THE APPLICATION OF BIOLOGICALLY ACTIVE COMPOUNDS AND ANTIBACTERIAL PROPERTIES EXPRESSING SURFACES FOR THE CONTROL OF CAMPYLOBACTER JEJUNI IN POULTRY PROCESSING

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LITHUANIAN UNIVERSITY OF HEALTH SCIENCES VETERINARY ACADEMY

Gintarė Zakarienė

THE APPLICATION OF BIOLOGICALLY

ACTIVE COMPOUNDS AND

ANTIBACTERIAL PROPERTIES

EXPRESSING SURFACES FOR THE

CONTROL OF CAMPYLOBACTER JEJUNI

IN POULTRY PROCESSING

Doctoral Dissertation

Agricultural Sciences, Veterinary (02A)

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Dissertation has been prepared at the Department of Food Safety and Quality of Veterinary Academy of Lithuanian University of Health Sciences during the period of 2012–2017.

Scientific Supervisor

Prof. Dr. Mindaugas Malakauskas (Lithuanian University of Health Sciences, Agricultural Sciences, Veterinary – 02A)

Dissertation is defended at the Veterinary Research Council of the Lithuanian University of Health Sciences.

Chairperson

Prof. Dr. Rasa Želvytė (Lithuanian University of Health Sciences, Agricultural Sciences, Veterinary – 02A).

Members:

Prof. Dr. Algimantas Paulauskas (Vytautas Magnus University, Biomedical Sciences, Biology – 01B);

Assoc. Prof. Dr. Modestas Ružauskas (Lithuanian University of Health Sciences, Agricultural Sciences, Veterinary – 02A);

Prof. Dr. Arūnas Stankevičius (Lithuanian University of Health Sciences, Agricultural Sciences, Veterinary – 02A);

Dr. Ilma Tapio (Natural Resources Institute Finland, Agricultural Sciences, Zootechnics – 03A).

Dissertation will be defended at the open session of the Veterinary Research Council of Lithuanian University of Health Sciences, Veterinary Academy, Dr. S. Jankauskas Auditorium, at 10:00 a.m. on the 6th_{of April,} 2018.

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LIETUVOS SVEIKATOS MOKSLŲ UNIVERSITETAS VETERINARIJOS AKADEMIJA

Gintarė Zakarienė

BIOLOGIŠKAI AKTYVIŲ MEDŽIAGŲ BEI

ANTIBAKTERINĖMIS SAVYBĖMIS

PASIŽYMINČIŲ PAVIRŠIŲ TAIKYMAS

CAMPYLOBACTER JEJUNI KONTROLEI

PAUKŠTIENOS GAMYBOS GRANDINĖJE

Daktaro disertacija

Žemės ūkio mokslai, Veterinarija (02A)

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Disertacija rengta 2012–2017 metais Lietuvos sveikatos mokslų universitete, Veterinarijos akademijos Maisto saugos ir kokybės katedroje.

Mokslinis vadovas

prof. dr. Mindaugas Malakauskas (Lietuvos sveikatos mokslų universitetas, žemės ūkio mokslai, veterinarija – 02A)

Disertacija ginama Lietuvos sveikatos mokslų universiteto Veterinarijos mokslo krypties taryboje:

Pirmininkė

prof. dr. Rasa Želvytė (Lietuvos sveikatos mokslų universitetas, žemės ūkio mokslai, veterinarija – 02A).

Nariai:

prof. dr. Algimantas Paulauskas (Vytauto Didžiojo universitetas, biomedicines mokslai, biologija – 01B);

doc. dr. Modestas Ružauskas (Lietuvos sveikatos mokslų universitetas, žemės ūkio mokslai, veterinarija – 02A);

prof. dr. Arūnas Stankevičius (Lietuvos sveikatos mokslų universitetas, žemės ūkio mokslai, veterinarija – 02A);

dr. Ilma Tapio (Suomijos natūraliųjų išteklių institutas, žemės ūkio mokslai, zootechnika – 03A).

Disertacija bus ginama viešame Veterinarijos mokslo krypties tarybos posėdyje 2018 m. balandžio mėn. 6 d., 10 val., dr. S. Jankausko auditorijoje.

Disertacijos gynimo vietos adresas: Tilžės g. 18, LT-47181 Kaunas, Lietuva.

