Bottler Quality Training

International Quality
Module 15: Microbiology

Bottler Quality Training: July 30, 2005 Module 15, Page 1

 Master Process Flowchart
WAREHOUSING,
RAW MATERIALS PRODUCTION DISTRIBUTION,
AND RETAIL

2. Sensory 2. Sensory 2. Sensory

3. Quality 3. Quality 3. Quality

Assurance Assurance Assurance

4. Water 7. Concentrate &

Batching
5. Sweeteners
9. Bottle Washing
6. CO2

7. Concentrate & 10. CO2 Gas

Batching Volumes
11. Degassing
8. Packaging
12. Brix

13. Titratable
Acidity

14. Filling
15. Microbiology
Learning Objectives

What’s the Benefit for Me?

Upon completion of this module, you will be able to:

 Describe the fundamentals of cleaning and sanitizing.
 Identify organisms of concern.
 Identify where organisms can be found in the plant.
 Explain where and how samples are collected.
 Explain isolation techniques.
 Understand the need for a plant specific sampling
program.
Bottler Quality Training: July 30, 2005 Module 15, Page 3
Learning Objectives

Agenda

 5 Step Sanitation
 Plant Sanitation Program
 Microorganisms
 Sample Collection
 Membrane Filtration

Bottler Quality Training: July 30, 2005 Module 15, Page 4

Sanitation

Purpose of Sanitation Program

 A plant sanitation program removes dirt and

kills microorganisms to prevent:
 Unsanitary conditions
 Product spoilage
 Package failure
 Prevent cross-contamination

Bottler Quality Training: July 30, 2005 Module 15, Page 5

 Sanitation

Five-Step Sanitation Process

1 2 3 4 5
Rinse Clean Rinse Sanitize Rinse
Soil Cleaner Sanitizer

Minimum flow rate or velocity for each of the five steps

of the CIP is 5 feet per second, 1.5 meters per second.
Bottler Quality Training: July 30, 2005 Module 15, Page 6
 Sanitation

Step One: Rinse Soil

1 2 3 4 5
Rinse Clean Rinse Sanitize Rinse
Soil Cleaner Sanitizer

 Removes loose soil and softens remaining soil

 Improves effectiveness of cleaner
 Types of soils:
 Organic (sweetener, juices)
 Inorganic (calcium scale, alkaline film)
 Hydrocarbon( grease, oil)
 Microbial (bacteria, yeast, mold)

Bottler Quality Training: July 30, 2005 Module 15, Page 7

 Sanitation

Step 2: Cleaning

1 2 3 4 5
Rinse Clean Rinse Sanitize Rinse
Soil Cleaner Sanitizer
• Time
• Temperature
• Concentration

 Cleaning removes soil through physical and chemical

action.
 Cleaning process consists of the
 Removal of all soil from the equipment
 Dispersion of the soil into the cleaning solution
 Prevention of re-depositing the soil onto cleaned surfaces
Bottler Quality Training: July 30, 2005 Module 15, Page 8
Sanitation

Types of Cleaners

ALKALINE: Tri-sodium phosphate, caustic soda

Temperature: 50-60oC
pH: 11.0+
Time: 20 minutes
Concentration: As recommended by manufacturer

Bottler Quality Training: July 30, 2005 Module 15, Page 9

Sanitation

Types of Cleaners

ACID: Phosphoric Acid

Temperature: Not greater than 60oC
pH: <2.2
Time: 20 minutes
Concentration: As recommended by
manufacturer

Bottler Quality Training: July 30, 2005 Module 15, Page 10

 Sanitation

Step 3: Rinse Cleaner

1 2 3 4 5
Rinse Clean Rinse Sanitize Rinse
Soil Cleaner Sanitizer

phenolphthalein

or thymolphthalein
at high pH

 Removes cleaning solution and any remaining residue

 Prepares lines for sanitizing
 Minimum flow rate or velocity for each of the five steps of the CIP
is 5 feet per second, 1.5 meters per second.
Bottler Quality Training: July 30, 2005 Module 15, Page 11
 Sanitation

Step 4: Sanitize

1 2 3 4 5
Rinse Clean Rinse Sanitize Rinse
Soil Cleaner Sanitizer
• Strength
• Temperature
• Contact Time

 Sanitation is the treatment of a cleaned surface to kill

microorganisms
 Effectiveness depends on:
 Strength
 Temperature
 Contact time
Bottler Quality Training: July 30, 2005 Module 15, Page 12
Sanitation

Step 4: Sanitize

 Types of sanitizers
 Heat (>85°C for 15 minutes)
 Chlorine (100 ppm for 20 minutes at pH 6.0-7.5)
 Caustic (1-2% at 80°C for 30 minutes)

Minimum flow rate or velocity for each of the five steps

of the CIP is 5 feet per second, 1.5 meters per second.

