食品安全研究进展实用教案

1、第1页/共43页第一页，共43页。lThe objective of this study was to investigate the antimicrobial activity of xoconostle pears (Opuntia matudae) against Escherichia coli O157:H7. lDirect extract of xoconostle pears was tested against four strains of E. coli O157:H7 in brain heart infusion (BHI:脑心浸出液肉汤).lThe direct

2、 extract had a significant (P 0.05) inhibitory effect at 4, 6, and 8% (v/v) concentrations and complete inhibitory effect at 10% (v/v) during 8 h of incubation at 37C. Minimum inhibitory volume (MIV) was 400 LmL1(v/v) and minimum lethal volume (MLV) was 650 LmL1(v/v). The inhibitory effect of found

3、to be concentration dependent and not strain dependent. lThus, xoconostle pears extract has the potential to inhibit the growth of E. coli O157:H7 and could provide a natural means of controlling pathogenic contamination, thereby mitigating food safety risks.Abstract第2页/共43页第二页，共43页。目录目录(ml)(ml)1.In

4、troduction2. Materials and Methods3.Results4. Discussion第3页/共43页第三页，共43页。1Introduction引言引言(ynyn)(ynyn)仙人掌果仙人掌果(Opuntia matudae )为仙人掌属（为仙人掌属（Opuntia）植物的果实，果肉含）植物的果实，果肉含有丰富的微量元素、蛋白质、氨基酸、维生素、多糖类、黄酮类和果胶等。有丰富的微量元素、蛋白质、氨基酸、维生素、多糖类、黄酮类和果胶等。中药大辞典记载，仙人掌果具有中药大辞典记载，仙人掌果具有(jyu)行气活血、祛湿退热、生肌等作行气活血、祛湿退热、生肌等作用；印第安人

5、和墨西哥人将其作为食物和药物使用也有几千年的历史。用；印第安人和墨西哥人将其作为食物和药物使用也有几千年的历史。第4页/共43页第四页，共43页。第5页/共43页第五页，共43页。大肠大肠(dchng)(dchng)埃埃希菌希菌 Escherichia Escherichia coli O157:H7 coli O157:H7 毒素(d s)型致病菌肠聚集(jj)型肠产毒素型肠侵袭型出血性肠炎病原群溶血性尿毒综合症血性腹泻肠致病型肠出血型第6页/共43页第六页，共43页。 Escherichia coli O157:H7 大肠埃希菌的抗原结构较复杂，包括菌体O抗原、鞭毛H抗原及被膜K抗原。0抗

6、原超过(chogu)170种，是血清学分型的基础，H抗原超过(chogu)56种，K抗原在100种以上。根据耐热性不同，K抗原又分L、A、B3类，致病性大肠埃希菌的K抗原主要为B类。一个菌株中，一般只含一个类别的K抗原。表示大肠埃希氏菌血清型的方式是按0：K：H排列，易引起食物中毒的致病性大肠埃希菌的血清型有0157：H7、0111：B4、055：B5、026：B6和0124：B17等。第7页/共43页第七页，共43页。 Foodborne pathogens (食源性致病菌)are major concern to consumers, food, industry, and food re

7、gulatory agencies. The yearly cost of foodborne illnesses in the United States as reported in 2010 was about $152 billion including $993 million caused by Escherichia coli O157:H7 in healthcare. E. coli O157:H7 is one of the most important foodborne pathogens in the United States, having been first

8、identified in 1982. From 1982 to 2002, E. coli O157:H7 caused 73,000 illnesses annually in the United States including 8,598 infection cases and 40 deaths.第8页/共43页第八页，共43页。Therefore, the control and prevention of E. coli O157:H7 in food products is an area that is receiving worldwide attention.Chemi

9、cal preservatives have been used for years, later considered to be unhealthy.Therefore, natural antimicrobials demand has been increased.Natural antimicrobials can be found in a variety of plants including herbs, spices, fruits, vegetables, and tropical plants.Food industries are very motivated to r

