一株茶渣分解菌Fb的分离及其应用

缪伏荣; 陈鑫珠; 李忠荣; 刘景

doi:10.19303/j.issn.1008-0384.2021.04.015

一株茶渣分解菌Fb的分离及其应用

doi: 10.19303/j.issn.1008-0384.2021.04.015

福建省农业科学院畜牧兽医研究所，福建　福州　350013

基金项目: 福建省农业科学院自由探索科技创新项目（ZYTS2019022）；福建省科技计划公益类专项（2020R1026003）

详细信息

作者简介:
缪伏荣（1969−），男，硕士，副研究员，主要从事饲料资源和特种微生物的研究与应用（E-mail：miaofr@sina.com）

通讯作者:
刘景（1967−），男，副研究员，主要从事饲料资源的研究（E-mail：fjliuj@163.com）

中图分类号: Q 939.9
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出版历程
- 收稿日期: 2020-09-02
- 修回日期: 2021-02-28
- 网络出版日期: 2021-04-20
- 刊出日期: 2021-04-30

Isolation and Application of Microbes Capable of Decomposing Tea Dregs

Institute of Animal Husbandry and Veterinary Medicine, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian　350013, China

摘要

摘要: 目的筛选高温高效分解茶渣的菌株，为茶渣开发利用提供理论依据。方法利用高温特殊生境分离方法，从废弃茶渣中分离筛选较高产纤维素酶和蛋白酶的高温菌，对其中高产酶活力的菌株进行形态学、生理生化特性以及生长特性等研究；并克隆其16S rDNA基因序列和菌株gyrB基因测序，进行系统发育学分析；同时对该菌株分解茶渣效果进行验证。结果试验表明，分离筛选获得分解茶渣的高温菌5株，其中Fb菌产生的纤维素酶、蛋白酶的活力均最高。Fb属贝莱斯芽孢杆菌（Bacillus velezensis），可耐受55℃高温，最适生长条件：42～45 ℃、pH 5.0～7.0、16 h、盐度1.0%～6.0%、装液量0.12 mL·min⁻¹、摇床转速120 r·min⁻¹。茶渣添加Fb菌发酵7 d后，与对照组比，粗蛋白提高14.88%（P＜0.05）；除胱氨酸、蛋氨酸和组氨酸发酵外，其他14种氨基酸含量均显著提高（P＜0.05），且氨基酸总量提高5.98%；粗纤维下降9.69%，其中性洗涤纤维、酸性洗涤纤维和木质素分别下降10.72%、4.47%和11.37%（P＜0.05）。结论首次报道贝莱斯芽孢杆菌Fb能高效分解茶渣，提高茶渣的营养价值。
- 茶渣 /
- 贝莱斯芽孢杆菌 /
- 分离 /
- 蛋白质 /
- 纤维素
Abstract: Objective Microorganisms that can efficiently decompose tea dregs at high temperature were screened for potential waste treatment application. Method A high temperature special habitat separation method was employed to isolate cellulase and protease-producing bacteria on tea dregs. Morphological, physiochemical, and growth characteristics of selected strains were examined prior to 16S rDNA cloning for sequencing. Gene sequences of the candidates, along with gyrB, were subjected to phylogenetic analysis for species identification. Decomposing capacity of the isolated strains on tea dregs was determined for final selection. Results Five thermophiles exhibiting the desired properties were isolated. Among them, Fb showed the highest enzymatic activities and was found to be a strain of Bacillus velezensis. It was further characterized with a high temperature tolerance up to 55 ℃ and the optimal culture conditions of 42-45 ℃, pH 5.0-7.0, 16 h incubation, 1.0-6.0% salinity, 30 mL 250 min⁻¹ filling volume, and 120 rpm shaker speed. After Fb inoculation and incubation for 7 d, the crude protein in the resulting tea dregs significantly increased by 14.88% (P＜0.05), the contents of 14 amino acids, except cystine, methionine, and histidine, significantly increased (P＜0.05), the total amino acids increased by 5.98%, the crude fiber decreased by 9.69%, and the neutral detergent fiber, acid detergent fiber, and lignin decreased by 10.72%, 4.47%, and 11.37%, respectively (P＜0.05). Conclusion For the first time, B. velezensis Fb was identified to be capable of efficiently decomposing tea dregs with a significantly improved nutritional profile on the substrate. It could conceivably become a bioagent for treating the waste material.
- Tea dregs /
- Bacillus velezensis /
- isolation /
- protein /
- cellulose

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图 1 FB菌落形态

Figure 1. Colony morphology (FB)

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图 2 菌体形态

注：Fb菌培养16 h（未发现芽孢）。

Figure 2. Thallus morphology

Note: Morphology of Fb strains cultured for 16 h (no spores found).

