1、第1頁/共43頁第一頁，共43頁。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:腦心浸出液肉湯).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頁/共43頁第二頁，共43頁。目錄目錄(ml)(ml)1.In

4、troduction2. Materials and Methods3.Results4. Discussion第3頁/共43頁第三頁，共43頁。1Introduction引言引言(ynyn)(ynyn)仙人掌果仙人掌果(Opuntia matudae )為仙人掌屬（為仙人掌屬（Opuntia）植物的果實，果肉含）植物的果實，果肉含有豐富的微量元素、蛋白質、氨基酸、維生素、多糖類、黃酮類和果膠等。有豐富的微量元素、蛋白質、氨基酸、維生素、多糖類、黃酮類和果膠等。中藥大辭典記載，仙人掌果具有中藥大辭典記載，仙人掌果具有(jyu)行氣活血、祛濕退熱、生肌等作行氣活血、祛濕退熱、生肌等作用；印第安人

5、和墨西哥人將其作為食物和藥物使用也有幾千年的歷史。用；印第安人和墨西哥人將其作為食物和藥物使用也有幾千年的歷史。第4頁/共43頁第四頁，共43頁。第5頁/共43頁第五頁，共43頁。大腸大腸(dchng)(dchng)埃埃希菌希菌 Escherichia Escherichia coli O157:H7 coli O157:H7 毒素(d s)型致病菌腸聚集(jj)型腸產毒素型腸侵襲型出血性腸炎病原群溶血性尿毒綜合癥血性腹瀉腸致病型腸出血型第6頁/共43頁第六頁，共43頁。 Escherichia coli O157:H7 大腸埃希菌的抗原結構較復雜，包括菌體O抗原、鞭毛H抗原及被膜K抗原。0抗

6、原超過(chogu)170種，是血清學分型的基礎，H抗原超過(chogu)56種，K抗原在100種以上。根據耐熱性不同，K抗原又分L、A、B3類，致病性大腸埃希菌的K抗原主要為B類。一個菌株中，一般只含一個類別的K抗原。表示大腸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腸埃希菌的血清型有0157：H7、0111：B4、055：B5、026：B6和0124：B17等。第7頁/共43頁第七頁，共43頁。 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頁/共43頁第八頁，共43頁。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頁/共43頁第九頁，共43頁。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頁/共43頁第十頁，共43頁。2Mate

12、rials and Methods實驗實驗(shyn)(shyn)材材料和方法料和方法a.a.實驗細菌準備實驗細菌準備 b. b.仙人掌果提取物制備仙人掌果提取物制備 c. c.細菌計數細菌計數 d. d.細菌增長曲線細菌增長曲線(qxin) e.(qxin) e.瓊瓊脂擴散分析脂擴散分析 f. f.統計學分析統計學分析第11頁/共43頁第十一頁，共43頁。a.實驗細菌(xjn)制備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種大腸桿菌分別從提取自苜蓿芽(F4546)、生菜(shngci)(43895)、牛肉(43895)和意大利香腸(944);采用免疫印跡法和PCR技術測定實驗菌的血清型；第12頁/共43頁第十二頁，共43頁。是一種將高分辨率凝膠電泳和免疫化學分析技術相結合的雜交技術。免疫印跡法分三個階段SDS-聚丙烯酰胺凝膠電泳；電轉移將在凝膠 中已經(y jing)分離的條帶轉移至硝酸纖維素膜上，選用低電壓（100V）和大電流（12A），

