WO2023040422A1 - 益生菌及其代谢产物（后生元）配方在制备缓解结直肠炎产品中的应用 - Google Patents
益生菌及其代谢产物（后生元）配方在制备缓解结直肠炎产品中的应用 Download PDFInfo
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- lactobacillus
- lactobacillus reuteri
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- reuteri
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Definitions
- the invention belongs to the field of microbes, and in particular relates to the application of a formula of probiotics and metabolites (postbiotics) in preparing products for relieving colorectal inflammation.
- IBD Inflammatory bowel disease
- Probiotics especially Lactobacillus and Bifidobacteria, have recently become a hot spot in the treatment of IBD due to their safety, effectiveness and strong ability to regulate the flora.
- most studies have rarely reported the effects of multi-strain combinations and their metabolites on IBD.
- the probiotic strain and its metabolites can play a protective role in the DSS-induced colitis model by reducing inflammation and regulating the disordered microbiota, and the intervention effect of the mixed strain and its metabolites is better than that of a single strain.
- the purpose of the present invention is to provide five self-isolated and identified probiotics, their fermentation metabolites and bacterial composition formulations for clinical prevention and treatment of colorectal-associated inflammatory bowel disease.
- GDMCC Guangdong Microbial Culture Collection Center
- GDMCC Guangdong Microbial Culture Collection Center
- GDMCC Guangdong Microbial Culture Collection Center
- GDMCC Guangdong Microbial Culture Collection Center
- GDMCC Guangdong Microbial Culture Collection Center
- the present invention provides above-mentioned Lactobacillus reuteri (Lactobacillus reuteri) Lactobacillus reuteri Lactobacillus gasseri Lactobacillus acidophilus Bifidobacterium lactis The application of one or more of them, it or their fermentation culture or it or their metabolites in the preparation of products for relieving colorectal inflammation.
- Said product can be food, health product or medicine for relieving colitis.
- Lactobacillus reuteri Lactobacillus reuteri Lactobacillus gasseri Lactobacillus acidophilus Bifidobacterium lactis The application of the mixture of five kinds of bacteria, their fermentation cultures or their metabolites in the preparation of products for relieving colorectal inflammation.
- said Lactobacillus reuteri (Lactobacillus reuteri) Lactobacillus reuteri Lactobacillus gasseri Lactobacillus acidophilus Bifidobacterium lactis
- the mixture is a bacterial liquid, and its bacterial content is more than 10 ⁇ 9 cfu/ml. Further preferred is 10 ⁇ 9 cfu/ml.
- the second object of the present invention is to provide above-mentioned Lactobacillus reuteri (Lactobacillus reuteri) Lactobacillus reuteri Lactobacillus gasseri Lactobacillus acidophilus Bifidobacterium lactis Use of one or more of them, it or their fermentation cultures or it or their metabolites in the preparation of products that increase the relative abundance of Bacteroidaceae Bacteroides and Lachnospiraceae Ruminococcus in the intestinal tract.
- the third object of the present invention is to provide the use of Bacteroidaceae Bacteroides and/or Lachnospiraceae Ruminococcus as biomarkers for identifying the recovery status of intestinal inflammation.
- the fourth object of the present invention is the application of the preparation for detecting the content of Bacteroidaceae Bacteroides and/or Lachnospiraceae Ruminococcus in the preparation of preparations for identifying the recovery status of intestinal inflammation.
- the present invention relates to the core components of five self-isolated, identified and patent-preserved probiotics and their fermentation metabolites (postbiotics) and their protective effects on relieving colitis induced by mouse dextran sodium sulfate (DSS) Applications. It also discloses the application mechanism of the probiotics and their fermentation metabolites (postbiotics) in mouse colitis.
- the application of this invention can reduce macrophage infiltration, reduce intestinal inflammation, regulate the microenvironment of intestinal flora and accelerate the repair of intestinal mucosa , is expected to be used in the clinical prevention and treatment of colorectal-associated inflammatory bowel disease.
- GDMCC Guangdong Microbial Culture Collection Center
- GDMCC Guangdong Microbial Culture Collection Center
- GDMCC Guangdong Microbial Culture Collection Center
- GDMCC Guangdong Microbial Culture Collection Center
- GDMCC Guangdong Microbial Culture Collection Center
- Figure 1 is Lactobacillus reuteri Whole-genome sequencing strain gene circular function map
- Figure 2 is Lactobacillus reuteri Whole-genome sequencing strain gene circular function map
- Fig. 3 is Lactobacillus gasseri (Lactobacillus gasseri) Whole-genome sequencing strain gene circular function map;
- Figure 4 is Lactobacillus acidophilus Whole-genome sequencing strain gene circular function map
- Figure 5 is the bifidobacterium lactis (Bifdobacterium lactis) Whole-genome sequencing strain gene circular function map;
- Fig. 6 is (A) Lactobacillus reuteri (Lactobacillus reuteri ) evolution map, (B) Lactobacillus gasseri (Lactobacillus gasseri ) evolution map, (C) Lactobacillus acidophilus ) evolution map, (D) Bifidobacterium lactis ) evolution map;
- Fig. 7 is a figure for identification of fermentation metabolites of 5 strains of probiotic bacteria
- Fig. 8 is a figure for identification of fermentation metabolites of 5 purified probiotic strains
- Figure 9 Mixed strains alleviated the pathological symptoms of DSS-induced experimental colitis.
- Figure 10 Strains and their metabolites restore inflammatory damage to the colonic mucosa.
- Figure 11 Mixed probiotic strains can regulate the intestinal flora structure of DSS-induced colitis model.
- FIG. 12 Gut microbiota strongly correlates with disease phenotypes in a mouse model of colitis.
- Figure 13 Identification of key microbial markers interacting with mixed strains and their metabolite interventions during recovery from intestinal inflammation.
