Effects of acetic acid on efficiency of collagen extraction from jellyfish Rhopilema hispdium (Vanhöffen, 1888)

Nội dung text: Effects of acetic acid on efficiency of collagen extraction from jellyfish Rhopilema hispdium (Vanhöffen, 1888)

1. Vietnam Journal of Marine Science and Technology; Vol. 21, No. 1; 2021: 57–65 DOI: Effects of acetic acid on efficiency of collagen extraction from jellyfish Rhopilema hispdium (Vanhửffen, 1888) Pham Thi Kha, Pham The Thu*, Tran Manh Ha, Chu Van Thuoc, Le Ba Hoang Hiep Institute of Marine Environment and Resources, VAST, Vietnam *E-mail: thupt@imer.vast.vn Received: 21 November 2020; Accepted: 28 March 2021 â2021 Vietnam Academy of Science and Technology (VAST) Abstract Collagen is an extremely important fibrous protein in the body. It is the main structural material of all tissues: Skin, bones, ligaments, tendons, cartilage. The source of traditional collagen production is mainly from the skin and bones of animals. However, marine animals are currently a promising alternative source of materials with low risk of disease transmission, no religious barriers to consumption, abundant raw materials, and high-efficiency extraction. In present study, the effects of several factors: Acetic acid concentration, the ratio of jellyfish and acetic acid solution (w:v), as well as the time of extraction on efficiency of collagen extraction process from jellyfish (Rhopilema hispidum Vanhoffen, 1888) were investigated. The results showed that the collagen which was extracted from jellyfish in 0.1 M acetic acid, with the ratio 1:3 between the jellyfish and acetic acid solution (w:v) in 5-day extraction had high extraction efficiency. Extracted collagen in present study was mainly type I collagen, consisting of 3 polypeptide chains: β chain (~ 250 kDa), α1 chain (~ 40 kDa) and α2 chain (~ 100 kDa). Keywords: Rhopilema hispidum, collagen, jellyfish, extraction. Citation: Pham Thi Kha, Pham The Thu, Tran Manh Ha, Chu Van Thuoc, Le Ba Hoang Hiep, 2021. Effects of acetic acid on efficiency of collagen extraction from jellyfish Rhopilema hispdium (Vanhửffen, 1888). Vietnam Journal of Marine Science and Technology, 21(1), 57–65. 57
2. Pham Thi Kha et al. INTRODUCTION jellyfish with 986,880 tons, which are Collagen is the most abundant protein in distributed in shallow water along the coast of vertebrates making up approximately 20–30% the Gulf of Tonkin, with depth < 20 m, salinity of total protein [1, 2]. Collagen is a major < 31.0‰ and temperature of surface seawater < structural material of all connective tissues: 26oC. The average density of white jellyfish in Skin, bones, ligaments, tendons, and cartilage, the western coastal area of the Gulf of Tonkin as well as the interstitial tissues of all reached 2,218 individuals per square meter parenchymal organs [3]. All collagens have a (38.1 tons/km2) and in the Central Coast was triple helical structure composed of three 506 individuals/km2 (5.8 tons/km2) [11]. polypeptide chains (a chains) with a repeated Therefore, this paper provided initial results sequence of three amino acids, glycine-X-Y, in on acetic acid’s effect on collagen extraction which X and Y are mostly proline and efficiency from white jellyfish (Rhopilema hydroxyproline [3]. Currently, collagen and its hispidum), contributing to establishing the denatured form (gelatin) have been widely used collagen extraction process from jellyfish in in the food, pharmaceutical, biotechnology, Vietnam. biomedical and cosmetic industries [4]. Traditional raw materials in the collagen MATERIALS AND METHODS production are mainly from the skin and bones Materials of terrestrial animals such as cows and pigs [5]. Jellyfish were collected directly from However, use of porcine and bovine collagen fishing boats in Do Son - Hai Phong area in poses the risk of transmitting diseases such as March 2019. Jellyfish were washed, bovine spongiform encephalopathy (BSE), refrigerated, and brought immediately to the transmissible spongiform encephalopathy laboratory for analysis. The species was (TSE), and foot and mouth disease (FMD) [4, identified based on references such as Mayer 6]. In addition, for religious reasons, Muslims (1910); Kramp (1961), Omori et al., (2001), and Hindus do not consume products from Nishikawa et al., (2008), Thu et al., (2009) hedgehog or cows. Therefore, collagen from [12–17]. marine organisms is a promising alternative, Methods because of a low risk of disease transmission, Characteristics of the jellyfish material no religious barriers to consumption, abundant Some characteristics of jellyfish material raw materials, and higher extracting efficiency were determined, consisting moisture content, compared to other raw materials [7]. In the protein content, lipid content, ash content, and world, research on isolation and extraction of carbohydrate content. collagen from jellyfish has been of interest Moisture (g/100 g) was determined since 2000 [8, 9]. Recently, studies have shown according to TCVN 3700-90; the sample was that collagen derivatives from jellyfish were dried at 105–110oC, then determined by weight effective in preventing and curing rheumatoid method [18]. arthritis, osteoarthritis and osteoporosis, high Protein content (g/100 g) was determined blood pressure and anti-fatigue effects [10]. according to TCVN 3705-90; the sample was However, there has not been any published distilled by Kjeldahl equipment to convert all result on collagen extraction from jellyfish in + + forms of nitrogen (N-T) to NH4 , NH4 is Vietnam so far. complex indophenol with sodium hypochlorite, On the other hand, there were abundant and phenol, and alkaline citrate in the presence of diverse jellyfish resources in Vietnam with 128 sodium nitroprusside as a catalyst. The + recorded species, in which four species had concentration of NH4 was measured by high economic value, consisting white jellyfish indophenol complexing spectroscopy. Protein (Rhopilema hispidum), red jellyfish (Rhopilema content = N-T ì 6.25 (consider protein esculentum) and two other species (Crambione containing 16% as nitrogen) [19]. mastigophora and Lobonema smithii). The The lipid content (g/100 g) was estimated reserve was mainly from white determined according to TCVN 3703-2009; the 58
3. Effects of acetic acid on efficiency of collagen sample was extracted by Soxhlet equipment, Carbohydrate content (g/100 g) = 100 – then the solvent was evaporated, dried at 100oC moisture – protein – lipid – ash and then the residue content was determined by weight method [20]. Experimental design The ash content (g/100 g) was determined Collagen from jellyfish was extracted according to TCVN 5105-2009; the sample was according to the method of Nagai et al., (1999) heated with moisture at 500–600oC and then [22] with some changes fitted with the determined by weight method [21]. experimental conditions, figure 1. Fresh jellyfish B1. Washing, cutting 5x5cm, immersion in NaCl NaCl 5% & 12% for 2 & 6 days → to reduce water Salted jellyfish B2. Desalted jellyfish, immerse in NaOH to remove non- NaOH collagen substances:  Concentration = 0,025M; 0,05M; 0,075M; 0,1M  Time = 12h, 24h; 36h and 48h Treated jellyfish with NaOH B3. Extracted by CH3COOH:  Concentration = 0,1M; 0,2M; 0,3M; 0,5M CH3COOH  Ratio acid : jellyfish = 1:2; 1:3; 1:4; 1:5  Time = 1 day; 3 days; 5 days and 7 days Extracted solution B4. Centrifuge to obtain suspended solution:  At 4000 rpm for 20 min in 40C Extracted solution B5. Centrifuge to obtain precipitate: NaCl  At 4000 rpm for 20 min in 40C Collagen precipitate s Quantify collagen precipitate:  Determine extraction efficient _ H (%) B6: Purification and lyophilization Collagen Figure 1. Diagram of research and experiment The experiment to remove non-collagen The experiment to extract collagen by acid substances by NaOH: acetic: After removing viscosity, de-salting, and After removal of non-collagen substances removing part of water, jellyfish was treated by NaOH, collagen was extracted from jellyfish with NaOH to remove non-collagen substances at different NaOH concentrations: 0.025 M; sample by CH3COOH with different 0.05 M; 0.075 M; 0.1 M and at different times: experiments to optimize the extraction 12 hours; 24 hours; 36 hours and 48 hours. efficiency (H) as follows: 59