Bottler Quality Training: July 30, 2005 Module 15, Page 13

 Sanitation

Step 5: Rinse Sanitizer

1 2 3 4 5
Rinse Clean Rinse Sanitize Rinse
Soil Cleaner Sanitizer

Sanitizer Carryover
Microbial Check

 Removes sanitizer
 Cools equipment, if heat is used
 If lines will be down, pack with chlorinated treated water
 Re-rinse before production

Bottler Quality Training: July 30, 2005 Module 15, Page 14

Sanitation

Effectiveness of Sanitation

 Process Measures
 Chemical concentrations
 Temperatures
 Flow rates -Minimum flow rate or velocity for each of the five
steps of the CIP is 5 feet per second, 1.5 meters per second.
 Contact times

 Microbiological Measures
 Rinse waters
 Swabs
 Finished product
Bottler Quality Training: July 30, 2005 Module 15, Page 15
Sanitation

Developing Plant Specific Procedures

 A plant-specific sanitation program that is defined and

documented must be followed in order to meet all local
and country guidelines as well as all Pepsi-Cola
guidelines to ensure the production of quality products.

 General sanitation procedures and practice outlined in

quality manual.

 Plant specific program must be developed.

 Consider frequencies, product sensitivities, equipment
materials and configuration

Bottler Quality Training: July 30, 2005 Module 15, Page 16

Sanitation

Key Elements to An Effective Sanitation Program

 Trained Staff
 Proper Tools
 Right Procedures
 Good Measurement
 Drain all valves, tubes, sampling cocks
 Avoid dead end piping, too many bends in your piping,
headspace in your filler bowl
 Never keep the same sanitation program all the time

Bottler Quality Training: July 30, 2005 Module 15, Page 17

Microbiology

Why Perform Microbiological Analysis?

 Purpose of microbiological testing:

 Measures the quality of raw materials
 Measures the effectiveness of the sanitation program
 Confirms safe level of
microorganisms in our product

Bottler Quality Training: July 30, 2005 Module 15, Page 18

Microbiology

Why Microbiology Is A Concern

 Soft drinks and manufacturing processes provide an

excellent environment for microorganism growth

Microbial
Water + Nutrients + pH
Growth

Bottler Quality Training: July 30, 2005 Module 15, Page 19

Microbiology

Categories of Microorganisms

 Spoilage organisms
 off-flavor
 sediments
 odor
 CO2 by-product

 Quality Indicators
 improper sanitation

Bottler Quality Training: July 30, 2005 Module 15, Page 20

Microbiology

LOGARITHM OF NUMBER OF CELLS

Microorganism Growth

Stationary
Phase
Logarithmic
Growth
Death
Phase

LAG
Phase

TIME
Bottler Quality Training: July 30, 2005 Module 15, Page 21
Microorganisms of Concern

Yeast

 Cause 90% of soft drink spoilage

 Grow in beverage, syrup, water and environment
 Cause off-odors, off-tastes, visible particles
 Increase package pressure (fermentation)
 Common types:
 Saccharomyces
 Zygosaccharomyces
 Torulopsis
 Hansenula
 Rhodotorula
 Candida
 Brettanonyces
Bottler Quality Training: July 30, 2005 Module 15, Page 22
Microorganisms of Concern

Mold

 Need oxygen for growth

 Normally do not grow in a carbonated beverage
 Spores and mycelium can survive
 CO2 loss can result in growth
 Cause off-odor, off-taste
 Commonly found in and on returnable bottles
 Common types:
 Aspergillus
 Penicillium
 Rhizopus

