Panel of Bacillus subtilis Reporter Strains Indicative of Various Modes of Action

2.6.1. STERILITY2.6.1 无菌检查法The test is applied to substances, preparations or articles which, according to the Pharmacopoeia, are required to be sterile. However,a satisfactory result only indicates that no contaminating micro-organism has been found in the sample examined in the conditions of the test.本检查方法适用于按照药典要求应当无菌的原料、制剂或其他物质。

但是，如果按照本无菌检查法的结果符合要求，仅表明在该检查条件下未发现微生物污染。

PRECAUTIONS AGAINST MICROBIAL CONTAMINATION微生物污染防范The test for sterility is carried out under aseptic conditions.In order to achieve such conditions, the test environment has to be adapted to the way in which the sterility test is performed. The precautions taken to avoid contamination are such that they do not affect any micro-organisms which are to be revealed in the test. The working conditions in which the tests are performed are monitored regularly by appropriate sampling of the working area and by carrying out appropriate controls.无菌检测试验应在无菌的条件下进行。

华支睾吸虫感染临床特征与防治进展发布时间：2021-03-22T14:58:40.677Z 来源：《医师在线》2020年9月17期作者：黄国秀吴苏疆罗惠芳劳盈盈仲婷婷[导读] 华支睾吸虫病感染率与男性、较低的教育水平、食用生河鱼、居住在河流沿岸等因素有关。

黄国秀吴苏疆罗惠芳劳盈盈仲婷婷（广西壮族自治区人民医院健康管理中心；广西南宁530000）摘要华支睾吸虫病感染率与男性、较低的教育水平、食用生河鱼、居住在河流沿岸等因素有关。

人类易感华支睾吸虫且容易再次感染和重复感染。

华支睾吸虫感染后常继发急慢性胆管炎、肝实质炎症、肾损害等，并可导致胆管癌。

吡喹酮是强效的治疗药物，治愈率高，但研究发现，重复感染、多次使用吡喹酮治疗增加胆管癌的风险。

本文就华支睾吸虫感染流行病学特点、感染后临床特征、防治方面进行综述。

关键词华支睾吸虫；重复感染；胆管炎；综述华支睾吸虫（Clonorchis sinensisrg 又称肝吸虫）是食源性寄生虫，因其寄生于肝胆管故而又称肝吸虫。

其广泛分布于东亚，并在中国及邻近国家一些地方重度流行[1、2]。

近年来华支睾吸虫病出现了新的流行特点，部分患者治疗后多次重复感染，部分地区感染率出现不降反升趋势，目前华支睾吸虫病仍是亟待解决的重要公共卫生问题。

1.华支睾吸虫病流行情况研究现状华支睾吸虫的生物学与生活史研究已基本成熟。

在流行病学方面的研究，主要围绕重流行区域进行感染率调查和感染总量推测。

近年来发病率较高的国家有中国、越南、韩国和俄罗斯远东地区。

韩国研究了5条主要河流沿岸居民的感染率，男性总患病率为11.2%，女性为6.2%，50-59岁年龄段的患病率最高[3]。

对越南的研究则发现，华支睾吸虫病的感染率与男性、较低的教育水平、食用生河鱼、邻近水体、居住的地理位置（如河流沿岸）等因素有关，其中，食用生淡水鱼为至关重要的因素[4]。

我国寄生虫病第二次流行病学调查发现目前全国约有1300万感染者，以广东、广西感染率居前，黑龙江、辽宁部分地区也是高流行区[5.6]。

芽孢杆菌属（Bacillus）芽孢杆菌属(Bacillus)BacillusCells are straight, rod-shaped, 0.5 to 2.5 m m * 1.2 to 10 mu, often arranged in pairs or chains, with rounded ends or square ends.Bacillus sp.Latin name: (Bacillus, Cohn, 1872)2 morphological description editing cells are straight, rod-shaped, 0.5 to 2.5 mu m x 1.2 to 10 mu m, often arranged in pairs or chains, with rounded ends or square ends. Most of the cells stained in young cultures were gram positive and had flagella with peripheral flagella. Spores oval, oval, cylindrical, round, resistant to many adverse environments. Each cell produces a spore, which is not inhibited by oxygen. Aerobic or facultative anaerobic with a variety of physiological properties of heat, pH, and salt. Heterotrophic bacteria with a fermentation or respiratory metabolism type. Usually, the enzyme is positive. Found in different habitats; a few species are pathogenic to vertebrates and non vertebrates.Type subtilis: Bacillus subtilis (Bacillus).3 detailed information on the compilation of Bacillus spp.Bacillus1 genera of the genus Bacillus; Gram-positive bacteria. Producing spores, aerobic or facultative anaerobic, mostly dynamic, withoutcapsule, mostly hemolytic, usually catalase positive. G+C molar content in DNA was 32 ~ 62%. The genus includes Bacillus anthracis that is pathogenic to humans and animals, Bacillus cereus causing food poisoning, and nearly 50 species of non pathogenic Bacillus subtilis, Bacillus cereus, and Bacillus cereus.Gram positive bacteria. Producing spores; aerobic or facultative anaerobic. Most powerful, without capsule, mostly hemolytic, usually catalase positive. Bacillus anthracis including pathogenic Bacillus anthracis, food poisoning causing Bacillus cereus, non pathogenicBacillus subtilis, Bacillus cereus, Bacillus cereus and the like.Bacillus anthracis capsule, no flagella in artificial culture bacteria showed a long chain arrangement, the formation of oval spore; Bacillus bacteria in the central, but not greater than the cell width; aerobic; spore resistance is very strong, can survive for decades in the dry state; the bacteria mainly herbivores disease, disease a sharp, high mortality; the susceptibility of bacteria by only herbivores, broken skin, gastrointestinal or respiratory tract invades the body, high mortality; available antibiotics and sulfa treatment.4 lactic acid production, a common feature of lactic acid in the genus BacillusThere are also a number of lactic acid producing bacteria in the genus bacillus, among which are economically important species. Forthese lactic acid bacteria, Bacillus should belongto the category of lactic acid bacteria. The common feature of these species is that the cells are rod-shaped and form an endophytic spore. Lactic acid production is usually the same type of fermentation. Most of the contact enzymes were positive.(two) the species of lactic acid in BacillusBacillus subtilis 1.Bacillus coagulans originally developed by Hammer from canned milk rancidity were isolated and described as a new species. Then from the rancidity of food preservation in isolation of these bacteria, they produce L- lactic acid with high concentration, caused by carbohydrate containing canned food rancidity, which caused the people's attention. For some isolates, differences in the morphology of their cells, spores, and cysts between strains were also observed in many studies.These highly differentiated polymorphic strains cause many synonyms. Then through some physiological tests and observed that these strains can be divided into different clusters. Blumenstock from the phenotypic and genotypic characteristics of Bacillus coagulans will be divided into two groups. Nakamura and Blumenstock analyzed the phenotype similar to previously identified as Bacillus coagulans strain. The refolding rate of them with UV spectrophotometer for the determination of DNA chain to regroup, get the strains between the similarity values of DNA. The results show that 30 strains were divided into DNA group, which is associated with two. The 1 group of Bacillus coagulans on behalf of another group of strains of Bacillus andrelated species is similar to the DNA values are low, and the phenotypic characteristics of Bacillus coagulans are different, therefore, as a new species, named Smith bacillus (Bacillus smithii).2. Smith BacillusIf the above is the original identified as Bacillus coagulans strain by re classification and the establishment of the. The strains were isolated from milk, canned food, cheese, milk sugar beet juice.3. Bacillus stearothermophilusBacillus stearothermophilus rod-shaped cells, usually 0.6-1.0um*2.0-3.5um. Spore ellipse, secondary end or end, variable size. Gram reaction variable motion. In the glucose medium most strains can grow actively under anaerobic conditions, until the pH is reduced to 5.3-4.8 is not active. A few strains could not grow under anaerobic conditions. Anaerobic fermentation products are mainly L (+) lactic acid, formic acid, acetic acid and a small amount of ethanol, the ratio of 2:1:1. In some strains of nitrate can promote the anaerobic growth and gas production. Some strains had no such effect or reduction to nitrite. The nutritional needs of the lowest significant differences between strains.The heat resistance of spores and resistance to other adverse environments than other Bacillus in any kind of high temperature. Obviously the vegetative cells sensitive to adverse conditions, such as cooling to room temperature theactivity may lose. (the lowest growth temperature of 30-45 DEG C). Note that this feature must be identified so.This can be found in the following places: bacteria in habitat soil and desert sand, hot springs, marine sediments, compost and food etc.. The vegetative cells in many foods, such as ph>5.0 and suitable temperature can quickly germination and growth, can cause the "flat acid canned food".4. L - lactic acid bacillus (Bacillus laevolacticus)The cell width 0.4-0.7um, Bacillus 0.6-0.8*0.8-1.2um oval cystslightly swollen, movement.The growth of glucose and other carbohydrates, acid production from glucose without gas production, the main production of D (-) lactic acid.Peptidoglycan side chain amino acid linked directly with two more. Methyl quinone is mainly MK-7. Cellular fatty acid - ISO C15:0 and C17:0 fatty acids. Isolated from plant rhizosphere.5. racemic lactic acid bacillus (Bacillus racemilacticus)Production of DL- lactic acid type fermentation with glucose.L lactic acid bacillus and racemic lactic acid bacillus is two Nakayama and Yanoshi (1967) reported that included bacillus in Berger's bacteriology manual position behind the undetermined species within the genus.6. balloon bacillus (Bacillus vesicuiferous)Trinkumuite (1987) reported a new species of lactic acid formationof spore of pneumatocyst. This kind of strains isolated from the gut of lobster.The genus Bacillus (5) identification of 5 Bacillus spore cells, baculovirus, editing, strictly anaerobic, micro aerobic decomposition, lignin, halophilic, decomposition of thiamine, containing more than10%NaCL Omega, alicyclic acid and 515F probe and 1741F probe hybridization, hybridization, and decomposition of pyrite of Bacillus subtilis [1]6 is (Bacillus) two retrieval table editing Bacillus widely exist in nature, many of the hydrolyzed starch, protein, pectin,alginate decomposition, industrial useIn the extraction of amylase, protease, pectinase. Some of them have some antibiotics, can cause human and animal diseases, and some can cause plantCorruption. They are found in soil, water and air, and the nature of material transformation, soil fertility and environmental health are closely related, fromWhat are the major microbial workers will be exposed to, identify problems in the genus bacillus. Liao Yanxiong, Fu Xiaochong (Jiangxi Academy of Sciences Nanchang 330029) Abstract] according to "Berger's Manual of Systematic Bacteriology", proposed the genus Bacillus (Bacilus) of the two index table of species and the genus Bacillus and related genera.Atlas of entryMore pictures*。

