Food processing by heating, albeit economical and efficient, has damaging effects on heat labile compounds [1
], affecting the color, flavor, and texture [2
]. Non-thermal food processing methods are thus of special interest and high pressure processing (HPP) is one such method. Since HPP involves minimal heat, inactivates microbes, and maintains nutritional and sensory qualities, it has found favor in the food industry. HPP can be broadly classified into high hydrostatic pressure (HHP) processing and continuous high pressure (CHP) processing. HHP is a batch process in which the food is sealed in a flexible container, and the container put in a pressure vessel filled with pressure transmitting fluid such as water or oil. This vessel is then pressurized and the pressure transmitting fluid then in turn applies pressure to the food. An important aspect of HHP is that the pressure on the food is applied equally from all directions and, as a result, the foods maintain their shape. The pressure applied in HHP ranges from 100 to 900 MPa [3
]. On the other hand, CHP, as the name implies, is a continuous process and is synonymous with high pressure homogenization (HPH). Conventional homogenization, such as that used in the dairy industry, operates at 20–60 MPa. HPH operates at higher pressures, up to 200 MPa. Ultra high pressure homogenization (UHPH) employs even greater pressures, up to 400 MPa. Specialized homogenization valves, which can withstand such high pressures, are required for CHP. CHP is being increasingly researched as a technique for reducing the size of particle in emulsions for making stable emulsions as well as modifying the viscosity [4
The University of Georgia, GA, USA [5
] developed a novel system called continuous flow high pressure throttling (CFHPT) by modifying the homogenizing valve to throttle instead of having a fixed opening as normally used by other researchers. This valve regulates the fluid flow under high pressure via an adjustable orifice. The process of regulating the fluid flow under high pressure though an extremely narrow orifice has been referred to as throttling, and the homogenizing valve referred to as the throttling valve. Like homogenization, CFHPT employs turbulence, cavitation, and shear forces, which are generated as the food under pressure flows through a highly constricted opening of the throttling valve [6
]. A noteworthy aspect of this process is a rise in temperature as the food throttles. The rise in temperature at the point of throttling is due to the frictional heat generated as a result of very high fluid velocities [8
], and is directly proportional to the pressure. This temperature rise can be exploited for microbial reduction [9
The conventional soy milk production method results in a loss of about 35% of the soybean solids in the form of okara [12
], leading to a poor yield. This loss is a result of the removal of insoluble and coarse solids from soy milk during the filtration step. Additionally, soy milk produced in this way has a paint-like odor and flavor [13
]. Particle size distribution (PSD), an important property of soy milk, is indicative of the changes that take place during processing [14
] as well as of the formation of particle agglomerates [15
]. Sivanandan et al.
] used CFHPT attached with a micro-metering valve to process soy milk made from whole dehulled soybeans. Their method is special in that there is no filtration step and practically all the soybean solids are recovered in the final product.
Lipoxygenase (LOX) is an enzyme naturally present in soy milk, and catalyzes the oxidation of polyunsaturated fatty acids (chiefly linoleic acid), which soybeans are rich in. LOX is difficult to inactivate by pressure alone, and supplementing the treatment with heat greatly reduces LOX activity [16
]. The pH of fresh soy milk, which usually ranges between 6.5 and 7.7, is lowered during storage [18
]. Even though CHP reduces the initial bacterial counts in soy milk, the bacterial load tends to increase upon storage [11
]. Bacterial spores are the most resilient microbial entity under pressure and usually require a combination of pressure and temperature for their inactivation [1
]. The majority of soy milk manufacturers add flavors and mouth-feel improving agents such as sugar, cocoa powder, vanilla flavor, gums, etc.
, to improve its overall appeal [21
]. Very few studies have been conducted on the descriptive sensory analysis of continuous high pressure (CHP) processed soy milk [22
In the current study, soy milk was prepared from whole dehulled soybeans leading to negligible wastage. The effect of UHPH on particle size distribution, lipoxygenase activity, pH, microbial, and sensory qualities of the soy milk was investigated, and the changes in these parameters during storage at 4 °C were monitored for four weeks.
2. Materials and Methods
2.1. Preparation of Soy Milk
The soybeans (Woodruff variety) were provided by the Georgia Seed Development Commission, Athens, GA, USA and stored at 4 °C and 20% relative humidity (RH). Soy milk was prepared according to the method developed by Sivanandan et al.
