbon Dioxide

Fermentation(anaerobic, glycolysis)

S. cerevisciae

342 mw 18 mw 2 x 46 mw 2 x 44 mw

S. Pombe

Fructose

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The Wine making process


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The beer-brewing process


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DISTILLERY


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TYPES OF FERMENTATION IN DISTILLERY• MOLASSES BASED - BATCH TYPE

- CONTINUOUS FERMENTATION

» ALFA LAVAL CONTINUOUS FERMENTATION-BIOSTILL

» CASCADE TYPE

» SEMI CONTINUOUS

• GRAIN/STARCH BASE - BATCH TYPE


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Typical view of distillery plant


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Continuous fermentation plant


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GRAIN BASED FERMENTATION-BATCH TYPE

Ferm. #1 Ferm. #2 Ferm. #3 Ferm. #4

Mash from

Liquefaction

Yeast

Propagator

Mash

Coolers

To Beerwelland Distillation

Spirizyme Fuel/Plus

Tech.,Yeast

Water

CO2 to Scrubber

90 - 93oF (32 – 34 C)pH 3.8 - 5.0

88 - 90oF (31 - 32C)

pH 3.6 - 4.0

Batch

Fermentation

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Molasses based fermentation


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The Molasses Process

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Propagation System :

100/ 1000 ML

90 Liter2000 Litre

�Complete Sterile operation

�Propagation under controlled conditions: Air, Temp, Pressure

�Minimum cell count of 250 X 106 per ml in each stage

�Normally takes 10 to 12 hours in each stage

LAB Prop I Prop II


100/ 1000 ML

Propagation System : Alfa Laval System

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Air

Cooling WaterOut

Mash Headerto Fermenters

Cooling Water In

Steam

Process WaterMash

Yeast Product Yeast Propogation ion ProcessYeast Propagation

yeast

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FERMENTATION PROCESS


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Parameters for Control :

Yeast Cell Count

Checked under microscope

Controlled by the air flow rate

to the fermenter

Residual Sugars

Checked by titration

Control of unfermented sugars

in molasses

Fermenter Dissolved Solids

Checked by Oven Maintained for check on infection

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Parameters for Control….

Yeast + Sludge concentration

Checked by spin test in lab centrifuge

To have better performance on Yeast Separation

Alcohol Concentration

Checked by Ebulliometer / Distillation

To have consistent feed to distillation plant

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Yeast Recycling1. Maintains cell concentration

(Efficient conversion)

1. Saves Sugars (More Ethanol Yield)

2. Quick conversion to ethanol (Less residence time)

Exhausted Wash Recycle

1. Maintains dissolved solids concentration

(Fights infection)

2. Decreases water requirement (Savings in Water)

3. Decreases spent wash quantity (Less effluent problem)

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Parameters Batch Fermentation

Cascade

4 Ferm. Sys

Alfa Laval cont.

2 Ferm System

Fermentation Efficiency

84% to 85% 86% to 87% 90% guaranteed

Yield at 46%FS @ 96%v/v

258 Liters/Mt 261 Litres/Mt 273 Litres/Mt

Sturdiness Prone to infection

Prone to Infection 100% infection free

Fermenter Alcohol %v/v

7.5 to 8.5% 7.5 to 8% 7.5 % - 8.5 %

Fermenter DS%w/w

9 to 10% 10 to 11% 14 to 16%

Yeast Count 100 X 106 100 X 106 500 X 106

Effluent Generation

15 Litre/Litre 10 to 12 Litre/Litre 5 Litre/Litre

Technology Comparison:

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Continuous Fermentation Plant

Flexibility: the GREATEST Advantage of Alfa laval Process�Raw Material : Molasses

�Process Water : NO treatment

�Yeast Strain : Schizosaccharomyces Pombe OR Saccharomyces Cerevisease.

�Fermenter Alcohol: 6.5 to 8%

�Dissolved Solids : 10% to 16%

�Spent Wash Generation: 11 – 5 litres

�Turn down ratio : below 40%

�Shutdowns & Start-up’s : Even for period of 15 days

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Stress factors affecting yeast & fermentation efficiency.-Sugar Concentration

-PH

-Temperature

-Alcohol Tolerance

-Neutrients

-Yeast concentration


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Bacterial Contamination

What Happens if the Fermenter is Infected?

Effects�Low yield�High impurities –fusel oil formation�Bad quality of product�Excessive foaming�Loss in production due to stoppages.�……

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Bacterial Contamination

• SOURCES

• Stock culture

• Raw Material

• Process water

• Environment

• Air

• Additives

• Hygienic conditions

• Defective designing

• Yeast & Bacteria


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Common contaminants in yeast fermentationsContaminants Effects

Acetobacter Prod.of Acetic Acid,Robiness

Lactobacillus Production of lactic acid,Mousy occur

Leconostoc mesenteroidesLeuconostoc dextranicm

Production of Dextran, a Polysaccharide from Sucrose

Saccharomyces pastorianus Turbidity in Beer

Saccharomyces turbidians Turbidity in Beer

Candida mycoderrna Formation of film on the surface, Breakdown of alcohol,organicacidsand other substances

Achromobacter anaerobicum Turbidity,Off flavour

Streptococci Sourness,turbidity,Ropiness

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Bacterial Growth Path

Bacteria Vs Yeast• Normally Bacterial

growth is faster than the Yeast.

• Temp factor


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Methods to control Contamination• Find out sources- Like Molasses,water,air supply

• Good yeast culture

• Propagation process errors/backers yeast.

• Unattended fermentation process.

• Unhygienic conditions: Immediately complete cleaning of

spillage.

• Sterilization of mash – practically not possible but during

propagation stages proper sterilization & care needs to be taken

• Good microbiological practices- Keen observation on the

process in microbiological point of view, like microscopic observation.

• CIP

• GOOD DESIGNS © Alfa Laval Slide 62

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Methods to control Contamination


“Cleaners bag of tricks” – Four T’s of cleaning

& Sanitizing.

-TIME

- TEMPERATURE

- TITRATIONS

-TURBULENCE

CIP- Cleaning in Place

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Methods to control Contamination


CIP- Cleaning in Place

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Designs Precautions

• Yeast propagators – good finishing

• Fermenters- finished surface

• Drainage layout

• Dead spots/crevices

• Piping/valves- no retention

• Sources include, but not limited to

– Tanks

– Transfer lines

– Heat Exchangers


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Turbulence

• Effective cleaning requires velocity min 1-1.5 to 3 m/s. forward flow & backward flow

• Flow 1.5 times the normal process flow.

• Dead ends

• Bypass valve© Alfa Laval Slide 66

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Cont….

• Elimination of dead end- branch lines from the

main line should have the valve installed not more than one dia form the wall of main pipe.(ensurence turbulancein branch)



• Tees : Dead end should be minimum.

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Fermenter Design

• Mash Filling Pipe line loop

- min velocity should be 3m/s


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• Fermenter CIP Sprayhead(pressure requirement)


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• External Heat Exchanger loop

- 1.5 times normal process flow.


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• Fermented product emptying pipeline.

– Min 3m/s velocity.


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Thank you!


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Microbiology in food processing presentation