芽孢杆菌的发酵流程英文回答：Bacillus subtilis is a type of spore-forming bacteria commonly used in fermentation processes. Its fermentation process involves several key steps, each contributing to the overall production of desired products.First, the fermentation process starts with the inoculation of a culture medium with Bacillus subtilis. This culture medium typically contains nutrients such as carbon sources (e.g., glucose or starch), nitrogen sources (e.g., ammonium salts or amino acids), and other essential minerals. The bacteria are then allowed to grow andmultiply in this nutrient-rich environment.During the growth phase, Bacillus subtilis utilizes the carbon and nitrogen sources in the culture medium to synthesize various metabolites, enzymes, and other cellular components. These components are essential for thebacteria's survival and growth. The growth phase typically lasts for a certain period, depending on factors such as temperature, pH, and oxygen availability.As the bacteria continue to grow, they reach a point where nutrient depletion and accumulation of waste products start to limit their growth. This signals the entry into the stationary phase of the fermentation process. In this phase, the bacteria undergo significant changes in their metabolism and physiology. They start to produce and secrete various secondary metabolites, such as antibiotics, enzymes, and organic acids.The secretion of these secondary metabolites is often a result of complex regulatory networks within the bacteria. For example, the production of antibiotics by Bacillus subtilis is regulated by a system called quorum sensing, where the bacteria communicate with each other through the secretion and detection of signaling molecules. This ensures that the production of antibiotics is coordinated and occurs at the appropriate time.After the stationary phase, Bacillus subtilis enters the sporulation phase. Sporulation is a survival mechanism employed by the bacteria when faced with unfavorable conditions, such as nutrient depletion or environmental stress. During sporulation, the bacteria undergo a series of morphological and physiological changes, ultimately leading to the formation of endospores.Endospores are highly resistant structures that protect the bacteria's genetic material and other essential components. They can survive harsh conditions, such as high temperatures, desiccation, and exposure to chemicals or radiation. This ability to form endospores is one of the reasons why Bacillus subtilis is widely used in industrial fermentation processes.Once the fermentation process is complete, the desired products can be harvested from the culture medium. This can be done through various methods, such as filtration, centrifugation, or precipitation. The harvested products can then undergo further processing, purification, and formulation before being used in various applications.中文回答：芽孢杆菌是一种常用于发酵过程的孢子形成菌。

