胶质芽孢杆菌（<i>Bacillus mucilaginosus</i>）复合抗热保护剂优化研究

陈郑榕; 陈龙照; 程扬健; 张秉涯; 赵思睿; 陈建民; 陈勇红; 陈晓晨; 林日珲

doi:10.19303/j.issn.1008-0384.2023.03.013

胶质芽孢杆菌（Bacillus mucilaginosus）复合抗热保护剂优化研究

doi: 10.19303/j.issn.1008-0384.2023.03.013

1.
福州大学先进制造学院，福建　晋江　362200
2.
中国冶金地质总局第二地质勘查院，福建　福州　350108
3.
尤溪县农田建设与土肥技术推广站，福建　尤溪　365100
4.
仙游县农业农村局，福建　仙游　351200
5.
福州大学环境与安全工程学院，福建　福州　350108
6.
漳平市农业农村局，福建　漳平　364400

基金项目: 福建省科技计划引导性项目（2022N0024）

详细信息

作者简介:
陈郑榕（1997−），男，硕士，研究方向：矿物联合微生物重金属土壤修复（E-mail：450641381@qq.com）

通讯作者:
陈龙照（1968−），男，高级工程师，研究方向：土壤修复（E-mail：187036416@qq.com）

中图分类号: Q939.36
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- 被引次数: 0
出版历程
- 收稿日期: 2022-12-01
- 录用日期: 2022-12-01
- 修回日期: 2022-12-28
- 网络出版日期: 2023-03-28
- 刊出日期: 2023-03-28

Thermal Protection of Encapsulated Bacillus mucilaginosus

1.
School of Advanced Manufacturing, Fuzhou University, Jinjiang, Fujian　362200, China
2.
Second Geological Exploration Institute, China Metallurgical Geology Bureau, Fuzhou, Fujian　350108, China
3.
Youxi Farmland Construction and Soil Fertilizer Technology Promotion Station, Youxi, Fujian　365100, China
4.
Xianyou County Agriculture and Rural Bureau, Xianyou, Fujian　351200, China
5.
College of Environment & Safety Engineering, Fuzhou University, Fuzhou, Fujian　 350108, China
6.
Zhangping Agriculture and Rural Bureau, Zhangping, Fujian　364400, China

摘要

摘要: 目的针对胶质芽孢杆菌（Bacillus mucilaginosus）直投式菌剂菌体存活率低及易失活等问题，对喷雾干燥复合抗热保护剂进行优化研究，以期为其菌剂生产提供理论与技术支持。方法以直投式菌剂菌体存活率为指标，利用喷雾干燥生产工艺，考察海藻糖、脱脂奶粉、麦饭石改性材料（H-MS）对菌体的抗热保护效果；并利用响应面法对复合抗热保护剂配方进行优化。结果不添加抗热保护剂的菌剂菌体存活率仅有（34.15±1.07）%；添加单一保护剂时，以海藻糖保护效果最佳，麦饭石改性材料（H-MS）及脱脂奶粉次之，菌体存活率分别可达（52.17±1.08）%，（51.64±0.77）%，（43.67±1.62）%，海藻糖和H-MS对菌体存活率具有极显著影响（P＜0.01），脱脂奶粉对菌体存活率具有显著影响（P＜0.05）。利用响应面优化得到最佳复合抗热保护剂配方为海藻糖4.33%、脱脂奶粉2.90%和麦饭石改性材料（H-MS）7.57%。在此最佳添加配比下，菌体存活率为（73.32±0.76）%，活菌数最高可达1.12×10⁹ CFU·g⁻¹。结论添加3种抗热保护剂均能提高直投式菌剂菌体存活率，且三者之间存在交互作用，复合使用的抗热效果优于单独使用。扫描电镜观察显示海藻糖和脱脂奶粉将菌体包埋成微胶囊固定在麦饭石改性材料（H-MS）的孔道中，能有效保护菌体细胞，提高菌剂菌体存活率。
- 麦饭石 /
- 胶质芽孢杆菌 /
- 响应面优化 /
- 喷雾干燥 /
- 抗热保护剂
Abstract: Objective Effect of encapsulation to protect Bacillus mucilaginosus from thermal shock was evaluated for formulation optimization. Method Rates of survival and activation of B. mucilaginosus encapsulated with trehalose, skimmed milk powder, and/or modified H-MS medical stone after spray-drying were determined. Optimal formulation for the encapsulation was obtained using the response surface method. Result The survival rate of the naked B. mucilaginosus was (34.15±1.07)%. In contrast, the encapsulation with trehalose significantly improved the rate to (52.17±1.08)% (P＜0.01), while with H-MS, (51.64±0.77)% (P＜0.01) and with skimmed milk powder, (43.67±1.62)% (P＜0.05). Furthermore, a combined use of trehalose at 4.33%, skimmed milk powder at 2.90%, and H-MS at 7.57% for the encapsulation, the survival reached (73.32±0.76)% with a microbial count of 1.12×10⁹ CFU·g⁻¹. Conclusion The three encapsulation materials, especially, when they were applied in combination, significantly raised the B. mucilaginosus survival rate under a heat treatment. The scanning electron microscopic image revealed that trehalose and skimmed milk powder immobilized the bacteria cells in H-MS pores producing an effective thermal insulation that sheltered the encapsulated microbes.
- Medical stone /
- Bacillus mucilaginosus /
- response surface optimization /
- spray-drying /
- anti-thermal protectants