4. Pham Thi Kha et al. Investigation on the effect of acetic acid without b-mercaptoethanol and heated for 5 concentration (factor A) with experiments at min at 95oC. Each sample (20 àg protein) was different concentrations: A1 = 0.1 M; A2 = 0.2 loaded into a well and run at 80 V for 10 min, M; A3 = 0.3 M; A4 = 0.5 M. followed by 120 V for 1.5 h. Following Investigation on the effect of the ratio of electrophoresis, the gel was stained in a 0.05% jellyfish mass to volume of acetic acid solution (w/v) Coomassie brilliant blue R-250 solution - w:v (factor B) with experiments at different in 15% (v/v) methanol and 5% (v/v) acetic ratios: B1 = 1:2; B2 = 1:3; B3 = 1:4; B4 = 1:5. acid. The molecular weight and composition of Investigation on the effect of the jellyfish collagen protein were determined when extraction time (factor C) with experiments at compared with the standard protein (marker) different times: C1 = 1 day, C2 = 3 days, C3 = whose size ranged from 75 kDa to 250 kDa. 5 days, C4 = 7 days. Data analysis Sodium dodecyl sulfate polyacrylamide gel All experiments were performed in electrophoresis (SDS-PAGE) triplicate, and data were presented as means ± SDS-PAGE was performed according to SD. All experimental data were processed on the method of Laemmli (1970) [23] using a 8% Microsoft Excel software. A probability value resolving gel and a 4% stacking gel on vertical of P ≤ 0.05 was considered to be significant electrophoresis (omniPAGE WAVE Maxi and analysis of variance (ANOVA) was System) from Cleaver Scientific in a buffer of performed to test for significant differences 0.1% SDS, 0.025 M Tris and 0.192 glycine. between experiments. The samples were then mixed with buffer at the The f collagen extraction efficiency (H) sample buffer ratio of 1:1 (v/v) with and was calculated by the following formula: Weight of wet collagen H %100 (gram) collagen Weight of treated jellyfish RESULTS AND DISCUSSION Removing non-collagen substances by NaOH Characteristics of jellyfish material De-salted jellyfish materials were The characteristics of jellyfish materials experimented to remove non-collagen were shown in table 1. substances by NaOH at different concentrations (0.025 M; 0.05 M; 0.075 M; 0.1 M). The ratio Table 1. The characteristics of jellyfish materials of jellyfish:NaOH solution was 1:10 (w/v), experimental time was from 12 to 48 hours No. Ingredients Content (% wet weight) with stirring at the temperature from 5oC to 1 Moisture 97.100 ± 0.099 10oC to limit the growth of microorganisms and 2 Lipid 0.150 ± 0.014 denatured collagen. Finally, the jellyfish 3 Protein 1.960 ± 0.015 materials were washed by distilled water and 4 Ash 0.790 ± 0.007 examined for the sensory properties; the results 5 Carbohydrate Not detected were shown in table 2. The process of treating jellyfish with salt and NaOH solution is an essential step, NaOH The results in table 1 showed that jellyfish not only removes non-collagen substances but was composed mainly of water with also breaks down H-H bonds between 3 helix proximately 97% content, followed by protein chains of collagen, to increases the solubility of (proximately 2%), meanwhile fat (lipid), ash collagen in solvents. The experimental results and carbohydrate content was very low. This in table 2 showed that the NaOH concentration result was also consistent with previous of 0.5 M and processing time of 36 hours gave researches; jellyfish mainly consisted of water the best efficiency in treating jellyfish samples and protein [12]. with the characteristics: Yellowish and soft. 60
