鄂尔多斯地区酸粥细菌多样性分析及乳酸菌的分离鉴定

史学敏; 刘珊娜; 杜茜

doi:10.13386/j.issn1002-0306.2022060261

鄂尔多斯地区酸粥细菌多样性分析及乳酸菌的分离鉴定

doi: 10.13386/j.issn1002-0306.2022060261

史学敏^1,,
刘珊娜^{1, 2,*, ,},
杜茜¹

1.
天津农学院食品科学与生物工程学院，天津 300392
2.
天津市农副产品深加工技术工程中心，天津 300392

详细信息

作者简介:
史学敏（1997−），女，硕士研究生，研究方向：食品加工与安全，E-mail：2395568435@qq.com

通讯作者:
刘珊娜（1984−），女，博士，副教授，研究方向：食品生物技术，E-mail：lsn_nxy@aliyun.com

中图分类号: TS201.3
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出版历程
- 收稿日期: 2022-06-28
- 刊出日期: 2023-05-01

Bacterial Diversity Analysis of Sour Porridge from Ordos and Isolation and Identification of Lactic Acid Bacteria

SHI Xuemin^1
,,
LIU Shanna^{1, 2,*
, ,},
DU Xi¹

1.
College of Food Science and Bioengineering, Tianjin Agricultural University, Tianjin 300392, China
2.
Tianjin Engineering and Technology Research Center of Agricultural Products Processing, Tianjin 300392, China

摘要

摘要: 以采集自内蒙古鄂尔多斯地区的8个酸粥样品为研究对象，利用Illumina MiSeq高通量测序技术，对样品进行细菌菌群结构及多样性研究，分离鉴定其优势菌株。结果表明：8个样品中的细菌主要来自厚壁菌门（Firmicutes）和变形菌门（Proteobacteria），乳酸杆菌属（Lactobacillus）和醋酸杆菌属（Acetobacter）为优势菌属。乳酸杆菌属在各样品中占比最高，为42.63%~97.49%。SZ1、SZ2样品与其他样品的菌群结构差异较大，可能与不同采集时间有关。对上述样品中的乳酸菌进行分离纯化，通过生理生化试验结合分子生物学方法，鉴定获得1株干酪乳杆菌（Lactobacillus casei），1株发酵乳杆菌（Lactobacillus fermentum），2株植物乳杆菌（Lactobacillus plantarum）和4株副干酪乳杆菌（Lactobacillus paracasei）。上述研究结果为酸粥工业化生产提供菌种资源和理论依据。
- 酸粥 /
- 高通量测序 /
- 细菌多样性 /
- 乳酸菌 /
- 分离鉴定
Abstract: The bacterial community structure and diversity of eight sour porridge samples collected from Ordos, Inner Mongolia were studied by Illumina Miseq high-throughput sequencing technology, and the dominant strains were isolated and identified. The results showed that the bacteria in the eight samples mainly belonged to Firmicutes and Proteobacteria. Lactobacillus and Acetobacter were the dominant genera. Lactobacillus was the highest abundance genus in all samples accounting for 42.63% to 97.49%. The structures of bacterial community in SZ1 and SZ2 differed from those in other samples, which was probably related to different sampling time. One strain was identified as Lactobacillus casei, one strain was identified as Lactobacillus fermentum, two strains were identified as Lactobacillus plantarum and four strains were identified as Lactobacillus paracasei by physiological and biochemical experiments as well as molecular biological method. The above research results provide strain resources and theoretical basis for the industrialized production of sour porridge.
- sour porridge /
- high-throughput sequencing /
- bacterial diversity /
- lactic acid bacteria /
- isolation and identification

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图 1 酸粥样品的稀释性曲线（A）和香农曲线（B）

Figure 1. Rarefaction curves (A) and Shannon index curves (B) of sour porridge samples

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图 2 在门水平（A）和属水平（B）上的细菌群落结构

Figure 2. Bacterial community structure at phylum level (A) and genus level (B)

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图 3 细菌群落PCoA分析

Figure 3. PCoA analysis of bacterial community

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图 4 乳酸菌菌落形态

Figure 4. Colony morphology of lactic acid bacteria

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图 5 乳酸菌革兰氏染色结果

Figure 5. Gram staining of lactic acid bacteria

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表 1 酸粥样品高通量测序结果

Table 1. High-throughput sequencing data of sour porridge samples

样品名称	原始序列数	高质量序列数	有效序列数	样品平均序列长度（bp）	GC（%）	Q20（%）	Q30（%）
SZ1	79915	79554	75123	423	52.76	98.92	95.70
SZ2	80121	79763	78951	416	52.77	99.00	95.89
SZ3	80129	79938	76016	429	51.70	99.29	96.80
SZ4	79925	79767	78243	422	53.00	99.38	97.23
SZ5	79652	79492	78233	428	52.14	99.31	96.95
SZ6	80224	80047	77922	419	53.40	99.39	97.21
SZ7	80359	80179	76030	428	51.99	99.30	96.90
SZ8	80173	80019	76593	422	51.97	99.40	97.20

