Article(id=1307013489042682396, tenantId=1146029695717560320, journalId=1306274286668972124, issueId=1307013423896753139, articleNumber=null, orderNo=null, doi=10.11843/j.issn.0366-6964.2026.03.044, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1734451200000, receivedDateStr=2024-12-18, revisedDate=null, revisedDateStr=null, acceptedDate=null, acceptedDateStr=null, onlineDate=1789545898924, onlineDateStr=2026-09-16, pubDate=1774195200000, pubDateStr=2026-03-23, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1789545898924, onlineIssueDateStr=2026-09-16, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1789545898924, creator=13701087609, updateTime=1789545898924, updator=13701087609, issue=Issue{id=1307013423896753139, tenantId=1146029695717560320, journalId=1306274286668972124, year='2026', volume='57', issue='3', pageStart='1177', pageEnd='1776', issueExtLink='null', onlineDate='null', pubDate='1774195200000', pubDateStr='2026-03-23', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=1, specialIssue=null, createTime=1789545883392, creator='13701087609', updateTime=1789609930841, updator='13701087609', preIssue=null, nextIssue=null, articleTotal=null, ext={EN=IssueExt(id=1307282058464743901, tenantId=1146029695717560320, journalId=1306274286668972124, issueId=1307013423896753139, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1307282058464743902, tenantId=1146029695717560320, journalId=1306274286668972124, issueId=1307013423896753139, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null, downloadFileDto=null}, startPage=1697, endPage=1710, ext={EN=ArticleExt(id=1307013489483084317, articleId=1307013489042682396, tenantId=1146029695717560320, journalId=1306274286668972124, language=EN, title=The Effects of Bacillus subtilis Combined with Different Lactic Acid Bacteria on the Quality of Fermented Astragalus and HD11 Cells Function, columnId=1307007406035198078, journalTitle=Acta Veterinaria et Zootechnica Sinica, columnName=BASIC VETERINARY MEDICINE, runingTitle=null, highlight=null, articleAbstract=

The aim of this study was to investigate the effect of Bacillus subtilis (KC) in combination with different lactic acid bacterias on the quality and active effects of fermented Astragalus. In which the different lactic acid bacterias inludes Lactobacillus acidophilus (SS), Lactobacillus rohitaensis (LY), Lactobacillus plantarum (ZW), and Streptococcus alactolyticus (FGM). Four kinds of fermented Astragalus were prepared by the mixed probiotics (SS+KC, LY+KC, ZW+KC, and FGM+KC), respectively. The negative control group (UNF) was the same preparation process with fermented Astragalus, but no probiotics. The quality of 4 fermented products was evaluated by measuring general physicochemical indicators (pH, ethanol content), characteristic physicochemical indicators (total acid, organic acid, crude polysaccharide content), total flavonoids, total saponins, total polyphenols, aflatoxin B1 (AFB1), and deoxynivalenol (DON) content. HD11 cells were stimulated with different concentrations of 4 fermented products and UNF samples, respectively, and the cell proliferation activity was determined by the CCK8 method, and the phagocytic ability and cytokine expression were determined by immunofluorescence and ELISA methods. The results showed that the general physicochemical indicators and characteristic physicochemical indicators of LY+KC group, FGM+KC group, and ZW+KC group all met the requirements of plant enzyme (QB/T5323-2018). Compared with UNF group, the total saponin content of LY+KC group was significantly reduced (P<0.05), and the total flavonoid and total saponin contents of FGM+KC group and ZW+KC group were significantly increased (P<0.05). Compared with UNF group, the AFB1 and DON contents of FGM+KC group, ZW+KC group, and LY+KC group were significantly reduced (P<0.05). At the concentration of 10 µg·mL-1, four kinds of fermented Astragalus could promote the viability of HD11 cells, which was determined as the subsequent working concentration. After treated with ZW+KC and FGM+KC fermented Astragalus, the phagocytosis capacity and the expression of pro-inflammatory factors (IL-1β and IL-12) in HD11 cells were significantly higher than those in CON, UNF, SS+KC and LY+KC groups (P<0.05). In conclusion, the selection of probitics is closely related to the quality and activity of fermented Astragalus. Among them, Astragalus membranaceus fermented by Bacillus subtilis combined with Lactobacillus plantarum or Streptococcus alactolyticus FGM has better quality, which can increase the content of total flavonoids and total saponins, degrade AFB1 and DON, and improve the function of HD11 cells.

, authors=Jiahui LIU, Guowei XU, Kang ZHANG, Jianxi LI, Zhiting GUO, Songwei HAN, Fubin CHEN, Haiyu CHEN, Xueyan GU, Jingyan ZHANG, Lei WANG, authorsList=Jiahui LIU, Guowei XU, Kang ZHANG, Jianxi LI, Zhiting GUO, Songwei HAN, Fubin CHEN, Haiyu CHEN, Xueyan GU, Jingyan ZHANG, Lei WANG, authorCompany=null, correspAuthors=Jingyan ZHANG, Lei WANG, authorNote=null, correspAuthorsNote=
ZHANG Jingyan, E-mail:
WANG Lei, E-mail:
, copyrightStatement=null, copyrightOwner=null, extLink=null, articleAbsUrl=null, sourceXml=null, magXml=null, pdfUrl=null, pdf=null, pdfFileSize=null, pdfExtLink=null, richHtmlUrl=null, mobilePdfUrl=null, reviewReport=null, pdfFirstPage=null, abstractGraph=null, abstractGraphContent=null, abstractVideo=null, citation=null, cebUrl=null, magXmlContent=null, mapNumber=null, fund=null), CN=ArticleExt(id=1307013493232792110, articleId=1307013489042682396, tenantId=1146029695717560320, journalId=1306274286668972124, language=CN, title=枯草芽孢杆菌联合不同乳酸菌制备黄芪酵素的质量评价及其对HD11细胞功能的影响, columnId=1307007406223941760, journalTitle=畜牧兽医学报, columnName=基础兽医, runingTitle=null, highlight=null, articleAbstract=