10、eplace chemical preservatives with natural antimicrobials.第9页/共43页第九页，共43页。Xoconostle pearss (Opuntia matudae) strong anticancer, antidiabetic, and antioxidant characteristics have attracted the attention of researchers around the world. rich source of soluble phenolics, ascorbic acid, and betalains

11、effective antimicrobial activityThus xoconostle pears have great potential as natural antimicrobial.The objective of this study was to examine the antimicrobial activity of xoconostle pears direct extract against E. coli O157:H7 in brain heart infusion (BHI) laboratory medium.第10页/共43页第十页，共43页。2Mate

12、rials and Methods实验实验(shyn)(shyn)材材料和方法料和方法a.a.实验细菌准备实验细菌准备 b. b.仙人掌果提取物制备仙人掌果提取物制备 c. c.细菌计数细菌计数 d. d.细菌增长曲线细菌增长曲线(qxin) e.(qxin) e.琼琼脂扩散分析脂扩散分析 f. f.统计学分析统计学分析第11页/共43页第十一页，共43页。a.实验细菌(xjn)制备Bacterial Culture Activation and Preparation Four strains of E. coli O157:H7, F4546 (alfalfa sprout isolate

13、), H1730 (lettuce isolate), 43895 (beef isolate), and 944 (salami isolate) were used stored at 80C freezer stock storage Immunoblot using Protran nitrocellulose membranes (BA85, Whatman, Schleicher and Schuell, Sanford, ME) was performed to identify the E. coli O157:H7 strains Polymerase Chain React

14、ion (PCR) assay was also conducted to identify the serogroup of E. coli O157:H7 strain4种大肠杆菌分别从提取自苜蓿芽(F4546)、生菜(shngci)(43895)、牛肉(43895)和意大利香肠(944);采用免疫印迹法和PCR技术测定实验菌的血清型；第12页/共43页第十二页，共43页。是一种将高分辨率凝胶电泳和免疫化学分析技术相结合的杂交技术。免疫印迹法分三个阶段SDS-聚丙烯酰胺凝胶电泳；电转移将在凝胶 中已经(y jing)分离的条带转移至硝酸纤维素膜上，选用低电压（100V）和大电流（12A），

15、通电45min ；酶免疫定位将印有蛋白质条带的硝酸纤维素膜（相当于包被了抗原的固相载体）依次与特异性抗体和酶标第二抗体作用后，加入能形成不溶性显色物的酶反应底物，使区带染色。免疫(miny)印迹法广泛应用于分析抗原组分(zfn)及其免疫活性，并可用于疾病的诊断.在艾 滋病病毒感染中此法作为确诊试验第13页/共43页第十三页，共43页。a.实验(shyn)细菌制备Bacterial Culture Activation and Preparation The strains were activated in BHI (Becton Dickinson, Sparks, MD, USA) bro

16、th by transferring 100 L from the stored culture to 10mL BHI broth and incubating at 37C for 24 h and were stored in a refrigerator at 4C Prior to each experimental replication, each individual bacterial strain was streaked on BHI agar and incubated for 24 h at 37C One isolated colony was transferre

17、d to 10mL BHI broth, and incubated at 37C for next day use100 L实验(shyn)菌+ 10mL BHI， 37C 培养24 h， 4C保存；将每种菌株在BHI琼脂培养基上培养24 h， 37C；取一个菌落转移至10mL BHI培养液中， 37C培养，隔天使用；第14页/共43页第十四页，共43页。b.仙人掌果提取物制备(zhbi) Xoconostle Extract Preparation Xoconostle pears were obtained from a local grocery market in Greensbor

18、o, NC For each experiment replication, 450 g of fresh xoconostle pears were rinsed under running tap water, blotted with paper towel, sliced into small pieces, and blended in a kitchen blender for 4min This preparation was placed in 50mL tubes and centrifuged at 7800 g for 10 min The supernatant was

