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Beautiful glass bottle in purple with a swing stopper. Straight-sided. Ideal for home-made cordials, flavoured water, infused oils, vinegards, sloe gin, wine, damson vodka and all sorts!Capacity: 500ml. Measurements: 28cm high x 6.5cm diameter. Hand wash only.Also available in yellow, red, blue and green.
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All orders are processed within 1 working day. Orders are sent with Royal Mail 48, or heavier items are sent with DHL. First-class orders are sent with Royal Mail 24, or with overnight DHL. We can track and trace most orders - please call us on 01903 233299 with any delivery issues.

subset: commoncrawl


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Van Loi hotel is ideally located on sand-bank of Thu Bon River, allowing you to get open to the river, with romantic views and local scenery, normal life of farmers also. The hotel, on the area of 2500 sqm has 82 rooms built in 5 floors with panorama view. We also have a meeting room for 70 people.
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Model Mayhem Admin
Corrie Dawn SwarisModel Female Guelph, Ontario, Canada
My Website: http://www.modelmayhem.com/corrieswarisMy MM URL: http://www.modelmayhem.com/corrierobley
Mayhem # 576238
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I have been a model mayhem member for a few years now. I have enjoyed being able to test the modeling waters. I have had the joy with working with many talented photographers, and thrilled with the results. I do feel that my portfolio needs a little more edge some more character. I am extremely versitile from serious lifestyle to dramamic endless imagination. I feel that every photograph should have a story, a reason for existance.
I am a certified interior decorator, i have been working with colour for over ten years now. I strongly feel that my knowledge in colour contrasts could benefit the emotional attraction in photograghs. Art has always been a passion of mine. Since i have an artistic eye i am able to blend my vision with the photographers which develops a unique and classy outcome. I don't have much professional modeling experience but i do take pride in my work and do want the best turn out possible. Art is the best way to express yourself, it can never be wrong.
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Since i do have a interest in the bold and beautiful, i'm waiting to develop this in more photos to come. Thank you to all the photographers i have worked, i'm looking forward to my modeling future.
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My Favorite Flirts is a feature that lets you reach your favorite Flirts easily from My Account or on 1-800-TO-FLIRT.Manage My Favorites
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How to make a bow
1,795pages onAdd New Page
this wiki
this wiki
get a stick, get some time, get some string, some skewers for arrows, tape.
bend the stick a little bit and tie the string on the ends. It should look like this.
l \
l \
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make 3 fins with tape


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The MSIG-led excess D&O consortium at Lloyd's is set to provide an increased line size in 2015 of $75 million, representing a capacity increase of $13 million.
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MSIG, Lloyd's, D&O, Europe, ArgoGlobal, Chaucer, Channel Syndicate, AG Doré, Andrew Pecover


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Sending postcards and packages to the folks back home needn't be difficult for vacationers who understand a bit about the postal services in the Caribbean.
Post offices hours vary depending on the island, but generally speaking, most open between 7:30 a.m. and 9:00 a.m. and close between 4:00 p.m. and 5:00 p.m. A post office is the best place to mail a parcel, but if you'd like to send a postcard or letter, traveling to the post office is not necessary. Most hotels and resorts sell stamps and postcards and will also send these items out for you. Post rates vary from island to island.
When sending mail from any island in the Caribbean to another country, expect the following delivery times:
Arrival Time
|Airmail: 3-4 weeks
|Airmail: 1-2 weeks
|New Zealand
|Airmail: 3-4 weeks
|United Kingdom
|Airmail: 2-3 weeks
|United States
|Airmail:1-2 weeks
These times are subject to change; check with a local island post office for the most current rates and delivery times.
If you require overnight mail services, some, but not all, islands have Airborne, FedEx or UPS branches. Note that the term "overnight service" does not always ensure next-day delivery; the planes that carry mail from the Caribbean are in short supply. Plan on two- or three-day delivery when utilizing an overnight service in the Caribbean.
Help us improve! We welcome your corrections and suggestions.


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He's fading.
This is a short poem about the downfall of a boy.
Once there was a boy, but isn't there always?
Lingering in the mind like a simple cloud.
Curiosity, overtaking the brain.
Always consumed in these thoughts.
What happens when their presence lingers away?
The thoughts fade to gray.
All the questions unanswered.
Millions of possibilities no more.
The boy is gone.
But the girl is stranded in her mind.
Fading to gray as well.


text:
Originally written on NESN.com | Last updated 10/30/14
Sorry ladies, but you won't be getting a Kim Kardashian or Brooke Burke-shaped rump by simply lacing up a pair of weird-looking Sketchers. But you will be getting your money back. According to The Associated Press, the shoe company owes $40 million "to settle charges by the Federal Trade Commission that the footwear company made unfounded claims that its Shape-ups shoes would help people lose weight and strengthen their butt, leg and stomach muscles." The shoes were designed with a "rocker" sole that was supposed to promote weight loss and enhance muscle strength. The toning shoes cost about $100. Check...
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Days That Wane
... the Lord's hand was guiding his life and his presence was all that mattered. Even if it seemed his life continued downward, even if he face trouble at every turn, even if he did not understand why things were happening to him, David was aware that his relationship with the Lord was the only reality he desired. His life was complete if he had the Lord. That was the one thing he was seeking to have in life above all else.
Psalm 143:8
Let me hear of your ...
Tarsus (Acts 9:11, 30; 11:25; 21:39; 22:3)
... the ancient city from Paul's time in the 1st century. In addition, just up the road a little ways there is the remains of a large bath house with archways still standing from the Roman period. Also, a large stone inscription was found dating from the time of the Roman Emperor Severus Alexander (222-235 A.D.) and is now standing near Cleopatra's Gate. The inscription is for the blessing/protection of the Imperial Temple and a benediction for the greatness of ...
Turkey by Tenere: Day 5
... The lushness of the lowlands disappears. The trees look smaller and older somehow. There is more exposed rock, the clouds are closer. The air felt damp and chilly. The towns were spaced further apart and as the peak of Mt Hasan Kucuk loomed closer, clouds began to move in and obscure it. In my perusal of both Google Maps and OpenStreetMap I found that there was a track up over the aforementioned saddle, I just had to go to Kitrelli ...
Not quite Turkish Delight
... cup of cold beer. Except it's not a winnings tray. It's the floor.
I've not moved so fast for a while.
Final night in Turkey. It's surprised me how friendly everyone is, I've not been hassled, I feel very safe, I've not been ripped off - on the contrary, I've found people to be exceptionally generous. But, like Boxing Day, I've had my fill of Turkey. It's all a bit "samey" and apart from Istanbul it's not quite Turkish Delight.
... and company of a pretty girl on my nook, I'd turned to this - what was I doing? After Another days ride the cities began to break up in patches of greenery, traffic improved but roads worsened as I neared ever looming mountains.
My nerves were settled with my first taste of Turkish welcome. Passing a petrol station a man called out with the mime of a stirring cup - I pulled over for cay. That night i ate with the petrol pump-men and slept on the ...


