HPLC-UV法检测微生物降解体系中西维因含量

采用高效液相色谱-紫外检测(HPLC-UV)法测定微生物降解体系中西维因含量。根据细菌和霉菌降解体系的不同性质选用适宜前处理方式,以Sepax GP-C_(18)柱(150 mm×4.6 mm,5.0μm)为色谱柱,V(乙腈)∶V(水)=(56∶44)为流动相,流速0.5 m L/min,用紫外检测器于220 nm处检测西维因。结果表明,西维因标准品保留时间为7.922 min,在0.5~100 mg/L范围内线性关系良好,相关系数0.999 1。细菌培养液中西维因平均回收率为102.01%,RSD为0.29%;霉菌培养液中平均回收率为100.76%,RSD为1.22%;最低检测限为0.4mg/L。运用该法测得实验室保藏的细菌菌株A4和B-1在LB培养基中培养2 d对20 mg/L西维因降解率分别为35.72%、38.97%;霉菌菌株M-4和YAT在MM培养基中培养7 d对20 mg/L西维因降解率分别为38.69%、51.40%。该法简单,快速,回收率和精密度高,稳定性好。     

培养条件和果汁种类对脂环酸芽孢杆菌生长的影响

脂环酸芽孢杆菌是造成巴氏灭菌果汁腐败的重要细菌之一。鉴于目前脂环酸芽孢杆菌的培养方法尚未统一且污染特征不明确,本研究考察了pH、7种培养基和9种果汁对其生长的影响,同时采用固相微萃取(SPME)和气相色谱(GC)-质谱(MS)技术检测了该菌的主要代谢产物愈创木酚和2,6-二溴苯酚在果汁中的释放情况。结果表明,脂环酸芽孢杆菌在pH4.0时生长情况最好,pH2.5时生长较差,pH6.0~7.0时最差。AAM培养基和BAT培养基较其他5种培养基(YSG培养基、K氏培养基、SK培养基、PDB培养基和营养肉汤培养基)更适合用来培养脂环酸芽孢杆菌。脂环酸芽孢杆菌接种到9种果汁中后生长差异明显,在葡萄汁和橙汁中生长情况最好,细菌数量分别为1.5×104 CFU/mL和8.5×103 CFU/mL;在菠萝汁和西柚汁中生长缓慢,细菌数量分别为4.4×102 CFU/mL和1.9×102 CFU/mL。此外,9种果汁接种脂环酸芽孢杆菌后均未检测出2,6-二溴苯酚;但是,除葡萄汁外的8种果汁均检测出愈创木酚,浓度在8.6~23.9 μg/L之间。     

细菌ZY-H产灵菌红素发酵参数优化

通过正交实验,考察了培养基组成和发酵条件对细菌ZY-H产灵菌红素的影响。结果表明,ZY-H菌最优培养基和发酵条件为:蔗糖10g/L、蛋白胨15g/L,氯化钙0.025mol/L;发酵液初始pH6,接种量为4%,装液量30mL/250mL三角瓶,在温度为28℃,180r/min培养48h时,发酵液中灵菌红素的含量达到28.37g/L,分别是PDA培养基和牛肉膏蛋白胨培养基的19.57、43.11倍,且差异极显著(p<0.01)。研究结果得到了细菌ZY-H高效表达灵菌红素的培养条件,为灵菌红素的工业化生产提供技术资料。     

政策法规

正《卫生纸(含卫生纸原纸)》等4项生活用纸制品相关国家标准批准发布近日,国家标准委新发布了4项生活用纸制品相关国家标准,其中《纸尿裤和卫生巾用高吸收性树脂》《卫生巾(护垫)》《卫生纸(含卫生纸原纸)》为修订标准,《生活用纸和纸制品化学品及原料安全评价管理体系》为制定标准。新修订的GB/T 22875-2018《纸尿裤和卫生巾用高吸收性树脂》,主要提高了残留单体(丙烯酸)安全指标要求,调整了吸收速度指标要求和挥发物含量的测定方法,增加了返黄值、可萃取物含量及相应测定方法。     