19、 filtered using a 0.45m Nalgene filter (Nalge Nunc International Corp, Rochester, NY, USA) to collect the xoconostle direct extract which was stored at 4C until used within 1216 h450 g新鲜仙人掌果洗净(x jn)擦干切碎后，搅拌机搅拌4min;将果泥于50mL离心杯7800G中离心10min;取上清液过0.45m 的滤膜；提取液储存于4C条件，在1216 h内使用；第15页/共43页第十五页，共43页。c.细菌(

20、xjn)计数 Bacterial Enumeration Bacterial populations were determined by plating onto BHI agar. In this procedure, samples were individually diluted into serial of 9mL 0.1% peptone water solution; (pH 7.25 0.08); then 100 L of appropriate dilutions were surface plated onto triplicates BHI agar plates a

21、nd incubated at 37C for 24 h Plates with colonies ranging between 30300 were considered for colony counting to determine the bacterial populations细菌(xjn)数量测定采用BHI琼脂平板法；将样品用9mL 0.1%蛋白胨水溶液(pH 7.25 0.08)逐级稀释；取100 L 进行BHI琼脂平板培养， 37C /24 h；使菌落数范围在30300间，即可采用菌落计数法确定细菌(xjn)数量；第16页/共43页第十六页，共43页。d.细菌(xjn)生长

22、分析 Growth Over Time Assay Overnight activated bacterial strains were individually diluted into serial of 9mL 0.1% peptone water solution to obtain a bacterial population of approximately 4 log CFU mL1 For each individual strain, batches of sterilized tubes containing 9mL BHI broth were mixed with xo

23、conostle extract to obtain different concentrations (4, 6, 8, and 10% v/v) and inoculated with 1mL of the previously diluted individual bacterial strains将隔夜培养的实验菌菌株用9mL 0.1%蛋白胨水溶液逐级稀释至光密度为4 log CFU mL1；将稀释液与不同(b tn)浓度的仙人掌果提取液混合，并加入相应1mL已计数菌种；第17页/共43页第十七页，共43页。d.细菌(xjn)生长分析 Growth Over Time Assay Co

24、ntrol samples without xoconostle extract for each individual bacterial strain and blank samples without bacterial inoculation for each treatment level were included Samples were incubated with shaking at 37C for 8 h, and bacterial growth was monitored by measuring the optical density (O.D. 610 nm) a

25、t 2 h interval using Thermo Scientific Genesys 10S UV-Vis spectrophotometer The final bacterial populations were determined at the end of the incubation period样品对照、空白对照；混合培养(piyng)液摇床培养(piyng)37C 8 h；细菌生长量采用测定610 nm处培养(piyng)液光密度表示；每隔2h测定一次，在培养(piyng)结束后最后一次测定；第18页/共43页第十八页，共43页。e.琼脂扩散(kusn)分析 Agar

26、Well Diffusion Assay Individual strain was grown overnight then serially diluted into 9mL 0.1% peptone water solution to obtain bacteria populations of 6 log CFU mL1approximately Diluted strains at 10mL each were mixed in a sterilized container BHI agar at 500mL with 0.2% of Tween 80 was prepared an

27、d sterilized at 121C for 15min Prepared BHI agar was placed in a water bath at 48C and allowed to cool down then inoculated with 20mL of previously mixed culture to achieve a bacterial population of 4-5 log CFU mL1将隔夜培养的菌株用9mL 0.1%蛋白胨水溶液逐级稀释至光密度约为6 log CFUmL1； 500mL BHI琼脂+2%Tween 80 ， 121C下溶解(rngji)

28、灭菌15min；将20mL上述BHI琼脂液水浴冷却至48C +10mL稀释菌液混合，测定光密度4-5 log CFU mL1；第19页/共43页第十九页，共43页。e.琼脂(qingzh)扩散分析 Agar Well Diffusion Assay Inoculated BHI agar was poured into Petri dishes (15 100mm2) at approximately 50mL each and allowed to solidify in 30min under biohazard cabinet A sterile cork borer (8.0mm di