旨在评价枯草芽孢杆菌(KC)与不同乳酸菌联合使用对黄芪酵素质量和活性的影响,不同乳酸菌包括嗜酸乳杆菌(SS)、罗伊氏乳杆菌(LY)、植物乳杆菌(ZW)和非解乳糖链球菌(FGM)。分别按照工艺制备以SS+KC、LY+KC、ZW+KC、FGM+KC为混合菌种的4种黄芪酵素,以不加混合菌的含黄芪药性的发酵培养基为阴性对照组(UNF),分别通过测定一般理化指标(pH、乙醇含量)、特征性理化指标(总酸、有机酸、粗多糖含量)、总黄酮、总皂苷、总多酚、黄曲霉毒素B1(AFB1)、呕吐毒素(DON)的含量,评价黄芪酵素质量;分别用不同浓度的4种黄芪酵素、UNF样本刺激HD11细胞,采用CCK8法测定细胞增殖活力,采用免疫荧光法、ELISA方法测定黄芪酵素对HD11细胞吞噬能力、细胞因子表达的影响。结果发现,4种黄芪酵素中LY+KC组、FGM+KC组、ZW+KC组的一般理化指标和特征性理化指标均符合植物酵素(QB/T 5323—2018)要求;与UNF组相比,LY+KC组的总皂苷含量显著降低(P<0.05),FGM+KC组和ZW+KC组的总黄酮、总皂苷含量均显著升高(P<0.05);与UNF组相比,FGM+KC组、ZW+KC组、LY+KC组的AFB1和DON均显著降低(P<0.05);4种黄芪酵素在10 µg·mL-1时均可促进HD11细胞活力,确定为后续工作浓度,经ZW+KC、FGM+KC黄芪酵素处理后,HD11细胞的吞噬能力、促炎因子(IL-1β、IL-12)表达量均显著高于CON组、UNF组、SS+KC组和LY+KC组(P<0.05)。综上所述,发酵菌种选择与黄芪酵素质量、活性作用均密切相关,其中以枯草芽孢杆菌联合植物乳杆菌或非解乳糖链球菌为混合发酵菌种的黄芪酵素质量较优,可促进总黄酮、总皂苷含量提升,降解AFB1、DON,提高HD11细胞功能。

, authors=刘佳惠, 徐国伟, 张康, 李建喜, 郭志廷, 韩松伟, 陈富斌, 陈海瑜, 古雪艳, 张景艳, 王磊, authorsList=刘佳惠, 徐国伟, 张康, 李建喜, 郭志廷, 韩松伟, 陈富斌, 陈海瑜, 古雪艳, 张景艳, 王磊, authorCompany=null, correspAuthors=张景艳, 王磊, authorNote=

刘佳惠,硕士生,主要从事兽医学研究,E-mail:

, correspAuthorsNote=
张景艳,主要从事中兽医及免疫研究,E-mail:
王磊,主要从事中兽医及免疫研究,E-mail:
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A, B, C, D, E. Represent pH, ethanol content, total acid content, organic acid content, and crude polysaccharide content, respectively. No letters or the same lowercase letters indicate that the difference is not significant (P>0.05), and different lowercase letters indicate significant differences (P<0.05), The same as below

, figureFileSmall=pF3BvqRLZwfg0xm3GmEgAA==, figureFileBig=SiRWwLdNa/RpcIlxxumwJQ==, tableContent=null), ArticleFig(id=1307281641974559072, tenantId=1146029695717560320, journalId=1306274286668972124, articleId=1307013489042682396, language=CN, label=图1, caption=不同菌株对黄芪酵素理化指标的影响

A、B、C、D、E.分别代表pH、乙醇含量、总酸含量、有机酸含量、粗多糖含量。柱上无字母或相同小写字母表示差异不显著(P>0.05),不同小写字母表示差异显著(P<0.05),下同

, figureFileSmall=pF3BvqRLZwfg0xm3GmEgAA==, figureFileBig=SiRWwLdNa/RpcIlxxumwJQ==, tableContent=null), ArticleFig(id=1307281642062639457, tenantId=1146029695717560320, journalId=1306274286668972124, articleId=1307013489042682396, language=EN, label=Fig.2, caption=Effects of different bacteria strains on the active components in fermented Astragalus

A, B, C. Represent total flavonoid content, total saponin content and total polyphenol content, respectively

, figureFileSmall=ar2Vy8ve55BiZ34h7TBk/w==, figureFileBig=dbQ3CRzGsNS70Q27zJc9kQ==, tableContent=null), ArticleFig(id=1307281642163302754, tenantId=1146029695717560320, journalId=1306274286668972124, articleId=1307013489042682396, language=CN, label=图2, caption=不同菌株对黄芪酵素活性成分的影响

A、B、C.分别代表总黄酮含量、总皂苷含量、总多酚含量

, figureFileSmall=ar2Vy8ve55BiZ34h7TBk/w==, figureFileBig=dbQ3CRzGsNS70Q27zJc9kQ==, tableContent=null), ArticleFig(id=1307281642242994531, tenantId=1146029695717560320, journalId=1306274286668972124, articleId=1307013489042682396, language=EN, label=Fig.3, caption=Aflatoxin and vomitoxin content in fermented Astragalus

A. Aflatoxin B1 levels; B. Vomitoxin levels

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A.黄曲霉毒素B1含量;B.呕吐毒素含量

, figureFileSmall=yYB7WMEXG3XDpIHzMWJQ9A==, figureFileBig=PXEiwt3u66Ym5Ix/FwJ0Bw==, tableContent=null), ArticleFig(id=1307281642377212261, tenantId=1146029695717560320, journalId=1306274286668972124, articleId=1307013489042682396, language=EN, label=Fig.4, caption=Effect of fermented Astragalus on HD11 cell viability, figureFileSmall=YNEFJsLg7Szfh8aX4BVFyA==, figureFileBig=PIGhMza4BOj8LvNMw3iPIg==, tableContent=null), ArticleFig(id=1307281642448515430, tenantId=1146029695717560320, journalId=1306274286668972124, articleId=1307013489042682396, language=CN, label=图4, caption=黄芪酵素对HD11细胞活力的影响, figureFileSmall=YNEFJsLg7Szfh8aX4BVFyA==, figureFileBig=PIGhMza4BOj8LvNMw3iPIg==, tableContent=null), ArticleFig(id=1307281642540790119, tenantId=1146029695717560320, journalId=1306274286668972124, articleId=1307013489042682396, language=EN, label=Fig.5, caption=Effect of fermented Astragalus on phagocytosis of HD11

A. Representative fluorescent images captured by laser scanning confocal microscopy at a magnification of 200×, with a scale bar of 20 µm. B. The statistical bar chart generated by calculating the mean fluorescence intensity (MFI) based on the fluorescent images in Panel A

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A.激光共聚焦显微镜拍摄的代表性荧光照片,拍摄倍数为200×,比例尺为20 µm;B.基于图A的荧光图像,计算平均荧光强度(MFI)后生成的统计图

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A. The cell migration observed under the microscope; B. The cell migration rate (%)=[(0 h area-6 h/12 h/18 h/24 h area)/0 h average area]×100

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A.显微镜下观察细胞迁移;B.细胞迁移率(%)=[(0 h面积-6 h/12 h/18 h/24 h面积)/0h平均面积]×100

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A, B, C. IL-1β, IL-12, and TNF-α content, respectively

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A、B、C.分别代表IL-1β、IL-12、TNF-α含量

