Extraction of Starches From Wheat and Corn

1.3.2. Starch Extraction from Wheat

• 1.3.2.1. Alkaline Steeping Method for Starch Extraction

The method (McDonald & Stark, 1988) was modified for extraction of wheat starch. Kernels were cracked, steeped for 18 hours at 4°C, and neutralized with 0.02M NaOH. The aqueous solution was drained and centrifuged for solids recovery. The solid material was rubbed in pestle and mortar and sieved through 125μm screen. The remaining residue was homogenized in blender and filtration was done. The filtered starch layer then centrifuged and purified with a toluene shaking procedure as described earlier (McDonald & Stark, 1988) using 5 volume of NaCl and 1 volume of toluene. Then the pallet was washed with distilled water and centrifuged. The process was repeated until brown layer disappeared. Then the starch was washed with acetone to remove water (Raeker, Gaines, Finney, & Donelson, 2017).

• 1.3.2.2. Traditional Method for Starch Extraction

According to traditional method wheat flour was mixed with distilled water and blended followed by centrifugation.  The supernatant and brown protein layer was removed carefully. Then washing was done with distilled water. The process was repeated until brown layer disappeared. The starch pallet was re suspended in deionized water and passed through sieve to remove all the insoluble material and washed with ethyl alcohol then dried to get the starch powder (Kim & Huber, 2008).

• 1.3.2.3. Acid Steeping for Starch Extraction

In another method flour was suspended in hydrochloric acid by adding sodium metabilsulfite and thiomersal and pH was adjusted to 7.6. The protease solution was added to the starch slurry and incubated while stirring. Washing was done with cesium chloride and centrifuged. The starch pallet was re-suspended in deionized water and pass through sieve to remove all the insoluble material and wash with ethyl alcohol then dry to make the starch powder (Kim & Huber, 2008).

Another method was reported for extraction of starch directly from grains by soaking in sodium azide containing de ionized water at 4 ^°C for 16 hours. The grains were softened in pestle and mortar. The slurry was filtered through four layers of cheese cloth to remove fibrous material. Centrifugation was done to separate the starch layer. Supernatant was discarded. By re-suspending the pallet in cesium chloride it was again centrifuged and washed with deionized water. Starch pallet was washed with deionized water and ethanol until all the residues removed. Then it was dried to form powdered starch (Peng, Gao, Båga, Hucl, & Chibbar, 2000).

1.4. Applications of Starch

The industrial application of starch is governed by its texture, viscosity, gel formation, product homogeneity, film formation, moisture retention, binding, and adhesion. It is the most important ingredient of soups, sauces, gravies, bakery products, dairy, confectionary, snacks, batter, coatings and meat products (Ba et al., 2013). The use of starch is also getting popular in pharmaceuticals, textiles, alcohol based fuels, adhesives, low calorie substitutes, biodegradable packaging materials as well as thin films and plastic materials (B. Kaur, Ariffin, Bhat, & Karim, 2012).

1.5. Uses of Starch in Food Industry

Starch and its value added products (dextrins and syrups etc) are widely used in food industry. It provides many benefits in bakery, confectionery, extruded products, ice cream, mayonnaise, preserves and baby foods. It acts as thickening agent, increase stability, prolong shelf life and improves texture of these products. Low caloric and high fiber food is the growing necessity of busy man therefore, starch and its products are good choice and hence their use is towards rise (Kraak, 1992).

1.6. Starch Modification

There is abundance of native starch and its production is economical but their limited functional properties are limited that make them unsuitable for modern food and industrial processes. So there is a need to find new better ways of starch production with desired functional properties. Two major starches wheat and corn are improving the rheological, performance and storage characteristics in food and non-food systems. The starch is rarely consumed in its native form; this form is also not commonly used in industry because native starches have restricted solubility in water, which limits industrial applications (BeMiller & Whistler, 2009). There are some disadvantages of native starches as they are insoluble in cold water, loss of thickening and viscosity after cooking. The shortcomings are easily be removed by their modification (Ashogbon & Akintayo, 2014).

The native starch is modified to diminish its negative attributes or to enhance the acceptability of starch in industry. The modified starches are environment and consumer friendly. They are modified by physical, chemical and enzymatic methods. Physical modification is the easiest and safest because it doesn’t need any chemicals or biological agents. Chemical modification include the addition of functional group in the starches such as derivatization (etherification, esterification, crosslinking, oxidation, cationzation and grafting of starch) to alter the composition and properties. Enzymatic modification is done by hydrolyzing enzymes for the production of glucose syrup and high fructose corn syrup (Bemiller, 1997).

1.7. Amylolysis

Amylolysis means hydrolysis of starch. In 1811, the German scientist Kirchhoff devised a method by using diluted acid to hydrolyze starch into syrup (Van Der Maarel, Van Der Veen, Uitelehaag, Leemhuis, & Dijkhuizan, 2002). Acids such as HCl and H₂SO₄ were used to prepare soluble starch for many years (Hoover, 2000). Acid hydrolysis decreases the viscosity of starches (Kim & Ahn, 1996) as well as their amylose content as compared to native starches (Wang, Truong, & Wang, 2003). But acid hydrolysis of starch produces some unwanted products and undesirable characteristics in maltodextrins (Moore, Cante, Amanto, & Soldi, 2005).