15、通電45min ；酶免疫定位將印有蛋白質條帶的硝酸纖維素膜（相當于包被了抗原的固相載體）依次與特異性抗體和酶標第二抗體作用后，加入能形成不溶性顯色物的酶反應底物，使區帶染色。免疫(miny)印跡法廣泛應用于分析抗原組分(zfn)及其免疫活性，并可用于疾病的診斷.在艾 滋病病毒感染中此法作為確診試驗第13頁/共43頁第十三頁，共43頁。a.實驗(shyn)細菌制備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實驗(shyn)菌+ 10mL BHI， 37C 培養24 h， 4C保存；將每種菌株在BHI瓊脂培養基上培養24 h， 37C；取一個菌落轉移至10mL BHI培養液中， 37C培養，隔天使用；第14頁/共43頁第十四頁，共43頁。b.仙人掌果提取物制備(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新鮮仙人掌果洗凈(x jn)擦干切碎后，攪拌機攪拌4min;將果泥于50mL離心杯7800G中離心10min;取上清液過0.45m 的濾膜；提取液儲存于4C條件，在1216 h內使用；第15頁/共43頁第十五頁，共43頁。c.細菌(

20、xjn)計數 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細菌(xjn)數量測定采用BHI瓊脂平板法；將樣品用9mL 0.1%蛋白胨水溶液(pH 7.25 0.08)逐級稀釋；取100 L 進行BHI瓊脂平板培養， 37C /24 h；使菌落數范圍在30300間，即可采用菌落計數法確定細菌(xjn)數量；第16頁/共43頁第十六頁，共43頁。d.細菌(xjn)生長

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將隔夜培養的實驗菌菌株用9mL 0.1%蛋白胨水溶液逐級稀釋至光密度為4 log CFU mL1；將稀釋液與不同(b tn)濃度的仙人掌果提取液混合，并加入相應1mL已計數菌種；第17頁/共43頁第十七頁，共43頁。d.細菌(xjn)生長分析 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樣品對照、空白對照；混合培養(piyng)液搖床培養(piyng)37C 8 h；細菌生長量采用測定610 nm處培養(piyng)液光密度表示；每隔2h測定一次，在培養(piyng)結束后最后一次測定；第18頁/共43頁第十八頁，共43頁。e.瓊脂擴散(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將隔夜培養的菌株用9mL 0.1%蛋白胨水溶液逐級稀釋至光密度約為6 log CFUmL1； 500mL BHI瓊脂+2%Tween 80 ， 121C下溶解(rngji)

28、滅菌15min；將20mL上述BHI瓊脂液水浴冷卻至48C +10mL稀釋菌液混合，測定光密度4-5 log CFU mL1；第19頁/共43頁第十九頁，共43頁。e.瓊脂(qingzh)擴散分析 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將50mL接種后的BHI瓊脂培養基倒入15 100mm2培養皿中，在無菌柜中凝固30min;無菌打孔器在瓊脂平板打孔，將不同(b tn)濃度的仙人掌果提取液注入孔中；將培養名置于無菌柜中預擴散30min，37 C恒溫 培養12 h；測定

31、抑菌圈的生長情況；第20頁/共43頁第二十頁，共43頁。e.瓊脂(qingzh)擴散分析 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抑菌圈測定直徑長度精確度為0.1mm，總直徑打孔直徑；持續(chx)培養3d，以確定最小抑菌濃度(MLV) ；最小抑菌濃度為37C下培養12 h時對細菌生長具有顯著抑制影響時的最低抑制劑濃度；當抑菌圈的直徑大于3mm時可認為有顯著抑制活性；第21頁/共43頁第二十一頁，共43頁。f.統計學分析(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每組實驗3個平行(pngxng)；平行(pngxng)實驗結果取均值、標準差；采用SAS version 9.2軟件進行顯著性分析、因素方差分析；顯著性水平為

36、P 0.05；第22頁/共43頁第二十二頁，共43頁。3Results結果結果(ji (ji gu)gu)分析分析實驗證實：仙人掌果對O157:H7血清型大腸(dchng)埃希菌具有顯著抑菌活性。第23頁/共43頁第二十三頁，共43頁。Figure 1 細菌生長曲線(qxin)分析Growth Over Time Assay生菜(shngci)意大利香腸(xingchng)第24頁/共43頁第二十四頁，共43頁。Figure 1 細菌生長(shngzhng)曲線分析Growth Over Time Assay苜蓿(m xu)芽牛肉(ni ru)第25頁/共43頁第二十五頁，共43頁。 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 圖1展示的是不同濃度仙人掌果提取物對BHI液體培養基中不同類型大腸埃希菌O157:H7生長影響的狀況； In control samples, optical density readings reached absorbance in the range of 0.6540.812 (O.D. 610 nm) 測定的培