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ABBREVIATIONS

EFSA – European Food Safety Authority

GMP – Good Manufacturing Practice

HACCP – Hazard Analysis and Critical Control Points DLC – Diamond Like Carbon

GRAS – Generally Recognized As Safe MAP – Modified Atmosphere

EPA – Environmental Protection Agency

ECDC – European Centre for Disease Prevention and Control

mCCDA – Campylobacter blood free medium base with Campylobacter Charcoal Cefoperazone Desoxycholate Agar (CCDA) supplement ALOA – Agar Listeria Ottavani & Agosti

CFU – Colony Forming Unit

ATCC – American Type Culture Collection

NCTC – National Collection of Type Cultures (a culture collection of Public Health England)

SPSS – Statistical Package for Social Sciences ANOVA – Analysis of Variance

PMA – Propidium Monoazide

qPCR – Quantitative Real Time Polimerase Chain Reaction BHI – Brain Heart Infusion

PBS – Phosphate Buffered Saline CIN – Cinnamaldehyde

LIN – Linalool LA – Lactic acid

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INTRODUCTION

Campylobacter spp. is one of the most prevalent causes of bacterial human gastroenteritis worldwide [1, 2] with C. jejuni species being the most common cause of human infection [3]. Campylobacteriosis is usually defined by sporadic cases related to consumption of undercooked meat (mainly poultry), traveling abroad, environmental contamination and cross-contamination between raw and cooked food [4–7]. Poultry, especially broiler meat, is considered to be the main food-related source of human campylobacteriosis [2, 8]. Despite that, consumers value poultry meat for nutritional properties and relatively low price [9, 10] and increasing demand will likely spur continued growth. With this rising supply comes the risk of an accompanying increase of human campylobacteriosis cases. Currently there are no measures to ensure that broiler flocks are free from campylobacters [11] and further slaughtering of broilers results in contamination of their carcasses with campylobacters up to 54% of all carcasses and 3 log10CFU/g counts [12, 13]. It is estimated that the reduction of campylobacter numbers by 2 log units on chicken carcasses might lower the incidence of chicken meal related campylobacteriosis by 30 times [14]. Therefore, it is necessary to look for new effective measures to reduce campylobacter numbers in food and thus reduce the incidence of Campylobacter infection in humans.

Strict biosecurity at the pre-harvest level of poultry production, GMP/HACCP during slaughtering and various post-harvest control measures like the removal of fecal residues from carcasses, irradiation, freezing, steam pasteurisation and the use of chemical agents on meat are considered as possible control measures for the reduction of human infection [3, 15–17]. Chemical substances like trisodium phosphate, chlorine and acidified sodium chlorite [18–20] are used in various developed countries, while EU has not authorised chemical decontaminants for poultry meat, however, lactic acid was approved for decontamination of bovine carcasses [21]. European Food Safety authority (EFSA) published scientific opinions based on the evaluation of antimicrobial effect of lactic acid and peroxyacetic acid solutions which underlines further need of strong evidence studies to prove the positive effect of antimicrobials applied on the carcass, especially for Campylobacter [22, 23]. Currently, only potable water is permitted for poultry decontamination in the EU [24].

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The aim of the study

The aim of this study was to evaluate different control measures against C. jejuni on poultry products and contact surfaces with a purpose of reducing the risk to acquire campylobacteriosis

The objectives of the study

1. To select bioactive compounds, most effective against C. jejuni, and to evaluate their antimicrobial effect on poultry product.

2. To compare the antimicrobial effect of commercial marinade and natural marinades with spices against C. jejuni during the shelf-life of poultry product.

3. To determine the ability of bacterial species, expressing antagonistic activity against other bacteria, to reduce C. jejuni numbers on poultry product packed under modified atmosphere.

4. To evaluate the potential of DLC (diamond like carbon) based Ag nanoparticles and silver layers to reduce the risk of acquiring campylobacteriosis.

The scientific novelty and practical usefulness

This dissertation is based on a variety of tested control measures against C. jejuni bacteria: bioactive compounds, reformulation of broiler meat product preparation technology, storage of meat products and antimicrobial activity of food preparation surfaces. The antimicrobial effect of cinnamaldehyde and linalool has not been explored in purified forms as marinating ingredients against Campylobacter on poultry products previously and the antimicrobial effect of lactic acid was uncertain due to high variations in results from different studies. Additionally, newly formed natural marinades expressed significant antimicrobial activity against C. jejuni bacteria and was also efficient in the reduction of other microorganisms like yeasts. In the mean time, application of tested thyme-based marinade under real poultry processing facility conditions allowed the evaluation of marinade efficiency in practice. For the first time Lactobacillus delbrueckii subsp. lactis was applied for the reduction of campylobacters under modified atmosphere packaged broiler meat and showed good antibacterial properties. In addition, up to our knowledge, there was no published research regarding the effect of Diamond like carbon with embedded silver nanoparticles against C. jejuni.

