Analysis of solid-state anaerobic fermentation process of mackerel processing by-products by high-throughput sequencing
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摘要: 为了实现易产组胺的鲭鱼 (Pneumatophorus japonicus) 加工副产物综合利用产业化技术开发，对鲭鱼加工副产物进行了不灭菌的乳杆菌固态厌氧发酵，尝试生产饲料用新型活性蛋白源。系统监测了发酵过程中组胺、酸溶蛋白、挥发性盐基氮 (Total volatile basic nitrogen, TVB-N)、pH的变化，通过高通量测序技术分析了发酵过程微生物群落的变化，对比分析了发酵前后抗原蛋白、游离氨基酸、脂肪酸成分的变化，以全面评价发酵过程中营养、活性成分和安全性的变迁。结果显示，植物乳杆菌 (Lactobacillus plantarum) HSCC-LP121在提高酸溶蛋白含量、降低组胺积累、减少挥发性盐基氮产生方面综合效果最好。发酵30 d时组胺降低到0.629 g·kg−1。这一数据和高通量测序结果表明，HSCC-LP121具有组胺降解能力，同时可抑制嗜冷杆菌属 (Psychrobacter) 和假单胞菌属 (Pseudomonas) 等潜在产组胺微生物的生长。该菌种还可抑制曲霉菌属 (Aspergillus) 等潜在危害真菌的生长，同时诱使一些生香酵母的生长。Abstract: In order to develop commercial technology for the comprehensive utilization of mackerel (Pneumatophorus japonicus) processing by-product, we carried out the non sterilized solid-state anaerobic fermentation of mackerel by-products to produce a novel protein source for feed with bioactivity. In addition, we monitored the content of histamine, acid-soluble protein and other indexes such as pH, total volatile basic nitrogen and antigenic protein; and investigated the evolution of microbial community during the fermentation by high-throughput sequencing technology, comparing the free amino acids and fatty acids before and after the fermentation. The results show that Lactobacillus plantarum HSCC-LP121 had the best comprehensive effect in increasing acid soluble protein content, reducing histamine accumulation and reducing volatile base nitrogen production. After 30 d of fermentation, the histamine content decreased to 0.629 g·kg−1, which indicates that HSCC-LP121 had the ability to degrade histamine. Moreover, HSCC-LP121 inhibited the growth of potential histamine-producing microorganisms such as Psychrobacter and Pseudomonas. Besides, the strain could also inhibit the growth of potentially harmful fungi such as Aspergillus, and induce the growth of some aroma-producing yeasts.
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图 1 不同乳杆菌菌株发酵对鲭鱼加工副产物理化指标的影响
注：L0、L1、L2和L3分别为无任何处理的对照组、接种嗜酸乳杆菌、接种植物乳杆菌和接种贝氏乳杆菌的实验组；不同字母之间表示存在显著性差异 (P<0.05)，图 2 同此。
Figure 1. Effects of different lactic acid bacteria fermentation on physicochemical indexe of processed by-products of mackerel
Note: L0, L1, L2 and L3 are the control group without any treatment, groups inoculated with L. acidophilus, L. plantarum and L. beijerinck, respectively. Different letters indicate significant difference (P<0.05). The same case in Fig. 2.
图 2 灭菌与不灭菌发酵对鲭鱼加工副产物理化指标的影响
注：S0、S1、S2 分别为灭菌不接种乳杆菌、灭菌接种乳杆菌、不灭菌接种乳杆菌；组胺相对含量由每一组组胺实际含量除以第 0 天未发酵的组胺实际含量计算得出。
Figure 2. Effects of sterilization and non-sterilization fermentation on physicochemical indexes of processed by-products of mackerel
Note: S0, S1 and S2 represent sterilization without inoculation of lactic acid bacteria, and non-sterilization with inoculation of lactic acid bacteria, respectively. The relative histamine content is calculated by the actual histamine content of each group by the actual histamine content of the sterilization group on 0th day.