HTML全文

图 1 胶质芽孢杆菌生长曲线

Figure 1. Growth curve of B. mucilaginosus

下载: 全尺寸图片幻灯片

图 2 不同抗热保护剂处理下菌剂含水率

不同小写字母表示差异显著（P＜0.05）；CK：空白；A：海藻糖；B：脱脂奶粉；C：H-MS；D：复合保护剂。

Figure 2. Moisture content of B. mucilaginosus encapsulated with various protectants

Data with different lowercase letters indicate significant differences (P<0.05); CK: blank control; A: trehalose; B: skimmed milk powder; C: H-MS; D: combination protectant.

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图 3 3种抗热保护剂的交互作用对菌剂菌体存活率影响的响应面

Figure 3. Response surface of effect by interactions of 3 protectants on B. mucilaginosus survival rate

下载: 全尺寸图片幻灯片

图 4 菌剂的扫描电镜图

Figure 4. SEM images of spray-dried encapsulated B. mucilaginosus on unmodified medical stone and H-MS

下载: 全尺寸图片幻灯片

表 1 Box-Behnken试验设计因素水平

Table 1. Factor levels in Box-Behnken experiment design

水平 Level	因素 Factor
水平 Level	A 海藻糖 Trehalose/%	B 脱脂奶粉 Skimmed milk powder/%	C H-MS/%
−1	2	2	6
0	4	3	8
1	6	4	10

下载: 导出CSV

表 2 单因素试验结果

Table 2. Results of single factor experiment

抗热保护剂 Anti-thermal protectant	添加质量分数 Appending proportion/%	菌体存活率 Bacterial survival rate/%
空白 Blank	0	34.15±1.07
海藻糖 Trehalose	2	41.65±2.66 c
	4	52.17±1.08 a
	6	50.84±1.22 ab
	8	48.17±1.95 b
脱脂奶粉 Skimmed milk powder	1	36.25±2.27 b
	2	39.78±1.73 ab
	3	43.67±1.62 a
	4	41.39±2.25 a
H-MS	6	47.18±1.72 c
	8	51.64±0.77 a
	10	50.68±0.86 ab
	12	48.96±1.30 bc
同一处理组内不同小写字母表示差异显著（P＜0.05）。 Different lowercase letters within the same numbered group indicate significant differences (P<0.05).

下载: 导出CSV

表 3 Box-Behnken试验设计及结果

Table 3. Box-Behnken experimental design and results

试验号 Test number	A因素 A-factor level	B因素水平 B-factor level	C因素 C-factor level	菌体存活率 Bacterial survival rate/%
1	−1	−1	0	54.86
2	−1	1	0	60.14
3	1	−1	0	64.43
4	1	1	0	55.38
5	−1	0	−1	56.23
6	1	0	−1	67.53
7	−1	0	1	59.16
8	1	0	1	56.24
9	0	−1	−1	62.47
10	0	1	−1	64.61
11	0	−1	1	64.47
12	0	1	1	58.12
13	0	0	0	73.54
14	0	0	0	72.84
15	0	0	0	71.84

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表 4 回归模型方差分析

Table 4. Analysis of variance of regression model

方差来源 Variance source	自由度 Freedom	离差平方和 Sum of squares	均方 Mean square	F值 F value	P值 P value
修正模型　Model	9	570.65	63.41	64.05	0.0001
A	1	21.75	21.75	21.97	0.0054
B	1	7.96	7.96	8.04	0.0364
C	1	20.64	20.64	20.85	0.0060
AB	1	51.34	51.34	51.86	0.0008
AC	1	50.55	50.55	51.07	0.0008
BC	1	18.02	18.02	18.20	0.0080
A²	1	256.36	256.36	258.98	＜ 0.0001
B²	1	120.17	120.17	121.40	0.0001
C²	1	78.72	78.72	79.53	0.0003
残差 Residual	5	4.95	0.99
失拟项 Lack of fit	3	3.49	1.16	1.59	0.4080
纯误差 Pure error	2	1.46	0.73
总计 Total	14	575.60

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