22 PRESERVATIVES
Lactobacillus brevis (L. brevis), requiring almost ten times the concentration to inhibit its growth in comparison to Escherichia coli or Staphylococcus aureus (entry 2 and 4). L. brevis is a species of bacteria that is
commercialised as a probiotic. A probiotic can be defined as a live bacteria or yeast that has health benefits. There are reports that L. brevis and other probiotics may promote the breakdown of food, encourage absorption of nutrients as well as protect against more harmful microorganisms. Probiotics are well established in the food
industry and have been added to fermented foods such as yogurt, or sold as dietary supplements. Therefore, L. brevis has been labelled a ‘friendly bacteria’. It Is thought that adding probiotics to a cosmetic product for rinse-off or leave-on applications may encourage a healthy and harmonious skin microbiome by adding ‘friendly bacteria’ to your skin. In vitro testing of the fruit acid preservative
directly against L. brevis was performed using a Zone of Inhibition (ZOI) method. In this test, a ZOI for Iscaguard IAF was compared with that of a traditional type of preservatives: ■ Sample 1: 1.5% Iscaguard IAF ■ Sample 2: 0.5% Phenoxyethanol/Parabens (INCI name: phenoxyethanol, methylparaben, ethylparaben, butylparaben, propylparaben, isobutylparaben) ZOI is a semi-quantitative test to assess the
ability of a preservative to inhibit the growth of bacteria or fungi. Although quantitative data can be extracted in some cases by measuring the diameter of the inhibition zone, it is widely regarded as a qualitative method used for screening potential antimicrobial active ingredients. Test plates were streaked evenly with a
swab containing pure L. brevis culture. A well was bored out of the agar and the preservative under test was placed in the centre. The plates were incubated at 32°C for 48 hours. The plates were then assessed for bacterial growth: plates with a large zone of inhibition
Figure 2: Analysing microbial growth using binocular microscope at ISCA’s microbiology department
surrounding the preservative demonstrated a strong antibacterial effect against the test species. If no zone is observed around the preservative, then that ingredient has minimal effect against the bacteria. After 48 hours, the plate containing 1.5%
of Iscaguard IAF did not contain a zone of inhibition, indicating very little inhibitory effect on the L. brevis. However, the plates containing the phenoxyethanol/parabens preservative
contained a significant zone around the central well. As expected, these preservatives showed a measurable activity against L. brevis bacteria (Figure 3).
The obstacle to generating probiotic
cosmetics is that the preservative must work to ensure no unwanted or harmful bacteria thrive, whilst allowing the friendly bacteria to remain intact. Due to its high MIC value for L. brevis, it was hypothesised that probiotic cosmetics for both rinse-off and leave-on applications could be preserved using Iscaguard IAF. When used as a preservative, the
recommended dosage for Iscaguard IAF is 0.5 - 1.5%. Theoretically, a dosage of 1.5% should be sufficiently high to preserve the formulation against pathogens, whilst not interfering with the survival of L. brevis. Cultures of L. brevis were obtained from
TCS Biosciences. After checking for viability, the cultures were grown on suitable media (Lactobacillus MRS Agar), before being inoculated into cosmetic formulations. Simultaneously, Lactobacillus acidophilus
Figure 3: Preservative ZOI against L. brevis. Left: phenoxyethanol / paraben (0.5%). Right: Iscaguard IAF (1.5%)
TABLE 4: INOCULATION LEVELS IN COLONY FORMING UNITS PER G (CFU/G) FOR THREE EXAMPLE FORMULATIONS. INOCULUM: SODIUM CHLORIDE IN DEIONISED WATER (0.9%)
Entry 1
2 Formulation Hand Cream Vitamin C Serum 3 Hand Wash
Initial inoculum (cfu/g) 1 x 106 1 x 106 1 x 106
PERSONAL CARE July 2023
Count-2 h (cfu/g) 7 x 103 5 x 104 2 x 101
Count-2 days (cfu/g) 1.29 x 104 2.6 x 104 <10
Count-7 days (cfu/g) 7.2 x 103 2.0 x 103 <10
commercial food supplement powder was purchased, but unfortunately failed to grow despite several attempts under varying conditions. Thus, parallel work for the Lactobacillus acidophilus was discontinued as it was proved to be unculturable. As shown in 4, the fruit acid preservative
allows survival of the L. brevis in two of the example formulations (entry 1 and 2). The hand cream formulation proved very robust for
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TABLE 3: MINIMUM INHIBITORY CONCENTRATIONS (MIC, %) FOR THE AQUEOUS BLEND OF FRUIT ACIDS AGAINST A RANGE OF MICROORGANISMS
MICROORGANISM
Entry 1
2 3
4 5 6
Bacteria (gram-negative)
Pseudomonas aeruginosa Escherichia coli
Lactobacillus brevis Bacteria (gram-negative)
Staphylococcus aureus Yeasts
Candida albicans Moulds
Aspergillus brasilliensis 0.1
0.2 2.0
0.2 0.3 0.3
MIC (%)
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