29、ameter) was used to punch wells in the inoculated agar The agar plugs were removed using a sterilized wire loop Xoconostle extract at different volumes (2001000 L with 25L unit increase) were adjusted with sterilized distilled water to 1mL to obtain different concentrations (v/v) and poured into the

30、 wells to the top Plates were kept under a biohazard cabinet for 30min for prediffusion to occur, incubated at 37C for 12 h then examined for the development of clear inhibitory zone将50mL接种后的BHI琼脂培养基倒入15 100mm2培养皿中，在无菌柜中凝固30min;无菌打孔器在琼脂平板打孔，将不同(b tn)浓度的仙人掌果提取液注入孔中；将培养名置于无菌柜中预扩散30min，37 C恒温 培养12 h；测定

31、抑菌圈的生长情况；第20页/共43页第二十页，共43页。e.琼脂(qingzh)扩散分析 Agar Well Diffusion Assay Observed inhibitory zones were measured to the nearest 0.1mm and reported after subtracting the well diameter from the observed zone diameter Minimum inhibitory volume (MIV) was determined at this point Incubation of the plates w

32、as continued for three days to determine the minimum lethal volume (MLV) MIV was defined as the lowest volume concentration that caused significant inhibitory effect during 12 h of incubation at 37C and MLV was defined as the lowest volume concentration that showed significant inhibitory effect afte

33、r three days of incubation Inhibitory zone at 3mm or larger was considered significant抑菌圈测定直径长度精确度为0.1mm，总直径打孔直径；持续(chx)培养3d，以确定最小抑菌浓度(MLV) ；最小抑菌浓度为37C下培养12 h时对细菌生长具有显著抑制影响时的最低抑制剂浓度；当抑菌圈的直径大于3mm时可认为有显著抑制活性；第21页/共43页第二十一页，共43页。f.统计学分析(fnx) Statistical Analysis Each experimental test was conducted thr

34、ee times to determine the effect of xoconostle pears on the growth of E. coli O157:H7. Mean values and standard deviations were calculated from the triplicate samples Statistical analysis system (SAS) version 9.2 was used to determine significant antimicrobial activity at different concentrations of

35、 xoconostle extract and significant differences among strains at the same concentration of xoconostle extract using the data means by a factorial analysis of variance of triplicate samples at a significant level of P 0.05每组实验3个平行(pngxng)；平行(pngxng)实验结果取均值、标准差；采用SAS version 9.2软件进行显著性分析、因素方差分析；显著性水平为

36、P 0.05；第22页/共43页第二十二页，共43页。3Results结果结果(ji (ji gu)gu)分析分析实验证实：仙人掌果对O157:H7血清型大肠(dchng)埃希菌具有显著抑菌活性。第23页/共43页第二十三页，共43页。Figure 1 细菌生长曲线(qxin)分析Growth Over Time Assay生菜(shngci)意大利香肠(xingchng)第24页/共43页第二十四页，共43页。Figure 1 细菌生长(shngzhng)曲线分析Growth Over Time Assay苜蓿(m xu)芽牛肉(ni ru)第25页/共43页第二十五页，共43页。 Figu

37、re 1 shows the growth of E. coli O157:H7 in BHI broth treated with different volumes of xoconostle extract during 8 h of incubation at 37C 图1展示的是不同浓度仙人掌果提取物对BHI液体培养基中不同类型大肠埃希菌O157:H7生长影响的状况； In control samples, optical density readings reached absorbance in the range of 0.6540.812 (O.D. 610 nm) 测定的培

38、养液光密度在0.6540.812范围内； When E. coli O157:H7 strains were grown in BHI broth treated with 4, 6, 8, and 10% (v/v) xoconostle extract, optical density readings reached ranges of 0.5120.668, 0.3390.440, 0.2200.259, and 0.0360.103 (O.D. 610 nm) respectively 加入4, 6, 8, and 10% (v/v) 仙人掌果提取物的培养液，最终(zu zhn)光密