利用谷物进行类胡萝卜素固态发酵

研究了 7种谷物培养基及相关因素对青霉PT95菌株进行类胡萝卜素固态发酵的影响作用。结果表明 ,谷物培养基的组成对PT95菌株在固态发酵条件下的菌核生物量和菌核中的类胡萝卜素含量都有明显的影响 ;接种方式和接种量能明显影响谷物培养基上的菌核生物量 ,但对菌核中的类胡萝卜素含量没有明显的影响 ;在谷物培养基里添加麸皮有利于菌核的形成。在选择最佳接种方式、接种量 ,并在培养基里添加 2 0 %麸皮 (干重 )的固态发酵条件下 ,大米培养基上得到的菌核生物量最高 ,达到 1 5 0 0 g/1 0 0 g(干料 ) ;荞麦培养基上 ,菌核中的类胡萝卜素含量最高 ,达到82 6μg/g(干菌核 ) ;而在谷子培养基上 ,类胡萝卜素产率最高 ,达到 1 1 45 7μg/1 0 0 g(干料 )。     

γ-氨基丁酸产生菌的选育及发酵条件优化

目的:从自然界中筛选γ-氨基丁酸产生菌,研究其最佳发酵条件;方法:采用乳酸菌分离纯化方法挑出γ-氨基丁酸产生菌,对其形态和生理生化特征进行鉴定.随后分别采用正交试验及单因素法对菌株产γ-氨基丁酸的培养基组成和培养条件进行优化;结果:根据伯杰细菌鉴定手册,所筛菌株初步确定为乳酸链球菌(Streptococcus laetis).试验优化的培养基组成为:蔗糖10g/L,丁二酸钠10 g/L.胰蛋白胨5g/L,酵母膏5g/L;优化后的最佳发酵条件为:培养基初始pH为6.5、培养温度为32℃、培养时间为48h;结论:在最佳培养基组合和发酵条件下,发酵液中γ-氨基丁酸的含量达4.23g/L,菌种可作为γ-氨基丁酸产生菌.     

氧化石墨烯对土壤中细菌和固氮菌量的影响

氧化石墨烯(GO)是石墨烯的重要衍生物,经过氧化处理后,GO仍保持石墨的层状结构,但在每一层的石墨烯单片上引入了许多氧基功能团,这使得GO有良好的生物相容性。本文将四种浓度(0mg/kg、10mg/kg、20mg/kg、30mg/kg)的GO分别加入四种养分含量不同的土壤中,培养20天,利用选择性培养基对土壤中的细菌和固氮菌含量进行检测。实验结果表明:30mg/kg的GO在培养后的第7天对细菌的数量有明显促进作用;20mg/kg的GO在培养后的第7天对固氮菌量有明显促进作用。     

食品微生物能力验证霉菌酵母菌计数——检验方法比较

目的:探讨能力验证中霉菌酵母菌计数检验方法及注意事项。方法:采用不同培养基(孟加拉红培养基倾注法、马铃薯-葡萄糖-琼脂培养基倾注法)、不同放置培养方式(正置培养法、倒置培养法)比较分析霉菌酵母菌计数结果的差异性。结果:在两种培养基中,霉菌和酵母菌数量差异不大;比较不同培养方式,在孟加拉红培养基中,霉菌、酵母菌数量正置培养方式比倒置培养方式分别高10.3%、20.0%;而在马铃薯-葡萄糖-琼脂培养基中,霉菌、酵母菌数量在正置培养方式比倒置分别高7.1%、12.5%。结论:孟加拉红培养基和马铃薯-葡萄糖-琼脂培养基对霉菌酵母菌计数结果差异不明显,霉菌酵母菌采用正置培养方式检出率比倒置培养方式高。     

