7 mL H
2
SO
4
were added, also boiling chips which pass No. 10 sieve. Test portion was digested 1 h after all H
2
O is distilled and acid comes to true boil. Minimum volume of H
2
O was added to dissolve solids, cooled, and thin film of petroleum jelly placed on rim of flask. Digested and boiling chips
transferred to distillation apparatus and flask rinsed 6 times with 2 mL portions H
2
O. Under condenser with tip extending below surface of solution was placed 125 mL Erlenmeyer contained 5 mL saturated H
3
BO
3
solution and 4 drops indicator Added 10 mL NaOH-Na
2
S
2
O
3
solution to still, collected ca 15 mL distillate, and diluted to ca 50 mL. Titrated to end point. Made blank
determination and calculate. Nitrogen content calculated by the equation:
Nitrogen wet basis =
normality HCl × corrected acid volume mL × . g Nmol
weight of wet sample g
× Nitrogen dry basis =
normality HCl × corrected acid volume mL × . g Nmol
weight of dried sample g
× Protein = N × Protein factor
= N × 5.45 combination of coconut and rice protein factor
3.4 Determination of lipid contents, soxhlet extraction
AOAC 963.15, 2007
Fat flask were dried in a hot air oven at 105
o
C for 1 hour and then cooled in desiccator and weighed. Weighed 2 g of sample used fat-free cotton and
wrapped by filter paper Whatman® no 1 then tied with a white thread. Placed thimble containing test sample in soxhlet, supported it with spiral or glass beads.
Rinsed digestion beaker, drying beaker, and watch glass with three 50 mL portions petroleum ether, and added to wash the thimble. Reflux digested test
sample 4 h adjusted heat so that extractor siphons
≥30 times. Removed flask, and evaporated solvent on steam bath. Dried flask at 105°C until it reached its
constant weight. Cooled in desiccator to room temperature and weighed. Calculated fat content by the following equation:
Fat wet basis = a
‒ b c Fat wet basis =
Fat wb ‒ Moisture content wb
× Description :
a = weight of flask and fat g b = weight of flask g
c = weight of wet sample g 2.4.5 Determination of carbohydrate, by difference
Carbohydrate content of the sample was calculated by deducting 100 nutrient samples with moisture content, ash content, protein content, fiber content
and fat content. Its value could be determined by using the following formula:
8 Carbohydrate levels = 100
‒ MC + A + P + F Description:
MC = moisture content A = ash content
P = protein content F = fat content
4 Sensory evaluation Lawless and Heymann, 1998
Sensory analysis was carried out to determine the best sample using thirty five untrained panelists. Three formulations were given to the panelists in frozen
condition. Then, 9-point hedonic scale was used for testing. Es puter was evaluated on the basis of acceptance of appearence, color, aroma, bitterness,
sourness, taste, texture, and overall liking.
5 Consumer test Meilgaard et al., 2007
Consumer test was done to determine the acceptance of es puter final product. The location of consumer test was in the central location of Mae Fah
Luang University, Thailand, with 100 responses.
6 Statistical analysis Meilgaard et al., 2007
SPSS 17.0 and manual calculation were used for data analysis. Total phenolics, anthocyanins, and antioxidant activities data were reported as means +
standard deviations for triplicate analyses of the same sample. Statistic manual calculation was used for paired-samples t-test unfermented black glutinous rice
and fermented black glutinous rice and SPSS was used for One-way Analysis of Variance ANOVA at 95 confidence level formulations of es puter
and results of sensory evaluation.
