Feeding of Brown Seaweed Ecklonia, Ecklonia stolonifera Eisenia bicyclis to Abalone for Accumulation of Phlorotannin

Abstract

Abstract Abalone is one of the most prized sea delicacies worldwide capable of generating high export income. Entirely comprised in the genus Haliotis, these herbivorous marine gastropods have long been utilized as a food source by humans. To further enhance abalone production and to improve the value of the product, some aspects of the quality of the product need to be addressed. In particular, our survey effort in this area have led to three main themes and three sub-themes.These include: (1) feeding of Ecklonia cava to abalone Haliotis discus hannai for accumulation of phlorotannin, (2) feeding of Ecklonia stolonifera to abalone for accumulation of phlorotannin and, (3) feeding of Eisenia bicyclis to abalone for accumulation of phlorotannin. The three sub-themes of our survey are (a) distribution of phlorotannin in the different tissues of abalone (b) diminishing of phlorotannin in the muscle of ablone, and (c) enzymatic degradation of phlorotannins. In attempt to know phlorotannin which could be accumulated in abalone, we isolated phlorotannin from E. cava, E. stolonifera and E. bicyclis by Reverse Phase High Performance Liquid Chromatography (RP-HPLC), and identify them by comparing MS, and 1 H- NMR with published spectral data of known phlorotannin prior to abalone feeding. In attempt to accumulate phlorotannin in abalones, abalones were starved for four days and then fed with E. cava, E. stolonifera, and E. bicyclis. RP-HPLC enables the isolation of phlorotannin compounds from the muscle of abalone H. discus hannai and identification of each phlorotannin by comparing its MS, and 1H - NMR with published spectral of known compound as P13, 7-phloroeckol , and eckol. The first part of this study based on accumulation of phlorotanni in the abalone by feeding with E. cava has shown accumulation of phlorotannin in abalone ranged from 0.278±0.095 to 1.617±0.300 mg/g dw for P13, from 0.021±0.021 to 0.831？0.260 mg/g dw for 7-phloroeckol, and from 0.096±0.096 to 0.30±0.036 mg/g dw for eckol. Abalone fed with L. japonica as reference has only showed the accumulation of P13 at very low rate. Investigation on distribution of phlorotannin in the different tissues of abalone has shown accumulation of the compound P13 (1.617±0.300 mg/g dw), 7-phloroeckol (0.831±0.260 mg/g dw), and eckol (0.300±0.036 mg/g dw) in the muscle of E. cava fed abalone. In the gut, compound P13 (0.347±0.042 mg/g dw) was accumulated, except 7-phloroeckol and ecklol. In the gonad, P13 (0.515±0.040 mg/g dw), and 7-phloroeckol (0.076±0.010 mg/g dw), were accumulated except eckol. In the heart, compound P13 (0.072±0.090 mg/g dw) was accumulated, except, 7-phloroeckol and eckol. In the study of diminishing of phlorotannin in the muscle of abalone during 20 days feeding with E. cava and then replaced with L. japonica for 7 days, has revealed diminishing of all accumulated phlorotannini. L. japonica fed abalone has shown a diminishing of compound P13, from 1.617±0.300 mg/g dw to 0.075±0.030 mg/g dw during 7 days, 7-phloroeckol from 0.831±0.260 mg/g dw to 0.031±0.031mg/g dw during 3 days, and eckol from 0.30±0.036 mg/g dw to 0.057？0.057 mg/g dw during 5 days respectively. The 20 days feeding of abalone and starved for 7 days has shown diminishing of compound P13 from 1.617±0.300 to 0.105±0.008mg/g dw during 7 days, 7-phloroeckol from 0.831±0.026 to 0.022±0.008 mg/g dw during 5 days, and eckol from 0.300±0.036 to 0.011±0.011 mg/g dw during 5 days respectively. The second part of our study on accumulation of phlorotannin in the abalone by feeding with E. sotlonifera has showed accumulation of compound P13, 7-phloroeckol, and eckol. Accumulation of phlorotannin ranged from from 0.329±0.110 to 1.720±0.120 mg/g dw for compound P13, from 0.021±0.021 to 0.580±0.130 mg/g dw for 7-phloroeckol, and from 0？