Highly efficient extraction technology of chitooligosaccharides in the chrysalis shell of Hermetia illucens

doi:10.3969/j.issn.1004-1524.20250315

Acta Agriculturae Zhejiangensis ›› 2026, Vol. 38 ›› Issue (2): 225-238.DOI: 10.3969/j.issn.1004-1524.20250315

• Animal Science • Previous Articles Next Articles

Highly efficient extraction technology of chitooligosaccharides in the chrysalis shell of Hermetia illucens

LIU He¹^,²(), KOU Rui¹^,², LI Junli¹^,², PENG Shiliang¹^,², LIAN Tianjing¹^,², MAI Liwen¹^,², YANG Xia¹^,², WANG Dingmei¹^,², SHAO Mingying³^,^*()

1. Environmental and Plant Protection Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, China
2. Hainan Danzhou Tropical Agro-ecosystem National Observation and Research Station, Danzhou 571737, Hainan, China
3. Hainan Vocational University, Haikou 570216, China

Received:2025-04-17 Online:2026-02-25 Published:2026-03-24

Abstract

Abstract:

To address the environmental concerns associated with traditional extraction methods for chitooligosaccharides from black soldier fly (Hermetia illucens) chrysalis shells, this study aimed to develop an environmentally friendly pretreatment and enzymatic hydrolysis process for the efficient conversion of these shells into chitooligosaccharides. Using black soldier fly chrysalis shells as the raw material, physical pretreatment was performed via ultrafine grinding combined with ultrasonication. Organic acids, including formic acid, oxalic acid, and citric acid, were used for demineralization. Subsequently, enzymatic hydrolysis was conducted using various chitin-hydrolyzing enzymes, such as keratinase, and the effects of temperature, enzyme dosage, and metal ion cofactors on hydrolysis efficiency were investigated. The feasibility of using acidic cellulase as a substitute for chitinase was also explored. The results showed that citric acid was most effective in removing calcium carbonate from the chrysalis shells; ultrasonication did not significantly enhance subsequent enzymatic hydrolysis efficiency; chitinase hydrolysis efficiency increased with higher temperature and enzyme dosage; metal ions exhibited either promotive or inhibitory effects on the activity of different chitin-hydrolyzing enzymes; and acidic cellulase could effectively degrade chitin in the shells, performing comparably to chitinase. This study established a green extraction process for chitooligosaccharides from black soldier fly chrysalis shells based on organic acid demineralization and enzymatic degradation. It confirmed that acidic cellulase can serve as an effective alternative to chitinase, providing a feasible pretreatment strategy and enzyme selection basis for the high-value utilization of black soldier fly chrysalis shells. This contributes to enhancing the utilization efficiency of black soldier fly biomass and promoting the circular development of agricultural organic waste.

Key words: Hermetia illucens L., chrysalis shell, chitin, chitooligosaccharide, organic acid, enzymatic degradation, acidic cellulase

CLC Number:

TS201.2
S816

LIU He, KOU Rui, LI Junli, PENG Shiliang, LIAN Tianjing, MAI Liwen, YANG Xia, WANG Dingmei, SHAO Mingying. Highly efficient extraction technology of chitooligosaccharides in the chrysalis shell of Hermetia illucens[J]. Acta Agriculturae Zhejiangensis, 2026, 38(2): 225-238.

Figures/Tables 20

Table 1 Plotting of the reducing sugar standard curve

编号 Serial number	葡萄糖体积/mL Volume of glucose/mL	葡萄糖质量浓度/(mg·mL^-1) Concentration of glucose/(mg·mL^-1)
1	0	0
2	0.4	0.2
3	0.8	0.4
4	1.2	0.6
5	1.6	0.8
6	2.0	1.0

Table 2 Orthogonal experimental factors and levels

水平 Level	(A)几丁质酶质量/g Chitinase mass/g	(C)超声时间/min Ultrasonic time/min	(D)酶解时间/d Enzymatic hydrolysis time/d
1	0.1	10	1
2	0.5	30	2
3	1.0	60	3

Fig.1 Chrysalis shell of the black soldier fly(a) and chrysalis shell powder of the black soldier fly after ultramicro grinding(b)

Fig.2 Standard curve of reducing sugar

Fig.3 Reducing sugar content under different treatments Bars marked without the same lowercase letter indicated significant differences at p<0.05. The same as below.

Fig.4 Scanning electron microscopy image of chitin powder a, Chitin powder before ultrasound; b, Chitin powder after ultrasound; c, Chitin after enzymatic and ultrasound.

Fig.5 Reducing sugar content under different ultrasonic crushing conditions

Table 3 Orthogonal experimental design and the results

试验号 Test No.	A	C	D	空列 Blank column	降解率/% Degradation rate/%
1	1	1	1	1	11.0 g
2	1	2	2	2	5.0 i
3	1	3	3	3	7.0 h
4	2	1	2	3	29.0 e
5	2	2	3	1	42.5 d
6	2	3	1	2	26.7 f
7	3	1	3	2	58.0 a
8	3	2	1	3	53.5 b
9	3	3	2	1	54.7 c

Table 4 Results of range analysis

因素 Factor	k₁/%	k₂/%	k₃/%	R	最优水平 The optimal level
A	7.67	32.72	55.40	47.73	3
C	32.67	33.67	29.46	4.20	2
D	30.39	29.57	35.83	6.27	3

Fig.6 Reducing sugar content with different metal cofactors

Fig.7 Degradation rate of chitin by acidic cellulase in the presence of different metal cofactors

Fig.8 Degradation rate of chitin by chitinase in the presence of MnSO4 The values without the same lowercase letters indicate significant (p<0.05) difference. The same as below.

Fig.9 Chitin degradation rate by neutral cellulase

Fig.10 Degradation rate of chitin by acidic cellulase

Fig.11 Reducing sugar content of chitin by formic acid, keratinase, and ultrasonic treatment

Fig.12 Reducing sugar content produced by chitinase hydrolysis following demineralization using oxalic acid or citric acid

Fig.13 Influence of different masses of citric acid on the reducing sugar content in chitin degradation by acidic cellulase

Fig.14 Absorbance of filtrate at 474 nm (D474) under different acid leaching time

Fig.15 Content of reducing sugars produced from chitin degradation with varying masses of chitinase

Fig.16 HPLC analysis results of the degradation products a, Detection results of the sample, peak 1 is chitotriose, and peak 2 is the enriched main mixture of heterosaccharides; b, Detection results of chitobiose-chitoheptaose standards.

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Highly efficient extraction technology of chitooligosaccharides in the chrysalis shell of Hermetia illucens

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