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The director of BBC North, Peter Salmon, has been given a new role as the corporation's director, England, encompassing its centres in Bristol and Birmingham which will become home to the BBC's training and HR departments.
Salmon, who has overseen the growth of BBC North in Salford, will lead "creativity, collaboration and local partnerships" across the three cities.
The BBC's Birmingham centre has lost a large part of its production base in recent years, including BBC1's Countryfile, but will now be home to its BBC Academy training centre and its entire HR department, currently based in west London.
The BBC said a total of 190 jobs would move to Birmingham by the end of next year. The BBC's Arts Council tie-up, The Space, will also move out of London to Birmigham.
Salmon will work alongside Alice Webb, the BBC's chief operating officer for England and David Holdsworth, the BBC's controller of English regions, who will continue to lead the BBC's local and regional services across the country.
Despite production cutbacks, Birmingham remains home to Radio 4's The Archers, its local news facilities and the Drama Village production centre which makes BBC1's Father Brown.
The BBC's director general, Tony Hall, told staff in an email that when he arrived the Birmingham centre was "half empty – there was simply no sense of energy, the warmth and diversity of Birmingham".
Salmon, who will not receive a pay rise beyond the £387,900 he already earns, said: "Over the last few years the BBC has helped build a strong broadcasting and production community in Salford and the north of England.
"Now I'm looking forward to working with BBC teams in Birmingham and Bristol to forge even better relationships with our audiences and build new partnerships across the country to make sure our key bases keep making great content, develop great people and have a thriving future."
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DeadlineNow: Will 'Dawn Of The Planet Of The Apes' Heat Up Tepid Summer Box Office? (Video)
11 Comments
To answer you question, "Hell Yes" (this movie is poised to blow up the box office!)
Thank to movie price,it is cheaper to wait until the DVD become available and either rent the movie or buy it.
Movie tickets are still pretty cheap entertainment, whether you go to an early show for $10 or less, or see a 3-D IMAX for $15 or so. Compare that to seeing a sporting event (especially pro), a concert or a Broadway show.
I believe you mean it's cheaper to wait until the movie is available streaming. It is 2014, after all.
Apes and Guardians will salvage the summer. Insterstellar, i'm not so sure about.
Interstellar isn't coming out in the summer lol
Dawn of the planet of the apes will dominate the summer. I can see it spending 3 weeks at #1 at the box office.
too many guns in this one
I believe the potential is there, but the marketing I have seen seems too heavily weighted to the low hanging fruit of ticket buyers: young, male, action. That is not, however, the picture's strong suit, which could cause an otherwise excellent product to face some backlash. The marketing needs to expand the potential audience by instead emphasizing the emotional element while still indicating the action component. That is the sweet spot. Think "Gravity" : a mix of peril/action/fx and gravitas. The ads I have seen seem to have given up on expanding to older and female audiences, and that's a shame. As an "action" picture, "Dawn of the Planet of the Apes" is not particularly notable. But as a studio tentpole, it stands out by having a strong emotional component, first rate execution, and something on its mind other than just blowing things up.
where can I see SWV reunited clips
Great movie. I like how the trailers actually showed alternate takes of certain scenes, so it still feels fresh when you see them again in the theater.