- the present invention obtains 5 strains of probiotics through screening and separation, through 16S sequencing (16S full-length amplification primer, 16S_Forw: AGAGTTTGATCCTGGCTCAG, 16S_Rev: GGTTACCTTGTTACGACTT), whole genome sequencing circular functional map (extracting strain fermentation sludge accounting, building a library Carry out next-generation sequencing, the sequencing platform is Ilumina Hiseq X10) and phylogenetic tree construction, and obtain Lactobacillus reuteri (Lactobacillus reuteri) Lactobacillus reuteri Lactobacillus gasseri Lactobacillus acidophilus Bifidobacterium lactis Specific information is as follows:
- GDMCC Guangdong Microbial Culture Collection Center
- GDMCC Guangdong Microbial Culture Collection Center
- Lactobacillus gasseri It was deposited in Guangdong Microbial Culture Collection Center (GDMCC) on October 24, 2019, address: 5th Floor, Building 59, Compound, No. 100 Xianlie Middle Road, Guangzhou City, postcode: 510070, and deposit number: GDMCC No: 60830.
- the nucleotide sequence of its 16s rDNA is shown in SEQ ID NO.3, the gene circular function map of the whole genome sequencing strain is shown in Figure 3, and the evolution map is shown in Figure 6.
- GDMCC Guangdong Microbial Culture Collection Center
- GDMCC Guangdong Microbial Culture Collection Center
- Lactobacillus reuteri, Lactobacillus gasseri and Lactobacillus acidophilus use optimized MRS medium peptone (10g/L), beef extract powder (8g/L), yeast extract powder (4g/L), glucose (20g /L), dipotassium hydrogen phosphate (2g/L), diamine hydrogen citrate (2g/L), sodium acetate (5g/L), magnesium sulfate (0.2g/L), manganese sulfate (0.04g/L)
- the solvent is water
- the specific preparation method is to mix the components evenly, and sterilize for future use.
- Bifidobacterium lactis uses optimized bifidobacterium medium: peptone (15g/L), glucose (20g/L), yeast extract powder (2g/L), soluble starch (0.5g/L), sodium chloride (5g /L), L-cysteine (0.5g/L), tomato extract powder (5g/L), liver extract powder (2g/L), the solvent is water, and the specific preparation method is to mix the ingredients evenly, bacteria spare.
- Fermentation and preparation process of probiotics Inoculate Lactobacillus or Bifidobacterium into 5ml sterilized MRS liquid medium or Bifidobacterium liquid medium respectively, and enrich the bacteria by anaerobic culture for 24 hours, then inoculate 5ml of the bacterial liquid, Transfer to 1 L medium for anaerobic fermentation and amplification for 48 hours. After the fermentation, the bacteria sludge was collected by centrifugation, and the supernatant bacteria liquid was filtered through a 0.22 ⁇ m filter membrane to obtain the filtered supernatant for later use.
- step B and step D Repeat step B and step D three to four times. At this time, it can be observed that the upper liquid is yellowish brown and transparent, and the lower liquid is brown.
- the upper strata liquid is the oil phase: the substance soluble in ethyl acetate in the supernatant; the lower floor liquid is the water phase: the substance insoluble in ethyl acetate in the supernatant).
- the liquid in the sample bottle is evaporated to no longer decrease, turn off the main switch and take off the sample bottle. At this time, the liquid in the bottle is the substance initially extracted from the supernatant of 500ml bacterial liquid.
- H&E staining mouse colon was fixed, dehydrated, embedded, sliced, and dewaxed at 68°C for 50 minutes. The specific steps for dewaxing and hydration were toluene dewaxing I dewaxing 5 minutes; xylene dewaxing II dewaxing 5 minutes ; Xylene dewaxing III dewaxing 5min; 100% ethanol dewaxing 2min; 95% ethanol I hydration 2min; 95% ethanol II hydration 2min; 80% ethanol hydration 2min; , wash three times.
- the steps are: dipping with hematoxylin for 2 minutes; washing with tap water for 1 minute, 3-5 times (2 dyeing boxes are filled with tap water, and two times in sequence, 1 dyeing box is trickled under tap water and overflowed naturally for 1 minute/ Or wash for 1 minute, change the box and repeat 5 times); 0.3% hydrochloric acid alcohol differentiation for a few seconds (intestinal 1-2s, liver, BAT soak 3 times, iWAT soak 1 time, each time 1s); run tap water once; change the box and wash twice time, 1 min each time; blue solution for 10 min; 0.5% eosin aqueous solution: intestinal 4 h; liver: 1-2 h; BAT, iWAT: 5 h; 80% ethanol I dehydration 2 min; 90% ethanol II dehydration 2 min; 95 Dehydrate with % ethanol II for 2 min; dehydrate with absolute ethanol I for 2 min; dehydrate with absolute ethanol II for 2 min; xylene I for 2 min; xylene II
- H.&E stained colon tissue was observed under a microscope and then scored for pathology. The scoring was evaluated by other experimenters who did not know the details of the experiment. The scoring criteria were epithelial loss, crypt damage, and goblet cells. The comprehensive score of exhaustion and inflammatory cell infiltration, the specific score is shown in Table 1:
- Fecal DNA extraction, sequencing library construction and 16S rRNA sequencing use MoBio PowerSoil DNA Extraction Kit (QIAGEN, USA) to extract DNA from mouse feces, extract the kit (QIAGEN, USA), and measure its concentration with a nano titrator (Thermo Fisher, USA). 50ng of DNA was used for the construction of 16S rRNA sequencing library, using Q5 high-fidelity DNA polymerase (NEB), targeting the V3-V4 region of bacterial 16S rRNA (Forword primer: 5'-CCTACGGGNGGCWGCAG-3'; Reverse primer: 5' -GACTACHVGGGTATCTAATCC-3'), and then purified with AMPure XP (Beck).
- NEB Q5 high-fidelity DNA polymerase
- mice 14-week-old C57BL/6J mice to establish a DSS-induced colitis model: add 2.5% DSS to sterile water Free feeding was given for 7 days, followed by free access to sterile water for the next 7 days.