图 3 不同发酵阶段的细菌群落相对丰度
注：保藏 270 d 指发酵后保存在常温下 270 d；图 4 同此。
Figure 3. Relative abundance of bacterial communities at different fermentation stages
Note: Storage of 270 d means storing at room temperature for 270 d after fermentation; the same case in Fig. 4.
图 4 不同发酵阶段真菌群落的相对丰度
Figure 4. Relative abundance of fungal communities at different fermentation stages
图 5 乳杆菌发酵降解抗原蛋白的SDS-PAGE蛋白电泳图谱
Figure 5. SDS-PAGE electrophoretic pattern of lactobacilli protein degraded by fermentation
表 1 植物乳杆菌发酵鲭鱼加工副产物的Alpha多样性指数
Table 1. Alpha diversity index of processed by-products of mackerel fermented by L. plantarum
样品
SampleACE 指数
ACE indexChao1指数
Chao1 index香农指数
Shannon index覆盖率
Goods coverage对照组
Control group第1天 1st day 123.735 123.438 0.905 1 第30天 30th day 151.349 149.636 1.072 1 发酵组
Fermentation group第1天 1st day 128.206 121.5 1.371 1 第30天 30th day 114.553 110.286 1.668 1 第270天 270th day 57.176 56.0 0.634 1 
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表 2 乳杆菌发酵鲭鱼加工副产物游离氨基酸组成的变化
Table 2. Changes of free amino acid composition of processed by-products of mackerel fermented by lactic acid bacteria
mg·g−1 氨基酸名称
Amino acid name发酵前
Before fermentation对照组第30天
Control group on 30th day乳杆菌发酵第30天
Lactic acid bacteria fermentation on 30th day天冬氨酸 Asparagine 3.742 4.674 4.701 苏氨酸 Threonine 1.420 1.565 1.493 丝氨酸 Serine 1.828 2.079 2.152 谷氨酸 Glutamic acid 7.111 8.030 7.897 甘氨酸 Glycine 2.874 2.863 3.034 丙氨酸 Alanine 2.315 2.411 2.567 胱氨酸 Cystine 0.357 0.313 0.410 缬氨酸 Valine 1.814 1.702 1.953 蛋氨酸 Methionine 0.186 0.118 0.184 异亮氨酸 Isoleucine 1.529 1.489 1.881 亮氨酸 Leucine 2.799 3.049 2.358 酪氨酸 Tyrosine 0.971 1.131 1.246 苯丙氨酸 Phenylalanine 1.896 2.036 1.893 赖氨酸 Lysine 1.107 1.197 1.081 组氨酸 Hlstidine 2.182 1.881 2.266 精氨酸 Argnine 2.940 2.898 2.938 脯氨酸 Proline 2.082 2.407 3.275 总和 Total 37.153 40.175 41.331
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表 3 乳杆菌发酵前后脂肪酸组成变化
Table 3. Changes of fatty acid composition before and after lactic acid bacteria fermentation
% 脂肪酸种类
Fatty acid type发酵前占比
Proportion before fermentation乳杆菌发酵第30天占比
Proportion of lactic acid bacteria fermentation on 30th day十三烷酸 C13:0 9.34 7.51 棕榈酸 C16:0 24.13 25.13 棕榈油酸 C16:1 7.32 7.13 油酸 C18:1 2.79 3.02 亚油酸 C18:2 12.95 15.11 亚麻酸 C18:3 2.81 2.96 γ-亚麻酸 C18:3 8.15 9.53 花生酸 C20:0 2.16 2.15 二十碳二烯酸 C20:2 3.74 3.27 二十碳五烯酸 C20:5 8.83 7.75 芥子酸 C22:1 0.56 0.55 二十二碳二烯酸 C22:2 4.28 3.89 木蜡酸 C24:0 9.11 8.33 二十二碳六烯酸 C22:6 3.84 3.67 单不饱和脂肪酸 MUFA 10.67 10.70 多不饱和脂肪酸 PUFA 44.60 46.18 饱和脂肪酸 SFA 44.74 43.12
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