植物乳杆菌(Lactobacillus plantarum)8-6产细菌素发酵条件的优化

对植物乳杆菌(Lactobacillus plantarum)8-6产细菌素的发酵条件进行了优化,分别研究了培养时间、温度、接种量、培养基起始pH值、培养基碳源、氮源等因素对细菌素产生的影响,通过单因素水平试验和正交试验,确定产细菌素的最佳培养基组合和最佳发酵条件为葡萄糖3%,胰蛋白胨2%,蛋白胨1%,酵母膏1%,硫酸镁0.058%,吐温-80 0.2%,30℃培养24h,培养基起始pH值为6.5,接种量2%。乳杆菌8-6优化后效价为1825.56IU/mL,比优化前提高了373.15%。     

L-丙氨酸转化菌发酵条件的优化

产L-天冬氨酸-β-脱羧酶(L-aspartate-β-decarboxylase,Asd)菌株以L-天冬氨酸(L-aspartic acid,L-Asp)为底物转化生产L-丙氨酸,通过单因素及正交设计试验对睾丸酮丛毛单胞菌HY-08D培养基组成及发酵条件进行优化。结果表明,最佳培养基组成:富马酸1.0%、L-Asp 1.0%、谷氨酸1.0%、玉米浆干粉0.6%、酵母粉0.8%、氯化钠0.7%、磷酸二氢钾0.15%、硫酸镁0.1%,pH 6.0。产酶菌株HY-08D转化用培养液采用三级扩培,一级接二级使用0.1%低接种量,二级接三级使用10%的接种量,最适通气量为10 L/min。优化后培养液中HY-08D菌体量和酶活较初始提高约1倍,培养周期缩短6 h~8 h。     

活性乳酸菌制品中乳酸菌计数培养条件探讨

为快速、准确地计数活性乳酸菌制品中的乳酸菌，用CO2烛缸法代替现行国家标准GB／T16347－1996活性乳酸菌饮料中乳酸菌微生物检验方法中的有氧培养法，CO2烛缸法操作简便，计算准确可靠，在菌种对比实验中，CO2烛缸法的乳酸菌计数结果是国家标准方法的8倍，在试样的对比实验中，CO2烛缸法的乳酸菌计数结果是国家标准方法的20倍，CO2烛缸法较目前国家标准方法更能真实施反映活性乳酸菌制品中乳酸菌的数量。     

Comparison of dry culture medium and conventional plating techniques for enumeration of bacteria in pasteurized fluid milk

Standard plate counts, psychrotrophic bacterial counts, and coliforms were determined by conventional plating techniques and by Petrifilm TM plates, a dry culture medium, for 48 commercially processed milk samples (24 whole milk and 24 skim milk). The Petrifilm SM plate counts were compared with counts on standard methods agar for the standard plate count, psychrotrophic bacterial count, and rapid psychrotrophic bacterial count. The Petrifilm violet red bile plate counts were compared with counts on violet red bile agar for coliform test with a solid medium and the preliminary incubation method for detection of coliforms. Standard plate counts were determined within 24 h of packaging and after 7, 10, and 14 d of storage at 6.1 degrees C. Psychrotrophic bacterial counts and coliform counts were determined with 24 h of packaging and after 7 d storage. There was a strong linear relationship between Petrifilm SM and standard methods agar plates (excluding counts on samples plated within 24 h of packaging) and for the psychrotrophic bacterial count method. Petrifilm SM had a weak linear relationship with Standard Methods Agar plates for the rapid psychrotrophic bacterial count. Coliform counts determined on Petrifilm violet red bile plates were generally within the same range as counts on violet red bile agar plates. The positive predictive values for the Petrifilm violet red bile plates and violet red bile agar plates were essentially the same for samples plated within 24 h of packaging.     