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枯草芽孢杆菌联合不同乳酸菌制备黄芪酵素的质量评价及其对HD11细胞功能的影响
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刘佳惠 , 徐国伟 , 张康 , 李建喜 , 郭志廷 , 韩松伟 , 陈富斌 , 陈海瑜 , 古雪艳 , 张景艳 , 王磊
畜牧兽医学报 | 基础兽医 2026,57(3): 1697-1710
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畜牧兽医学报 |基础兽医 2026 , 57 (3) : 1697 -1710
枯草芽孢杆菌联合不同乳酸菌制备黄芪酵素的质量评价及其对HD11细胞功能的影响
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刘佳惠 , 徐国伟, 张康, 李建喜, 郭志廷, 韩松伟, 陈富斌, 陈海瑜, 古雪艳, 张景艳 , 王磊
作者信息
  • 中国农业科学院兰州畜牧与兽药研究所 中泰中兽医药学技术联合实验室,兰州 730050
通讯作者:
张景艳,主要从事中兽医及免疫研究,E-mail:
王磊,主要从事中兽医及免疫研究,E-mail:
作者简介:

刘佳惠,硕士生,主要从事兽医学研究,E-mail:

The Effects of Bacillus subtilis Combined with Different Lactic Acid Bacteria on the Quality of Fermented Astragalus and HD11 Cells Function
Jiahui LIU , Guowei XU, Kang ZHANG, Jianxi LI, Zhiting GUO, Songwei HAN, Fubin CHEN, Haiyu CHEN, Xueyan GU, Jingyan ZHANG , Lei WANG
Affiliations
  • China-Thailand Joint Laboratory for Traditional Chinese Veterinary Medicine Technology Lanzhou Institute of Animal Husbandry and Veterinary Medicine,Chinese Academy of Agricultural Sciences,Lanzhou 730050,China
  • Corresponding Author:
    ZHANG Jingyan, E-mail:
    WANG Lei, E-mail:
出版时间: 2026-03-23 doi: 10.11843/j.issn.0366-6964.2026.03.044
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旨在评价枯草芽孢杆菌(KC)与不同乳酸菌联合使用对黄芪酵素质量和活性的影响,不同乳酸菌包括嗜酸乳杆菌(SS)、罗伊氏乳杆菌(LY)、植物乳杆菌(ZW)和非解乳糖链球菌(FGM)。分别按照工艺制备以SS+KC、LY+KC、ZW+KC、FGM+KC为混合菌种的4种黄芪酵素,以不加混合菌的含黄芪药性的发酵培养基为阴性对照组(UNF),分别通过测定一般理化指标(pH、乙醇含量)、特征性理化指标(总酸、有机酸、粗多糖含量)、总黄酮、总皂苷、总多酚、黄曲霉毒素B1(AFB1)、呕吐毒素(DON)的含量,评价黄芪酵素质量;分别用不同浓度的4种黄芪酵素、UNF样本刺激HD11细胞,采用CCK8法测定细胞增殖活力,采用免疫荧光法、ELISA方法测定黄芪酵素对HD11细胞吞噬能力、细胞因子表达的影响。结果发现,4种黄芪酵素中LY+KC组、FGM+KC组、ZW+KC组的一般理化指标和特征性理化指标均符合植物酵素(QB/T 5323—2018)要求;与UNF组相比,LY+KC组的总皂苷含量显著降低(P<0.05),FGM+KC组和ZW+KC组的总黄酮、总皂苷含量均显著升高(P<0.05);与UNF组相比,FGM+KC组、ZW+KC组、LY+KC组的AFB1和DON均显著降低(P<0.05);4种黄芪酵素在10 µg·mL-1时均可促进HD11细胞活力,确定为后续工作浓度,经ZW+KC、FGM+KC黄芪酵素处理后,HD11细胞的吞噬能力、促炎因子(IL-1β、IL-12)表达量均显著高于CON组、UNF组、SS+KC组和LY+KC组(P<0.05)。综上所述,发酵菌种选择与黄芪酵素质量、活性作用均密切相关,其中以枯草芽孢杆菌联合植物乳杆菌或非解乳糖链球菌为混合发酵菌种的黄芪酵素质量较优,可促进总黄酮、总皂苷含量提升,降解AFB1、DON,提高HD11细胞功能。

黄芪酵素  /  枯草芽孢杆菌  /  质量分析  /  活性作用  /  细胞功能

The aim of this study was to investigate the effect of Bacillus subtilis (KC) in combination with different lactic acid bacterias on the quality and active effects of fermented Astragalus. In which the different lactic acid bacterias inludes Lactobacillus acidophilus (SS), Lactobacillus rohitaensis (LY), Lactobacillus plantarum (ZW), and Streptococcus alactolyticus (FGM). Four kinds of fermented Astragalus were prepared by the mixed probiotics (SS+KC, LY+KC, ZW+KC, and FGM+KC), respectively. The negative control group (UNF) was the same preparation process with fermented Astragalus, but no probiotics. The quality of 4 fermented products was evaluated by measuring general physicochemical indicators (pH, ethanol content), characteristic physicochemical indicators (total acid, organic acid, crude polysaccharide content), total flavonoids, total saponins, total polyphenols, aflatoxin B1 (AFB1), and deoxynivalenol (DON) content. HD11 cells were stimulated with different concentrations of 4 fermented products and UNF samples, respectively, and the cell proliferation activity was determined by the CCK8 method, and the phagocytic ability and cytokine expression were determined by immunofluorescence and ELISA methods. The results showed that the general physicochemical indicators and characteristic physicochemical indicators of LY+KC group, FGM+KC group, and ZW+KC group all met the requirements of plant enzyme (QB/T5323-2018). Compared with UNF group, the total saponin content of LY+KC group was significantly reduced (P<0.05), and the total flavonoid and total saponin contents of FGM+KC group and ZW+KC group were significantly increased (P<0.05). Compared with UNF group, the AFB1 and DON contents of FGM+KC group, ZW+KC group, and LY+KC group were significantly reduced (P<0.05). At the concentration of 10 µg·mL-1, four kinds of fermented Astragalus could promote the viability of HD11 cells, which was determined as the subsequent working concentration. After treated with ZW+KC and FGM+KC fermented Astragalus, the phagocytosis capacity and the expression of pro-inflammatory factors (IL-1β and IL-12) in HD11 cells were significantly higher than those in CON, UNF, SS+KC and LY+KC groups (P<0.05). In conclusion, the selection of probitics is closely related to the quality and activity of fermented Astragalus. Among them, Astragalus membranaceus fermented by Bacillus subtilis combined with Lactobacillus plantarum or Streptococcus alactolyticus FGM has better quality, which can increase the content of total flavonoids and total saponins, degrade AFB1 and DON, and improve the function of HD11 cells.