Enzymatic hydrolysis of starch for the production of sweeteners and syrups, at commercial level, was started in 1921 when Newkirk discovered a new process. The acid hydrolysis of starch was then replaced with the use of amylolytic enzymes to yield maltose syrup, glucose syrup, maltodextrin derivatives and cyclodextrins (Van Der Maarel, Van Der Veen, Uitelehaag, Leemhuis, & Dijkhuizan, 2002). These sweeteners are very useful in food industry therefore; the use of enzymes to obtain sugars from agricultural produce is ever increasing (Crabb & shetty, 1999). 

1.8. Amylolytic Enzymes

A variety of different enzymes are used for the production of sugars from starches. There are four basic groups of amylolytic enzymes i.e. endoamylases, exoamylases, debranching enzymes and transferases.

• 1.8.1. Endo-amylases

Endo-amylases cleave α-(1 → 4) glycosidic bonds present in the inner part of amylose and amylopectin chain and found in a variety of microorganisms belonging to archea as well as bacteria. The end products obtained are oligosaccharides (Van Der Maarel, Van Der Veen, Uitelehaag, Leemhuis, & Dijkhuizan, 2002). Alpha amylase is an example of endo-amylases. The most widely used commercially available α-amylase is derived from Bacillus licheniformis. The drawback of this enzyme is that it requires high level of calcium and pH above 6.3. Since the starches have the pH of 3.2-3.6 therefore extra expenses occur to adjust pH and meet calcium requirment (Crabb & shetty, 1999).

• 1.8.2. Exo-amylases

Exo-amylases act on the non reducing end of the polysaccharides and release successive glucose and maltose residues from the termini.  These enzymes can cleave both α-(1 → 4) glycosidic bonds and α-(1 → 6) glycosidic bonds as in case of amyloglucosidase or glucoamylase or they can only cleave α-(1 → 4) glycosidic bonds e.g., β amylases. (Van Der Maarel, Van Der Veen, Uitelehaag, Leemhuis, & Dijkhuizan, 2002). Unfortunately, these enzymes are not stable at high temperature (Crabb & shetty, 1999).

• 1.8.3. Debranching enzymes

This group of enzymes can hydrolyze α-(1 → 6) glycosidic bonds i.e. isoamylases and pullulanases. Pullulanase hydrolyze α-(1 → 6) glycosidic bond in pullulan and amylopectin while isoamylase hydrolyze α-(1 → 6) glycosidic bond in amylopectin. The difference in them is the ability to hydrolyze pullulan which isoamylase cannot do (Van Der Maarel, Van Der Veen, Uitelehaag, Leemhuis, & Dijkhuizan, 2002).

1.8.4. Tranferases

Tranferases cleave α-(1 → 4) glycosidic bond of the donor molecule and transfer part of the donor to gycosidic acceptor with the formation of a new glycosidic bond i.e. amylomaltase. Amylomaltases are found in the microorganisms which utilize maltose and degrade glycogen (Van Der Maarel, Van Der Veen, Uitelehaag, Leemhuis, & Dijkhuizan, 2002). The general classification of amylolytic enzymes is given in Figure1.4. (Crabb & shetty, 1999).

There has always been a need to explore and discover new enzymes for starch hydrolysis which are thermostable, energy efficient and cost effective. In this regard Ahmad and coworkers (2014) discovered a novel pullulanase from Thermococcus kodakarensis (TK-PUL) which is highly thermostable, work in the absence of Ca⁺² and convert starches into sugars in single step unlike other enzymes that complete the task in two steps i.e. liquefaction and saccharification (Ahmad, Rashid, Haider, Akram, & Akhtar, 2014).

Figure 1.4: General classification of amylolytic enzyme.

1.9. Pullulanase from Thermococcus kodakarensis (TK-PUL)

Pullulan is the polymer of maltotriose linked via α-1,6 glycosidic linkage and some of α-1,4 glycosidic linkages (Duan, Chen, & Wu, 2013). Pullulanases (E.C. 3.2.1.41) are the enzymes which attack on pullulan and hydrolyze it into smaller saccharides. The reported pullulanases are of five types (Hii, Tan, Ling, & Ariff, 2012).

Pullulanase from Thermococcus kodakarensis (TK-PUL) is a novel thermo-acidophilic type III pullulan hydrolase that can efficiently hydrolyze starch in the absence of any metal ions under industrial conditions. This enzyme can liquefy and saccharify starch in one step for the production of maltose syrup (Ahmad, Rashid, Haider, Akram, & Akhtar, 2014).

1.10. Value Added Products of Amylolyis for Food Industry

Starch has many applications in making value added products in food industry. These products are either made by acid or enzymatic hydrolysis. Few of the products obtained are glucose syrup, maltose syrup, high protein flour, maltodextrins, high maltose syrup, corn syrup, cyclodextrins, high fructose corn syrup, sweeteners etc. These products are used in different food products i.e. confectionery, soft drinks, meat, packed products, ice creams, sauces, baby foods, canned fruits and preserves (Synowiecki, Polaina, & Mac, 2007).