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From acclaimed author Melissa Storm comes the first in a brand-new series of sweet and wholesome small-town love stories with the community church at its center…
Summer Smith is at a crossroads in life. Fresh out of college with no idea what comes next, she agrees to take over her aunt's flower shop for the season. She arrives in the small, close-knit community of Sweet Grove, Texas, hoping to find some answers.
Ben Davis has lived in the shadow of his family's mistakes for years. Forced to give up everything he ever wanted for himself, he begins to consider taking his own life in a final effort to end all the pain. A desperate plea sent to the God he isn't even sure he believes in is soon answered by a series of miracles that bring Summer and Ben crashing into each other.
Love's Prayer offers a dramatic story about two people who must find a way to believe in each other and in themselves in order to finally find the place where they belong. This novel of finding faith, hope, and—ultimately—love in the darkest of times is sure to tug at your heartstrings and leave you craving more from the First Street Church Romances!
About Melissa
Melissa Storm is a mother first, and everything else second. She used to write under a pseudonym, but finally had the confidence to come out as herself to the world. Her fiction is highly personal and often based on true stories. Writing is Melissa's way of showing her daughter just how beautiful life can be, when you pay attention to the everyday wonders that surround us.
Melissa loves books so much, she married fellow author Falcon Storm. Between the two of them, there are always plenty of imaginative, awe-inspiring stories to share. Melissa and Falcon also run the business Novel Publicity together, where she works as publisher, marketer, editor, and all-around business mogul. When she's not reading, writing, or child-rearing, Melissa spends time relaxing at home in the company of her two dogs, five parrots, and rescue cat. She never misses an episode of The Bachelor or her nightly lavender-infused soak in the tub.
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Using a specific tool for almost every aspect of the mechanical CAD drafting process can save you time. Intelligent mechanical drafting tools enable you to re-edit features without having to remove and recreate the original feature. For example, you can easily resize a chamfer or fillet through the original dialog box parameters by double-clicking the chamfer or fillet.
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2016 Official United Methodist Program Calendar App FAQs and Help
We hope this list of frequently asked questions is helpful. Need answers to a question you're not seeing below? Email email@example.com
Q. Will this app work on my device?
A. There are two versions of the app: Android and Apple.
The Apple app is available on the iTunes store and will work on all Apple devices running iOS 6.0 or above, including these legacy devices: the iPhone 3GS, iPhone 4, iPhone 4S, iPad 2, iPad 3, and iPod touch 4th generation.
The Android app is available on the Google Play store and works on all Android devices running Android 2.3.3 or above.
There are no current plans to make the app available for Windows-based phones or tablets.
Q. I see a star on the event page. What does the star mean?
A. When you open an event, tapping the star allows you to add a bookmark. You can view all of your bookmarks from the main menu. You must be logged in for bookmarking to be available. If you're not logged in, you will be prompted to log in or create a free account on UMCom.org.
Q. I downloaded the free trial today but the first entry I see is not until Sunday. Why don't I see all the days of the week?
A. While technically the application is a calendar, the only dates currently showing are those with events in the church or secular year. When you open the calendar the date showing will be the next date with information available, not necessarily today's date. We are considering an update that would have a "true" calendar view with all days and the ability to bookmark and note additional days. If you would like this type of update to the app, please let us know.
Q. Why don't I have the option to see a week or a month at a time?
A. We will be releasing a new version of the app within a few weeks that includes a simple monthly calendar view.
Q. How do I sync this calendar to my MS Outlook or Google Calendar?
A. The current version of the Official Program Calendar app will not sync with MS Outlook/Exchange or Google Calendar. We are currently testing an update that includes integration with both calendar options. If you would like to see other updates to the app, please let us know.
Q. How do I share events from this calendar?
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text: Turkish Journal of Biology Turk J Biol (2014) 38: 412-419 © TÜBİTAK doi:10.3906/biy-1310-69 http://journals.tubitak.gov.tr/biology/ Research Article Inhibitory effect of enterocin KP in combination with sublethal factors on Escherichia coli O157:H7 or Salmonella Typhimurium in BHI broth and UHT milk 1, 1 1 2 2 Zeliha YILDIRIM *, Yaselin İLK , Metin YILDIRIM , Kader TOKATLI , Nilgün ÖNCÜL 1 Department of Food Engineering, Faculty of Engineering, Niğde University, Niğde, Turkey 2 Department of Food Engineering, Gaziosmanpaşa University, Tokat, Turkey Received: 28.10.2013 Accepted: 25.02.2014 Published Online: 14.04.2014 Printed: 12.05.2014 Abstract: The effects of physical and chemical sublethal treatments on the antibacterial activity of enterocin KP produced by Enterococcus faecalis KP against Escherichia coli O157:H7 and Salmonella Typhimurium were investigated. Enterocin KP was not active against intact cells of E. coli O157:H7 or S. Typhimurium. However, the use of enterocin KP together with ethylenediaminetetraacetic acid (50 mM), sodium tripolyphosphate (50 mM), sublethal heating (60 °C for 10 min), cold shock (–20 °C for 2 h), or acid stress (mixture of 40% lactic acid, 16% propionic acid, 16% acetic acid) in BHI medium decreased the cell number of E. coli O157:H7 by 7.27, 6.28, 3.39, 3.06, 4.20 log and S. Typhimurium by 7.21, 6.20, 3.64, 3.38, 3.98 log cfu/mL, respectively. The combination of enterocin KP with ethylenediaminetetraacetic acid decreased the cell number of E. coli O157:H7 in UHT milk to undetectable level, enterocin KP plus sodium tripolyphosphate or enterocin KP plus sublethal heating caused a reduction by 6.07 and 5.68 log cycles. The results of this study showed that enterocin KP could be applied as a biopreservative to inhibit E. coli O157:H7 and S. Typhimurium in combination with physical and food grade chemical hurdles. Key words: Bacteriocin, enterocin KP, sublethal injury, gram-negative bacteria, UHT milk 1. Introduction Over the past decades, food safety has become an important issue in many countries. Inhibition of food spoilage and foodborne pathogenic bacteria by natural, biological, or food-grade compounds is of great interest to the food industry due to public health and economic concerns. In food safety, gram-negative foodborne pathogenic and spoilage bacteria are especially problematic because of their inherent resistance to some natural antimicrobials (Helander and Mattila-Sandholm, 2000; Belfiore et al., 2007). Biopreservation in the food industry has attracted great attention in recent years. Biopreservation can be defined as the extension and improvement of shelf life and safety of foods by natural or controlled microbiota and/or their antimicrobial compounds (Stiles, 1996). Bacteriocin producing lactic acid bacteria (LAB) and bacteriocin extracts fall within this concept. Bacteriocins are proteinaceous antimicrobial substances produced by many bacterial species. Bacteriocins produced by LAB have been widely studied due to their potential use in food preservation as natural biopreservatives. The use of *Correspondence: [email protected] 412 bacteriocins in the food industry can help to reduce the addition of chemical preservatives as well as the intensity of heat treatments, resulting in foods that are more naturally preserved and richer in organoleptic and nutritional properties (Cleveland et al., 2001; Drider et al., 2006). Although LAB bacteriocins have created considerable interest for use as food biopreservatives, they have several limitations. Their inhibitory activity is generally narrowed to gram-positive bacteria. Most LAB bacteriocins are not active against gram-negative bacteria. To be an effective food biopreservative, the bacteriocins preferably should have antibacterial activity against important gram-positive and gram-negative spoilage and pathogenic bacteria. Therefore, food application of bacteriocins relies on the use of multiple hurdles. The concept of hurdle technology is defined as the intelligent use of combinations of 2 or more antimicrobial factors or techniques acting synergistically and more effectively at suboptimal levels than each of them alone at the optimal level. This approach is particularly useful not only because it improves the stability and safety of foods but also because the acceptability of foods is enhanced (Leistner, 2000). The application of LAB YILDIRIM et al. / Turk J Biol bacteriocins as part of hurdle technology has received great attention in recent years. It has been verified that the antibacterial activities of LAB bacteriocins such as nisin and pediocin against gram-positive bacteria are greatly increased and their inhibitory spectrum also extends to gram-negative bacteria when used in combination with physical and chemical treatments damaging the bacterial outer membrane, which acts as a barrier against diffusion of bacteriocin molecules to the cytoplasmic membrane (Stevens et al., 1991; Kalchayanand et al., 1992, 1994; Schved et al., 1994; Cutter and Siragusa, 1995a, 1995b; Boziaris et al., 1998, Ananuo et al., 2005; Osmanağaoglu, 2005). Therefore, bacteriocins in combination with other antimicrobial factors may be useful tools for the implementation of methods intended to significantly reduce the load of food spoilage and foodborne pathogenic bacteria. Enterocin KP produced by Enterococcus faecalis KP exhibits bactericidal activity against some of the gram-positive pathogenic and spoilage bacteria such as Listeria monocytogenes, L. ivanovii, E. faecium, and E. faecalis (Isleroglu et al., 2012). Researchers reported that enterocin KP did not cause a clear inhibition zone against E. coli O157:H7 or Salmonella Typhimurium on BHI agar. However, a decrease in cell density was observed at where enterocin KP was applied as a spot assay in solid medium. The objective of the present study was to determine the efficacy of enterocin KP on Salmonella Typhimurium or Escherichia coli O157:H7 in combination with physical and chemical treatments that cause damage to the outer membrane in buffer and UHT milk. 2. Materials and methods 2.1. Organisms and culture conditions Enterococcus faecalis KP was used as enterocin KP producer strain (Isleroglu et al., 2012). Salmonella Typhimurium RSSK 95091 and E. coli O157:H7 RSKK 232 were supplied by the Refik Saydam Hıfzıssıhha Culture Collection (Turkey). Enterococcus faecalis KP was propagated in De Mann Rogosa and Sharp (MRS) (Fluka, Germany) broth at 30–32 °C and the other bacteria were grown in Brain– Heart Infusion (BHI) (Fluka, Germany) broth at 35–37 °C. Enterococcus faecalis KP was maintained frozen at –80 °C in MRS containing 20% (v/v) glycerol, S. Typhimurium, and E. coli O157:H7 in BHI broth containing 20% (v/v) glycerol. 2.2. Preparation of enterocin KP Enterocin KP was prepared by using the method given by Moreno et al. (2002). After the culture of E. faecalis KP grown in MRS broth at 32 °C for 18 h was centrifuged (8000 × g at 4 °C, 20 min), the pellet was discarded. The pH of cell-free culture supernatant was adjusted to 6.5 by the addition of 10 N NaOH and then it was filter- sterilized (0.45 µm). Filter-sterilized supernatant was subjected to ammonium sulfate precipitation (50% of saturation) and organic solvent precipitation (a methanol/ chloroform mixture, 1:2, v/v). Finally, the pellet obtained by centrifugation was stored at –80 °C until used. The activity of enterocin KP against Lactobacillus plantarum in cell-free sterile supernatant and the pellet was 800 and 102,400 AU/mL, respectively. 2.3. Bacteriocin bioassay Antimicrobial activity of enterocin KP was determined by using the agar spot test. For the bacteriocin assay test, Lactobacillus plantarum DSM 2601 was used as indicator bacterium, which is one of the most sensitive bacteria to enterocin KP. The antimicrobial activity was defined as the reciprocal of the highest serial 2-fold dilution showing a clear zone (at least 2 mm) of growth inhibition of the indicator strain and expressed as one arbitrary unit (AU) per milliliter of the bacteriocin preparation (AU/mL) (Bhunia et al., 1988). 2.4. Antimicrobial activity assays of enterocin KP to E. coli O157:H7 or S. Typhimurium Enterocin KP (1600 AU/mL) was added to exponential phase cultures of E. coli O157:H7 or S. Typhimurium strains (about 107 cfu/mL) incubating in BHI broth at 25 °C. At desired intervals, samples were removed and serially diluted into sterile saline solution (NaCl 0.85%). The appropriate dilutions were plated on duplicate BHI plates, and the average numbers of colonies (cfu/mL) obtained after different periods of incubation at 25 °C were used to establish the growth and survival curves. In addition, the antimicrobial activity of enterocin KP against E. coli O157:H7 or S. Typhimurium was determined by agar spot test. Soft BHI agar (0.8% agar) inoculated with E. coli O157:H7 or S. Typhimurium at the level of about 107 cfu/mL was poured on BHI agar (1.5% agar) pre-poured plates. After solidification, 20 µL of enterocin KP (1600 AU/mL) was spotted on soft BHI agar and incubated at 35–37 °C for 24 h. At the end of the incubation, the plates were checked for inhibitory zones. 