fermented Astragalus  /  Bacillus subtilis  /  quality analysis  /  active function  /  cell function
刘佳惠, 徐国伟, 张康, 李建喜, 郭志廷, 韩松伟, 陈富斌, 陈海瑜, 古雪艳, 张景艳, 王磊. 枯草芽孢杆菌联合不同乳酸菌制备黄芪酵素的质量评价及其对HD11细胞功能的影响. 畜牧兽医学报, 2026 , 57 (3) : 1697 -1710 . DOI: 10.11843/j.issn.0366-6964.2026.03.044
Jiahui LIU, Guowei XU, Kang ZHANG, Jianxi LI, Zhiting GUO, Songwei HAN, Fubin CHEN, Haiyu CHEN, Xueyan GU, Jingyan ZHANG, Lei WANG. The Effects of Bacillus subtilis Combined with Different Lactic Acid Bacteria on the Quality of Fermented Astragalus and HD11 Cells Function[J]. Acta Veterinaria et Zootechnica Sinica, 2026 , 57 (3) : 1697 -1710 . DOI: 10.11843/j.issn.0366-6964.2026.03.044
中药发酵的历史可追溯至六七千年前的酒曲酿酒时代,是传统中药加工炮制的重要方法。由于传统发酵工艺时间长,微生物的数量及种类不受控制[1],中药发酵产品容易产生黄曲霉毒素[2⁃3]。在中药发酵过程中还会受多种客观因素的影响,发酵效果很不稳定,进而产品的功效较难保持。此外,中药发酵产品一直以来缺乏系统的质量标准研究。因此,这种加工炮制方法有待发展。现代研究表明,发酵过程不仅可以改变药材原有的性能,而且可以增强其功效。此外,新的活性成分还会通过发酵过程得以催生。近年来,国家高度重视中医药行业的振兴发展,明确提出加快推进中药现代化创新研究的战略方针。在此背景下,基于现代生物工程技术的中药发酵再次引发国内外广大学者和相关技术人员的关注。如Yang等[4]采用枯草芽孢杆菌对银杏叶进行固态发酵,发现黄酮类、多糖、蛋白质、总氨基酸、蛋白酶和β-葡萄糖苷酶的含量显著增加。如姚晓云等[5]利用混合乳酸菌发酵枇杷(也被称之为枇杷酵素),该酵素富含多种维生素、矿物质和抗氧化物质,具有提高免疫力、改善肠道菌群、促进消化、消炎抗菌、降低血糖等作用。目前,我国有关中药发酵的行业标准尚处于空白,但在2018年颁布的中华人民共和国轻工业行业标准《植物酵素》(QB/T 5323—2018)为中药发酵或中药酵素的产品研发提供了参考依据。
黄芪主要包含多糖、皂苷类、黄酮类以及氨基酸、微量元素等多种化学成分,具备促进畜禽生长、减轻氧化应激、增强免疫力和抗炎等特性[6]。黄芪活性成分和乳酸菌联合使用可通过减少肠道损伤和炎症反应,改善肠道菌群结构、免疫力及氧化应激,增强雏鸡抗大肠杆菌感染能力,有替代抗生素的作用[6]。本实验室用含有黄芪提取液的培养基驯化鸡肠道分离菌,得到1株可提高黄芪发酵后多糖含量的鸡肠道乳杆菌FGM[7]。此外,Qiao等[8]研究发现,植物乳杆菌固体发酵黄芪能够提升黄芪活性成分的提取率以及有机酸的生产率。Sheih等[9]利用黑曲霉菌固态发酵黄芪3 d,成功提高了黄芪中酚类物质含量,并且发酵黄芪的抗氧化活性显著高于未发酵的黄芪。
相比于单菌发酵,混合菌发酵过程菌种之间存在相互协同作用,能够扩大对原料的适应性和防杂菌的能力。周瑶等[10]利用植物乳杆菌和鼠李糖杆菌混菌发酵番木瓜汁提高了活菌总数。胡羚等[11]研究发现,经混合菌发酵,马铃薯汁水中氨基酸质量分数比单菌株发酵高。然而,有关混合菌发酵黄芪的文献报道鲜少。芽孢杆菌类主要有枯草芽孢杆菌、双歧芽孢杆菌和地衣芽孢杆菌等,具有较强的耐受性,可以在高温条件下生存和繁殖,适用于一些高温发酵过程。芽孢杆菌还能产生多种酶类,有助于降解复杂底物,提高发酵效率[12]。因此,本研究利用前期建立的黄芪液态发酵工艺,依据《植物酵素》(QB/T 5323—2018)中养殖业用植物酵素的相关要求,并考虑到黄芪药材中主要活性成分在发酵前后的变化,进一步探索枯草芽孢杆菌联合不同乳酸菌菌株对黄芪酵素产品中主要成分及活性作用的影响,旨在为黄芪酵素产品的研发和发酵中药的炮制提供技术支持,为中兽药的现代化创新提供研究策略。
膜荚黄芪,购自甘肃省兰州市黄河中药材市场,粉碎60目过筛备用。发酵菌株:嗜酸乳杆菌(批号:211230)、罗伊氏乳杆菌(批号:211205)、植物乳杆菌(批号:BNCC185392)和枯草芽孢杆菌(批号:ATCC6633)购自河北北纳生物科技有限公司;非解乳糖链球菌FGM(GenBank登录号:JX435470,中国专利号:20120141827.5)由中国农业科学院兰州畜牧与兽药研究所中兽医临床创新团队分离并保存。
MRS肉汤培养基和LB肉汤培养基购自广东环凯生物科技有限公司;磷酸氢二钾购自天津市光复科技发展有限公司;硫酸钠购自北京西林布克网络科技有限公司;葡萄糖购自天津市百世化工有限公司;葡萄糖标准品,芦丁标准品和黄芪甲苷标准品购自中国食品药品检定研究院;AFB1和DON检测试剂盒购自河北伊莱莎生物技术有限公司;CCK-8试剂盒(细胞增殖及毒性检测试剂盒)购自北京索莱宝科技有限公司;鸡白细胞介素12(IL-12)ELISA检测试剂盒、白细胞介素1β(IL-1β)ELISA检测试剂盒和鸡肿瘤坏死因子α(TNF-α)ELISA检测试剂盒购自上海酶联生物科技有限公司。苯酚、浓硫酸、NaNO2、NaOH、正丁醇、香草醛、冰醋酸、无水乙醇、酚酞指示剂、亚硝酸钠、硝酸铝、香草醛、冰醋酸、没食子酸、福林酚试剂、甲醇、碳酸氢钠和高氯酸购自国药集团化学试剂有限公司。
