Commercial flint corn was obtained from the local market in Mysore. The corn moisture was measured in ambient air conditions and was stored at 4 °C. Before milling, the maize grains were hand-picked to remove broken and damaged kernels and foreign materials. The sodium meta bi-sulfite and lactic acid used for steeping were from Sisco Research Laboratories, Mumbai, India. α-tocopherol, γ-tocopherol, δ-tocopherol, and γ-tocotrienol were purchased from Sigma Chemicals Co. (St. Louis, MO, USA). Lutein and zeaxanthin were purchased from Cayman Chemicals Ltd., Michigan, USA. Campesterol, stigmasterol, β-sitosterol, and pyridine (anhydrous 99.8 %) were procured from Sigma Chemicals Co. (St. Louis, MO, USA). N, O-Bis (trimethylsilyl) trifluoroacetamide (BSTFA), and trimethylchlorosilane (TMCS) were procured from Fluka, Madrid, Spain. All other solvents and reagents were of analytical grade (Merck, Madrid, Spain) and were used without further purification. Methanol, acetonitrile, dichloromethane, and hexane were of HPLC grade, and ethanol and ammonium acetate were purchased from Spectrochem, Mumbai.
Lab-scale corn wet milling process
Several experiments were conducted during the process development work, which led to a final procedure for recovering steep solubles, Germ, Fiber, Starch, and Gluten. The modified lab corn wet mill process flow was designed after these experiments, considering the previous lab-scale corn wet milling experiments(Dowd 2003; N. Singh and Eckhoff 1996). The process flow chart shows the steps followed to separate these products (Figure 1.).
[Figure 1 near here]
The process begins with steeping of corn kernels for 24 h in a solution of sodium metabisulfite solution (0.2 %) and lactic acid (0.5 %), at 53 °C in a 1 L screw-top glass bottle, which facilitates kernel hydration, lactic acid fermentation, and sulfur dioxide reactions within the endosperm. The bottle was kept in an orbital shaker for efficient mixing and soaking of the kernels. After steeping, the steep water was strained from the bottle containing the steeped grains. The steeped corn was then subjected to coarse grinding in a blender (Sai mill) with blunt-edged blades to render corn de-germination. The inner white germ separated from the corn was manually isolated using forceps by flotation and skimming method. Corn Germs were rinsed with water on a sieve (pore size-354 microns) to remove the bound starch and fiber. The remaining ground corn slurry and the rinse volume were finely ground with sharp-edged blades at high rpm in the blender to make a fine slurry. Corn fiber was isolated from the slurry by washing with water on a sieve (pore size-75 microns) using a mechanical sieve shaker. The flow-through starch-gluten slurry was subject to centrifugation at 8000 rpm to isolate gluten from starch. This step was followed by drying the fractions, i.e., germ, fiber, gluten, and starch, in a hot air oven at 55 °C for 90 minutes. The yield of the fractions was calculated for 100 g of corn kernels utilized.
The corn wet mill by-products were obtained as mentioned in Figure 2.
[Figure 2 near here]
Sample preparation for analysis
The dried corn germ, fiber, gluten fractions, and whole corn were pulverized in a small-sized, high rpm blender mill (KRUPS 75 TYP 203, Solingen, Germany)with sharp-edged blades. Samples (germ, fiber, gluten, and whole corn) were stored at -20°C for further analysis.
All the samples were tested for their moisture content estimated by AACC (2000) method by keeping in an air oven at 130 °C for 2 h or until constant weight is achieved(A.A.C.C. 2000).
Vitamin-E (Tocols: Tocopherols & Tocotrienols) extraction
In this process, a redesigned method of direct solvent extraction was employed for extracting tocopherols (TP) and tocotrienols (TT) from the samples, as described by(Jayadeep and Malleshi 2011).Summarily, 10 ml of HPLC grade methanol was used to extract tocols from each gram of pulverized corn germ, fiber, gluten, and whole corn flour separately and mixed in a tube mixer (IKA™ Loopster Digital, fisher scientific) at 27 °C. Centrifugation at 5000 g and 27 °C (room temperature) facilitated the separation of supernatant from the solution. The supernatant was filtered using Whatman 0.45 μm membrane filter (Polyvinylidene difluoride). These tocol extract samples were stored at −20 °C until HPLC analysis.