2.5. Effect of sublethal heating and cold shock on activity of enterocin KP The overnight grown culture (10 mL) of E. coli O157:H7 or S. Typhimurium was harvested by centrifugation and resuspended in 100 mL of sterile peptone water (0.1%). Cell suspension of E. coli O157:H7 or S. Typhimurium was divided into 4 portions of 9 mL. The cell suspensions supplemented with 1 mL of enterocin KP preparation or supplemented with 1 mL of sterile water were heated at 60 °C for 10 min and then cooled immediately in water at 4 °C (Ananou et al., 2005). The final concentration of enterocin KP in the cell suspension was 1600 AU/mL. To determine the effect of cold shock on inhibitory activity of enterocin KP, cell suspensions of E. coli O157:H7 413 YILDIRIM et al. / Turk J Biol 414 determined by sampling on BHI agar plates in duplicate at 37 °C for 24–48 h. The UHT milk used in this study was obtained a retail market in Tokat (Turkey). 2.9. Statistical analyses Data were analyzed using analysis of variance (ANOVA) performed with the Statistical Package for Social Sciences for Windows (version 11.0; SPSS, Chicago, IL, USA). The application of treatments (enterocin KP, sublethal heating, cold shock, acid stress, EDTA, STPP) was used as the factor. Differences were considered significant at P < 0.05. All experiments were performed 3 times. 3. Results 3.1. Inhibitory activity of enterocin KP against E. coli O157:H7 or S. Typhimurium The growth of E. coli O157:H7 or S. Typhimurium in BHI broth at 25 °C was inhibited by enterocin KP (Figure 1). The decrease in the number of E. coli O157:H7 or S. Typhimurium cells was 0.66 and 0.45 log in the first hour of incubation and over the 24-h incubation period the reduction in viable cell numbers reached 1.27 and 0.98 log (P < 0.05), respectively. Although enterocin KP reduced the viable cell numbers, it did not give a clear inhibition zone against E. coli O157:H7 or S. Typhimurium when 12 Log cfu/mL 10 a 8 6 4 2 0 12 Ec 0 1 3 6 Time (h) 9 Ec-Enterocin 12 24 b 10 Log cfu/mL or S. Typhimurium with/without enterocin KP were frozen at –20 °C for 2 h and then thawed immediately. The viable bacterial counts of the treated cells were determined by sampling on BHI plates in duplicate at 37 °C for 24–48 h (Boziaris and Adam, 2001). For both analyses, the initial concentration of E. coli O157:H7 or S. Typhimurium was about 4 × 108 cfu/mL. 2.6. Effect of chelating agents on activity of enterocin KP An overnight culture of E. coli O157:H7 or S. Typhimurium cells was individually inoculated into 12 tubes containing sterile fresh BHI broth and then incubated at 35–37 °C until their OD values reached 0.1 at 600 nm. After that, each bacterial culture was centrifuged (15 min at 6000 × g) and supernatants were removed. The cell pellets were resuspended in 1 mL of the following solutions: (a) sterile peptone water, (b) disodium ethylenediaminetetraacetic acid (Na2-EDTA) (50 mM), (c) sodium tripolyphosphate (STPP) (50 mM), (d) enterocin KP (1600 AU/mL), (e) EDTA plus enterocin KP, and (f) STPP plus enterocin KP. Cell suspensions were incubated at 35–37 °C for 30 and 60 min, centrifuged (at 6000 × g for 15 min), washed with sterile peptone water, and resuspended again in 1 mL of sterile peptone water. Serial dilutions were made in sterile peptone water and plated in duplicate in BHI agar. After incubation at 35–37 °C for 24–48 h, the survivor cells were enumerated and bacterial counts were given as cfu/ mL (Ananua et al., 2005). For both analyses, the initial concentration of E. coli O157:H7 or S. Typhimurium was about 4 × 108 cfu/mL. 2.7. Effect of acid stress on activity of enterocin KP A cell suspension of E. coli O157:H7 or S. Typhimurium (about 4 × 108 cfu/mL) in sterile peptone water (0.1%) was exposed to a combination of enterocin KP (1600 AU/ mL) and an acid solution containing 40% lactic acid, 16% propionic acid, and 16% acetic acid (pH 5.5). The acid solution was used at the 1% level, which gives a final acid concentration of 0.7%. The treated samples with enterocin KP plus acid solution and the control samples (just treated with acid solution or enterocin KP) were stored at 4–5 °C for 7 days. During the storage, samples were taken and the viable bacterial cells were determined by sampling on BHI agar plates in duplicate at 37 °C for 24–48 h (Kalchayanand et al., 1992). 2.8. Effect of enterocin KP alone or in combination with heat, EDTA, or STTP on E. coli O157:H7 in UHT milk Cell suspensions of E. coli O157:H7 (about 2 × 108 cfu/mL) inoculated into UHT milk were exposed to the following treatments: (i) enterocin KP (1600 AU/mL), (ii) EDTA (50 mM), (iii) EDTA plus enterocin KP, (iv) STPP (50 mM), (v) STPP plus enterocin KP, (vi) sublethal heating (60 °C/10 min), and (vii) sublethal heating plus enterocin KP. During incubation at room temperature for 24 h, samples were periodically taken and viable cell counts were 8 6 4 2 0 Sal 0 1 3 6 9 Time (h) Sal-Enterocin 12 24 Figure 1. Effect of enterocin KP against E. coli O157:H7 (a) and S. Typhimurium (b) in BHI broth at 25 °C. Ec, E. coli O157:H7; Sal, S. Typhimurium. YILDIRIM et al. / Turk J Biol Ec Heat a Heat-En b Log cfu/mL 10 9 8 7 6 5 4 3 2 1 0 a Heat Heat-En b c E. coli Sal cd S. Typhimurium Figure 2. Effect of enterocin KP alone or in combination with sublethal heating on E. coli O157:H7 or S. Typhimurium. Ec, E. coli O157:H7; En, enterocin KP; Sal, S. Typhimurium. 10 9 8 7 Log cfu/mL tested by the agar spot test in solid medium (results not shown). 3.2. Effect of sublethal heating and cold shock on activity of enterocin KP against E. coli O157:H7 or S. Typhimurium The effect of sublethal heating on survival of E. coli O157:H7 and S. Typhimurium in the presence or absence of enterocin KP is shown in Figure 2. A heat treatment of 60 °C for 10 min reduced the initial numbers of E. coli O157:H7 or S. Typhimurium by 1.09 and 1.21 log cfu/ mL (P < 0.05), respectively. It was observed that surviving cells after heat treatment became sensitive to enterocin KP (P < 0.01). The combined effect of sublethal heating and enterocin KP reduced the cell counts of E. coli O157:H7 or S. Typhimurium by 3.39 and 3.64 log cfu/mL (P < 0.01), respectively. Figure 3 shows the effect of cold shock with or without enterocin KP on the survival of E. coli O157:H7 and S. Typhimurium. The combination of enterocin KP and cold shock decreased the cell number of E. coli O157:H7 by 3.06 log cycles and S. Typhimurium by 3.38 log cycles (P < 0.01). It was observed that statistically there was not a significant difference between the sensitivity of these bacteria to treatment of enterocin KP together with cold shock (P > 0.05). 3.3 Effect of chelating agents on the activity of enterocin KP against E. coli O157:H7 or S. Typhimurium The combination of enterocin KP with EDTA or STPP had a synergistic effect on the inhibitory activity of enterocin KP against E. coli O157:H7 or S. Typhimurium. Treatment of E. coli O157:H7 with EDTA or STTP in the presence of enterocin KP resulted in 7.27 and 6.28 log reduction in the viable counts after 60 min of incubation, respectively (P < 0.01) (Figure 4). The use of enterocin KP together with EDTA or STTP decreased the cell number of S. Typhimurium by 7.21 and 6.20 log at the end of incubation (P < 0.01), respectively (Figure 5). Addition of enterocin 6 Ec Freezing Freezing-En Sal Freezing Freezing-En a a ab c ab cd 5 4 3 2 1 0 E. coli O157:H7 S. Typhimurium Figure 3. Effect of enterocin KP alone or in combination with freezing on E. coli O157:H7 or S. Typhimurium. Ec, E. coli O157:H7; En, enterocin KP; Sal, S. Typhimurium. KP alone at 1600 AU/mL resulted in a decrease in the cell population of E. coli O157:H7 or S. Typhimurium by 0.71 and 0.50 log (P > 0.05), respectively (Figures 4 and 5). The application of EDTA or STTP alone did not cause any reduction in the number of cells (P > 0.05). Cell numbers of positive control samples containing only E. coli O157:H7 or S. Typhimurium increased during the incubation period (Figures 4 and 5). 3.4. Effect of acid stress on activity of enterocin KP against E. coli O157:H7 or S. Typhimurium The combination of enterocin KP and acid stress decreased the cell numbers of E. coli O157:H7 or S. Typhimurium by 2.1 and 1.8 log cycles on day 1 of storage, and 4.20 and 3.98 log cycles on day 7 of storage, respectively (P < 0.01) (Figure 6). The cell numbers of E. coli O157:H7 and S. Typhimurium subjected to acid stress did not decrease significantly on day 1 of storage (0.42 and 0.37 log cycles), and the cell counts showed a further decrease with the extension of storage time (1.31 and 0.95 log cycles, P > 0.05) (Figure 6). 3.5. Effect of combination of enterocin KP with heat, EDTA, or STTP on E. coli O157:H7 in UHT milk Application of enterocin KP or heat treatment alone caused only 0.50 and 0.92 log cycle decreases in the cell counts of E. coli O157:H7 in UHT milk after 1 h of incubation at 37 °C, respectively (Figure 7). Over the 24-h incubation period, the decrease in enterocin KP containing sample reached 1.11 log cfu/mL. EDTA or STTP did not reduce the cell counts of E. coli O157:H7 in UHT milk. However, the combination of enterocin KP with EDTA, STTP, or heat treatment resulted in a significant cell reduction in UHT milk. Treatment of E. coli O157:H7 cells with enterocin KP plus EDTA caused 6.95 log cycles reduction after 12 h of incubation, and the viable cell number was 415 YILDIRIM et al. / Turk J Biol Ec-EDTA-En Ec 10 9 Ec-En a abcd cd Ec-STPP-En a cd 10 a ba 9 cd 7 6 5 4 3 ef 2 e ef g 0 30 Time (min) 60 Figure 4. Effect of enterocin KP alone or in combination with EDTA or STTP on E. coli O157:H7. Ec, E. coli O157:H7; En, enterocin KP. undetectable at the end of the incubation period (Figure 7). When E. coli O157:H7 was treated with enterocin KP plus STTP or enterocin KP plus heat treatment, the cell count decreased by 6.07 and 5.68 log cycles (P < 0.01) at the end of the incubation period, respectively (Figure 7). 4. Discussion Enterocin KP decreased the viable cell number of E. coli O157:H7 and S. Typhimurium by only 1.27 and 0.98 log in BHI broth at the end of the incubation period. Although enterocin KP reduced the cell densities of both bacteria to a certain level, this reduction was not enough to cause a clear inhibition zone on BHI agar applied by the agar spot test. A similar result was reported by Isleroglu et al. (2012). Hence, it was assumed that both bacteria were resistant to enterocin KP because of their outer membrane. Therefore, in order to impair the integrity of the outer membrane and increase sensitivity of E. coli O157:H7 and S. Typhimurium to enterocin KP, both bacteria were exposed to some chemical and physical treatments. The outer membrane of gram-negative bacteria acts as a permeability barrier to the action of antimicrobial compounds such as LAB bacteriocins, preventing their penetration to their site of action, the cytoplasmic membrane (Gao et al., 1999; Yethon and Whitfield, 2001). If the permeability of the outer membrane is altered by chemical or physical treatments, gram-negative bacteria can show sensitivity to bacteriocins (Kalchayanand et al., 1992, 1994; Boziaris et al., 1998). EDTA and STPP, food-grade chelators, are used in a wide variety of food products to prevent oxidation and other deteriorative reactions catalyzed by metal ions. They 416 Sal-EDTA-En Sal-EDTA Sal-En Sal-STPP Sal-STPP-En a a a a a a b a cb 7 6 5 4 3 def 2 d de fg 1 0 1 0 Sal 8 Log cfu/mL Log cfu/mL 8 Ec-STPP a Ec-EDTA 0 30 Time (min) 60 Figure 5. Effect of enterocin KP alone or in combination with EDTA or STTP on S. Typhimurium. En, enterocin KP; Sal, S. Typhimurium. also have antimicrobial activity by enhancing the activity of antimicrobials and antibiotics, especially against gramnegative bacteria (Alakomi et al., 2003; Schrödter et al., 2008). There was a comparable loss of viability of E. coli O157:H7 and S. Typhimurium when they were treated with both enterocin KP and EDTA or enterocin KP and STTP. The use of enterocin KP together with EDTA or STTP decreased the cell number of these 2 gram-negative bacteria about 7 and 6 log cfu/mL, respectively. These results indicated that permeabilized cells became sensitive to enterocin KP. The combined effect of enterocin KP and EDTA offered a better result than enterocin KP and STPP did. Treatment of gram-negative bacteria with chelating agents generally results in removal of Ca2+ and Mg2+ cations from the lipopolysaccharide layer by chelation, destabilizing the outer membrane structure, altering its permeability, and allowing bacteriocins to reach the cytoplasmic membrane (Vaara, 1992; Helander et al., 1997; Yethon and Whitfield, 2001; Hancock and Rozek, 2002; Alakomi et al., 2003). As a result, EDTA and STPP overcome the penetration barrier in gram-negative bacteria, rendering these species sensitive to hydrophobic antibiotics and bacteriocins (Alakomi et al., 2003; Sampathkumar et al., 2003; Belfiore et al., 2007). The enhanced effect of chelators such as EDTA, disodium pyrophosphate, trisodium phosphate, hexametaphosphate, or citrate against gram-negative bacteria has been demonstrated for nisin both under laboratory conditions and in foods (Stevens et al., 1991; Cutter and Siragusa, 1995a, 1995b; Carneiro De Melo et al., 1998; Boziaris and Adams, 1999; Fang and Tsai, 2003). Brochrocin C, enterocin AS-48, pediocin P, cerein 8A, carnocyclin A, and carnobacteriocin BM1 also showed an increased antimicrobial activity on EDTA or STTP-treated gram-negative bacteria (Abriouel et al., 1998; Gao et al., YILDIRIM et al. / Turk J Biol Ec Ec-Enterocin a Log cfu/mL Ec-Acid-Enterocin acb ab 7 Ec-Acid 6 a d 5 a de d 4 f 3 2 1 0 9 Log cfu/mL 8 7 1 Sal Sal-Enterocin a Sal-Acid ab abc Sal-Acid-Enterocin a def 6 7 Storage (day) cd cde 5 b g 4 3 2 1 0 1 Storage (day) 7 Figure 6. Effect of enterocin KP alone or in combination with acid stress on E. coli O157:H7 (a) and S. Typhimurium (b). Ec, E. coli O157:H7; En, enterocin KP; Sal, S. Typhimurium. 