Abstract Present study elaborates the temperature dependent kinetics of fatty acids of fortified biscuit

Abstract
Present study elaborates the temperature dependent kinetics of fatty acids of fortified biscuit. The study reflected that the significant (p 0.05). effect on the minerals.

1. Introduction
Biscuit is widely most consumable and affectionate bakery product. The major ingredients of the biscuit are wheat, lipid, sugar and milk powder 1-3. However, due to increase in the consumer health consciousness towards gluten-free, bad & good lipid and low in sugar, from past few years, it became a trend to replace these major ingredients with a healthy one. Among them primarily, fortification of fiber-rich ingredient to replace the wheat flour is most considerable 4,5. Additionally, advancement in technologies, developing functional & healthy ingredients, screening & characterization of healthier foods is also under considerations 6-9. Bottle gourd (Lagenaria siceraria) is herbaceous plant globally grown in the tropical and subtropical regions. Mature fruit contains a substantial amount of seeds which is rich in fiber (4.29%), ash (3.91%), and several essential minerals such as K (3001 mg/100g), Mg (568 mg/100g) and Fe (57.4 mg/100g) 10-12. Additionally, seeds are a rich source of lipid content (39.22%) and mostly used in pharmaceutical oil industries 13,14. Secondly, replacement of margarine and saturated fatty acids rich oils with vegetable oils have been also under consideration 15. Among the healthy and alternative one flaxseed oil is one of the most selectively and extensively utilized source of healthy oil in bakery industries 15. Oil from flaxseed contains substantial amount of polyunsaturated and monounsaturated fatty acid (PUFA) mainly linolenic acid (55.0%), linoleic acid (16.2%) and oleic acid (17.5%) 16 and support the metabolic system fight against several chronic diseases such as coronary heart disease, inflammation, cancer and problems related to inadequate 17. Studies say that the fortification of healthy ingredients into the food product intend to be processed by exposing extensive temperature, oxidative stress, radiation, and pressure can be limited 18-20.
This study was mainly focused on the effect of different baking temperature on the unsaturated fatty acids and mineral content biscuit made up of the previously optimized ratio of de-oiled bottle gourd seed cake powder (BGSCP) and whole wheat flour 4, additionally fortified with flaxseed oil.
2. Materials and methods
2.1. Ingredients and chemical standards
De-oiled bottle gourd seed cake was procured from the Department of Farm Engineering, Institute of Agricultural Science, BHU, Varanasi, India. Mechanically pressed flaxseed oil, wheat flour, milk powder, baking powder, baking soda, and sugar was purchased from the local supplier of the Varanasi. Chemical for the analysis in this experiment was ordered from the Sigma-Aldrich, India. Analytical standards viz., Calibration mix majors and Calibration mix2 (ICP-OES Wavecal Conc.) for mineral profile analysis was procured from Agilent Technologies, USA and Fatty Acid Methyl Esters (FAME) kit for the fatty acid analysis was procured from Sigma-Aldrich, Germany.
2.2. Preparation of biscuit
Dough for the biscuit was made following our previous method 4. Additionally, molded and shaped dough was baked at a different baking temperature of 180, 190, 200, 210 and 220 °C for the baking period of 15 minutes. After baking biscuits were precooled at room temperature and packed into air-tight polyethylene bag for further characterization.
2.5. Analysis of fatty acids
Extraction of oil from the sample was carried out by dissolving 10 g of the sample in 50 mL of n-hexane, centrifuged for 5 min at 10 °C and 6000 rpm, the supernatant removed, the solvent evaporated using a vacuum concentrator and stored under refrigerated conditions till further analysis. The extracted oil was esterified to their corresponding fatty acid methyl esters (FAME) using a method suggested by 16, which involved refluxing of a mixture of 0.5g oil with 5 mL of H2SO4:toluene: methanol at a ratio of 1:10:20. The refluxed mixture was subsequently cooled and diluted with the sequential addition of 5 mL of water and 7 mL n-hexane. The supernatant in n-hexane was isolated using a separating funnel, dried over Na2SO4 to remove moisture traces if any, and stored in vials under refrigerated conditions till further analysis.
Fatty acid profile of the esterified oil was determined using GC with FID detector (Varian 450, USA) equipped with GsBP-Inowax column (30m × 0.25mm). Helium was used as the carrier gas and the column pressure was maintained at 24 kPa. One µL of the aliquot was injected into the GC using a split ratio of 40:1. During the entire runtime of 36 minutes, an initial column temperature of 60 °C was maintained for 1 min. Subsequently, the temperature was ramped to 180 °C @ 8 °C/min, 180 °C maintained for 5 min, further ramped to 230 °C @ 12 °C/min and a temperature of 230 °C was maintained for 10 min During the entire separation process, the detector and injector temperatures were maintained at 320 °C and 275 °C, respectively.
The composition of fatty acids was calculated using peak normalization method assuming equal detector response. The peaks were identified by co-eluation of fatty acid methyl ester (FAME) standards procured from Sigma-Aldrich, processed under similar conditions. All experiments were carried out in triplicate and the average value was used for analysis. The values were quantified as a percentage of total fatty acids present in the samples.
2.6. Analysis of the mineral composition
Mineral analysis of samples was performed using a method suggested by Liman et al. 21 with some modifications. About 0.5g of the defatted sample was digested using DK 20 Heating Digester unit (VELP Scientifica, Italy) in 10 mL of concentrated HNO3 at 200 °C until the solution became translucent and a smoke stopped being released, which indicated the complete digestion of the organic matter. Samples were allowed to cool at room temperature. Then, the digested samples were transferred to 100 mL volumetric flasks. The transfer was accomplished using ash-free quantitative filter paper Whatman No. 41. The volume of the solutions was made up to 100 mL using deionized water and stored for further analysis. Digested samples were analyzed using a simultaneous multi-analyte Microwave Plasma-Atomic Emission Spectrometer (MPAES – Agilent Technologies 4100, USA), constituting of microwave-induced plasma (produced using nitrogen gas) and interfaced with an atomic emission spectrophotometer (AES). Elemental determinations were performed using a coupled solid-state CCD detector in MP-AES system. High purity (99.998%) argon (Sigma, USA) was used as the plasma source. The MP-AES instrument operational parameters were as follow: RF (radio frequency) power – 1550 W; plasma gas flow – 20 L/min; auxiliary gas flow 1.5 L/min for ease of or each of the operation. Nebulizer gas flow was maintained at 1.0 L/min. An autosampler (Agilent SPS 3, USA) was used to feed the sample. Mineral concentrations were expressed in mg/100g of the sample. Classification of minerals into essential and non-essential categories was done as suggested by Pinto et al. 22.
2.8. Statistical analysis
Statistical analyses were carried out using PROC RSREG of SAS (version, USA). To avoid the analytical and manual error samples were analyzed in triplicate (n =3) and a consequent least significant difference (P < 0.05) test was applied for multiple sample assessment.
3. Results and Discussion
3.3. Effect of temperature on fatty acid composition
Analysis of fatty acids composition was investigated to understand the thermal dependent kinetics of essential fatty acids (especially PUFAs – poly-unsaturated fatty acid & MUFAs – mono-unsaturated fatty acids) during the baking process. Results depicted that the fatty acid composition of the fortified biscuit was significantly (P < 0.05) influenced by baking temperature. Table 1 displayed that the flaxseed oil contained more than 86.70% unsaturated