Topic 3 : Sanitation

Question 1

What is the difference between cleaning, sanitising and disinfecting?

Answer 1

Cleaning: Removes dirt and reduces microbes.
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Sanitising : kills most microorganisms (MO)** to a safe level** using chemical / physical means and may not target viruses effectively.

Disinfecting : higher level of killing of MO, including viruses

Question 2

What is the definition of cleaning?

Answer 2

Physical removal of soil and MO in soil, aided by chemical agents.

Question 3

What are the 3 types of inorganic soil (residues)?

Answer 3

1. Hard water deposits, minerals found in water such as calcium and magnesium
2. Metallic deposits
3. Alkaline deposits

Question 4

What type of cleaning compound is required to remove inorganic soil (hard water deposit / metallic deposit / alkaline deposit)?

Answer 4

Acid-type cleaner

Question 5

What are the 3 different types of organic soil and examples?

Answer 5

1. Food deposits → food residues such as lipids and protein
2. Petroleum deposits → from lubricating agents/ lubricating oils / grease (for machinery)
3. Non-petroleum deposits → animal fats, vegetable oils (lipids), proteins

Question 6

What kind of cleaning compounds are used to clean non-petroleum deposits (lipids/proteins)?

Answer 6

Alkali-type cleaner

Question 7

Why can’t acid type cleaning compounds be used for hydrolysing protein deposits, instead of alkaline ones?

Answer 7

Usage of acid type cleaning compounds means pH will be low, making it nearer to the isoelectric point of the proteins. Proteins will thus coagulate and making it harder to remove the deposits.

Question 8

What are the 3 steps in removing soil (residues) from a surface, and how to achieve it?

Answer 8

1. Separate soil from surface using physical / chemical means
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2. Dissolve / disperse soil in cleaning solution → use surfactants to disperse soil as a colloid
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3. Prevent dispersed soil from reattaching to surface → achieved through rinsing

Question 9

Types of cleaning compounds

What kinds of applications are strong alkaline cleaners used for?

Answer 9

CIP systems, as strong alkaline cleaners are not suited for manual cleaning.

Question 10

Types of cleaning compounds

What are some examples of weak alkaline cleaners?

Answer 10

Na₂CO₃, Na₃PO₄

Note : phosphates are sequestrants too → bind to metal ions

Question 11

Types of cleaning compounds

What 2 kinds of deposits do cleaners with active chlorine get rid of, and what is their mechanism of action?

Answer 11

Chlorinated cleaners get rid of carbohydrate / protein deposits (macromolecules)
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Mechanism of action:
- Reacts with polymers to make them shorter chain / more water soluble
- Helps in peptization (process of turning a precipitate into a colloid by mixing it with an electrolyte)

i.e. Peptization : breaking down larger molecules into smaller ones and helping to disperse the smaller molecules into the continuous phase (cleaning / sanitising agent)

Question 12

Types of cleaning compounds

Give examples of highly corrosive acidic cleaning compounds, and state their applications [3].

Answer 12

Strong inorganic acids (no C) such as HCl, HF.

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Applications:

• removing of minerals and rust (inorganic metals)
• floor maintenance
• brightening of steel surfaces

Note : acidic cleaners react with inorganic metals. Recall : acid + metal → salt + hydrogen

Question 13

Types of cleaning compounds

Give examples of mildly acidic cleaning compounds, and what are they used for?

Answer 13

Mildly acidic cleaning compounds are typically organic acids such as citric acid.

• They are used for removal of water scales (buildup of dissolved minerals that forms on surfaces that are in contact w water)

Question 14

Types of cleaning compounds

What are the 3 types of surfactants?

Answer 14

1. Cationic surfactants
2. Anionic surfactants
3. Nonionic surfactants

Question 15

Types of cleaning compounds

Cationic surfactants are usually used as cleaning agents to clean surfaces. True or False?

Answer 15

False, cationic surfactants are not typically used as cleaning agents, but rather bactericidal sanitisers

Bactericidial = “bacteria” + “suicide” = kill bacteria

Question 16

Types of cleaning compounds

What are anionic surfactants used for?