- the mice in the 6 experimental groups need to be fed with the corresponding single strain or mixed strain resuspended in the filtered supernatant every day, while the mice in the control group are fed with PBS.
- body weight, changes in stool hardness, bloody stool and other experimental indicators were recorded every day.
- gut microbiota is strongly correlated with disease phenotypes in colitis mouse models.
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Abstract
罗伊氏乳杆菌（Lactobacillus reuteri） PLBK®1、罗伊氏乳杆菌（Lactobacillus reuteri） PLBK®2、加氏乳杆菌（Lactobacillus gasseri） PLBK®3、嗜酸乳杆菌（Lactobacillus acidophilus） PLBK®4、乳双歧杆菌（Bifdobacterium lactis） PLBK®5中的一种或多种菌株及其发酵培养物或代谢物在制备缓解结直肠炎产品中的应用，这种应用能够降低巨噬细胞浸润、降低肠道炎症、调节肠道菌群微环境并加速肠粘膜修复，有望用于结直肠相关炎症性肠炎的临床预防与治疗。
Description
本发明属于微生物领域，具体涉及一种益生菌及其代谢产物(后生元)配方在制备缓解结直肠炎产品中的应用。
炎症性肠病(IBD)由于发病率高且治愈率低，现已成为全球公共卫生问题。虽然该病的发病机制不明确，但肠道微生物群与炎症信号之间存在的潜在关联已被建立，可以成为研究该病的切入点。益生菌，尤其是乳杆菌及双歧杆菌，由于其安全有效性且强大的菌群调控能力，最近成为IBD的治疗热点。但是大多研究对于多菌株组合与其代谢产物对IBD作用的评估还很少被报道。在这项研究中，我们探究了实验室自主分离和专利保护的益生菌，罗伊氏菌，格氏乳杆菌，嗜酸乳杆菌和乳双歧杆菌及其代谢物对葡聚糖硫酸钠(DSS)诱导结肠炎的小鼠的保护作用。结果表明，混合菌株及其代谢物不仅改善了疾病表型，比如降低体重变化、DAI评分和组织病理学分数等。还通过增加有益菌和降低病原菌来改善肠道菌群的结构组成，且效果比单菌株好。此外，还发现了肠道菌群与表型之间具有复杂且密切的互作网络。总之，该益生菌菌株及代谢产物能通过减轻炎症和调节紊乱的微生物群对DSS诱导的结肠炎模型发挥保护作用，且混合菌株及其代谢物的干预效果要优于单菌株，为未来益生菌对结肠炎的临床治疗提供策略。
发明内容：
本发明的目的是提供用于结直肠相关炎症性肠炎的临床预防与治疗的五株自主分离、鉴定的益生菌及其发酵代谢产物和菌剂组合物配方。
本发明的五株自主分离的益生菌，具体如下：
罗伊氏乳杆菌(Lactobacillus reuteri)
其于2019年10月24日保藏于广东省微生物菌种保藏中心(GDMCC)，地址：广州市先烈中路100号大院59号楼5楼，邮编：510070，保藏编号：GDMCC No:60828。
罗伊氏乳杆菌(Lactobacillus reuteri)
其于2019年10月24日保藏于广东省微生物菌种保藏中心(GDMCC)，地址：广州市先烈中路100号大院59号楼5楼，邮编：510070，保藏编号：GDMCC No:60829。
加氏乳杆菌(Lactobacillus gasseri)
其于2019年10月24日保藏于广东省微生物菌种保藏中心(GDMCC)，地址：广州市先烈中路100号大院59号楼5楼，邮编：510070，保藏编号：GDMCC No:60830。
嗜酸乳杆菌(Lactobacillus acidophilus)
其于2019年10月24日保藏于广东省微生物菌种保藏中心(GDMCC)，地址：广州市先烈中路100号大院59号楼5楼，邮编：510070，保藏编号：GDM CC No:60831。
乳双歧杆菌(Bifdobacterium lactis)
其于2019年10月24日保藏于广东省微生物菌种保藏中心(GDMCC)，地址：广州市先烈中路100号大院59号楼5楼，邮编：510070，保藏编号：GDMCC No:60832。
本发明提供了上述罗伊氏乳杆菌(Lactobacillus reuteri)
罗伊氏乳杆菌(Lactobacillus reuteri)
加氏乳杆菌(Lactobacillusgasseri)
嗜酸乳杆菌(Lactobacillus acidophilus)
乳双歧杆菌(Bifdobacterium lactis)
中的一种或数种、它或它们的发酵培养物或它或它们的代谢产物在制备缓解结直肠炎产品中的应用。
所说的产品可以是缓解结直肠炎的食品、保健品或药物。
优选，罗伊氏乳杆菌(Lactobacillus reuteri)
罗伊氏乳杆菌(Lactobacillus reuteri)
加氏乳杆菌(Lactobacillusgasseri)
嗜酸乳杆菌(Lactobacillus acidophilus)
乳双歧杆菌(Bifdobacterium lactis)
五种菌的混合物、它们的发酵培养物或它们的代谢产物在制备缓解结直肠炎产品中的应用。
优选，是罗伊氏乳杆菌(Lactobacillus reuteri)
罗伊氏乳杆菌(Lactobacillus reuteri)PLBK
加氏乳杆菌(Lactobacillusgasseri)
嗜酸乳杆菌(Lactobacillus acidophilus)
乳双歧杆菌(Bifdobacterium lactis)
