产细菌素植物乳杆菌的筛选及其细菌素对大黄鱼保鲜效果

许华娣; 高腾琪; 崔子杭; 柳春胜; 盘赛昆; 杨杰

doi:10.13386/j.issn1002-0306.2022050162

产细菌素植物乳杆菌的筛选及其细菌素对大黄鱼保鲜效果

doi: 10.13386/j.issn1002-0306.2022050162

江苏海洋大学食品科学与工程学院，江苏连云港 222005

基金项目: 江苏省高等学校自然科学研究面上项目（920KJB550008）。

详细信息

作者简介:
许华娣（1990−），女，硕士，研究方向：食品微生物和食品安全，E-mail：huadixu@126.com

通讯作者:
杨杰（1988−），男，博士，副教授，研究方向：微生物天然产物发掘与生物合成、水产品加工与贮藏，E-mail：yangjie@jou.edu.cn

中图分类号: TS254.4
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- 被引次数: 0
出版历程
- 收稿日期: 2022-05-17
- 网络出版日期: 2022-09-15
- 刊出日期: 2022-10-24

Identification and Screening of Bacteriocin-producing Lactobacillus plantarum and Its Effects on Preservation of Large Yellow Croaker

College of Food Science and Engineering, Jiangsu Ocean University, Lianyungang 222005, China

摘要

摘要: 本研究首先利用富集培养、稀释涂布和琼脂平板扩散实验从传统发酵食品中筛选乳酸菌，采用生理生化实验、16S rDNA基因序列同源性分析对菌株进行鉴定。其次，通过排酸实验、过氧化氢排除实验、蛋白酶水解实验明确该乳酸菌所产的主要抑菌物质。最后，通过醇沉水提、Sephadex LH-20层析获得细菌素粗提物，并将其应用于大黄鱼整鱼保鲜。每3 d取样进行感官评价、pH测定、菌落总数测定、总挥发性盐基氮测定和质构分析，评价其保鲜效果。结果表明，所筛选获得的菌株为植物乳杆菌，命名为MMB-11。植物乳杆菌MMB-11所产细菌素对蛋白酶敏感，具有良好的酸耐受性和热稳定性。将该细菌素应用于大黄鱼保鲜发现，在保藏12 d时，MMB-11细菌素处理组、对照组和Nisin处理组菌落总数分别为3.50×10⁴、1.10×10⁵和4.50×10⁴ CFU/g，挥发性盐基氮分别为20.91±0.66、31.37±0.21和21.44±0.45 mg/100 g。因此，相较于Nisin处理组和对照组，MMB-11细菌素粗提液对大黄鱼具有更好的保鲜效果。本研究可为大黄鱼安全无害的生物保鲜剂的开发奠定理论基础。
- 植物乳杆菌 /
- 分离鉴定 /
- 细菌素 /
- 大黄鱼 /
- 保鲜
Abstract: In this study, the screening of lactic acid bacteria from traditional fermented food was carried out by enrichment culture, dilution separation, and agar plate diffusion experiments. Physiological and biochemical and 16S rDNA gene sequence homology analysis were used to identify the lactic acid bacteria. Then, the main antibacterial substances produced by the lactic acid bacteria were determined by acid excretion, hydrogen peroxide exclusion, and protease hydrolysis. Finally, the bacteriocin crude extract was obtained by alcohol precipitation and Sephadex LH-20 chromatography, and then was applied to the preserving the whole large yellow croaker. To evaluate the preservation effect of the bacteriocin crude extract on the large yellow croaker, sensory evaluation, pH determination, viable cell counts, TVB-N determination and texture profile analysis were systematically carried out every 3 days. The experimental results showed that the lactic acid bacteria isolated from shrimp paste was Lactobacillus plantarum and named MMB-11. Meanwhile, the bacteriocin produced by MMB-11 was sensitive to protease and had notable acid tolerance and thermal stability. With the application of the bacteriocin on the preservation of large yellow croaker, the viable cell counts in MMB-11 group, control group and Nisin-treated group after 12 d of storage were 3.50×10⁴, 1.10×10⁵ and 4.50×10⁴ CFU/g, respectively. Furthermore, after 12 d of storage, the TVB-N treated with MMB-11 group, control group and Nisin group were 20.91±0.66、31.37±0.21 and 21.44±0.45 mg/100 g, respectively. Overall, the results showed that the crude bacteriocin extract of MMB-11 had a better preservation effect on large yellow croaker than the Nisin-treated group and control group. This study provides a fundamental knowledge for developing safe and harmless biological preservatives for large yellow croaker.
- Lactobacillus plantarum /
- isolation and identification /
- bacteriocin /
- large yellow croaker /
- preservation

