Utilization of Dates in Biotechnology .I
Production of Alginic Acid by Solid State Fermentation

Introduction

               Low quality date cultivars and low grades of dates are considered as suitable raw materials for a wide range of modern biotechnology production processes due to their unique characteristics which could be summarized below:

1. They are available in large quantities as by-products of dates packing processes.
2. They have the same composition of high quality cultivates and low grades of dates (i.e. high sugar content and low moisture content).
3. They are of much lower cost than the dates which are used in packing, paste production and date syrup production.
4. They are readily extractable by hot water.

                    Biotechnology has been known since the early days of industrial fermentations as the field which converts low cost and low grade agricultural products into valuable bio-products (e.g. antibiotics, enzymes, organic acids, bakery yeast and high protein feed). Although solid state fermentations (SSF) have been known since several thousand years in the Far East, their industrialization started to be visualized during the last third of the past century. These fermentations are conducted in media where no free water exists (i.e. all water is in the bound form). This feature is vitally important in the arid and semi-arid regions of the world (e.g. most Arab countries). The following characteristics gave SSF increasing economic significance over the submerged type fermentations:

1.  Lower investment cost (construction and equipment) due to fermenter size     reduction
2. Lower water consumption
3. Overall reduction of upstream and downstream processes size
4. Direct utilization of raw materials without pretreatment processes
5. Higher concentration of the targeted bio-product
6. Overall reduction of the operational cost (e.g. less homogenization, less Electricity)
7. The best technical and economic solution for shear-sensitive organisms and bio-products

What and Why is Alginic Acid?
         Alginic acid is a heteropolysaccharide composed of tow types of linear polysaccharide chains:
Poly-β-D-mannuronic acid (M-Blocks)
Poly-α-L-guluronic acid (G-Blocks) 
    The molecular size of this bio-polymer depends on the microbial source and production process where it could be between 105 to over 106 Dalton.
      In addition to the usual thickening effect of most polysaccharides, alginic acid exhibits a unique gelling (gel formation) process where its clear solution turn into transparent gel when calcium ions (e.g. calcium chloride) are added to the solution. The gel formation requires only few seconds to set due to a rapid and temperature independent ion exchange process. 
  Industrial Applications of Alginic Acid
    Approximately 50% of the industrial applications of alginic acid is in the food industry followed by the pharmaceuticals industry. The applications could be summarized as follows:

1. Gelling agent in cooked pies, pastry fillings, milk candies, gelatin candies   pharmaceutical products, formulated animal feed and children toys.
2. Stabilizing and thickening agent in salad dressings, sauces, foam stability in juices and milk shakes, particle suspender in fruit juices, paint and polish emulsions, detergents foam stabilizer and solids suspension in ceramics
3. Film formation in sausages and processed meat products
4. Water entrapment and prevention of ice crystals formation in frozen foods
5. Microbial cells entrapment (immobilized cells) for bioreactors in the continuous production processes of bio-products

Alginic Acid Sources

1. Marine brown algae from Phaeeophycae family (available in certain parts of  the world only)
2. Pseudomonas aeruginosa  (not approved for industrial production due to its high pathogenesity)
3. Azotobacter vinelandii (soil bacteria co-existing with plant roots capable of nitrogen fixation )

            Since alginic acid biosynthesis pathway (both types of building blocks) in A. vinelandii starts with fructose (glucose is isomerized enzymatically by the bacteria), dates represent excellent raw material for the production medium due to their high content of both sugars in a readily extractable form.
SSF Process of Alginic Acid (Upstream processes)
      The optimal production conditions are as summarized below:

1.  Production Medium 
   1. The production medium is composed of two major components
2. Solid part : wheat bran
   1. Wetting liquid : Date extract of 4% total soluble solids containing: 0.05% potassium monohydrogen phosphate and 075% commercial yeast with a final pH of 7.0

The two components are mixed in a ratio of 5 volumes of the wetting liquid with each 1 volume of the solid part. The mixture is heat sterilized inoculated with the bacteria and incubated at 28◦C for 6 days with occasional homogenization (4 times /day, 10 min periods)under low aeration rate.

1. Alginic Acid Recovery  (Downstream processes)

        The production medium is extracted with 5 volumes of warm water at 50◦C followed by liquid/solid separation (filtration or centrifugation). The solid matter represents rich supplement in feed and bio-fertilizers. The extracted alginic acid is purified by adding either ethanol or iso-propanol (3:1, v/v), separation of the precipitated alginic acid by centrifugation and drying by hot air stream (55-60◦C).

1. Basic Biochemical Characteristics of A. vinelandii

         The dried alginic acid was further purified with absolute ethanol, dissolved in warm water and subjected to ion-exchange chromatography (DEAE-cellulose column) and molecular sieving chromatography (Sephacryl-S-1000 column) where the following in comparison with the algal product. The summarized results are as follows:
Ion-Exchange Chromatography:
 - Both preparations have 4 alginic acid components (3 major and 1 minor) which reflects differences in charge density and charge distribution
-  The first two components in the bacterial preparation were 30-50% higher than those of the algal preparation. Since the charge density of alginic acid is a key property in its functional characteristics, the bacterial preparationwould have better quality.
Molecular Sieving Chromatography
- Both preparations showed one peak with a molecular weight exceeding 106 Dalton which indicates similar functional characteristics.

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