是按数量比1:1:1:1:1混合的。
进一步优选，所述的罗伊氏乳杆菌(Lactobacillus reuteri)
罗伊氏乳杆菌(Lactobacillus reuteri)
加氏乳杆菌(Lactobacillus gasseri)
嗜酸乳杆菌(Lactobacillus acidophilus)
乳双歧杆菌(Bifdobacterium lactis)
的混合物是菌液，其含菌量是10
^9cfu/ml以上。进一步优选是10
^9cfu/ml。
本发明的第二个目的是提供上述罗伊氏乳杆菌(Lactobacillus reuteri)
罗伊氏乳杆菌(Lactobacillus reuteri)
加氏乳杆菌(Lactobacillusgasseri)
嗜酸乳杆菌(Lactobacillus acidophilus)
乳双歧杆菌(Bifdobacterium lactis)
中的一种或数种、它或它们的发酵培养物或它或它们的代谢产物在制备升高肠道中Bacteroidaceae Bacteroides和Lachnospiraceae Ruminococcus的相对丰度产品中的应用。
本发明的第三个目的是提供Bacteroidaceae Bacteroides和/或Lachnospiraceae Ruminococcus作为鉴定肠道炎症恢复状况的生物标志物中的应用。
本发明的第四个目的是检测Bacteroidaceae Bacteroides和/或Lachnospiraceae Ruminococcus含量的制剂在制备鉴定肠道炎症恢复状况的制剂中的应用。
本发明涉及五株自主分离、鉴定和专利保藏的益生菌及其发酵代谢产物(后生元)的核心成分及其在 缓解小鼠葡聚糖硫酸钠(DSS)诱导结肠炎的小鼠的保护作用的应用。还公开了该益生菌及其发酵代谢产物(后生元)环节小鼠结肠炎应用机理，应用该发明能够降低巨噬细胞浸润、降低肠道炎症、调节肠道菌群微环境并加速肠粘膜修复，有望用于结直肠相关炎症性肠炎的临床预防与治疗。
罗伊氏乳杆菌(Lactobacillus reuteri)
其于2019年10月24日保藏于广东省微生物菌种保藏中心(GDMCC)，地址：广州市先烈中路100号大院59号楼5楼，邮编：510070，保藏编号：GDMCC No:60828。
罗伊氏乳杆菌(Lactobacillus reuteri)
其于2019年10月24日保藏于广东省微生物菌种保藏中心(GDMCC)，地址：广州市先烈中路100号大院59号楼5楼，邮编：510070，保藏编号：GDMCC No:60829。
加氏乳杆菌(Lactobacillusgasseri)
其于2019年10月24日保藏于广东省微生物菌种保藏中心(GDMCC)，地址：广州市先烈中路100号大院59号楼5楼，邮编：510070，保藏编号：GDMCC No:60830。
嗜酸乳杆菌(Lactobacillus acidophilus)
其于2019年10月24日保藏于广东省微生物菌种保藏中心(GDMCC)，地址：广州市先烈中路100号大院59号楼5楼，邮编：510070，保藏编号：GDMCC No:60831。
乳双歧杆菌(Bifdobacterium lactis)
其于2019年10月24日保藏于广东省微生物菌种保藏中心(GDMCC)，地址：广州市先烈中路100号大院59号楼5楼，邮编：510070，保藏编号：GDMCC No:60832。
图1是罗伊氏乳杆菌(Lactobacillus reuteri)
的全基因组测序菌株基因环状功能图谱；
图2是罗伊氏乳杆菌(Lactobacillus reuteri)
的全基因组测序菌株基因环状功能图谱；
图3是加氏乳杆菌(Lactobacillus gasseri)
的全基因组测序菌株基因环状功能图谱；
图4是嗜酸乳杆菌(Lactobacillus acidophilus)
的全基因组测序菌株基因环状功能图谱；
图5是乳双歧杆菌(Bifdobacterium lactis)
的全基因组测序菌株基因环状功能图谱；
图6是(A)罗伊氏乳杆菌(Lactobacillus reuteri
)进化图谱、(B)加氏乳杆菌(Lactobacillus gasseri
)进化图谱、(C)嗜酸乳杆菌(Lactobacillus acidophilus
)进化图谱、(D)乳双歧杆菌(Bifdobacterium lactis
)进化图谱；
图7是5株益生菌菌株发酵代谢产物鉴定图；
图8是纯化的5株益生菌菌株发酵代谢产物鉴定图；
图9：混合菌株减轻了DSS诱导的实验性结肠炎的病理症状。
图10：菌株及其代谢物可恢复结肠黏膜的炎症损伤。
图11：混合益生菌菌株可调节DSS诱导结肠炎模型的肠道菌群结构。
图12：肠道菌群与结肠炎小鼠模型的疾病表型具有强相关性。
图13：鉴定在肠道炎症恢复期间与混合菌株及其代谢物干预相互作用的关键微生物标志物。
以下实施例是对本发明的进一步说明，而不是对本发明的限制，本发明的保护范围不限于以下具体实施例。
实施例1：
一、实验方法
1、益生菌菌株类别及专利保藏编号
本发明通过筛选和分离获得了5株益生菌，通过16S测序(16S全长扩增引物，16S_Forw:AGAGTTTGATCCTGGCTCAG,16S_Rev:GGTTACCTTGTTACGACTT)、全基因组测序环状功能图谱(提取菌株发酵菌泥核算，建库进行二代测序，测序平台为Ilumina Hiseq X10)以及进化树构建，获得罗伊氏乳杆菌(Lactobacillus reuteri)
罗伊氏乳杆菌(Lactobacillus reuteri)
加氏乳杆菌(Lactobacillus gasseri)
嗜酸乳杆菌(Lactobacillus acidophilus)
乳双歧杆菌(Bifdobacterium lactis)
具体信息如下：
罗伊氏乳杆菌(Lactobacillus reuteri)