] with some modifications. Soybeans were left overnight (16 h) in loosely covered HDPE (high density polyethylene) buckets to equilibrate them to room temperature (23–28 °C). The beans were put into perforated SS trays (1 kg per tray) and roasted at 154 °C for 5.5 min in an air impingement oven (Lincoln Impinger Model 1450, Lincoln Foodservice Products, Inc., Fort Wayne, IN, USA). They were cooled for 15–20 min and dehulled in a plate mill (Quaker City Mill Model 4-E, QCG Systems, LLC, Phoenixville, PA, USA). The cotyledons were separated from the hulls by air classification. Deionized water (DW) was used to blanch the soybeans (1:5 kg dehulled soybeans:kg DW) at 60 °C for 2.5 h, then rinsed three times with DW, covered, and stored overnight at 4 °C, 20% (RH). The following day, DW (three times the mass of blanched soybeans) was weighed and divided into two equal parts. The first part was used to grind the blanched soybeans in a food processor (Robot Coupe Model RSI 10 V, Robot Coupe UGA, Inc., Jackson, MS, USA) for 2.5 min at 3000 rpm followed by 2.5 min at 3500 rpm. The paste was then ground in a super mass-collider (Super Mass Collider Model MK CA6-3, Masuko Sangyo Co. Ltd., Kawaguchi-city, Saitama-pref, Japan) using a sanitary stone pair (E6–46). To maintain consistent grinding speed, the electrical current to the equipment was kept between 2 and 3 amperes. The paste was passed through the mass collider eight times, after which the remaining water was mixed and the soy milk passed through the equipment four more times. To prevent clogging of the extremely small opening of the throttling valve during high-pressure processing, the soy milk was filtered using a 254 µm filter. Only 200–250 g residue was obtained from a batch of about 20 L of soy milk. Finally, a vacuum was applied to the soy milk for 20 min to remove the entrapped air. This was the control sample.
2.2. Ultra High Pressure Homogenization (UHPH) of Soy Milk (Figure 1)
The soy milk was fed pneumatically into the Stansted high pressure equipment (Model nG7900, Stansted Fluid Power Ltd., Stansted, Essex, UK) at room temperature (23–28 °C) with an inlet pressure of 700 kPa. Soy milk was pressurized to 207 or 276 MPa using two alternately acting pressure intensifiers (Hydropax P60-03CXS, Stansted Fluid Power Ltd., Stansted, Essex, UK). A heat exchanger between the intensifiers and the throttling valve preheated the pressurized soy milk so as to achieve the target temperatures (121 °C and 145 °C) as measured at the end of the holding tube. The temperature of soy milk after preheating and after throttling was also monitored. The difference between the two was calculated as the temperature rise after throttling. A holding tube was placed after the throttling valve to allow time for microbial destruction. Two flow rates, 0.75 L/min and 1.25 L/min, were studied and they corresponded to a residence time of 20.8 s and 12.48 s, respectively, in the holding tube. Since the temperature of soy milk after throttling was above its boiling point, a back pressure valve (a minimum of 400 kPa) was placed at the end of the holding tube to prevent splashing. The soy milk was quickly cooled to room temperature in a heat exchanger prior to collection. Soy milk was collected in 15-mL sterile Polypropylene tubes (Corning, Inc., Corning, NY, USA) for physical, chemical, and microbial analyses, and in 946-mL HDPE jugs with lined caps for sensory analysis. The entire experiment was duplicated. The samples were stored at 4 °C until analyzed. All the analyses were done on day 1, and weeks 1, 2, 3, and 4 (except sensory analysis).
Aerobic plate counts (APC) and total psychrotrophs were determined. The method of Smith et al.
] was followed. All samples were analyzed in duplicated and the values averaged. One milliliter soy milk samples were serially diluted in peptone water of the following concentration: 0.1 g peptone (BactoTM Peptone, Becton, Dickinson and Company, Sparks, MD, USA) per 100 mL DW for a dilution factor of 1/10 per dilution. Exactly 0.1-mL aliquots of appropriate dilutions were spread plated onto tryptic soy agar (Difco Tryptic Soy Agar, Becton, Dickinson and Company, Sparks, MD, USA) plates. If the number of colonies at these dilutions were too few to detect, 0.1-mL aliquots of soy milk samples were plated directly onto the plates. The plates were incubated at 30 ± 1 °C for 48 h (APC) and at 4 °C for 7–10 days (psychrotrophs). Results were recorded as colony forming units per milliliter (CFU/mL).
Triplicate pH readings of samples were taken (Accumet Basic AB 15, Fisher Scientific Company L.L.C., Pittsburgh, PA, USA) and the averages recorded.
2.5. Dry Solids Content
As there was no loss of water or soybean solids during UHPH, the total solids remained unchanged. Thus, the control soy milk samples were analyzed for total solids using Halogen Moisture Analyzer (Model HR73, Mettler-Toledo, Inc., Columbus, OH, USA) at a temperature of 115 °C. The total solid content also served as an indicator of the control over soy milk preparation.
2.6. Particle Size Distribution (PSD)
The PSD was measured using a Malvern Laser Particle Size Analyzer, Mastersizer S with 300 mm lens (Malvern Instruments, Southborough, MA, USA). Soy milk samples were dispersed in 150 mL DW and the pump speed of the dispersion chamber was kept at 2100 rpm. The obscuration in the diffractometer cell was maintained at 16% ± 0.5%. The predicted scattering was calculated based on the following refractive index (RI) information fed into the software: real RI = 1.47; imaginary RI = 0.00; RI of water = 1.33. The software calculated the average volume-weighted diameter, D[4,3] = Σnidi4/Σnidi3 (where ni is the number of particles in a class of diameter di), the surface-weighted mean diameter; D[3,2] = Σnidi3/Σnidi2; and the D(v,0.9) value, which is the diameter below which 90% of the particles (by volume) are found [23
]. All soy milk samples were analyzed in duplicate and averages were recorded.
