9 Figure 2 represents the effect of different time
–temperature combinations on quality retention and microbial inactivation for thermal process. It can be seen
from the graph that the acceptable combinations of time and temperature for thermal process fall within the sterile area. Heating of a product in non-sterile
area was unacceptable because the microbiological requirements was not achievable.
Figure 2 Diagram of t-T relationship for microbial destruction F and cooking
C Holdsworth 1985.
10
3 METHODS
3.1 Materials
Yellow-seeded soybean Glycine max L. was obtained from Organization of Tempe and Tofu Producers KOPTI Bogor, Indonesia. A commercial
powdered tempe starter Raprima brand, Bandung, Indonesia was used for tempe making. This was stored at 5
o
C, dry and in the dark. All chemicals used were pro- analysis grade purchased from Sigma Chemical Co. St. Louis, Mo., U. S. A and
Merck KgaA Darmstadt, Germany. The scheme of research is given in Figure 3.
Figure 3 The scheme of tempe research.
3.2 Tempe Preparation
Clean soybean grains 300 g were soaked with an acetis acid solution pH 3.5 650 mL for 22 hours at 27
o
C. The soybean was then dehulled manually, and the separated cotyledons were boiled 95
o
C in tap water 2 L for 40 min. After cooling down to room temperature, solid state fermentation was performed by
inoculating the mixed culture Raprima 0.03 into cooked soybean in perforated polypropylene bags 25 cm × 12 cm, and then incubated for 40 hours at 30
o
C.
Production of Tempe Heat Penetration Test
Determination of lag time during heating
Thermal Treatments
Heating tempe at 75, 85, 95
o
C isothermal condition for specific interval of time
depending on the tempe properties
Chemical Physical Analysis
Antioxidant capacity, total penolic content, total flavonoid
content, texture, color
Kinetic Modeling
Determination of kinetic parameters k and Ea
Information about kinetic changes of antioxidant capacity
and physical quality of tempe during heating as the basic to design of optimal thermal processing
11
3.3 Sample Preparation
Fresh tempe was cut into cylindrical shape with diameter of 12 mm and height of 20 mm. Three tempe cuts ± 6 g were placed in vial tubes d: 20 mm,
h: 75 mm considering to minimize lag time. Before closing the tube, 2 of aqueous salt NaCl solution ± 20 mL was added, which was usually used as
heating medium in thermal process.
Figure 4 Thermocouple position in the vial tube.
3.4 Temperature Measurement
Coper thermocouple wires were introduced into the center of the cylinders such that the thermocouple junction was placed at the geometrical center of the vial
where it was a coldest point Figure 4. Thermocouples were connected to a thermorecorder to record the temperature readings at regular intervals of 1 min for 1 h.
A water bath
Gesellschaft für Labortechnik, Germany
set at three different temperatures
75, 85, and 95
o
C
was used. Heating profiles of instrument and samples were used to evaluate the process lag time.
3.5 Thermal Treatments
Samples were heated in water bath Gesellschaft für Labortechnik, Germany set at three different temperature 75, 85, and 95
o
C after lag time in isothermal condition. Process time for chemical, textural, and color analysis was
120, 30, and 12 min, respectively. Following the heat treatments, samples were immediately cooled in ice water to minimize quality deteration during cooling.
For chemical analysis tempe and the salt solution were separated and freeze-dried, whereas for physical analysis only tempe were determined.
3.6 Extraction of Samples
The freeze-dried samples were accurately weighed into centrifuge tubes and extracted with 50 acetone and 80 acetone 1:5, wv. Solvents were
selected according to a solvent comparison study by Xu and Chang 2007, which
Thermocouple junction 2 of Salt solution
Sample
12 found 50 acetone extracts exhibited the highest TPC values and 80 acetone
the highest DPPH scavenging capacity and TFC values for yellow soybean. The sample flour-solvent mixtures were vortexed 16 times during 4 h of extraction
according to Ferreira et al. 2011. The tubes were then centrifuged by 5810R Centrifuge Hamburg, Germany at 3000 rpm for 10 min, and the extracts were
stored at 5
o
C in the dark for use.
3.7 Radical DPPH Scavenging Capacity
The DPPH 2,2-diphenyl-1-picrylhydrazyl power of samples were evaluated according to the method of Xu and Chang 2007. The sample extract
0.2 mL was added to 3.8 mL ethanol solution of DPPH radical 0.1 mM. The mixture was vortexed vigorously for 1 min and then kept at room temperature in
the dark for 30 min. The absorbance of samples was measured using a UV-Visible Spectrophotometer U-2900, Hitachi, Tokyo, Japan at 517 nm against ethanol as
a blank. A negative control was mixture of DPPH solution and extraction solvent. The inhibitory percentage of DPPH was calculated according to the following
equation:
Inhibition = [1 – A
sample
A
control
] × 100 The DPPH free radical scavenging capacity was expressed as millimoles
of ascorbic acid equivalent per gram of freeze-dried sample mmol of AAEg. The standard calibration curve range was 10 to 1000
μM R
2
= 0.996.
3.8 Total Phenolic Content TPC
Total soluble phenolics in the extracts were determined using a Folin- Ciocalteu assay as described by Xu and Chang 2007. The sample extract 50
μL, distilled water 3 mL, Folin-
Ciocalteu’s reagents 250 μL, and 7 NaCO3 750
μL were vortexed and allowed to stand for 8 min at room temperature. Thereafter, distilled water 950
μL was added to the mixture and the absorbance was read using the U-2900 Spectrophotometer at 765 nm after 2 h of incubation
against distilled water blank. The TPC was expressed as milligrams of gallic acid equivalents per gram of freeze-dried sample mg of GAEg. The TPC was
expressed as milligrams of gallic acid equivalents per gram of freeze-dried sample mg of GAEg. The standard calibration curve range was 10 to 1000
μM R
2
= 0.998.
3.9 Total Flavonoid Content TFC
The TFC was determined using a colorimetric method described by Xu and Chang 2007. The sample extract 0.25 mL, distilled water 1.25 mL, and
5 NaNO
2
solution 75 μL were mixed in a test tube and kept for 6 min of
incubation. Then, 150 μL of 10 AlCl
3
·6H
2
O solution was added to the mixture. After 5 min, a 0.5 mL of 1 M NaOH and 2.5 mL of distilled water were added and
