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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参考文献(47)

[1]	朱飞, 苏娣, 冉雷, 等. 黑曲霉固态发酵改善茶渣营养价值的研究 [J]. 动物营养学报, 2018, 30(10):4269−4278. doi: 10.3969/j.issn.1006-267x.2018.10.054 ZHU F, SU D, RAN L, et al. Nutritional improvement of tea residue by solid-state fermentation with Aspergillus niger [J]. Chinese Journal of Animal Nutrition, 2018, 30(10): 4269−4278.(in Chinese) doi: 10.3969/j.issn.1006-267x.2018.10.054
[2]	郑清梅, 陈昆平, 钟艳梅, 等. 4类茶叶及其茶渣主要成分的测定与分析 [J]. 广东农业科学, 2015, 42(6):14−20. doi: 10.3969/j.issn.1004-874X.2015.06.003 ZHENG Q M, CHEN K P, ZHONG Y M, et al. Determination and analysis of main components of four kinds of tea and their tea wastes [J]. Guangdong Agricultural Sciences, 2015, 42(6): 14−20.(in Chinese) doi: 10.3969/j.issn.1004-874X.2015.06.003
[3]	叶勇. 茶饲料的应用及发展前景 [J]. 中国茶叶, 2000(4):14−15. YE Y. Application and development prospects of tea feed [J]. China Tea, 2000(4): 14−15.(in Chinese)
[4]	刘红云, 梁慧玲. 茶渣用作饲料的研究 [J]. 饲料研究, 2004(9):19−20. doi: 10.3969/j.issn.1002-2813.2004.09.006 LIU H Y, LIANG H L. Research on the use of tea residue as feed [J]. Feed Research, 2004(9): 19−20.(in Chinese) doi: 10.3969/j.issn.1002-2813.2004.09.006
[5]	佐野满昭, 孙希琪. 茶添加到家畜饲料中的利用效果 [J]. 农业新技术新方法译丛, 1996, 0(2):32−33. SANO MANZHAO, SUN X Q. Utilization effect of tea added to livestock feed [J]. Translation of new agricultural technologies and methods, 1996, 0(2): 32−33.(in Chinese)
[6]	徐瑞, 王改琴, 邬本成, 等. 茶叶渣对育肥猪生产性能及猪肉品质的影响 [J]. 黑龙江畜牧兽医, 2017(10):73−75, 286. XU R, WANG G Q, WU B C, et al. Effect of tea residue on fattening pig performance and pork quality [J]. Heilongjiang Animal Science and Veterinary Medicine, 2017(10): 73−75, 286.(in Chinese)
[7]	吴慧敏. 茶渣、茶末对蛋鸡生产性能及鸡蛋品质的影响研究[D]. 福州: 福建农林大学, 2016. WU H M. Study on the effect of tea residue and the tea dust on production performance and egg quality[D]. Fuzhou: Fujian Agriculture and Forestry University, 2016. (in Chinese)
[8]	舒庆龄, 赵和涛. 茶渣饲养肉用鸡效果研究 [J]. 安徽农业科学, 1995(4):355−356. SHU Q L, ZHAO H T. Research on the effect of tea residues in breeding chicken for meat [J]. Journal of Anhui Agricultural Sciences, 1995(4): 355−356.(in Chinese)
[9]	潘发明, 李发弟, 郝正里, 等. 茶渣单宁含量对绵羊养分消化利用与氮代谢参数的影响 [J]. 畜牧兽医学报, 2012, 43(1):71−81. PAN F M, LI F D, HAO Z L, et al. Effects of tannin content in residue of tea-leaves on digestion and utilization of nutrients and metabolic parameters of nitrogen in sheep [J]. Chinese Journal of Animal and Veterinary Sciences, 2012, 43(1): 71−81.(in Chinese)
[10]	傅志民, 吴永福. 废弃茶渣综合再利用研究进展 [J]. 中国茶叶加工, 2011(1):17−20. FU Z M, WU Y F. Research on multi-purpose utilization of used waste tea leaves [J]. China Tea Processing, 2011(1): 17−20.(in Chinese)
