Article pubs.acs.org/JAFC
Preparation and Physicochemical Properties of Whole-Bean Soymilk Hsin-Yu Kuo,† Shih-Hsin Chen,‡ and An-I Yeh*,† †
Graduate Institute of Food Science and Technology, National Taiwan University, 1 Roosevelt Road, Section 4, Taipei 10617, Taiwan Department of Food Science, National Ilan University, Ilan, 1 Shen-Lung Road, Section 1I-Lan 260, Taiwan
‡
ABSTRACT: Whole-bean soymilk has been prepared by using media-milling. Some characteristics of media-milled soymilk have been determined and compared with filtered soymilk (similar to commercial ones) and whole-bean soymilk prepared by blending. There existed particles in the nano/submicrometer scale in both media-milled and filtered soymilk. The particles in blended soymilk were greater than 1 μm. Media-milled soymilk was the most stable among three samples, even after autoclaving. Solid recovery (98.44 ± 0.16%), viscosity (160.59 ± 4.26 cps), dietary fiber (22.68 ± 0.97% on dry basis), total polyphenol recovery (95.15 ± 7.09%), and isoflavone content (4.42 ± 0.03 mg/g dry solid) of media-milled samples were greater than those of filtered ones. Aglycones, the most bioactive form of isoflavone, in autoclaved media-milled soymilk were more than 2-fold those in autoclaved filtered soymilk. With almost no okara generated, the media-milled soymilk retained fiber in soybeans which would be beneficial to human health. KEYWORDS: soymilk, media milling, isoflavone, whole bean
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INTRODUCTION Soymilk, an aqueous extract of soybeans, is a traditional beverage consumed for centuries in Asia. Soymilk provides high-quality proteins and essential fatty acids containing no cholesterol, gluten, and lactose. Thus, it is a good dietary protein source for general consumers or vegetarians and is a suitable substitute of cow’s milk for lactose-intolerant populations.1 In addition, there exist various biologically active phytochemicals such as isoflavones, polyphenols, phytate, saponins, lecithin, phytosteroids, and tocopherol.2 Isoflavones, a plant derived phytoestrogen, have caught considerable attention due to its biological activities including antioxidation and prevention of cardiovascular diseases, type 2 diabetes, cancers, osteoporosis, as well as relief of menopausal syndromes.3−8 Isoflavones can be divided into three types, daidzein, genistein, and glycitein, and each type consists of four different chemical forms which are β-glucoside, 6″-O-acetyl-βglucoside, 6″-O-malonyl-β-glucoside, and aglycone.9 During soymilk production, some processes such as soaking, grinding, filtering, and heating result in loss of isoflavones to different degrees.10,11 These processes generally result in high loss of glucoside and malonylglucoside but increase in aglycone.11−13 Via traditional methods, soymilk is produced after soaked soybeans are ground and filtered.14 To process 1 kg soybean for soymilk manufacture, about 8−10 kg water are added and about 0.3 kg of okara (dry basis) is generated.15,16 Okara is rich in protein (25.4−28.4%), oil (9.3−10.9%), dietary fiber (52.8− 58.1%), and isoflavone (0.14%).17,18 Jackson et al.11 have found that the proportion of aglycone in okara is greater than that in soymilk. However, okara is generally considered as waste. It putrefies very quickly due to high water activity, which results in an issue of plant management for commercial production. Whole-bean soymilk can retain original nutrients and is environmentally friendly. A media mill is derived from a stirred ball mill which is utilized to produce nano/submicrometer particles via a topdown concept and has been applied in paint and © 2013 American Chemical Society
pharmaceutical industries. During media-milling, material is comminuted among the stirring media, the grinding chamber wall, and the material itself by friction, impact, compression, and shearing forces.19 Yeh et al.20 have revealed that mediamilling can significantly decrease crystallinity and average particle size (to submicrometer scale) and effectively enhance the enzymatic hydrolysis of microcrystalline cotton cellulose. Nano/submicrometer corn starch has been prepared by using media-milling and exhibits unique pasting properties.21 Mediamilling appears to be an attractive method for utilizing whole edible materials. The objectives of this study were to explore the feasibility of preparing whole-bean soymilk by using media-milling and to understand the physicochemical properties of media-milled soymilk.