Answer 16

Removal of calcium and magnesium salts. (Anionic surfactants work well with alkaline type cleaners)

Anionic —> attract cations (metal cations)

Question 17

Types of cleaning compounds

What are non-ionic surfactants used for?

Answer 17

Surface cleaning (including walls), as non-ionic surfacants have tendency to form foams which is good for cleaning a wide surface area.

Question 18

Types of cleaning compounds

How do surfactants work?

Answer 18

1. Individual surfactant molecules consist of a hydrophilic head and a hydrophobic tail. In water, surfactant molecules self-assemble into micelles, where hydrophobic groups cluster in the centre.
2. The hydrophobic groups attach themselves to the soil / residues, while hydrophilic head interacts with the surrounding water / detergent.
3. This reduces surface tension between the soil and the surface, allowing the surfactant to lift and suspend the soil away from the surface.
4. The soil particles are trapped inside the micelles, preventing them from reattaching to the cleaned surface.

See diagram in notes

Question 19

Types of cleaning compounds

Surfactants reduce the surface tension of liquids and this improves cleaning efficiency. What does this mean?

Answer 19

Surfactants reduce the cohesive forces between liquid molecules (e.g. detergent molecules). This allows the detergent to spread more easily across surfaces, increasing the area of contact between the cleaning solution and the surface, which improves cleaning efficiency.

Question 20

Auxillary agents

What are auxillary agents in cleaning agents?

Answer 20

Auxillary agents are substances that improve the perfomance of a cleaning agent.

Question 21

Auxillary agents

What is the purpose of chelators / sequestrants? Give some example(s).

Answer 21

Sequestrants prevent precipitation between alkaline cleaning agents and minerals from hard water (Ca²⁺, Mg²⁺)

• e.g. : citric acid, phosphonates, EDTA, polyphosphates

Question 22

Auxillary agents

What are suspending agents and their purpose? Give examples.

Answer 22

They are thickeners that prevent dispersed soil from reattaching back to surfaces
- example : hydrocolloids

Question 23

Auxillary agents

What are scouring agents?

Answer 23

Abrasives that help to remove stubborn soil from surfaces physically.
- abrasive powder, like finely ground minerals like pumice, calcium carbonate (chalk), or quartz

Question 24

Auxillary agents

What is the purpose of corrosion inhibitors and what kind of cleaning agents are they found in? Give examples.

Answer 24

Corrosion inhibitors prevent corrosion of surfaces, and can be found in (strong or weak) acid / alkali cleaners.

• Example : silicates

Question 25

# **Sanitisers** What are the 6 factors that affect the effectivness of sanitisers?

Answer 25

1. Exposure time (longer time = more effective) 2. Temperature (higher temp = more effective BUT some sanitisers are volatile) 3. Presence of soil (present = lowered effectiveness as residue/soil can shield microbes) 4. pH (affects stability of sanitisers ; and microbial reduction dependent on pH) 5. Microbes present (high microbial load reduce effectiveness) 6. Presence of biofilms (resuce effectiveness of sanitsers as MOs are more resistant)

Question 26

# **Sanitisers** What are the 2 main types of physical sanitation methods?

Answer 26

1) Thermal sanitation 2) Radiation - non-ionising radaition : UV - ionising radiation : gamma rays

Question 27

# **Sanitisers** What is used for thermal sanitation?

Answer 27

Hot water / steam

Question 28

# **Sanitisers** What are the disadvantages for thermal sanitation? [2]

Answer 28

1. Dangerous for manual cleaning 2. Steam quickly loses effectiveness when it becomes cold

Question 29

# **Sanitisers** Radiation sanitation can be categorised into non-ionising and ionising radiation. What is non-ionising radiation and how does it kill microbes?

