Faisal, Md (2019) Impact of different processing methods on immunogenicity of prawn. PhD thesis, Victoria University.

Abstract

Prawn allergy is one of the most common seafood-borne allergies affecting more than 2% of the global population resulting in significant socio-economic problems to the society and prevention is by avoidance. Not only are some consumers affected by prawn allergy, but also the seafood industry workers (7 to 36%) related to different processing stages. The impact of different processing techniques was assessed to attenuate antigenicity of banana prawn tropomyosin (Fenneropenaeus merguiensis). Frying and boiling at different temperatures, acid treatment using different acids, and storage at - 20 °C up to 3 months were investigated. Untreated prawn sample was used as control. Frying significantly increased antigenicity (6 to 8 times) in temperature dependent manner, whereas the trend was fundamentally reversed with boiling. Boiling at 121 °C resulted in the lowest antigenicity (12.99 mg mL-1) among all heat-treated samples, yet higher than the control (5.06 mg mL-1). Freezing had initially very minor impact, although prolonged storage at -20 °C increased antigenicity slightly (2.29 mg mL-1) compared to control. Antigenicity was impacted the most by reduction in pH independent on type of acid, since both acetic and HCl acids significantly reduced antigenicity of tropomyosin by ~90% compared to control. This could be considered as a new approach in processing that may potentially reduce tropomyosin derived antigenicity in prawns and prawn products. Furthermore, changes in tropomyosin derived antigenicity of banana prawn due to high pressure processing (HPP) at 600 MPa for 5 and 10 min at various temperatures (40, 80, 120 °C) were also investigated. HPP of prawn samples at 40 and 80 °C for 5 min increased tropomyosin derived antigenicity by almost double, whereas HPP at 120 °C for 10 min decreased antigenicity by 65%, detected using enzyme linked immunosorbent assay (ELISA) kit. A significant (P ≤ 0.05) reduction of tropomyosin antigenicity after pepsin digestion was noticeable in prawns after HPP, but not in control prawn sample. However, further digestion of the control and HPP sample with pancreatin enzyme decreased antigenicity to ~0 mg mL-1. The combination of HPP and high temperature (120 °C) in the current study can potentially reduce tropomyosin- derived antigenicity in whole prawn muscle, whereas Simulated Intestinal Fluid digestion with pancreatin enzyme may present a new prospective method to produce hypo- antigenic, enzymatically digested prawn products. Moreover, the current research implementing a new methodology using unstained gel, confirmed the actual molecular weight of banana prawn tropomyosin to be 37 kDa. Finally, the research has explored the immunogenicity (T-helper cell-mediated immune response in vitro) of processed banana prawn proteins using human peripheral blood mononuclear cells (PBMCs) and then characterized these allergenic proteins using Liquid chromatography with tandem mass spectrometry (LC/MS/MS). Prawn muscles were treated with acetic acid and high-pressure processing (600 MPa) separately to analyse their antigenicity and immunogenicity. The protein fractions were separated and isolated using preparative high-performance liquid chromatography (Prep-HPLC), and their antigenicity was analysed using IgG ELISA kit. Out of 39 protein fractions only four (A10, A11, B10 and C9) were detected with antigenic potentials. The immunogenicity of these protein fractions was analysed using human PBMCs, and supernatants were collected at multiple times from 0 to 144 h. The treated fractions (B10 and C9) showed significantly (P < 0.05) lower pro- and anti-inflammatory cytokine production compared to control (A10). The allergenic fractions were characterized using an LC/MS/MS, which identified 9 proteins. Among these, six proteins (tropomyosin, arginine kinase, hemocyanin, enolase, vitellogenin and 14-3-3 zeta) have been established as allergenic in prawn muscle and ovaries. Other three proteins (Beta-1,3- glucan binding protein, translationally controlled tumor protein and farnesoic acid O- methyltransferase short isoform protein) identified in this study, need further investigation for their immunogenic properties.

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