5. Effects of acetic acid on efficiency of collagen Table 2. Characteristics of jellyfish materials after removing non-collagen substances by NaOH NaOH concentration 0.025 M 0.05 M 0.075 M 0.1 M Time 12 h Yellowish, tough Yellowish, tough Yellowish, tough Yellowish, viscous 24 h Yellowish Yellowish, tough Yellowish, hard Yellowish, viscous Yellowish, Yellowish, 36 h Yellowish Yellowish, soft slightly viscous viscous Yellowish, Yellowish, Yellowish, 48 h Yellowish slightly viscous slightly viscous viscous Extraction of collagen by acetic acid of jellyfish:acetic acid solution and extraction The experimental results of assessment of time on collagen yield efficiency were shown the effect of acetic acid concentration, the ratio in figure 2. Figure 2. Effect of acetic acid concentration, the ratio of jellyfish:acetic acid solution (w:v) and extraction time on collagen yield efficiency Figure 2 showed that collagen extraction efficiency because collagen precipitates at the efficiency which gradually increased over isoelectric point (pH = pI); when the collagen extraction time with 1 day, 3 days, 5 days, and molecule is not electrically charged, without 7 days was 0.79, 4.94, 7.80 and 7.89%, electrostatic repulsion it will be easy to respectively. However, the extraction coagulate or precipitate. When pH ≠ pI, efficiency from fifth day to seventh day collagen molecules which are electrically increased slightly (0.09%). In particular, the charged with the same sign would repel each results of ANOVA test (table 3) also showed other, cause challenge to precipitate. Therefore, that the extraction efficiency in 5 days and 7 it is imperative to find the right concentration days was not significantly different (P > 0.05), of acetic acid in collagen extraction. The acetic while the extraction efficiency in 1 day and 3 acid concentrations were tested (figure 2), the days was significantly different from other variation of extraction efficiency tended not to experiments. Therefore, the 5-day extraction be much. However, the extraction efficiency time should be selected for the collagen was highest in the experiment at the acetic acid extraction from jellyfish. concentration of 0.1 M and extraction time of 5 The acetic acid solution concentration days (11.83%), although no significant significantly affected collagen collection difference was found between experiments 61
6. Pham Thi Kha et al. (ANOVA, P > 0.05). But, minimization of and 7 days was 11.83 and 12.01%, respectively. acidity in extraction is also consistent with the On the other hand, the collagen extraction economic and environmental criteria. Thus, efficiency at the 1:3 ratio was also significantly the 0.1 M acetic acid concentration should different from other experiments (ANOVA, P also be selected for the collagen extraction < 0.05, table 3). Therefore, the ratio 1:3 from jellyfish. between jellyfish and the acetic acid solution The results of experiments at four levels of should also be selected for collagen extraction the ratio between the jellyfish and the acetic from jellyfish. acid solution (w:v) (figure 2) showed that the Thus, the concentration of acetic acid 0.1 collagen extraction efficiency was the highest M, the ratio 1:3 (w:v) between the jellyfish and at the 1:3 ratio with the average efficiency the acetic acid solution, and the extraction time ranging from 6% to 7%, especially the collagen of 5 days were suitable for collagen extraction extraction efficiency at extraction time 5 days from jellyfish. Table 3. Results of one way ANOVA test between experiments (P-value) Extraction time (day) P-value C = 1 day C = 3 days C = 5 days C = 7 days C = 1 day + + + C = 3 days 2.7E-06 + + C = 5 days 3.16E-06 0.000641547 - C = 7 days 2.76E-07 7.55467E-05 0.845355976 The ratio of jellyfish:acetic acid (w:v) P-value 1:2 1:3 1:4 1:5 1:2 + - - 1:3 0.000631 + + 1:4 0.031371 0.002802476 - 1:5 0.044921 0.003260603 0.901289334 The molecular weight of collagen by SDS- chains (β, α1, α2), in which the molecular PAGE weight of α1, α2 chains was higher than that SDS-PAGE could be used to determine the of β chain, this indicated that extracted composition, molecular weight of collagen, collagen from jellyfish was type I collagen. type of collagen, and the purity of product. The Besides, several other studies also showed that analysis results of the extracted collagen in