1Bacillus Subtilis Expression Vectors Product Information and InstructionsNovember 20053generally regarded as safe); (ii) it has no significant bias in codon usage; (iii)it is capable of secreting functional extracellular proteins directly into the culture medium (at present, about 60% of the commercially available enzymes are produced by Bacillus species); (iv) a large body of information concerning transcription, translation, protein folding and secretion mechanisms, genetic manipulation and large-scale fermentation has been acquired.But there are also two obstacles reducing the use of B. subtilis : (i) production of a number of extracellular proteases which recognize and degrade heterologous proteins, and (ii) stable vector plasmids. The first obstacle has been largely solved by the construction of protease-deficient strains. And the second has been completely overcome by introducing plasmids using the theta-mode of replication such as those derived from the natural plasmids pAM β1 and pBS72 (Jannière et al., 1990; Titok et al., 2003).Quite recently, the construction and use of four different expression vectors based on the E. coli - B. subtilis shuttle vector pMTLBS72 exhibiting full structural stability was published (Nguyen et al., 2005).The two new vectors pHT01and pHT43 allow high-level expression of recombinant proteins within the cytoplasm, where pHT43 directs the recombinant proteins into the medium. Both vectors are based on the strong σA -dependent promoter preceding the groE operon of B. subtilis which has been converted into an efficiently controllable (IPTG-inducible) promoter by addition of the lac operator. Derivatives of pHT01 are available either with a 8xHis tag (pHT08), a Strep tag (pHT9) or a c-Myc tag (pHT10).II Aat IIXba IBam HI Xho I5II Xba IBam HI Xho IApa I P grac: P grac promoter (consisting of the gro E promoter; the lac O operator and the gsi B SD sequence)ColE1 ori: ColE1 origin Amp R : ampicillin resistance lacI: lacI gene (lac repressor)Cm R : chloramphenicol resistance SamyQ: amyQ signal sequenceComplete DNA sequence is available on request.tgcgcggaagccatcaccatcaccatcaccatcacGGATCC TCTAGA gtcgacgtcCCCGGGLocation of the Strep tag in pHT09:Strep tag Bam H I Xba I Sma IP grac -lacO-RBS-atgaattggagccatccgcaatttgaaaaaGGATCC TCTAGA gtcgacgtcCCCGGG Location of the c-Myc tag in pHT10:Bam H I Xba I c-Myc tagP grac -lacO-RBS-GGATCC TCTAGA gtcgacgtcgaacaaaaacttattagcgaagaagatctttaataacacgtc3. ProtocolsDetailed protocols for E. coli and Bacillus molecular genetic handling (growth, transformation etc.) can be found in the relevant laboratory manuals such as Sambrook and Russell (2001). For transformation of B. subtilis we recommend the protocol of Anagnostopoulos and Spizizen (1961), slightly modified:1.20 ml LS medium (Spizizen’s medium* supplemented with 0.5 %glucose, 5 µg/ml DL-tryptophane, 5 µg/ml uracil,0.01% casein hydrolysate, 0.1% yeast extract [Difco], 1 mM MgS04,2.5 mM MgCl 2, 0.5 mM CaCl 2) are inoculated with 1 ml of an 5 ml overnight culture grown in HS medium (Spizizen’s medium*supplemented with 0.5 % glucose, 50 µg/ml DL-tryptophane,50 µg/ml uracil, 0.02% casein hydrolysate, 0.1% yeast extract [Difco], 8µg/ml arginine, 0.4 µg/ml histidine, 1 mM MgSO 4) at 37°C, shaking slowly at 30°C for 3 to 4 hours.2.Incubate 1 ml of this HS culture (late log/early stationary phase; OD 578)with 10 µl of 0.1 M EGTA at room temperature for 5 minutes and add 1to 2 µg plasmid DNA.3.After shaking at 37°C for 2 hours for development of antibiotic resistance,the cells are plated on selective plates.*Spizizen's medium: 2 g (NH 4)2SO 4, 14 g K 2HPO 4, 6 g KH 2PO 4, 1 g sodium citrate;add 100 ml distilled water, autoclave, then add 0.1 ml 1 M MgSO 4.7。

·科普论坛·生物技术通报BIOTECHNOLOGY BULLETIN2019, 35(7):230-232收稿日期： 2019-07-08基金项目：固态发酵资源利用四川省重点实验室开放基金项目（2018GTY008），中央高校基本科研业务费资助项目（FRF -TP -18-012A1；FRF -BR -18-009B）作者简介：刘洋，男，博士，副教授，研究方向：微生物生态学与分类学；E -mail ：liuyang@近年来，国内外科学家相继从植物根际土壤［1］、养殖池塘［2］、深海［3］、大曲［4］分离得到大量的贝莱斯芽孢杆菌，并通过研究发现许多贝莱斯芽孢杆菌菌株在促进植物生长、拮抗病原菌［5］及白酒酿造［6］等方面发挥一定的作用，具有十分重要的理论研究与实践应用价值。

然而贝莱斯芽孢杆菌的“身份”一直以来都是细菌分类学家的争论焦点，其作为独立的分类单元及其所具有的有效的物种名称经历了一番波折（图1），直到2016年其在学界公认的分类学地位才暂且尘埃落定。

为了让读者清晰了解贝莱斯芽孢杆菌物种名称的由来与更迭历程，从而更加规范与科学的使用贝莱斯芽孢杆菌及其“曾有分类瓜葛”物种的名称，本文采用按时间顺叙的方式对贝莱斯芽孢杆菌物种名称变化过程中的“关键节点事件”进行系统阐述，以期客观、简要地还原贝莱斯芽孢杆菌名称演变发展的历程。

1943年，日本科学家Fukumoto ［7］在研究过程中分离获得了一株具有高产淀粉酶活力的细菌菌株，并将此研究成果发表于日本农业化学学会学报（Journal of the Agricultural Chemical Society of Japan ）上，并将其命名为解淀粉芽孢杆菌（Bacillus amyloliquefaciens ）。

但由于种种原因，该细菌名称贝莱斯芽孢杆菌（Bacillus velezensis ）物种名称的“前世今生”刘洋1 刘晓昆1 陈文浩2（1. 北京科技大学化学与生物工程学院，北京 100083；2. 宜宾学院固态发酵资源利用四川省重点实验室，宜宾 644000）摘 要： 贝莱斯芽孢杆菌（Bacillus velezensis ）是环境中常见的具有广泛功能性的细菌种类，而其分类地位与物种名称的确定却是细菌分类学家一直以来争论的焦点，直到最近贝莱斯芽孢杆菌有效的物种名称才得到确定。

摘要：利用生物药剂防治番茄灰霉病可降低致病菌的抗药性，且对果品安全健康，对环境无污染。

为了研究枯草芽孢杆菌和哈茨木霉菌对番茄灰霉病的防治效果，在室内条件下研究枯草芽孢杆菌和哈茨木霉对番茄灰霉病致病菌灰葡萄孢菌拮抗作用的基础上，在田间条件下研究了二者不同用量对番茄灰霉病的防治效果。

室内对峙试验结果显示，枯草芽孢杆菌和哈茨木霉均对灰葡萄孢菌具有拮抗作用，且均对灰葡萄孢菌菌丝有直接的破坏作用；枯草芽孢杆菌发酵无菌滤液的抑菌效果随着稀释倍数的增大而逐渐降低，其中10倍稀释液的抑菌率达到80.95%，50倍稀释液的抑菌率仅为28.57%。

田间防效试验结果显示，枯草芽孢杆菌和哈茨木霉均对番茄灰霉病具有防治作用，其中枯草芽孢杆菌用量为1125g/hm 2、哈茨木霉用量为18000g/hm 2时防效最好，对叶片的防病效果分别为84.91%和85.85%，对果实的防病效果分别为90.00%和96.00%。

生物药剂枯草芽孢杆菌和哈茨木霉菌均可用于番茄灰霉病的防治。

关键词：番茄灰霉病；枯草芽孢杆菌；哈茨木霉菌；拮抗作用；防治效果中图分类号：S436.412文献标识码：A 文章编号：1008-1631（2024）01-0066-08收稿日期：2023-08-28基金项目：河北省蔬菜产业技术体系冀南高品质蔬菜技术提升岗位项目（HBCT2023100205）；石家庄市科技计划项目（229490132N ）作者简介：王丹丹（1991-），女，河北沧州人，农艺师，硕士，主要从事设施蔬菜栽培与生理研究。