Answer 29

Radiation that does not break chemical bonds, through the use of UV light. It kills microbes by : - causing pyrimidine bases in DNA (cytosine / thymine) to dimerise - repair of DNA leads to mutation of microbial cell or cell death

Question 30

# **Sanitisers** What are some examples of ionising radiation in sanitation?

Answer 30

Gamma radiation, X-ray, e-beam (shorter wavelength than UV, higher energy)

Question 31

# **Sanitisers** What are the 2 mechanism of action that ionising radiation damage microbial cells?

Answer 31

1. Direct damage, by breaking the double stranded helix of DNA and lead to cell death 2.Indirect damage : breaking of chemical bonds causes formation of radicals / reactive oxygen speicies which damages DNA, or causes lipid / protein oxidation, eventually leading to cell death

Question 32

# **Sanitisers** What can ionising radiation do to the organoleptic properties of food? What is ionising radiation usually used for?

Answer 32

Formation of free radicals in ionising radiation may change the taste of food (break covalent bonds in foods ; esp lipid peroxidation). It is usually used for sterilising food packaging.

Question 33

# **Sanitisers** What are the disadvantages of using UV radiation? [4]

Answer 33

1. Hazard to workers 2. Limited penetration depth, easily blocked by soil 3. UV lamp may violate glass policy of the plant (no glass in factory) 4. Radiation may not be effective (endospores resistant to UV an ionising radiation)

Question 34

# **Sanitisers** What are the 4 main mechanisms in which chlorine based sanitisers kill microbes

Answer 34

1. Reacting / oxidising nucleic acids (e.g. DNA) 2. Damage chromosomes 3. Inhibit oxidative phosphorylation (respiration) 4. Disrupt protein synthesis

Question 35

# **Sanitisers** What are iodophors (sanitiser)? Why must they be rinsed off at higher concentrations (i.e. cannot be "no-rinse" at higher conc)

Answer 35

Iodophors are **complexes** of *elemental iodine* with a *non-ionic surfactant* - need to be rinsed off at higher conc bc iodine is brown and can stain surfaces

Question 36

# **Sanitisers** What are the advantages of iodophors over chlorine based disinfectants? [3]

Answer 36

**1. Less irritating to human skin and mucous membrane 2. Stable at low pH** 3. Less reactive : do not react with other substances, and do not degrade as easily.

Question 37

# **Sanitisers** What are some disadvantages of iodophors over chlorine based disinfectant? [3]

Answer 37

1. **Stronger smell** which can contribute to off-flavours 2. Stain porous materials 3. Less effective in endospore inactivation → iodide down the periodic table compared to chloride, it is a weaker oxidising agent

Question 38

# **Sanitisers** Quarternary ammonium compounds are what kind of surfactants?

Answer 38

Cationic surfactants

Question 39

# **Sanitisers** What are some characteristics of quarternary ammonium compounds? [2]

Answer 39

- Have surfactant properties, **foams easily** Forms a **thin bacteriostatic residual layer** on surface

Question 40

# **Sanitisers** What are some advantages of quarternary ammonium compounds? [5]

Answer 40

1. Low toxicity, non-corrosive, non-irritating 2. Safe, easily removed from skin with anionic detergent (aka soap) 3. Colourless, odourless, 4. Stable against organic compounds 5. Little loss in effectiveness over time (unike chlorine based sanitisers)

Question 41

# **Sanitisers** Why shouldn't you use quartnery ammonium compounds to sanitise fermentation tanks?

Answer 41

The residual film could inhibit growth of bacteria, and fermentation requires bacterial growth.

Question 42

# **Sanitisers** Quartnery ammonium compounds are not compatible with which 2 types of cleaning agents?

Answer 42

1) anionic detergents - ammonium : NH₄⁺, will precipitate with anions 2) phosphate sequestrants

Question 43

# **Sanitisers** Acid sanitisers are usually available as a mix of which 2 acids?

Answer 43

Phosphoric Carboxylic

Question 44

# **Validation, monitoring, verification of cleaning** What is validation of the cleaning method? How can validation be carried out? [4]

Answer 44

Validation is to prove that the SSOP will be effective in reducing microbial load to a safe level.
**Ways to carry out validation: ** 1. Literature review **2. Field trial (most impt! Irreplaceable!)** 3. Regulations - e.g. FDA / CODEX 4. Lab tests

