One hour enzymatic synthesis of structural lipid enriched - PowerPoint PPT Presentation

One ‐ hour enzymatic synthesis of structural lipid ‐ enriched unsaturated fatty acids from silkworm pupa oil under microwave irradiation Fuan Wu School of Biotechnology, Jiangsu University of Science and Technology Sericultural Research Institute, Chinese Academy of Agricultural Sciences Zhenjiang 212018, PR China E-mail: fuword@163.com

Background Previous study Present study Conclusions Acknowledg- ments

Background 01 High fat diet Large amounts of saturated fatty acids are ingested and stay in the body Fat composition needs to be adjusted

Transesterification α‐ Linolenic acid triglyceride Oleic acid Linoleic acid Novel structured lipids

Previous study 02 Silkworm pupae biorefinery Structured lipids 1 Biodiesel 2 Silkworm pupa oil Silkworm Unsaturated fatty acid 3 pupae Yeast oil Cultivate yeast Silkworm pupa residue [1]Zhao X Y, Wang X D, Liu X, et al. European Journal of Lipid Science and Technology, 2015, 117(6): 879 ‐ 889. [2]Manzano ‐ Agugliaro F, Sanchez ‐ Muros M J, Barroso F G, et al. Renewable and Sustainable Energy Reviews, 2012, 16(6): 3744 ‐ 3753. [3] Yang L I F, Siriamornpun S, Li D. Journal of Food Lipids, 2006, 13(3): 277 ‐ 285.

APA ‐ style human milk fat analogue from silkworm pupae oil: Enzymatic production and improving storage stability using alkyl caffeates P P P Liu X, Wang J*, et al. Scientific Reports. 2015, 5: 17909.

Structured lipids enriched with unsaturated fatty acids produced by enzymatic acidolysis of silkworm pupae oil using oleic acid R 1 R 3 R 2 Zhao XY, Wang J*, et al. European Journal of Lipid Science and Technology. 2015, 117(6): 879 ‐ 889.

Present study 03 Enzymatic reaction need several hours ！ How to enhance the reaction ? Novel SLs Silkworm pupa oils Problem: Reduce the time of enzymatic transesterification

1h Microwave Lipozyme RM IM irradiation Free FAs 50W 81.04% UFAs glyceride SA PA Po SA ALA LnOLn LLLn Lipozyme LA RM IM LLLn OA OLL Ln ‐α‐ Linolenic acid (ALA) L ‐ Linoleic acid (LA) OOO PA Po SA O ‐ Oleic acid (OA)

Table 1 The content of fatty acids under different treatment. Optimum condition: Fatty acid C16:0 C18:1 C18:2 C18:3 Total fatty acid content (%) 24.55 ± 0.23 34.72 ± 0.06 5.58 ± 0.02 35.15 ± 0.15 SPOSLs a Substrate molar ratio: 1:5 24.71 ± 1.04 35.76 ± 1.23 8.32 ± 0.25 31.20 ± 0.43 MSLs b 24.84 ± 0.65 34.58 ± 0.23 5.54 ± 0.05 35.04 ± 0.37 ESLs c Enzyme loading: 7 % CSLs d 11.85 3.86 51.61 12.79 21.07 ± 0.54 32.52 ± 0.22 6.56 ± 0.31 30.96 ± 2.2 Microwave power: 50 W SSLs e Sn ‐ 2 fatty acid content (%) Microwave temperature: 60 ° C 4.77 ± 2.13 40.65 ± 0.39 5.31 ± 0.16 49.27 ± 1.62 SPOSLs a 3 h 18.41 ± 2.53 38.52 ± 2.62 4.78 ± 0.85 34.65 ± 0.79 MSLs b 3.86 ± 0.41 40.49 ± 1.02 5.53 ± 0.14 50.12 ± 0.73 Time: ESLs c CSLs d 3.59 2.22 42.06 42.38 1 h 3.46 ± 0.23 36.32 ± 1.23 6.61 ± 0.74 54.02 ± 0.61 SSLs e a The extracted oil from silkworm pupa. Sn ‐ 1, 3 fatty acid content (%) B Fatty acids composition of silkworm pupae oil 34.43 ± 1.39 31.75 ± 0.27 5.72 ± 0.10 28.09 ± 1.03 SPOSLs a transesterification product SLs under the optimum condition in microwave reactor. 28.35 ± 3.13 33.47 ± 0.01 10.10 ± 0.43 29.30 ± 1.20 MSLs b c Adding the enzyme (RM IM) without oleic acid into the silkworm pupae oil under microwave irradiation. 35.33 ± 1.05 31.62 ± 0.78 5.54 ± 0.09 27.50 ± 0.60 ESLs c d Fatty acids composition of silkworm pupae oil transesterification product SLs under the optimum CSLs d 16.43 3.68 48.09 28.03 condition from Zhao et al, 2015. 30.49 ± 0.74 28.72 ± 2.21 6.63 ± 0.75 22.93 ± 1.35 e The silkworm pupae oil used in shaker bath reactor. SSLs e