Lessons from the organization of a proficiency testing program in food microbiology by interlaboratory comparison: analytical methods in use, impact of methods on bacterial counts and measurement uncertainty of bacterial counts

The proficiency testing program in food microbiology RAEMA (Réseau d'Analyses et d'Echanges en Microbiologie des Aliments), created in 1988, currently includes 450 participating laboratories. This interlaboratory comparison establishes proficiency in detection of Salmonella and Listeria monocytogenes, as well as enumeration of aerobic micro-organisms, Enterobacteriaceae, coliforms, beta-glucuronidase-positive Escherichia coli, anaerobic sulfito-reducing bacteria, Clostridium perfringens, coagulase-positive staphylococci, and L. monocytogenes. Twice a year, five units samples are sent to participants to assess their precision and trueness for enumeration and detection of micro-organisms. Most of participating laboratories use standard or validated alternative methods, they were 50-70% in 1994 and, for 5 years, they are 95%. An increasing use of alternative methods was also observed. This phenomenon is all the more significant as standard methods are laborious and time consuming; thus, 50% of the laboratories use alternative methods for the detection of Salmonella and L. monocytogenes. More and more laboratories use ready-to-use media and although the percentage is variable according to the microflora, we can consider that, today, 50-60% of the laboratories participating to the proficiency program only use ready-to-use media. The internal quality assurance programs lead also to an increasing use of media quality controls. The impact of analytical methods on bacterial counts was assessed by grouping together the results obtained by participating laboratories during the 10 last testing schemes from 1999 to 2003. The identified significant factors influencing enumeration results are variable from one microflora to another. Some of them significantly influence many microflora: the plating method (spiral plating or not) is influential for aerobic micro-organisms, Enterobacteriaceae, coliforms, and staphylococci, the type of culture medium and the medium manufacturer is influential for aerobic micro-organisms, Enterobacteriaceae, coliforms, E. coli, anaerobic sulfito-reducing bacteria, staphylococci, and L. monocytogenes. Others are specific of some micro-organisms: the resuscitation broth for L. monocytogenes, the mode of medium preparation for staphylococci and the incubation temperature for C. perfringens. These effects lead generally to small differences of about 0.1 log10 cfu g(-1), except for the enumeration of anaerobic sulfito-reducing bacteria, where the difference reaches 0.7 log10 cfu g(-1). These results, although difficult to extrapolate to all actual situations, which associate numerous food constituents and physiological states of bacteria to detect or numerate, allow nevertheless the quantification of interlaboratory variations linked to the methods in use. The analysis of bacterial counts obtained by the laboratories participating to the RAEMA proficiency testing program allowed also to validate a formula to calculate the repeatability of bacterial counts and to estimate the between-laboratory uncertainties for the majority of micro-organisms enumerated in food microbiology. The repeatability uncertainty is only indirectly affected by the method in use but depends essentially on the number of counted colonies. On the other hand, the between-laboratory uncertainty varies with the enumeration method in use, this variability is relatively small for the enumerations calling for methods without colony confirmation, i.e. for the enumeration of aerobic micro-organisms, Enterobacteriaceae, 'total' and thermotolerant coliforms, beta-glucuronidase-positive E. coli and coagulase-positive staphylococci with the technique using the rabbit-plasma fibrinogen agar. For these methods, the average between-laboratory standard deviation is 0.17 log10 cfu g(-1). The between-laboratory uncertainty is, on the contrary, larger for more complex techniques. For the enumeration of coagulase-positive staphylococci with the Baird-Parker agar, the between-laboratory standard deviation is equal to 0.23 log10 cfu g(-1), it is equal to 0.28 log10 cfu g(-1) for the enumeration of L. monocytogenes, to 0.34 log10 cfu g(-1) for the enumeration of C. perfringens, and to 0.47 log10 cfu g(-1) for the enumeration of anaerobic sulfito-reducing bacteria.     