其于2019年10月24日保藏于广东省微生物菌种保藏中心(GDMCC)，地址：广州市先烈中路100号大院59号楼5楼，邮编：510070，保藏编号：GDMCC No:60828。其16s rDNA的核苷酸序列如SEQ ID NO.1所示，全基因组测序菌株基因环状功能图谱如图1所示，进化图谱如图6所示。
罗伊氏乳杆菌(Lactobacillus reuteri)
其于2019年10月24日保藏于广东省微生物菌种保藏中心(GDMCC)，地址：广州市先烈中路100号大院59号楼5楼，邮编：510070，保藏编号：GDMCC No:60829。其16s rDNA的核苷酸序列如SEQ ID NO.2所示，全基因组测序菌株基因环状功能图谱如图2所示，进化图谱如图6所示。
加氏乳杆菌(Lactobacillus gasseri)
其于2019年10月24日保藏于广东省微生物菌种保藏中心(GDMCC)，地址：广州市先烈中路100号大院59号楼5楼，邮编：510070，保藏编号：GDMCC No:60830。其16s rDNA的核苷酸序列如SEQ ID NO.3所示，全基因组测序菌株基因环状功能图谱如图3所示，进化图谱如图6所示。
嗜酸乳杆菌(Lactobacillus acidophilus)
其于2019年10月24日保藏于广东省微生物菌种保藏中心(GDMCC)，地址：广州市先烈中路100号大院59号楼5楼，邮编：510070，保藏编号：GDM CC No:60831。其16s rDNA的核苷酸序列如SEQ ID NO.4所示，全基因组测序菌株基因环状功能图谱如图4所示，进化图谱如图6所示。
乳双歧杆菌(Bifdobacterium lactis)
其于2019年10月24日保藏于广东省微生物菌种保藏中心(GDMCC)，地址：广州市先烈中路100号大院59号楼5楼，邮编：510070，保藏编号：GDMCC No:60832。其16s rDNA的核苷酸序列如SEQ ID NO.5所示，全基因组测序菌株基因环状功能图谱如图5所示，进化图谱如图6所示。
2、发酵培养基：
罗伊氏乳杆菌，加氏乳杆菌和嗜酸乳杆菌使用优化的MRS培养基：蛋白胨(10g/L)，牛肉浸粉(8g/L)，酵母浸粉(4g/L)，葡萄糖(20g/L)，磷酸氢二钾(2g/L)，柠檬酸氢二胺(2g/L)，乙酸钠(5g/L)，硫酸镁(0.2g/L)，硫酸锰(0.04g/L)，溶剂为水，具体制备方法是将各成分混合均匀，灭菌备用。
乳双歧杆菌使用优化的双歧杆菌培养基：蛋白胨(15g/L)，葡萄糖(20g/L)，酵母浸粉(2g/L)，可溶性淀粉(0.5g/L)，氯化钠(5g/L)，L-半胱氨酸(0.5g/L)，番茄浸粉(5g/L)，肝浸粉(2g/L)，溶剂为水，具体制备方法是将各成分混合均匀，灭菌备用。
3、益生菌发酵和制备工艺：将乳杆菌或双歧杆菌分别接种于5ml灭菌的MRS液体培养基或双歧杆菌液体培养基中，厌氧培养增菌24小时，然后将5ml菌液，转移至1L的培养基进行厌氧发酵扩增48小时。发酵结束后，离心收取菌泥，上清菌液通过0.22μm滤膜过滤获得过滤上清液备用。通过涂板计数益生菌cfu后，用过滤上清液重悬菌泥获得10
^9cfu/ml单菌株菌液，然后将5个单菌株菌液按照体积比
的比例混合获得混合益生菌菌液，用于以下实验。
4、益生菌代谢产物制备工艺：菌种发酵液，离心，上清液过滤(0.22μm)，由此得到各菌株的发酵代谢产物，再按照体积等比例混合五株菌发酵代谢产物，获得混合益生菌发酵代谢产物，4℃保藏。
5、益生菌代谢产物纯化工艺：
A、将500ml 0.22μm滤膜过滤益生菌发酵液上清液倒于分液漏斗中，随后再向其中倒入500ml的乙酸乙酯；
B、水平摇动分液漏斗2分钟以充分混匀两种液体；
C、将分液漏斗转移至通风橱中，室温静置5分钟，使液体充分分层；
D、重复步骤B和步骤D三至四次，此时可观察到上层液体呈黄棕色透明状，下层液体呈棕色。(上层液体为油相：上清液中可溶于乙酸乙酯的物质；下层液体为水相：上清液中不可溶于乙酸乙酯的物质)。
E、在分液漏斗的下口处放置一个500ml三角瓶，用来盛放下层液体；
F、打开分液漏斗的上活塞，使漏斗内外气压保持一致，随后拧开分液漏斗的下活塞使下层液体排放 至三角瓶内。此时可将较少的上层液体也一同排放至三角瓶，以排除液体分层处的干扰。
G、排放结束后，关闭下活塞。下层液体从上口倒出至旋转瓶内；
H、将旋转瓶连接旋转蒸发仪，将旋转瓶的1/4瓶身浸泡于水浴锅中，调整旋转蒸发仪的水浴锅数值为45℃，打开旋转蒸发仪的总开关。
I、待旋转瓶内的液体蒸发至不再减少时，关闭总开关，取下旋转瓶；并向旋转瓶内加入2-3ml的乙酸乙酯，充分混匀，用移液管将旋转瓶内的液体转移至样本瓶；
J、将样本瓶连接旋转蒸发仪，将样本瓶的1/4瓶身浸泡于水浴锅中，调整旋转蒸发仪的水浴锅数值为45℃，打开旋转蒸发仪的总开关。
K、待样本瓶内的液体蒸发至不再减少时，关闭总开关，取下样本瓶，此时瓶内的液体即为500ml菌液上清液初步萃取的物质。
由此获得每种菌的浓缩代谢产物(水相)。
6、结肠炎小鼠模型构建：14周龄C57BL/6J饮用的无菌水中添加质量分数2.5％葡聚糖硫酸钠(DSS，LOT NO：S2839，分子量为36000-50000Da；MP生物医学有限责任企业，美国索伦俄亥俄州)，自由饮用7天来建立结肠炎模型。设立6个实验组(罗伊氏乳杆菌
-代号LR1、罗伊氏乳杆菌
-代号LR2、加氏乳杆菌
嗜酸乳杆菌
-代号LA、乳双歧杆菌
-代号BL和混合益生菌组MIX)和一个对照组(CONTROL)，即5个单菌株菌液处理组和1个混合益生菌菌液处理组，对照组是PBS灌胃组，每组6-10只小鼠。在DSS造模后，每天进行菌液灌胃，每次200μL，每天一次，共灌胃14天，每天记录老鼠体重变化和状态，并在第8天和第14天收取粪便，在第15天将老鼠全部处死，取出结肠组织拍照并测量长度，另外还需要截取肠道组织进行福尔马林固定，菌液干预实验中还需要用玻片刮取剩余的肠内容物以便进行后续实验操作。
