INTRODUCTION:

A cosmetic product is defined as any substance or preparation intended to be placed in contact with the various external parts of the human body (epidermis, hair system, nails, lips and external genital organs) or with the teeth and the mucous membranes of the oral cavity with a view exclusively or mainly to cleaning them, perfuming them, changing their appearance and/or correcting body odors and/or protecting them or keeping them in good condition. Cosmetics products are basically non-sterile and most of them are prone to microbiological attack^1-3.

Contamination of cosmetics products by some gram negative bacteria are well known. Certain yeasts and molds have also been reported to degrade the microbiological quality of such products. The extent of microbial contamination largely depends on the unhygienic handling of bulk ingredients during manufacturing as well as due to insufficient in-process check and defective storage or distribution^4-7. The level of contamination in cosmetic products with aerobic bacteria should not exceed the BIS microbial limit for cosmetic finished products ⁸. If the limit exceeds, serious skin problem can occur. Several types of diseases including scabies, acne, eczema, dyschromia and other skin diseases have been reported upon usage of cosmetics. A regular microbiological monitoring during manufacturing, packaging and storage of the cosmetics products sold in market is required to ensure public health safety of consumers. Although, attempts have been made in microbiological quality control of pharmaceutical products in our country, however, studies relating to microbiological profiling of the cosmetics products is still lacking. The present study attempts to analyse some readily available cosmetic products and assess the bacterial and fungal load as well the presence of specific pathogenic bacteria using the standard microbiological methods. Some Cosmetic raw materials were also tested for microbial content on the lines of above samples.

MATERIALS AND METHODS:

Principle:

The analysis consist of plating a known amount of sample on selected culture media specifically suitable for the growth of bacteria and fungi and incubating them for specified period to permit the development of visual colonies for counting. The number of colonies seen is taken as a measure of the number of microorganisms present in the sample.

Equipments and glasswares:

Bio safety cabinet , Electronic balance, BOD Incubator (25 ^oC), Bacteriological Incubator (37^oC), Autoclave for sterilization at 121^oC, Hot air oven for dry sterilization at 165^oC, Thermostatically controlled water bath at 45^oC, colony counter, Sterile glassware such as conical flasks, petri dishes, test tubes .Micro pipets with sterile micro tips.

Media and reagents:

Plate count agar, Peptone, Tween 80, Isopropyl myristate. Nutrient Broth , Macconkey Agar., Eugon LT 100 broth, cetrimide agar, pseudomonas agar.

Procedure for total bacterial count and total yeast and mould:

Weigh 10 gm of the sample and place it in a sterile conical flask, containing sterile inactivating diluents containing appropriate neutralizer to give a 1:10 dilution of initial suspension. Cover the conical flask with cotton plug and mix thoroughly. In case of cream, 2 % isopropyl myristate is added to facilitate proper emulsification. Serial dilution is prepared by adding 1 ml of initial suspension of serial diluents.1 ml of diluted suspension of desired dilution is transferred to two sterile petri dishes. Pour 15-20 ml of sterilized molten plate count agar into petri dishes..Transfer 1 ml of diluted suspension of desired dilution in two sterile Petri dishes. About 15- 20 ml of sterilized molten sabbouraud dextrose agar is poured into petri dishes. The plate is rotated gently on a flat surface to ensure thorough mixing. The media in the plates is allowed to solidify at room temperature. Invert the plates of plate count agar and incubate at 30 ⁰ C for 48-72 hours. for bacterial enumeration. Subsequently the plates of sabouraud dextrose agar are inverted and incubated at 25 ⁰C for 5 days for enumeration of yeast and mould.. The numbers of colonies are counted in duplicate plates. The number of colonies in cfu/gm is calculated as follows:

Total bacterial count =

Number of colonies (mean count observed) x Dilution factor

Weight of sample

Total Yeast and Mould count =

Number of colonies (observed mean count) x Dilution factor

Weight of sample

Procedure for Pseudomonas aeruginosa, E.coli, S. aureus

Weigh 1 gm of sample and add to 9 ml of enrichment broth (Eugon LT 100 broth).Incubate the tube overnight at 37˚C for 20 hours.

For Pseudomonas aeruginosa: From the incubated enrichment broth tube take a loop full of incubated enrichment broth and streak on surface of cetrimide agar plate. Incubate the inoculated cetrimide agar plates at 37˚C for 24hours. After 24hr, if no growth is seen on the plate, Pseudomonas aeruginosa was absent. If colonies with yellow green pigment which fluoresces under UV light, proceed for the confirmation tests for Pseudomonas aeruginosa, such as gram staining, oxidase test.

For E. coli :

From the incubated enrichment broth tube take a loop full of incubated enrichment broth and streak on surface of Macconkey Agar plate. Incubate the inoculated Macconkey Agar plates at 37˚C for 24hours. After 24hrs, if no growth is seen on the plate, E.coli was absent. If colonies of brick red colour surrounded by zone of bile precipitation proceed for the confirmation tests, such as gram staining, colony characters on selective agar such as EMB agar.

For S. aureus :

From the incubated enrichment broth tube take a loop full of incubated enrichment broth and streak on surface of Baird parker Agar plate. Incubate the inoculated Baird parker Agar plates at 37˚C for 24hours. After 24hrs, if no growth is seen on the plate, S.aureus is absent.

