Extraction and composition characterisation of amino acids from tung meal

The most desirable content of amino acids (AAs) in the extracted products from tung (Aleurites fordii) meal was 93.88%, which was obtained from shelled tung meal at a hydrolysis temperature of 45°C and a isoelectric precipitation pH value of 4.4. Furthermore, the cytotoxic activity of extracted AAs was also evaluated by MTT assay. Antioxidant activity of extracted AAs was also measured by the DPPH assay. As a result, the high yield of extracted AAs exhibited so low cytotoxic and high antioxidant activity that had the potential use as a functional ingredient.


Introduction
Tung tree ( Aleurites fordii)i se ssentially an ative plant produced from oriental countries, especially China. It can yield tung oil consisting of highly unsaturated fatty acids and af ew saturated fatty acids. The applicationo ft ung oil was increasingly developed in manyfi elds (Wang &P adua 2005; Chene ta l. 2010; Li et al. 2010;Shang et al. 2010;Kundu&Larock 2011).
Free amino acids (AAs)couldb eextracted from somep lants such as potato (Galdó ne ta l. 2010), agrocybe chaxingu (Lee et al. 2011), rice bran (Sereewatthanawutetal. 2008) and gum karaya (Anderson et al. 1985). However, the reported results showed that the highest yield of total AAs was less than 50% of the dried product. To the best of our knowledge, the study on the extraction of high content of AAs from tung meal as the nutritiveadditive in feedstuffs has rarely been reported.

2R esults anddiscussion 2.1 Composition analysis of deoiled tung meal
According to the results, the percentage content of crude proteini nt unnelling microscopy (STM) reached 40.20%,w hile the percentage content of crude protein in ultrafast scanning tunneling microscope (USTM) was only 17.30%.T he contents of crude proteini nS TM were obviously higher than those in USTM.

Effectoftemperature and pH value
From Figure S1a it can be seen that the contentsoftotal AAs extracted from STM increased with the increaseo fp Hv aluef rom 3.0 to 4.4, especially the content of AAs hydrolyseda t4 5 8 C increased and the percentages of AAs reached 93.88% at ap recipitation pH value of 4.4.T he contentsoftotal AAs extracted from USTM showed the same changedtrend as thoseextracted from STM with the increaseofpHvalue as seen in Figure S1b. The reason that the amount of AAs was highest at ahydrolysistemperature of 458 Cmay be that Maillardreaction couldoccur betweencarbonyl of sugar and amidogenofAAs during the hydrolysis of tung meal at ahigher hydrolysing temperature (Koutinas et al. 2007;Goldmann et al. 2009) and therefore couldn ot provide enough energy for hydrolysis of crude protein at al ower hydrolysing temperature. In addition,wecan see from FigureS2that the hydrolysis temperature and precipitation pH had obvious effect on yield of AAs extracted from tung meal. Table S1 shows the composition of AAs extracted from STM and USTM at ah ydrolysis temperature of 458 Cand aprecipitation pH value of 4.4. It can be seen that the contents of AAs exceptfor Cys extracted from STM were generally high, and especially percentages of Thrand Arg exceeded 10%. However, at the same extraction condition the content of total AAsextracted from USTM was reduced to 72.45%.I ts uggests that the composition of extracted AAs was directly influenced by the shelloftung kernels containing crude fibre during hydrolysis (Buň ka et al. 2009).

Composition of AAs
In order to further analyse the composition of extracted products, Figure S3 shows GC-MS chromatograms of the fatty acids in tung meala nd extracted AAs. According to the area calculation of chromatographic peaks, the contents of a -C 18:3 and unsaturated fatty acids in fatty acids of STM were 80.30% and 95.05%,respectively. However, it cannot be obviouslyseen that the peaks of residualfatty acids in AAs extracted from STM from FigureS3a. By contrast, the content of residual fatty acids in AA extracted from USTM reached 2.59% as seen in Figure S3b. In addition,t he contents of a -C 18:3 and unsaturated fatty acids in fatty acids of USTM were 75.64% and 93.84%,r espectively. Figure S4 shows viability of Caco-2 cells incubated with AAsextracted from STM and USTM at at emperature of 458 Ca nd pH value of 4.4. There is evidence that the cell viability increased before 8h incubation with AAsand reduced subsequently. Viability of Caco-2 cells incubated with AAs extracted from STM at 8and 48 hwas 122% and 86%. However, the viabilityofcells incubated with AAs extracted from USTM was lower than that incubated with AAs extracted from STM at the same incubated time.For example, the cell viability increased from 106% to 109% with the increase of incubated time from 4t o8h, and decreasedt o7 3% after 48 h incubation.

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X. Zheng et al. 850 2.5 Antioxidant activityofA As As showninFigureS5, DPPH free radical scavenging activity and hydroxyl radical scavenging activityofAAs extracted from STM at atemperature of 458 Cand pH valueof4.4 was 78.5% and 70.3%.However, the two scavenging activity of AAs extracted from USTM at atemperature of 458 Ca nd pH value of 4.4 was 70.7% and 60.4%,r espectively.
In addition, antioxidant activitya ssay of AAs extracted from tung meala so ne of the byproducts of tung tree showst he important result that the extracted AAsa re particularly suitable for use as nutritivea dditive in feeds or foods, also as ingredient in medicine and cosmetics (Carmona-Jimé nez et al. 2014). Results of the DPPH assayf reer adical scavenging activitya nd hydroxyl radical scavenging activity of AAs extracted from STM and USTM at a temperature of 458 Ca nd pH value of 4.4 all showed the antioxidant activity.

Conclusion
According to our results, the percentages of total AAs and essential AAs in the products extracted from STM at ah ydrolysis temperature of 458 Ca nd ap recipitationp Hv alue of 4.4 reached 93.88% and 45.46%,r espectively. Moreover,b ased on the desirable cytotoxicity and antioxidant activityofthe extracted AAs, tung meal should be regarded as agood byproduct of tung tree for supplying the functional ingredient.

Supplementarymaterial
Supplementary materials associated with this article are available online at http://dx.doi.org/ 10.1080/14786419.2015.1062007.

Disclosures tatement
No potential conflict of interest was reported by the authors.