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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

Acknowledg- ments Present study Background Previous study Conclusions

01

Background

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

α‐Linolenic acid Oleic acid Linoleic acid

Transesterification

Novel structured lipids triglyceride

Silkworm pupae biorefinery Silkworm pupae Silkworm pupa oil Silkworm pupa residue

[3] Yang L I F, Siriamornpun S, Li D. Journal of Food Lipids, 2006, 13(3): 277‐285.

Biodiesel2 Structured lipids1

[2]Manzano‐Agugliaro F, Sanchez‐Muros M J, Barroso F G, et al. Renewable and Sustainable Energy Reviews, 2012, 16(6): 3744‐3753.

Unsaturated fatty acid3

[1]Zhao X Y, Wang X D, Liu X, et al. European Journal of Lipid Science and Technology, 2015, 117(6): 879‐889.

Cultivate yeast

Yeast oil

02

Previous study

P P P

APA‐style human milk fat analogue from silkworm pupae oil: Enzymatic production and improving storage stability using alkyl caffeates

Liu X, Wang J*, et al. Scientific Reports. 2015, 5: 17909.

R1 R2 R3

Structured lipids enriched with unsaturated fatty acids produced by enzymatic acidolysis of silkworm pupae oil using oleic acid

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！ Silkworm pupa oils Problem: Reduce the time of enzymatic transesterification How to enhance the reaction ? Novel SLs

UFAs glyceride

Lipozyme RM IM

ALA LA OA

OOO LnOLn OLL LLLn LLLn

PA Po SA SA PA Po SA

1h 50W

Microwave irradiation

Free FAs

Lipozyme RM IM

81.04%

Ln‐α‐Linolenic acid (ALA) L‐Linoleic acid (LA) O‐Oleic acid (OA)

Fatty acid C16:0 C18:1 C18:2 C18:3 Total fatty acid content (%) SPOSLsa 24.55±0.23 34.72±0.06 5.58±0.02 35.15±0.15 MSLsb 24.71±1.04 35.76±1.23 8.32±0.25 31.20±0.43 ESLsc 24.84±0.65 34.58±0.23 5.54±0.05 35.04±0.37 CSLsd 11.85 3.86 51.61 12.79 SSLse 21.07±0.54 32.52±0.22 6.56±0.31 30.96±2.2 Sn‐2 fatty acid content (%) SPOSLsa 4.77±2.13 40.65±0.39 5.31±0.16 49.27±1.62 MSLsb 18.41±2.53 38.52±2.62 4.78±0.85 34.65±0.79 ESLsc 3.86±0.41 40.49±1.02 5.53±0.14 50.12±0.73 CSLsd 3.59 2.22 42.06 42.38 SSLse 3.46±0.23 36.32±1.23 6.61±0.74 54.02±0.61 Sn‐1, 3 fatty acid content (%) SPOSLsa 34.43±1.39 31.75±0.27 5.72±0.10 28.09±1.03 MSLsb 28.35±3.13 33.47±0.01 10.10±0.43 29.30±1.20 ESLsc 35.33±1.05 31.62±0.78 5.54±0.09 27.50±0.60 CSLsd 16.43 3.68 48.09 28.03 SSLse 30.49±0.74 28.72±2.21 6.63±0.75 22.93±1.35

aThe extracted oil from silkworm pupa. B Fatty acids composition of silkworm pupae oil

transesterification product SLs under the optimum condition in microwave reactor.

cAdding the enzyme (RM IM) without oleic acid into the

silkworm pupae oil under microwave irradiation.

d Fatty acids composition of silkworm pupae oil

transesterification product SLs under the optimum condition from Zhao et al, 2015.

eThe silkworm pupae oil used in shaker bath reactor.

Optimum condition: Substrate molar ratio: 1:5 Enzyme loading: 7 % Microwave power: 50 W Microwave temperature: 60 °C Table 1 The content of fatty acids under different treatment.

1 h 3 h Time:

TAG molecular speciesa The relative content of SLs (%) MSLs CSLs LnLnLn 11.82 2.03 LnLLn 4.90 3.65 LLLn 12.48 8.42 LnOLn 14.25 2.28 LnLnP 2.98 14.50 LLL 5.58 8.90 OLL 17.03 11.90 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 OLO 7.45 LnLP 1.48 16.84 SLnP 0.41 5.97 SLP 0.41 0.61

aTAG names do not illustrate positional location of fatty

acids in the triacylglycerols.

• Fig. 1 The HPLC‐ELSD chromatogram of MSLs (A) and CSLs (B). The
• rdinate on the left is the absorbance of the standard TAGs (PPP, LLL,

OOO, LnLnLn). The ordinate on the right is the absorbance of the products (MSLs, CSLs).

CSLs: 51.9% MSLs: 81.04% UFAs glycerides 29.14% Table 2 Relative content of MSLs and 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

• il.

Sample

• NO. of transition

Transition temperature (°C) Weight loss at transition (%) Ti Tf MSPOSLsa 1 27.25 154.68 5.97 2 155.61 295.81 72.80 3 295.81 484.29 18.07 4 485.22 694.22 2.67 CSPOSLsb 1 26.98 143.09 37.54 2 144.04 342.42 42.13 3 343.37 706.13 17.68 SPOSLsc 1 27.44 106.64 14.70 2 107.57 697.87 82.16

Table 3 Results of TGA analysis different SLs.

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.

Better Thermal stability

• Fig. 4 Fourier infrared spectroscopy of Lipozyme RM IM in different
• reactors. RM IM without microwave represents for the unused

Lipozyme RM IM. RM IM with microwave represent for the Lipozyme RM IM which was used under the optimum condition in microwave reactor; RM IM after washing represent for the Lipozyme RM IM 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.

Sample Secondary structure type and content (%) 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 bath c 2.22 ‐ 87.53 10.25 RM IM after washing d ‐ 0.38 99.62 ‐

Table 4 FT‐IR was used to detect the secondary structure

• f the Lipozyme RM IM with microwave reactor and

without microwave reactor.

a Original Lipozyme RM IM. b The Lipozyme RM IM was used in the

microwave reactor.

c The Lipozyme RM IM was used in shaking bath

reactor.

d The Lipozyme RM IM washed with petroleum

ether.

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%

04

Conclusion

One hour was required for the enzymatic synthesis

• f structural‐lipid‐enriched UFAs from silkworm

pupa oil under microwave irradiation. The OOO‐ and OPO‐type products increased 2.06‐fold and 6‐fold

05

Acknowledgments

The Key Research and Development Program (Modern Agriculture)

• f

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).