Bottler Quality Training: July 30, 2005 Module 15, Page 23

Microorganisms of Concern

Bacteria

 Grow in beverage, bottles, water, syrup, equipment,

environment
 Cause off-odor, off-taste, package pressure, sediment,
rings, turbidity and “slimy” texture
 High counts reflect unsanitary conditions
 Sometimes difficult to remove (for example, Pseudomonas)
 Common spoilage types:
 Lactobacillus
 Leuconostoc
 Gluconobacter

Bottler Quality Training: July 30, 2005 Module 15, Page 24

Microorganisms of Concern

Coliform Bacteria

 Indicate unsanitary conditions

 Should not be anywhere in the process
 Very serious problem
 Typical sources:
 Pollution
 Very poor sanitation
 Fecal contamination

Bottler Quality Training: July 30, 2005 Module 15, Page 25

Microbiology

Sources of Microorganisms
 Water system
 Sweetener system
 Syrup process
 Filling processes
Microorganisms
 Empty packaging are
 Environment Everywhere!
 Process equipment
 People

Bottler Quality Training: July 30, 2005 Module 15, Page 26

Microbiology

Sampling

Get Right Samples,

From Right Places,
at Right Times

Bottler Quality Training: July 30, 2005 Module 15, Page 27

Microbiology

Types of Samples
I. After 5-Step Sanitation Procedure:
 Sanitation rinse water
 Filler valve rinse water:
 Should be taken from a pressurized filler (15-25 psi) and collected in
beverage containers with closures applied
 This simulates the actual beverage filling process
 A minimum of 10% of filler valves must be sampled per filler, per week
 Swabs from equipment:
 Valve snifts
 Rubber seals
 Syrup transfer pumps
 Undercover gas ports

Bottler Quality Training: July 30, 2005 Module 15, Page 28

Microbiology

Types of Samples

II. Raw Material & Containers

 Raw and treated water
 Sweeteners / syrups
 Empty containers

Bottler Quality Training: July 30, 2005 Module 15, Page 29

Microbiology

More Types of Samples

III. Production and Environmental

 Finished beverage
 Environmental air samples:
 Syrup room
 Filling room
 Micro lab
 Pre/Post mix area

Bottler Quality Training: July 30, 2005 Module 15, Page 30

Microbiology

Sampling Techniques and Equipment

 Sampling Techniques
 Samples must be collected without
introducing microbial
contamination

 Equipment
 Sterile sample containers
(e.g., whirlpaks)
 Steriles sampling devices (spoons,
scoops, pipettes, swabs)
 70% alcohol or ethanol (no flaming)

Bottler Quality Training: July 30, 2005 Module 15, Page 31

Microbiology

Sampling Tips and Precautions

 Agitate bulk samples and drums

 Remove any gross surface contamination from bulk
samples and drums
 Flush sampling port, wipe with alcohol, re-flush before
collecting sample
 Sterilize sampling device when conducting open
reservoir sampling
 Do not touch inside of sampling containers
 Obtain random, representative sample

Bottler Quality Training: July 30, 2005 Module 15, Page 32

Microbiology

Plating of Microorganisms

 Purpose
 To estimate the total viable yeast, mold or bacterial SAMPLE
population of a given sample

 Equipment MONITOR
& MEDIA
 47mm, 55mm monitors
 Vacuum pump
 Culture media VACUUM
 Incubator(25ºC and 35ºC)
 0.45µ membrane

Bottler Quality Training: July 30, 2005 Module 15, Page 33

Microbiology

Test Method

1. Aseptically collect sample.

2. Place monitor on vacuum.
3. Filter sample, with vacuum, through monitor.
4. Add culture medium.
5. Turn vacuum on (if media top load only)
6. Remove from vacuum and incubate.
7. Identify growth (with microscope if necessary).
8. Count colonies.
Bottler Quality Training: July 30, 2005 Module 15, Page 34
Microbiology

Isolation of Microorganisms

Test Media Description Temperature Time

Bacteria TGE Broth Nonselective nutrient 35C 48 hours
(total count) medium for general
use with membrane
filter technique ± 10C ±3hrs
Coliform mEndo Selective differential 35C 24 hours
(presumptive) medium used with
membrane filter
technique. Colonies ± 10C
show typical green
sheen.
Yeast/Mold mGreen Yeast Nonselective media 25C 120 hours
and and Mold for general use to
Aciduric determine total
Bacteria yeast/mold. Aciduric ± 10C ±3hrs
bacteria, yeast, or
mold growth.