marine invertebrates including the sea urchin present study was shown in figure 3. (Paracentrotus lividus) [27], starfish The SDS-PAGE patterns in figure 3 (Acanthaster planci) [28] and squid showed that in the extracted collagen sample, (Uroteuthis duvauceli) [28, 29] consisted of there were 4 types of proteins with molecular type I collagen. weights of about 250 kDa, 140 kDa, and 100 In addition, the results of SDS-PAGE kDa, which were β chain (band 250 kDa), α also showed that there were still faint colored chain (band α1 - 140 kDa and α2 - 100 kDa) lines along the lands because the extracted and γ chain (over 250 kDa), respectively. This collagen was not purified yet, no or few result was also consistent and similar to some bands had weight less than 100 kDa. The previously published results [24–26]. SDS-PAGE results also indicated that the Moreover, SDS-PAGE results showed that the purity of the extracted collagens was extracted collagen samples consisted of 3 relatively good. 62
7. Effects of acetic acid on efficiency of collagen REFERENCES [1] Lee, C. H., Singla, A., and Lee, Y., 2001. Biomedical applications of collagen. International Journal of Pharmaceutics, 221(1–2), 1–22. S0378-5173(01)00691-3. [2] Addad, S., Exposito, J. Y., Faye, C., Ricard-Blum, S., and Lethias, C., 2011. Isolation, characterization and biological evaluation of jellyfish collagen for use in biomedical applications. Marine Drugs, 9(6), 967–983. Doi: 10.3390/md9060967. [3] Gelse, K., Pửschl, E., and Aigner, T., 2003. Collagens–structure, function, and biosynthesis. Advanced Drug Delivery Reviews, 55(12), 1531–1546. /10.1016/j.addr.2003.08.002. [4] Ogawa, M., Portier, R. J., Moody, M. W., Bell, J., Schexnayder, M. A., and Losso, J. Figure 3. The electrophoretic patterns of N., 2004. Biochemical properties of bone collagen extracted from the jellyfish and scale collagens isolated from the (Rhopilema hispidum): M - Molecular weight subtropical fish black drum (Pogonia marker; 1, 2, 3 were extracted collagen samples cromis) and sheepshead seabream (Archosargus probatocephalus). Food CONCLUSION Chemistry, 88(4), 495–501. The jellyfish that was treated to remove /10.1016/j.foodchem.2004.02.006. non-collagen substances by NaOH at a [5] Jongjareonrak, A., Benjakul, S., concentration of 0.05 M with a processing time Visessanguan, W., Nagai, T., and Tanaka, of 36 hours gave good results. M., 2005. Isolation and characterisation of After treating non-collagen substances with acid and pepsin-solubilised collagens from NaOH, the jellyfish material which was the skin of Brownstripe red snapper extracted at 0.1 M acetic acid, with the ratio 1:3 (Lutjanus vitta). Food Chemistry, 93(3), between the jellyfish and acetic acid solution 475–484. (w:v) in 5-day extraction had high collagen j.foodchem.2004.10.026. extraction efficiency. [6] Song, E., Kim, S. Y., Chun, T., Byun, H. Extracted collagen from jellyfish J., and Lee, Y. M., 2006. Collagen Rhopilema hispidum was a type I collagen, consisting of 3 polypeptide chains: β chain (~ scaffolds derived from a marine source 250 kDa), α1 chain (~ 40 kDa) and α2 chain (~ and their biocompatibility. Biomaterials, 100 kDa). 27(15), 2951–2961. Doi: 10.1016/ j.biomaterials.2006.01.015. Acknowledgements: This work was partially [7] Senaratne, L. S., Park, P. J., and Kim, S. supported by the projects coded: K., 2006. Isolation and characterization of DT.07.19/CNSHCB (funded by project on collagen from brown backed toadfish development and application of biotechnology (Lagocephalus gloveri) skin. Bioresource in the food processing industry to 2020), Technology, 97(2), 191–197. DT.TS.2019.840 (funded by Hai Phong 10.1016/j.biortech.2005.02.024. Department of Science and Technology) and [8] Nagai, T., Worawattanamateekul, W., HNQT/SPéP/15.19 (funded by Ministry of Suzuki, N., Nakamura, T., Ito, T., Fujiki, Science and Technology). K., Nakao, M., and Yano, T., 2000. 63