0 to 0.272±0.080 mg/g dw for eckol respectively. Investigation of distribution of phlorotannin in the different tissues of abalone by feeding with E. stolonifera has shown the accumulation of compound P13 (1.640±0.170 mg/g-dw), 7-phloroeckol (0.021±0.021 mg/g-dw), and eckol (0.246±0.071 mg/g dw) in the muscle of abalone respectively. In the gut compound P13 (0.525±0.110 mg/g dw) was accumulated, except 7-phloroeckol and eckol. In the gonad compound P13 (0.477±0.070 mg/g dw), and 7-phloroeckol (0.076±0.010 mg/g dw) were accumulated except eckol. In the heart compound P13 (0.115±0.010 mg/g dw) was accumulated, except 7-phloroeckol and eckol. In the study of diminishing of phlorotannin in the muscle of abalone during 20 days feeding with E. stolonifera and then replaced with L. japonica for 7 days, has revealed diminishing of all accumulated phlorotannini. L. japonica fed abalone has shown a diminishing of compound P13 from 1.640±0.170 to 0.075±0.039 mg/g dw during 7 days, 7-phloroeckol from 0.021±0.021 to 0.013±0.013 mg/g dw during 2 days, and eckol from 0.246？0.071 mg/g dw to 0.017±0.017mg/g dw during 3 days respectively. The 20 days feeding of abalone and starved for 7 days has shown a diminishing of compound P13 from 1.640±0.017 to 0.114？0.030 mg/g dw during 7 days 7-phloroeckol from 0.021±0.021 to 0.013±0.013 mg/g dw during 2 days, and eckol from 0.246±0.071 to 0.004？0.004 mg/g dw during 4 days respectively. The third part of this study was focusing on the feeding of E. bicyclis to abalone for accumulation of phlorotannin and has shown accumulation of compound P13, 7-phloroeckol, and eckol in the muscle of abalone. Accumulation rate of each phlorotannin compound in the muscle of abalone ranged from 0.24±0.037 to 1.610±0.016 gmg/g-dw for compound P13, from 0.021±0.021 to 0.021±0.021 mg/g dw for 7-phloroeckol, and from 0.008±0.008 to 0.208±0.051 mg/g dw for eckol. Investigation on distribution of phlorotannin in the different tissues of abalone by feeding with E. bicyclis has revealed accumulation of compound P13 (1.610？0.016 mg/g-dw), 7-phloroeckol (0.021±0.021 mg/g dw), and eckol (0.208？0.051 mg/g dw) in the muscle of abalone. In the gut compound P13 (0.236±0.09 mg/g dw) was accumulated except 7-phloroeckol and eckol. In the gonad compound P13 (0.496±0.020mg/g dw) was accumulated except 7-phloroeckol and eckol. In the heart compound P13 (0.082 ±0.011 mg/g dw) was accumulated except, 7-phloroeckol, and eckol. In the study of diminishing of phlorotannin in the muscle of abalone during 20 days feeding with E. bicyclis and then replaced with L. japonica for 7 days, has shown a diminishing of compound P13 , 7-phloroecklol, and eckol. L. japonica fed abalone has also shown a diminishing of compound P13 from 1.610？0.016 to 0.090±0.080 mg/g dw during 7 days, 7-phloroeckol from 0.021±0.021 to 0.013±0.013 mg/g dw during 2 days, and eckol from 0.208±0.051 to 0.013±0.101 mg/g dw during 3 days. The 20 days feeding of abalone and starved for 7 days has showed diminishing of compound P13 from 1.610±0.016 to 0.157±0.052 mg/g dw during 7 days, 7-phloroeckol from 0.021±0.021 to 0.003±0.003 mg/g dw during 2 days, and eckol from 0.208±0.051 to 0.014？0.014 mg/g dw during 4 days respectively. In the study of enzymatic degradation of phlorotannin, the highest phlorotannin content seaweed E .cava was fed to abalone as source of enzyme in the study of enzymatic degradation of phlorotannin from E. cava, E. stolonifera and E bicycis. Phlorotannin poor seaweed L. japonica was also fed to abalone as source of enzyme. In the case of the enzyme from the muscle of E. cava fed abalone, the 4 hours of enzyme activities has revealed decreasing of the amount ofcompound P13 from 2.476±0.296 to 1.615±0.296 mg/mL during 4 h, 7-phloroeckol from 0.088±0.012 to 0±0 mg/mL during 1 h, and eckol from 0.441±0.004 to 0.236±0.118 mg/mL during 4 h. Enzyme from the muscle of L. japonica fed abalone has shown the decreasing of the amount of compound P13 from 2.355±0.086 to 0.828±0.014 mg/mL during 4 h , 7-phloroeckol from 0.050±0.025 to 0±0 mg/mL during 2 h, and eckol from 0.294±0.147 to 0.092±0.046 mg/mL during 4 h. According to overcome of the current study, it can be concluded that accumulation of phlorotannin in the abalone is possible by feeding abalone with phlorotannin-rich brown seaweeds.

Keywords: Abalone, accumulation, diminishing, distribution, Enzyme, E. cava, E. bicyclis, E. stolonifera, H.discus hannai, high performance liquid chromatography (HPLC), phlorotannin.