Question 45

# **Validation, monitoring, verification of cleaning** What are some areas the validation process should cover? [4] | **note : non-exhaustive**

Answer 45

1. Optimum cleaning frequency 2. SSOP can clean equipment and environment thoroughly, even **difficult to reach areas** **3. Establish the achievable level of cleaniliness** 4. Establish frequency of glove change/handwashing/shoe dips

Question 46

# **Validation, monitoring, verification of cleaning** What does monitoring mean? At what is the frequency is it done?

Answer 46

Monitoring : **ongoing, real-time observation** and **recording** of activities or parameters to ensure that processes are operating within established limits. - tests that are quick and easy to carry out, done on a daily basis

Question 47

# **Validation, monitoring, verification of cleaning** What is verification? At what frequency is it carried out?

Answer 47

Verification is the process of confirming that the monitoring system and SSOP is working effectively. - carried out on a longer interval basis, once every few months (e.g. audits of cleaning records) - some are carried out daily (cross signing by supervisor on cleaning records daily)

Question 48

# **Validation, monitoring, verification of cleaning** ATP monitoring can be used to monitor food. How does it work?

Answer 48

- use a swab to collect a sample from a surface - insert the swab into a luminometer which measures the amount of ATP present by triggering a chemical reaction that produces light - intensity of light is directly proportional to the amount of ATP

Question 49

# **Validation, monitoring, verification of cleaning** Since ATP is found in microbial cells, ATP monitoring is indicative of microbial contamination. True or False?

Answer 49

False. All organic material, including food (with live cells) contain ATP, as it is the universal energy currency of all cells. Thus, ATP monitoring is only indicative of cleaning effectiveness and not microbial contamination. (Also, food usually contains more ATP than microbes)

Question 50

# **Validation, monitoring, verification of cleaning** How does protein monitoring work?

Answer 50

It is based on the principle of BCA (bicinchoninate assay) / Biuret reaction, where Cu²⁺ is reduced to Cu⁺ by specific amino acids

Question 51

# **Validation, monitoring, verification of cleaning** When testing for microorganisms, what are the 2 main groups of microorganisms that can be tested?

Answer 51

1. Indicator organisms (total plate count, Enterobacteriaceae) 2. Industry specific pathogens

Question 52

Microbial tests can be done on ____ to ensure effective sanitation and cleaning ? [2]

Answer 52

1. Food contact surfaces 2. CIP rinse water

Question 53

When should environmental samples be collected and tested for microbes? [2] Give the rationale ## Footnote **Rinse → clean → rinse → sanitise → rinse **

Answer 53

1. Just after cleaning, before sanitising → to ensure all soil is removed before proceeding to sanitising ;; sanitizers may interfere with ATP / protein / allergen assay
2. Samples can be collected after sanitising → gives assurance for the next run ;; so you know the food processed in the next run would be safe.

Question 55

What should the sanitation SOP (standard operating procedure) contain? | **Note : non-exhaustive**

Answer 55

1) **When** : Cleaning frequency - e.g. once every 24 h / every product change-over / change of allergen type 2) **What** : Safety procedures, PPE required 3) **What** : Lock out / tag out procedures → to ensure equipment is not operating when someone is cleaning it 4) **What** : Chemical safety procedures - SDS (safety data sheet) - how to mix cleaning chemicals - first aid measures 5) Disassembly procedure → machinery, especially for cleaning out of place (COP) 6) Cleaning procedure - General steps : rinse → clean → rinse → sanitise / disinfect → rinse (only applies for those that need to be washed off) 7) Validation - ensure that cleaning agents will work with field trials 8) Monitoring procedure - environmental swabbing (ATP / protein monitoring / visual inspection / rapid protein or allergen tests) 9) Verification procedure - Cleaning logs and supervisor sign-off 10) Corrective Action and Preventive Action - Re-train staff - Re-clean surface and re-test with environmental swabbing to confirm if the cleaning agent is working

Question 56

As a general rule of thumb, how frequent should sanitation occur? [3]

Answer 56

- At least once every 24h - Every change in product type - Every change in allergen type

Question 57

What are the general steps for a cleaning procedure?