Bottler Quality Training: July 30, 2005 Module 15, Page 35

Microbiology

Incubation Conditions

Yeast Example

206
151
25°C

30°C
C.F.U.s

21
17

72 hours 120 hours

Time

Bottler Quality Training: July 30, 2005 Module 15, Page 36

Microbiology

Differentiating Colonies With M-Green

 Scope—yeast , mold, yeast like fungi, acid tolerant

bacteria
 Yeasts
 white to tan, pink, brown or black creamy
 generally round or oval (1-20 in size)
 Molds
 fuzzy gray-green or white
 usually long filaments (1-3 in width)
 Bacteria
 almost colorless, turquoise green, yellow (0.5-1 in size)

Bottler Quality Training: July 30, 2005 Module 15, Page 37

Microbes

Yeast

Bottler Quality Training: July 30, 2005 Module 15, Page 38

Microbes

Yeast

Bottler Quality Training: July 30, 2005 Module 15, Page 39

Microbes

Mold

Bottler Quality Training: July 30, 2005 Module 15, Page 40

Microbes

Mold

Bottler Quality Training: July 30, 2005 Module 15, Page 41

Microbiology

Differentiating Colonies With TGE Broth

 Scope—mainly bacteria

 Bacteria is colorless to brightly pigmented

 Usually smaller than yeast and mold organisms

 1 - 10 in size

 Cocci, bacilli, spirilla type shapes

 Unpleasant colony odor

Bottler Quality Training: July 30, 2005 Module 15, Page 42

Microbes

Bacteria

Bottler Quality Training: July 30, 2005 Module 15, Page 43

Microbes

Bacteria

Bottler Quality Training: July 30, 2005 Module 15, Page 44

Microbiology

Differentiating Colonies With M-Endo

 Scope: selectively differentiate coliform bacteria

 Pink to dark red color with a metallic green-gold
surface sheen
 Count at 24 hours, since non-coliform bacteria may
overgrow monitor
 Any presumptive coliforms should be confirmed

Bottler Quality Training: July 30, 2005 Module 15, Page 45

Microbiology

Tips and Precautions

 Use culture medium within shelf-life; store in dry place

away from direct sunlight
 Maintain clean working area
 Always use aseptic techniques
 Culture at proper temperature and length
 Use blanks to confirm culture media not contaminated
 Be careful in counting
 identify correct microorganism
 count spreader colony as 1 CFU
 Review results over time, look for trends

Bottler Quality Training: July 30, 2005 Module 15, Page 46

Microbiology

Good Lab Practices

 Use separate room, if possible
 with separate air control and microbial filters
 use laminar flow hood, if separate room not available
 Use stainless steel work surfaces
 Control and limit access to work area
 Make surroundings hostile to microbes
 eliminate cracks/crevices
 smooth walls
 smooth floors
 Regularly clean and sanitize work surfaces and equipment
 Use lab coats just for microbiology area

Bottler Quality Training: July 30, 2005 Module 15, Page 47

Microbiology

Video: Microbiology

Bottler Quality Training: July 30, 2005 Module 15, Page 48

Wrap-Up

Wrap Up Module 15 - Microbiology

 Microbiological evaluation indicates sanitation

program effectiveness.
 Each plant must prepare customized sanitation
program.
 Accurate results are dependent upon proper aseptic
and representative sample collection, monitor
preparation, incubation, and counting.

Bottler Quality Training: July 30, 2005 Module 15, Page 49

Examination

Demonstrating Comprehension of Key Points is Critical

Bottler Quality Training: July 30, 2005 Module 15, Page 50

Table Buzz

Internalizing Key Information is Critical

Bottler Quality Training: July 30, 2005 Module 15, Page 51

Lab Practices and Procedures

In-Lab Practical for Key Methods

Bottler Quality Training: July 30, 2005 Module 15, Page 52

Lab Practices and Procedures

In-Lab Practical for Key Methods

Bottler Quality Training: July 30, 2005 Module 15, Page 53