生物安全柜(HF safe-1500型,Heal Force),电子分析天平(BSA224S型,赛多利斯),电子天平(HX2002T型,慈溪市天东衡器厂),电热恒温培养箱(HZQ-F160,哈尔滨市东连电子技术开发有限公司),全自动多功能酶标仪(SYNERGYLX型,美国伯腾仪器有限公司),高速冷冻离心机(5804R型,德国Eppendorf公司),摇摆式高速中药粉碎机(DFY-500,温岭市大机械有限公司),不锈钢筛网(标准60目,浙江上虞市金鼎标准筛具厂),台式冷冻离心机(Allegra X-15R,贝克曼库尔特有限公司),数显恒温水浴锅(HH-S6,江苏正基仪器有限公司),分析天平(BS224S,赛多利斯科学仪器有限公司),恒温培养振荡器(ZHWY-2102C,上海智城分析仪器制造有限公司),厌氧培养箱(1029,北京昊诺斯科技有限公司),超纯水系统(NW30,上海康雷分析仪器有限公司)。
1.4.1.1 菌种活化和准备:将-80 ℃冻存的嗜酸乳杆菌(SS)、罗伊氏乳杆菌(LY)、植物乳杆菌(ZW)、非解乳糖链球菌(FGM)和枯草芽孢杆菌(KC)迅速解冻,分别接种于高压灭菌的MRS肉汤培养基和LB肉汤培养基中,37 ℃、120 r·min-1传代培养24 h。然后分别按2%的接种量将F1代菌液接种于MRS肉汤培养基,37 ℃、120 r·min-1培养24 h,即得F2代菌液。相同操作步骤,分别制备4种菌株的F3代菌液,作为黄芪发酵的种子液备用。
1.4.1.2 含黄芪的药性发酵培养基的准备:参照文献[13],配置含黄芪的药性发酵培养基。精密称取13.5 g黄芪、0.3 g酵母粉、0.5 g硫酸钠、0.2 g磷酸氢二钾,加入94 mL蒸馏水溶解并121 ℃高压灭菌20 min,再称取6 g葡萄糖加入6 mL蒸馏水溶解,混匀后105℃高压灭菌30 min,将两者充分搅拌均匀,制得含黄芪的药性发酵培养基。
1.4.1.3 不同发酵菌种的黄芪酵素制备:参照文献[14]中发酵黄芪制备方法,并略作改动。具体试验方法如下:试验设置为5组,每组设3个平行样。第1组为不加混合菌的含黄芪药性的发酵培养基的阴性对照组(UNF组),在无菌环境下,分别按2%和6%的体积比在含黄芪的药性发酵培养基中加入MRS肉汤和LB肉汤;第2组为嗜酸乳杆菌联合枯草芽孢杆菌发酵组(SS+KC组),在无菌环境下,分别在含黄芪的药性发酵培养基中加入6%嗜酸乳杆菌和2%枯草芽孢杆菌的菌悬液;第3组为罗伊氏乳杆菌联合枯草芽孢杆菌发酵组(LY+KC组),在无菌环境下,分别在含黄芪的药性发酵培养基中加入6%罗伊氏乳杆菌和2%枯草芽孢杆菌的菌悬液;第4组为植物乳杆菌联合枯草芽孢杆菌发酵组(ZW+KC组),在无菌环境下,分别在含黄芪的药性发酵培养基中加入6%罗伊氏乳杆菌和2%枯草芽孢杆菌的菌悬液;第5组为非解乳糖链球菌联合枯草芽孢杆菌发酵组(FGM+KC组),在无菌环境下,分别在含黄芪的药性发酵培养基中加入6%非解乳糖链球菌和2%枯草芽孢杆菌的菌悬液。将各组样品放置于37 ℃、120 r·min-1的振荡培养箱中发酵42 h。发酵后产物在3 000 r·min-1离心10 min,取上清液;沉淀物用60 mL蒸馏水溶解后,在80℃提取60 min,以3000 r·min-1离心10 min,取上清液,并将两次提取的上清液合并,浓缩后定容至100 mL。
1.4.1.4 黄芪酵素理化指标测定:参照《植物酵素》(QB/T 5323—2018)中养殖业用植物酵素一般理化指标和特征性指标要求,分别检测由不同发酵菌种液态发酵制成的黄芪酵素产品中pH、乙醇含量、总酸含量、有机酸含量、粗多糖含量。根据国家标准水果和蔬菜产品pH的测定方法(GB/T 10468)测定黄芪酵素的pH;参考文献[15],采用重铬酸钾比色法测定黄芪酵素产品中乙醇含量;根据食品安全国家标准食品总酸的测定(GB/T 12456)、食品中有机酸的测定(GB/T 5009.157),分别测定黄芪酵素产品中总酸和有机酸含量;根据出口植物源食品中粗多糖的测定苯酚-硫酸法(SN/T4260)测定黄芪酵素中粗多糖含量。
1.4.1.5 黄芪酵素的主要活性成分含量测定:参考文献[16],采用铝盐显色法测定黄芪酵素中总黄酮含量;参考文献[17],采用香草醛-冰醋酸-高氯酸显色法测定黄芪酵素中总皂苷含量;参考文献[18],采用福林-酚法测定黄芪酵素中总多酚含量。
1.4.1.6 黄芪酵素AFB1和DON含量测定:各组黄芪酵素中AFB1和DON的含量测定分别采用对应的ELISA检测试剂盒进行测定,将所需试剂从冷藏环境中取出,置于室温平衡30 min以上,注意每种液体试剂使用前均须摇匀,取出需要数量的微孔板,将不用的微孔板放进原锡箔袋中并且与提供的干燥剂一起重新密封,保存于2~8 ℃。不要冷冻,洗涤工作液在使用前也需回温,将样本和标准品对应微孔按序编号,每个样本和标准品做2孔平行,并记录标准孔和样本孔所在的位置,加标准品/样本50 µL到对应的微孔中,加入酶标物50 µL·孔-1,再加入抗试剂50 µL·孔-1,轻轻振荡混匀,用盖板膜盖板后置室温避光环境中反应30 min,小心揭开盖板膜,将孔内液体甩干,用洗涤工作液300 µL·孔-1,充分洗涤5次,每次间隔30 s,用吸水纸拍干,加入底物液A液50 µL·孔-1,再加底物液B液50 µL·孔-1,轻轻振荡混匀,用盖板膜盖板后置室温避光环境反应15~20min,加入终止液50 µL·孔-1,轻轻振荡混匀,设定酶标仪于450 nm处,测定每孔OD值。
1.4.2.1 细胞活力测定:配制多糖浓度为2 000 µg·mL-1的黄芪酵素母液,0.22 µm滤膜过滤,分装于1.5 mL EP管,-20 ℃保存备用,用含97% DMEM的高糖培养液将黄芪酵素稀释至2.5、5、10、20、40 µg·mL-1(按多糖浓度计)。取对数生长期的HD11细胞调整密度为2×105 cells·mL-1,每孔100 µL加入96孔板中,置于37 ℃、5% CO2培养箱中培养6 h,弃去孔中培养液后,分别加入不同浓度的黄芪酵素完全培养液100 µL,空白对照组(CON组)则加入100 µL完全培养液,每组3个重复,培养24 h,弃去孔中培养液后,每孔加入10 µL CCK-8溶液,在细胞培养箱内继续孵育1.5 h。用酶标仪测定在450 nm处的吸光值并参照公式计算细胞活力。
细胞活力(%)=[OD(加药)-OD(空白)]/
[OD(0加药)-OD(空白)]×100。
OD(加药):具有细胞、CCK-8溶液和药物溶液的孔的吸光度;
OD(空白):具有培养基和CCK-8溶液而没有细胞的孔的吸光度;
