Amaranth Starch Isolation, Oxidation, Heat-Moisture Treatment and Application in Edible Film Formation

Starch was isolated from amaranth grains and subjected to modification treatments. Oxidation of isolated starch was done using sodium hypochlorite and heat-moisture treatment was done at 85°C for 6hr keeping the moisture content 30% during treatment. Native and modified starches of amaranth were used for preparation of edible films and different characteristics of films were evaluated. Both the modification treatments increased tensile strength of amaranth starch films. Heat moisture treatment increased water vapour permeability while oxidation had contrary effects on amaranth starch films. Water solubility of films of amaranth starches was reduced by modification treatments of starches. Heat moisture treatments increased yellowness of starch films.

. Starches are modified by different treatments to overcome the shortcomings of native starch and to enhance the suitability for specific application. Various modification techniques such as physical, chemical, enzymatic, and genetic, or a combination of treatments have been developed to alter the properties of starches. Alteration of properties including functional, mechanical and organoleptic characteristics of starch films is possible by addition of certain amounts of various chemicals in Filmogenic solution (Mali et al., 2004), however, it is not desirable to mix chemicals to starch film for the reason that they are edible. Therefore, to improve the quality of starch films, modification of starch itself is preferable. developed oxidised and heat-moisture treated potato starch Studies conducted on development of edible films using native and modified starches of different sources revealed significant changes in various properties of films (Zavareze et al., 2012;Fonseca et al., 2015;Biduski et al., 2017). Amaranth (Amarnathus spp.) is a dicotyledonous plant comes under the category of pseudocereals and grown in Himalayan area and few states of India. It is well-known for good nutritional quality of its leaves and grains. Amaranth can serve as excellent source of starch due to high starch content in grains. Extremely small size of granules with diameter ranging from 1.05 to 1.78µm amaranth starch has gained attention for applications (Sindhu and Khatkar, 2016). No previous studies have been reported on the production of edible film prepared from amaranth starches modified physically and/or chemically. Therefore, present investigation was aimed on development of edible films from amaranth starch and evaluation of effects of modification treatments including heat moisture treatment and oxidation of starch on film properties. grains were screened to remove foreign matter and stored in sealed container at room temperature. The flour was prepared by grinding seeds on laboratory mill and stored in polyethylene bags at 10º C.

Starch Isolation
Starch was isolated from amaranth grains according to the alkaline steeping method (Choi et al., 2000). Grains were steeped in 0.25% aqueous NaOH solution for 18 hr at room temperature and stirred three times during this period. After steeping, the grains were washed with distilled water and ground in a blender at full speed for 2 min, and slurry was filtered step wise through 100 mash (150µm), 270 mesh (53µm) and 400 mesh (38 µm) sieves. The starch was isolated from the filtrate by centrifugation at 25,000g for 20 min. The supernatant was discarded, and the top yellowish layer of protein was removed. This step was repeated to obtain a white starch layer. The starch layer was re-suspended in distilled water, shaken and centrifuged as described above. Thereafter, the isolated starch was dried in hot air oven at below 40ºC for 8 to 10 hr and stored at room temperature in sealed container.

Heat Moisture Treatment of Starch
The heat moisture treatment of amaranth starch was carried out according to the method of Franco et al. (1995) with minor modifications. The moisture level of starch was adjusted to 30% by adding appropriate volume of distilled water (the moisture level of native starch was predetermined).The addition of distilled water was done slowly and simultaneously mixed for uniform distribution of water in starch powder. Sample was sealed in polyethylene pouch and equilibrated at 10°C overnight. After the incubation, starch was filled in air tight glass container and heated for 6 hr at 85°C. The container was shaken occasionally for uniform distribution of heat. The sample was cooled to room temperature and dried at 40° C for 6 to 8 hr and equilibrated at room temperature for 4hr. The dried starch powder was sealed in polyethylene bag, labelled and stored at room temperature for further analysis.