Maltose syrup is purified and concentrated starch hydrolysate that has great applications in food and pharmaceutical industry (Govindasamy, Campanella, & Oates, 1995). Maltose syrup yields 40-90% pure maltose (Lin et al., 2013). Maltose syrup is directly used in bakery, confectionery and brewery industry because of its functional properties e.g. low hygroscopicity, resistance to crystallization, low sweetness, reduced browning capacity and good heat stability (Gaouar, Zakhia, Aymard, & Rios, 1998). Pure maltose, obtained by crystallizing maltose from maltose syrup is employed in the pharmaceutical industry for the manufacture of antibiotics, vaccines and maltitol as well as in peritoneal dialysis as an osmotic agent (Lin et al., 2013).

The starches of corn, wheat and potatoes are used to make glucose syrup. It provides economic benefits and functional properties. It is widely used in industry as a sweetener (Hull, 2010). Glucose syrup is widely used as yeast fermentable and non-yeast fermentable source. One of its most important reactions is Millard reaction for providing color and flavor in baking industry. Glucose syrup is known for the production of polydextrose with low caloric value. Polydextrose is non-sweet and contain a small amount of sorbitol and citric acid. It is used in reducing calories from fat or sugar including preparation of (low caloric or light foods) dairy drinks, yogurt, sauces and dressings (Fuisz, Pyne, & Sekula, 1994). Glucose syrup is also used in the production of sorbitol at elevated temperature with hydrogen under pressure and used as a cryoprotectant for meat (Guzmán‐Maldonado, Paredes‐López, & Biliaderis, 1995).

The usefulness and properties of wheat and corn starches are abundant therefore the current study revolves around the utilization of wasted wheat and corn for starch extraction and value addition of starch by amylolysis to make syrups. There are numerous causes of wastage of commodities. The most common cause is the qualitative, quantitative and economic losses each year all over the world (Ahmedani et al., 2011) by inadequate post harvest handling (Tefera, 2012). The roots of post harvest losses include storage-insect pests, improper storage practices for grains, post harvest handling, weather conditions at harvesting and storage length (Gitonga, De Groote, Kassie, & Tefera, 2013). Therefore, isolation of starch from wasted materials (wheat and corn) and subsequent production of syrups (from isolated starch) is a great step towards the utilization of low grade, non marketable wheat and corn. The present study was therefore planned to devise cost effective strategy for utilization of nonmarketable wheat and corn by isolating starch and amylolysis with the help of novel enzyme TK-PUL.

Objectives

The main objectives of the study were as follows;

• Extraction of starches from wheat and corn
• Physicochemical analysis of raw materials and fractions obtained during starch extraction
• Amylolysis of various fractions of wheat and corn

CHAPTER 2

Materials and Methods

The work was based on the extraction of starches from corn and wheat. Physicochemical analysis of various fractions obtained during starch extraction was also done and finally these fractions were hydrolyzed enzymatically. The details are given in this chapter.

2.1. Chemicals and Reagents

Corn and wheat grain samples were procured from a local village. Themro-acidophilic pullunase type III from Thermococcus kodakarensis KOD1 (TK-PUL) was provided by Doctor. All other chemicals and reagents used in this study were of high purity. These were purchased either from Fluka (Buchs, Switzerland) or Merck (Darmstadt, Germany), or Fisher Scientific (Leicestershire, UK). Maltose was from Sigma (Taukkirchen, Germany).

2.2. Proximate Analysis

Proximate analysis was performed according to the official methods of AACC (AACC, 2000). All determinations were performed in triplicate and average values were considered. Samples to be analyzed were grains, residues obtained during starch isolation and powdered starch.

• 2.2.1. Moisture Content

The moisture content of the samples was measured according to the procedure given in method No.44-15A (AACC, 2000). It was performed by taking 5-6 g of grinded and homogenized sample which was placed in already weighed pre-dried petri plate and dried in an oven at 100 ± 5 °C, over 24 h, 2hr, 1hr intervals, respectively till constant weight. The moisture percentage was calculated by using the following formula:

• 2.2.2. Fat Content

Moisture free sample was taken for analysis of crude fat following AACC method No.30-25 using Soxhlet apparatus (AACC, 2000). For this, moisture free sample (4-5g) was taken in paper thimble and extracted with n-hexane at a condensation rate of 2-3 drops/sec for 3-4 hours. Then the thimble was removed from Soxhlet extractor and solvent was evaporated till dryness in oven at 70 ± 5 °C.  Thimble was weighed again and crude fat was calculated using following formula;

• 2.2.3. Crude Fibre

The crude fibre content was determined by following AACC method No 32-10 (AACC, 2000). Defatted moisture free sample (3-5 g) was taken in flask containing 0.255 N H₂SO₄ (200 mL) and boiled for 30 minutes. Any loss because of evaporation was compensated by adding hot distilled water to adjust constant volume. Hot material was filtered through linen cloth followed by 2-3 washings with hot water till acid free residue was obtained. Residue was collected from linen cloth, transferred into a flask containing 0.313 N NaOH (200 mL) and boiled for 30 minutes. The contents were again filtered through linen cloth. Residue was washed thoroughly with distilled water and ethyl alcohol till it became alkali free. The content was dried at 100 ± 5 °C till constant weight in oven. Cooled in desiccator and weighed (W1) then transferred to muffle furnace at 550 ºC for 4 hours till grey ash was obtained. Crucible was then cooled in desiccator and weighed (W2). The loss in weight was considered as crude fibre. 