Practical usefulness is reflected by the application of microbiologically safer for consumers broiler meat processing and preservation technology (marinating, modified atmosphere packaging, etc.) and the usage of antibacterial surfaces for the reduction of cross-contamination risk. Experimental spice-based marinades and bacteria cultures, expressing

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antagonistic acitivity against C. jejuni, could be used for the enhanced safety of contaminated broiler meat by reducing Campylobacter jejuni numbers. Modified atmosphere packaging could also extend the shelf life of broiler meat product. Additionally, a wide range of possible applications of Diamond like carbon based silver nanocomposites may be considered: coating of domestic and commercial food preparation surfaces, slaughterhouse equipment and the surfaces of poultry processing facility. Therefore, these control measures may reduce public health risk to acquire campylobacteriosis through miss-handling and consumption of broiler meat and satisfy consumers demand for more natural broiler meat products.

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1. LITERATURE REVIEW

1.1. Campylobacter morphology, ecology and epidemiology

Campylobacters are small (0.2–0.8 μm × 0.5–5 μm), Gram-negative bacteria, characterised by spiral, curved, “S” or rod shape [25, 26]. The favourable conditions are microaerophilic, however, some strains may survive for considerable periods under atmospheric level of oxygen [25, 27, 28]. Campylobacters are considered to be thermophilic bacteria with an optimum temperature of 41.5 °C and the ability to grow well at 37–42 °C [25]. Out of 17 species and 6 subspecies assigned to the genus Campylobacter [25, 29], Campylobacter jejuni is the dominant cause of human campylobacteriosis [30].

Campylobacters are widespread bacteria in animals and environment with the gastrointestinal tract of wild and domestic mammals and birds being the main source of infection [25, 31–33]. It is usually part of a normal microbiota in animals [34, 35] and clinical cases are rare [25]. However, C. jejuni and C. coli often cause clinical cases of campylobacteriosis in humans with young children from 1 to 5 years being the most susceptible to infection [36, 37]. Additionally, older than 65 year and immunocompromised people are also less resistant to infection and may require antibiotic treatment [38]. Young adults (20–29 years) are also considered an important age group for campylobacteriosis infection [39]. It must be mentioned that C. jejuni accounts for approximately 80–93% of human campylobacteriosis cases [39– 41]. Infection may occur after a contact with domestic animals like poultry, cattle, pigs, sheep, cats and dogs during working and/or living in a farm, visiting it for recreational or educational purposes, drinking untreated or contaminated water and milk, handling of contaminated meat of animals used for food [32, 33, 42–44]. Also, an increased incidence of human campylobacteriosis cases during summer time was reported in literature [45, 46], however specific meals could be attributed to campylobacteriosis peaks in winter months in certain countries [47].

1.2. Global view of campylobacteriosis

Campylobacteriosis is 1 of 4 main global causes of diarrhoeal illnesses worldwide [26]. Although, disease is usually self-limiting [48], rare complications due to campylobacteriosis may cause a life-threatening damage to human health. For example, Campylobacter-induced Gullain-Barre syndrome (GBS) can be the cause of death in humans [49]. This syndrome

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was reported in a wide range of regions: Europe and North America [50, 51], China [52, 53], Japan [54, 55], Mexico [56], Argentina [57], Bangladesh [58] and it was estimated that approximately one third of GBS syndrome could be attributed to campylobacter infection [59]. Additionally, reactive arthritis was registered in 4.3 cases per 100,000 population of high-income countries [60] and it occurs in 1–5% of patients infected with campylobacters [49]. On the other hand, irritable bowel syndrome (IBS) is even more common complication between campylobacter infected patients as it occurred within 1–2 years in 36% of people as a consequence of acute campylobacteriosis [61].

Approximately 13 cases of campylobacteriosis are reported per 100,000 population in the USA each year [62] with an estimated cost of 1.3–6.8 billion dollars [63, 64]. Although this number is smaller in comparison to European Union level (65.5 cases per 100,000 population) due to accepted control measures [62], further steps should be considered for the reduction of campylobacteriosis incidence. Additionally, Kirk et al. [65] summarised the data from national studies and studies, calculating the incidence and mortality rate from regions not supplying the national data. The results estimating the number of campylobacteriosis cases per 100,000 population in certain region, caused by Campylobacter spp., are presented in Figure 1.2.1.