Oxidation of Starch
Oxidation of isolated starch was done by following the method of Forssel et al. (1995). Starch sample was weighed 100g (db) and dispersed in 500ml distilled water. The pH of the suspension was adjusted to 9.5 with 2.0M NaOH. Sodium hypochlorite solution (4% active chlorine available) of volume 25ml was slowly added to the starch slurry over a period of 30min with constant stirring while maintaining the pH in the range from 9.0 to 9.5 with 1M H2SO4. The reaction was allowed for 10min after all the sodium hypochlorite has been added. The pH of slurry was adjusted to 7 with 1M H2SO4 and centrifuged at 4000rpm for 10 min. The starch cake obtained was washed 4 to 5 times with distilled water and dried at 40°C in hot air oven. The dried oxidized starch was ground and passed through 75µm sieve, packed in polyethylene bags, labelled and stored at room temperature for further analysis.

Carboxyl content of starch
The carboxyl content of the oxidized starch was determined by following the method described by Smith (1967) and employed by Parovuori et al. (1995). In 500 mg starch (db) sample, 30 ml of 0.1 M HCl was mixed at room temperature to acidifying the carboxyl groups of the samples and maintained under the magnetic stirring for 30 min. Subsequent to this, the starch was exhaustively washed and recovered by centrifugation (2,000g) until the pH raised to neutrality. This procedure was named demineralization by Smith (1967). The starch was dispersed in 300 ml of distilled water and heated at 98°C for 30 min under agitation for complete starch gelatinization. While still hot, the samples were titrated with 0.002 M NaOH solution until pH 8.3, using the phenolphthalein as indicator. Complete experiment was performed with native starch instead of oxidised starch and treated as blank. The carboxyl content was calculated using the following equation: wt. of sample (g) Where Vs is the volume of NaOH used for the sample (ml); Vb is the volume of NaOH used for the blank (ml); M is the molarity of NaOH.

Preparation of Starch Films
Starch films using native and modified starches of amaranth were prepared by following the method described by Chandla et al. (2017) with minor modifications. Filmogenic solutions were prepared by dispersion of 5g starch in 100 ml distilled water with continuous stirring at magnetic stirrer for 15min. Glycerol at rate of 3g/100g starch was added as plasticizer and mixed thoroughly. The solution was magnetically stirred for 15min at 85°C. The resulting solution was cooled at room temperature to avoid air bubbles during pouring. Casting technique was used to prepare films. The prepared solution was poured onto the polypropylene round trays of diameter 12.5cm and dried at 40°C for 16hr in hot air oven with circulating air in chamber. 2.6 Analysis of Films 2.6.1 Thickness The thickness of starch films was determined using Digital micrometer with an accuracy of ±0.001mm. The average value of 10 thickness measurement at different locations on each film was used in all calculations. 2.6.2 Moisture Content Moisture content of starch films was determined by drying the pre-weighed pieces of films at 110°C for 6 to  Gontard et al. (1994). Preweight piece of starch film was immersed in water at room temperature for 24hr. The immersed film piece was removed from water and dried in oven at 110°C for 4 to 5 hr, cooled and weighed. The water solubility of starch film was measured as the difference in weight of dried piece of film before and after immersion in water.

Color Parameters
Color of native and modified starch films was measured using CR-300 Chroma meter (Minolta, Japan). The system determines the L*, a* and b* values, where L* represents lightness and darkness; a* represents the opposition between green and red color ranging from positive (red) to negative (green) values; and b* is the yellow/blue opposition ranging from positive (yellow) to negative (blue) values. The average value of three measurements were calculated and used. 2.6.5 Water vapour permeability Water vapour permeability of starch films was determined by following the E96-95 ASTM standard method (ASTM, 1995). Each film sample was sealed over the circular opening of a permeation cell containing anhydrous CaCl2 (0% RH) and weighed. These cells were placed on desiccators with a saturated NaCl solution (75% RH) at 25°C. The weight of each permeation cell was recorded after 24hr and water vapour permeability of films was calculated using following formula-

