The residue of Macadamia after extracting oil were obtained from Key Laboratory of Tropical Crop Products Processing Ministry of Agriculture in China (our own laboratory). Hydrochloric acid (HCl) and sodium hydroxide (NaOH) were procured from Guangdong Guanghua Chemical Factory Co., Ltd. (China). HH-W600 electric heated water bath, 752N spectrophotometer, electronic balance, PHS-25 acidity meter, circulating water vacuum pump and low-speed desktop centrifuge were used in this experiment. All other chemicals used were analytical grade for the experiments and analysis.

Box-Behnken Design (BBD) was used to estimate and optimize the experiment for improving the yield of protein. The optimum parameters were materials to water ratios 1: 91, extraction time 2.5 h, extraction temperature 55˚C and pH 9.0. Under the optimized conditions, macadamia protein was extracted and centrifuged at 5000 rpm for 10 min. Then the supernatant was collected and and pH was adjusted to 4.6 to precipitate protein. After incubation at room temperature for one hour, the content was centrifuged again at 5000 rpm for 10 min and the supernatant was discarded. The protein left in the centrifuge tubes was freezing dried. Parallel experiments were conducted with extraction at pH 11. Extracted proteins before and after freezing dried could be found in Figure 2.

The analysis was carried out in a column packed with 4.6 mm ID * 60 mm Hitachi custom

ion exchange resin and connected with EZChrom Elite software. Analysis time: 30 minutes approximately; Reproducibility of peak retention time: CV 0.3% (Arg), 0.5% (Ala); Reproducibility of peak area: CV 1.0% (Gly, His); Detection limit: 3 pmol (S/N = 2, Asp); Spectrophotometer: Aplanatic concave diffraction grating with 570 nm and 440 nm Analyzer; CPU: 32 bits OS, Windows xp; Operating temperature range: 15˚C to 35˚C; Power supply: 100 - 115 V AC/220 - 240 V AC, 800 VA and over, w/in 50/60 Hz ± 0.5 Hz; N2 gas source must be prepared. Amino Acid Analyzer is composed of these parameter and components.

All experiments were performed in triplicate, and statistical analysis of the biological replicates was conducted using Excel or OriginPro 8.5 (www.originlab.com).

The content of amino acids was explained by the following formula:

where A₁, A₀ represent the peak area of amino acid in sample and standard, respectively; C₀ is the concentration of amino acids in sample (nmol/20μl); N: dilution multiple of sample; M: molecular weight of amino acid; W: mass of sample.

The amino acids scores was explained according to the following formula:

where A: content of essential amino acid in sample; B: content of essential amino acid in the FAO/WHO pattern (1973) [15] .

There was no significant difference in the amino acids analysis of the first and second protein samples from extraction at pH 9 or pH 11. The content of amino acids of macadamia protein and the impact of pH on amino acids composition were investigated in Table 1 and Table 2. The cation-exchange chromatography analysis of the protein samples of pH 9 revealed the AA profile with contribution of 18 AA including 7 essential (Tryptophan was not detectable) and rest non-essential amino acids. The content of 18 AA quantified by cation-exchange chromatography ranged between 6.31 and 164.35 mg/g according to formula 1 (Table 1). The highest content was of Glu 164.35 mg/g followed by Arg 117.77 mg/g, Asp 96.72 mg/g and Gly 67.19 mg/g. It is noticeable that both the acidic amino acids, Glu and Asp and basic amino acid, Arg were among the dominant contributors. And the essential AA, Leu (64.84 mg/g) is present in almost twice amount than the other essential AA quantified (Thr 30.36, Val 37.14, Met 6.31, Ile 29.60, Phe 28.32, Lys 28.06 mg/g). Even though the amount of Trp was not detectable for the method of determination, the relatively high content of AA is much more than amaranth’s [11] . SD of the data was analyzed by Microsoft Excel software and P-value <

^aWM: Molecular weight of amino acid; ^bSD: Standard deviation; ^cESTD: External standard method; ^dRT: Retention Time; *: Essential amino acids; ND: Not detectable; *P-value < 0.05. Data represent mean ± SD (3 biological replicates).

0.05.

The cation-exchange chromatography analysis of the protein samples of pH 11 revealed the content of 18 AA quantified by cation-exchange chromatography ranged between 4.38 and 117.18 mg/g ,and the highest content was of Glu 117.18 mg/g followed by Arg 78.72 mg/g, Asp 63.58 mg/g and Gly 45.47 mg/g (Table 2). Also it is noticeable that both the acidic amino acids, Glu and Asp and basic amino acid, Arg were among the dominant contributors. And the essential AA, Leu (44.45 mg/g) is present in almost twice amount than the other essential AA quantified (Thr 22.31, Val 25.34, Met 4.38, Ile 20.45, Phe 21.51, Lys 19.63 mg/g). The content of all AA reducted substantially, compared with reported in above-mentioned studies. These data support the results that the degradation of AA of macadamia protein for extraction of pH 11 is caused by the impact of pH.

Conformationally, constrained amino acids are a useful way of tailoring the rigidity of peptides [16] . Rafiemanzelat et al. (2012) have developed a degradable monomer based

^aWM: Molecular weight of amino acid; ^bSD: Standard deviation; ^cESTD: External standard method; ^dRT: Retention Time; *: Essential amino acids; ND: Not detectable; *P-value < 0.05. Data represent mean ± SD (3 biological replicates).

on α-amino acid to accelerate hard segment degradation [17] . Additional amino acids in cyclic tetrapeptides are supposed to play important role for effectively inhibiting the histone deacetylases [18] . Thus, the synthes and evaluation of AA play a crucial role in the utilization of AA.

In order to evaluate the degradation of AA of macadamia protein caused by the impact of pH, we introduced this new bubble chart for explaination of AAS. The absence of some essential AA in protein causes other AA difficult to be fully utilized and the overall protein digestibility was reduced. Therefore, nutritional value of protein in food depends on species, quantity and proportion of essential AA. The amount of essential AA, nonessential AA and total AA was also shown in the Figure 3. The amount of essential AA, nonessential AA and total AA from extraction at pH 9 was 22.463%, 63.796% and 86.259%, respectively. And the ratio of amount of essential AA and nonessential AA, total AA was 0.35 and 0.26, respectively. From Figure 3, for pH 9, the AAS of Met + Cys, Ile, Thr, Leu, Val + Tyr was 55.3, 74, 75.9, 92.6, 160.1 according to formula 2. And the corresponding data for pH 11 was 40.9, 51.1, 55.8, 63.5, 111.1. Even though the amount of the latter was less than the former, these related data was better than the AAS of FAO/WHO protein pattern. Moreover, the total AAS of pH 9 and pH 11 sample was 64.2, 56.6 respectively. Although compared with AA from extraction at pH 9, the amount of essential AA, nonessential AA and total AA from extraction at pH

11 was only 19.882%, 39.672% and 59.554%, the ratio of amount of essential AA and nonessential AA, total AA was 0.50 and 0.33. The latter was more close to the FAO/ WHO protein pattern of reference.