[11]	谢月琴, 罗君谊, 陈婷, 等. 茶渣资源再利用研究概况 [J]. 中国农学通报, 2019, 35(33):141−145. doi: 10.11924/j.issn.1000-6850.casb18060083 XIE Y Q, LUO J Y, CHEN T, et al. Reuse of tea residue resources: A review [J]. Chinese Agricultural Science Bulletin, 2019, 35(33): 141−145.(in Chinese) doi: 10.11924/j.issn.1000-6850.casb18060083
[12]	刘姝, 涂国全. 茶渣经微生物固体发酵成饲料的初步研究 [J]. 江西农业大学学报, 2001(1):130−133. doi: 10.3969/j.issn.1000-2286.2001.01.029 LIU S, TU G Q. A preliminary study on the microbial feed by solid fermentation of tea residue [J]. Acta Agriculturae Universitis Jiangxiensis, 2001(1): 130−133.(in Chinese) doi: 10.3969/j.issn.1000-2286.2001.01.029
[13]	胡桂萍, 杨广, 欧阳雪灵. 茶渣发酵蛋白饲料辅料优选及成本分析 [J]. 重庆理工大学学报(自然科学), 2017, 31(5):86−90, 142. HU G P, YANG G, OUYANG X L. Optimation of supplemental material in microbial fermentation of tea residue and its costs analysis [J]. Journal of Chongqing University of Technology (Natural Science), 2017, 31(5): 86−90, 142.(in Chinese)
[14]	倪星虹. 茶渣提取蛋白及饲料化利用的初步研究[D]. 南京: 南京农业大学, 2011. NI X H. Studys of the protein extract and utilization of producing protein feed from tea residues[D]. Nanjing: Nanjing Agricultural University, 2011. (in Chinese)
[15]	方呈祥. 中国高等学校菌种目录[M]. 北京: 化学工业出版社, 2009.
[16]	JAIN D K, COLLINS-THOMPSON D L, LEE H, et al. A drop-collapsing test for screening surfactant-producing microorganisms [J]. Journal of Microbiological Methods, 1991, 13(4): 271−279. doi: 10.1016/0167-7012(91)90064-W
[17]	PINCHUK I V, BRESSOLLIER P, SOROKULOVA I B, et al. Amicoumacin antibiotic production and genetic diversity of Bacillus subtilis strains isolated from different habitats [J]. Research in Microbiology, 2002, 153(5): 269−276. doi: 10.1016/S0923-2508(02)01320-7
[18]	WANG L T, LEE F L, TAI C J, et al. Bacillus velezensis is a later heterotypic synonym of Bacillus amyloliquefaciens [J]. International Journal of Systematic and Evolutionary Microbiology, 2008, 58(Pt 3): 671−675.
[19]	王伟, 李术娜, 李红亚, 等. 番茄灰霉病拮抗细菌的筛选与X-75菌株鉴定 [J]. 园艺学报, 2010, 37(2):307−312. WANG W, LI S N, LI H Y, et al. Screening of antagonistic bacteria against Botrytis cinerea and identification of strain X-75 [J]. Acta Horticulturae Sinica, 2010, 37(2): 307−312.(in Chinese)
[20]	王青华, 孙晓晖, 唐旭, 等. 深海贝莱斯芽孢杆菌DH82的筛选、鉴定及其抗菌粗蛋白性质分析 [J]. 海洋通报, 2019, 38(1):63−69. WANG Q H, SUN X H, TANG X, et al. Screening and identification of Bacillus velezensis strain DH82 and the characterization of the crude antimicrobial protein [J]. Marine Science Bulletin, 2019, 38(1): 63−69.(in Chinese)
[21]	杜淑涛, 李术娜, 朱宝成. 白菜黑斑病拮抗细菌Bacillus velezensis DL-59的筛选鉴定及田间防效实验 [J]. 河北农业大学学报, 2010, 33(6):51−56. doi: 10.3969/j.issn.1000-1573.2010.06.011 DU S T, LI S N, ZHU B C. Screening and identification of antagonistic strain DL-59 of Bacillus velezensis against Alternaria brassicae and biocontrol efficiency [J]. Journal of Agricultural University of Hebei, 2010, 33(6): 51−56.(in Chinese) doi: 10.3969/j.issn.1000-1573.2010.06.011
