Natural Preservatives For A Cosmetic Company

Published Date: 02 Nov 2017

Cosmetics are commercially available products that are used to improve the appearance of the skin (Mary and Lupo, 2001). Cosmetic products become easily contaminated by bacteria and fungi. Containing water, oils, peptides, and carbohydrates cosmetics are a very good medium for growth of microbes. All these factors contribute to the fact that cosmetic products need preservation to prevent microbial growth and spoiling of the cosmetic product and also infection of the skin (Kostarelos and Rheins, 2002). The last few decades have witnessed a great demand for herbal cosmetic products, away from synthetics. This is so because these herbal and natural cosmetics are safe to use and do not have any side effects.

With the increasing concern of public health and consumers’ awareness, non-chemical cosmetics are booming worldwide. Plants are important in formulating natural and non-chemical cosmetic products, thus making the plant materials highly potential to be developed into cosmetic formulating ingredients.

Our on-going research into safer, more environmentally friendly and natural preservatives include the development of innovative products based on biomimetic polymers, antimicrobial peptides, and novel plant extracts.

NATURAL ANTIMICROBIALS AND ANTIOXIDANTS IN PLANTS

Preservatives are added to all manner of personal care and cosmetic products for the primary purpose of inhibiting the development of microorganisms. By preventing the growth of harmful bacteria, fungi and yeasts, preservatives help to maintain product safety, protect against contamination in use, and extend a product’s shelf life.

Antioxidants are ingredients that naturally inhibit oxidative reactions. They help to condition the skin as well as stop oxidation that causes products to become rancid and spoil. Anti-oxidants extend the shelf life of your products by reducing the rate of oxidation of your oils. Use an antioxidant in any formulation, which contains fragile oils such as sweet almond, hemp, avocado, flax, or evening primrose. You can add antioxidants directly to your oils to help keep them fresh, or you can add the antioxidant to the oil phase of your recipe. Lip balms, lotion bars, creams, lotions, scrubs and any other product containing oils can benefit from the addition of an antioxidant.

An anti-microbial is an ingredient or substance that helps to destroy unwanted microorganisms such as bacteria. In the context of handmade skin care products; an anti-microbial helps preserve a product by keeping the product free of these unwanted microorganisms.

The following are examples of natural preservatives that can be used in making cosmetics products:

Grapefruit Seed Extract

Grapefruit Seed Extract (GSE) is a citrus seed based anti-microbial used as a preservative in skin care products. GSE is made with the extracts of citrus seeds and pulp. It is blended with vegetable glycerin to make it non-irritating to the skin and mucous membranes when used in formulations. ItCan be added to cold or hot cosmetic formulas and the usual concentration is 0.5-1 percent (for more complex formulas 2-3 percent may be necessary). For highly effective or long-term preservation, combine with broad spectrum preservatives.

INCI Nomenclature: Grapefruit (Citrus Grandis) Extract (and) Glycerin

Honey

This is known for being highly stable against microbial growth because of its low moisture content and water activity, low pH and anti-microbial constituents.

Coriander oil

Coriander oilis resistant to a range of toxic bacteria and thereby works well with other natural oils and extracts as a preservative. Coriander is a known skin allergen.

Germaben II

Germaben II is a convenient, ready-to-use broad-spectrum anti-microbial preservative for personal care products such as shampoos, conditioners, lotions, creams, body sprays and other formulations. It is highly effective against gram positive and gram-negative bacteria, yeasts and molds and does not need any additional preservatives. It is a clear, viscous liquid with mild odour. It is soluble in both oil/water emulsions and aqueous formulations up to a level of 1.0%. At 1%, Germaben II provides 0.30% Germall II, 0.11% methylparaben, 0.03% propylparaben, and 0.56% propylene glycol.

INCI Nomenclature: Propylene Glycol (and) Diazolidinyl Urea (and) Methylparaben (and) Propylparaben

Germaben II-E

Germaben II-E was developed to protect formulations that contain ingredients that inactivate parabens. It is a liquid preservative system that contains 20% Germall II, 10% methylparaben, 10% propylparaben, and 60% propylene glycol. It is used to preserve water-in-oil and oil-in-water emulsions but should not be used in aqueous formulations. It is readily soluble at 1.0% and should be added to the emulsified product under gentle agitation before the addition of fragrance. Germaben II-E is a complete preservative effective against gram positive and gram negative bacteria, yeasts and molds. It is compatible with almost all cosmetic ingredients including surfactants and proteins.

INCI Nomenclature: Propylene Glycol (and) Diazolidinyl Urea (and) Methylparaben (and) Propylparaben

Lichen extract

Lichen extract is a water-free, natural preservative that can be used to prevent the growth of certain types of bacteria, fungi and yeast. It also benefits other preservatives by enhancing their activity. Lichen extract is nontoxic and nonirritating to skin.

