Characterization, ultrafiltration, depolymerization and gel formulation of ulvans extracted via a novel ultrasound-enzyme assisted method

Abstract

Sea lettuce, or Ulva spp., dominates global algal biomass and significantly contributes to "green tides.", representing a sustainable source for biomaterials. This study explores an innovative ultrasound-enzyme assisted extraction method with the novel Cellic® CTEC3 enzyme cocktail, applied for the first time in Ulva spp. succesfully enhancing ulvan release and extraction efficiency. Various processing methods, including ultrafiltration and dialysis, were employed to achieve higher ulvan purity. Dialyzation of ulvan resulted in a more purified product with a carbohydrate content up to 55.34 %, a sulfate content up to 21 %, and no glucose contamination. Liquid extracts were fractionated through ultrafiltration, with a 3 kDa MWCO yielding 93.51 % ulvan precipitate, representing 50.28 % of the total extractable ulvan. Sequential ultrafiltration concentrated ulvans but only partially modified their molecular weight distribution. Depolymerization using microwave and H₂O₂ shifted ulvans towards lower molecular weights, reducing high molecular weight residue. HPSEC confirmed pH-dependent aggregation behavior, with all isolated ulvans having molecular weights above 786 kDa. Hydrolysis methods were compared, with 2-hour 1 M TFA hydrolysis at 121 °C providing the best monosaccharide profile of ulvan. FTIR and NMR analyses showed preservation of sulfation. Rheology indicated biopolymeric behavior and stable gel formation. Ulvans demonstrated nutraceutical potential, being suitable for a low Na⁺ and high K⁺ diet, with a Na⁺:K⁺ ratio as low as 0.14, and were rich in Mg²⁺.

Keywords: Carbohydrates; Enzyme; Seaweed; Ultrasound; Ulva; Ulvan.

Graphical abstract

Fig. 1

Flow diagram of preliminary extraction configurations for ulvan obtention with ultrasound and or enzyme-assisted extraction. T.1 and T.2 correspond to a 60 min treatment with or without Cellic® CTec3 cellulase-hemicellulase enzyme mix, respectively; T3 and T4 correspond to a 90 min treatment, also with and without the same enzyme mix.

Fig. 2

Flow diagram illustrating various ulvan obtention pathways, based on the best yielding preliminary treatment (T.3), showing experimental procedures and different processing pathways with corresponding mass balances. BP-3B-R10, BP-3B-R3 and BP-3B-R1, refer to ulvan precipitated from retentate fractions of > 10, 3–10, and 1–3 kDa (MWCOs), respectively.

Fig. 3

Surface morphology of Ulva spp. a-b) show dehydrated Ulva spp. samples, c-f) show solid residues after ultrasound treatment without (c, d) or with (e, f) simultaneous Cellic®CTec3 digestion.

Fig. 4

Ulvan extraction yields from treatment 3 (BP-3B) with centrifugation prior to precipitation, comparing dialysis, enzyme medium, and water extraction solutions. The inset white box in the non-dialyzed ulvan indicates cold-precipitated ulvan. Different letters denote significant mean differences; error bars represent standard deviations.

Fig. 5

A. Flow diagram of membrane fractionation. B. Percentages of precipitated ulvan showing ulvan in fraction relative to d.w. of fraction (in black) or relative to total extractable ulvan from the liquid extract (in grey) or ulvan yield relative to dry weight of Ulva spp. (in white). Different letters indicate significant differences among means of the same group. Bars indicate standard deviation. C. Ethanol-precipitated ulvan from different fractionated liquors, illustrating successful fractionation of ulvans. R: retentate, P: permeate.

Fig. 6

Molecular size distributions based on HPSEC analysis. A: HPSEC spectra of membrane fractions with mobile phase at two different pH. B: HPSEC molecular size distributions of ulvan submitted to various degradation assays. Control: Ulvan BP-3A submitted to no treatment; Acid: pH = 3, t = 10 min; Microwave: 200 °C, H₂O₂ 2 %, t = 20 min; pH of mobile phase = 3.

Fig. 7

Helix-coil transition analysis of the BP-3A and BP-3B ulvans in presence of different NaOH concentrations.

Fig. 8

Antioxidant capacity and total phenolic content of obtained liquors (left) and fractions (right). LFA/B: Liquid fraction from processing option A or B. SE-A/B: Saline extract from processing option A or B. R: Retentate, P: Permeate; 10, 3 and 1 kDa MWCOs. Bars represent standard deviations.

Fig. 9

Fourier transform infrared spectra from ulvans after different extraction procedures, all involving US-assisted extractions at 37 kHz. A. BP-1: 60 min US-treatment, combined with enzymatic digestion, BP-2: 60 min US-treatment, without enzymatic digestion, BP-3A: 90 min US-treatment with enzymatic digestion, BP-4: 90 min US-treatment without enzymatic digestion. B. BP-3B corresponds to ulvan which included a centrifugation step of prior to precipitation. R/P10, R/P3, R/P1 correspond to retentate or permeates from the membrane fraction assay, with MWCOs of 10, 3 and 1 kDa. Continuous and discontinuous lines correspond to non-dialyzed and dialyzed samples, respectively.

Fig. 10

¹H NMR spectra of ulvans extracted from Ulva spp. Relevant peaks are pointed out with arrows. A. BP-1: 60 min US-treatment, combined with enzymatic digestion, BP-2: 60 min US-treatment, without enzymatic digestion, BP-3A: 90 min US-treatment with enzymatic digestion, BP-4: 90 min US-treatment without enzymatic digestion. B. BP-3B corresponds to ulvan of identical extraction method as BP-3A but which included a centrifugation step of prior to precipitation. BP-3B-R3 correspond to ulvan precipitated from the 3 kDa retentate of the membrane fraction assay.

Fig. 11

Representative example of ¹H–¹³C HSQC spectra of crude ulvan BP-3A.

Fig. 12

A. Dispersions of ulvan samples, at different concentrations. BP-3A corresponds to ulvan obtained without a centrifugation step of the extract. BP-3B corresponds to ulvan precipitated after centrifugation of the extract. BP-3B-R1 and −R3 correspond to a 1–3 kDa and 3–10 kDa retentate from a membrane fractionation process, respectively. B. Circles represent BP-3A ulvan, triangles illustrate BP-3B ulvan, and squares show BP-3B ulvan with addition of KCl 0.1 M. Note here that closed symbols represent the loss modulus and open symbols the storage modulus. All samples contain ulvan at 3 % (w/w).