Characterization of tocols by RP-HPLC (Reverse phase High-Performance Liquid Chromatography)
The HPLC system consisted of a Waters 2690 Alliance Separations Module, a Waters PDA detector, and a Waters 474 Scanning Fluorescence Detector (Millipore, Bedford, MA). The Millennium 32 Ver. 3.20 Chromatography Software (Waters) did the recording and preparation of Chromatograms. The purified sample was injected through a Kromasil C18(Octadecylsilane) column (5μ, 100 A, 4.6 mm* 250mm). Gradient mode of HPLC was conducted for high throughput detection. The RP-HPLC method used the initial mobile phase conditions of 45% acetonitrile, 45 % methanol (MeOH), 5 % isopropanol (IsOH), and 5 % of aq. Acetic acid (1%), for 6 min. The mobile phase of Acetonitrile: MeOH: IsOH was changed linearly at the ratio of 25:70:5 (v/v/v) over the next 10 min and held there for 12 min before returning to the initial conditions. The flow rate of the mobile phase was 1ml/min with a run time of 30 min. The tocopherols (TPs) and tocotrienols (TTs) in the samples were detected at excitation and emission wavelengths of 298 and 328 nm by a fluorescence detector, respectively(Chen and Bergman 2005). The concentrations of tocols stock and working standards solutions were determined using a spectrophotometer (GENESYS 150, Thermofisher Scientific)using the specific extinction coefficients-75.8 (α-S, Absmax- 292 nm), 91.4 (γ-TP, Absmax- 298 nm), 87.3 (δ-TP, Absmax- 298 nm), and 90.5 (γ-TT, Absmax- 296 nm) (Chen and Bergman, 2005).The TPs and TTs in the samples were identified by comparing retention times with authentic standards.A standard curve was plotted each for α-TP (1.14- 12.1 ng, r2=0.95), γ-TP (1.13- 5.63 ng, r2=0.964), δ-TP (0.94– 9.39 ng, r2=0.999), and γ-TT (1 – 33.15 ng, r2=0.985) respectively.
The standard tocol concentration corresponding to the peak area was used to determine the respective tocol component concentration in the samples. The content of α-TT and δ-TT in the samples were calculated corresponding to standard α-TP and δ-TP. TheHPLC chromatograms of Tocol (Vitamin-E) standards and tocols extracted from corn germ, fiber, gluten, and whole corn flour are shown in Figure 3.
[Figure 3 near here]
Carotenoid extraction, analysis, and characterization by RP-HPLC
A redesigned method for total carotenoid extraction described by(Alarcão-E-Silva et al. 2001)from the wet milling by-products was used. The samples (1 gm each) were mixed with extraction solvents (acetone: petroleum ether (b.p. 60–80 °C), at 1:1(v/v) ratio) in centrifuge tubes for 1 hr using a tube mixer (IKA™ Loopster Digital, fisher scientific). After mixing, the samples were centrifuged (Remi centrifuge C‐30BL, Mumbai, India) at 3000 g at room temperature (27 °C) to separate the supernatant. The supernatant was extracted multiple times till the extraction solvent became colorless. After centrifugation, the supernatant layer was mixed and shaken well with distilled water, four times in volume of acetone, to separate the upper petroleum ether layer from the lower acetone layer using a separatory funnel. The upper carotenoid layer was transferred to a graduated cylinder through a filter paper containing Na2SO4 (sodium sulfate) anhydrous to remove residual water and noted the volume for further calculation. A suitable petroleum ether volume was made (b.p. 60–80 °C), and the absorbance of the samples was recorded at a wavelength of 454 nm (λmax) using a UV/Vis spectrophotometer (GENESYS 150, Thermofisher Scientific). The total carotenoid content of the by-product samples was calculated using the specific extinction coefficient() of 2500 at 454 nm.
The carotenoids dissolved in solvents were dried in a vacuum concentrator (Eppendorf concentrator plus). The dried residue was then dissolved in ethanol and stored at -20 °C until RP-HPLC analysis. An aliquot of the sample solution was used for injection into the HPLC system.The experiment was carried out in dim light, and glassware was covered sufficiently with aluminum foil to protect carotenoids from light.The carotenoids were segregated on a C-18 (Octadecylsilane)column, of 250 mm*4.6 mm i.d.(internal diameter), five μm (particle size), 100 A° (pore size) (Kromasil, Supelco, USA).The mobile phase used for the process wasacetonitrile/methanol/dichloromethane (60:20:20 v/v/v) containing 0.1% ammonium acetate. Samples were injected into the system with an isocratic condition maintained at a 1 ml/min flow rate. All the carotenoids were observed at 450 nm (λmax) with a UV-visible detector (Agilent, USA). The individual carotenoid peak identities and λmax values were confirmed by their retention times and characteristic spectra of standard chromatograms, respectively. They were quantified from their peak areas compared to the respective reference standards. The HPLC method was based on(Lakshminarayana et al. 2005). HPLC chromatograms of carotenoid standards and carotenoids extracted from corn germ, fiber, gluten, and whole corn flour are shown in Figure 4.