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图 3 芽孢形态

注：Fb菌培养25 h（发现芽孢，以红色圆圈标记）。

Figure 3. Strain morphology

Note: Morphology of Fb strains cultured for 25 h (spores marked by red circle).

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图 4 基于16S rDNA的相似菌株系统发育树

Figure 4. Phylogenetic tree of strains based on 16S-rDNA differentiation

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图 5 基于gyrB的相似菌株系统发育树

Figure 5. Phylogenetic tree of strains based on gyrB homologies

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图 6 培养温度对菌株生长的影响

Figure 6. Effect of temperature on Fb growth in culture

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图 7 装液量对菌株生长的影响

Figure 7. Effect of liquid volume on Fb growth in culture

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图 8 摇床转速对菌株生长的影响

Figure 8. Effect of flask shaking speed on Fb growth in culture

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图 9 培养时间对Fb菌生长的影响

Figure 9. Growth curve of cultured Fb

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图 10 初始pH对Fb菌生长的影响

Figure 10. of initial pH on enzyme production of Fb

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图 11 盐度对菌株生长的影响

Figure 11. Effect of salinity on Fb growth in culture

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表 1 菌株生长条件单因素试验

Table 1. Single factor test on growth conditions of bacteria

因素　　 Factors	添加水平　　 Level
培养温度 Incubation temperature/℃	30、33、36、39、42、45、48、51、54、57、60
装液量 Liquid loading quantity/mL	10、15、20、25、30、35、40、45、50、55、60
摇床转速 Shaking speed/r·min⁻¹	100、120、140、160、180、200
培养时间 Fermentation time/h	2、4、6、8、10、12、14、16、18、20
初始pH值 The initial pH	4.0、4.5、5.0、5.5、6.0、6.5、7.0、7.5、8.0、8.5、9.0
盐度 Salinity/%	0、0.5、1.0、1.5、2.0、2.5、3.0、3.5、4.0、4.5、5.0、5.5、6.0、6.5、7.0
注：盐度是指海水中溶解物质质量与海水质量的比值。 Note: Salinity refers to the ratio of dissolved substances in water to water.

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表 2 菌株酶活力比较

Table 2. Enzyme activities of selected strains

菌号 The fungus	CL活力（U） Cellulose enzyme	ACP活性（U） Acid protease activity	NP活性（U） Neutral protease activity	AKP活性（U） Alkaline protease activity
DJ	191.58	21.59	40.23	23.35
Fb	198.33	22.90	41.24	44.36
8116	178.46	23.08	28.25	33.06
8106	189.95	24.44	30.90	27.85
HLH	154.34	16.61	54.17	18.76

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表 3 Fb菌株生理生化特性

Table 3. Characteristics of Fb

项目 Items	结果 Result	项目 Items	结果 Result	项目 Items	结果 Result	项目 Items	结果 Result
β-木糖苷酶 β-xylosidase	+	L-赖氨酸芳胺酶 L-lysine aromatase	−	L-天冬氨酸芳胺酶 L-aspartate aromatase	−	亮氨酸芳胺酶 Leucine aromatase	−
苯丙氨酸芳胺酶 Phenylalanine aramidase	+	L-脯氨酸芳胺酶 L-proline aramidase	−	β-半乳糖苷酶 β-galactosidase	−	L-吡咯烷酮芳胺酶 L-pyrrolidone aramidase	+
α-半乳糖苷酶 α-Galactosidase	+	丙氨酸芳胺酶 Alanine Aramidase	−	酪氨酸芳胺酶 Tyrosine Aramidase	−	β-N-乙酰氨基葡糖苷酶 β-N-Acetylglucosaminidase	−
丙氨酸-苯丙氨酸-脯氨酸芳胺酶 Alanine-phenylalanine-proline arylaminease	+	环糊精 Cyclodextrin	−	D-半乳糖 D-galactose	−	糖原 Glycogen	−
肌醇Inositol	−	甲基-α-D-吡喃葡萄糖苷酸化 Methyl-α-D-glucopyranosylation	+	ELLMAN 试剂	−	甲基-D-木糖苷 Methyl-D-xylosid	−
α-甘露糖苷酶 α-mannosidase	−	麦芽三糖 Maltotriose	−	甘氨酸芳胺酶 Glycine arylaminease	−	D-甘露醇 D-mannitol	+
D-甘露糖 D-mannose	+	D-松三糖 D-matsutriose	−	N-乙酰-D-氨基葡萄糖 N-acetyl-D-glucosamine	−	古老糖 Ancient sugar	+
L-鼠李糖 L-Rhamnose	−	β-葡糖苷酶 β-glucosidase	+	β-甘露糖苷酶 β-mannosidase-phos	−	磷酰胆碱 Phorylcholine	−
丙酮酸盐 Pyruvate	+	α-葡萄糖苷酶+ α-glucosidase	−	D-塔格糖 D-tagatose	−	D-海藻糖 D-trehalose	+
菊粉 Inulin	−	D-葡萄糖 D-glucose	+	D-核糖 D-ribose	+	腐胺同化 Putrescine assimilation	−
吲哚 Indole	+	卡那霉素耐药 Kanamycin resistance	−	竹桃霉素耐药 Oleander resistance	−	七叶苷水解 Aescin hydrolysis	+
红四氮唑 Red tetrazolium	−	多粘菌素B耐药 Polymyxin B resistance	−
注：“+”表示阳性，“-”表示阴性；ELLMAN试剂用于检测游离的巯基（-SH）。 Note: 1: + positive; − negative. 2: ELLMAN reagent for free sulfhydryl (-SH) detection.