LiquaPar Oil

LiquaPar Oil is a clear, liquid blend of isopropyl, isobutyl and n-butyl esters of parahydroxybenzoic acid. It is a very stable and effective preservative against gram positive and gram-negative bacteria, yeast and mold. LiquaPar Oil is readily incorporated into various types of formulations, including anhydrous products, without heating. It is a good choice for salt scrubs and bath oils where no water is present but may be inadvertently introduced to the container during regular use. The recommended usage rate is 0.3 - 0.6% however, in complex formulations, 0.1% Germall II may be required for adequate preservation.

T-50 Vitamin E Oil

Vitamin E contains natural antioxidants, which extend the life of your products. Gamma tocopherol, a component of Vitamin E, is a great antioxidant for protecting cosmetic formulations. T-50 has a larger amount of gamma tocopherols than other forms of Vitamin E oil. While the alpha tocopherol in the 250, 1000, and 1400IU/g oils is wonderful as an in vitro antioxidant, studies show that the gamma tocopherol in the Vitamin E T-50 oil is a better antioxidant for oils/lipids in cosmetic formulations. T-50 has a higher content of gamma tocopherols and can be used at a rate of .04% or 400ppm to adequately protect your oils.

INCI Nomenclature: Tocopherols

Rosemary Oil Extract

Rosemary oil extract (ROE) also acts as a highly concentrated natural antioxidant that inhibits free-radical induced reactions such as oxidation and can thereby extend the shelf life of cosmetic preparations. Rosemary also adds fragrance to formulations and should be used at a ratio of 0.1 percent or less. Consider combining it with grapefruit seed extract and vitamin E oil for potent, all-natural preservative action.The rosemary oil extract is a 100% pure extract. It has not been diluted in a vegetable oil.

INCI Nomenclature: RosmarinusOfficinalis (Rosemary) Leaf Extract

Citric acid

Citrus acid is derived from citrus fruit and can be used as a preservative. It is also recognized for its astringent and antioxidant properties and has a low sensitizing potential. However it can be irritating when applied to chapped, cracked or inflamed skin.

Aloe vera

Aloe verais a natural product that is nowadays frequently used in the field of cosmetologyAloevera is a stem less plant species of succulent plant .The common varieties are Aloe barbadensisMiller,Aloesaponaria, Aloe variegata, Aloe forex, Aloe lalifolia, Curacao Aloe.Out of all these,the most popular is Aloe barbadensis Miller which has most therapeutic value and referred to as ‘True Aloe’.It is the most potent and it is therefore the plant we use.Aloevera L. (A. barbadensisMill.)belongs to Aloaceaefamily.It is native to tropical and southern Africa. It has been cultivated for its thick exudates that contain many active compounds with known therapeutic properties(Park and Lee 2006). It is animportantcommercial plant because of the secondary metabolites produced (phenolic compounds) such as aloesin, aloin and aloe-emodin (oxidative product of aloin) not only due to it’s cosmetic properties but also due to their physiological role(Acur ero 2008).Aloe vera extract is found in the leaves of the aloe vera plant, the pulp or gel is known as aloe veraextract.This extract (taken from the leaves) acts as a humectant, providing moisture replacement.In other aloe species of Aloe the active phenolic compounds were most concentrated in the peripheral regions of the plant leaves (Chauser-Volfson 2002) (Gutterman 2000). The top third of the leaf and the leaf edges have the highest concentration because those parts are most susceptible to consumption by herbivores (Chauser-Volfson 2002). Aloe vera juice (made from grinding the pulp) is also an extract from the plant and may be considered as a type of aloe vera plant extract.Extracts from Aloe vera are widely used in the cosmetics and industries, being marketed as variously having rejuvenating, healing or soothing properties. It can be use for the production of Aloe Jelly, Aloe Vera Gel,all Purpose Cream, Body Lotion, Body Wash,Shampoo, Hair Gel, Aloe Vera Powder, Aloe Oil and many more.

IDENTIFICATION OF THE ACTIVE INGREDIENT IN ALOE VERA

Active components of aloe verawith its properties: Aloe vera contains 75 potentially active constituents: vitamins, enzymes, minerals, sugars, lignin, saponins, salicylic acids and amino acids.(Shelton 1991 )

Vitamins: It contains vitamins A (beta-carotene), C and E, which are antioxidants. It also contains vitamin B12, folic acid, and choline. Antioxidant neutralizes free radicals.

Enzymes: It contains 8 enzymes: aliiase, alkaline phosphatase, amylase, bradykinase, carboxypeptidase, catalase, cellulase, lipase, and peroxidase. Bradykinase helps to reduce excessive inflammation when applied to the skin topically, while others help in the breakdown of sugars and fats.