1999; Ananou et al., 2005; Osmanağaoğlu, 2005; Lappe et al., 2009; Martin-Visscher et al., 2011). Application of enterocin KP together with sublethal heating caused the cell reduction of E. coli O157:H7 or S. Typhimurium by 3.39 and 3.64 log cycles, respectively. Based on the data obtained in this study, sublethally heated S. Typhimurium was more sensitive to enterocin KP than sublethally heated E. coli O157:H7, but this difference was not statistically significant (P > 0.05). Sublethal heat treatment causes some changes in the outer membrane, including morphological and structural changes, involving damage or release of lipopolysaccharides. These changes can alter the permeability barrier of the outer membrane, allowing the bacteriocin molecule to reach to its target side, the cytoplasmic membrane (Boziaris and Adams, 2001). Some researchers reported that gram-negative bacteria injured by heat treatment became highly sensitive to nisin (Kalchayanand et al., 1992; Boziaris et al., 1998; Boziaris and Adam, 2001), pediocin AcH (Kalchayanand et al., 1992), bacteriocin AS-48 (Abriouel et al., 1998), pediocin P (Osmanagaoglu, 2005), and enterocin AS-48 (Ananou et al., 2005). 13 12 11 10 9 8 7 6 5 4 3 2 1 0 Ec Heat Heat-En En EDTA EDTA-En STTP STTP-En Log cfu/mL 9 8 0 1 Time (h) 12 24 Figure 7. Effect of enterocin KP alone or in combination with EDTA, STTP, or sublethal heat treatment on E. coli O157:H7 in UHT milk. Ec, E. coli O157:H7; En, enterocin KP. Cold shock treatment alone did not result in a significant decrease in the counts of E. coli O157:H7 (0.55 log cfu/mL) or S. Typhimurium (0.41 log cfu/mL) (P > 0.05). However, E. coli O157:H7 and S. Typhimurium became sensitive to enterocin KP when these cells were frozen along with enterocin KP application, and their viable cell numbers were decreased by 3.06 and 3.38 log cycles, respectively. Similar to sublethal heating, S. Typhimurium was more sensitive to the combination of enterocin KP and cold shock than E. coli O157:H7, but it was observed that statistically there was not a significant difference between the 2 bacteria (P > 0.05). Freezing causes some morphological and structural changes in the outer membrane of gramnegative bacteria, crystallizing the liquid-like lipids within membranes and creating channels (Haest et al., 1972). When the outer membrane has lost its integrity, bacteriocins may bind the phospholipid head groups and form transient transmembrane pores. These changes can cause sensitivity to hydrophobic compounds, efflux of periplasmic enzymes, and small molecules (Ter steeg et al., 1999; Boziaris and Adams, 2000; Cao-Hoang et al., 2008). The enhanced effect of cold shock and bacteriocins against gram-negative bacteria such as Aeromonas hydrophila, S. Typhimurium, Yersinia enterocolitica, E. coli 0157:H7, and Pseudomonas fluorescens has been demonstrated for nisin and pediocin AcH (Kalchayanand et al., 1992; Boziaris and Adams, 2000; Boziaris and Adams, 2001) and pediocin P (Osmanağaoğlu, 2005). Cao-Hoang et al. (2008) reported that the combining effect of rapid chilling and nisin application caused a dose-dependent reduction in the population of E. coli cells in both exponential and stationary growth phases. A reduction of 6 log of exponentially growing cells was achieved with rapid chilling in the presence of 100 IU/mL (1 IU/mL = 100 AU/ 417 YILDIRIM et al. / Turk J Biol mL). Cells were more sensitive if nisin was present during stress. Weak organic acids including lactic acid and acetic acid have been used for centuries to preserve foods. Lactic acid and acetic acid are able to cause sublethal injury to gram-negative bacteria such as E. coli, P. aeruginosa, and S. Typhimurium. Such injury involved disruption of the lipopolysaccharide layer (Roth and Keenan, 1971; Przybylski and Witter, 1979; Alakomi et al., 2000). In the present study, it was observed that E. coli O157:H7 and S. Typhimurium exposed to acid stress with the acid mixture containing lactic acid, propionic acid, and acetic acid gained sensitivity to enterocin KP and the level of surviving populations of these 2 gram-negative bacteria decreased with the extension of storage time. Our data are in accord with earlier findings that organic acids can sensitize gramnegative bacteria to bacteriocins by causing sublethal injury. Therefore, bacteriocins became active against gram-negative bacteria when used together with organic acids (Kalchayanand et al., 1992; Alakomi et al., 2000; Helander and Mattila-Sandholm, 2000; Osmanağaoğlu, 2005; Ananou et al., 2007). The combination of enterocin KP with EDTA, STTP, or sublethal heat treatment caused a significant reduction in the cell number of E. coli O157:H7 in UHT milk. Treatment of enterocin KP together with EDTA decreased the cell number of E. coli O157:H7 in UHT milk to an undetectable level, while enterocin KP plus STTP or enterocin KP plus sublethal heat treatment caused a reduction by 6.07 and 5.68 log cycles at the end of the incubation period, respectively. These results indicate that the combination of enterocin KP with EDTA is more effective against E. coli O157:H7 in UHT milk than that of enterocin KP with STTP or with heat treatment. Ananou et al. (2005) reported that the combined application of enterocin AS48 with 0.5% STPP or a sublethal heat shock to E. coli O157:H7 in apple juice caused the cells to become very sensitive to enterocin AS-48. The researchers reported that in both cases the survival cells were completely eliminated after 24-h incubation (initial E. coli population was 103 cfu/L). From this work, we can conclude that the antimicrobial activity of enterocin KP against E. coli O157:H7 or S. Typhimurium, which are normally resistant to enterocin KP, can be enhanced by combination with one of the physical or chemical stresses. Consequently, the use of food-grade chelators, mild heat treatment, cold shock, or acid stress in combination with enterocin KP would be an ideal approach for the application of hurdle technology to inhibit the proliferation of gram-negative foodborne pathogens in foods, and enhance the shelf life and safety of foods. Acknowledgment This research was supported by the Scientific and Technological Research Council of Turkey (TÜBİTAK) (Project no. 102O282 TOVAG). References Abriouel H, Valdivia E, Gálvez A, Maqueda M (1998). Response of Salmonella choleraesuis LT2 spheroplasts and permeabilized cells to the bacteriocin AS-48. Appl Environ Microbiol 64: 4623–4626. Boziaris IS, Adams MR (1999). Effect of chelators and nisin produced in situ on inhibition and inactivation of Gram negatives. Int J Food Microbiol 53: 105–113. Alakomi H, Saarela M, Helander J (2003). Effect of EDTA on Salmonella enterica serovar Typhimurium involves a component not assignable to lipopolysaccharide release. Microbiol 149: 2015–2021. Boziaris IS, Adams MR (2000). Transient sensitivity to nisin in coldshocked Gram negatives. Lett Appl Microbiol 31: 233–237. Alakomi HL, Skytta E, Saarela M, Mattila-Sandholm T, Latva-Kala K, Helander IM (2000). Lactic acid permeabilizes gram-negative bacteria by disrupting the outer membrane. Appl Environ Microbiol 66: 2001–2005. Ananou S, Gálvez A, Martinez-Bueno M, Maqueda M, Valdivia E (2005). Synergistic effect of enterocin AS-48 in combination with outer membrane permeabilizing treatments against Escherichia coli O157:H7. J Appl Microbiol 99: 1364–1372. Boziaris IS, Adams MR (2001). Temperature shock, injury and transient sensitivity to nisin in Gram negatives. J Appl Microbiol 91: 715–724. Boziaris IS, Humpheson L, Adams MR (1998). Effect of nisin on heat injury and inactivation of Salmonella enteritidis PT4. Int J Food Microbiol 43: 7–13. Bhunia A, Johnson MC, Ray B (1988). Purification, characterization and antimicrobial spectrum of a bacteriocin produced by Pediococcus acidilactici. J Appl Bacteriol 65: 261–268. Ananou S, Maqueda M, Martinez-Bueno M, Gálvez A, Valdivia E (2007). Bactericidal synergism through enterocin AS-48 and chemical preservatives against Staphylococcus aureus. Lett Appl Microbiol 45: 19–23. Cao-Hoang L, Dumont F, Marechal PA, Gervais P (2008). Rates of chilling to 0 degrees C: implications for the survival of microorganisms and relationship with membrane fluidity modifications. Appl Microbiol Biotechnol 77: 1379–1387. Belfiore C, Castellano P, Vignolo G (2007). Reduction of Escherichia coli population following treatment with bacteriocins from lactic acid bacteria and chelators. Food Microbiol 24: 223–229. Carneiro De Melo AMS, Cassar CL, Miles RJ (1998). Trisodium phosphate increases sensitivity of gram-negative bacteria to lysozyme and nisin. J Food Protect 61: 839–844. 418 YILDIRIM et al. / Turk J Biol Cleveland J, Montville TJ, Nes IF, Chikindas ML (2001). Bacteriocins: safe, natural antimicrobials for food preservation. Int J Food Microbiol 71: 1–20. Cutter CN, Siragusa GR (1995a). Population reductions of gram negative pathogens following treatments with nisin and chelators under various conditions. J Food Protect 58: 977–983. Cutter CN, Siragusa GR (1995b). Treatments with nisin and chelators to reduce Salmonella and Escherichia coli on beef. J Food Protect 58: 1028–1030. Drider D, Fimland G, Hechard Y, McMullen LM, Prévost H (2006). The continuing story of class IIa bacteriocins. Microbiol Mol Biol Rev 70: 564–582. Fang TJ, Tsai HC (2003). Growth patterns of Escherichia coli O157:H7 in ground beef treated with nisin, chelators, organic acids and their combinations immobilized in calcium alginate gels. Food Microbiol 20: 243–253. Gao Y, van Belkum MJ, Stiles ME (1999). The outer membrane of gram negative bacteria inhibits antibacterial activity of brochocin-C. Appl Environ Microbiol 65: 4329–4333. Leistner L (2000). Basic aspects of food preservation by hurdle technology. Int J Food Microbiol 55: 181–186. Martin-Visscher LA, Yoganathan S, Sit CS, Lohans CT, Vederas JC (2011). The activity of bacteriocins from Carnobacterium maltaromaticum UAL307 against Gram-negative bacteria in combination with EDTA treatment. FEMS Microbial Lett 317: 152–159. Moreno FMR, Leisner JJ, Tee LK, Ley C, Radu S, Rusul G, Vancanneyt M, De Vuyst L (2002). Microbial analysis of Malaysian tempeh, and characterization of two bacteriocins produced by isolates of Enterococcus faecium. J Appl Microbiol 92: 147–157. Osmanağaoğlu Ö (2005). Sensitivity of sublethally injured gramnegative bacteria to pediocin P. J Food Safety 25: 266–275. Przybylski KS, Witter LD (1979). Injury and recovery of Escherichia coli after sublethal acidification. Appl Environ Microbiol 37: 261–265. Roth LA, Keenan D (1971). Acid injury in Escherichia coli. Can J Microbiol 17: 1005–1008. Haest CWM, De Gier J, Van Es GA, Verkleij AJ, Van Deenen LM (1972). Fragility of the permeability barrier of Escherichia coli. Biochim Biophys Acta 288: 43–53. Sampathkumar B, Khachatourians G, Korber D (2003). High pH during trisodium phosphate treatment causes membrane damage and destruction of Salmonella enterica serovar enteritidis. Appl Environ Microbiol 69: 122–129. Hancock R, Rozek A (2002). Role of membranes in the activities of antibacterial cationic peptides. FEMS Microbiol Lett 206: 143– 149. Schrödter K, Bettermann G, Staffel T, Wahl F, Klein T, Hofmann T (2008). Phosphoric acid and phosphates. Ullmann's Enc Ind Chem, 26: 679–721. Helander IM, Mattila-Sandholm T (2000). Permeability barrier of the Gram-negative bacterial outer membrane with special reference to nisin. Int J Food Microbiol 60: 153–161. Schved F, Henis Y, Juven BJ (1994). Response of spheroplasts and chelator-permeabilized cells of Gram-negative bacteria to the action of the bacteriocins pediocin SJ-1 and nisin. Int J Food Microbiol 21: 305–314. Helander IM, von Wright A, Mattila-Sandholm TM (1997). Potential of lactic acid bacteria and novel antimicrobials against Gramnegative bacteria. Trends Food Sci Technol 8: 146–150. Isleroglu H, Yildirim Z, Tokatli M, Nilgun O, Yildirim, M (2012). Partial characterisation of enterocin KP produced by Enterococcus faecalis KP, a cheese isolate. Int J Dairy Technol 65: 90–97. Kalchayanand N, Sikes T, Dunne CP, Ray B (1994). Hydrostatic pressure and electroporation have increased bactericidal efficiency in combination with bacteriocins. Appl Environ Microbiol 60: 4174–4177. Kalchayanand N, Hanlin MB, Ray B (1992). Sublethal injury makes gram-negative and resistant gram-positive bacteria sensitive to the bacteriocins, pediocin AcH and nisin. Lett Appl Microbiol 15: 239–243. Lappe R, Motta AS, Sant'anna V, Brandelli A (2009). Inhibition of Salmonella Enteritidis by cerein 8A, EDTA and sodium lactate. Int J Food Microbiol 135: 312–316. Stevens KA, Sheldon BW, Klapes NA, Klaenhammer TR (1991). Nisin treatment for inactivation of Salmonella species and other gram-negative bacteria. Appl Environ Microbiol 57: 3613–3615. Stiles ME (1996). Biopreservation by lactic acid bacteria. Antonie van Leeuw 70: 331–345. Ter steeg PF, Hellemons JC, Kok AE (1999). Synergistic actions of nisin, sublethal ultrahigh pressure, and reduced temperature on bacteria and yeast. Appl Environ Microbiol 65: 4148–4154. Vaara M (1992). Agents that increase the permeability of the outer membrane. Microbiol Rev 56: 395–411. Yethon J, Whitfield C (2001). Lipopolysaccharide as a target for the development of novel therapeutics in Gram-negative bacteria. Curr Drug Targets Infect Disord 1: 91–106. 419