Answer 57

Rinse → clean → rinse → sanitise / disinfect → rinse* ## Footnote ***Not all sanitisers need to be rinsed off. depends**

Question 58

# **Special topic : biofilms** What are biofilms?

Answer 58

A microbial community characterised by its **adhesion to a solid surface** and by the production of an **extracellular polymeric matrix** in which MOs are embedded.

Question 59

# **Special topic : biofilms** What 2 factors of surfaces can encourage biofilm formation?

Answer 59

1. Rough surfaces (e.g. presence of scratches) 2. Hydrophobic surfaces

Question 60

# **Special topic : biofilms** Formation of biofilms involve production of polysaccharides by vacteria. How does the microbe know when to start producing these polysaccharides, mate and disperse?

Answer 60

Through quorum sensing, whereby cell to cell communication of bacteria cells allows them to share information about cell density and adjust gene expression accordingly.

Question 61

What is the general mechanism of quorum sensing?

Answer 61

1. **Autoinducers (small, diffusible signal molecules**) are produced and secreted at a basal level during bacterial growth 2. **Concentration of signal increases** as bacteria population increases 3. Bacteria display different **phenotypes** depending on population density. ## Footnote **example L acylhomoserine lactone (AHL)** AHL is produced at basal level * AHL diffuses and is uptake by bacteria through active transport. * Above a certain threshold, AHL binds to transcription factors (R proteins). This complex binds to DNA and causes changes in gene transcription

Question 62

What are some strategies to manage biofilms? [2]

Answer 62

1. Thorough cleaning and sanitation 2. Surface coatings (hydrophilic) to discourage biofilm formation, since they grow on rough and hydrophobic surfaces 3. Sanitisers that specifically inhibit quorem sensing? 4. Additives that inhibit quorum sensing? ## Footnote **Point 3 and 4 : ongoing research needed**

Question 63

# **Sanitation SSOP** Outline what should be included in the santiation SSOP. [6 + 4]

Answer 63

**1) When : establish frequency of sanitation procedure** - every product change / once every 24h **2) What : chemical safety procedures (safety)** - Chemical safety datasheet - How to mix chemicals - First aid measures **3) What : PPE and safety procedures (personnel) (safety)** **4) What : lock out / tag out procedures (safety)** **5) How : Disassembly proceures (for COP) (safety)** **6) How : cleaning procedure** - Rinse → clean → rinse → santise / disinfect → rinse* (not required all the time) ------------------ **7) Validation** **8) Monitoring** - Periodic environmental sampling - ATP / protein (BCA assay) monitoring **9) Verification** - To verify that sanitation has been done in accordance w SSOP, cleaning records & logs to note who did sanitation, at what time and to be signed off by supervisor **10) Corrective action Preventive Action (CAPA)** - To be done if SSOP fails in maintaining clean and hygienic environment

Question 64

# **Sanitation SSOP** Under the sanitation SSOP, what are the 3 key main elements to ensure safety, and what is covered under each?

Answer 64

**1) Personnel safety** - covers safety procedures and PPE to be worn **2) Chemical safety** - Chemical safety data sheet (SDS) - How to mix chemicals - First aid **3) Lock out procedures** - Procedures to lock equipment to ensure that it is not operating while someone is cleaning it

Question 65

# **Sanitation SSOP** Validation is a key step in the sanitation SSOP. What is there to be validated? [4]

Answer 65

1. Optimum cleaning frequency 2. Frequency of glove change / handwashing / shoe dips 3. Calidate that SSOP can clean equipment and environment thoroughly, even in difficult to reach areas 4. Establish the **achievable level of cleanliness**(baseline level) ## Footnote Also maybe can validate effectiveness of cleaning agents in ensuring cleanliness

Question 66

# **Sanitation SSOP** If SSOP fails in maintaining a clean and hygienic environment, what corrective actions / preventive actions can be taken? [2]

Answer 66

1. Re-clean surface, re-test with environmental swabbing to confirm if cleaning agent is working 2. Re-train staff