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Effects of Soaking and Grinding Conditions on Anti-nutrient and Nutrient Contents of Soy Milk

Humyra Nowshin
Department of Food Technology and Rural Industries, Faculty of Agricultural Engineering and Technology, Bangladesh Agricultural University, Mymensingh- 2202, Bangladesh

Kumkum Devnath
Department of Food Technology and Rural Industries, Faculty of Agricultural Engineering and Technology, Bangladesh Agricultural University, Mymensingh- 2202, Bangladesh

Anjuman Ara Begum
Department of Food Technology and Rural Industries, Faculty of Agricultural Engineering and Technology, Bangladesh Agricultural University, Mymensingh- 2202, Bangladesh

Md. Anisur Rahman Mazumder
Department of Food Technology and Rural Industries, Faculty of Agricultural Engineering and Technology, Bangladesh Agricultural University, Mymensingh- 2202, Bangladesh

Abstract/ Executive Summary:

Although soy milk is a very good source of nutrient with high biological value, the presence of antinutritional factors affects its nutrition quality and limits bio-availability of the nutrients. The effects of soaking duration and combination of soaking and grinding (hot or cold) on phytate, lipoxygenase, urease, trypsin inhibitor activity, protein solubility and other nutrient contents were investigated. Soaking alone at 55 and 60^oC for different durations was found effective for the reduction of lipoxygense activity. Combination of soaking, blanching (80^oC for 10 min) and hot grinding (100^oC) significantly (P>0.05) reduced urease activity, more than 80% phytate activity and deactivated trypsin inhibitor, but did not affect protein solubility. Meanwhile, protein solubility (10–15%) was increased due to hot grinding. Soy milk extracted from soaking at 55 and 60^oC for 2, 4 and 6 h with hot grinding provided higher protein content compared to cold grinding. Increase in soaking temperature from 55 to 60^oC increased the extracted solid content having a potential fraction of lipid. Increasing soaking time from 4 to 6 h did not show any significant difference in terms of phytate inhibition, urease activity reduction, trypsin inhibition and protein solubility except lipoxygenase activity. The results suggested that soaking of soybean at 60^oC for 6 h and hot grinding (100^oC) with blanching at 80^oC for 10 min is the best for reducing anti-nutrient and retaining nutrient activity for soy milk and other soy-based products.

Keywords: Soy milk, Soaking, Grinding, Phytate, Protein solubility

Location: Department of Food Technology and Rural Industries, Faculty of Agricultural Engineering and Technology, Bangladesh Agricultural University, Mymensingh- 2202, Bangladesh

Start Date:

End Date:

Program Area: Quality and Nutrition

Commodity: Milk

Objectives:

This research aimed to evaluate the effects of soaking time-temperature and hot grinding on the anti-nutritional factors such as phytate, lipoxygenase, urease activity, protease and trypsin inhibitor, protein solubility and other nutrient content of soy milk.

Methodology:

Soy milk extraction Soy milk was extracted by the modified method described by Mazumder (2016). Prior to grinding, the soybeans were soaked in 0.5% sodium bicarbonate solution at 55 and 60^oC for 2, 4 and 6 h in a water bath (JSSB-30T, Korea). The ratio of soybean and water for soaking was 1:2. The beans were drained well after wards. After discarding water, the soaked soybean was dehulled manually with hands to remove unwanted substances before grinding. Hydrated beans were blanched in 0.5% sodium bicarbonate solution at 80^oC for 10 min. The solution was then drained well and washed with potable water for three times. The blanched soybean was grinded with addition of hot water (100°C) or cold water by using super mass colloider and a basket centrifuge. The ratio of soybean to water was 1:4. Soy milk was obtained after filtering through double layers of cheese cloths. The soy milk was stored at -18°C for further studies. Soy milk prepared by different methods was analyzed for total solid, protein, fat, protein solubility, phytae, lipoxygenase, urease activity and tripsin inhibitor. Moisture content for total solid, protein and fat content was determined following AOAC (1999). Determination of lipooxygenase (LOX) assays of soy milk One hundred milliliter (100 ml) of soy milk was centrifuged at 25,000 x g for 20 min at room temperature. The supernatant was used for the crude enzyme extract. LOX activity was measured as the change in absorbance at 234 nm with a linolenic acid substrate solution according to Gokmen et al. (2002). A double beam spectrophotometer (Photolab 7600UVVIS) and 1 cm path-length cuvette were used for enzyme measurement. Determination of phytate activity of soy milk Extraction of phytate was done for 1 h in a 20 ml vial with 0.5 N HCl in a ratio of 1:20 (w/v) while stirring. 0.5 ml of soy milk and 10 ml of 0.5 N HCl was used throughout. Approximately 2 ml of crude extract of each sample was centrifuged at 18,000 x g for 10 min in a micro-centrifuge. An aliquot of 1 ml of supernatant containing phytate was then filtered with a 1 ml tuberculin syringe and a 13-mm/0.45-µm syringe filter. Filtered samples could be stored at 4°C for several days prior to HPLC analysis. Phytate was determined by the modified method as described by Rounds and Nielsen (1993). Elution of phytate for HPLC analysis was achieved by using a 30 min linear gradient of 0.01 M 1-methylpiperazine at pH 4.0 and 0.5 M NaNO₃ in 0.01 M 1-methylpiperazine at pH 4.0 with a flow rate of 1 ml/min. Wade’s color reagent consisting of 0.015% (w/v) FeCl₃ and 0.15% (w/v) 5-sulfosalicylic acid was prepared based on Wade and Morgan (1955) having a flow rate of 1 ml/min. Phytate eluted from the column were mixed with Wade’s reagents in a mixing tee with inline check valves for both eluents installed prior to the mixing tee to prevent backflow. The post column reaction was allowed to take place in 0.05 × 210 cm poly ether ketone tubing at a combined flow rate of 2 ml/min. The absorbance was monitored at 500 nm and the detector signals and/or phytate peaks were processed and integrated by chromatographic data acquisition system. Determination of urease activity of soy milk The urease activity was determined by an assay method described by Croston et al. (1955). The pH of the urease-urea reaction should maintain 6.8 to prevent a decrease in urease activity with increasing alkalinity. All reactions were carried out in a constant temperature bath at 40°C. The reagents used were 0.1 N HCl, 0.1 N NaOH, phosphate buffer of pH 6.8 made up of 0.025 mole of K₂HPO₄, 0.025 mole KH₂PO₄ and 0.8 g of glutathione per liter and the buffered urea solution was prepared daily by dissolving 6 g urea in 100 ml of buffer solution without the glutathione. Twenty milliliter (20 ml) of soy milk was added to 5 ml of the buffer solution containing glutathione, and was allowed to stand for 30 min at 40°C. Five milliliter (5 ml) of the buffered urea solution was then added to initiate the reaction. The pH of the reaction was maintained at approximately 6.8 by slowly adding 0.1 N HCl and using bromthymol blue in its green color range toindicate the desired end-point. At the end of 30 min, the reaction was terminated by rapidly adding additional 0.1 N HCl to a total of 10 ml or more. The system was then titrated with 0.1 N NaOH to pH 4.7. A control was run parallel with each sample. For the control, the urease was inactivated by adding HCl to the sample before adding phosphate buffer and buffered urea solution. The difference between the control and sample in ml of 0.1 N HCl or its ammonia equivalent was taken as the urease activity of the meal. Determination of trypsin inhibitor in soy milk Trypsin inhibitor activity was determined according to the modified method described by Erlanger et al. (1961). Fifty milligram (50 ml) of soy milk and 5 ml of 0.1 M Tris-HCl buffer were mixed together to adjust pH 8.2. The solution was homogenized in an Erlenmeyer containing 20 mM CaCl₂ and agitated for 3 h followed by centrifugation for 3600 x g for 20 min. One hundred milliliter (100 ml) of soy milk was added with 450 µl of buffer and 50 µl of trypsin solution in a test tube and homogenized followed by keeping the suspension at room temperature for 10 minutes. 500 µl of the homogenate was transferred to a new test tube containing 500 µl of buffer and 500 µl of D, L-BApNA solution. The solution was agitated for few minutes and left at room temperature for 10 minutes; the reaction was stopped by adding 300 µl of 60% acetic acid. Absorbance of the solution was determined at 410 nm using a spectrophotometer (Photolab 7600UV-VIS) and the results were converted in milligram of inhibited trypsin per gram of total protein in the sample. Determination of protein solubility of soy milk Protein solubility of soy milk was determined based on nitrogen (N) solubility. Fifty milliliter (50 ml) soy milk was centrifuged at 10,000 x g for 10 min at room temperature to separate solid and liquid. The suspension was pipetted and N solubility was determined by using Lowry method (Lowry et al., 1952). Protein solution (0.3 ml) was added in test tubes with 0.3 ml 2 M NaOH. The solution was heated at 100oC for 10 min and cooled at room temperature (25^oC). Three milliliter (3 ml) of complex forming agent (2% sodium carbonate, 1% copper sulphate and 2% sodium potassium tartarate) was added to the solution. The solution was then mixed well. This solution was incubated at room temperature for 10 min. Then 0.3 ml of Folin-Ciocalteau solution (reagent solution) was added to each tube and incubated for 30 min (not more than 60 min). Absorbance of the solution was determined at 550 nm using a spectrophotometer (Photolab 7600UV-VIS). The absorbance was plotted against protein concentration to get a standard calibration curve and calculate percent nitrogen solubility.

Source of Information: J Bangladesh Agril Univ 16(1): 158–163, 2018 ISSN 1810-3030 (Print) 2408-8684 (Online)

Article Link (Online Journal): doi: 10.3329/jbau.v16i1.36497

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

Soy milk is a good source of protein, fat, and mineral contents. Soaking time and temperature along with hot or cold grinding reduced the anti-nutrient factors in soy milk. Soaking conditions did not considerably affect nutrient contents of soy milk; however, soaking temperature affected the nutrient content of soy milk. Hot grinding (100^oC) was found sufficient to reduce 100% of urease activity, more than 85% phytate and trypsin inhibitor, and increase protein solubility. It is concluded that soaking at 60^oC for 6 h in combination with hot grinding (100^oC) with blanching at 80^oC for 10 min is the best practice for producing high quality soy milk and other soy products.

Knowledge Management: Journal