39、度分别为0.5120.668, 0.3390.440, 0.2200.259, 0.0360.103 ，对照组为0.6540.812 ；第26页/共43页第二十六页，共43页。 An optical density reading of 0.1 (O. D. 610 nm) or less was previously defined as the division between visual growth and no growth 当光密度小于0.1 时，可认为细菌处于可见生长和未生长中间的状态(zhungti)； Xoconostle extract at 4, 6, and 8% (

40、v/v) concentrations was able to slow down the growth of E. coli O157:H7 strains whereas 10% was enough to cause no growth 当仙人掌果提取液浓度在4, 6, and 8% (v/v) 范围时，具有减缓O157:H7 血清型大肠埃希菌生长的作用，当浓度达到10% 时，足以抑制其生长；第27页/共43页第二十七页，共43页。Table 1 Final population of E. coli O157:H7 strains in BHI broth in the presenc

41、e of xoconostle extract at different concentrations % (v/v)after 8 h of incubations at 37C. Data represent the average of three replicates with standard error.仙人掌果提取物浓度%(v/v)Final population (Log CFU/mL) of E. coli O157:H7 strainsH1730944F4546438950 (control)9.19aA0.168.66aA 0.129.12aA 0.209.31aA 0.

42、1348.09bA 0.097.92bA 0.148.08bA 0.178.25bA 0.1566.79cA 0.19 6.75cA 0.146.78cA 0.17 7.04cA 0.1585.32dA 0.155.16dA 0.18 5.32dA 0.11 5.90dA 0.14103.08eA 0.162.98eA 0.163.07eA 0.124.02eB 0.09 Averages with different lower case letters in the same column are significantly different (P 0.05). Averages wit

43、h different upper case letters in the same row are significantly different (P 0.05).第28页/共43页第二十八页，共43页。 Table 1 shows the final population of E. coli O157:H7 strains grown in BHI broth treated with different concentrations (v/v) of xoconostle extract after 8 h of incubation at 37C 表1显示，不同(b tn)浓度仙人

44、掌提取液处理，8h培养后BHI培养基中O157:H7血清型大肠埃希菌的最终数量； In control samples, E. coli O157:H7 continued to grow and reached the stationary phase 在对照组的O157:H7血清型大肠埃希菌生长达到平稳期； The additions of xoconostle extract at 4, 6, 8, and 10% (v/v) caused significant (P 0.05) reductions in E. coli O157:H7 populations at averages

45、 of 0.990.17, 2.230.35, 3.660.22, and 5.780.41 log CFU mL1, respectively 加入仙人掌提取液显著降低了O157:H7血清型大肠埃希菌的数量，0.990.17, 2.230.35, 3.660.22, and 5.780.41 log CFU mL1；第29页/共43页第二十九页，共43页。 Samples treated with 10% (v/v) xoconostle extract caused final bacterial populations to remain within the initial count

46、 range (about 3 log CFU mL1) 用10%仙人掌提取液处理的样品最终细菌数量在起始(q sh)统计菌数范围内(约为3 log CFU mL1)； These results indicate that xoconostle pears have a significant inhibitory effect on E. coli O157:H7, and 10% (v/v) concentration of xoconostle extract is required to achieve complete growth inhibition 结果显示，仙人掌提取液对O

47、157:H7血清型大肠埃希菌具有显著的抑制活性，当提取物浓度为10%时，能够完全抑制其生长；第30页/共43页第三十页，共43页。Table 2 Inhibitory zones in BHI agar inoculated with mixture of E.coliO157:H7 strains that formed around the wells due to the present of xoconostle extract at different concentrations (v/v) after 12 h of incubation at 37C.Concentration

48、s are in L adjusted to 1mL by distilled water and inhibitory zone = diameter of the zone 8mm(diameter of the well). Data represent the average of three replicates with standard errorConcentration (L/mL) 2003004005006007008009001000Inhibitory zone (mm)01.4 0.32.9 0.24.3 0.455.7 0.36.6 0.367.7 0.359.0