• 2.2.4. Ash Content

The ash content of samples was determined according to the method 08-01 (AACC, 2000). Oven dried sample (2 g) was ignited over flame till it became smokeless in a pre-weighed crucible. The crucible was placed in muffle furnace for 6 hours at 550 ± 5°C till the sample become greyish white residue. The sample was cooled in a desiccator and weighed. The ash percentage in the sample was calculated as under;

• 2.2.5. Crude Protein

The percentage of nitrogen content in each sample was determined by Kjeldahl’s method as described in AACC method No. 46-10 (AACC, 2000). The nitrogen content was converted to crude protein by applying formula.

Moisture free homogenized sample (2-3 g) and 5g digestion mixture (containing copper sulfate, ferric sulfate, potassium sulfate; 4:1:94) were taken in micro Kjeldahl digestion flask. Added 30mL sulfuric acid and digestion was allowed for 5-6 hours till light green color appeared. The volume of digested sample was then made up to 100 mL. Diluted sample (10 mL) was taken in micro kjeldahl distillation unit and distilled in the presence of 10 mL NaOH solution (50% w/v). Ammonia gas thus generated was trapped in boric acid solution (4% w/v) containing 2-3 drops of methyl red indicator. The distillate was then titrated with 0.1 N sulfuric acid. Pink color was the indication of end point. The protein content was determined by the following formula;

*The nitrogen content was multiplied with 6.25 for corn and 5.78 for wheat to calculate crude protein content.

• 2.2.6. Nitrogen Free Extract (NFE)

Nitrogen free extract was calculated by the following formula;

2.3. Determination of Reducing Sugars

Reducing sugars were determined by the method of Bernfeld 1955.

• 2.3.1. Preparation of 3, 5- Dinitro Salisylic Acid (DNS) Reagent

For analysis of reducing sugars DNS reagent was prepared by dissolving DNS (1g) in 20 mL of 2M NaOH and added 15 mL distilled water. While heating (avoided boiling) sodium potassium tartrate (30g) was added pinch by pinch. After cooling the volume was made up to 100 mL. The solution was filled in amber color bottle and stored at cool dark place until used.

• 2.3.2. Standard Curve for Reducing Sugars

In order to determine of reducing sugars maltose was used as standard. Stock solution of maltose (10 mM) was prepared by dissolving 0.035g of maltose in 1mL distilled water. Various dilutions of maltose stock solution were then made. Aliquots (250 μL) from different dilutions of maltose were then mixed with equal volumes (250 μL) of DNS reagent in eppendorf tubes and incubated in boiling water for 5 minutes. After cooling at room temperature dilution was done with 1 mL distilled water and absorbance was measured at 540 nm wavelength. Absorbances were plotted against their respective concentrations (µmoles/mL) to get standard curve.

• 2.3.3. Determination of Reducing Sugars

Sample (1mL) from various fractions obtained during starch extraction was taken in eppendorf tubes and centrifuged at 13000 rpm for 5 minutes. Pallets were discarded and supernatants (250μL) were mixed with DNS reagent (250μL) in new eppendorf tubes. The color was developed by incubation in boiling water for 5 minutes. After cooling down to room temperature, distilled water (1mL) was added and absorbance was measured at 540 nm.

2.4. Determination of Amylose and Amylopectin Content

• 2.4.1. Amylose and Amylopectin Standard Curve

Amylose and amylopectin content of extracted starches was determined by the method of Hoover and Ratnayake described in Current Protocols in Food Analytical Chemistry (Hoover and Ratnayake, 2001). Amylose and amylopectin (SIGMA) were used as standard. Amylose and amylopectin solutions of various concentrations (i.e. 0:100, 10:90, 20:80, 30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:10, 100:0) were reacted with iodine solution (0.0025 M I₂/0.0065 M KI ) and optical density of the resultant blue complex was determined at 600 nm.

• 2.4.2. Amylose and Amylopectin Content

Defatted moisture free sample (20 mg) was taken in 2 mL of 90 % DMSO (90 mL DMSO: 10 mL distilled water), incubated at 85 °C for 15 minutes and cooled down to room temperature. Volume was made up to 10 mL in volumetric flask. Diluted solution (0.1 mL) was taken in a test tube, added 4.4 mL distilled water, 0.5 mL 10% diluted iodine solution (0.0025 M I₂/0.0065 M KI ) and volume was made up to 25 mL in volumetric flask. Stay time of 15 minutes was given at room temperature and absorbance was measured at 600 nm.

2.5. Qualitative Test for Protein Estimation

Ninhydrin solution (0.1% w/v) was prepared (0.1 g ninhydrin in 100 mL distilled water). Sample (3 mL) was taken with 3 mL 0.1% ninhydrin solution in test tube and incubated at 100 °C for 2 minutes. The development of blue color indicated the presence of protein. The color development was because of the fact that ninhydrin reacts with the primary or secondary amines present in proteins.