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表 2 酸粥样品的Alpha多样性指数分析

Table 2. Analysis of Alpha diversity indexes of sour porridge samples

样品名称	OTU数	ACE	Chao1	Simpson	Shannon	覆盖率（%）
SZ1	60	128.64	93.33	0.69	1.91	99.97
SZ2	59	123.70	84.30	0.70	2.04	99.97
SZ3	171	187.89	192.75	0.44	1.46	99.96
SZ4	203	205.35	205.40	0.83	3.21	99.99
SZ5	203	205.44	207.50	0.17	0.95	99.99
SZ6	210	210.16	210.00	0.75	3.04	100.00
SZ7	189	197.01	199.06	0.57	1.80	99.97
SZ8	171	204.95	202.89	0.69	1.92	99.94

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表 3 酸粥pH及乳酸菌的菌落总数

Table 3. The pH of sour porridge samples and the total number of lactic acid bacteria colonies

样品名称	pH	乳酸菌菌落总数（lg CFU/mL）
SZ1	3.49±0.02^c	6.02±0.56^d
SZ2	3.51±0.01^c	6.08±0.47^d
SZ3	4.03±0.02^b	6.65±0.38^c
SZ4	4.01±0.03^b	6.78±0.26^a
SZ5	4.52±0.03^a	4.18±0.71^e
SZ6	3.98±0.01^b	5.20±0.64^e
SZ7	4.01±0.03^b	6.70±0.39^b
SZ8	4.01±0.01^b	5.51±0.46^e
注：同列不同小写字母表示差异显著（P<0.05）。

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表 4 乳酸菌的生理生化鉴定结果

Table 4. Physiological and biochemical identification results of lactic acid bacteria

菌株名称	来源	七叶苷	纤维二糖	麦芽糖	甘露醇	水杨苷	山梨醇	蔗糖	棉子糖	菊糖	乳糖
L140	SZ1	+	+	+	+	+	+	+	−	+	+
L24	SZ2	+	+	+	+	+	+	+	+	+	+
L218	SZ2	+	+	+	+	+	+	+	+	+	+
L353	SZ3	+	+	+	+	+	+	+	−	+	+
L520	SZ5	+	+	+	+	+	+	+	−	+	+
L74	SZ7	+	+	+	+	+	+	+	−	+	+
L813	SZ8	+	+	+	−	+	−	+	−	+	+
A117	SZ1	+	+	+	+	+	+	+	−	+	+
注：“+”表示结果呈阳性；“−”表示结果呈阴性。

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表 5 乳酸菌的16S rDNA序列同源性分析

Table 5. 16S rDNA sequence homology analysis of lactic acid bacteria

菌株名称	鉴定结果	同源性（%）	NCBI登录号
L140	Lactobacillus casei	99.79	9W0ZB1H3016
L24	Lactobacillus plantarum	99.79	EHFNJHFC016
L218	Lactobacillus plantarum	99.93	9W1GPU97016
L353	Lactobacillus paracasei	99.79	EHG55D2H013
L520	Lactobacillus paracasei	99.97	9W1YJJPB01R
L74	Lactobacillus paracasei	99.86	9W22CWAC016
L813	Lactobacillus fermentum	99.73	EHGAFDAN013
A117	Lactobacillus paracasei	100	9W2A3P4501R

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参考文献(30)