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表 4 茶渣常规营养成分的变化（n=9）

Table 4. Changes in nutritional composition of tea dregs with Fb inoculation (dry base, n=9) （单位：%）

项目 Items	水分 Moisture	粗蛋白 Crude protein	粗纤维 Crude fiber	灰分 Ash
对照组 Control group	63.12±1.96 a	18.42±1.13 b	17.75±1.09 a	5.21±0.79 a
发酵组 Fermentation group	36.57±1.72 b	21.16±1.23 a	16.03±1.43 b	5.17±0.51 a
注：粗蛋白、粗纤维和灰分为干基所测值；同列数据后相同字母表示差异不显著（P＞0.05），相邻或相隔字母分别表示差异显著（P＜0.05）或差异极显著（P＜0.01）。下表同。 Note: Crude protein, crude fiber and ash are measured on a dry basis；the same letter in the same column indicates that the difference is not significant（P＞0.05）, and the adjacent or spaced letters indicate that the difference is significant（P＜0.05）or extremely significant（P＜0.01）. The same as follows.

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表 5 茶渣纤维成分的比较（干基, n=9）

Table 5. Change on fiber composition of tea dregs by treatment (dry base, n=9) （单位：%）

项目 Items	中性洗涤纤维 Neutral detergent fiber	酸性洗涤纤维 Acid washing fiber	木质素 Lignin
对照组 Control group	50.75±1.62 a	44.12±1.62 a	2.11±1.62 a
发酵组 Fermentation group	45.31±1.38 b	42.15±1.16 b	1.87±1.21 b

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表 6 茶渣处理前后氨基酸含量的比较（干基, n=9）

Table 6. Change on amino acid composition of tea dregs by treatment （单位：%）

项目 Items	对照组 Control group	发酵组 Fermentation group
天冬氨酸 Asp	2.14±0.18 b	2.29±0.13 a
苏氨酸 Thr	1.09±0.05 b	1.14±0.03 a
丝氨酸 Ser	1.14±0.11 b	1.20±0.02 a
谷氨酸 Glu	2.76±0.12 b	2.95±0.13 a
甘氨酸 Gly	1.25±0.07 b	1.34±0.02 a
丙氨酸 Ala	1.36±0.04 b	1.43±0.05 a
胱氨酸 Cys	0.11±0.01 a	0.11±0.02 a
缬草氨酸 Val	1.38±0.03 b	1.47±0.03 a
精氨酸 Arg	1.34±0.05 b	1.41±0.06 a
甲硫（蛋）氨酸 Met	0.09±0.01 a	0.11±0.01 a
异亮氨酸 Iso	1.11±0.05 b	1.16±0.04 a
赖氨酸 Lys	1.65±0.02 b	1.77±0.06 a
亮氨酸 Leu	2.14±0.02 b	2.24±0.01 a
酪氨酸 Tyr	0.85±0.02 b	0.91±0.03 a
苯丙氨酸 Phe	1.33±0.03 b	1.38±0.01 a
组氨酸 His	0.05±0.01 a	0.05±0.01 a
脯氨酸 Pro	1.11±0.04 b	1.20±0.03 a
总量 Total	20.92 b	22.17 a

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