E-mail ：yuwenhanzhu@126.com 。

通讯作者：耿晓彬（1973-），男，河北赵县人，高级农艺师，主要从事农业技术推广工作。

E-mail ：*****************。

Control Effects of Bacillus subtilis and Trichoderma harzianum on Tomato Gray MoldWANG Dan-dan 1，ZHANG Qing-yin 1，LI Yan 1，TIAN Dong-liang 1，CHEN Chang 2，QI Lian-fen 1，SHI Jian-hua 1，GAO Xi-biao 3，GENG Xiao-bin 1*（1.Shijiazhuang Academy of Agriculture and Forestry Sciences ，Shijiazhuang 050041，China ；2.Institute of Coastal Agriculture ，Hebei Academy of Agriculture and Forestry Sciences ，Caofeidian 063200，China ；3.Zhao County Agri-cultural Technology Service Center ，Zhao County 051530，China ）Abstract ：The use of biological agents to control tomato gray mold can reduce the resistance of pathogenic bac-teria ，and is safe and healthy for fruits ，without polluting the environment.In order to study the control effects of Bacillus subtilis and Trichoderma harzianum on tomato gray mold ，the antagonistic effects of B.subtilis and T.harzianum on the pathogenic bacterium Botrytis cinerea were studied under indoor condition.The control effects of different dosages of B.subtilis and T.harzianum on tomato gray mold was studied under field condition.The in-door confrontation test results showed that both B.subtilis and T.harzianum had antagonistic effects on B.cinerea ，and both had a direct destructive effect on its mycelium.The antibacterial effects of sterile filtrate from B.subtilis fermentation gradually decreased with the increasing of dilution ratio.Among them ，the antibacterial rate of 10fold dilution solution reached 80.95%，while the antibacterial rate of 50fold dilution solution was only 28.57%.The results of field control tests showed that both B.subtilis and T.harzianum had control effects ontomato gray mold.Among them ，the best control effect was achieved when the dosage of B.subtilis was 1125g/hm 2and T.harzianum was 18000g/hm 2.The control effectson leaves were 84.91%and 85.85%，respectively ，andon fruits were 90.00%and 96.00%，respectively.Bio-logical agents B.subtilis and T.harzianum can both control tomato gray mold ，reduce the resistance of pathogenic bacteria ，ensure the safety and health of fruits ，and have no pollution to environment.Key words ：Tomato gray mold ；Bacillus subtilis ；Trichoderma harzianum ；Antagonism ；Control effect王丹丹1，张庆银1，李燕1，田东良1，陈昶2，齐连芬1，师建华1，高西彪3，耿晓彬1*（1.石家庄市农林科学研究院，河北石家庄050041；2.河北省农林科学院滨海农业研究所，河北曹妃甸063200；3.赵县农业技术服务中心，河北赵县051530）枯草芽孢杆菌和哈茨木霉菌对番茄灰霉病的防治效果DOI ：10.12148/hbnykx.20240022河北农业科学，2024，28（1）：66-73，80Journal of Hebei Agricultural Sciences第1期番茄（Lycopersicon esculentum）是世界范围内栽培面积很广的蔬菜作物之一，也是中国设施栽培的主要蔬菜之一[1]。

WATER,PURIFIED纯化水H2O M r18.12 DEFINITIONWater for the preparation of medicines other than those that are required to be both sterile and apyrogenic,unless otherwise justified and authorized.定义制药用水不同于其它用水，要求它是无菌的、无热源的，除非另有调整或授权。

Purified water in bulk散装纯化水PRODUCTIONPurified water in bulk is prepared by distillation,by ion exchange,by reverse osmosis or by any other suitable method from water that complies with the regulations on water intended for human consumption laid down by the competent authority.Purified water in bulk is stored and distributed in conditions designed to prevent growth of micro-organisms and to avoid any other contamination.生产：散装纯化水是经合格的当局规定的适宜人类使用的水经蒸馏、离子交换、反渗透膜或其他任何适合的方法制备。

散装纯化水存储和分配于可防止微生物生长和可避免其他任何污染的条件下。

Microbiological monitoring During production and subsequent storage, appropriate measures are taken to ensure that the microbial count is adequately controlled and monitored.Appropriate alert and action levels are set so as to detect adverse trends.Under normal conditions,an appropriate action level is a microbial count of100CFU/mL,determined by filtration through a membrane with a nominal pore size not greater than0.45μm,using R2A agar and incubating at30-35°C for not less than5days.The size of the sample is to be chosen in relation to the expected result.微生物监测在生产和其后的存储过程中，采取适当的方式以确保水的微生物数受到足够的控制和监测。

- 0 T -I-I IC-T-l-l华北农学报·2011。

26(增刊)：481—483D O N[A L l·81N IC A细菌素抗菌作用机理及其应用研究彭子欣，胡婷，王安如，王丹玉，王洪彬，宋维平(北京大北农科技集团股份有限公司，北京100193)摘妻：很多细菌素是由乳酸菌在代谢过程中通过核糖体途径合成的一类具有抗菌活性的多肽物质。

细菌素具有生产来源安全、易被蛋白酶降解、热稳定及不易产生耐药性等优点，引起人们的广泛关注．主要阐述了细菌素的分类、蛋白结构、抗菌机制、在畜牧业的应用以及发展前景．关奠词：细菌素：抗菌机制；研究进展；应用前景A d va n c e in mechanisms an d ap plic atio n of bacte riocinsPENG Z i—x i n，H U Ting，WANG An—ru，WA NG Dan—yu，WANG Ho n g—b i n，S O N G Wei—ping木(Beijing D a Be i N o n g Tec hn o lo gy G r o u p Co．Ltd．，Beij ing 100193．Ch ina)Abstr act：Bact erio cins a r e rib os o ma l ly sy n t he s iz e d a n t i m ic r o b i al p e p ti d e s usually p rod uce d by food—grade l ac ti c acid bacteria．Si nce bactefiocins have t h e n a t u r e of p ro te in ac e ou s a n d heat—st abl e，th ey d r a w a w id e ly p u b l i c attention w itho ut t h ee m e r g e n c e of bacteria ac qu i ri ng resistance．Here，recent studies o n bacteriocins a r e s u m m a r i z e d in the i r structural diversities，antimicrobialmechanisms，application o n an im a l hu sba ndr y and future pe rspe cti ves．Keywo rd：Bact erio cins；An tiba cter ial mechanism；R esea rch p r o g r es s；A p p l i c a t i o n p ro s p e c t细菌素是微生物在代谢过程中通过核糖体途径1．1羊毛硫抗生素合成的一类具有抗菌活性的多肽物质，对细菌素产生I类细菌素羊毛硫抗生素(含羊毛硫氨酸的抗生菌自身没有抑制作用。

鞭毛运动及胞外聚合物介导生物膜形成的研究进展发布时间：2022-07-21T07:55:51.877Z 来源：《科学与技术》2022年30卷第5期第3月作者：王树东[导读] 细菌在面对环境压力时，通常会主动调节自身的生理行为以聚集生长或形成生物膜来抵御恶劣环境的影响王树东（合肥工业大学，安徽省合肥市，230009）摘要：细菌在面对环境压力时，通常会主动调节自身的生理行为以聚集生长或形成生物膜来抵御恶劣环境的影响。

普遍存在于冷却水塔、食品加工厂或饮用水系统中的生物膜会导致微生物污染和系统堵塞，造成社会经济损失、威胁公众健康安全。

生物膜形成包含一系列复杂的变化过程，由表面附着开始，随后是微菌落的形成和分化结构的演化。

本文综述了细菌鞭毛介导的运动以及分泌的胞外聚合物（extracellular polymeric substances，EPS）在生物膜初期形成中的作用，并在此基础上为探究生物膜形成的内在机制提出了建议，以期为饮用水系统和其它水环境的微生物污染控制提供理论指导和新的思路。

关键词：鞭毛运动、胞外聚合物、表面附着、生物膜以往的研究表明，细胞-细胞和细胞-固体表面间的相互作用决定了细菌附着和生物膜的形成过程，然而这一过程依赖于细菌和固体表面的物理化学性质，以及其所处的微观环境[1]。

根据微观环境条件的改变，细菌会响应调节其生理行为（如鞭毛的合成和运动，以及胞外聚合物、信号分子的生成）以抵御不利环境对其的影响[2]。

细菌的个体行为会干预细胞-细胞间和细胞-基质间的交互作用，使得细菌在悬浮和附着状态之间切换，这也是细菌抵御不利环境的生存保护机制。

因此，细菌鞭毛介导的运动和分泌的EPS对细菌附着和生物膜形成至为重要，本文综述了生物膜的形成以及运动和EPS介导生物膜形成的方式。

1生物膜形成过程及优缺点生物膜是细菌附着在固体表面后分泌粘性胞外聚合物（extracellular polymeric substances，EPS）并在其包裹下生长繁殖而形成的细菌菌落[3]。