38、養液光密度在0.6540.812范圍內； 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養液，最終(zu zhn)光密

39、度分別為0.5120.668, 0.3390.440, 0.2200.259, 0.0360.103 ，對照組為0.6540.812 ；第26頁/共43頁第二十六頁，共43頁。 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 當光密度小于0.1 時，可認為細菌處于可見生長和未生長中間的狀態(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 當仙人掌果提取液濃度在4, 6, and 8% (v/v) 范圍時，具有減緩O157:H7 血清型大腸埃希菌生長的作用，當濃度達到10% 時，足以抑制其生長；第27頁/共43頁第二十七頁，共43頁。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.仙人掌果提取物濃度%(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頁/共43頁第二十八頁，共43頁。 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顯示，不同(b tn)濃度仙人

44、掌提取液處理，8h培養后BHI培養基中O157:H7血清型大腸埃希菌的最終數量； In control samples, E. coli O157:H7 continued to grow and reached the stationary phase 在對照組的O157:H7血清型大腸埃希菌生長達到平穩期； 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 加入仙人掌提取液顯著降低了O157:H7血清型大腸埃希菌的數量，0.990.17, 2.230.35, 3.660.22, and 5.780.41 log CFU mL1；第29頁/共43頁第二十九頁，共43頁。 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%仙人掌提取液處理的樣品最終細菌數量在起始(q sh)統計菌數范圍內(約為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 結果顯示，仙人掌提取液對O

47、157:H7血清型大腸埃希菌具有顯著的抑制活性，當提取物濃度為10%時，能夠完全抑制其生長；第30頁/共43頁第三十頁，共43頁。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不同濃度(nngd)仙人掌果提取液處理后37C培養12 h 的抑菌圈的生長情況；第31頁/共43頁第三十一頁，共43頁。 Bacterial growth developed a greenish cloud all over the agar whereas distinguishable clear zone

50、remained around the well 在打孔周圍可見清晰的微綠色抑菌圈； The lowest concentration that shows a clear inhibitory zone was 275 LmL1 (v/v) with an average of 1.0 0.2 mm 抑菌圈可見的最低抑制劑濃度為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、菌濃度為有顯著(xinzh)抑菌效果時抑菌劑濃度，400 LmL1 (v/v) ，抑菌圈2.90.2 mm；第32頁/共43頁第三十二頁，共43頁。 When xoconostle extract without dilution was transferred to the well, the average inhibitory zone reached 9.8 1.01 mm 未經稀釋的仙人掌果提取液抑菌圈為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 經過3d的持續培養， MLV達到顯著抑菌效果(xiogu)時抑菌劑濃度為650 LmL1 (v/v) ，抑菌圈2.8 0.25 mm; Xoconostle pears had significant inhibitory effect on E. coli O157:H7 仙人掌果對O157:H7血清型大腸埃希菌具有顯著抑菌活性；第33頁/共43頁第三十三頁，共43頁。4Discussion討論討論(toln)(toln)第34頁/共43頁第三十四頁，共43頁。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消費者對低加工程度的或含有天然成分的產品需求逐漸增長；食品產業尋求新的天然抗菌劑；仙人掌果的抗菌活性與其含有的酚類物質、Vc、色素類物質密切相關；已有研究

56、(ynji)報道仙人掌果含有多種多酚組分，包括五倍子酸、香草酸、4-羥基苯甲酸、兒茶酸、表兒茶素、香蘭素等。第35頁/共43頁第三十五頁，共43頁。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酚類物質通常被認為是具有抗菌能力的主要活性物質；其多用于細菌的細胞膜，影響膜蛋白的結構和功能；不同多酚組分間具有協同作用；仙人掌果中含有高含量(hnling)的抗壞血酸，能有效清除化學或生物體系中的自由基，同