Analysis of Metal Cations and Inorganic Anions

The cations are quantitatively determined by atomic-absorption spectroscopy (AAS), whereas the anions are detected by ion chromatography (Arienzo M. and Capasso R., 2000).

9Archer M. H., Dillon V. M., Cambell-Platt G., and Owens J. D. (1994) The partitioning of diacetyl between food oils and water. Food Chem, 50, 407-409.

Attempts to separate soluble anions from OMWW by ion-exchange or to remove the oil fraction by solid-phase or solvent extractions were not completely satisfactory and erratic results were observed. Buldini P.L. et al. (2000) presented a simple and accurate procedure for the determination of inorganic anions in OMWW using on-line microdialysis of OMWW directly followed by the ion chromatography analysis of soluble chloride, nitrate, phosphate, and sulfate with conductimetric detection. OMWW is first of all sonicated at room temperature to make it homogeneous, then diluted and microdialized. Most of the organic load of the effluents is removed in a few minutes without using reagents, while soluble anion quantitation remains unaffected. The clear solution is analyzed for the inorganic anions content by direct injection on to an ion chromatograph equipped with a conductivity detector. In the absence of standards, the separation efficiency of microdialysis has been investigated by spiking wastewater samples as well as standard oil emulsions with varying amounts of inorganic anions and subjecting them to microdialysis for different periods of time prior to performing instrumental analysis. Excellent spike recoveries and low relative standard deviations are obtained for all the anions if a 10 min microdialysis time is overcome. Chloride, nitrite, nitrate, phosphate, and sulfate are not affected by the microdialysis procedure and their recovery is between 96 and 104% in wastewater as well as in standard oil emulsion. The dialysis membrane has been replaced after more than 100 analyses. The UV photolysis pretreatment of the same sample demonstrates the different information that can be obtained by the two sample pretreatment procedures.

Antioxidant Activity

Antioxidant activity has been assessed in many ways. In general, the antioxidant effectiveness is measured by monitoring the inhibition of a suitable substrate. After the substrate is oxidized under standard conditions, the extent of oxidation is measured by chemical, instrumental, or sensory methods. Hence, the essential features on any test are a suitable substrate, an oxidation initiator, and an appropriate measure of the end product. Antolovich M. et al. (2002) reviewed the major methodologies for the determination of antioxidant activity used by the food industry, with the diphenylpicrylhydrazyl (DPPH) radical assay being one of the more utilized due to its relative simplicity; it is, however, a lengthy procedure.

The limitation of many newer methods is the frequent lack of an actual substrate in the procedure. The combination of all approaches with the many test methods available explains the large variety of ways in which results of antioxidant testing are reported. The measurement of antioxidant activities, especially of antioxidants that are mixtures, multifunctional, or are acting in complex multiphase systems, cannot be evaluated satisfactorily by a simple antioxidant test without due regard to the many variables influencing the results. Several test procedures may be required to evaluate such antioxidant activities. A general method of reporting antioxidant activity independent of the test procedure has been proposed by Antolovich M. et al. (2002).

The antioxidant and anti-inflammatory activity properties of OMWW have been measured by Visioli F. et al. (1999). OMWW obtained by employing a benchtop mill were fractionated by liquid-solid extraction and further processed to yield three extracts. Extract 1 was obtained by fractionation of lyophilized OMWW on a chromatographic column filled with DUOLITE® XAD 1180 resin particles and elution with ethanol. Extract 2 was obtained by ethyl acetate extraction of hexane-washed OMWW. Extract 3 was obtained following a fractionation of extract 2 on a Sephadex LH-20 column. Multiple antioxidant assays (LDL oxidation, DPPH radical scavenging activity, superoxide anion scavenging, and protection of catalase against hypochlorous acid) and an anti-inflammatory activity assay (leukotriene B4 production by human neutrophils) were performed. Extract 1 contained a complex mixture of phenolics including many polymers responsible for a high background absorption at 254 nm and exhibited low antioxidant activity and no anti-inflammatory activity. Extract 2 contained mainly low and medium molecular weight phenolics with elenolic acid as the principal constituent and showed good antioxidant and excellent anti-inflammatory activities. Extract 3 comprised hydroxytyrosol, tyrosol, and the unidentified derivative of the former and exhibited the most potent antioxidant activity and reasonable anti-inflammatory activity. The authors suggested that the extracts acted mainly as metal chelators and also had a potent free radical scavenging activity — see also Chapter 10: "Uses", section "Antioxidants".

Amro B. et al. (2002) investigated the antioxidative activity of different butanol extract fractions of olive cake. The residue left after evaporation of the ethanolic extract was dissolved in water and sequentially extracted with hexane, chloroform, and butanol. The butanol extract was fractionated in a silica gel column and nine fractions were collected. The fractions were examined using various measures of antioxidant activity [iron(III) reduction; inhibition of oxidation in refined soyabean oil; DPPH radical scavenging] and, consistent with previous studies, the antioxidant activity varied according to the test method. The first four fractions showed marked antioxidative activity in comparison with BHT(butylhydroxy toluene). Fractions tested also showed good hydrogen donating abilities, indicating that they had effective activities as radical scavengers.

Chemiluminescence is an alternative detection technique used for the determination of antioxidant activity, having the advantages of low detection limits, wide linear dynamic ranges, and speed of response. Luminol and lucigenin have been widely used for the determination of reactive oxygen species in a variety of biological systems and have been used indirectly to evaluate antioxidant activities. The chemiluminescene reactions provide a more rapid approach for measuring antioxidant activities when compared with standard methods (Atanassova D. et al., 2005a).

Atanassova D. et al. (2005a) described a rapid, simple and sensitive procedure for estimating the total phenolic/antioxidant levels of OMWW and 2POMW

samples, using Co(II)ethylenediaminetetracetic acid (EDTA)-induced luminol chemiluminescence. A fair linear relationship was observed between the total phenolic content (measured by the classic Folin-Ciocalteu test and expressed as caffeic acid) and the antioxidant activity (measured by the luminol Co(II)/EDTA-enhanced chemiluminescence technique) for both samples.

Using thermogravimetric analysis (TGA), it is possible to estimate oil resistance to oxidation, by measuring weight gain percent due to reaction of a sample with oxygen during oxidation, and initial and final oxidation temperature.

Identification of Bacteria

Isolated bacteria can be identified using (Jones C.E. et al., 2000; EU project: FAIR-CT96-1420 "IMPROLIVE"):

• Standard microbiological tests;

• Biochemical growth differences (API);

• Polar lipid composition;

• Fatty acid composition;

• Molecular biological analyses;

• PCR-based 16S rRNA sequence analysis;

• Restriction fragment length polymorphism (RFLP);

• Single-stranded conformational polymorphism (SSCP).

Animal Feed Analysis

Two main types of laboratory analysis of nutritive value of feeds are used:

• Chemical evaluation;

• Near infrared reflectance (NIR);

Weende System

After water is eliminated, feed is divided into five chemically defined components:

1. Crude fiber (CF), which approximates structural carbohydrate content.

2. Crude protein (CP) (=Nx6.25), which approximates true protein content.

3. Ash, which approximates mineral content.

4. Ether extract (EE), which approximates lipid content.

5. Nitrogen-free extract (NEE), which approximates non-structural carbohydrate content. This is estimated by difference between total dry matter and the sum of the other four chemical components.

Detergent System (van Soest)

Extraction with neutral detergent recovers major plant cell wall components (cellulose, hemicellulose, lignin) and removes all other organic constituents.

Extraction of residue with strong acid detergent recovers cellulose; lignin and lignin-N-complexes and removes hemicellulose and fiber-bound protein.

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