OD(0加药):具有细胞、CCK-8溶液而没有药物溶液的孔的吸光度。
1.4.2.2 细胞吞噬能力测定:取对数生长期的HD11细胞调整密度为2×105 cells·mL-1,每孔2 mL加入玻底细胞培养皿中,置于37 ℃、5% CO2培养箱中培养6 h。弃去孔中培养液后,分别加入工作浓度的黄芪酵素完全培养液2 mL,空白对照组(CON组)则加入2 mL完全培养液,每组3个重复,培养18 h。分别弃去上清液并加入100 µL的100 µg·mL-1 FITC-葡聚糖生理盐水溶液,于37 ℃避光孵育30 min;弃去上清液,每孔加入200 µL的预冷PBS溶液,重复洗涤细胞2次,最后加入50 μL的4%多聚甲醛固定细胞,加入少量DAPI染色液,室温放置3~5 min。吸除DAPI染色液,用生理盐水洗涤2~3次,每次3~5 min。最后置于激光扫描共聚焦显微镜下观察荧光并记录拍照,并用软件Image pro plus 6.0进行荧光强度的计算。
1.4.2.3 细胞迁移试验:取对数生长期的HD11细胞调整密度为2×105 cells·mL-1,每孔1 mL加入24孔板中,置于37 ℃、5% CO2培养箱中培养,等待细胞长到80%后,用200 µL移液器吸头沿24孔板中央垂直45度方向划痕。用PBS冲洗划痕后的孔板2次,轻轻吹打,不要将贴壁细胞冲洗下来。分别加入工作浓度的黄芪酵素完全培养液1 mL,空白对照组则加入1 mL完全培养液,每组分别于0、6、12、18、24 h观察拍照。每组采集5个视野,用软件Image pro plus 6.0进行前后划痕面积的计算。各组细胞迁移率(%)=[(0 h面积-6 h/12 h/18 h/24 h面积)/0 h平均面积]×100。
1.4.2.4 细胞因子含量检测:取对数生长期的HD11细胞调整密度为2×105 cells·mL-1,按1 mL每孔接种于24孔板中,37 ℃、5%CO2培养6 h,弃上清。分别加入工作浓度的黄芪酵素完全培养液2 mL,空白对照组则加入2 mL完全培养液,每组3个重复,培养18 h,收取上清液用于后续ELISA测定细胞因子含量。
本次试验数据通过采用Microsoft Office Excel 2019软件进行初步整理,试验数据采用IBM SPSS Statistics 26软件对数据进行One-way-ANOVA分析,并绘图。P<0.05表示显著差异。
枯草芽孢杆菌联合不同乳酸菌发酵制备的黄芪酵素pH、乙醇、总酸、有机酸及多糖的含量测定结果见图1。图1A所示,SS+KC、LY+KC、FGM+KC、ZW+KC组的pH均低于《植物酵素》(QB/T 5323—2018)中养殖业用植物酵素标准(pH≤7.5),且各黄芪酵素组的pH均显著低于UNF组(P<0.05)。图1B所示,各黄芪酵素组的乙醇含量均低于《植物酵素》(QB/T 5323—2018)中养殖业用植物酵素标准(乙醇含量≤0.8 g·L-1)。图1C所示,除SS+KC组外,其它3个黄芪酵素组(LY+KC、FGM+KC、ZW+KC组)的总酸含量均高于《植物酵素》(QB/T 5323—2018)中养殖业用植物酵素标准(总酸≥5 g·L-1),各黄芪酵素组的总酸含量均显著高于UNF组(P<0.05)。图1D所示,各黄芪酵素组的有机酸含量均高于《植物酵素》(QB/T 5323—2018)中养殖业用植物酵素标准(有机酸≥1 g·L-1),且三个黄芪酵素组(LY+KC、FGM+KC、ZW+KC)的有机酸含量显著高于UNF组、SS+KC组(P<0.05)。图1E所示,除了SS+KC组,其他各酵素组(UNF、LY+KC、FGM+KC、ZW+KC)的粗多糖含量均高于《植物酵素》(QB/T 5323—2018)中养殖业用植物酵素标准(粗多糖≥10 g·L-1),且ZW+KC、FGM+KC的粗多糖含量显著高于UNF组和SS+KC组(P<0.05)。上述结果表明,枯草芽孢杆菌联合3种乳酸菌(罗伊氏乳杆菌、非解乳糖链球菌、植物乳杆菌)发酵制备的黄芪酵素符合《植物酵素》(QB/T 5323—2018)中养殖业用植物酵素标准中理化指标要求,其中KC联合ZW发酵的黄芪酵素质量较优。
枯草芽孢杆菌联合不同乳酸菌发酵制备的黄芪酵素的总黄酮、总皂苷、总多酚含量结果见图2。图2A所示,枯草芽孢杆菌联合乳酸菌发酵的各组(SS+KC、LY+KC、FGM+KC、ZW+KC组)中总黄酮的含量均显著高于UNF组(P<0.05),且ZW+KC组的总黄酮含量最高,为0.288 mg·mL-1。图2B所示,FGM+KC、ZW+KC组的总皂苷含量显著高于UNF组(P<0.05),且FGM+KC组的总皂苷含量最高,为4.380 mg·mL-1。图2C所示,LY+KC组的总多酚含量显著高于UNF组(P<0.05)。结果表明,枯草芽孢杆菌联合不同乳酸菌制备的黄芪酵素中总黄酮、总皂苷的变化与乳酸菌种类有较大关联,其中KC联合ZW、FGM发酵的黄芪酵素质量较优。
枯草芽孢杆菌联合不同乳酸菌发酵制备的黄芪酵素中AFB1和DON含量测定见图3。图3A所示,UNF组的AFB1含量为10.035 ng·mL-1,而添加乳酸菌发酵后,各黄芪酵素中AFB1含量均显著降低(P<0.05),其中ZW+KC组和FGM+KC组的AFB1含量显著低于SS+KC、LY+KC组(P<0.05)。图3B所示,UNF组的AFB1含量为0.984 ng·mL-1,而添加FGM+KC发酵后,黄芪酵素中DON含量显著低于UNF组(P<0.05);而SS+KC发酵后,黄芪酵素中DON含量显著高于UNF组(P<0.05)。结果表明,枯草芽孢杆菌联合不同乳酸菌制备的黄芪酵素中AFB1、DON的变化与乳酸菌种类有较大关联,其中KC联合ZW、FGM发酵的黄芪酵素质量较优。
枯草芽孢杆菌联合不同乳酸菌发酵制备的黄芪酵素对HD11细胞活力的影响结果见图4,各组黄芪酵素浓度(按多糖浓度计)在2.5~10 µg·mL-1范围逐渐升高时,HD11细胞活力逐渐提升;UNF、SS+KC、LY+KC、ZW+KC、FGM+KC组的黄芪酵素浓度在10 µg·mL-1时,细胞活力平均最大值分别为141.9、161.6、146.6、180.8、150.3%。依据上述结果,细胞实验中各组黄芪酵素的工作浓度确定为10 µg·mL-1。
