黑水虻蛹壳几丁寡糖的高效提取技术

doi:10.3969/j.issn.1004-1524.20250315

浙江农业学报 ›› 2026, Vol. 38 ›› Issue (2): 225-238.DOI: 10.3969/j.issn.1004-1524.20250315

黑水虻蛹壳几丁寡糖的高效提取技术

刘和¹^,²(), 寇枘¹^,², 李君丽¹^,², 彭实亮¹^,², 连天境¹^,², 麦力文¹^,², 杨霞¹^,², 王定美¹^,², 邵明英³^,^*()

1.中国热带农业科学院环境与植物保护研究所, 海南海口 571101
2.海南儋州热带农业生态系统国家野外科学观测研究站, 海南儋州 571737
3.海南职业技术学院, 海南海口 570216

收稿日期:2025-04-17 出版日期:2026-02-25 发布日期:2026-03-24
作者简介:刘和,研究方向为发酵技术与微生物。E-mail:shadowshade@163.com
通讯作者: ^*邵明英,E-mail: mingshao2013@aliyun.com
基金资助:
海南省重点研发项目(ZDYF2025XDNY097);海南省重点研发项目(ZDYF2023XDNY033);海南省自然科学基金面上项目(324MS107);中国热带农业科学院基本科研业务费专项(1630042025003)

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

摘要/Abstract

摘要：

针对黑水虻(Hermetia illucens)蛹壳几丁寡糖传统提取方法环境压力大的问题,本研究旨在建立一种环境友好的预处理与酶解工艺,实现将黑水虻蛹壳高效转化为几丁寡糖。以黑水虻蛹壳为原料,采用超微粉碎结合超声进行物理预处理;选用甲酸、草酸、柠檬酸等有机酸进行脱矿处理;随后使用角蛋白酶等不同几丁质水解酶进行酶解,并考察温度、酶添加量与金属离子辅助因子对酶解效果的影响;同时探究酸性纤维素酶替代几丁质酶的可行性。结果表明:柠檬酸对蛹壳中碳酸钙的脱除效果最佳;超声处理对后续降解效率无显著提升;几丁质酶的降解效率随温度与酶添加量的增加而提高;金属离子对不同几丁质水解酶的活性具有促进或抑制作用;酸性纤维素酶可有效降解蛹壳几丁质,其效果与几丁质酶相当。本研究建立了一种基于有机酸脱矿与酶法降解的黑水虻蛹壳几丁寡糖绿色提取工艺,证实酸性纤维素酶可作为几丁质酶的有效替代,为黑水虻蛹壳的高值化利用提供了可行的预处理策略与酶选用依据,有助于提升黑水虻生物资源利用率,推动农业有机废弃物的循环发展。

关键词: 黑水虻, 蛹壳, 几丁质, 几丁寡糖, 有机酸, 酶解, 酸性纤维素酶

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

中图分类号:

TS201.2
S816

刘和, 寇枘, 李君丽, 彭实亮, 连天境, 麦力文, 杨霞, 王定美, 邵明英. 黑水虻蛹壳几丁寡糖的高效提取技术[J]. 浙江农业学报, 2026, 38(2): 225-238.

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.

图/表 20

表1 还原糖标准曲线绘制

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

表2 正交试验的因素和水平

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

图1 黑水虻蛹壳(a)和超微粉碎后的黑水虻蛹壳粉(b)

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

图2 还原糖标准曲线

Fig.2 Standard curve of reducing sugar

图3 不同处理的还原糖含量柱上无相同小写字母表示差异显著(p<0.05)。下同。

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.

图4 几丁质粉末的电镜图 a,超声前的几丁质粉末;b,超声后的几丁质粉末;c,酶解超声后的几丁质粉末。

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.

图5 不同超声波粉碎条件下的还原糖含量

Fig.5 Reducing sugar content under different ultrasonic crushing conditions

表3 正交试验设计与结果

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

表4 极差分析结果

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

图6 不同金属辅助因子作用下的还原糖含量

Fig.6 Reducing sugar content with different metal cofactors

图7 不同金属辅助因子催化酸性纤维素酶条件下几丁质的降解率

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

图8 MnSO4催化几丁质酶条件下几丁质的降解率不同数值间没有相同小写字母表示差异显著(p<0.05)。下同。

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.

图9 中性纤维素酶对几丁质的降解率

Fig.9 Chitin degradation rate by neutral cellulase

图10 酸性纤维素酶对几丁质的降解率

Fig.10 Degradation rate of chitin by acidic cellulase

图11 甲酸、角蛋白酶与超声处理黑水虻蛹壳粉后的还原糖含量

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

图12 草酸和柠檬酸脱矿后几丁质酶水解产生的还原糖含量

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

图13 不同柠檬酸质量对酸性纤维素酶降解几丁质的还原糖含量的影响

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

图14 不同酸浸时间下滤液在474 nm的吸光度(D474)

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

图15 不同质量几丁质酶降解几丁质产生的还原糖含量

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

图16 降解产物的HPLC检测结果 a为样品检测结果,峰1为几丁三糖,峰2为富集的主要杂糖混合物;b为几丁二糖至七糖标准品检测结果。

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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黑水虻蛹壳几丁寡糖的高效提取技术

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

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