2.6. Starch Extraction From Wheat and Corn

Starch extraction was done following the method of (Vasanthan, 2001). Extraction of starch was carried by taking 5 kg of each wheat and corn. Washed and soaked the samples in water for 18 hours. The material was grounded and blended with water. Ice cold water was used to avoid heat generation and preservative (Sodium metabisulphite) was added (0.05% w/v) to avoid contamination and discoloration due to enzymatic activity. Mixture was filtered using a muslin cloth with several washes until the washed water became clear. The starch milk was allowed to sediment and decant was drained out.  Obtained starch slurry was centrifuged in falcon tubes for further separation of fractions like wet crude starch, brown layer (proteins, fat and other solids) and supernatant. Centrifugation was done at 6500 rpm, for 20 min at 4 °C. Purification of crude starch was done by washing it with 0.05 N NaOH. Centrifuged the crude starch containing 0.05 N NaOH solution at 6500 rpm, 4 °C for 20 min in falcon tubes. After centrifugation discarded the supernatant and the washed pure starch was air dried on filter paper and blotting paper at room temperature. Pure starch was then oven dried at 35 ± 5 °C. The temperature should not exceed 40 °C because high temperature causes disruption of starch granules. The dried starch was then ground to powder using pestle and mortar.

Purified dried starch was stored in air tight container for further use. The flow diagram for extraction of wheat and corn starches is given in Figures 2.1 and 2.2.

Figure 2.1: Flow Diagram For Extraction

Figure 2.2: Flow diagram for the extraction of corn starch.

2.7. Amylolysis of Wheat and Corn

The extracted starches (corn and wheat), fibrous residues (wheat and corn), whole wheat and corn were hydrolyzed with TK-PUL for 26 hours. Samples were prepared with 30% (w/v) substrate concentration in case of starches (wheat and corn) and powders (of whole wheat and corn). The substrate concentration in case of fibrous residues was set to 20% (w/v). Samples were suspended in 50 mM citrate buffer (pH 4.2) and TK-PUL (0.3 g) was added. The mixtures were placed in water bath at 90 °C. Sampling was done at every 30 minutes interval till 180 minutes then the samples were taken after every 2 hours till 18^th hour and then after every 4 hours till 26^th hours. The samples were collected in pre-weighed eppendorfs and these collected samples were diluted to 30 times. Vortex and spin the tubes at 13000 rpm for 1 minute. After spinning 250 μL supernatant was collected and 250μL DNS was added in the eppendorf tubes. Heated the samples for 5 minutes in boiling water and cooled at room temperature. Before taking the OD’s added 1 ml distilled water in each. OD’s were taken at 540 nm.

CHAPTER 3

Results and Discussions

3.1. Proximate Analysis of Whole Wheat and Corn

Proximate compositions of ground corn and wheat samples (used in this study) are presented in Table 3.1. The results were in close agreement with the previous reports on the proximate compositions of wheat and corn. Dziedzic and Kearsley (2012) reported 14% moisture content for wheat and 16% in corn. Fat percentage was 2% (in wheat) and 4% (in corn), fibre content was 3.5% (in wheat) and 2% (in corn) (Dziedzic & Kearsley, 2012).  Fistes and coworkers reported 1.57% ash in wheat (Fistes et al., 2014). Ash content for corn ranged between 1.10-2.95% (Enyisi, Umoh, Whong, Alabi, & Abdullahi, 2014).

Table 3.1: Proximate analysis whole wheat and corn used as raw materials for starch extraction.

Source	Moisture (%)	Fat (%)	Fibre (%)	Ash (%)	Protein (%)	NFE (%)
Wheat	13.78 ± 0.48	1.07 ± 0.21	3.15 ± 0.20	1.33 ± 0.23	11.42 ± 0.51	69.25*
Corn	14.24 ± 0.24	3.25 ± 0.29	2.08 ± 0.71	2.2 ± 0.88	8.31 ± 0.19	69.92*

* Since Nitrogen Free Extract (NFE) was calculated from the mean values of other  fractions therefore standard deviation for its values was not calculated

3.2. Starch Extraction

The starch from wheat and corn was extracted as described in Materials and Methods (section 2.6). Typical range for yield starch from wheat is 45-60% (Van Der Borght, Goesaert, Veraverbeke, & Delcour, 2005). The yields obtained during our studies was 32.3% for wheat  and 53.8% for corn. The yield of corn starch was in line with the figures reported by others who obtained the extraction yield between 43-64% (Paraginski et al., 2014; Malumba et al., 2009).

Table 3.2: Starch yield and fractions obtained during starch extraction.

Fractions	Wheat	Corn
Raw material	1kg	1kg
Residue (pulp)	239 g	344 g
Filtrate, slurry	2000ml	3000ml
Starch milk	455ml	850ml
Decant	1545ml	2150 ml
Supernatant	40ml	60 ml
Brown layer	30ml	45 ml
NaOH wash	20ml	35ml
Powdered Starch	323g	538g
Yield	32.3%	53.8%

3.3. Physico-chemical Analysis of Various Fractions Obtained During Starch Extraction

In order to make the process cost effective there was need to devise a strategy for extraction of valuables as much as possible. Fractions obtained at various steps were therefore analyzed for suspected components in them.