7、H&E染色：小鼠结肠固定、脱水、包埋，切片，68℃烤片脱蜡50分钟，进行脱蜡水化具体步骤为甲苯脱蜡Ⅰ脱蜡5min；二甲苯脱蜡Ⅱ脱蜡5min；二甲苯脱蜡Ⅲ脱蜡5min；100％乙醇脱蜡2min；95％乙醇Ⅰ水化2min；95％乙醇Ⅱ水化2min；80％乙醇水化2min；水洗3min，换盒子洗，每次1min，洗三次。之后进行染色，步骤为：苏木素浸染2min；自来水洗1min，3-5遍(2个染盒装好自来水，依次简单过两下，将1染盒在自来水下细流接水并自然溢出1min/或者清洗1分钟，换盒子重复5次)；0.3％盐酸酒精分化数秒(肠道1-2s、liver、BAT泡3下，iWAT泡1下，每下1s)；自来水过一下；换盒子洗两次，每次1min；蓝化液返蓝10min；0.5％伊红水溶液：肠道4h；liver：1-2h；BAT、iWAT：5h；80％乙醇Ⅰ脱水2min；90％乙醇Ⅱ脱水2min；95％乙醇Ⅱ脱水2min；无水乙醇Ⅰ脱水2min；无水乙醇Ⅱ脱水2min；二甲苯Ⅰ透明2min；二甲苯Ⅱ透明2min；中性树胶封片。
8、组织学评分：H.&E.染色后的结肠组织经显微镜下观察后进行病理学评分，评分由不知实验详情的 其他实验人员进行评估，评分标准为上皮缺失，隐窝损伤，杯状细胞耗竭和炎性细胞浸润的综合评分，具体评分见表1：
表1葡聚糖硫酸钠(DSS)诱导结肠炎的组织学评分
9、粪便DNA提取、测序文库构建和16S rRNA测序：利用MoBio PowerSoil DNA提取试剂盒(美国QIAGEN公司)提取小鼠粪便中的DNA，提取试剂盒(QIAGEN，美国)，用纳米滴定仪测量其浓度(ThermoFisher，USA)。50ng的DNA被用于16S rRNA测序文库的构建，使用Q5高保真DNA聚合酶(NEB)，针对细菌16S rRNA的V3-V4区(Forword primer：5'-CCTACGGGNGGCWGCAG-3'；Reverse primer：5'-GACTACHVGGGTATCTAATCC-3')，然后用AMPure XP(Beck)纯化。
10、16S rRNA扩增子测序和生物信息学统计：采用Qiime 2标准化流程进行分析，通过DADA2算法获得高质量的扩增子序列变体(ASVs).根据silva数据库(2019年12月发布)，进行分类剖析，并转化为系统、类、目、科、属和种水平的相对丰度。相对丰度低于0.001或在所有组别中出现率低于70％的微生物数据被过滤掉，以获得核心细菌类群用于进一步分析。在R软件包EasyMicroPlot上进行了Alpha 多样性、Beta多样性、共发生网络图分析、结构图、随机森林模型、冗余分析(RDA)和PICRUSt分析等。
11、统计分析：所有实验的统计学细节和样本数在每张图的图例中都有描述，结果以平均值±标准误表示。对于两组之间的显著性比较采用双尾非配对学生t检验。多组之间的显著性测试采用单因素方差分析然后进行Tukey's和LSD的事后检验。统计学意义在图例中描述为：*p<0.05，**p<0.01，***p<0.001。
二、实验结果
1、5株益生菌菌株发酵代谢产物鉴定结果如图7所示。5株益生菌在发酵后，通过0.22μM滤膜过滤后，获得发酵上清液，MRS培养基作为空白对照，进行LC-MS/MS质谱分析其中代谢物，代谢物成分和含量通过与MRS空白对照对比，获得益生菌发酵产生的代谢物。
2、纯化的5株益生菌菌株发酵代谢产物鉴定如图8所示。5株益生菌在发酵后，通过0.22μM过滤乙酸乙酯浓缩，收集下层(水相)的代谢物，通过低温浓缩蒸干，获得浓缩1000x的代谢物，通过LC-MS/MS质谱分析其中代谢物，代谢物成分和含量通过与MRS空白对照对比，获得益生菌发酵产生的代谢物。
3、为了解五种益生菌及其混合益生菌及其代谢物对结肠炎的干预作用，我们用14周龄C57BL/6J小鼠建立DSS诱导的结肠炎模型：在无菌水中添加2.5％DSS自由喂食7天，随后7天继续自由引用无菌水。同时，6组实验组的老鼠需要每天灌胃相对应的过滤上清液重悬的单菌株或者混合菌株，而对照组灌胃的则是PBS。在整个实验过程中，每天记录体重、粪便硬度变化、血便情况等实验指标，另外，在第8天和第14天收集粪便后通过16S rRNA测序进行肠道微生物群分析，老鼠处死后还需要收集结肠组织以便进行后续H.&E.染色和IHC实验等(图9A)。通过分析发现，与对照组相比，混合菌株及其代谢物组能改善DSS引起的结肠缩短(图9B)，除此之外，与对照组相比，菌株及其代谢物的体重变化曲线更平缓，体重减轻明显减少(图9C)且DAI评分也显著较低(图9D)，其中9C和9D中的右侧柱状图从左至右分别是CONTROL、MIX、LG、LR1、LR2、LA、BL。然而，单菌株组对于体重变化和DAI评分的降低效果却不如混合益生菌菌株组显著，综上可见，混合菌株减轻了DSS诱导的实验性结肠炎的病理症状。
4、我们对结肠切片进行H.&E.染色以评估结肠粘膜损伤情况，从对照组的结肠切片可发现肠道结构和屏障发生了改变，具体表现为粘膜层破坏，隐窝损伤和炎症细胞浸润(图10A)。相比之下，混合菌株及其代谢物的处理对结肠具有保护作用，表现为完整的隐窝和较少的炎症细胞浸润，对结肠切片进行组织学评估发现。巨噬细胞已被确定为评估结肠炎严重程度的标志物，为了进一步研究混合菌株及其代谢物对结肠炎模型的影响，我们利用免疫组织化学实验(IHC)以检测与炎症有关的巨噬细胞标志物(F4/80)(图10B)，处理结果与H.&E.染色结果一致，表现为菌株及其代谢物的F4/80水平显著降低，且与对照组相比，单菌株处理组F4/80的表达水平也相对较低。综上可见，菌株及其代谢物可恢复结肠黏膜的炎症损伤。