[1]	王强. 糜米粉对面团特性及其面包品质影响研究[D]. 天津: 天津科技大学, 2019. WANG Q. Studies on the effect of proso millet flour on dough properties and bread qualities[D]. Tianjin: Tianjin University of Science and Technology, 2019.
[2]	马薇薇. 糜米馒头关键技术及蛋清粉对其品质影响研究[D]. 天津: 天津科技大学, 2020. MA W W. Studies on the key technology of proso millet steamed bread and the effect of egg white powder on its quality[D]. Tianjin: Tianjin University of Science and Technology, 2020.
[3]	王吉力特, 刘聪, 夏美茹, 等. 糜米对高脂饮食大鼠血脂的影响[J]. 粮食与油脂,2022,35(1):30−34. [WANG J L T, LIU C, XIA M R, et al. Effects of Panicum miliaceum L. on blood lipid in rats fed with high-fat diet[J]. Cereals & Oils,2022,35(1):30−34. doi: 10.3969/j.issn.1008-9578.2022.01.008
[4]	王凤玲, 刘爱国, 李娜, 等. 糜米发酵液中优势乳酸菌分离鉴定[J]. 食品科学,2007,28(11):342−345. [WANG F L, LIU A G, LI N, et a. Isolation and identification of properties of dominant biology lactic acid bacteria form mimi fermented liquid[J]. Food Science,2007,28(11):342−345. doi: 10.3321/j.issn:1002-6630.2007.11.078
[5]	薛建岗, 陈永福, 于海静, 等. 内蒙古西部地区自然发酵酸粥化学成分及微生物组成分析[J]. 食品科技,2013,38(7):10−14. [XUE J G, CHEN Y F, YU H J, et al. Analysis of chemical and microbiological composition in naturally fermented acidic-gruel from western areas of Inner Mongolia[J]. Food Science and Technology,2013,38(7):10−14. doi: 10.13684/j.cnki.spkj.2013.07.035
[6]	张悦, 张川, 李中媛, 等. 传统酸粥制备工艺的优化[J]. 食品工业,2020,41(1):128−132. [ZHANG Y, ZHANG C, LI Z Y, et al. Optimization of traditional suanzhou preparation process[J]. The Food Industry,2020,41(1):128−132.
[7]	王炜宏, 杜晓华, 张家超, 等. 内蒙古鄂尔多斯地区酸粥发酵液中乳酸菌的分离鉴定[J]. 食品与生物技术学报,2010,29(2):266−270. [WANG W H, DU X H, ZHANG J C, et al. Isolation and identification of LAB from naturally fermented congee in Ordos area of Inner Mongolia[J]. Journal of Food Science and Biotechnology,2010,29(2):266−270.
[8]	王琪, 刘聪, 景彦萍, 等. 晋西北酸粥发酵液中乳酸菌的潜在益生特性[J]. 食品与发酵工业,2019,45(2):60−66. [WANG Q, LIU C, JING Y P, et al. Probiotic properties of LAB isolated from fermentation broth of sour porridge in Northwestern Shanxi[J]. Food and Fermentation Industries,2019,45(2):60−66.
[9]	KAVITAKE D, SURYAVANSHI M V, KANDASAMY S, et al. Bacterial diversity of traditional fermented food, Idli by high thorough-put sequencing[J]. Journal of Food Science and Technology, 2022.
[10]	张青, 郭淑文, 苏靖, 等. 基于高通量测序技术巴彦淖尔市酸粥中细菌多样性分析[J]. 中国酿造,2020,39(9):101−105. [ZHANG Q, GUO S W, SU J, et al. Bacterial diversity in sour porridge from Bayannur based on high-throughput sequencing[J]. China Brewing,2020,39(9):101−105. doi: 10.11882/j.issn.0254-5071.2020.09.019
[11]	张青, 石晶红. 山西河曲与内蒙古五原酸粥细菌类群结构及其基因功能的比较分析[J/OL]. 食品与发酵工业, 2022. DOI: 10.13995/j.cnki.11-1802/ts.032385. ZHANG Q, SHI J H. Comparative analysis of bacterial group structure and gene function between Hequ sour porridge and Wuyuan sour porridge[J/OL]. Food and Fermentation Industries, 2022. DOI: 10.13995/j.cnki.11-1802/ts.032385.
[12]	中华人民共和国卫生部. GB 4789.35-2016食品微生物学检验乳酸菌检验[S]. 北京: 中国标准出版社, 2016. Ministry of Health of the People's Republic of China. GB 4789.35-2016 Food microbiological examination Lactic acid bacteria[S]. Beijing: Standards Press of China, 2016.
[13]	王素华. 新平腌菜中乳酸菌的筛选、特性及应用研究[D]. 天津: 天津农学院, 2020. WANG S H. Screening, characterization and application of lactic acid bacteria from Xinping pickles[D]. Tianjin: Tianjin Agricultural University, 2020.
[14]	虞任莹, 彭思佳, 李艺, 等. 梅干菜和腌制雪菜细菌菌群多样性分析[J]. 食品工业科技,2021,42(18):134−141. [YU R Y, PENG S J, LI Y, et al. Analysis of bacterial diversity of the pickled and dried mustard and the fermented potherb mustard[J]. Science and Technology of Food Industry,2021,42(18):134−141. doi: 10.13386/j.issn1002-0306.2021010065
[15]	ZHANG X M, DANG X J, WANG Y B, et al. Diversity and composition of microbiota during fermentation of traditional Nuodeng ham[J]. Journal of Microbiology,2021,59(1):20−28. doi: 10.1007/s12275-021-0219-4