硼酸滴耳液微生物限度检查方法学验证发布时间：2021-03-22T09:19:19.520Z 来源：《医师在线》2021年2期作者：李静芳胡小林[导读] 目的建立医院制剂硼酸滴耳液的微生物限度检验方法李静芳胡小林云南省保山市食品药品检验检测中心 678000摘要：目的建立医院制剂硼酸滴耳液的微生物限度检验方法。

方法按《中国药典》2015年版四部微生物限度检查法规定，采用平皿法对云南省保山市人民医院3个批次的硼酸滴耳液进行微生物限度检查方法适用性试验。

结果本研究建立的检查方法对金黄色葡萄球菌、铜绿假单胞菌、枯草芽孢杆菌、白色念珠菌、黑曲霉五种验证菌株的回收率均在0.5～2；控制菌试验组、阳性对照组都为阳性，供试品对照组、阴性菌对照组、阴性对照组为阴性，试验结果有效。

结论硼酸滴耳液进行微生物限度检查时，可取供试品1:10,1:100的稀释液，采用平皿法检查需氧菌总数、霉菌和酵母菌总数；控制菌采用常规法。

可有效的反映药物中微生物的污染情况。

关键词 :硼酸滴耳液；微生物限度；验证；常规法Verification of microbial limit test methodology for boric acid ear dropsAbstract: Objective To establish a microbial limit test method for boric acid ear drops from hospital preparations. Methods According to the four microbial limit test methods of Chinese Pharmacopoeia (2015 edition), the applicability of microbial limit test method for 3 batches of boric acid ear drops from Baoshan People's Hospital of Yunnan Province was tested by plate method. Results The recoveries of Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Candida albicans and Aspergillus Niger were 0.5-2. The control bacteria test group and the positive control group were positive, while the test product control group, the negative bacteria control group and the negative control group were negative, and the test result was effective. Conclusion When boric acid ear drops were used for microbial limit test, diluents of 1:10 and 1:100 samples were recommended. The total number of aerobic bacteria, molds and yeasts was detected by plate method. Routine methods were used to control the bacteria. It can effectively reflect the contamination of microorganisms in drugs.Key words: Boric acid ear drops; Microbial limits; Validation; The conventional method引言硼酸滴耳液广泛用于治疗慢性中耳炎，具有防腐消毒作用。

一、湿热灭菌法本法系指将物品置于灭菌柜内利用高压饱和蒸汽、过热水喷淋等手段使微生物菌体中的蛋白质、核酸发生变性而杀灭微生物的方法。

该法灭菌能力强，为热力灭菌中最有效、应用最广泛的灭菌方法。

药品、容器、培养基、无菌衣、胶塞以及其他遇高温和潮湿不发生变化或损坏的物品，均可采用本法灭菌。

流通蒸汽不能完全杀灭细菌孢子，一般可作为不耐热无菌产品的辅助灭菌手段。

湿热灭菌条件通常采用121℃*15min、121℃*30min、或116℃*40min的程序，也可采用其他温度和时间参数，但必须保证物品灭菌后的SAL《10-6。

对热稳定的物品，可采用过度杀灭法，其SAL应《10-12。

热稳定性较差产品的标准灭菌时间F0［指灭菌温度为121℃，生物指示菌的耐热参数D值为1分，灭菌温度系数Z值为10.0℃时的标准灭菌时间（121℃下计算的微生物等效灭活率）］一般不低于8min。

如产品的热稳定性很差时，可允许湿热灭菌的F0低于8，此情况下，应在生产全过程中，对产品中污染的微生物严加监控，并采取各种措施降低微生物污染水平，确保被灭菌产品达到无菌保证要求。