RESULTS AND DISCUSSION:

Cosmetics are not supposed to be sterile as they contain nutrients which support the growth of variety of microorganisms. However, cosmetics products must be free from pathogens and total aerobic bacterial load should be low so that it could not impair skin and mucous membrane defense mechanisms. As the occurrence of skin diseases is much more frequent in developing countries due to the unhygienic dense environment, improper sanitation, and the usage of microbiologically contaminated water, maintenance of good microbiological quality in cosmetic products is important. It is therefore necessary to carry out microbiological analysis of the raw materials and final products of cosmetics for obtaining products with good microbiological quality. The present investigation was attempted to analyze various cosmetic products available in the urban population. Lotion spray foam, Skin care cream face pack, Neem orange Face wash, Lemon scrub, and Oil free skin cream were analysed for total microbial count, yeast and mould and gram negative pathogens as per IS 14648:2011⁸

Of the various categories of samples studied, all the samples exhibited a load of total viable bacteria in the range of 10 to 330 cfu/gm (Table 1). The fungal load was less than 1 cfu/gm. Among the pathogenic bacteria identified (Tables 1), Staphylococcus aureus, E. coli and salmonella and pseudomonas were totally absent in all the samples.

In order to minimize the chance of contaminated finished product, it is necessary to control the microbial content of cosmetic raw materials along with other physical and chemical attributes. Cosmetic manufacturers should evaluate the microbiological quality of their raw materials and establish appropriate specifications based on the best available scientific information. Some of the raw materials such as Xanthan gum and veegum ultra used in cosmetics were received in our laboratory for microbial analysis and the results of analysis are summarized in Table 2.

Xanthan gum is an anionic, high molecular weight polysaccharide, which is used as a stabilizer and viscosity modifier in aqueous based systems. Xanthan gum solutions are pseudoplastic, i.e. they show shear thinning flow behavior. This pseudoplasticity imparts a smooth texture to the final product and provides a pleasant application. Xanthan gum is soluble in cold and hot water and shows a high degree of viscosity even in low concentrations. The high viscosity of xanthan gum at low shear rates effectively stabilizes creams and lotions which are primarily oil-in-water emulsions. Xanthan gum keeps emulsions stable over a broad temperature and pH-range. It also assures lotion flowability even upon ageing. Furthermore xanthan gum stabilizes the oil phase of creams and lotions and delivers the active ingredients to the skin in a uniform manner. With its high viscosity at rest xanthan gum effectively suspends insoluble ingredients in cosmetics. Due to the shear thinning flow behavior of xanthan gum highly pigmented products like liquid make-up can be spread easily and uniformly. Xanthan gum provides effective stabilization and suspension to hair care products and enhances the lathering quality of shampoos. Xanthan gum is a natural product which is non-toxic and non-irritating. It can be used in combination with other thickeners and stabilizers to improve texture, flow behavior, stability and appearance. All these properties make xanthan gum a very effective thickening and stabilizing agent for cosmetics and toiletries. Ten samples of xanthan gum were analysed for total bacterial count, total yeast and mould and the pathogens. The results are summarized in Table 2. The mean bacterial count was 307 cfu/gm, Standard deviation 18.3 and RSD 5.9. Total yeast and mould for xanthan gum was <1. The pathogens were absent in all the samples.

Another raw material Veegum ultra (also known as Magnesium Aluminum Silicate) .is extensively used in cosmetics .It is unique acidic smectite clay. It produces dispersions in the 4.2 to 5.2 pH range, making it particularly suitable for topical. It is especially easy to hydrate, taking no more than 15 minutes in most cases. It is whiter and brighter than other clays, which carries over to the finished formula. Typical use levels are between 0.5% and 2%. Because of its unique nature, Ultra clay is an exception. It is relatively unaffected by changes in hydration parameters. Some applications for this product are lotions creams, cleansers, shaving creams and liquid foundations.The results of microbial analysis is summarized in Table 2. It is observed that the mean of total microbial count for xanthan gum samples was 75 cfu/gm, Standard deviation 18.1 and RSD 24.2. In case of Veegum clay the total yeast and mould mean was 16.4, standard deviation 2.75 and RSD 16.7.In case of veegum clay the pathogens are absent in the samples.

Table 1.Microbial examination of Cosmetic samples

Sample	Total microbial count cfu/gm	Yeast and mould	E.coli	Salmonella	Staphylococcus aureus	Pseudomonas
Lotion spray foam	<10	<1	Absent	Absent	Absent	Absent
Skin care cream facepack	330	<1	Absent	Absent	Absent	Absent
Neem orange Face wash	80	<1	Absent	Absent	Absent	Absent
Lemon scrub	210	<1	Absent	Absent	Absent	Absent
Oil free skin cream	70	<1	Absent	Absent	Absent	Absent

Table 2. Microbial analysis of some raw materials used in cosmetics

Sample

Total microbial count cfu/gm

N=10

Yeast and mould

N=10

E.coli.

Salmonella

Staphylococcus aureus

Pseudomonas

Xanthan gum

Mean= 307

SD= 18.3

RSD= 5.9

Absent

Veegum ultra

Mean= 75

SD= 18.1

RSD= 24.2

Mean= 16.4

SD= 2.75

RSD= 16.7

Absent

CONCLUSION:

Cosmetics products must be free from pathogens and total aerobic bacterial load should be low so that it could not impair skin and mucous membrane defense mechanisms. As the occurrence of skin diseases is much more frequent in developing countries due to the unhygienic dense environment, improper sanitation, and the usage of microbiologically contaminated water, maintenance of good microbiological quality in cosmetic products is important. Cosmetic manufacturers should evaluate the microbiological quality of their raw materials and establish appropriate specifications based on the best available scientific information. A regular microbiological monitoring during manufacturing, packaging and storage of the cosmetics products sold in market is required to ensure public health safety of consumers.

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Received on 09.04.2015 Accepted on 12.05.2015

Research J. Topical and Cosmetic Sci. 6(1):Jan.–June 2015 page 48-51

DOI: 10.5958/2321-5844.2015.00008.4