在本次研究中,采用激光共聚焦技术对鸡巨噬细胞HD11吞噬FITC标记的葡聚糖的量进行观察与量化分析,以此作为评估HD11细胞吞噬能力的依据。枯草芽孢杆菌联合不同乳酸菌发酵制备的黄芪酵素对HD11细胞吞噬能力的调节作用结果见图5。枯草芽孢杆菌联合乳酸菌发酵制备的黄芪酵素刺激HD11的吞噬能力显著高于CON组(P<0.05),发酵质量较优的ZW+KC组、FGM+KC组黄芪酵素刺激HD11的吞噬能力显著高于其他组(CON、UNF、SS+KC、LY+KC)(P<0.05)。
枯草芽孢杆菌联合不同乳酸菌发酵制备的黄芪酵素对HD11细胞迁移能力的调节作用结果见图6,观察0、6、12、18、24 h各个时间点划痕愈合面积,黄芪酵素刺激后巨噬细胞迁移率升高,24 h时ZW+KC组和FGM+KC组的HD11细胞划痕愈合率显著高于CON组(P<0.05)。
枯草芽孢杆菌联合不同乳酸菌发酵制备的黄芪酵素对鸡巨噬细胞HD11细胞因子表达的影响见图7。图7A所示,ZW+KC、FGM+KC组细胞中IL-1β的表达显著高于UNF、CON、SS+KC和LY+KC组(P<0.05)。图7B所示,ZW+KC、FGM+KC组细胞中IL-12的表达显著高于UNF、CON、SS+KC和LY+KC组(P<0.05)。由图7C可知,UNF、SS+KC、LY+KC、ZW+KC、FGM+KC组细胞中TNF-α的表达显著高于CON组(P<0.05),但各药物干预组之间无显著性差异(P>0.05)。
中药的发酵是涉及生物转化的过程,其本质是微生物以中药组分及其它培养基为底物的利用、转化和自身增殖的过程。在这一过程中,发酵是否成功、发酵产品的质量是否合格可以通过发酵菌种在自身增殖过程中产生的代谢物含量的高低来判断。研究表明,乳酸菌在一些发酵食品的生产中扮演着关键角色,它们能够产生乳酸并促进产品风味的形成,所以酸度是评估发酵食品质量的一个重要指标[19]。在乳酸菌发酵过程中,pH的显著降低有助于防止食源性致病菌的生长,从而确保发酵产品的安全性[20]。乙醇是酵素无氧发酵过程中生成的一种代谢产物,它不仅影响酵素产品风味口感,还是衡量酵素产品质量的一个重要指标[21]。在刺五加叶发酵的研究中,通过使用干酪乳杆菌、嗜酸乳杆菌和植物乳杆菌进行发酵,发现所有类型的乳酸菌发酵都能显著增加刺五加叶中多糖的含量[22]。乳酸菌发酵可显著提升了燕麦和荞麦饮料有机酸含量,从而抑制有害微生物来提高食品安全[23]。因此,本研究参照《植物酵素》(QB/T 5323—2018)中食用植物酵素一般理化指标和特征性指标要求,分别通过检测pH,测定乙醇、粗多糖、总酸和有机酸的含量,评价了枯草芽孢杆菌联合不同乳酸菌发酵黄芪制备的黄芪酵素的质量。结果表明,枯草芽孢杆菌联合罗伊氏乳杆菌、枯草芽孢杆菌联合植物乳杆菌、枯草芽孢杆菌联合FGM发酵制备的三种黄芪酵素,其pH均显著低于对照组,总酸、有机酸和粗多糖含量显著高于对照组。因此,以枯草芽孢杆菌联合罗伊氏乳杆菌、枯草芽孢杆菌联合植物乳杆菌、枯草芽孢杆菌联合非解乳糖链球菌FGM为混合发酵菌种的黄芪酵素符合《植物酵素》(QB/T 5323—2018)中养殖业用植物酵素一般理化指标和特征性指标要求,其中枯草芽孢杆菌联合植物乳杆菌发酵制备的黄芪酵素质量较优。
乳酸杆菌发酵能够转化生物活性成分,增强其有益特性,并提高植物性食品的营养价值和风味[24]。研究发现,乳酸菌发酵红海藻Bangia fuscopurpurea上清液中总黄酮含量在0至36 h的发酵期间显著增加,这可能是由于复杂多酚在发酵过程中被酶分解成简单的黄烷醇化合物[25];由于乳酸杆菌对红藻Bangia fuscopurpurea中不溶性糖基化酚类的脱糖作用,总多酚含量在发酵过程中有所增加。乳酸菌发酵增强了黄芪肉汤中的活性成分,其中总皂苷含量在发酵期间增加了72.81%[26]。众所周知,黄芪药材中主要的活性成分为总黄酮、总皂苷、总多酚等,其具有多种药理活性,如抗菌和抗炎特性、增强机体免疫力以及治疗心脑血管疾病的有效性等作用[27]。本研究发现,枯草芽孢杆菌联合乳酸菌发酵制备的黄芪酵素显著提升了总黄酮、总皂苷的含量,其中枯草芽孢杆菌联合植物乳杆菌发酵制备的黄芪酵素对于总黄酮的发酵性能最优,枯草芽孢杆菌联合非解乳糖链球菌FGM发酵制备的黄芪酵素对于总皂苷的发酵性能最优。可见发酵菌种对黄芪酵素的活性成分释放或产生有较大影响。
AFB1和DON作为两种主要的真菌毒素,广泛分布于花生、玉米、水果、蔬菜和草药等原料中,被机体摄入后可造成严重的免疫抑制并有致癌作用,严重影响人类和动物的健康[28⁃29]。自1960年发现以来,研究人员设计了许多方法来帮助去除和降解农作物中的AFB1,包括物理、化学和生物方法,其中生物方法具有污染小、耗能低等优点。为了延长食品的保质期,Bangar等[30]在皮塔饼面包中添加了含有植物乳植杆菌的发酵乳清,发酵乳清对扩展青霉和短青霉菌株表现出显著的抗真菌作用。由植物乳杆菌发酵的米糠培养基表现出优异的抗真菌效果并产生更高浓度的抗真菌代谢物[31]。本研究发现,枯草芽孢杆菌联合植物乳杆菌、非解乳糖链球菌发酵制备的黄芪酵素均显著降低了黄曲霉毒素含量,枯草芽孢杆菌联合非解乳糖链球菌发酵制备的黄芪酵素显著降低了DON含量。这一结果可能与乳酸菌通过其生物代谢作用,降解了部分霉菌毒素相关。
巨噬细胞在组织稳态、体内平衡、组织修复和对病原体的防御过程中具有独特且不可取代的地位,也是先天免疫防御中的关键成员[32⁃33]。巨噬细胞作为主要的天然免疫效应细胞,可根据微环境的变化而发生极化状态的改变,从而发挥多样的功能(吞噬、促炎、趋化、抗原递呈等)[34]。因此,本研究分别通过吞噬功能试验、促炎因子测定试验、细胞迁移试验对4种黄芪酵素的活性作用进行了综合比较。
CCK-8法是用于测定细胞增殖或细胞毒性试验中活细胞数目的一种高灵敏度,无放射性的比色检测法。在药物对细胞干预作用的研究中,通常被作为药物工作浓度确定的试验方法和结果判定依据[35]。本研究发现,枯草芽孢杆菌联合植物乳杆菌、非解乳糖链球菌发酵制备的黄芪酵素显著提升了HD11细胞的细胞活力,并发现各黄芪酵素在10 µg·mL-1刺激HD11细胞呈现最高的细胞活力。为了在同一工作浓度下,比较枯草芽孢杆菌联合不同乳酸菌发酵黄芪制备的黄芪酵素优劣,在后续试验中以10 µg·mL-1(按多糖含量计)为不同黄芪酵素组的工作浓度。