采用湿热灭菌时，被灭菌物品有适当的装载方式，不能排列过密，以保证灭菌的有效性和均一性。

湿热灭菌法应确认灭菌柜在不同装载时可能存在的冷点。

当用生物指示剂进一步确认灭菌效果时，应将其置于冷点处。

本法生物指示剂为嗜热脂肪芽孢杆菌孢子（spores of Bacillus stearothermophilus）。

二、干热灭菌法本法系指将物品置于干热灭菌柜、隧道灭菌器等设备中，利用干热空气达到杀灭微生物或消除热原物质的方法。

适用于耐高温但不宜用湿热灭菌法灭菌的物品灭菌，如玻璃器具、金属材质容器、纤维制品、固体试药、液状石蜡等均可采用本法灭菌。

干热灭菌条件一般为160~170℃*120min以上、170~180℃*60min以上或250℃*45min以上，也可采用其他温度和时间参数。

应保证物品灭菌后的SAL《10-6。

中国畜牧兽医 2024,51(4):1511-1519C h i n aA n i m a lH u s b a n d r y &V e t e r i n a r y Me d i c i n e 枯草芽孢杆菌的生理功能及其在畜禽生产中的应用张关锋1,杨 泰2,3,宋先凡4,徐伟伟1,阳治康1,陈清华1,2(1.湖南农业大学动物科学技术学院,长沙410128;2.长沙市生物饲料工程技术研究中心,长沙410209;3.湖南百宜科技集团有限公司,长沙410128;4.湖南德邦牧业有限公司,长沙410000)摘 要:枯草芽孢杆菌是一类可形成芽孢的具有多种生理功能且对动物机体无毒副作用的理想益生菌㊂在饲料中添加枯草芽孢杆菌具有调节畜禽肠道功能㊁平衡微生物区系,提高免疫力和促生长等作用㊂枯草芽孢杆菌还可以作为饲料发酵菌种,经发酵后的饲料会降低抗营养因子的含量,提高营养物质的含量,从而促进畜禽对饲料养分的消化和吸收㊂枯草芽孢杆菌内生芽孢的特性,可以保证其在饲料生产和畜禽内环境条件下的功能稳定性以及作为疫苗表达载体的独特优势㊂因此,枯草芽孢杆菌在畜禽生产中具有十分广阔的应用前景㊂作者综述了枯草芽孢杆菌的生物学特性㊁生理功能及其在畜禽生产中的应用,以期为枯草芽孢杆菌在畜禽生产中的应用提供一定的参考㊂关键词:枯草芽孢杆菌;肠道菌群;发酵菌种;疫苗表达载体中图分类号:S 816.3文献标识码:AD o i :10.16431/j .c n k i .1671-7236.2024.04.019 开放科学(资源服务)标识码(O S I D ):收稿日期:2023-10-12基金项目:湖南省 双一流 建设专项(k x k 201801004)联系方式:张关锋,E -m a i l :1335073795@q q .c o m ㊂通信作者陈清华,E -m a i l :c h qh 314@163.c o m P h y s i o l o g i c a l F u n c t i o n s o f B a c i l l u s s u b t i l i s a n d I t s A p p l i c a t i o n i nL i v e s t o c ka n dP o u l t r y Pr o d u c t i o n Z HA N G G u a n f e n g 1,Y A N G Ta i 2,3,S O N G X i a n f a n 4,X U W e i w e i 1,Y A N GZ h i k a n g 1,C H E N Q i n gh u a 1,2(1.C o l l e g e o f A n i m a lS c i e n c e a n dT e c h n o l o g y ,H u n a nA g r i c u l t u r a lU n i v e r s i t y ,C h a n gs h a 410128,C h i n a ;2.C h a n g s h aB i o l o g i c a lF e e dE n g i n e e r i n g T e c h n o l o g y Re s e a r c hC e n t e r ,C h a n g s h a 410209,C h i n a ;3.H u n a nB a i y iT e c h n o l o g y G r o u p C o .,L t d .,C h a n gs h a 410128,C h i n a ;4.H u n a nD e b a n g A n i m a lH u s b a n d r y C o .,L t d .,C h a n g s h a 410000,C h i n a )A b s t r a c t :B a c i l l u s s u b t i l i s i sa ni d e a l p r o b i o t i cs p e c i e st h a tc a nf o r m s p o r e s w i t hav a r i e t y of p h y s i o l og i c a l f u n c t i o n sa n di sn o n -t o x i ca n dh a sn osi d ee f f e c t so na n i m a lo r g a n i s m s .A d d i n gB a c i l l u ss u b t i l i s t o f e e d c a n r e g u l a t e i n t e s t i n a lf u n c t i o n ,b a l a n c e m i c r o b i a lf l o r a ,e n h a n c e i m m u n i t y ,a n d p r o m o t e g r o w t h .B a c i l l u s s u b t i l i s c a na l s ob eu s e da sa f e e df e r m e n t a t i o ns t r a i n .T h e f e r m e n t e d f e e dw i l l r e d u c e t h e c o n t e n t o f a n t i -n u t r i t i o n a l f a c t o r s a n d i n c r e a s e t h e c o n t e n t o fn u t r i e n t s ,t h e r e b y p r o m o t i n g t h ed i g e s t i o na n da b s o r p t i o n o ff e e d n u t r i e n t sb y li v e s t o c ka n d p o u l t r y .T h ec h a r a c t e r i s t i c s o f B a c i l l u ss u b t i l i s e n d o p h y t i cs p o r e sc a n e n s u r ei t sf u n c t i o n a l s t a b i l i t y i n f e e d p r o d u c t i o n a n dl i v e s t o c k a n d p o u l t r y i n t e r n a le n v i r o n m e n ta n di t s u n i qu e a d v a n t a g e s a s a v a c c i n e e x p r e s s i o n v e c t o r .T h e r e f o r e ,B a c i l l u s s u b t i l i s h a s a v e r y b r o a d a p p l i c a t i o n p r o s p e c t i n l i v e s t o c ka n d p o u l t r yp r o d u c t i o n .T h ea u t h o r s r e v i e w s t h eb i o l o gi c a l c h a r a c t e r i s t i c s ,p h y s i o l o g i c a l f u n c t i o n s ,a n d a p p l i c a