Minerals: It provides calcium, chromium, copper, selenium, magnesium, manganese, potassium, sodium and zinc. They are essential for the proper functioning of various enzyme systems in different metabolic pathways and few are antioxidants.

Sugars: It provides monosaccharides (glucose and fructose) and polysaccharides: (glucomannans/polymannose). These are derived from the mucilage layer of the plant and are known as mucopolysaccharides. The most prominent monosaccharide is mannose-6-phosphate, and the most common polysaccharides are called glucomannans [beta-(1,4)-acetylated mannan]. Acemannan, a prominent glucomannan has also been found. Recently, a glycoprotein with antiallergic properties, called alprogen and novel anti-inflammatory compound, C-glucosylchromone, has been isolated from Aloe vera gel.7,8

Anthraquinones: It provides 12 anthraquinones, which are phenolic compounds traditionally known as laxatives. Aloin and emodin act as analgesics, antibacterials and antivirals.

Fatty acids: It provides 4 plant steroids; cholesterol, campesterol, β-sisosterol and lupeol. All these have anti-inflammatory action and lupeol also possesses antiseptic and analgesic properties.

Hormones:Auxins and gibberellins that help in wound healing and have anti-inflammatory action.

Others: It provides 20 of the 22 human required amino acids and 7 of the 8 essential amino acids. It also contains salicylic acid that possesses anti-inflammatory and antibacterial properties. Lignin, an inert substance, when included in topical preparations, enhances penetrative effect of the other ingredients into the skin. Saponins that are the soapy substances form about 3% of the gel and have cleansing and antiseptic properties.

Aloe Vera Gel

The Aloe Vera Gel is a colorless, odorless, hydrocolloid with several natural beneficial substances. The effectiveness of Aloe Vera gel as a cosmetic skin care product is indisputable. The voluminous research shows clearly that the gel reduces scarring in burns, skin ulcers and other lesions.It has also been shown to have an invigorating effect on skin when applied on a regular basis. Major chemical constituents of Aloe Vera Gel consist primarily of water and polysaccharides (pectins, hemicelluloses, glucomannan, acemannan, and mannose derivatives). It also contains amino acids, lipids, sterols (lupeol, campesterol, and β-sitosterol), tannins, and enzymes. Mannose 6-phosphate is a major sugar component.

http://bestaloevera.co.uk/wp-content/uploads/2008/12/fresh_aloe_vera_gel1.jpg

Cut aloe vera showing the Gel

http://www.superfoods-scientific-research.com/wp-content/uploads/2013/03/aloe-barbadensis-miller.jpg

Aloe Vera (Barbadensis Miller)

ALOIN

Aloin is the major anthraquinonic component of Aloe vera(Groom and Nolds 1987.). It is a mixture of two diastereoisomers, aloin A and aloin B with numerous functions(MontiD and Speranza 1990.).Aloin is an anthraquinone that has been used because of its cathartic and purgative effect and is involved in plant defence mechanism against herbivores (Esteban et al. 2001). Aloin is synthesized in the plastids of the assimilating tissue, released to the apoplast through exocytosis, and finally stored in the aloin cell, adjacent to the vascular bundle sheath cell (16).Thus, aloin is the major phenolic compound in A. vera leaves, and it is mainly contained in the bitter, smelly exudates seeping out from freshly cut leaves, while very low amounts of aloin exist in the gel(Martínez-Romero et al. 2013).

Aloe barbadensis is an important medicinal plant belonging to the family Liliaceae and has applications in pharmaceutical, food and cosmetic industries. The plant is traded in the medicinal drug market for flavoring liquid formulations(Reynolds 2004). The gel present inside the leaves of Aloe plant contains phenolic compounds like aloin-A (barbaloin), aloesin, isoaloeresin D and aloeresin E used in the treatment of tumors, diabetes, ulcers and cancer ( Ishii et al., 1990, Okamura et al., 1996 and Park et al., 1998). Aloin-A (barbaloin, C21H22O9) is the major phenolic compound reported from the plant (Groom and Reynolds 1987). Earlier reports have shown the effect of arbuscularmycorrizal fungi and Azotobacter on the growth and barbaloin content of the plant ( Tawaraya et al., 2007 and Pandey and Banik, 2009). In the present study, the effect of PSB on TCP amended soil is reported for biometric parameters and the aloin-content in A. barbadensis.

http://upload.wikimedia.org/wikipedia/commons/thumb/0/09/Aloin_structure.png/220px-Aloin_structure.pnghttp://upload.wikimedia.org/wikipedia/commons/thumb/d/d8/Aloin.jpg/220px-Aloin.jpg

The Structure of Aloin

PROBLEMS

Synthetic preservatives are widely used in making cosmetics products due to the fact that they have broad-spectrum of activity against bacteria and fungi, have low concentrations required to effectively preserve product and have relatively low costs. Also, they have a very long shelf life of 2 or 3 years.