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图 1 菌株MMB-11菌落形态及革兰氏染色图

Figure 1. Colony morphology and Gram staining of MMB-11 strain

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图 2 MMB-11的系统发育树

Figure 2. Phylogenetic tree of MMB-11

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图 3 MMB-11排酸实验结果

Figure 3. Results of MMB-11 acid removal experiment

注：不同大写字母表示组内差异显著，不同小写字母表示组间差异显著，P<0.05；图4~图8同。

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图 4 MMB-11排过氧化氢实验结果

Figure 4. Results of MMB-11 hydrogen peroxide removal experiment

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图 5 MMB-11蛋白酶水解实验结果

Figure 5. Results of MMB-11 protease hydrolysis experiment

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图 6 MMB-11 醇沉水提组分抑菌活性

Figure 6. Antibacterial activities of the components of MMB-11 by alcohol precipitation

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图 7 MMB-11细菌素粗提液温度耐受性

Figure 7. MMB-11 bacteriocin crude extract temperature tolerance

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图 8 MMB-11细菌素粗提液pH耐受性

Figure 8. MMB-11 bacteriocin crude extract pH tolerance

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图 9 感官评价分值随着贮藏时间的变化

Figure 9. The change of organoleptic evaluation scores in the process of storage

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图 10 pH随着贮藏时间的变化

Figure 10. The change of pH in the process of storage

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图 11 菌落总数随着贮藏时间的变化

Figure 11. The change of TAC in the progress of strorage

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图 12 TVB-N随着贮藏时间的变化

Figure 12. The change of TVB-N in the progress of strorage

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表 1 大黄鱼的感官评价标准

Table 1. Sensory evaluation criteria of large yellow croaker

品质参数	参数评分（分）
品质参数	10~9	8~6	5~3	2~0
气味	鱼本身的鲜味	有异味	有轻微腥臭味	不愉快的腐臭味
肌肉组织	致密完整，纹理清晰	紧密，纹理较清晰	松散，基本无纹理	塌陷，无纹理
弹性	快速回弹，富有弹性	回弹快，较有弹性	回弹慢，弹性不佳	不回弹，无弹性
颜色	色泽鲜亮	色泽较淡	颜色发暗，色泽变淡	色泽灰暗
粘液	清爽不粘	有轻微渗出液	渗出液多，有粘感	发粘感重
总分	（0~50）

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表 2 不同菌株的抑菌圈直径

Table 2. Diameters of inhibition zone of different strains

编号	抑菌圈直径（mm）
编号	金黄色葡萄球菌	希瓦氏腐败菌
X8	10.15±0.05	10.50±0.10
X9	10.00+0.00	10.90±0.10
X14	10.50±0.10	10.75±0.05
X15	10.80±0.20	11.80±0.20
A16	10.51±0.25	11.30±0.01
L73	10.82±0.33	10.79±0.01
S49	11.73±0.01	10.20±0.07
MMB-11	11.70±0.05	11.40±0.05

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表 3 MMB-11生理生化鉴定结果

Table 3. Physiological and biochemical identification results of MMB-11 strain

特征	结果	特征	结果
葡萄糖产气	−	木糖	+
明胶液化	−	七叶灵	+
从精氨酸产NH₃	−	纤维二糖	+
葡萄糖	+	麦芽糖	+
阿拉伯糖	+	甘露醇	+
果糖	+	山梨醇	+
甘露糖	+	蔗糖	+
蜜二糖	+	棉籽糖	+
水杨苷	+	菊糖	+
海藻糖	+	乳糖	+
苦杏仁苷	+	半乳糖	+
注：表中的−表示阴性，+表示阳性。

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表 4 Sephadex LH-20层析凝胶收集管抑菌活性

Table 4. Antibacterial activity of Sephadex LH-20 gel chromatography collection tube