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To make comments, please initiate or continue a conversation in the Speak Out forum.
Speak Out 6/20/05
Help from the city
WHEN YOUR neighbors let their yard grow up in grass and weeds, call the city, which will see to it that the yard gets mowed.
College funding
IF THE state government can spend $2.4 million on building an entrance to the university and six figures to tear down a communications tower, please don't come whining to me that the state education system needs more of my tax dollars to survive. No wonder Southeast Missouri State University has to balance its budget on the backs of the students by raising tuition.
Dangerous cats
THERE ARE mountain lions in this area. I shot one 10 years ago in self defense. I didn't tell anybody because 20 years ago I took plaster tracks of a mountain lion, and the Missouri Department of Conservation tried to tell me it was a dog. Imagine what a 150-pound cat can do to rabbits and birds. It's their nature to kill, and they're very effective because of survival. They kill for meat and for pleasure. The conservation department is playing this down so irresponsible folks won't go out looking for cats. As a hunter, I can tell you they will find you first. These animals are dangerous and need to be dealt with promptly.
Spending money
BOONDOGGLE No. 1: the Lexington Avenue area with miles of sidewalk along trees and bushes, not houses. The money could have been better used to resurface Independence Street, which is one of the roughest streets in town. Boondoggle No. 2: Southeast Missouri State University's useless and expensive change on Henderson Avenue. All that was needed was a stop sign. Now the university wants to increase student fees. SEMO and the city council always find money to spend but at other times, moan and groan about needing new funds. Look out, taxpayers.
Major issues
WHAT DO you think of all the birds flying around in retail stores? This is unsanitary. Why doesn't the health department do something about this? Also, America needs to start taking care of its own people. We have people living in poverty all around us, especially in the Appalachian Mountains and the Ozarks. We can't continue to take care of the world. It's time our politicians take care of the people who elected them. Stand up, America, for what is right. Bring our jobs home and have decent health care for people.
Different party now
A SPEAK Out comment compared Howard Dean to Harry Truman. Dean, who now speaks for the Democratic Party, is about as far from Truman's values and political views as you can get. I'm old enough to have voted for Harry Truman before Democrats with an agenda like Dean's took over our party and left those who believe in God's values and decency with no party we could respect. Dean is especially what this party is: hate and socialism together.
Think for yourself
WE'RE HEARING so much talk about the poor treatment of the prisons at Guantanamo. I've heard little criticism of the criminals' barbaric military treatment of our people. I haven't read where we've cut off any heads or hands. I believe our military could be home already if it wasn't for all this effort to try to make the president look like a failure. I pray that people think a little bit for themselves.
Judging others
I WOULD like to point out that many of this area's backbiting gossips are Christians. They think that gives them the right to point out everybody's shortcomings and talk about them.
Smoother pavement
WHEN ARMSTRONG Drive is constructed in western Cape Girardeau, I hope the pavement is smoother than Siemers Drive, which is bumpier than most county gravel roads.