[Figure 4 near here]
Phytosterol extraction, analysis, and characterization by GC (Gas chromatography)
Phytosterols (PSs) were extracted from corn germ, fiber, gluten, and whole corn flour using a modified technique as outlined by Hossain and Jayadeep (2020)(Hossain and Jayadeep 2020; Jiménez-Escrig et al. 2006). Initially, the samples were subjected to acid hydrolysis, followed by lipid extraction and saponification to extract the unsaponifiables. 1 g of sample and 4 ml of internal standard (IS) solutions in a leak-proof screw-capped glass tube (0.1 mg di-hydrocholesterol in 1 ml of absolute ethanol) were mixed thoroughly. Each sample in the tube was mixed with hydrochloric acid (6 M) and heated in the water bath at 80 °C for an hour to facilitate acid hydrolysis. Hexane and diethyl ether in the ratio of 1:1 was added to the cooled mixture. The two phases were subject to accelerated separation by centrifugation for 5 min. The lipidic upper phase was transferred to 15 ml centrifuge tubes, and the whole solvent content was evaporated in a speed vac concentrator. The dried residues were saponified using pyrogallol-ethanol (3% w/v) solution and dissolved. Saturated potassium hydroxide solution was added to the solution and mixed properly. The sample tubes were heated in a water bath at 80 °C for 30 minutes, mixed vigorously at 2-minute intervals, and cooled.Cyclohexane and distilled water were added to each tube and shaken for 10 minutes to mix the contents thoroughly. After this step, the saponified sample and lipid-containing solvent phases were separated by centrifugation at 5000 g for 5 minutes.The upper, unsaponifiable lipid phase was then transferred to centrifuge tubes by passing it through anhydrous sodium sulfate (Na2SO4) to remove residual moisture. The sample solvent was vacuum evaporated and dried in a speed-vac concentrator (Eppendorf concentrator plus). The dried residue was then silylated to produce sterol ester derivatives.
Production of trimethylsilyl (TMS) ester derivatives of phytosterols was done by a modified procedure(Jiménez-Escrig et al. 2006). Pyridine (500 μL) was immediately added to the dried PS in the round bottom flask. In addition, BSTFA (N, O- Bis (trimethylsilyl) trifluoroacetamide) containing 1 % TMCS (Trimethylchlorosilane) (500 μL) was added and mixed properly and stored overnight at room temperature for silylation. The excess reagent was then removed with N2 gas, and the dry PS ester was dissolved in di-chloromethane: hexane (1:1), and the aliquots were stored at -4 °C until analysis.
Gas Chromatography of the standard (stigmasterol, sitosterol, and campesterol) and the sample phytosterols (corn germ, fiber, gluten, and whole corn flour) was performed to obtain standard chromatograms. The GC analysis of PSs was carried out as previously reported(Jiménez-Escrig et al. 2006). The TMS ester derivative sample (1 µl) was injected to flow through the biphenyl polysiloxane column (30 m × 0.25 mm i.d.× 0.25 μm f.t.) (Elite-5, PerkinElmer, Waltham, Massachusetts, USA) built into a gas chromatograph (Shimadzu GC2010) for detection in a flame ionization detector (300 °C). The column temperature program started at 245 °C, was held for 1 minute and increased to 275 °C at the rate of 3 °C/min to be held for 28.5 min. The carrier gas, nitrogen with a flow rate of 1.08 ml/min, flowed with a split ratio of 1:22. Three major PSs—Campesterol, Stigmasterol, and β-Sitosterol were determined by their respective retention times of the standards.. The GC chromatograms of the by-product phytosterol extracts are shown in Fig. 5. All experiments were performed at least twice with triplicate samples each time.
[Figure 5 near here]
The values in the tables are represented as Mean± SD (standard deviation) of three independent replicates. The results were analyzed by a two-way ANOVA (p< 0.001) test followed by a post-hoc Tukey test (multiple comparison test) using GraphPad prism application (Inc. La Jolla, CA, USA).