Table 2 Relative content of MSLs and CSLs The relative content of SLs (%) TAG molecular species a MSLs CSLs LnLnLn 11.82 2.03 UFAs glycerides LnLLn 4.90 3.65 LLLn 12.48 8.42 LnOLn 14.25 2.28 MSLs: 81.04% LnLnP 2.98 14.50 LLL 5.58 8.90 OLL 17.03 11.90 29.14% LLP 3.20 0.51 OOPo 1.77 7.04 OLP 1.65 1.27 OOO 14.98 7.27 OPO 5.63 0.93 SLnS 1.46 0.44 CSLs: 51.9% OLO 0 7.45 LnLP 1.48 16.84 SLnP 0.41 5.97 SLP 0.41 0.61 Fig. 1 The HPLC ‐ ELSD chromatogram of MSLs (A) and CSLs (B). The a TAG names do not illustrate positional location of fatty ordinate on the left is the absorbance of the standard TAGs (PPP, LLL, acids in the triacylglycerols. OOO, LnLnLn). The ordinate on the right is the absorbance of the products (MSLs, CSLs).

Fig. 2 Thermogravimetric map of SLs in different reactors. Condition : (A) TAG product in microwave reactor with substrate ratio 1:5 at 60 ° C 50 W and under enzyme loading 7% (w/w) in 1 h; (B) TAG product in Shaker bath reactor with substrate ratio 1:5 at 60 ° C and under enzyme loading was 7% in 3 h; (C) Original silkworm pupa oil.

Table 3 Results of TGA analysis different SLs. Transition temperature ( ° C) Weight loss at Sample NO. of transition transition (%) T i T f Better 1 27.25 154.68 5.97 2 155.61 295.81 72.80 Thermal MSPOSLs a 3 295.81 484.29 18.07 stability 4 485.22 694.22 2.67 1 26.98 143.09 37.54 CSPOSLs b 2 144.04 342.42 42.13 3 343.37 706.13 17.68 1 27.44 106.64 14.70 SPOSLs c 2 107.57 697.87 82.16 a Fatty acids composition of silkworm pupae oil interesterified product SLs under the optimum condition in microwave reactor. b Fatty acids composition of silkworm pupae oil interesterified product SLs under the shaking bath reactor. Conditions, Substrate ratio, 1:5; Temperature, 60 ° C; Enzyme loading, 7%; Time, 3 h. c The silkworm pupa oil extracted from silkworm pupa.

Table 4 FT ‐ IR was used to detect the secondary structure of the Lipozyme RM IM with microwave reactor and without microwave reactor. Secondary structure type and content (%) Sample Random α‐ helix β‐ turn β‐ sheet RM IM a ‐ 0.55 99.45 ‐ RM IM in MW b 0.35 0.66 59.25 27.48 RM IM in shaking 2.22 ‐ 87.53 10.25 bath c RM IM after ‐ 0.38 99.62 ‐ washing d a Original Lipozyme RM IM. b The Lipozyme RM IM was used in the Fig. 4 Fourier infrared spectroscopy of Lipozyme RM IM in different microwave reactor. reactors. RM IM without microwave represents for the unused c The Lipozyme RM IM was used in shaking bath Lipozyme RM IM. RM IM with microwave represent for the Lipozyme reactor. RM IM which was used under the optimum condition in microwave d The Lipozyme RM IM washed with petroleum reactor; RM IM after washing represent for the Lipozyme RM IM ether. washed with petroleum ether; RM IM in shaker bath represent for the Lipozyme RM IM which was used under the optimum condition in shaker bath.

Conclusion 04 Novel SLs Thermal stability ， Melting temperature ， Enrichment with unsaturated fatty acids, Peroxide value Relative content of UFAs glyceride increased to 81.04% from 51.9% The OOO ‐ and OPO ‐ type products increased 2.06 ‐ fold and 6 ‐ fold One hour was required for the enzymatic synthesis of structural ‐ lipid ‐ enriched UFAs from silkworm pupa oil under microwave irradiation.

Acknowledgments 05 The Key Research and Development Program (Modern Agriculture) of Jiangsu Province (BE2017322) The Qing Lan Project of Jiangsu Province (Year 2014) The Six Talent Peaks Project of Jiangsu Province (2015-NY-018) The Shen Lan Young scholars program of Jiangsu University of Science and Technology (Year 2015).