However, whether accurate or not, studies pointing to potential health risks of certain synthetic preservatives have sent companies scrambling to find natural alternatives. Synthetic preservations are often petroleum based. Also, people are interested in products that contain natural preservatives instead of chemical or synthetic ones. People are becoming aware and troubled by the fact that cosmetic products contain ingredients that are harmful to our health.

Therefore, natural preservatives are now used and mostly preferred by people for the making of cosmetics. Many natural ingredients are healing for the skin, since they contain numerous vitamins and nutrients. They can improve the health of the skin (if the product is properly formulated) and actually nourish the skin (the skin can absorb the nutrients). Natural cosmetics usually contain much higher amounts of actives than conventional products.

PROJECT AIM

The aim of this project includes-

Identifying plants with natural preservatives that can be used for production of cosmetics.

Theuse of genetic modification approach to enhance the yield of a target natural preservative (Aloin).

To increase soil available P, P uptake in plants and plant growth

To increase aloin-A content due to higher plant biomass.

PROJECT OBJECTIVES

The hopes for this project is to makenatural preservatives present in plants available for the production of cosmetics that are both natural and safe for the masses eliminating or minimizing the use of harmful preservatives.This has many advantages such as cutting costs for the company in the production of cosmetics thus increasing profits.

There are also many other benefits derived from natural preservatives such as allowing "natural" and "organic" product labeling.Natural preservatives function over a broad pH range. It confers many natural fragrances to products.It is also used in producing skin friendly cosmetics.

For this project, we would have to identify possible natural preservative sources and come up with strategies to enhance the yield. We also need to have a contingency plan if one aspect fails such as changing of the method of enhancing yield of our natural preservative.

BASIC RESEARCH PLAN

To increase the P uptake by the plants by increasing soil available P.

To get maximum yield of aloin-A and also the highest increase in the aloin-content per plant using PSB consortium treated plants grown in TCP amended soil(Gupta et al. 2012 ).

EXPERIMENTAL OUTLINE

Isolation and screening of P solubilizing bacteria

Production of indole acetic acid and siderophore

Morphological, biochemical and molecular identification of bacteria

Pot experiments

Quantitative high-performance liquid chromatography (HPLC) analysis of aloin-A

Analysis of soil available P content and P uptake in plants

Statistical analysis

EXPERIMANTAL OUTCOMES

1)Isolation and identification and characterization of PSB

The amount of P-solubilization by PSB varied from 150 to 340 μg ml−1. Maximum P solubilization bacteria was observed by A1 (340 μg ml−1) followed by A6 (276 μg ml−1), A51 (212 μg ml1) and A20 (150 μg ml−1). All PSB isolates were Gram negative unicellular rods. Bacterial colonies were circular and smooth. The colony color was cream (A1 and A20), brownish yellow (A6) and red (A51). All PSB were catalase positive. All were non lactose fermenters except A51. All PSB were oxidase negative except A1 isolate which is an oxidase positive (Table 1). All PSB showed indole negative, methyl red negative and simmon citrate positive characteristics. Isolate A1 and A6 were negative for Voges-proskauer whereas isolate A20 and A51 were positive for Voges-proskauer (Table 1). The G + C content of DNA of PSB isolates was 59.2% (A1), 65.5% (A6), 58.3% (A20) and 58% (A51). The results of the BLAST search of the 16S rRNA gene sequences indicated A1, A6, A20 and A51 isolates as closely related to P. synxantha, B. gladioli, E. hormaechei and S. marcescens, respectively. Based on the NJ phylogenetic tree done with the 16S rDNA similarity (%), the nearest taxas of PSB isolates were identified as Pseudomonas synxantha IAM 12356 for A1; Burkholderia gladioli R406 for A6 Enterobacterhormaechei EN 314 for A20 and Serratiamarcescens A3 for A51 isolate.

Table 1.Biochemical characterization of PSB isolates.

PSB isolates

Catalase

Oxidase

Lactose fermentation

Indole

Methyl red

Voges-proskauer

Simmons citrate

A1

+ve

+ve

NLF

−ve

−ve

−ve

+ve

A6

+ve

−ve

NLF

−ve

−ve

−ve

+ve

A20

+ve

−ve

NLF

−ve

−ve

+ve

+ve

A51

+ve

−ve

LF

−ve

−ve

+ve

+ve

Full-size table

The selected bacterial isolates will be further tested for siderophore and IAA production. Isolates, A1, A6, A20 and A51 produced siderophore 94.47%, 95.22%, 27.86% and 86.67% respectively. Isolate A6, A20 and A51 should produce IAA 6.93 μg ml−1, 4.33 μg ml−1, 28.2 μg ml−1 respectively). Whereas, isolate A1 should produce no IAA.