t i o no f B a c i l l u s s u b t i l i s i n l i v e s t o c ka n d p o u l t r yp r o d u c t i o n ,i n o r d e r t o p r o v i d es o m er e f e r e n c e f o r t h ea p p l i c a t i o no f B a c i l l u s s u b t i l i s i nl i v e s t o c ka n d p o u l t r y中国畜牧兽医51卷p r o d u c t i o n.K e y w o r d s:B a c i l l u s s u b t i l i s;i n t e s t i n a lm i c r o b i o t a;f e r m e n t a t i o n s t r a i n;v a c c i n e e x p r e s s i o nv e c t o r枯草芽孢杆菌(B a c i l l u s s u b t i l i s)是一类广泛存在于自然界中的革兰阳性菌,在土壤㊁植物根系㊁水生环境和动物胃肠道中都可以分离得到[1]㊂1872年,枯草芽孢杆菌被C o h n命名为B a c i l l u s s u b t i l i s (E h r e n b e r g)C o h n[2]㊂经过多年的研究和应用,目前枯草芽孢杆菌在基因工程㊁微生物合成㊁发酵工程㊁畜禽生产中都有重要的应用[3]㊂大量研究表明,枯草芽孢杆菌在调节畜禽肠道健康㊁促进畜禽生长等方面有良好的应用效果[4],但由于其作用机理不够明确,从而限制了其在畜禽生产中的应用㊂作者旨在明确枯草芽孢杆菌的生理功能及生物学特性,总结枯草芽孢杆菌在畜禽生产中的应用研究进展,以期为枯草芽孢杆菌的进一步研究和应用提供一定的参考㊂1枯草芽孢杆菌的生物学特性枯草芽孢杆菌菌体呈梭状,单个细胞在(0.7~ 0.8)μmˑ(2~3)μm,无荚膜,周身具有鞭毛,具有一定运动能力㊂可形成内生芽孢,芽孢大小在(0.6 ~0.9)μmˑ(1.0~1.5)μm,生于菌体内部,不影响菌体本身大小[5-6]㊂枯草芽孢杆菌形成的菌落呈污白色或微黄色,粗糙不透明㊂枯草芽孢杆菌能够根据外界环境的变化,主动启动多种生存机制,如运动性㊁外源D N A摄取㊁形成生物膜和生成芽孢等[7]㊂生成芽孢的主要目的是产生一种在很大程度上没有代谢活性的休眠细胞[8],它能够在恶劣的环境条件下生存,直到恢复有利的生长条件,发芽并且恢复其营养细胞周期㊂枯草芽孢杆菌安全性高,对人和动物无毒性㊁无病原性,虽然在胃肠道大量存在,但并未被视为造成畜禽肠道疾病的病原菌,其多种生物功能还被认为是调节肠道㊁保障畜禽健康的重要基础㊂2枯草芽孢杆菌的生理功能2.1调节畜禽肠道菌群肠道微生物被称为机体的 第五器官 ,兼顾着接收㊁传递各类信号,调节包括消化吸收㊁抵御病原入侵和刺激免疫系统发育在内的多种生理过程[9]㊂枯草芽孢杆菌作为动物肠道微生物区系中重要的部分,也具有很强的菌群调节作用,可以促进益生菌丰度的提高,抑制有害菌的定植,进而保障机体健康[10-12]㊂M a等[13]在1日龄白羽肉鸡饲粮中添加1.0ˑ109C F U/g枯草芽孢杆菌D S M32315发现,肉鸡盲肠厚壁菌丰度增加,同时吸血弧菌㊁志贺氏菌和副拟杆菌等潜在的有害菌的丰度降低㊂众多研究表明,枯草芽孢杆菌作为需氧型微生物,在进入肠道后,借助肠道中适宜的温度和p H,快速地从休眠的芽孢形态萌发㊂短时间内大量耗氧增殖,制造厌氧的肠道环境,促进丁酸梭菌和乳酸菌等肠道益生菌的增殖和定植[14-15]㊂与此同时,大量增殖的枯草芽孢杆菌代谢产生乙酸和丁酸等短链脂肪酸,进一步造成肠道酸性环境,为乳酸菌等益生菌提供良好的生长环境;持续降低肠道中酸度,形成酸性和厌氧的肠道环境,有效地限制了病原菌的增殖,达到菌群调节的作用[12,16]㊂另一方面,枯草芽孢杆菌可以分泌多种细菌素,其广谱抗菌特性也可对肠道有害菌产生颉颃或杀灭的效应(图1)[17]㊂基于枯草芽孢杆菌具有安全性(已被收录在饲料添加剂目录中)㊁益生性以及广谱抗菌特性的功效,在畜禽生产中越来越多饲料企业以及养殖场倾向于将枯草芽孢杆菌作为一种饲料添加剂添加在畜禽饲粮中㊂2.2提高养分利用率,促进畜禽生长肠道作为机体重要的消化吸收器官,在畜牧健康养殖中占有特殊的地位[18]㊂大量研究表明,枯草芽孢杆菌可以提高饲料养分利用率,提高畜禽生产性能[19-21]㊂其主要作用机制可以总结为以下3个方面㊂首先,枯草芽孢杆菌可以产生短链脂肪酸,对肠道绒毛结构的生长和再生都起到促进作用,保障了肠道消化吸收的物理接触面积,提高消化效率[22];同时,枯草芽孢杆菌颉颃病原微生物增殖的作用也间接保护了肠道绒毛形态,实现对于肠道屏障和吸收功能的双重保障㊂其次,枯草芽孢杆菌可以代谢产生或促进多种生物活性酶的分泌,如淀粉酶㊁蛋白酶㊁纤维素酶和一些黏膜二糖酶等㊂这些生物酶可以降解饲料原料中的抗营养因子和有毒有害物质[23-24],改善饲料品质,也可以提高其他营养成分的高效利用,提高养分利用率[25-27]㊂最后,枯草芽孢杆菌不仅可以代谢产生生物酶,还可以合成如氨基酸㊁多肽和维生素等营养成分,为机体生长发育提供营养物质[28]㊂因此,枯草芽孢杆菌可以通过自身代谢产物和改善畜禽肠道形态来提高畜禽对养分的利用率,从而促进畜禽生长㊂21514期张关锋等:枯草芽孢杆菌的生理功能及其在畜禽生产中的应用图1 枯草芽孢杆菌调节畜禽肠道菌群的作用机制F i g .1 M e c h a n i s mo f B a c i l l u s s u b t i l i s r e g u l a t i n g g u tm i c r o b i o t a i n l i v e s t o c ka n d p o u l t r y2.3 调节畜禽机体免疫,减少肠道炎症研究表明,在畜禽饲粮中添加枯草芽孢杆菌可以提高其免疫水平,促进免疫器官发育㊁激活固有免疫和适应性免疫[29-31]㊂一方面枯草芽孢杆菌可以促进肠道相关淋巴组织生长,提高T ㊁B 淋巴细胞数量,刺激肠道黏膜层中分泌型免疫球蛋白A 和整体的免疫球蛋白水平提高,从而提高机体免疫能力[32];另一方面,枯草芽孢杆菌作为病原体相关分子模式进入体内,被树突状细胞等识别细胞捕获,刺激一系列免疫细胞的增殖和分化,进而提高机体内免疫因子的水平[33]㊂但过高的免疫因子水平会导致体内的促炎症因子大量积累,产生炎症的反馈调节,最终出现炎症因子风暴,导致机体组织功能受损,畜禽生长受到抑制㊂S e l v a m等[34]研究表明,枯草芽孢杆菌可以通过分泌表面活性物质来抑制炎症反应中的关键酶(磷脂酶A 2)活性,达到抑制炎症的效果㊂也有研究表明,芽孢杆菌可以通过调控肠道机械屏障,减少外界病原侵染,降低抗原与T o l l 样受体的特异性结合,抑制炎性信号通路下游蛋白表达,从而阻止炎症反应进程(图2)[35]㊂总之,枯草芽孢杆菌在提高畜禽机体免疫力的同时,又能够抑制肠道炎症的发生,从而促进畜禽机体健康㊂3 枯草芽孢杆菌在畜禽生产中的应用3.1 作为饲料添加剂由于枯草芽孢杆菌具有产生孢子的能力,使其具有很强的抗逆性,对高温㊁强酸㊁胆盐的耐受性强,因此,枯草芽孢杆菌可作为益生菌饲料添加剂应用于畜禽生产㊂研究表明,枯草芽孢杆菌具有良好的抑菌性能㊂G u o 等[36]从自然界中筛选出750株枯草芽孢杆菌,其中枯草芽孢杆菌MA139对4种致病菌(金黄色葡萄球菌㊁鼠伤寒沙门菌㊁大肠杆菌K 88㊁大肠杆菌K 99)抗菌活性最高,而这4种致病菌正是引起仔猪发生腹泻的主要病原体[37-38]㊂P a r k等[39]研究表明,在罗斯308肉鸡饲粮中添加1.0ˑ105C F U /g 枯草芽孢杆菌B2A ,可以显著降低肉鸡大肠和小肠中沙门菌的含量㊂陈锁[40]研究发现,在罗斯308肉鸡饲粮中添加200㊁400g /t 枯草芽孢杆菌B -1165制剂能够提高肠道免疫功能,调节肠道菌群,并且对产气荚膜梭菌和球虫引起的肉鸡坏死性肠炎具有良好的治疗作用㊂其抑菌机理可能是:枯草芽孢杆菌是一种耐酸碱的好氧性益生菌,能以消耗肠内氧气的方式竞争性抑制好氧病原菌的增殖,也可通过产生抑菌物质来抑制或杀死产气荚膜梭菌等病原菌,进而调控畜禽肠道菌群平衡,降低宿主感染病原体的风险[41-42]㊂此外,H u 等[43]研究发现,在断奶仔猪饲粮中添加300m g /t (1.0ˑ109C F U /g)3151中 国 畜 牧 兽 医51卷枯草芽孢杆菌C -3102,能够显著提高仔猪对饲粮中干物质㊁粗蛋白质和能量的消化率,减少仔猪肠道大肠杆菌数量㊁增加乳酸菌数量㊂Z h a n g 等[44]研究表明,在母猪妊娠第90天到仔猪断奶期间的饲粮中添加4.0ˑ108C F U /k g 枯草芽孢杆菌PB 6,可缩短仔猪出生间隔,提高仔猪生长性能,改善母猪妊娠后期肠道健康㊂还有研究表明,在罗曼粉壳蛋鸡饲粮中添加0.5g /k g (2.0ˑ1010C F U /g)枯草芽孢杆菌,可以通过调节蛋鸡的肠道菌群和相关代谢物来抑制肠道炎症和氧化应激[45]㊂可见,枯草芽孢杆菌作为饲料添加剂在畜禽饲粮中应用,可以抑制肠道有害菌的生长,调节肠道菌群和机体免疫,减少肠道炎症,提高饲粮中养分的利用率,从而改善畜禽机体健康,提高生产性能㊂图2 枯草芽孢杆菌调节畜禽机体免疫、减少肠道炎症的作用机制F i g .2 M e c h a n i s mo f B a c i l l u s s u b t i l i s r e g u l a t i n g t h e i m m u n e s y s t e ma n d r e d u c i n g i n t e s t i n a l i n f l a m m a t i o n o f l i v e s t o c k a n d p o u l t r y3.2 作为饲料发酵菌种枯草芽孢杆菌可作为微生物发酵饲料的优良菌种㊂一方面,枯草芽孢杆菌发酵后可产生表面活性素㊁环脂肽和内切酶等物质[46-47];另一方面,混合饲料或饲料原料经枯草芽孢杆菌等益生菌发酵后能够提高营养物质的含量㊁降低抗营养因子和有毒有害物质[48-50],从而促进畜禽对于营养物质的消化和吸收㊂Z h a n g 等[51]研究表明,通过枯草芽孢杆菌B S 12固态发酵豆粕,发酵后豆粕中的抗营养因子大豆球蛋白和β-伴大豆球蛋白分别减少92.36%和88.44%,同时还提高了粗蛋白质㊁灰分和总磷的含量;在仔猪饲粮中添加10%该发酵豆粕,可以显著提高仔猪的日增重,显著降低促炎细胞因子I L -4㊁I L -6的m R N A 水平和仔猪空肠㊁回肠中p 38MA P K ㊁I κB -α和p 65N F -κB 的磷酸化水平㊂S h i等[52]采用枯草芽孢杆菌Z J 12-1和粪肠球菌N C I M B10415固态发酵玉米豆粕混合饲料,发酵后混合饲料中大豆抗原蛋白(β-伴球蛋白和甘氨酸)的含量显著降低,中性洗涤纤维㊁半纤维素和植酸酶的含量分别下降了38%㊁53%和46%,另外,发酵后小肽和游离氨基酸的含量增加了6.5倍㊂Z h a n g等[53]研究发现,在仔猪饲粮中添加10%经枯草芽孢杆菌和粪肠球菌发酵的玉米-豆粕混合饲料,可以提高仔猪平均日采食量㊁日增重以及空肠绒毛高度㊁绒毛高度/隐窝深度㊁胰蛋白酶活性和淀粉酶活性,此外,仔猪对于干物质㊁粗蛋白质和磷的表观消化率分别提高了3.01%㊁3.5%和15.38%㊂K o n k o l 等[54]采用枯草芽孢杆菌67固态发酵菜籽粕,发酵后菜籽粕中干物质㊁粗灰分㊁粗脂肪的含量和代谢能显著升高,同时,粗纤维和硫代葡萄糖甙含量显著降低;在41514期张关锋等:枯草芽孢杆菌的生理功能及其在畜禽生产中的应用罗斯308肉鸡饲粮中添加3%该发酵菜籽粕,可以显著提高肉鸡的生长性能㊁改善肉品质㊂另外,赵洁羽等[55]利用枯草芽孢杆菌G J00141和酿酒酵母菌G J00079固态发酵棉粕,显著降低了棉粕中棉酚的含量;在白羽肉鸡饲粮中用18%的发酵棉粕等量替代豆粕,可以促进肉鸡生长㊁降低饲料成本㊂综上,在畜禽生产中可以利用枯草芽孢杆菌的发酵特性发酵各种饲料或饲料原料,提高饲料营养价值,再按照一定比例添加到畜禽饲粮中,通过改善畜禽肠道健康㊁提高畜禽对营养物质的消化率来提高畜禽的生长性能㊁降低饲养成本㊂但要注意发酵过程中菌种的类别㊁发酵参数㊁发酵底物以及根据畜禽的种类和所处生长阶段来确定最适添加量㊂3.3作为疫苗表达载体随着D N A重组技术的迅速发展,枯草芽孢杆菌孢子已被用作治疗和预防由细菌㊁病毒和寄生虫引起的各种疾病的潜在疫苗表达载体,因为它可以有效地引发体内的免疫应答[56-57]㊂枯草芽孢杆菌作为疫苗表达载体具有许多优点:①本身无毒且具有安全性和稳定性[58-59];②抗逆性强,枯草芽孢杆菌的休眠孢子对胃肠道的恶劣环境具有极强的耐受力,这一特性使其成为口服给药抵抗胃内酸性环境并在肠道中释放的理想载体[60];③免疫原性强,通过口服㊁舌下和鼻腔给药均可以引起畜禽免疫反应[61];④可作为颗粒佐剂,孢子具有亚微米级的纳米结构,使它们能够作为有效的颗粒佐剂[62],颗粒佐剂能够充分靶向抗原呈递细胞(A P C),激发机体免疫应答[63]㊂研究表明,枯草芽孢杆菌孢子与吸附在孢子外壳表面上的蛋白抗原共同施用时,可观察到强烈的免疫辅助作用[62]㊂另外,孢子疫苗有许多优点,如通过提高输送的简易性和速度来进行大规模接种疫苗,通过省去净化步骤来降低成本,通过黏膜或口腔途径灵活给药,可提供 无针 和 无冷藏 的疫苗输送系统[64-65]㊂L i等[66]通过枯草芽孢杆菌R B孢子构建了一种能够预防猪圆环病毒2型(P C V2)的新型口腔黏膜疫苗,并在小鼠模型中进行了免疫性能测定,结果表明,该疫苗能激活较强的特异性黏膜和体液免疫应答,同时,孢子表现出良好的黏膜免疫佐剂功能,促进C D3+㊁C D4+和C D8+T细胞等免疫细胞的增殖㊂L e e等[67]将枯草芽孢杆菌H B3孢子作为抗禽流感病毒H9N2的佐剂诱导雏鸡产生特异性抗体和T细胞反应,并对雏鸡进行免疫接种,结果表明,枯草芽孢杆菌孢子作为佐剂,不仅能够增强H9N2病毒特异性I g G产生,还增强了C D4+和C D8+T细胞反应,增加了促炎细胞因子的产生,比传统疫苗佐剂更加有效㊂L v等[68]通过枯草芽孢杆菌W B800N研发了一种能够防御肠炎沙门菌的口服黏膜疫苗,并对雏鸡进行口服免疫,结果表明,试验组能够通过提高雏鸡体内I g A㊁I g G以及I L-2水平和T细胞介导的免疫水平,从而有效抵抗肠炎沙门菌和减少其对雏鸡脾脏㊁空肠造成的损伤㊂综上,由于枯草芽孢杆菌孢子优良的抗逆性使其在疫苗表达载体方面有着巨大的应用潜力㊂3.4枯草芽孢杆菌在畜禽生产中应用存在的问题虽然枯草芽孢杆菌在畜禽生产中应用较为广泛,但仍然存在一些问题㊂如不同菌株在畜禽饲粮中的添加量还未形成具体的应用标准;抑菌作用的分子机制还不够明确,抑菌效果的评定标准不够完善;发酵参数㊁工艺还需要进一步优化;枯草芽孢杆菌孢子作为疫苗表达载体的关键调控因子和信号传递等方面的研究尚浅;关于枯草芽孢杆菌单独或与其他益生菌配伍使用的颉颃效应研究很少等㊂4小结饲料端的抗生素禁用,让中国饲料和添加剂行业面临一个机遇和挑战并存的时期,寻找安全有效的抗生素替代物对于畜牧业生产的稳定发展至关重要㊂枯草芽孢杆菌作为一类优质的益生菌,具有多种生理功能和生物学特性,且在畜牧行业已进行多方面应用㊂在畜禽饲粮中添加适量的枯草芽孢杆菌可以调节肠道菌群,改善胃肠道内环境,提高饲粮中养分的利用率,促进畜禽生长㊂同时,通过其发酵性能可以提高饲料营养价值㊁促进畜禽对饲料养分的消化和吸收,其内生芽孢的特性,可以保证其在饲料生产和畜禽内环境条件下的功能稳定性以及作为疫苗表达载体的独特优势㊂因此,枯草芽孢杆菌在畜禽生产中具有巨大的应用潜力㊂目前枯草芽孢杆菌应用虽然较为普遍,但存在品质差异较大㊁单一菌种功能特性较弱㊁代谢产物作用机制不明确等问题㊂在未来,基于优良枯草芽孢杆菌菌种研发的同时,强化单一菌种的功能特性并明确其作用机制,充分利用枯草芽孢杆菌自身的生物学特性来拓宽其在畜牧生产中的应用,可能会成为枯草芽孢杆菌的研发方向㊂参考文献(R e f e r e n c e s):[1]杨敏敏,车育彦,王睿,等.枯草芽孢杆菌对脂多糖应激肉仔鸡生长性能㊁免疫性能和血清抗氧化性能的5151中国畜牧兽医51卷影响[J].动物营养学报,2022,34(4):2347-2360.Y A N G M M,C H E Y Y,WA N G R,e t a l.E f f e c t so fB a c i l l u ss u b t i l i s o n g r o w t h p e r f o r m a n c e,i m m u n ep e r f o r m a n c e,a n ds e r u m 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