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MATERIALS AND METHODS
Samples. Soybean (Kaohsiung no. 1 cultivar, moisture content 9.24 ± 0.13%) was provided by the Kaohsiung District Agricultural Research and Extension Station and was stored at 4 °C until use. Soybean Flour Preparation. Soybeans were milled in a commercial pulverizing machine (RT-04, Hsin An Instrument Co. Ltd., Taipei, Taiwan) for 1−2 min through a 20-mesh sieve. Soymilk Preparation. Soybeans (60 g) were soaked in deionized water (440 g) overnight at 4 °C and then ground by using a laboratory blender (MX-7012S, Waring Laboratory Science, New Hartford, CT, USA) for 3−4 min. The soybean slurry was used to prepare filtered, blended, and media-milled soymilk. Filtered Soymilk (FS). The soybean slurry was filtered through a double-layered cheese cloth. The filtrate was designated as filtered soymilk, which was similar to the commercial products in Taiwan. The pulp was considered as okara. Received: Revised: Accepted: Published: 742
October 4, 2013 December 3, 2013 December 31, 2013 December 31, 2013 dx.doi.org/10.1021/jf404465w | J. Agric. Food Chem. 2014, 62, 742−749
Journal of Agricultural and Food Chemistry
Article
Blended Soymilk (BS). The soybean slurry was further minced by using a high-speed blender (PT 3100, Kinematica, Lucerne, Switzerland) at 2000 rpm for 10 min to obtain the blended soymilk. As no okara was produced, its composition was similar to whole soybeans. Media-Milled Soymilk (MS). The media-milled soymilk was prepared by using a media mill (Minipur, Netzsch-Feinmahltechnik GmbH, Germany) with a driving motor of 0.94 kW. Media (0.8 mm, yttria-stabilized tetragonal zirconia, YTZ) at 70% v/v filling ratio was loaded in the milling chamber (200 mL). The soybean slurry was loaded into a jacket-cooling tank and then fed at a flow rate of 350 mL/min into the milling chamber by a circulating pump. The agitation speed was set at 3000 rpm, and the milling was conducted for 60 min. During milling, particles smaller than the gap (0.30 mm) of the media separator were driven back to the stirred tank for cooling by the circulation system. The temperature of the soymilk was maintained below 30 °C during milling. Generally, soymilk is cooked or autoclaved before consumption. To minimize the effect of concentration, the solid contents of three soymilks were adjusted to 7% (w/w) and then heated by an autoclave (TM-325, Tomin Medical Equipment Co. Ltd., Taipei, Taiwan) at 121 °C for 3 min.22 The effects of heating on pH, viscosity, and stability of soymilk were explored. Particle Size Distribution (PSD). Particle size distribution of samples was measured by using a laser diffraction particle size analyzer (LS 230, Beckman Coulter, Fullerton, CA) with a detection range of 0.04−2000 μm. The instrument was calibrated with deionized water. All samples were diluted 10-fold with deionized water and vortexed for 10 s. Average diameters of particles were obtained using the following equations.
detected every 5 min for 12 h. The stability was expressed by the difference (ΔIt−0) in the light intensity at time of t (It) and initial state (0 min) (I0) along with time. A positive ΔIt−0 indicates the occurrence of precipitation. Once floating or foaming occurred, ΔIt−0 drops with time. For a stable suspension, ΔIt−0 does not change significantly with time. Composition Analysis. After freeze-drying, the lyophilized powder of soymilk was used for composition analysis. The contents of moisture, ash, crude fat, crude protein, and dietary fiber (including soluble, insoluble, and total fiber) were determined according to the methods of the Association of Official Analytical Chemists (AOAC).23 Total Polyphenol Content. The content of total polyphenol was determined according to the method of Xu and Chang.24 Soybean flour and lyophilized soymilk powders (0.5 g) were extracted with 5 mL of acetone/water (50/50, v/v), and the mixture was stirred at 300 rpm for 3 h. After centrifuging at 3000 rpm for 10 min, the supernatant was collected. The precipitate was extracted again via the procedures described above. Both supernatants were combined and added with 50% acetone to be 10 mL and stored at 4 °C in the dark until analysis. The total polyphenol content was determined by a Folin−Ciocalteu assay25 using gallic acid as a standard.26 The mixture of 50 μL sample solution, 3 mL of distilled water, 250 μL of Folin−Ciocalteu’s reagents solution, and 750 μL of 7% sodium carbonate (Na2CO3) were vortexed and incubated for 8 min in the dark at 25 °C. Then, 950 μL of distilled water was added. The mixture was left in the dark for reaction for 2 h at 25 °C. The absorbance was measured at 765 nm. The total polyphenol content was obtained against the calibration curve of gallic acid and expressed as gallic acid equivalents (mg of GAE/g of dry sample or mg of gallic acid/100 g undiluted soymilk). The calibration curve of gallic acid was established with concentration of 12.5−1000 μg/mL. Isoflavone Content. Isoflavone Extraction. The