3.3.1. Proximate analysis of brown layers obtained during starch extraction

When starch milk was centrifuged to sediment crude starch the whole material was separated into three layers. Crude starch was collected in the bottom layer. Soluble solids were collected in the uppermost layer contained. The middle layer includes protein, fibre, mucilage’s and other insolubles. Since this layer can be seen as dark/ brown in color, therefore may be referred as brown layer (Figure 3.1).

Figure 3.1: Sedimentation of crude starch during centrifugation of starch milk (adapted from (Vasanthan, 2001).

Proximate analysis of brown layers obtained during our studies is shown in Table 3.5.

Table 3.3: Proximate analysis of brown layers collected during starch extraction from wheat and corn.

Source of Brown Layer	Moisture (%)	Fat (%)	Fibre (%)	Ash (%)	Protein (%)	NFE (%)
Wheat	13.29 ± 0.62	1.39 ± 0.15	2.92 ± 0.37	1.76 ± 0.31	3.76 ± 0.50	64.88*
Corn	9.84 ± 0.58	2.85 ± 0.18	3.98 ± 0.01	1.59 ± 0.17	4.23 ± 0.57	67.51*

* Since Nitrogen Free Extract (NFE) was calculated from the mean values of other  fractions therefore standard deviation for its values was not calculated

3.3.2. Reducing Sugars in Discarded Fractions

Liquid fractions obtained at various steps (filtrate, supernatant and NaOH wash) were analyzed for reducing sugars that represent damaged starch, dextrins and other saccharides washed with them. The concentration of reducing sugars was determined by DNS method as described in Materials and Methods (section 2.3). Maltose was used as standard and standard curve was made by plotting concentrations of maltose solutions against respective ODs (of maltose-DNS complex) at 540 nm.

Figure 3.2: Standard curve of maltose.

Reducing sugar content of various fractions is presented in Table 3.6. It was observed that bulk of sugars were washed with decant during the intial step of sedimentation.

Table 3.4: Reducing sugars of discarded fractions

Fractions	Volume (mL)	Reducing sugars* (μ moles /mL)	Total reducing sugars* (μ moles)
Wheat  filtrate	2000	8.04	16080.24
Wheat supernatant	40	8.12	324.93
Wheat NaOH wash	20	3.94	78.89
Corn filtrate	3000	2.18	6553.04
Corn supernatant	60	3.22	193.30
Corn NaOH wash	35	3.91	137.05

* Maltose was used as standard for reducing sugars.

3.4. Qualitative Estimation of Protein

In order to determine the fate of proteins during starch extraction qualitative estimation of proteins was done by ninhydrin (Kaiser, Colescott, Bossinger, & Cook, 1970).

Fractions obtained at various steps were analyzed for presence of proteins as described in Materials and Methods (section 2.4).

Figure 3.3: Qualitative estimation of proteins in various fractions obtained during starch extraction from wheat. WD: wheat decant (the upper layer of water drained out during initial settling of starch); WB: wheat brown layer obtained during centrifugation; Control: Milk sample. The test was performed in triplicates.

In case of decant and brown layers of wheat a deep blue color was developed upon reaction with ninhydrin which indicated the presence of proteins. So, it was concluded that most of the proteins were washed with decant and brown layer during starch exraction.

Figure 3.4: Qualitative estimation of proteins in various fractions obtained during centrifugation of starch extracted from wheat. WS: Wheat Supernatant (Obtained during centrifugation); WN: Wheat NaOH Wash (Obtained during centrifugation when washed with 0.05N NaOH); Control: Milk Sample. The test was performed in triplicates.

The wheat  supernatant showed a very light blue color indicating that it contains only traces of protein in it, while no color was observed in NaOH wash. The results indicated that most of the proteins were washed at previous steps. i.e. decant, brown layer and supernatant.

Figure 3.5: Qualitative estimation of proteins in various fractions obtained during centrifugation of starch extracted from corn. CS: Corn Supernatant (Obtained during centrifugation) CN: Corn NaOH Wash (Obtained during centrifugation when washed with 0.05N NaOH). The test was performed in triplicates.

In case of corn supernatant and  NaOH wash no color was observed. The results indicated that most of the proteins were washed at previous steps of starch extraction i.e brown layer.

Figure 3.6: Qualitative estimation of proteins in various fractions obtained during starch extraction from corn. CD: Corn decant (The upper layer of water drained out during the settling of starch);  MB: Corn brown layer (obtained during centrifugation); Control: Milk Sample. The test was performed in triplicates.

The brown layer of corn showed a deep blue color upon reaction with ninhydrin while there was no color observed in corn decant. The results indicated that most of the proteins were washed with brown layer.

3.5. Physico-chemical Analysis of Major Fractions

• 3.5.1. Proximate analysis of Wheat Starch and Corn Starch

The results obtained for the proximate composition of extracted starches (Table 3.3) were in accordance with the findings of previous reports on proximate analysis of wheat and corn starches. Dziedzic and Kearsley (2012) reported 14% moisture content for wheat and 13% for corn. Fat percentage during their studies ranged between 0.8-1.2 (in wheat) and 0.6-0.8 (in corn) whereas ash content was 0.15% and 0.12% for wheat and corn, respectively (Dziedzic & Kearsley, 2012). Waterschoot and coworkers reported 0.2-0.3% protein in wheat and 0.4% protein in corn (Waterschoot, Gomand, Fierens, & Delcour, 2015).