2) Effect of PSB on plant growth

The PSB treatments (applied individually or as a consortium) increased all parameters of A. barbadensis in un-amended soil with the consortium treatment yielding better results than each individual treatment. It significantly (P ≤ 0.05) increased leaf length by 39.5%, root length by 31.1%, total number of leaves by 48.1%, total gel volume by 143%, dry gel weight by 147% and dry rind weight by 95.2% as compared to the control plants (Table 2). In plants treated with single PSB strains, maximum stimulatory effects on various biometric parameters were obtained by P. synxantha followed by S. marcescens, B. gladioli and E. hormaechei (Table 2). The individual PSB treatment also showed significant stimulatory effects on plant growth. In case of total number of leaves, only inoculation with P. synxantha showed the significant increase (29.6%) over the control plants, whereas other PSB treatments did not show any effect on this parameter. The application of PSB as a consortium increased all biometric parameters of A. barbadensis plants grown in TCP amended soils more than individual PSB treatments (Table 2). Compared to control plants, an increase of over 243% in total gel volume was observed in plants treated with the PSB consortium. In individual treatments, the degree of stimulation varied with respect to the type of growth parameter. Maximum increase in leaf length (21.0%) and dry rind weight (51.8%) was shown by P. synxantha treated plants (Table 2). Whereas, in case of root length, total gel volume and dry gel weight, maximum stimulatory effects were shown by S. marcescens treated plants. The plants treated with B. gladioli and E. hormaechei showed smaller increases in growth parameters than P. synxantha and S. marcescens treated plants.

Table 2.Enhanced growth and P uptake of Aloe barbadensis through PSB inoculations

Treatment

Leaf length (cm)

Root length (cm)

Total no of leaves

Total gel volume (ml)

Dry gel weight (g)

Dry rind weight (g)

P content in soil (mg kg−1)

P uptake in leaves (gel + outer rind) (mg plant−1)

aS (control)

b30.5 ± 0.5 (a)

9.76 ± 0.6 (a)

5.4 ± 0.5 (a)

95.0 ± 4.3 (a)

0.41 ± 0.01 (a)

1.47 ± 0.03 (a)

1.35 ± 0.2 (a)

4.89 ± 0.5(a)

S + P. synxantha (A1)

36.9 ± 0.6 (b)

11.9 ± 0.4 (b d)

7.0 ± 0.7(b c)

173 ± 3.4 (b)

0.78 ± 0.01 (b)

2.16 ± 0.04 (b)

3.89 ± 0.2 (b)

8.84 ± 0.7(b)

S + B. gladioli (A6)

34.3 ± 0.9 (c)

10.9 ± 0.5 (b c)

6.4 ± 0.5 (a b)

152 ± 3.3 (c)

0.69 ± 0.06 (c)

1.81 ± 0.05 (c)

3.34 ± 0.1(c)

7.14 ± 0.7(b c)

S + E. hormaechei (A20)

35.3 ± 1.2 (b c)

10.7 ± 0.6 (a c)

6.6 ± 0.5 (a b)

150 ± 2.6 (c)

0.65 ± 0.01 (c)

1.78 ± 0.04 (c)

3.36 ± 0.1(c)

6.62 ± 0.9(c)

S + S. marcescens (A51)

34.4 ± 0.9 (c)

11.7 ± 0.1 (b c)

6.6 ± 0.5 (a b)

171 ± 4.8 (b)

0.77 ± 0.01 (b c)

1.85 ± 0.04 (c)

3.89 ± 0.1(b)

7.53 ± 0.2(b)

S + consortium

42.5 ± 1.7 (d)

12.8 ± 0.3 (d)

8.0 ± 0.7(c)

231 ± 4.5 (d)

1.02 ± 0.1 (d)

2.86 ± 0.04 (d)

7.17 ± 0.1 (d)

13.9 ± 0.5(d)

S + cTCP (control)

32.2 ± 1.1 (a)

9.98 ± 0.6 (a)

6.0 ± 0.7 (a)

125 ± 5.5 (a)

0.50 ± 0.02 (a)

1.63 ± 0.1 (a)

2.91 ± 0.2(a)

5.59 ± 0.4 (a)

S + TCP + P. synxantha (A1)

39.0 ± 0.5 (b)

12.1 ± 0.5 (b c)

7.4 ± 0.5 (a)

189 ± 5.0 (b)

0.92 ± 0.02 (b)

2.48 ± 0.04 (b)

6.04 ± 0.2 (b)

11.3 ± 0.9 (b)

S + TCP + B. gladioli (A6)

35.6 ± 0.6 (c)

11.5 ± 0.2 (b)

6.6 ± 0.5 (a)

179 ± 5.9 (b)

0.82 ± 0.02 (c)

1.98 ± 0.09 (c)

5.59 ± 0.2 (b)

8.98 ± 0.6 (c)

S + TCP + E. hormaechei (A20)