49、 0.459.8 1.01Table 2 shows the inhibitory zones (with 100 L unit increase) that were formed around the wells after 12 h of incubation at 37C表2不同浓度(nngd)仙人掌果提取液处理后37C培养12 h 的抑菌圈的生长情况；第31页/共43页第三十一页，共43页。 Bacterial growth developed a greenish cloud all over the agar whereas distinguishable clear zone

50、remained around the well 在打孔周围可见清晰的微绿色抑菌圈； The lowest concentration that shows a clear inhibitory zone was 275 LmL1 (v/v) with an average of 1.0 0.2 mm 抑菌圈可见的最低抑制剂浓度为275 LmL1 (v/v) ，抑菌圈1.0 0.2 mm； MIV was recorded for a significant inhibitory effect at 400 LmL1 (v/v) with an average of 2.90.2 mm 最小抑

51、菌浓度为有显著(xinzh)抑菌效果时抑菌剂浓度，400 LmL1 (v/v) ，抑菌圈2.90.2 mm；第32页/共43页第三十二页，共43页。 When xoconostle extract without dilution was transferred to the well, the average inhibitory zone reached 9.8 1.01 mm 未经稀释的仙人掌果提取液抑菌圈为9.8 1.01 mm； After three days of incubation, MLV was recorded for a significant inhibitory

52、effect at 650 LmL1 (v/v) with an average of 2.8 0.25 mm 经过3d的持续培养， MLV达到显著抑菌效果(xiogu)时抑菌剂浓度为650 LmL1 (v/v) ，抑菌圈2.8 0.25 mm; Xoconostle pears had significant inhibitory effect on E. coli O157:H7 仙人掌果对O157:H7血清型大肠埃希菌具有显著抑菌活性；第33页/共43页第三十三页，共43页。4Discussion讨论讨论(toln)(toln)第34页/共43页第三十四页，共43页。Discussion

53、Consumer demand for food products that are minimally p r o c e s s e d a n d c o n t a i n n a t u r a l i n g r e d i e n t s h a s increasedThis deman d has r e su lte d in an e f f or t b y the f ood industry to search for natural antimicrobialsThe antimicrobial activity of xoconostle pears was s

54、tudied using growth over time and well diffusion assaysD i r e c t e x t r a c t o f x o c o n o s t l e p e a r s w a s o b t a i n e d m e c h a n i c a l l y w i t h o u t a n y c h e m i c a l , h e a t i n g , o r concentration processingThe antimicrobial activity of xoconostle pears can be acc

55、ounted for several active compounds includin g Xoconostle pears have a combination of phenolic c o m p o u n d s i n c l u d i n g g a l l i c , v a n i l l i c , 4 -hydroxybenzoic acids, catechin, epicatechin, and vanillin消费者对低加工程度的或含有天然成分的产品需求逐渐增长；食品产业寻求新的天然抗菌剂；仙人掌果的抗菌活性与其含有的酚类物质、Vc、色素类物质密切相关；已有研究

56、(ynji)报道仙人掌果含有多种多酚组分，包括五倍子酸、香草酸、4-羟基苯甲酸、儿茶酸、表儿茶素、香兰素等。第35页/共43页第三十五页，共43页。Discussioncompounds are commonly known for their antimicrobial effects P h e n o l i c c o m p o u n d s interfere with membrane function and interact with membrane proteins, causing deformation in structure and functionality

57、A combination of phenolic c o m p o u n d s c a n p r o v i d e synergistic antimicrobial effects and can contribute to better antimicrobial reaction as compare to the reaction of an individual compound Xoconostle pears a have high amount of is well characterized as a reducing a g e n t w i t h f r

58、e e c h e m i c a l r a d i c a l s i n c h e m i c a l a n d biological systems As well, Has the ability to absorb oxygen which might p ro v i d e a ba r r i e r a g a i n s t available oxygen required for E. coli O157:H7酚类物质通常被认为是具有抗菌能力的主要活性物质；其多用于细菌的细胞膜，影响膜蛋白的结构和功能；不同多酚组分间具有协同作用；仙人掌果中含有高含量(hnling)的抗坏血酸，能有效清除化学或生物体系中的自由基，同