收集管管号	抑菌圈直径（mm）
收集管管号	金黄色葡萄球菌	希瓦氏腐败菌	嗜水气单胞菌
59	0.00±0.00	0.00±0.00	0.00±0.00
61	0.00±0.00	0.00±0.00	17.40±0.10
63	12.85±0.07	14.20±0.10	17.65±0.10
65	15.10±0.14	15.80±0.10	21.65±0.05
67	16.25±0.21	17.90±0.10	25.60±0.10
69	16.85±0.07	18.25±0.05	25.80±0.10
71	17.75±0.07	17.55±0.55	23.60±0.10
73	15.10±0.14	14.10±0.10	23.05±0.25
75	11.60±0.28	11.50±0.05	20.20±0.10
77	0.00±0.00	0.00±0.00	17.75±0.05
79	0.00±0.00	0.00±0.00	12.60±0.15
81	0.00±0.00	0.00±0.00	0.00±0.00

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表 5 不同贮藏时间的TPA

Table 5. The TPA in the process of storage

指标	处理方式	贮藏时间（d）
指标	处理方式	0	3	6	9	12
硬度	N组	60.50±1.41^Ab	45.43±5.71^Ad	36.70±1.31^Ce	51.60±3.21^Bc	75.40±11.82^Ba
	P组	60.50±1.41^Aa	57.53±10.21^Aa	75.73±15.2^Aa	60.80±4.36^Aa	77.27±4.71^Aa
	C组	60.50±1.41^Ab	49.60±6.80^Ac	47.27±13.93^Bd	46.83±5.06^Ce	66.97±4.73^Ca
弹性	N组	0.54±0.01^Aa	0.41±0.05^Ab	0.25±0.01^Ce	0.29±0.03^Ac	0.27±0.03^Ad
	P组	0.54±0.01^Aa	0.32±0.06^Ac	0.35±0.06^Bb	0.27±0.02^Ad	0.24±0.04^Ae
	C组	0.54±0.01^Aa	0.35±0.07^Ab	0.54±0.02^Aa	0.27±0.03^Ac	0.34±0.11^Ab
黏聚性	N组	0.13±0.04^Aa	0.16±0.01^Aa	0.17±0.03^Aa	0.17±0.02^Aa	0.14±0.01^Aa
	P组	0.13±0.04^Aa	0.12±0.03^Aa	0.17±0.04^Aa	0.14±0.01^Ba	0.13±0.00^Aa
	C组	0.13±0.04^Aa	0.14±0.01^Aa	0.16±0.02^Aa	0.17±0.01^Aa	0.20±0.06^Aa
胶黏性	N组	7.54±1.96^Ac	7.23±0.50^Ad	6.10±0.99^Ae	8.92±0.53^Ab	10.58±1.93^Aa
	P组	7.54±1.96^Aa	7.04±2.69^Aa	13.50±5.67^Aa	8.70±0.38^Aa	10.04±0.61^Aa
	C组	7.54±1.96^Ad	6.82±1.48^Ae	7.74±3.22^Ac	8.16±1.38^Ab	13.66±3.88^Aa
咀嚼性	N组	4.06±0.95^Aa	2.95±0.20^Ab	1.53±0.26^Ce	2.60±0.46^Ad	2.79±0.38^Ac
	P组	4.06±0.95^Aa	2.30±1.26^Aa	4.65±1.84^Aa	2.32±0.05^Aa	2.37±0.35^Aa
	C组	4.06±0.95^Aa	2.42±0.98^Aa	1.93±0.89^Ba	2.18±0.26^Aa	4.71±2.58^Aa
黏附性	N组	−0.35±0.07^Aa	−0.37±0.06^Ab	−0.60±0.17^Ac	−0.73±0.15^Ae	−0.67±0.06^Bd
	P组	−0.35±0.07^Aa	−0.43±0.15^Aa	−0.50±0.17^Aa	−0.37±0.25^Aa	−0.77±0.12^Ca
	C组	−0.35±0.07^Aa	−0.27±0.15^Aa	−0.47±0.12^Aa	−0.33±0.06^Aa	−0.37±0.06^Aa
注：不同大写字母表示纵向比较，不同小写字母表示横向比较，P<0.05。硬度、咀嚼性、胶黏性的单位是N，黏附性的单位是N·sec，黏聚性和弹性无单位。

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