35.6 ± 0.4 (c)

11.4 ± 0.1 (b)

7.0 ± 0.7 (a)

176 ± 5.1 (b)

0.76 ± 0.01 (c)

1.89 ± 0.03 (c)

4.49 ± 0.1 (c)

8.52 ± 0.7 (c)

S + TCP + S. marcescens (A51)

37.8 ± 0.4 (b)

12.8 ± 0.2 (c)

7.2 ± 0.4 (a)

218 ± 4.9 (c)

0.97 ± 0.01 (b)

2.46 ± 0.04 (b)

8.27 ± 0.1 (d)

12.3 ± 0.9 (b)

S + TCP + consortium

45.1 ± 0.8 (d)

14.0 ± 0.3 (d)

7.6 ± 0.5 (b)

430 ± 8.4 (d)

1.51 ± 0.06 (d)

4.98 ± 0.27 (d)

8.83 ± 0.1 (d)

22.6 ± 3.8 (d)

3)Effect of PSB on P content

Inoculation with PSB increased the available P content of the soil (Table 2). The results were again more pronounced in the presence of the bacterial consortium than with individual PSB additions. Un-amended soil with the inoculated consortium had 7.17 g of available P content 430% higher than the control. The TCP amended soil, had 8.83 g of P, available an increase of 203% compared to the TCP control. Single treatment with P. synxantha and S. marcescens showed a similar increase of available P in the un-amended soil. In the TCP amended soil, a maximum increase (184%) in available P yielded the treatment with S. marcescens followed by P. synxantha, B. gladioli and E. hormaechei.

4)Effect of PSB on aloin-A content

Aloe plants grown in TCP amended soil, showed higher aloin-A contents than plants grown in soil without TCP amendment (Fig. 1). The increase in the aloin-A content was due to both the increase in biomass of Aloe plants and the increased biosynthesis of aloin-A as compared to control plants. The PSB consortium treated plants grown in TCP amended soil showed maximum yield of aloin-A and also the highest increase in the aloin-A content per plant. The increases were 159% and 673% based on g−1 dry gel weight basis and plant−1 dry gel weight basis, respectively.

Full-size image (32 K)5409

Fig. 1. Influence of PSB treatment on the aloin-A content. Error bars represent standard deviation. Bars with same letter are not significantly different according to LSD at P≤ 0.05.

TIME FRAME

Project will be conducted in the greenhouse (uncontrolled conditions) and will take five months (for example from April to September).

APPLIED RESEARCH PLAN

Biological Details

We plan to isolate and screen P solubilizing bacteria. The soil adhering to the roots of Aloe barbadensis will be separated by gentle tapping, using sterilized forceps and collected in sterilized plastic bags. The soil samples will be processed on the same day. Samples of 1.0 g soil will be suspended in 9.0 ml of phosphate buffer saline (pH 7.2) and serial dilutions (1:10) spread on Pikovskaya's (PVK) agar containing TCP as the phosphate source. Plates will be incubated at 30 °C for 7 days and colonies with a clear halo will be marked positive for phosphates solubilization. The phosphate solubilizing potential of PSB in liquid medium will be estimated by inoculating separated colonies in 100 ml PVK broth containing 0.5%, TCP, adjusted to 7.0 and incubating at 30 °C on rotary shaker (130 rev min−1). 5.0 ml of the cultures will be taken every 24 h for 7 days, centrifuged, and the content of soluble-P estimated by colorimetry.

We plan to produce indole acetic acid and siderophore. PSB will be tested for their ability to produce IAA as assayed by the colorimetric method using Ferric chloride–perchloric acid (FeCl3–HClO4) (Gordon and Paleg 1957). PSB will be inoculated in the minimal medium (g l−1): KH2PO4, 1.36; Na2HPO4, 2.13; MgSO4·7H2O, 0.2, pH = 7.0, amended with 5.0 mM l-tryptophan solution (g 100 ml−1): glucose, 10; L-tryptophan, 1.0; Yeast extract 0.1) (Frankenberger and Poth 1988) at 30 °C on a rotary shaker (130 rev min−1). Culture supernatant will be analyzed for IAA production using Salper's reagent, Development of pink color will be assayed with a spectrophotometer at 530 nm. The production of siderophore will be analyzed using the method of Schwyn and Neilands (1987). PSB will be inoculated in iron deficient medium containing (g l−1): K2HPO4, 0.1; KH2PO4, 3.0; MgSO4·7H2O, 0.2; (NH4)2SO4, 1.0; succinic acid, 4.0 at 30 °C on a rotary shaker at 130 rev min−1 for 48 h. Culture supernatant will be analyzed for siderophore production using Universal Chrome Azurol-S (CAS) colorimetric assay. Absorbance will be read at 630 nm for the loss of blue color. The activity will be recorded in percentage siderophore units calculated as [(Ar − As) × Ar−1) ×100]. Where, ‘Ar’ is defined as absorbance of reference (un-inoculated media + CAS solution) and ‘As’ is absorbance of test (culture supernatant + CAS solution).