[16]	GRICE E A, KONG H H, CONLAN S, et al. Topographical and temporal diversity of the human skin microbiome[J]. Science,2009,324(5931):1190−1192. doi: 10.1126/science.1171700
[17]	CHEN W. Lactic acid bacteria[M]. Singapore: Springer, 2019: 181−209.
[18]	吴燕. 特色自然发酵浆水中乳酸菌的筛选、表征及应用[D]. 无锡: 江南大学, 2019. WU Y. Screening, characterization and application of lactic acid bacteria in characteristic natural fermented Jiangshui[D]. Wuxi: Jiangnan University, 2019.
[19]	牟俊. 山西老陈醋醋酸发酵阶段乳酸菌和醋酸菌相互作用研究[D]. 天津: 天津科技大学, 2018. MOU J. Interaction between lactic acid bacteria and acetic acid bacteria in the acetic acid fermentation process of Shanxi aged vinegar[D]. Tianjin: Tianjin University of Science and Technology, 2018.
[20]	GUO H, SUN Z, HAO Y, et al. Correlation between bacterial communities and organic acids in the fermentation stage of traditional Chinese sour porridge[J]. International Journal of Food Properties,2020,23(1):1430−1440. doi: 10.1080/10942912.2020.1801724
[21]	WANG Y R, HOU Q C, GUO Z, et al. Bacterial community structure in acidic gruel from different regions and its influence on quality[J]. Food Research International,2021,141:110130. doi: 10.1016/j.foodres.2021.110130
[22]	常健. 晋西北酸粥发酵过程中细菌种群分析及乳酸菌、醋酸菌的分离鉴定[D]. 太原: 山西大学, 2016. CHANG J. Bacterial population analysis, isolation and identification of lactic acid bacteria and acetic acid bacteria during the fermentation of Northwestern Shanxi acid porridge[D]. Taiyuan: Shanxi University, 2016.
[23]	LIU W J, ZHENG Y, KWOK L Y, et al. High-throughput sequencing for the detection of the bacterial and fungal diversity in Mongolian naturally fermented cow’s milk in Russia[J]. BMC Microbiology,2015,15:45. doi: 10.1186/s12866-015-0385-9
[24]	LI W, MUTUVULLA M, CHEN X, et al. Isolation and identification of high viscosity-producing lactic acid bacteria from a traditional fermented milk in Xinjiang and its role in fermentation process[J]. European Food Research and Technology,2012,235:497−505. doi: 10.1007/s00217-012-1779-7
[25]	TIAN H X, SHI Y H, ZHANG Y, et al. Screening of aroma-producing lactic acid bacteria and their application in improving the aromatic profile of yogurt[J]. Journal of Food Biochemistry,2019,43(10):12837.
[26]	陈雨, 吉巧琳, 李智昊, 等. 高产乳酸菌株的筛选、鉴定及发酵条件的研究[J/OL]. 云南民族大学学报(自然科学版), 2023(2): 1−14. https://kns.cnki.net/kcms/detail/53.1192.N.20220426.0827.002.html. CHEN Y, JI Q L, LI Z H, et al. Screening, identification and fermentation conditions of high yield kactic acid bacteria[J/OL]. Journal of Yunnan Minzu University (Natural Sciences Edition), 2023(2): 1−14. https://kns.cnki.net/kcms/detail/53.1192.N.20220426.0827.002.html.
[27]	孟宪刚, 李雪萍, 李建宏, 等. 浆水中乳酸菌分离鉴定及其代谢特性的初步研究[J]. 食品工业科技,2015,36(1):181−186, 210. [MENG X G, LI X P, LI J H, et al. Isolation and identification of lactic acid bacteria from Jiangshui and preliminary study on metabolic characteristics[J]. Science and Technology of Food Industry,2015,36(1):181−186, 210.
[28]	王琳珺, 潘道东, 杨媛, 等. 鱼类肠道嗜酸乳杆菌的分离及功能特性分析[J]. 中国食品学报,2014,14(5):20−25. [WANG L J, PAN D D, YANG Y, et al. Isolation and functional analysis of Lactobacillus acidophilus from fish intestine[J]. Journal of Chinese Institute of Food Science and Technology,2014,14(5):20−25.
[29]	张文平, 赵英杰, 罗晟, 等. 高产胞外多糖植物乳杆菌筛选及其发酵工艺优化[J]. 食品与发酵工业,2019,45(21):38−45. [ZHANG W P, ZHAO Y J, LUO S, et al. Screening of Lactobacillus plantarum with higher yield of exopolysaccharides and optimization of fermentation conditions[J]. Food and Fermentation Industries,2019,45(21):38−45. doi: 10.13995/j.cnki.11-1802/ts.021589
[30]	余萍, 曹蓝, 矫艳平, 等. 副干酪乳杆菌HCS17-040的筛选鉴定及其益生特性[J]. 食品与机械,2021,37(11):28−33, 129. [YU P, CAO L, JIAO Y P, et al. Screening of Lactobacillus paracasei HCS17-040 with triglyceride tunction and study on its probiotic characteristics[J]. Food & Machinery,2021,37(11):28−33, 129.