产植酸酶益生乳酸菌的筛选

doi:10.6048/j.issn.1001-4330.2024.09.025

新疆农业科学 ›› 2024, Vol. 61 ›› Issue (9): 2290-2298.DOI: 10.6048/j.issn.1001-4330.2024.09.025

• 植物保护·微生物·畜牧兽医·土壤肥料 • 上一篇下一篇

产植酸酶益生乳酸菌的筛选

王浩中¹^,²(), 林青², 曾军², 高雁², 赵燕慧³, 时红玲², 马贵军³, 马正海¹, 娄恺²(), 霍向东²()

1.新疆大学生命科学与技术学院,乌鲁木齐 830046
2.新疆农业科学院微生物应用研究所/新疆特殊环境微生物实验室,乌鲁木齐 830091
3.新疆天康饲料有限公司,新疆五家渠 750300

收稿日期:2024-02-14 出版日期:2024-09-20 发布日期:2024-10-09
通信作者: 娄恺(1968-),男,河南人,研究员,博士,研究方向为微生物学,(E-mail)loukai02@mail.tsinghua.edu.cn;
霍向东(1974-),男,甘肃人,副研究员,博士,研究方向为微生物资源,(E-mail)xiangdonghuo@163.com
作者简介:王浩中(1997-),男,甘肃人,硕士研究生,研究方向为微生物学,(E-mail)490335311@qq.com
基金资助:
新疆生产建设兵团重点研发计划专项(2022AB013);农业科技创新稳定支持项目(xjnkywdzc-2023005-6)

Screening of phytase producing probiotic lactic acid bacteria

WANG Haozhong¹^,²(), LIN Qing², ZENG Jun², GAO Yan², ZHAO Yanhui³, SHI Hongling², MA Guijun³, MA Zhenghai¹, LOU Kai²(), HUO Xiangdong²()

1. College of Life Science and Technology, Xinjiang University, Urumqi 830046, China
2. Xinjiang Laboratory of Special Environmental Microbiology / Institute of Microbiology, Xinjiang Academy of Agricultural Sciences, Urumqi 830091, China
3. Xinjiang Tiankang Feed Co., Ltd, Wujiaqiu Xinjiang 750300, China

Received:2024-02-14 Published:2024-09-20 Online:2024-10-09
Supported by:
Key R & D Program of Xinjiang Production and Construction Corps(2022AB013);Project for Stable Support to Agricultural Sci-Tech Renovation(xjnkywdzc-2023005-6)

摘要/Abstract

摘要：

【目的】分离筛选产植酸酶益生乳酸菌菌株。【方法】以发酵饲料、博扎、浆水为代表的发酵样品中,分离筛选出形态差异菌株,通过产酶筛选及16S rDNA分子鉴定,获得产植酸酶乳酸菌菌株,并通过耐酸、耐胆盐、耐肠胃液、自聚集力及抑菌分析对其进行评价。【结果】筛选出4株产植酸酶乳酸菌,其中菌株JS5植酸酶酶活高达0.83 U/mL,与植物乳杆菌菌株 DSM 20174相似性为100%,确定该菌株为植物乳杆菌(Lactiplantibacillus plantarum)。菌株JS5在pH值3条件下存活率高达100%,在胆盐浓度0.1%时JS5存活率为0.55%,在模拟肠液和胃液中,存活率分别为8.56%和0.38%,其活菌数均在1×10⁶CFU/mL以上,达到发挥益生作用的最小活菌数要求。菌株JS5自聚集力为(9.69%±0.44%),可促进菌株在肠道定殖。菌株JS5发酵液对大肠杆菌、沙门氏菌、金黄色葡萄球菌、白色念珠菌、铜绿假单胞菌均有抑制作用。【结论】植保乳杆菌JS5产植酸酶较高,可在模拟肠胃液中存活,具有良好的细胞黏附能力和抑制病原菌生长,可用于益生的畜禽微生态制剂。

关键词: 乳酸菌; 植酸酶; 筛选

Abstract:

【Objective】 To isolate and characterization phytase production probiotic lactic acid bacteria. 【Methods】 Morphologically different strains were isolated from fermented feed, boza and Jiangshui. Through enzyme production screening and 16S rDNA molecular identification, phytase producing lactic acid bacteria strains were obtained and evaluated through acid tolerance, bile salt tolerance, intestinal and gastric juice tolerance, self-aggregation ability, and antibacterial analysis. 【Results】 Four phytase producing lactic acid bacteria were identified. Among them, strain JS5, which was 100% similar to Lactobacillus paracasei JCM 1171, with a maximum phytase activity of 0.83 U/mL. The strain was designated as lactobacillus paracasei. The survival rate of strain JS5 under pH 3 was as high as 100%. At a bile salt concentration of 0.1%, the survival rate of JS5 was 0.55%. In simulated gastric juice and intestinal juice, the survival rates were 8.56% and 0.38%,respectively, and the number of viable bacteria was above 1×10⁶CFU/mL, which was the lowest viable bacteria number with beneficial effects. The self-aggregation ability of strain JS5 was (9.69±0.44), which can promote the colonization of strain JS5 in the intestine. It had inhibitory effects on Escherichia coli, Salmonella, Staphylococcus aureus, Candida albicans, and Pseudomonas aeruginosa. 【Conclusion】 Lactobacillus paracasei JS5 has a high phytase production and can survive in simulated gastrointestinal fluid. It has good cell adhesion ability and inhibits the growth of pathogenic bacteria and can be used as a probiotics for livestock and poultry.

Key words: lactic acid bacteria; phytase; screen

中图分类号:

S188

王浩中, 林青, 曾军, 高雁, 赵燕慧, 时红玲, 马贵军, 马正海, 娄恺, 霍向东. 产植酸酶益生乳酸菌的筛选[J]. 新疆农业科学, 2024, 61(9): 2290-2298.

WANG Haozhong, LIN Qing, ZENG Jun, GAO Yan, ZHAO Yanhui, SHI Hongling, MA Guijun, MA Zhenghai, LOU Kai, HUO Xiangdong. Screening of phytase producing probiotic lactic acid bacteria[J]. Xinjiang Agricultural Sciences, 2024, 61(9): 2290-2298.

图/表 10

参考文献 35

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菌株 Strain	透明圆直径 D (mm)	菌落直径 d (mm)	直径比 D (d)
F9	35.00±6.67	14.85±2.96	2.21±0.72
F10	35.15±3.40	17.00±0.77	1.87±0.48
T7	32.58±2.10	15.18±0.25	1.88±0.49
S1	12.05±0.69	7.38±0.13	1.50±0.27
J1	15.93±2.01	8.30±0.25	1.73±0.42
F4	27.85±1.82	16.28±0.15	1.58±0.31
JS2	16.13±0.62	9.53±0.34	1.54±0.30
JS5	11.45±0.15	7.23±0.11	1.44±0.23
J6	7.40±0.16	6.15±0.11	1.16±0.07
F1	21.55±2.06	14.13±0.36	1.44±0.27
F2	16.05±2.79	12.33±0.33	1.30±0.20
T8	11.28±0.69	8.05±0.72	1.36±0.09
F11	18.45±1.56	15.63±1.24	1.18±0.02
S2	9.10±0.07	6.95±0.11	1.27±0.08
F8	20.05±1.25	15.55±0.35	1.23±0.09
T4	9.30±0.16	7.30±0.12	1.22±0.08
SC1	14.00±0.14	12.43±0.37	1.10±0.04
T1	10.95±0.57	7.33±0.78	1.42±0.28
T3	7.73±0.80	6.75±0.27	1.12±0.09
F7	38.08±0.68	18.55±0.67	1.77±0.43
F6	22.43±2.46	14.23±0.73	1.48±0.30
T2	9.18±0.08	7.28±0.15	1.19±0.10

菌株 Strain	透明圆直径 D (mm)	菌落直径 d (mm)	直径比 D (d)
F9	35.00±6.67	14.85±2.96	2.21±0.72
F10	35.15±3.40	17.00±0.77	1.87±0.48
T7	32.58±2.10	15.18±0.25	1.88±0.49
S1	12.05±0.69	7.38±0.13	1.50±0.27
J1	15.93±2.01	8.30±0.25	1.73±0.42
F4	27.85±1.82	16.28±0.15	1.58±0.31
JS2	16.13±0.62	9.53±0.34	1.54±0.30
JS5	11.45±0.15	7.23±0.11	1.44±0.23
J6	7.40±0.16	6.15±0.11	1.16±0.07
F1	21.55±2.06	14.13±0.36	1.44±0.27
F2	16.05±2.79	12.33±0.33	1.30±0.20
T8	11.28±0.69	8.05±0.72	1.36±0.09
F11	18.45±1.56	15.63±1.24	1.18±0.02
S2	9.10±0.07	6.95±0.11	1.27±0.08
F8	20.05±1.25	15.55±0.35	1.23±0.09
T4	9.30±0.16	7.30±0.12	1.22±0.08
SC1	14.00±0.14	12.43±0.37	1.10±0.04
T1	10.95±0.57	7.33±0.78	1.42±0.28
T3	7.73±0.80	6.75±0.27	1.12±0.09
F7	38.08±0.68	18.55±0.67	1.77±0.43
F6	22.43±2.46	14.23±0.73	1.48±0.30
T2	9.18±0.08	7.28±0.15	1.19±0.10