In addition, we plan to identify the bacteria morphologically, biochemically and molecularly. PSB isolates grown on nutrient agar will be characterized for colony morphology, Gram staining and biochemical analysis. Isolates will also tested for catalase (Graham and Parker1964), oxidase (Kovaks 1956), and lactose fermentation (Snyder and Atlas 2006). The guanosine + cytosine content (mol% G + C) of genomic DNA will be determined by the thermal denaturation method (Marmur and Doty 1962).

The identification of PSB would be done on the basis of 16S rRNA gene sequencing. The genomic DNA of PSB isolates will be extracted by the QiagenDNeasy Plant Mini Kit (Qiagen, Valencia, CA). The primers fD1 (5′-AGAGTTTGATCCTGGCTCAG-3′) and rP2 (3′-ACGGCTACCTTGTTACGACTT-5′) will be used for amplification of 16S rRNA gene (Weisburg et al. 1991). The total PCR reaction mixture should be 50.0 μl comprising 200 μMdNTPs, 50 μM each primer, 1X PCR buffer, 3 U Taq polymerase, and 100 ng genomic DNA. The thermocycling conditions should involve an initial denaturation at

94 °C for 4 min, followed by 35 cycles of 94 °C for 1 min, 52 °C for 1 min, and 72 °C for 2 min and final extension at 72 °C for 8 min. We are going to purify the 16S rRNA gene using gel electrophoresis, ligated to pGEM-T vector (Promega, Madison) and transformed in E. coli JM109. The sequences of the insert will be determined using a Big-Dye Terminator Cycle Sequencer and an ABI Prism 310 Genetic Analyzer (Applied Biosystems, CA). The 16S rRNA gene sequences will be analyzed using the gapped BLASTn (http://www.ncbi.nlm.nih.gov) search algorithm and aligned to their nearest neighbors. The evolutionary distances among phosphate-solubilizing isolates and their related taxa will be calculated using TREECON software and Kimura's two-parameter model, after aligning the sequences with ClustalW.

We also plan to do pot experiments. Each PSB isolate will be grown in nutrient broth at 30 °C in an orbital shaker (150 rev min−1) for 24 h. Cultures will be centrifuged in 50 ml sterile plastic tubes at 6000 × g for 15 min. The pellets will be re-suspended in PBS to obtain a final concentration of 108 ml−1colony forming units (CFU). We plan to use the liquid cultures of each PSB isolate for individual inoculations. A consortium will be prepared by mixing all individual cultures of PSB of equal cell density 108 CFU ml−1 into 250 ml sterilized flask and will be used as a mixed inoculum(Mamta et al. 2010).

Experiments will be conducted in the greenhouse (uncontrolled conditions) for five months. Unsterile loamy soil (pH, 7.8; total organic C, 0.10%; available N, 46.8 mg kg−1; available P, 5 mg kg−1; available K, 14.8 mg kg−1; total Ca, 0.6 m Eq 100 g−1; total Mg, 0.12 m Eq 100 g−1;) will be thoroughly mixed, passed through a 2 mm sieve and dried at sunlight for 7 days. Ethanol disinfected earthen pots (24 cm diameter × 30 cm height) will be filled with 4.0 kg of soil. Roots of tissue cultured plantlets will be sterilized by dipping in 2% NaOCl solution for 10 min and then washed three times with sterile distilled water. Roots will be dipped into the selected bacterial suspension (108 CFU ml−1). Experiments will be performed in a completely randomized block design. Plantlets will be planted under two different soil conditions and six different treatments; (i) soil (control) or soil + TCP; (ii) P. synxantha; (iii) B. gladioli; (iv) E. hormaechei; (v) S. marcescens; (vi) mixture of all PSB.

Also, we will need to analysealoin-A using quantitative high-performance liquid chromatography (HPLC). Leaves of harvested A. barbadensis will be washed with running tap water and twice with distilled water. The inner gel will be mechanically separated from the outer cortex of the leaf with the help of a knife. Total gel will be extracted from all the leaves of a plant, then freeze dried (Heto dry winner, Model DW 1-0-110, Allerφd, Denmark).

The 0.5 g of each freeze-dried gel sample will be extracted with 10.0 ml methanol for 24 h at 4 °C(Okamura et al. 1996). The suspension will be centrifuged at 4000 × g for 10 min at 4 °C. The supernatant will be subjected to HPLC (WATER Corporation, USA, Lichrocart® C-18 coloumn, flow rate of 0.8 ml min−1, 800 psi, run time 35.0 min). The mobile phase will be 0.1% acetic acid/acetonitrile (60:40). Analytes will be detected at wavelength 290 nm with a photodiode array detector and identified by their retention time and by spiking the sample with standard aloin-A (Sigma-Chemical, USA). Quantification will be done by reference to the peak area percentage obtained for the standard compound.