巨噬细胞在先天免疫及适应性免疫中均发挥重要作用,在黏膜免疫中对病原微生物进行吞噬和消化,并进一步激活淋巴细胞或其他免疫细胞,在抗感染、抗肿瘤及调节机体免疫过程中扮演重要角色[36]。有研究已经证明,松花粉多糖(PPPS)可以刺激鸡腹腔巨噬细胞吞噬能力从而增强亚单位疫苗的免疫效果[37]。乳酸菌发酵可提升海带褐藻糖胶的免疫活性,中性红吞噬能力比未发酵的高1.15倍左右[38]。细胞划痕试验是一种体外研究创伤愈合的常用试验方法,创伤愈合是细胞增殖和迁移两种不同功能共同作用的结果[39]。董丽红等[40]研究发现,在40 µg·mL-1质量浓度时,发酵荔枝果壳原花青素作用细胞迁移率较发酵前提高了75.37%。本研究发现,枯草芽孢杆菌联合乳酸菌发酵黄芪可以显著提升HD11细胞的吞噬能力,发酵后的黄芪在吞噬能力上显示出比UNF组更强的增强效果,其中ZW+KC、FGM+KC组效果较优。发酵后的黄芪在迁移率上显示出比UNF组更强的增强效果,其中ZW+KC组效果较优。
细胞因子是一类由多种活化细胞产生的多功能、高活性的多肽或蛋白质类物质[41]。这些分子通过相互促进或抑制,以及相应受体表达的协调作用来发挥生物学效应。它们在信息传递中扮演着关键角色,调节机体内的动态平衡,并对先天免疫及适应性免疫具有重要的调控作用[42]。本研究主要检测了HD11细胞分泌的主要细胞因子:IL-1β、TNF-α和IL-12。其中,IL-1β对各种免疫细胞具有广泛的作用,可激活T细胞、B细胞、巨噬细胞等免疫细胞,促使它们增殖、分化和分泌免疫活性物质,进而增强机体的免疫应答。此外,IL-1β还能通过调节Th17和Th1细胞的免疫应答,参与适应性免疫应答的调节[43]。TNF-α对免疫细胞的功能具有重要的调节作用。它可以促进T细胞的活化和增殖,增强自然杀伤细胞(NK细胞)的杀伤活性,同时也能抑制B细胞的增殖和抗体分泌。在免疫应答过程中,TNF-α的适量分泌对于维持免疫系统的平衡和稳定具有重要意义,但过度分泌则会导致炎症反应失控和组织损伤[44]。此外,IL-12主要由树突状细胞、巨噬细胞等抗原呈递细胞分泌,其主要作用是促进T细胞向Th1细胞分化,并增强Th1细胞的免疫功能。Th1细胞主要参与细胞免疫应答,能够分泌干扰素-γ(IFN-γ)等细胞因子,进一步增强免疫细胞的杀菌活性和吞噬能力,从而有效地清除病原体[45]。王坤等[45]研究发现,发酵乳杆菌S1荚膜多糖在25~200 µg·mL-1范围内,可通过上调RAW264.7 细胞相关基因表达促进细胞因子IL-6、IL-1β和TNF-α等的释放。本研究发现,枯草芽孢杆菌联合不同乳酸菌发酵制备的黄芪酵素显著提升了HD11细胞中IL-1β、IL-12、TNF-α的表达,其中KC联合ZW、FGM发酵的黄芪酵素质量较优。
本研究分别从理化指标、活性成分、毒素及干预鸡巨噬细胞HD11功能等方面,综合评价了枯草芽孢杆菌联合4种不同乳酸菌发酵黄芪对黄芪酵素质量和作用的影响,明确发酵菌种选择与黄芪酵素质量、作用均密切相关,其中枯草芽孢杆菌联合植物乳杆菌或非解乳糖链球菌FGM作为混合发酵菌种,可显著提高黄芪酵素产品中总酸、有机酸、粗多糖、总黄酮、总皂苷,显著降低pH、AFB1、DON,显著提高鸡巨噬细胞功能,在提升中药功效和调节免疫功能方面具有较大潜力。
  • 益生菌发酵黄芪工艺研究及制剂开发(2022YFD1801104)
  • 甘肃省鲁甘协作项目(22CX8NA013)
  • 中国农业科学院创新团队项目(25-LZIHPS-06)
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2026年第57卷第3期
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doi: 10.11843/j.issn.0366-6964.2026.03.044
  • 接收时间:2024-12-18
  • 首发时间:2026-09-16
  • 出版时间:2026-03-23
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  • 收稿日期:2024-12-18
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益生菌发酵黄芪工艺研究及制剂开发(2022YFD1801104)
甘肃省鲁甘协作项目(22CX8NA013)
中国农业科学院创新团队项目(25-LZIHPS-06)
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    中国农业科学院兰州畜牧与兽药研究所 中泰中兽医药学技术联合实验室,兰州 730050

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张景艳,主要从事中兽医及免疫研究,E-mail:
王磊,主要从事中兽医及免疫研究,E-mail:
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2种不同金属材料的力学参数
科
Family
属数
Number of
genus
种数
Number of
species
占总种数比例
Percentage of
total species (%)
属
Genus
种数
Number of
species
占总种数比例
Percentage of total
species (%)
鹅膏菌科Amanitaceae 2 11 5.26 鹅膏菌属 Amanita 10 4.78
小菇科 Mycenaceae 2 12 5.74 丝盖伞属 Inocybe 5 2.39
多孔菌科 Polyporaceae 8 14 6.70 蜡蘑属 Laccaria 5 2.39
红菇科 Russulaceae 3 23 11.00 小皮伞属 Marasmius 6 2.87
小菇属 Mycena 11 5.26
光柄菇属 Pluteus 5 2.39
红菇属 Russula 17 8.13
栓菌属 Trametes 5 2.39
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