Experimental Design and Statistical Analysis
The experiment was replicated two times using different batches of fresh raw skim milk. Each replication was completed in 3 d. On the first day, milk was carbonated at 0 to 1°C to contain 0 (control), 600, 1200, 1800, and 2400 ppm added CO2 using a continuous tubular carbonation unit (230 ml/min). After overnight storage at 0 to 1°C, subportions of milk at each CO2 concentration were heated to 40, 56, 72, and 80°C, held at the desired temperature, and cooled to 0 to 1°C in a tubular heat exchanger at a flow rate of 372 ml/min. The 40 and 56°C processing was done on the second day, and the 72 and 80°C processing was done on the third day. At each temperature five pressures were applied: 69 (control without added pressure), 138, 207, 276, and 345 kPa. The pH of skim milk at each carbonation level, each temperature, and each pressure was measured directly inline at the end of the holding section as milk was being pumped through the heat exchanger.
Because it is known that milk pH decreases with increasing temperature (
), data from the experiment would be best presented by focusing on how milk pH changed during heating as a function of CO2
concentration and system pressure. The entire dataset was analyzed as four independent split-plot designs by setting temperature at a fixed level (i.e., at 40, 56, 72, or 80°C). The whole plot factor was CO2
concentration (five levels) and the subplot factor was pressure (five levels). The ANOVA model is listed in Table 1
. Analyses were done using
Table 1ANOVA model used for analysis of the effect of CO2 concentration and pressure on milk pH at the end of the holding section of a tubular heat exchanger at a fixed temperature (i.e., 40, 56, 72, or 80°C).
Carbonation was conducted at 0 to 1°C in a laboratory-scale continuous inline CO2 injection system at a milk flow rate of 230 ml/min. The injection system was a countercurrent stainless steel tubular heat exchanger (i.d. = 0.5 cm) cooled by circulating ice water. Milk was pumped through the system with a peristaltic pump (Amicon LP-1 pump, Beverly, MA, with Cole-Palmer Masterflex 7015-81 pump head, Vernon Hills, IL) and CO2 (beverage grade, Empire Air Gas, Radnor, PA) was injected (60 psi input pressure) through a stainless steel tube (i.d. = 0.08 cm) inserted through a Tee-fitting perpendicular to the milk flow immediately after the feed pump. The residence time of milk in the heat exchanger was approximately 60 s. Desired carbonation levels were achieved by adjusting the flow rate of CO2 while keeping the flow rate of milk constant. Milk with added CO2 was collected in plastic 2-L screw-cap jugs and stored overnight at 0 to 1°C in a walk-in cooler prior to processing. Preliminary work indicated that very little loss of CO2 from milk occurred under this storage condition.
Inline pH Measurement and Pressure Control
Milk pH was measured inline as milk was heated to 40, 56, 72, and 80°C in a laboratory-scale countercurrent stainless steel tubular heat exchanger (i.d. = 0.5 cm). The system consisted of 19 straight pieces of tube that were each 105 cm long, interrupted by eighteen 13-cm diameter U-turns with flow entering at the lowest point in the system and having an upward pitch on successive loops of tubes until reaching the system exit. This was done to promote turbulent flow. The heat exchanger consisted of eight sections, they were sequentially from inlet to exit: 1) a milk feed reservoir; 2) a peristaltic pump, which fed the milk (0 to 1°C) into the heat exchanger at a flow rate of 372 ml/min; 3) a heating section, which heated the milk to the target temperature; 4) a holding section, which kept the milk at the target temperature for a fixed time period; 5) a sanitary pressure gauge (Anderson Instrument Company, Inc., Fultonville, NY) that registered the system pressure at the end of the holding section; 6) pH probes (model HA 405 DXK-58/120 combination pH probe; Mettler Toledo, Columbus, OH) that were inserted perpendicular to the milk flow through Tee-fittings to measure milk pH continuously inline at the end of the holding section; 7) a cooling section, circulated with ice water that cooled the milk to 0 to 1°C for collection at the exit; and 8) a needle valve that was opened and closed for decreasing and increasing of system pressure. Throughout the system, several temperature probes were inserted inline to monitor the inlet, heating, holding, and exit temperature of the milk.
The heating and holding sections were circulated with hot water to achieve the desired temperature targets. For both replications, each of the four target temperatures was achieved and well maintained in the holding section for each of the five CO2
concentrations and the five pressures. For the first replication, the average temperatures (n = 25) were 40.2 ± 0.2°C, 56.0 ± 0.2°C, 72.2 ± 0.1°C, and 80.4 ± 0.2°C and for the second replications, temperatures were 40.5 ± 0.2°C, 56.5 ± 0.4°C, 72.1 ± 0.2°C, and 80.3 ± 0.2°C. For the 40, 56, and 72°C treatments, it took the milk 19.5 s (heating time) to reach the target temperature, and the milk was maintained at the target temperature for 31.2 s (holding time) before being cooled. For the 80°C treatment, the heating time and holding time were 29.6 and 20.9 s, respectively. The 72°C for 31.2 s and 80°C for 20.9 s heating conditions produced a comparable degree of heat denaturation of whey proteins as the conditions used for milk pasteurization in the fluid milk industry in New York (
Ma et al., 2000
- Ma Y.
- Ryan C.
- Barbano D.M.
- Galton D.M.
- Rudan M.A.
- Boor K.J.
Effects of somatic cell count on quality and shelf life of pasteurized fluid milk.
Both a pressure gauge and pH probe were inline next to each other and were both at the end of the holding section, just before the cooling section. Pressures were set at 69 (control, without added pressure and with the needle valve completely open), 138, 207, 276, and 345 kPa. Increasing pressure up to 345 kPa did not change the flow rate of milk (i.e., 372 ml/min).
The pH probe was calibrated first with buffers (Fisher Scientific, Fair Lawn, NJ) before being inserted inline. To obtain accurate pH measurement, the pH probe and the calibration buffers were tempered to the temperature at which the pH of the milk was measured. This tempering procedure ensured more rapid response and more stable readings of the pH probe. In addition, the appropriate reference pH for each calibration buffer at each temperature was used. The pH of the buffers were 6.97 and 4.03 at 40°C, 6.98 and 4.08 at 56°C, 6.98 and 4.14 at 72°C, and 6.98 and 4.16 at 80°C.
Raw milk was tested for percentage fat (Mojonnier method, AOAC, method number 989.05; 33.2.26), TS (AOAC, method number 990.20; 33.2.44), total nitrogen (AOAC, method number 991.20; 33.2.11), and NPN (AOAC, method number 991.21; 33.2.12). Percentage true protein was calculated as (total nitrogen-NPN) × 6.38. The CO2
concentration in milk before heating on each of the processing days (i.e., second and third day) was measured (
Ma et al., 2001
- Ma Y.
- Barbano D.M.
- Hotchkiss J.H.
- Murphy S.
- Lynch J.M.
Impact of CO2 addition to milk on selected analytical testing methods.
). To verify that no loss of CO2
occurred in the heat exchanger, the CO2
concentration of milk collected at the exit of the heat exchanger after heating and cooling was also determined at the four temperatures (i.e., 40, 56, 72, and 80°C) and three selected pressures (69, 207, and 345 kPa).