extraction procedure was conducted according to the method described by Murphy et al.27 Soymilk samples were freeze-dried and sieved through a 60-mesh (for FS and MS) or 20-mesh (for BS) sifter. Lyophilized powder (1.0 g) was extracted with 10 mL of acetonitrile, 7 mL of deionized water, and 2 mL of 0.1 N HCl by stirring (400 rpm) at room temperature for 2 h and then filtered through Whatman no. 1 filter paper. The filtrate was evaporated to remove solvent at vacuum conditions and then was dissolved in 2 mL of 80% methanol. The solution was seeped through a 0.45 μm nylon syringe filter. Fluorescein (1000 μg/mL) was added into filtrate as an internal standard with a ratio of fluorescein solution to a sample solution of 1:9 (v/v). After being analyzed by using HPLC, the content of isoflavone was calculated using an internal standard method reported by Kao and Chen.28 A calibration curve is required to quantitatively determine the concentration of isoflavone by utilizing the internal standard method. To establish calibration curves, each individual isoflavone compound was dissolved in 80% methanol at a concentration of 1000 μg/mL, which was further diluted at different concentrations (3.125, 6.25, 12.5, 25, 50, 100, 200 μg/mL) as standard solutions. Filtration and addition of fluorescein solution were conducted as described above for HPLC analysis. HPLC Analysis. The high-performance liquid chromatography (HPLC) method9 with minor modification was employed to determine the content of isoflavones. The HPLC used was equipped with a C18 column (4.6 mm × 250 mm, YMC-Pack ODS-AM, S-5 μm, YMC Co., Tokyo, Japan), a UV−vis detector (UV-2075 Plus, Jasco, Tokyo, Japan), and a chromatography data processor (EC 2000, Analab Co., Taipei, Taiwan). Linear concentration gradient in mobile phase was conducted by using two solutions: (A) 0.1% glacial acetic acid in deionized H2O and (B) 0.1% glacial acetic acid in acetonitrile. After a 20 μL sample was injected into the column, a percentage of solvent B was increased from 15% to 20% in 20 min, successively increased to 24% in 10 min, and held for 4 min then further increased to 35% in 10 min and held for 8 min, and finally reduced to 15% in 5 min and held for 3 min. The total time was 60 min. The flow rate of the mobile phase was 1.0 mL/min. Statistical Analysis. Data were reported as the mean of measurements in triplicate. One-way analysis of variance (ANOVA)
by volume
DV =
∑ i
nidi 4 nidi 3
by number
DN =
∑ i
nidi ni
where ni was the number of i particle with diameter of di. All of the measurements were done in triplicate, and the average data were reported. Morphology. Morphology of suspended particles in soymilk was examined by optical microscope (OM) (model TS100, Nikon Co., Tokyo, Japan) and scanning electron microscope (SEM) (Hitachi S800, Hitachi Co. Ltd., Tokyo, Japan). Diluted samples (1%, w/w) were used for OM observation. For SEM, critical-point drying was used to prepare samples. Ten-fold diluted soymilk (FS, BS, or MS, 1 mL) was mixed with 9 mL of acetone and then evaporated to 1 mL at 60 mmHg. After repeating the replacement process in triplicate, solids were suspended in 5 mL of acetone. The suspension was further dried using liquid CO2 in a critical-point drying apparatus (Hitachi HCP-2, Hitachi Co. Ltd., Tokyo, Japan) at 31.1 °C and 73.9 bar. After being dried, the sample was attached on a SEM stub using double-backed cellophane tape. The stub and sample were coated with gold− palladium and then were examined and photographed at 20 kV. Viscosity. A rheometer (AR 2000ex, TA Instruments, New Castle, NJ, USA) was employed to measure the viscosity of soymilk. Soymilk (30 mL) was transferred into a concentric cylinder system with a procedure of FLOW. The viscosity of soymilk was measured at 25 °C using steady mode with a shear rate at 100 s−1. In addition, the solid content of MS was adjusted to 7, 8, 9, and 10% (w/w) for understanding the effect of solid content on the viscosity of MS. pH. The pH of sample was determined by using a pH meter (SP2200, Suntex Instrument Co., Ltd., Taipei, Taiwan). Stability. The stability of soymilk was determined by using a demixing tester (LUMiCheck, L.U.M. GmbH, Berlin, Germany). About 3 mL of sample was loaded into the sample cell. The intensity of light (870 nm) backscattered from the tilted bottom of the cell was 743
dx.doi.org/10.1021/jf404465w | J. Agric. Food Chem. 2014, 62, 742−749
Journal of Agricultural and Food Chemistry
Article
Figure 1. Particle size distribution: (a−c) volume and (d−f) number of (a,d) filtered (FS), (b,e) blended (BS), and (c,f) media-milled (MS) soymilk.
Figure 2. (a−c) Optical microscopic observation and (d) appearance of (a) filtered (FS), (b) blended (BS), and (c) media-milled (MS) soymilk was used to determine the significance of treatment using the Statistical Analysis System (SAS version 9.1; SAS Institute Inc., Cary, NC), followed by a Duncan’s new multiple range test. Differences were considered as statistically significant when the P value was