Table 3.5: Proximate analysis of starches extracted starches from wheat and corn.

Starch Source	Moisture (%)	Fat (%)	Fibre (%)	Ash (%)	Protein (%)	NFE (%)
Wheat	12.06 ± 0.63	0.86 ± 0.12	1.05 ± 0.10	0.17 ± 0.12	0.4 ± 0.12	85.46*
Corn	13.0 ± 0.72	0.73 ± 0.14	1.595 ± 0.52	0.10 ± 0.08	0.4 ± 0.10	84.14*

* Since Nitrogen Free Extract (NFE) was calculated from the mean values of other  fractions therefore standard deviation for its values was not calculated

• 3.5.2. Proximate Analysis of Residues Obtained as by Product

Fibrous residues were obtained as by product during starch extraction from wheat and corn. Results obtained during proximate analysis of these residues are shown in Table 3.4.  Fibre content in wheat residues (15.53%) and corn residues (12%) was five to six times higher than the fibre contents of raw materials used (i.e. 3.15% in wheat and 2.08% in corn, Table 3.1).  NFE content provides an estimate of digestible carbohydrates including starch (Saura-Calixto, Canella, & Soler, 1983). NFE contents of both wheat and corn residues were significantly high. Their values were only slightly lower than the NFE contents of their respective raw materials used for starch extraction (Table 3.1).  From these findings it may be speculated that these fibrous residues still contain appreciable amounts of starch and other digestible carbohydrates.

Table 3.6: Proximate analysis of residues (fibrous material) collected during starch extraction from wheat and corn.

Source of Residue	Moisture (%)	Fat (%)	Fibre (%)	Ash (%)	Protein (%)	NFE (%)
Wheat	12.65 ± 0.51	1.02 ± 0.01	15.53 ±0.61	2.93 ± 0.32	2.56 ± 0.17	63.67*
Corn	12.95 ± 0.17	1.98 ± 0.24	12.00 ± 0.64	1.89 ± 0.74	3.06 ± 0.21	66.42*

* Since Nitrogen Free Extract (NFE) was calculated from the mean values of other fractions therefore standard deviation for its values was not calculated

 3.6. Determination of Amylose and Amylopectin Content in Extracted Starches

Properties of starches and their suitability for particular end uses depend upon their amylose and amylopectin ratios. Therefore amylose and amylopectin content in the extracted starches was determined as described in Materials and Methods (section 2.3). Amylose and amylopectin solutions of various concentration (i.e. 0:100, 10:90, 20:80,30:70, 40:60, 50:50, 60:40, 70:30, 80:20, 90:100 and 100:0) were reacted with iodine solution (Materials and Methods section 2.4.1.) and optical density of the resultant blue complex was determined at 600 nm. Standard curve for determination of amylose and amylopectin was drawn by plotting OD_600nm against respective concentrations (Figure 3.6)

Figure 3.7: Standard curve of amylose and amylopectin

 The amylose and amylopectin content of ground corn and wheat (used in this study) are presented in Table 3.6. The results were in close agreement with the previous reports on the amylose and amylopectin of wheat and corn. Schirmer (2013) reported 27.8% amylose for wheat and 22.7% in corn (Schirmer, Höchstötter, Jekle, Arendt, & Becker, 2013). Amylose was 25% (in wheat starch) and 29% (in corn) (Lin et al., 2013). In another study 29.1% amylose content for wheat starch was recorded (Singh, Singh, Kaur, Sodhi, & Gill, 2003; Jòzef, 2007).

Table 3.7: Amylose and amylopectin content of extracted starches.

Source	Wheat	Wheat starch	Wheat residue	Corn	Corn   starch	Corn residue
Amylose (%)	25.2	25.9	21.1	28.5	27.1	27.1
Amylopectin (%)	74.8	74.1	78.9	71.5	72.9	72.9

 3.7. Amylolysis of Fractions Obtained During Starch Extraction

The ultimate objective of the current study was to evaluate the potential of newly discovered thermostable pullulanase (TK-PUL) to hydrolyze crude and raw starches from wheat and corn. Since yields of extracted starches were lower than the reported values (Table 3.2) and NFE contents of the fibrous residues was almost equal to those of their respective raw materials (Table 3.6) therefore it was expected that appreciable amounts of starches remained entrapped in the fibrous residues. Keeping in view these facts fibrous residues were also utilized as substrates for hydrolysis with TK-PUL. The aim was to recover the valuables (dextrins and saccharides) from that entrapped starch. Powders of whole wheat and whole corn were also hydrolyzed with TK-PUL to explore its hydrolytic potential towards crude starch.

• 3.7.1. Amylolysis of Whole Wheat and Corn

The suspension was prepared by dispersing 30 g each of whole wheat and corn powders into 65 mL distilled water, pH was adjusted to 4.2 by 1M sodium citrate/citric acid and volume was made 100 mL. Partially purified TK-PUL (1mL ~ 0.3 mg) was then added and suspensions were incubated in water bath at 90 °C under occasional stirring. Samples were collected at regular intervals and reducing sugars were measured by DNS method (Bernfeld, 1955).  Results of hydrolysis are presented in Table 3.8 and Figure 3.7. It was observed that the hydrolysis rate of TK-PUL was higher towards corn than that of wheat. Final yield or reducing sugars in terms of maltose in corn hydrolyzates was 582.8 μmoles maltose/ml that was around four times higher than the final yield of wheat hydrolyzates (i.e. 155.5 μmoles maltose/ml).