5、结果发现，在不同的实验时期，7组肠道微生物群的组成在物种水平上存在差异，其中菌株及其代谢物组中Muribaculaceae Muribaculaceae的相对丰度在第8天较高，Bacteroidaceae Bacteroides的相对丰度在第14天较高(图11A,B)。与此同时，在第8天和第14天，分别对7组在物种水平上的α-多样性值(Simpson指数)进行了评价，其中，在第8天，菌株及其代谢物的Simpson指数与对照组有显著差异，而其他单菌株处理组则无显著差异(图11C)，而在第14天，对照组和菌株及其代谢物之间不存在差异(Simpson指数，图11D)，这些结果表明，混合菌株及其代谢物干预能对炎症持续期的微生物群落组成和多样性产生强烈的影响，从而进一步加速了炎症的恢复。此外，在第八天，基于Bray-Curtis度量距离主坐标分析(PCoA)的β-多样性显示出，只有对照组和混合菌株及其代谢物的肠道微生物群被明确地分为两个集群(图11E，G)，这意味着混合菌株及其代谢物组在第8天的物种水平上的微生物结构发生了明显改变。值得注意的是，在第14天，七个组的PCoA图与对照组都存在显著不同(图11F，H)。事实上，相比之下，混合菌株及其代谢物组在肠道炎症的恢复阶段显示出不同的微生物组成，例如，第8天的Muribaculaceae Muribaculaceae、Oscillospirales UCG.005和第14天的Bacteroidaceae Bacteroides、Lachnospiraceae Ruminococcus(图11I-J)。综上可见混合益生菌菌株可调节DSS诱导结肠炎模型的肠道菌群结构。
6、在我们的结肠炎和混合菌株及其代谢物处理模型中，疾病表型数据(如体重、DAI、结肠长度和组织学评分)和肠道菌群的改变(如α和β-多样性，相对丰度)表明宿主和肠道菌群可能共同作用于疾病的发生、进展和恢复。因此，我们利用冗余分析(RDA)来解锁肠道微生物组成与表型数据之间的关系。在物种水平上，7组的核心微生物与表型数据矩阵之间存在较强的相关性(图12A:model p-value:0.003,图12B:model p-value:0.025)。其中，种层级别的核心微生物数据组成的解释变量共同解释了七组样本中的体重，DAI，血便和粪便硬度高达52.68％的变异性，在经999次置换检验，V4，V9，V17，V36，V34和V42等细菌对该表型数据排布的约束具有统计学意义(V4：r2＝0.1152，p＝0.016；V9：r2＝0.1658，p＝0.001；V17：r2＝0.2226，p＝0.001；V36：r2＝0.2292，p＝0.001；V34：r2＝0.1557，p＝0.003；V42：r2＝0.1228，p＝0.009)。另外，种层级别的核心微生物数据对结肠长度和组织学评分的解释度高达89.13％,且V8，V39，V12和V42对该表型数据具有显著影响(V8：r2＝0.2082，p＝0.028；V39：r2＝0.1823，p＝0.028；V12：r2＝0.1673，p＝0.045；V42：r2＝0.1932，p＝0.026)。综上可见，肠道菌群与结肠炎小鼠模型的疾病表型具有强相关性。
7、为了在肠道炎症恢复期准确识别微生物标志物，我们主要对混合菌株及其代谢物和对照组分别在第8天和第14天采取随机森林分析结合5倍交叉验证来进一步识别特征细菌(图13A,C)。对两组的并集进行分析后发现有显著差异的菌为十一株，在八天有7株菌，在第14天有4株菌。其中在第八天的两组相互比较下，Parasutterella Burkholderiales和Lactobacillaceae Lactobacillus的相对丰度在对照组显著升高，而Muribaculaceae Muribaculaceae，Oscillospirales UCG.005，Dubosiella Firmicutes，Tissierellales Anaerovoracaceae和Clostridia Clostridia的相对丰度在mix组显著升高(图13B)。另外，在第十四天，在 对照组的处理下，Bacteroidales Tannerellaceae和Rikenellaceae Rikenellacea的相对丰度显著升高，经过混合菌株及其代谢物处理后的Bacteroidaceae Bacteroides和Lachnospiraceae Ruminococcus的相对丰度显著升高(图13D)。在益生菌干预组中，核心菌株且益生菌核心菌株Bacteroidales Tannerellaceae和Rikenellaceae Rikenellacea不管是在干预第8天还是第14天，都处于菌种网络的核心区域(图13E-G)。在益生菌干预这些数据表明，混合菌株及其代谢物处理通过对肠道微环境的特异性调节而发挥综合作用。
Claims (9)
- 罗伊氏乳杆菌(Lactobacillus reuteri)罗伊氏乳杆菌(Lactobacillus reuteri)
加氏乳杆菌(Lactobacillus gasseri)
嗜酸乳杆菌(Lactobacillus acidophilus)
乳双歧杆菌(Bifdobacterium lactis)
中的一种或数种、它或它们的发酵培养物，或，它或它们的代谢产物在制备缓解结直肠炎产品中的应用。
- 根据权利要求1所述的应用，其特征在于，所述的产品是缓解结直肠炎的食品、保健品或药物。
- 根据权利要求1所述的应用，其特征在于，罗伊氏乳杆菌(Lactobacillus reuteri)罗伊氏乳杆菌(Lactobacillus reuteri)
加氏乳杆菌(Lactobacillus gasseri)
嗜酸乳杆菌(Lactobacillus acidophilus)
乳双歧杆菌(Bifdobacterium lactis)
五种菌的混合物、它们的发酵培养物混合物或它们的代谢产物混合物在制备缓解结直肠炎产品中的应用。
- 根据权利要求3所述的应用，其特征在于，是罗伊氏乳杆菌(Lactobacillus reuteri)罗伊氏乳杆菌(Lactobacillus reuteri)
加氏乳杆菌(Lactobacillus gasseri)
嗜酸乳杆菌(Lactobacillus acidophilus)
乳双歧杆菌(Bifdobacterium lactis)