8. Pham Thi Kha et al. Isolation and characterization of collagen [18] TCVN 3700-90 - Aquatic products - from rhizostomous jellyfish (Rhopilema Method for the determination of moisture asamushi). Food Chemistry, 70(2), 205– content. 208. [19] TCVN 3705-90 - Aquatic products - 8146(00)00081-9. Method for determination of total nitrogen [9] Zhuang, Y., Hou, H., Zhao, X., Zhang, Z., and protein contents. and Li, B., 2009. Effects of collagen and [20] TCVN 3703-2009 - Fish and fishery collagen hydrolysate from jellyfish products - Determination of fat content. (Rhopilema esculentum) on mice skin [21] TCVN 5105-2009 - Fish and fishery photoaging induced by UV irradiation. products -Determination of ash content. Journal of Food Science, 74(6), H183– [22] Nagai, T., Ogawa, T., Nakamura, T., Ito, H188. Doi: 10.1111/j.1750- T., Nakagawa, H., Fujiki, K., Nakao, M., 3841.2009.01236.x. and Yano, T., 1999. Collagen of edible [10] Cao, H., and Xu, S. Y., 2008. Purification jellyfish exumbrella. Journal of the and characterization of type II collagen Science of Food and Agriculture, 79(6), from chick sternal cartilage. Food 855–858. Doi: 10.1002/(sici)1097- Chemistry, 108(2), 439–445. Doi: 10.1016/j.foodchem.2007.09.022. 0010(19990501)79:6 3.0.co;2-n. jellyfish resources in coastal areas of [23] Laemmli, U. K., 1970. Cleavage of Vietnam, propose solutions for structural proteins during the assembly of exploitation and protection. the head of bacteriophage T4. Nature, [12] Peggy, H. Y., Leong, F. M., and Rudloe, 227(5259), 680–685. Doi: J., 2001. Jellyfish as food. Hydrobiologia, 10.1038/227680a0. 451(1–3), 11–17. Doi: [24] Huang, C. Y., Kuo, J. M., Wu, S. J., and 10.1023/A:1011875720415. Tsai, H. T., 2016. Isolation and [13] Nishikawa, J., Thu, N. T., and Ha, T. M., characterization of fish scale collagen 2008. Jellyfish fisheries in northern from tilapia (Oreochromis sp.) by a novel Vietnam. Plankton and Benthos Research, extrusion–hydro-extraction process. Food 3(4), 227–234. Doi: 10.3800/pbr.3.227. Chemistry, 190, 997–1006. [14] Omori, M., and Nakano, E., 2001. /10.1016/j.foodchem.2015.06.066. Jellyfish fisheries in southeast Asia. [25] Sun, L., Hou, H., Li, B., and Zhang, Y., Hydrobiologia, 451(1), 19–26. Doi: 2017. Characterization of acid-and pepsin- 10.1023/A:1011879821323. soluble collagen extracted from the skin of [15] Mayer, A. G., 1910. Medusae of the Nile tilapia (Oreochromis niloticus). world, Vol III. The Scyphomedusae. International Journal of Biological Carnegie Institution of Washington, Macromolecules, 99, 8–14. Washington, DC. 10.1016/j.ijbiomac.2017.02.057. [16] Kramp, P. L., 1961. Synopsis of the [26] Zhang, F., Wang, A., Li, Z., He, S., and medusae of the world. Journal of the Marine Biological Association of the Shao, L., 2011. Preparation and United Kingdom, 40, 7–382. Doi: Characterisation of Collagen from 10.1017/S0025315400007347. Freshwater Fish Scales. Food and [17] Thu, N. T., Ha, T.M., Thu, P.T., Nutrition, 2, 818–823. Doi: Nishikawa, J., 2009. Species 10.4236/fns.2011.28112. composition of scyphozoa medusa and [27] Benedetto, C. D., Barbaglio, A., their distribution in the coastal water of Martinello, T., Alongi, V., Fassini, D., Vietnam. Vietnam Journal of Marine Cullorà, E., Patruno, M., Bonasoro, F., Science and Technology, 9(Suppl.), Barbosa, M. A., Carnevali, M. D. C., and 238–249. Sugni, M., (2014). Production, 64
9. Effects of acetic acid on efficiency of collagen characterization and biocompatibility of crown-of-thorns starfish (Acanthaster marine collagen matrices from an planci). International Food Research alternative and sustainable source: the sea Journal, 20(6), 3013–3020. urchin Paracentrotus lividus. Marine [29] Delphi, L., Sepehri, H., Motevaseli, E., Drugs, 12(9), 4912–4933. Doi:10.3390/ and Khorramizadeh, M. R., 2016. md12094912. Collagen extracted from Persian Gulf [28] Tan, C. C., Karim, A. A., Latiff, A. A., squid exhibits anti-cytotoxic properties on Gan, C. Y., and Ghazali, F. C., 2013. apple pectic treated cells: assessment in an Extraction and characterization of pepsin- in vitro bioassay model. Iranian Journal solubilized collagen from the body wall of of Public Health, 45(8), 1054–1063. 65