Analysis of soil available P content and P uptake in plants will also be conducted. The available P content of soil will be determined using the colorimetric sodium bicarbonate-extractable P method (Olsen et al. 1954). Phosphorus uptake in Aloe plants will be determined by using vanadomolybdo phosphoric yellow color method (Koenig and Johnson 1942).

Lastly, we will conduct statistical analysis. Statistical analysis will be conducted by using Analysis of Variance (ANOVA) statistical package for social sciences (SPSS) software, version 30 followed by comparison of multiple treatment levels with the control, using the posthocBonferroni least significant difference (LSD) at P ≤ 0.05.

CONTIGENCY PLAN

An alternative method to this technique used by applying ultraviolet (UV) treatments on the aloin content of Aloe vera L. gel. In 36 days the effect of UV-A treatment to A. vera plants will led to an increase in the aloin concentration in gel, rind tissue, and latex. Also to check the effect of the UV treatment ,Penicilliumdigitatum and Botrytis cinerea will be artificially inoculated on the leaf surface.This will be observed based on if there is decrease in the growth of the penicilliumdigitum and Botrytis cinerea in UV-A-treated leaves which might be attributed to the increase in the aloin concentration by the UV-A treatment. In addition, UV-C treatment to detached leaves also led to an increase in the gel aloin concentration, at higher levels than occurred with UV-A treatment.

CONCLUSION

P-solubilizershas potential to increase Aloe plant growth and aloin-A content under greenhouse conditions. The effect of an inoculation by a consortium is more pronounced than individual inoculations for all the mentioned parameters in both soil conditions. Increased P uptake in plants directly correlates with enhanced aloin-A production.

COMMERCIAL ASPECTS

Plants and plant derived ingredients are common and of major importance in the fields of cosmetics. The cosmetic industry is a fast moving market. Products have short lifecycles and the industry has to come up with innovative products. (Gaffar et al., 2008). Many cosmetic manufacturers have been using aloe extracts to supplement their products. Therefore, they may have an interest in increasing the yield of active compounds. Using aloe vera may be a marketing strategy but modified Aloe will make it a better product. Therefore, an Aloe veraplant that has been genetically modified having more yield of the chemical compound in the natural extract will be more marketable than the normal Aloe vera plants that are not modified.

The significant increase in the aloin-A content of PSB treated plants emphasizes the potential of an economical and eco-friendly means of achieving higher levels of aloin-A.

 

REFERENCE LIST

ACUR ERO, Án gela Ma tos (2008). CIENCIA, 16 (4), 389 - 395.

ESTEBAN, A, et al. (2001).. Plant Physiol. Bio chem. [online]. Last accessed 5 April 2013

GROOM, O.J and NOLDS, Rey T (1987.). Planta Med. [online]. Last accessed 5 April 2013

GUPTA, M, et al. ( 2012 ). Isolation and identification of phosphate solubilizing bacteria able to enhance the growth and aloin-A biosynthesis of Aloe barbadensis Miller. Microbiol Res, 167 (6), 358-63.

MAMTA, et al. (2010). Stimulatory effect of phosphate-solubilizing bacteria on plant growth, stevioside and rebaudioside-A contents of Stevia rebaudiana Bertoni. Applied Soil Ecology, 46 ( 2), 222-229.

MARTÍNEZ-ROMERO, Domingo, et al. (2013). Is It Possible To Increase the Aloin Content of Aloe vera by the Use of Ultraviolet Light? J. Agric. Food Chem, 61 (9), 2165–2170.

MONTID, Mannito P and SPERANZA, G.J ( 1990.). Chem. Soc. Perkin Trans. [online]. Last accessed 5 April 2013

OKAMURA, Nobuyuki, et al. ( 1996). High-performance liquid chromatographic determination of phenolic compounds in Aloe species. Journal of Chromatography , 746 ( 2 ), 225-231.

PARK, YI and LEE, SK (2006). New Perspectives on Aloe. [online]. Last accessed 5 April 2012

REYNOLDS, T (2004). Aloe chemistry. the genus Aloe, 39-74.

SHELTON, RM ( 1991 ). Aloe vera. Its chemical and therapeutic properties. Int J Dermatol. , 30 (10), 679-83.

SNYDER, James W and ATLAS, M Atlas (2006). Handbook of Media for Clinical Microbiology. [online]. Last accessed 5 April 2013 at: www.amazon.com ›. › Infectious Disease › Communicable Diseases

WEISBURG, W.G, et al. ( 1991). 16S ribosomal DNA amplification for phylogenetic study. Journal of Bacteriology, 173 ( 2), 697-703.