To obtain the best wool wax recovery using an optimal extraction time, a kinetic study was carried out using two different solvants of water and hexan (Fig. 1). The results highlight the importance of this parameter. When we used water at 70°C, a low proportion of wool wax was extracted during the first 20 min. However, when hexan was used, the extraction rate of lanolin increased. The solvant did not have any influence on the extraction rate after 50 min (20 and 10 µg of extract/g of wool using hexane and water respectively). The resultant extraction profiles suggest that the extraction rate is limited by the solubility of some wool wax components. This is logic because hexan is an apolar solavnt. This result is similar with those found by Dominguez et al. in 2003 [7].
Physiochemical characterization showed that wax presents a slightly acidic pH which extends from 6 to 6.5 and a high dry matter content of 60.2 to 62.3% (Table 1). This effluent is characterized by the absence of alcohol which could not therefore be at the origin of a possible toxicity.
Table 1
Wool wax physiochemical and total lipid content analysis
|
Water extract
|
Hexan extract
|
pH
|
6.5
|
6
|
Conductivity (ms/cm)
|
17.9
|
18.4
|
Salinity (g/l)
|
18.6
|
18.9
|
Protein (%)
|
0.13
|
0.08
|
Nitrogen (%)
|
0.1
|
0.07
|
Phosphate (%)
|
4.2
|
4.7
|
Alcohol test (%)
|
0
|
0
|
Water (%)
|
6
|
3
|
Dry matter (%)
|
48.2
|
62.3
|
Total lipid content (%)
|
60.7
|
95.6
|
The mineral composition of this effluent shows a low composition of water moisture (3 to 6%) coupled with a large amount of phosphate (4.2 to 4.7%) and conversely low doses of protein (0.08%) and nitrogen (0.07%). This disagree with previous work dealing with the the physiochimical characterization of baker yeast separation effluent [8].
By means of gaz chromatography-mass spectrometry, more than fifty compounds present in wax sample were identified in form of their methyl derivatives. 6 compounds were determinated comparing to NIST library. Cholesterol is strongly dominating followed by 2-MeO methyl ester of fatty acid with 18 carbon atoms (18:0) followed by methyl ester of 21:0, methyl ester of 16:0 and metyl ester of 15:0 (Table 2). 7-ketocholesterol, which is known to be present in lanolin especially as a product of aging, was not detected in our study. This is in conflict with conventional data of lanolin, where it was found in form of its degradation product 7-keto-3,5-cholestadiene [9]. Our results are in accordance with those found by the same authors in another publication [10].
Table 2
Fatty acids compsosition of wool wax
Peak number
|
Peak identification
|
10
|
FAME 15:0
|
13
|
FAME 16:0
|
16
|
FAME 17:0
|
18
|
MeO-FAME 18:0
|
24
|
FAME 21:0
|
47
|
Cholesteryl methyl ester
|
FAME : fatty acid methyl ester, MeO-FAME : Methoxy fatty acid methyl ester |
Lanolin consists of a complex mixture of esters and polyesters of high molecular weight alcohols and fatty acids [11]. Isolation of lanolin esters is extremely difficult and no conclusions were found that identify the individual esters which exist in lanolin. It has been reported from gas chromatographic investigations that the aliphatic alcoholic compounds in lanoline comprise 17.1% aliphatic nonalcohols, 8.7% aliphatic alkane-diols, 68.3% sterol and triterpene alcohols, and 5.9% unidentified and polyols [12].
The determination of the germination index of olive seeds during 15 days of treatment with different concentrations showed that this parameter is more important using diluted cream comparing by water control. This germination index reaches a maximum at a dose of 1.25 mg/g and then gradually decreases (Fig. 2). These results are in agreement with those found by Lan et al and Abida et al [13–14]. This can be explained, on the one hand by the richness of wax in nutritional elements as phosphate that stimulate germination and on the other hand by fatty acid content. In the same fashion, diethyl aminoethyl hexanoate (DA-6), a plant growth regulator, increases germination and seedling establishment from soybean seeds by increasing fatty acid metabolism and glycometabolism [15].
Irrigation with diluated wax showed an improvement of increasing average lengths of stems compared to the control, this growth in length reaches its maximum in plants irrigated by the dose set at 1.25 mg/g soil, the latter is of the order of 45.7 ± 2.52 cm (Fig. 3). Nevertheless, beyond this volumes irrigation, it causes an antagonistic effect resulting in a decrease of this length and confirming, the toxicity of the wax at high doses. To hypothesise the mode of action of wool wax and its fatty acids on phytohormones, auxin in treated plants was charactirized by gaz chromatographic analysis after derivatization step. Slight emprovement in auxin was noted in plantes treated with 1.25 mg of wax/g of soil according to control (0.9 ± 0.1 ng/ml and 0.7 ± 0.20 ng/ml respectively). Similar results were found by Ami et al. in sorghum plants using UPLC MS/%S assay [16]. For our best knowlege and lettirature survey, this is the first report on auxin enhancement in plants by wool wax fatty acids. Our results are similar to those found by Bach et al. in 2010 [17]. Auxin or indole-3-acetic acid (IAA) is a key plant growth hormone, involved in processes as diverse as branching, gravitropism, phototropism, and seed development [18]. However, the biosynthetic pathways leading to the main auxin in plants, are not well understood. Although there is good evidence that the amino acid tryptophane is an early precursor [19]. Wounding and/or stress of plant tissues can have a major impact on auxin biosynthetic route used by plants [20].