生长圈法应用于选育L-异亮氨酸菌株的条件优化

生长圈测定法是L-异亮氨酸高产菌筛选育种工作中较为简单、方便、经济、有效的一种测定方法。本文对影响生长圈法测定的检测培养基加入指示菌时温度、指示菌加入量、检测板琼脂浓度、琼脂块琼脂浓度等因素进行了研究。结果得到优化方法为测定平板和琼脂块均选用基本培养基;测定平板采用上层为10mL混菌培养基,下层为10mL基本培养基的双层平板,其琼脂浓度都为1%;琼脂块的琼脂浓度为1.5%;混菌平板中指示菌培养时间为18h,加入量为4%,混菌时最佳培养基温度为55℃。     

Novel method for estimating viable Salmonella cell counts using real-time PCR

A novel method for estimating viable Salmonella Enteritidis cell counts with 5'-nuclease real-time PCR was developed in this study. Our method was based on the increase kinetics of the target DNA region (invA) of the microorganism growing in a food/clinical sample in a culture medium during incubation. The index of increase in the target DNA region studied here was threshold cycle, CT. A test Salmonella strain was grown in buffered peptone water at the optimal temperature (39 degrees C). As Salmonella cells were grown, the value of CT decreased with time, generating a downward sigmoidal curve. The slope of the curve was constant at various initial cell concentrations. With higher initial cell concentration, the CT value evaluated from the slope at a given time was lower. With this relationship, a novel method for estimating the initial viable cell concentration of a sample was developed. Dead Salmonella cells or bacteria other than the target cell caused deviation in the CT curve. Incubation in a selective media suppressed the deviation caused by other bacterial cells. We think that this method could be applied to many other microorganisms cultivable in a suitable medium.     

乳酸菌菌粉定量计数方法研究

目的:以9株乳杆菌、6株双歧杆菌、3株球菌、1株凝结芽孢杆菌菌粉为研究对象,在国标GB 4789.35-2016的基础上,对稀释倍数、稀释液成分、培养基成分进行比较研究,考察对乳酸菌菌粉计数活菌数的影响。方法:采用稀释平板计数的方法,对不同的乳酸菌菌粉进行活菌计数。结果:初始乳酸菌菌粉样品稀释倍数对最终计数活菌数无明显影响,ISO稀释液对部分乳杆菌、双歧杆菌菌粉的活菌计数结果有显著提高(P<0.05);双歧杆菌菌粉采用TOS琼脂培养基的计数结果显著优于国标培养基(P<0.05);凝结芽孢杆菌菌粉采用改良芽孢计数培养基计数结果优于PCA和NA计数培养基。结论:平板计数方法中,稀释液中含有酪蛋白胨能提升双歧杆菌菌粉的活菌计数数量;TOS琼脂培养基更有利于双歧杆菌的增殖培养;改良芽孢计数培养基更有利于凝结芽孢杆菌的芽孢萌发增殖。     

Use of augmented cultural techniques in the diagnosis of the bacterial cause of clinical bovine mastitis.

Preculture incubation, preculture freezing, and increased plate inoculation volumes were tested in an attempt to increase the recovery rate of pathogens in milk from cases of clinical bovine mastitis. Culture of milk from 291 cases of clinical bovine mastitis was performed using standard milk culture techniques (.01 ml of fresh milk streaked on trypticase soy agar plates with 5% sheep blood and .1% esculin). The sensitivity of this method was compared with that of cultures performed using augmented techniques: 4 and 18 h of preculture incubation; preculture freezing of samples overnight at -20 degrees C; and increasing the plate inoculation volume to .05 and .1 ml for fresh, incubated, and frozen samples. Preculture incubation and larger plate inoculation volumes significantly increased the recovery rate of bacterial pathogens over the standard culture method. The greatest improvement in sensitivity without a concomitant increase in contamination was achieved when samples were incubated for 4 h and plates were inoculated with .1 ml of the sample. Recovery was enhanced significantly by this method for several organisms, including environmental streptococci and coliform bacteria. Freezing milk before culture yielded a significantly higher positive culture rate overall, but freezing did not affect the positive culture rate of any individual bacterial species.     