是按数量比1:1:1:1:1混合的。
- 根据权利要求4所述的应用，其特征在于，所述的罗伊氏乳杆菌(Lactobacillus reuteri)罗伊氏乳杆菌(Lactobacillus reuteri)
加氏乳杆菌(Lactobacillus gasseri)
嗜酸乳杆菌(Lactobacillus acidophilus)
乳双歧杆菌(Bifdobacterium lactis)
的混合物是菌液，其含菌量是10 ^9cfu/ml以上。
- 根据权利要求5所述的应用，其特征在于，所述的菌液是10 ^9cfu/ml。
- 罗伊氏乳杆菌(Lactobacillus reuteri)罗伊氏乳杆菌(Lactobacillus reuteri)
加氏乳杆菌(Lactobacillus gasseri)
嗜酸乳杆菌(Lactobacillus acidophilus)
乳双歧杆菌(Bifdobacterium lactis)
中的一种或数种、它或它们的发酵培养物，或它或它们的代谢产物在制备升高肠道中Bacteroidaceae Bacteroides和Lachnospiraceae Ruminococcus的相对丰度的产品中的应用。
- Bacteroidaceae Bacteroides和/或Lachnospiraceae Ruminococcus作为鉴定肠道炎症恢复状况的生物标志物中的应用。
- 检测Bacteroidaceae Bacteroides和/或Lachnospiraceae Ruminococcus含量的制剂在制备鉴定肠道炎症恢复状况的制剂中的应用。
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| CN202210397574.1A CN114984058A (zh) | 2022-04-15 | 2022-04-15 | 益生菌及其代谢产物（后生元）配方在制备缓解结直肠炎产品中的应用 |
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| NL2033398B1 (en) * | 2022-04-15 | 2023-11-06 | Inst Of Microbiology Guangdong Academy Of Sciences Guangdong Detection Center Of Microbiology | Formula of probiotics and metabolites thereof for relieving metabolic syndrome, and use thereof |
| CN117535175A (zh) * | 2023-10-12 | 2024-02-09 | 善恩康生物科技（苏州）有限公司 | 一种复合益生菌及其在制备预防或辅助治疗结直肠癌的产品中的应用 |
| CN117866854A (zh) * | 2024-03-08 | 2024-04-12 | 广州同康生物科技有限公司 | 修复胃肠黏膜损伤的罗伊氏粘液乳杆菌bn01及其后生元 |
| CN117982541A (zh) * | 2024-02-07 | 2024-05-07 | 中山大学肿瘤防治中心（中山大学附属肿瘤医院、中山大学肿瘤研究所） | 中华伊格尔兹氏菌在制备防治炎症性肠病和结直肠癌的产品中的应用 |
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| CN116369534A (zh) * | 2023-04-19 | 2023-07-04 | 黑龙江飞鹤乳业有限公司 | 磷脂酰丝氨酸的用途及基于其的营养组合物、食品及用途 |
| CN117402794B (zh) * | 2023-12-12 | 2024-02-27 | 四川厌氧生物科技有限责任公司 | 一种加氏乳杆菌及其应用 |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| NL2033398B1 (en) * | 2022-04-15 | 2023-11-06 | Inst Of Microbiology Guangdong Academy Of Sciences Guangdong Detection Center Of Microbiology | Formula of probiotics and metabolites thereof for relieving metabolic syndrome, and use thereof |
| CN117535175A (zh) * | 2023-10-12 | 2024-02-09 | 善恩康生物科技（苏州）有限公司 | 一种复合益生菌及其在制备预防或辅助治疗结直肠癌的产品中的应用 |
| CN117535175B (zh) * | 2023-10-12 | 2024-05-31 | 善恩康生物科技（苏州）有限公司 | 一种复合益生菌及其在制备预防或辅助治疗结直肠癌的产品中的应用 |
| CN117982541A (zh) * | 2024-02-07 | 2024-05-07 | 中山大学肿瘤防治中心（中山大学附属肿瘤医院、中山大学肿瘤研究所） | 中华伊格尔兹氏菌在制备防治炎症性肠病和结直肠癌的产品中的应用 |
| CN117866854A (zh) * | 2024-03-08 | 2024-04-12 | 广州同康生物科技有限公司 | 修复胃肠黏膜损伤的罗伊氏粘液乳杆菌bn01及其后生元 |
| CN117866854B (zh) * | 2024-03-08 | 2024-06-07 | 广州同康生物科技有限公司 | 修复胃肠黏膜损伤的罗伊氏粘液乳杆菌bn01及其后生元 |
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