Table 3.8: Reducing sugars released upon hydrolysis of whole wheat and corn with TK-PUL.

Time (Hours)	(μ moles maltose/mL)
	Corn	Wheat
0	6.4	1.9
0.5	13.3	15.4
1	19.5	16.7
1.5	20.6	19.1
2	42.4	23.9
3	49.2	26.9
4	62.7	28.6
6	88.1	30.4
8	89.3	51.8
10	149.8	59.5
12	307.7	59.9
14	493.9	112.0
18	534.0	159.1
22	560.0	144.5
26	582.8	155.5

Figure 3.8: Time course release of reducing sugars during hydrolysis of wheat (▲) and corn (●).

• 3.7.2. Amylolysis of Fibrous Residues of Wheat and Corn

For hydrolysis of fibrous residues, obtained during starch extraction from wheat and corn, suspensions (20% w/v) were made in distilled water (100 mL). pH was adjusted at 4.2 and hydrolysis was carried out at 90 °C in the presence of 0.3 mg TK-PUL. Samples were collected at regular intervals and analyzed for reducing sugars as described earlier. Results are presented in Table 3.9 and Figure 3.8. Corn residues were hydrolyzed at a greater extent than that of wheat. Keeping in view the successful hydrolysis of starch entrapped in fibrous residues as well as hydrolysis of whole wheat and corn (previous experiment) it can be concluded that TK-PUL has an efficient potential for hydrolysis of crude starches.

Table 3.9: Reducing sugars released upon amylolysis of residues (wheat and corn) with TK-PUL.

Time (Hours)	(μ moles maltose/mL)
	Corn	Wheat
0	5.4	5.0
0.5	10.1	9.7
1	15.8	12.2
1.5	30.0	23.0
2	26.8	25.9
3	35.1	31.3
4	73.2	31.4
6	61.8	32.4
8	157.9	64.1
10	167.3	69.4
12	183.6	70.3
14	186.1	83.8
18	200.0	123.4
22	206.2	141.1
26	231.5	155.4

Figure 3.9: Time course release of reducing sugars during hydrolysis of wheat residue (▲) and corn residue (●).

• 3.7.3. Application of TK-PUL in the Production of Maltose Syrups from Wheat and Corn Starch.

Maltose syrup is termed to a solution containing starch hydrolysates that include mixture of dextrins, oligosaccharides and smaller saccharides. However this syrup should contain a major proportion of maltose with respect to other saccharides (Ahmed, 2011). During our previous studies we had analyzed the potential of TK-PUL to synthesize maltose syrup from purified commercially obtained (from SIGMA) corn starch. In order to devise a cost effective process there was need to explore the hydrolytic potential of TK-PUL towards locally isolated raw starches.

For this purpose time course hydrolysis of wheat and corn starch extracted during current study was done with TK-PUL. It was performed by adding 1mL (~0.3 mg) of partially purified TK-PUL to 30% (w/v) starch suspension (100 mL) at pH 4.2 and 90 °C as described in Materials and Methods (section 2.7). Samples were collected at regular intervals and analyzed for reducing sugars by DNS method. Higher yield of reducing sugars (790.0 μmoles maltose/mL) observed in corn hydrolyzates as compared to the yield (161.4 μmoles maltose/mL) of wheat starch. It was therefore concluded that the corn starch was a preferred substrate of TK-PUL as compared to wheat starch.

Table 3.10: Reducing sugars released upon hydrolysis of starches (wheat and corn) with TK-PUL.

Time (Hours)	(μ moles maltose/mL)
	Corn starch	Wheat starch
0	10.2	6.1
0.5	17.4	14.0
1	18.2	17.7
1.5	20.5	22.4
2	27.0	24.6
3	69.0	27.1
4	75.2	31.0
6	117.4	35.8
8	157.7	57.8
10	336.8	61.8
12	392.0	89.7
14	553.8	105.2
18	554.9	152.2
22	771.2	160.7
26	790.0	161.4

Figure 3.10: Time course release of reducing sugars during hydrolysis of wheat starch (▲) and corn starch (●).

3.8. Conclusion

Keeping in view all the results it can be concluded that TK-PUL can efficiently hydrolyze raw and crude starches. Since it is a novel enzyme that hydrolyzes starches in a single step at extremely high temperature in the absence of any other enzyme (Ahmed, Rashid, Haider, & Akhtar, 2016) therefore, it can be speculated that the use of this enzyme in starch processing industry would be highly beneficial. Amylolysis of wasted corn and wheat can efficiently be done and resulting maltose syrup can serve as a feed stock for biotechnological synthesis of organic acids, vitamins, flavors and many more.

Future prospects

More research on amylolysis of wheat and corn is continued in our lab and future prospects are;

• Analysis of hydrolysis end products
• Preparation of high protein flours with the help of TK-PUL
• Use of starch hydrolyzates as feed stock for other biotechnological processes.

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