WVP = ΔW × X t × A × ΔP
Where WAP is the water vapour permeability (g.mm/m2.day.kPa); ΔW is the weight gain by descant (g); X is the film thickness (mm); t is the incubation period (days); A is the area of the exposed film surface (m2); and ΔP is the difference of partial pressure (kPa). 2.6.6 Tensile Strength Tensile strength of films was determined by a tensile test based on ASTM D-882-91 method (ASTM, 1996) using texture analyser (TA-XT 2i Stable Micro Systems, UK). The films were cut in strips (20mm × 50mm) and thickness of strips was measured at eight points. The strip was gripped from both the edges of width on 'tensile grip' probe and initial grip separation was set at 30mm. The force and distance were recorded during extension of strips at 0.8mm/s up to break. The tensile strength of films was calculated using following formula-

TS = F A
Where TS is the tensile strength (MPa); F is the maximum force (N); A is the area of film cross-section (thickness × width; m2).

Statistical Analysis
Analytical determinations were done in triplicate, and Duncan test was conducted to examine significant differenced among experimental mean values. The statistical significance was observed at p< 0.05. Data were analyzed using Statistical Analysis System SAS, version 8.2 and SPSS software version 16.0 (SPSS Inc).

III. RESULTS AND DISCUSSION
Oxidized starch showed 0.099% carboxyl content which was higher than that of 0.052% reported by Fonseca et al.  2017) reported tensile strength ranged from 2.30 to 2.61MPa for films of amaranth starches of different cultivars. The discrepancy in mechanical strength of films prepared from amaranth starch in present study and previous reports might be due to the differences in concentration of starch used for film formation, thickness, film formation conditions like heating temperature and drying rate. Oxidised starch showed tensile strength value of 1.42MPa which was higher than that of native starch film samples. Hydrothermally modified starch showed the highest value (2.51MPa) of tensile strength of film among tested samples. It has been reported by Zavareze et al. (2012) that oxidation and heat moisture treatment of potato starch increased the tensile strength of the films from 3.53 to 5.25MPa and 3.53 to 6.07MPa, respectively. Increment in tensile strength of film following oxidation of potato starch with 1.0% active chlorine while decrement in the value of tensile strength of films was noticed for potato starches oxidised with 0.5 and 1.5% active chlorine (Fonseca et al., 2015). The mechanical properties of the starch films depend on various factors such as polymeric chain arrangement, molecular chain interactions, film thickness, quantity and type of the plasticizer, and relative humidity of the environment. Additional interaction among amylose and amylopectin molecules resulted from heat-moisture treatments could be the reason for increment of tensile strength of films. Zhang et al. (2009) suggested that carbonyl and carboxyl groups present in oxidised starch can form hydrogen bonds with amylose and amylopectin chains, and these bonds offer larger structural integrity in the polymer matrix, thus causing increased tensile strength of film. al. (2015) for oxidised potato starch films than that of native starch films, and increasing level of oxidation resulted in raising water barrier capacity of film during oxidation. On further oxidation, these carbonyl groups were transformed to carboxyl groups (hydrophilic) and hydrophilicity of oxidized starch increased significantly. Therefore, lower water vapour permeability of oxidized starch films in the present investigation could be attributed to the higher carbonyl groups (hydrophobic) in oxidized starch than native starch. In heat-moisture treatment, retrogradation in starch gel takes place due to interaction in amorphous region at initial stage followed by interaction in crystalline domains. As amaranth starch is waxy type, heat moisture treatment increased stiffness in the starch granules and caused lesser retrogradation (due to absence of amylose) resulted in loose packing of gelatinised granules offering space for mobility of water molecules, consequently more hydrophilic films formed with more water vapour permeability and stiffness. Thickness of film is an another important factor affecting water vapour permeability and higher thickness of films in case of heat moisture treated starches might be the reason for more water vapour permeability than native starch film. Linearly increasing water vapour permeability with increasing thickness and hydrophilicity of starch films was recorded for different starches in literature (Cuq et   IV. CONCLUSION Amaranth starch was found interesting material for film formation. Native and modified starches of amaranth produced biodegradable films with different characteristics. Both the modification treatments resulted in increased tensile strength of films. Water vapour permeability of films increased in heat moisture treated while decreased in oxidised starch film samples. Overall, amaranth starch films were transparent, continuous and had good tensile strength.