Amino acid mixture

A stirred tank reactor-STR and SCBBR was bought from Bioengineering Inc, Germany. Spectrophotometer, GC column CP-WAX 58, Gas chromatography HP 5890 series II, where from Hewlett-Packard, Avondale, PA, USA. All other chemicals, including yeast extract nitrogen base without amino acid, ammonium acetate, amino acid mixture, sodium alginate, calcium chloride, sodium chloride, chitosan, hydrochloric acid, potassium sodium tartrate, dinitro salicylic acid (DNS), n-butanol, ethyl acetate, glucose, peptone, yeast extract, agar, sodium hydroxide were of analytical grades and purchased directly from Sigma (USA) and Applichem (Germany).

In short, 10 mg MRPI was hydrolyzed in 10 mL 6 M HCl at 110 °C for 22 h under nitrogen gas. Distilled water was added to the hydrolysate to adjust the volume to 50 mL. One milliliter of hydrolysate was then dried using a vacuum desiccator at 45 °C and redissolved in 2 mL water. This step was repeated twice. Ultimately, the hydrolyzed protein was dissolved in 1 mL sodium citrate buffer (pH 2.2), which was used for the determination of amino acid composition.
The identification and quantification of amino acids were carried out using an automatic amino acid analyzer (Biochrom 30+ series, Biochrom, Cambridge, UK) with an amino acid mixture (Sigma-Aldrich, St. Louis, MO, USA) as a standard. The amino acid content was calculated using the following formula: $X=\frac{c\times f\times V\times M}{m\times {10}^9}\times 100$
where X is the amino acid content (g/100 g protein), c is the amino acid concentration (nmol/μL), f is sample dilution factor, V is volume of hydrolyzed sample (mL), M is molecular weight of the amino acid, and m is the weight of sample (g).
The EAAI was calculated according to a previous study [30 (link)] using the following formula: $EAAI=\sqrt[n]{\frac{aa1}{AA1}\times \frac{aa2}{AA2}\times \cdots \times \frac{aan}{AAn}}$
where aan and AAn are the essential amino acid content over total protein content (mg/g) in the sample and FAO/WHO reference [29 ], respectively.