2.7. Visible Layer Separation/Sedimentation
All samples were inspected twice a week for any visible layer separation.
2.8. Lipoxygenase Activity
The method of Wang et al.
] was followed. First, a 0.2 M borate buffer of pH 9.0 was prepared using sodium borate (Sodium Borate, 10-Hydrate, Crystal, A.C.S. Reagent, J.T. Baker, Mallinckrodt Baker, Inc., Phillipsburg, NJ, USA) and boric acid (granular, A.C.S. Reagent, J.T. Baker, Mallinckrodt Baker, Inc.). Next, the substrate solution was made by mixing 0.01 mL linoleic acid (TCI America, Portland, OR, USA), 0.01 mL Tween 20 (Fisher Scientific, Fair Lawn, NJ, USA) and 4.0 mL of the borate buffer at 25 °C. This was homogenized using a Pasteur pipette by repeatedly taking in the solution and pushing it out. For clarification, 0.55 mL of 0.5 N NaOH (pellets, F.C.C., J.T. Baker, Mallinckrodt Baker, Inc.) was added and the volume made up to 60 mL using the borate buffer. To obtain the enzyme solution, soy milk was centrifuged at 30,000× g
for 30 min at 4 °C in a Sorvall RC6 Plus Centrifuge (Thermo Fisher Scientific, Inc., Waltham, MA, USA) using a Sorvall SM-24 rotor. Both were allowed to equilibrate to 4 °C for 1 h prior to centrifugation. Since the supernatant was cloudy, it was filtered through a 0.1 µm syringe filter. Prior to the assay, the filtered supernatant was diluted 5 times with DW. This comprised the enzyme solution. If some samples could not be analyzed at the designated time interval, they were stored at −65 °C. The assay mixture consisted of 2.0 mL of substrate solution, 0.9 mL of borate buffer, and 0.1 mL of enzyme solution. This mixture was prepared in a quartz cuvette and the cuvette shaken to start the reaction. The increase in absorbance at 234 nm was monitored using an Agilent 8453 spectrophotometer (Agilent Technologies, Santa Clara, CA, USA) for 5 min immediately after shaking the cuvette. The temperature of the lab and the reagents was maintained at 25 °C. The lipoxygenase activity was calculated from the linear portion of the absorbance-time curve as provided by the instrument software (UV-Visible Chem Station, Rev. B.04.01, Agilent Technologies). A blank (2.0 mL substrate solution and 1.0 mL borate buffer) was also prepared.
2.9. Sensory Analysis
Descriptive sensory analysis was conducted on two treatments: T6 (121 °C, 12.48 s, 276 MPa) and T8 (121 °C, 12.48 s, 207 MPa). Samples heated to 145 °C were found to have an excessive cooked flavor not favored by the panelists and were not evaluated. Data from 11 trained panelists were used for the analysis. All panelists were food science graduate students, experienced in sensory analysis, and were given seven 1-h training sessions. A 150-mm unstructured line scale, with indents marked at 12.5 mm from either end, was used for each attribute. The panelists were free to place a vertical mark anywhere on the line depending on their perception of that attribute’s intensity, which was then converted into millimeters. Six attributes were evaluated and these, in the order evaluated by the panelists, were: beany aroma, beany flavor, astringency, cooked flavor, bitterness, and chalkiness.
For the first few training sessions, the panel was calibrated for low and high intensity concentrations of each attribute. The panel, as a whole, came up with the intensities for each of these two concentrations. After calculating these consensual intensities, ‘x’ marks were put on the scales at appropriate distances signifying low and high concentrations. Next, medium-intensity concentrations of the reference samples were prepared and the panel calibrated for this concentration (Table 1
The final scale had two indentation marks and a box with an ‘x’ mark corresponding to the medium intensity for each attribute. Sensory evaluation was conducted in fluorescent-lit, individual sensory booths. Refrigerated soy milk and reference samples were served in clear plastic cups (59.15 mL) with lids. The soy milk samples were coded with three-digit random numbers and the order of presentation was randomized. The panelists were asked to cleanse their palates with crackers and water before tasting each sample. The evaluation sessions were held on days 1, 6, 13, and 20. Three commercial soy milk samples were also evaluated on two separate occasions, but no storage study was performed. These were Silk Organic Unsweetened Soy milk (White Wave Foods, Broomfield, CO, USA), SoyCow Unsweetened Soy milk (Well Luck Co., Inc., Jersey City, NJ, USA), and Vita Soy Unsweetened Authentic Asian Fortified Soy Beverage (Vitasoy USA, Inc., Ayer, MA, USA).
2.10. Data Analysis
The data were analyzed with JMP Pro Version 10.0.0 (SAS Institute Inc., Cary, NC, USA) and the results were considered to be significantly different if α < 0.05.