[22]	李生樟, 陈颖, 杨瑞环, 等. 一株拮抗黄单胞菌的贝莱斯芽孢杆菌的分离和鉴定 [J]. 微生物学报, 2019, 59(10):1969−1983. LI S Z, CHEN Y, YANG R H, et al. Isolation and identification of a Bacillus velezensis strain against plant pathogenic Xanthomonas spp [J]. Acta Microbiologica Sinica, 2019, 59(10): 1969−1983.(in Chinese)
[23]	KANJANAMANEESATHIAN M, WIWATTANAPATAPEE R, ROTNIAM W, et al. Application of a suspension concentrate formulation of Bacillus velezensis to control root rot of hydroponicallygrown vegetables [J]. New Zealand Plant Protection, 2013, 66: 229−234. doi: 10.30843/nzpp.2013.66.5556
[24]	张彩文, 程坤, 张欣, 等. 贝莱斯芽胞杆菌(Bacillus velezensis)分类学及功能研究进展 [J]. 食品与发酵工业, 2019, 45(17):258−265. ZHANG C W, CHENG K, ZHANG X, et al. Taxonomy and functions of Bacillus velezensis: A review [J]. Food and Fermentation Industries, 2019, 45(17): 258−265.(in Chinese)
[25]	PAN H Q, LI Q L, HU J C. The complete genome sequence of Bacillus velezensis 9912D reveals its biocontrol mechanism as a novel commercial biological fungicide agent [J]. Journal of Biotechnology, 2017, 247: 25−28. doi: 10.1016/j.jbiotec.2017.02.022
[26]	KIM S Y, LEE S Y, WEON H Y, et al. Complete genome sequence of Bacillus velezensis M75, a biocontrol agent against fungal plant pathogens, isolated from cotton waste [J]. Journal of Biotechnology, 2017, 241: 112−115. doi: 10.1016/j.jbiotec.2016.11.023
[27]	王金燕, 李彬, 王印庚, 等. 刺参养殖池塘一株贝莱斯芽孢杆菌的分离及其生理特性 [J]. 中国水产科学, 2018, 25(3):567−575. doi: 10.3724/SP.J.1118.2018.17383 WANG J Y, LI B, WANG Y G, et al. Screening and characteristic analysis of Bacillus velezensis from sea cucumber(Apostichopus japonicus) ponds [J]. Journal of Fishery Sciences of China, 2018, 25(3): 567−575.(in Chinese) doi: 10.3724/SP.J.1118.2018.17383
[28]	LIU X Y, REN B, CHEN M, et al. Production and characterization of a group of bioemulsifiers from the marine Bacillus velezensis strain H3 [J]. Applied Microbiology and Biotechnology, 2010, 87(5): 1881−1893. doi: 10.1007/s00253-010-2653-9
[29]	TOMAZ C T, QUEIROZ J A. Fractionation of Trichoderma reesei cellulases by hydrophobic interaction chromatography on phenyl-sepharose [J]. Biotechnology Letters, 2004, 26(3): 223−227. doi: 10.1023/B:BILE.0000013719.92024.04
[30]	中华人民共和国国家质量监督检验检疫局, 中国国家标准化管理委员会．饲料添加剂酸性蛋白酶活力的测定分光光度法: GB/T 28715—2012 [S]．北京: 中国标准出版社, 2012．
[31]	中华人民共和国国家技术监管局．蛋白酶制剂: GB/T 23527—2009［S］．北京: 中国标准出版社, 2009.
[32]	杨健, 何永林, 刘晔华. 微生物教学中常用细菌染色方法的改进 [J]. 川北医学院学报, 2003(4):165. doi: 10.3969/j.issn.1005-3697.2003.04.150 YANG J, HE Y L, LIU Y H. Improvement of commonly used bacterial staining methods in microbiology teaching [J]. Journal of North Sichuan Medical College, 2003(4): 165.(in Chinese) doi: 10.3969/j.issn.1005-3697.2003.04.150
[33]	布坎南 R E,吉本斯 N E.伯杰细菌鉴定手册[M].8版.中国科学院微生物研究所《伯杰细菌鉴定手册》翻译组,译.北京: 科学出版社,1984.