Strain	Bile salt (0%) viable count(CFU/mL)	Bile salt (0.1%) viable count(CFU/mL)	Survival rate (%)	Bile salt (0.2%) viable count(CFU/mL)	Survival rate (%)
J6	(2.85±0.05)×10⁸	(2.45±0.55)×10⁶	0.86	-	-
S1	(5.75±0.65)×10⁸	-	0	-	-
JS5	(8.25±0.35)×10⁸	(4.56±0.36)×10⁶	0.55	-	-
JS2	(4.31±0.41)×10⁸	-	0	-	-

Strain	Bile salt (0%) viable count(CFU/mL)	Bile salt (0.1%) viable count(CFU/mL)	Survival rate (%)	Bile salt (0.2%) viable count(CFU/mL)	Survival rate (%)
J6	(2.85±0.05)×10⁸	(2.45±0.55)×10⁶	0.86	-	-
S1	(5.75±0.65)×10⁸	-	0	-	-
JS5	(8.25±0.35)×10⁸	(4.56±0.36)×10⁶	0.55	-	-
JS2	(4.31±0.41)×10⁸	-	0	-	-

Strain	0 h viable count (CFU/mL)	6 h viable count (CFU/mL)	Survival rate (%)
J6	(0.53±0.15)×10⁸	(4.90±0.85)×10⁷	92.45
S1	(7.57±0.47)×10⁸	(3.93±0.13)×10⁸	51.92
JS5	(1.32±0.07)×10⁹	(1.13±0.06)×10⁸	8.56
JS2	(1.10±0.12)×10⁹	(0.80±0.14)×10⁷	0.73

产植酸酶益生乳酸菌的筛选

Screening of phytase producing probiotic lactic acid bacteria

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Strain	0 h viable count (CFU/mL)	3 h viable count (CFU/mL)	Survival rate (%)
J6	(7.05±0.35)×10⁸	(1.95±0.21)×10⁸	27.66
S1	(7.95±1.06)×10⁸	(1.40±0.28)×10⁷	1.76
JS5	(1.68±0.22)×10⁹	(6.33±1.15)×10⁶	0.38
JS2	(0.35±0.07)×10⁸	(1.85±0.35)×10⁷	52.86

菌株 Strains	Diameter of Bacteriostatic circle(mm)
菌株 Strains	大肠杆菌 Escherichia coli	沙门氏菌 Salmonella	金黄色葡萄球菌 Staphylococcus aureus	白色念珠菌 Candida albicans	铜绿假单胞菌 Pseudomonas aeruginosa
JS5-1	14.57±0.12^c	19.43±0.55^a	14.30±0.44^b	18.07±0.15^c	14.87±0.32^b
JS5-2	15.80±0.26^b	16.73±0.12^d	13.73±0.32^b	17.33±0.21^d	14.23±0.21^cd
JS2-1	15.83±0.30^b	17.23±0.12^c	12.87±0.32^c	17.63±0.12^d	13.67±0.21^e
JS2-2	14.43±0.20^c	17.80±0.26^b	16.30±0.20^a	16.73±0.32^e	14.97±0.06^b
S1-1	15.97±0.42^b	17.97±0.15^b	14.03±0.21^b	19.30±0.36^a	13.97±0.15^de
S1-2	16.77±0.38^a	18.13±0.35^b	13.73±0.64^b	18.83±0.12^b	14.60±0.26^bc
J6-1	11.50±0.17^e	16.00±0.10^e	-	13.33±0.21^f	12.59±0.40^f
J6-2	12.57±0.12^d	13.57±0.12^f	11.30±0.26^d	13.43±0.11^f	20.90±0.36^a

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