text: Welcome to new age of style in Indian wear by wearing this multicolor lehenga style saree. Fashioned from net material, low waist portion and one side pallu is embroidered with resham and pearl work in floral pattern. Inner lining of lehenga is enhanced with floral prints and beautiful colors are used to craft this piece. This outfit will definitely bring you loads of compliments when wore. This saree is paired with an unstitched brocade blouse fabric. The blouse in the picture is for photo purpose only. Note: Color may slightly vary due to digital photography. Standard shipping and handling is FREE in USA, UK, Australia, Canada, and World.


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BOS, one of the leading Haworth Preferred Dealers in the world, has been in business since 1948 and has created thousands of inspiring workspaces for clients in corporate, government, higher education, healthcare and hospitality. Come and experience BOS's BEST IN CLASS solutions.
Affordable Office Interiors (AOI) is one of the largest remanufacturers of office furniture in North America. Discounts provided for our pre-owned and new office furniture lines saves our clients thousands of dollars, year-after-year. With everything in-house, AOI controls the deliverables on projects to maximize furniture budgets and deliver success.
Florida Business Interiors (FBI), of Orlando, another premier BEST IN CLASS Haworth Preferred Dealer, was founded in 1992 and is a full service provider of office furniture in the Central Florida marketplace. FBI has had the privilege of serving some of the area's largest businesses, while offering creative blends of products to meet all budgets and requirements.
For over sixty years, Business Office Systems Installations (BOSI) has been a leading Union office furniture installation firm. The craftsmanship of our carpenters is unparalleled, showcasing quality installations of thousands of workstations, private offices and collaborative office environments for major corporations throughout the Midwest.
In January 2016 we opened the doors of our 150,000 S.F state-of-the-art Chicagoland facility.
Featuring public showrooms for both AOI and BOS, a massive warehouse, and green building.
BOS opened the doors to a new Chicago showroom in February 2016.
The 6,000 square foot West Loop space features state-of-the-art Haworth furniture, polished concrete floors, 13' high ceilings and a modern, urban loft feel. A comfortable mix of work, collaborative and lounge spaces seen in today's modern workplaces, BOS Chicago was designed in partnership with IA. As a working showroom, this new location at 365 S Jefferson Street will also be the new home of the growing BOS Chicago team, lead by Vice President and industry veteran Craig Shultz.
AOI proudly serves Madison and the surrounding area.
AOI Madison, located on Stoughton Road has been in Wisconsin for more than 12 years in a 10,000SF facility. Specializing in both new and used office furniture we have had the pleasure of working with many large entities in both applications.
Come see us at our public showroom in Brookfield.
AOI Brookfield, located on Barker Road in a 5,000 sf facility, has been in Wisconsin since 2010. Our experienced team of professional workspace experts can help you create a workspace you love.
Florida Business Interiors is a full service office interiors product and service provider in the middle-Florida marketplace.
Since 1992, Florida Business Interiors has served the commercial, government, education, health care and hospitality markets. We operate out of a public showroom in Lake Mary, Florida.
BOSI, Inc has been a leader in union office furniture installation for over sixty years.
With a staff of highly trained and qualified technicians, we have the ability to tackle any project, and do so with precise diligence. With the ability to provide any corporate environmental service, we make the process swift and painless. From the time we first sit down, face to face, with you to discuss the scope of your project, to the last bit of garbage being cleaned up, our overall goal is that, at the end of the day, you are 100% pleased with the experience you had working with us.


