ANTIMICROBIAL

PRESERVATION OVERVIEW

JIM MCELROY

THE BASICS

• THIS PRESENTATION IS FOR THE FORMULATION SCIENTIST THAT HAS LITTLE OR NO MICROBIOLOGY EXPERTISE

• IT IS ALSO AN INTRODUCTION TO BASIC PRESERVATIVE CHEMISTRY AND ACTION

COMPOSITION AND STRUCTURE OF MICROORGANISMSBACTERIA BASICALLY COME IN TWO TYPES:

• THOSE THAT HAVE A LOT OF PEPTOGLYCAN IN THEIR CELL WALLS AND THOSE THAT HAVE A LITTLE.

• WHEN BACTERIA ARE STAINED, IT IS DUE TO A CHEMICAL REACTION BETWEEN THE STAIN AND THE PEPTOGLYCAN. CRYSTAL VIOLET STAIN IS GRAM POSITIVE.

• SAFRANIN (WHICH IS RED) INDICATES GRAM NEGATIVE.

CELL MEMBRANES

• THE CELL ENVELOPE IS THE RATE LIMITING BARRIER FOR THE PARTITIONING OF :

• NUTRIENTS

• WASTE PRODUCTS

• CHEMICALS

• MAIN COMPONENTS ARE PHOSPHOLIPIDS AND PROTEINS

• MAJOR PROTEINS ARE HYDROPHOBIC

• THE PLASMA STRUCTURE IS STABILIZED BY HYDROGEN BONDING

• MG2+ AND CA2+ CATIONS STABILIZE THE MEMBRANE

COMPARISONProperty or Constituent Gram

PositiveGram Negative

1. Liposacharide Outer Layer Absent Present

2. Polysaccharide High Low

3. Lipids 2-4% 15% or more

4. Solubility of lipids in fat solvents

Resistant Less resistant

5. Amino Acids 3-4 Complete range

6. Optimal Growth pH Relatively high

Relatively low

7. Nature of toxin Exotoxin Endotoxin

YEAST AND MOLD• YEAST: CELL WALL IS COMPOSED OF TWO OR MORE

PROTEIN POLYSACHARIDE COMPLEXES HELD TOGETHER BY COVALENT BONDS

• YEAST: MANNAN IS A CELL WALL COMPONENT

• MOLD: CELL WALL IS COMPOSED OF TWO OR MORE PROTEIN POLYSACCHARIDE COMPLEXES HELD TOGETHER BY COVALENT BONDS

• MOLD: MANNAN IS NOT A CELL WALL COMPONENT

COMPARISON (BACTERIA, YEAST & MOLD)

Property Bacteria Yeast, Mold

Cell size (μm) 0.5-5 Yeast 20-50Mold ≥100

Cell wall Teichoic acids, muramic acid peptides

Chitin, glucans, manans, and diaminopimelic acid

Cytoplasmic membrane

No sterols Sterols

Cytoplasm No mitochondria and endoplasmic reticulum

Mitochondria and endoplasmic reticulum

Nucleus Prokaryotic (no membrane) Eukaryotic (membrane)

WHAT IS A PRESERVATIVE?

A CHEMICAL SUBSTANCE THAT IS USED TO PRESERVE PHARMACEUTICALS, FOOD OR OTHER ORGANIC MATERIAL FROM DECOMPOSITION OR FERMENTATION BY PREVENTING THE GROWTH OF MICROORGANISMS.

COMMONLY USED PRESERVATIVES

INGESTIBLE & TOPICAL

• PARABENS

• SORBIC ACID

• NA, K & CA SORBATE

• BENZOIC ACID

• NA, K & CA BENZOATE

• SODIUM METABISULFITE

• PROPYLENE GLYCOL (15-30%)

• BHT, BHA

• FLAVORS W/ BENZALDEHYDE

TOPICAL ONLY

• FORMALDEHYDE DONORS

• ESSENTIAL OILS

•MONOGLYCERIDE

• PHENOL

•MERCURY COMPOUNDS

PARABENS

• PARABENS ARE AMONG THE MOST COMMONLY USED PRESERVATIVES.

• THEY ARE RELATIVELY ACTIVE AGAINST A BROAD SPECTRUM OF MICROORGANISMS.

• THE METHYL ESTER IS MOST EFFECTIVE AGAINST BACTERIA AND MOLDS

• THE ETHYL, PROPYL & BUTYL ESTERS ARE MORE ACTIVE AGAINST YEAST AND MOLDS.

• PARABENS ARE MORE EFFECTIVE AGAINST GRAM NEGATIVE THAN GRAM POSITIVE ORGANISMS.

• PARABENS DISASSOCIATE AT HIGH PH (8 OR MORE) AND ARE RENDERED INEFFECTIVE.