Evaluation of dry sheet medium culture plate (Compactdry TC) method for determining numbers of bacteria in food samples

The Compactdry, a ready-to-use and self-diffusible dry medium sheet culture system, has been developed by the Nissui Pharmaceutical Co. Ltd. for enumerating bacteria in food. The Compactdry consists of special spread sheet with culture medium that is the same as standard method nutrients, a cold water-soluble gelling agent, and a unique plastic dish. The procedure for bacterial examination in a sample solution (1 ml) is to just inoculate a test solution into the center of the self-diffusible medium and incubate at 35 degrees C for 48 h. The Compactdry TC (CTC) for the enumeration of total aerobic bacteria from 97 food samples was compared with the standard plate count (SPC) method and 3M Petrifilm aerobic count plates (PAC). The correlation coefficients between the CTC and SPC method, the CTC and PAC, and the PAC and SPC method were 0.97, 0.99, and 0.97, respectively. The Compactdry system is useful for the enumeration of total aerobic bacteria in food and may be a possible suitable alternative to the conventional pour-plate or the Petrifilm plate methods.     

Assessment of the antibacterial activity of a triclosan-containing cutting board

Several studies have shown that consumers may not clean cutting boards properly between preparation of raw and cooked meat. Cutting boards may therefore act as sources for contamination of cooked meat or other ready-to-eat foods with pathogenic and spoilage bacteria. The aim of the work was to investigate if cutting boards containing the antimicrobial compound triclosan can reduce the viability of bacteria, thus acting as a hygiene barrier. Survival and growth of food pathogens and spoilage bacteria on two cutting boards without antimicrobials and a commercial cutting board containing triclosan were tested. No difference in bacterial counts on cutting boards without and with triclosan was found after exposure to naturally contaminated chicken filets for one hour. Pathogenic and spoilage bacteria were inoculated on coupons (6.7-7 log per coupon) of cutting boards and incubated at 25 °C at controlled relative humidity for 24 and 72 h. At a relative humidity of 100%, growth of Escherichia coli, Salmonella, Staphylococcus aureus, coagulase-negative staphylococci (CNS) and Serrratia spp. was observed and no antibacterial effect of the triclosan-containing board was found except for against Listeria monocytogenes. At lower humidity (70% RH) less growth was found on the triclosan-containing cutting board than untreated boards after 24 h. After 72 h of incubation, cell counts were reduced on triclosan-containing boards, with the most pronounced antibacterial effects observed against Salmonella, S. aureus and CNS. For S. aureus and Salmonella it was found that when a lower initial cell count was applied (3.5 log per coupon), the triclosan-containing board had an antibacterial effect under humid conditions, as well as a more pronounced antibacterial effect under dry conditions.An agar overlay assay showed that triclosan migrated out of the coupons. Repeated washing of the triclosan-containing cutting boards reduced the antibacterial effect, thus the amount of triclosan available on the surface seemed to be limited. In conclusion, using triclosan-containing cutting boards as a hygienic barrier may only work under certain conditions (low humidity, long exposure time, and clean conditions) and not against all genera of bacteria.     

复合微生物制剂制备与发酵白首乌二级浆检测

目的制备固态复合微生物制剂并进行发酵白首乌二级浆的成分检测。方法选用枯草芽孢杆菌、蜡状芽孢杆菌、保加利亚乳杆菌和乳酸菌等作为发酵菌种制备固态复合微生物制剂,然后利用其接种并进行白首乌二级浆发酵,并测定固态复合微生物制剂的营养成分和菌体生物量,同时分析发酵白首乌二级浆的氨基酸含量变化。结果固态复合微生物制剂的有效活菌总数为3.09×109 g~(-1),菌体生物量为5.69 g/L,菌体总蛋白得率为0.194%。发酵前白首乌二级浆粉氨基酸总量为0.788 g/100 g,发酵后白首乌二级浆粉氨基酸总量为1.193g/100 g,其中脯氨酸与丙氨酸含量比较高,分别达到0.387 g/100 g和0.085 g/100 g。结论制备的固态复合微生物制剂活细菌数量能够保证白首乌二级浆的发酵,发酵后白首乌二级浆粉的品质得到提高,从而为白首乌二级浆发酵饮料的制备提供科学依据。