[34]	沈萍. 微生物学实验[M]. 北京: 高等教育出版社, 2004.
[35]	东秀珠, 蔡妙英. 常见细菌系统鉴定手册[M]. 北京: 科学出版社, 2001: 364−398.
[36]	郝云婕, 韩素贞. gyrB基因在细菌系统发育分析中的应用 [J]. 生物技术通报, 2008(2):39−41. HAO Y J, HAN S Z. Application of gyrB gene in bacterial phylogenetic analysis [J]. Biotechnology Bulletin, 2008(2): 39−41.(in Chinese)
[37]	TANG X L, ZHAI L, LIN Y F, et al. Halomonas alkalicola sp. nov., isolated from a household product plant [J]. International Journal of Systematic and Evolutionary Microbiology, 2017, 67(5): 1546−1550. doi: 10.1099/ijsem.0.001757
[38]	PHENG S, LEE J J, EOM M K, et al. Paucibacter oligotrophus sp. nov., isolated from fresh water, and emended description of the genus Paucibacter [J]. International Journal of Systematic and Evolutionary Microbiology, 2017, 67(7): 2231−2235. doi: 10.1099/ijsem.0.001931
[39]	卫扬保. 微生物生理学[M]. 北京: 高等教育出版社, 1989.
[40]	王加启, 于建国. 饲料分析与检验[M]. 北京: 中国计量出版社, 2004.
[41]	于泓, 牟世芬. 氨基酸分析方法的研究进展 [J]. 分析化学, 2005(3):398−404. doi: 10.3321/j.issn:0253-3820.2005.03.028 YU H, MOU S F. Method development for amino acid analysis [J]. Chinese Journal of Analytical Chemistry, 2005(3): 398−404.(in Chinese) doi: 10.3321/j.issn:0253-3820.2005.03.028
[42]	BHATTACHARYA S S, GARLAPATI V K, BANERJEE R. Optimization of laccase production using response surface methodology coupled with differential evolution [J]. New Biotechnology, 2011, 28(1): 31−39. doi: 10.1016/j.nbt.2010.06.001
[43]	胡宝东, 邱树毅, 周鸿翔, 等. 酱香型大曲的理化指标、水解酶系、微生物产酶的关系研究 [J]. 现代食品科技, 2017, 33(2):99−106. HU B D, QIU S Y, ZHOU H X, et al. Relationships among physiochemical indices and hydrolyzing enzyme systems and enzymes-produced-ability in Jiangxiang daqu [J]. Modern Food Science and Technology, 2017, 33(2): 99−106.(in Chinese)
[44]	李芳蓉, 陈鑫, 张建军. 红芪药渣固态发酵生产蛋白饲料的探讨 [J]. 中央民族大学学报(自然科学版), 2019, 28(1):47−51. LI F R, CHEN X, ZHANG J J. Discussion on the production of protein feed by solid ferm entation of Hedysarum polybotrys hand. -Mazz. Residue [J]. Journal of Minzu University of China (Natural Sciences Edition), 2019, 28(1): 47−51.(in Chinese)
[45]	张沛, 杨国辉, 魏丽娟, 等. 稻壳粉固态发酵生产蛋白饲料的工艺研究 [J]. 中国饲料, 2015(8):25−27, 38. ZHANG P, YANG G H, WEI L J, et al. Study on the technology of protein feed frim rice husk powder fermented by solid slate [J]. China Feed, 2015(8): 25−27, 38.(in Chinese)
[46]	张楠, 张凌云. 茶渣在自然发酵与添加外源微生物发酵过程中的理化动态研究[C]//2009年中国茶叶科技创新与产业发展学术研讨会论文集. 重庆, 2009: 428-435.
[47]	陈奕业, 朱妮, 梁瑶, 等. 不同添加剂对凉茶渣发酵品质的影响 [J]. 家畜生态学报, 2020, 41(2):55−59. doi: 10.3969/j.issn.1673-1182.2020.02.010 CHEN Y Y, ZHU N, LIANG Y, et al. Effect of additives on quality of herbal tea residue fermentation [J]. Journal of Domestic Animal Ecology, 2020, 41(2): 55−59.(in Chinese) doi: 10.3969/j.issn.1673-1182.2020.02.010