PARABENS HAVE LIMITED SOLUBILITY IN H2O

Property Methylparaben sol. g/100mg

Ethylparabensol. g/100mg

Propylparabensol. g/100mg

Butylparabensol.g/100mg

Water 18ºC 0.16 0.08 0.023 0.005

Water 25ºC 0.25 0.11 0.04 0.015

Water 80ºC 3.2 0.86 0.45 0.15

Ethanol 22 25 26 110

PG 1.7 0.5 0.4 0.3

Peanut Oil 0.5 1 1.4 5

Mineral Oil 0.01 0.025 0.03 0.1

PARTITION COEFFICIENTS AND EMULSIONS

PAY CLOSE ATTENTION TO PRESERVATIVE PARTITIONING IN EMULSION SYSTEMS. PARABENS PREFER THE OIL PHASE THUS LEAVING LITTLE IN THE WATER PHASE WHERE IT IS NEEDED.

Oil dropletWater phase

Surfactant

PARTITIONING OF PARABENS

• PARABENS ARE LIPOPHILIC.

• PARABENS WILL PARTITION DISPROPORTIONATELY INTO THE OIL PHASE OF AN EMULSION.

• IF LARGE AMOUNTS OF THE PRESERVATIVE PARTITION INTO THE OIL PHASE, IT IS NOT AVAILABLE FOR ANTIMICROBIAL ACTION.

PARTITION COEFFICIENT MEASUREMENT

• CW IS THE PRESERVATIVE LEFT IN THE WATER PHASE

• C IS OVERALL % OF PRESERVATIVE

• KW IS THE PARTITION COEFFICIENT

• Ά IS PHASE VOLUME RATIO

Cw=C* ά + 1 / Kw + 1

PARTITION COEFFICIENTS FOR PARABENS & OTHER PRESERVATIVESPreservative Kw Oil

Methylparaben 7.5 Almond Oil

0.1 Mineral Oil

18.0 Isopropyl Myristate

200 Diethyl Adipate

Ethylparaben 26.0 Soya Oil

Propylparaben 87.0 Soya Oil

Butylparaben 280.0 Soya Oil

Benzoic Acid 6.10 Soya Oil

Phenol 5.60 Arachis Oil

0.07 Mineral Oil

Sorbic Acid 3.3 Almond Oil

0.21 Mineral Oil

CHELATING AGENTS AS PRESERVATIVE ENHANCERS

ALKALINE EARTH METALS SUCH AS CA+ AND MG+ ARE IMPORTANT FOR THE STABILIZATION OF THE OUTER MEMBRANE OF CELLULAR ORGANISMS. CHELATING AGENTS SEQUESTER THESE IONS.

EDTA IS A TYPICAL CHELATING AGENT USED IN FORMULATIONS.

CHELATING AGENTS ARE NOT PRESERVATIVES

• PROVIDE A SYNERGISTIC EFFECT FOR THE OVERALL PRESERVATIVE SYSTEM

• CONTRIBUTE TO THE PARTIAL SOLUBILIZATION OF THE CELL MEMBRANE WHICH ALLOW PRESERVATIVES A PATHWAY INTO THE CELL.

INGREDIENTS THAT ENHANCE PRESERVATIVE EFFICACY

• SOLUTES (SALTS & HIGH CONCENTRATION OF SUGARS)

• ESTERS

• CATIONIC AND ANIONIC SURFACTANTS

• HUMECTANTS (GLYCERIN, PROPYLENE GLYCOL)

• PHENOLIC ANTIOXIDANTS (BHT)

• CHELATING AGENTS (EDTA)

• FRAGRANCES

• LOW WATER ACTIVITY

INGREDIENTS THAT HINDER PRESERVATIVE EFFICACY

• SUGARS AND ALCOHOL SUGARS

• PROTEINS, PEPTIDES, YEAST EXTRACT

• NATURAL GUMS & CELLULOSE THICKENERS

• PLANT EXTRACTS (ALOE VERA, STARCH,…)

• VITAMINS

• CLAY COMPOUNDS

• HIGH WATER ACTIVITY

• SURFACTANTS (TWEEN 80)

MANUFACTURING STEPS THAT ENHANCE PRESERVATIVE EFFICACY• SANITARY CONDITION OF EQUIPMENT

• TREATMENT OF RAW MATERIALS

• HEAT TREATMENT OF SUB-PHASE OR FINAL BATCH

• SUB-MICRON FILTRATION

• HOT FILL

• ASEPTIC FILL

PACKAGING COMPONENTS AND PRESERVATIVE EFFICACY

• DESIGN OF OPENING OR DISPENSER

• SUSCEPTIBILITY TO CONTAMINATION FROM ENVIRONMENT

• SUSCEPTIBILITY TO CONTAMINATION FROM HUMAN CONTACT

CONCLUSION

IT IS IMPORTANT TO TAKE INTO CONSIDERATION ALL ASPECTS OF THE FORMULA BEFORE CONSIDERING A PRESERVATIVE SYSTEM. THIS INCLUDES:

• TYPE OF FORMULA (TOPICAL OR INGESTED)

• WATER ACTIVITY

• ORGANISMS LIKELY TO PROLIFERATE

• BULK HANDLING

• PACKAGING.