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jaygould necessitava di un nuovo design di logo e ha lanciato un contest su 99designs.
È stato scelto un vincitore tra 1 design presentati da 1 designer.
create reach
We are an advertising network for all demographics including men, women, tweens & kids & all ages for all vertical channels such as gaming, lifestyle, sports, food, etc.
We are fun yet professional company serving fortune 1,000 advertisers and marketers and website owners.
Fortune 1,000 advertisers such as Kraft, Build-a-Bear, Fruit of the Loom, Pepsi, Spalding, JC Penny, Nintendo, and more.
We need a professional logo with yellow, white and blue. The logo should be simple and clean.


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For sale by GUNMI & CO. PROPERTIES CONSULTING VERIFIED 1 Sep 4:41 pmIkorodu, Lagos
This 90% tastefully finished detached house of 6 units of 2 bedroom flats at Sulaimon-Kolawole Street, Grammar School, Ikorodu is now available for sale.
The house is designed with all en-suite bedrooms, living rooms the kitchen yet to be fitted with stylish storage cabinets, shelves and well tiled floors.
This house is lying on 2 plots of land and it is fenced completely with two gates.
Stay safe!
Watch out for:


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|by Amanda
|Date Added: Sunday 09 November, 2014
I love this product. I was nervous about the sizing as I have a very petite frame and many other reviews claim it runs small. I followed the recommended sizing chart (and bought a small in black) and it fit perfectly! The size can be somewhat adjusted because it is a lace up back and the ribbons given to tie it are very long (I actually had to trim mine down a bit or else it looked like I had a tail!) I had purchased this as part of my Halloween costume, but my boyfriend enjoyed it so much I'm sure it'll make more appearances ;) It looks identical to the image shown and the separate tutu was a perfect length, just enough to cover with a little peekaboo if you bend over too far.The cons I have are the thong that comes with it is plain and did not fit at all, the waistband was tight and the part between your legs was way too loose and has to basically be pulled up to my bellybutton (yes, I made sure I had it on correctly). I just threw it in the trash, I have better ones. And lastly, one of the spines within the corset broke. It is not uncomfortable and you can't notice, but it did snap in half which could have been my fault tying it too tightly or dancing a little too crazy :)For the price this product is fantastic, just remember you get what you pay for. It lasted through a 3 night college Halloween weekend of parties, and is still in the same condition that I received it aside from the one broken spine. It is comfortable as far as corsets go. I would definitely buy this product again and highly recommend it.
|by Rachelle Marie Essick
|Date Added: Monday 03 November, 2014
Great Quality. Arrived on time. Fits as expected.
|by Z. Causey
|Date Added: Monday 27 October, 2014
It was one of my Halloween outfits. Everyone loved it. The fit was great.
|by lori collins
|Date Added: Thursday 28 August, 2014
|by rawrah
|Date Added: Thursday 12 June, 2014
NO REFUNDS :( Throw out the panties and the petticoat bottom tutu thing extremely cheap. the only thing that might have worked would have been the top ( I bought black) the design was nice, but I got two sizes larger and it was still to big. disappoint. NO REFUNDS.
|by Marbles
|Date Added: Monday 27 January, 2014
I am 5' and 95 pounds. I bought this corset for a concert and it fit perfectly. I wore a strapless bra, so since the corset is so well-fitted my bra showed a little bit. It doesn't look cheap, and it's exactly what I wanted. I love it!


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One Dog's Incredible Journey
Description:His name was Stallone. Rescued during a dogfighting raid, this pitbull terrier won the love of everyone around him. The heartbreaking story of this victim sheds light on the extreme cruelty he, and other dogs like him, endured - fight after fight.
Added:3 years ago
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Is there a future for the Cactaceae genera Copiapoa, Eriosyce and Eulychnia? A status report of a prickly situation
- First Online:
- Received:
- Revised:
- Accepted:
DOI: 10.1007/s10531-014-0664-z
- Cite this article as:
- Larridon, I., Shaw, K., Cisternas, M.A. et al. Biodivers Conserv (2014) 23: 1249. doi:10.1007/s10531-014-0664-z
- Citations
- 312 Downloads
Many of Chile's iconic cactus species are threatened by human activities and global change. In order to safeguard them from extinction, both in situ and ex situ conservation actions are urgently needed. In this paper, an overview is given of the in situ and ex situ conservation status of the mainly Chilean cactus genera Copiapoa, Eriosyce and Eulychnia, including a worldwide survey of living ex situ collections of the species of these genera. From our results, we can conclude that although the threats to Chile's remarkable biodiversity are now recognized as an environmental problem, and efforts are underway to protect the threatened endemic flora, many of the most threatened species are currently not protected in situ. Although a higher percentage of living accessions of Copiapoa, Eriosyce and Eulychnia in ex situ collections are of known wild origin compared to results of previous studies on other plant groups, the number of available accessions is insufficient to adequately preserve the genetic diversity of the threatened species. Prospects to upscale both in situ and ex situ conservation of the studied genera are discussed.