Figure 1.2.1. The incidence of campylobacteriosis in different

0 500 1000 1500 2000 2500

Campylobacteriosis cases per 100,000 population

African region

Region of the Americas Eastern Mediterranean region

European region South-East Asian region Western Pacific region Global

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The highest incidence of campylobacteriosis was estimated in African region and the lowest – in European region. Additionally, the highest death and disability adjusted life years (DALYs) rates were determined in Eastern Mediteranean region – 1 and 90 per 100,000 population, respectively. Also, high rates were estimated in African region – 0.8 and 70, respectively. On the contrary, death and DALYs rates account only for 0.05 and 9 in European region and 0.04 and 10 in Western Pacific region, respectively [65]. These numbers may correspond for the absence of monitoring and surveillance systems in the majority of Eastern Mediterranean and African countries in comparison to European and Western Pacific region countries. Therefore, Campylobacter control measures applied by higher-income countries are effective in the reduction of campylobacteriosis incidence.

According to EFSA and European Centre for Disease Prevention and Control (ECDC) report, 229,213 human campylobacteriosis cases were confirmed in 2015 in the European Union (EU). This number was more than two-fold higher in comparison to confirmed human salmonellosis cases in the same year (94,625). An increasing trend over 2008–2015 year period was determined based on a 12-month moving average of confirmed human campylobacteriosis cases. Fortunately, the fatality rate remains low (0.03%). While most of the cases occurred sporadically, 8.9% out of 4,362 food-borne outbreaks were related to Campylobacter as a causative agent. Campylobacteriosis outbreaks were caused by the consumption of untreated and contaminated milk (56%) and broiler meat (24%) [66]. Therefore, broiler meat remains an important cause not only in sporadic campylobacteriosis cases but also in campylobacteriosis outbreaks. However, a progress in the reduction of campylobacter contamination on chicken meat from retail market was reported by Food Standards Agency (FSD) in 2016, when lower numbers (11%) of tested chicken meat products from retail market were found to be heavily contaminated (≥1000 CFU/g) in 2015 in comparison to 19% in 2014, promising future achievements in this area [67]. On the other hand, the underestimation, underreporting and under-ascertainment of campylobacteriosis remain important problems in the evaluation of morbidity assessment [1, 68]. For example, O’Kane and Connerton [28] stated that the number of 65,032 reported campylobacteriosis cases by England and Wales in 2012 [69] were significantly underated as O’Brien et al. [70] estimated that in 2009 the actual number was 280,400 cases of human campylobacteriosis, which cost economically approximately £50 million [71].

Campylobacteriosis in Lithuania

The number of confirmed campylobacteriosis cases showed an increasing tendency in Lithuania during 2001–2012, varying from 10 cases per 100,000 population in 2001 to 30 cases per 100,000 population in 2012 [72]. In 2013,

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1,139 confirmed human campylobacteriosis cases were reported, corresponding to the rate of 38.3 per 100,000 population and 6.3% of all gastrointestinal tract diseases caused by bacteria and viruses in Lithuania [73]. It should be mentioned that causative agent of 36.3% of bacterial gastrointestinal tract diseases was unidentified, therefore, suggesting a possibly higher number of campylobacteriosis cases [72]. However, even with only accurately identified cases of campylobacteriosis, it was the most frequently reported cause of bacterial gastroenteritis in Lithuania, slightly exceeding the number of confirmed salmonellosis cases (5.8%) [72]. Additionally, a slightly higher number of confirmed campylobacteriosis cases was reported in 2014 (1,184) and 2015 (1,186) corresponding to 40.2 and 40.6 cases per 100,000 population, respectively [8, 66].

Legaudaite-Lydekaitiene et al. [74] has recently estimated that 59.3% of tested broiler flocks in Lithuania were campylobacter-positive. Additionally, it was determined that campylobacters may survive in poultry farm surrounding environment and spread into broiler houses by workers shoes, clothes and wildlife [75]. Also, some campylobacter species may survive in the slaughterhouse environment after cleaning and disinfection [76]. As there are no effective control measures for the elimination of campylobacters in the slaughterhouse environment, bacteria spread to meat products. Therefore, the prevalence of Campylobacter spp. in Lithuania’s retail market was found to be similar to EU level with 46.8% positive broiler meat product samples [77]. This prevalence rate is almost identical to 46.5% level reported in Lithuania in earlier study [78]. However, it is significantly higher in comparison to the 12.3% prevalence reported in Estonia [79] and lower than 56.3% prevalence determined in Latvia [80]. It must be mentioned that besides broiler reservoir being one of the most important risk factors to acquire campylobacteriosis, environmental sources may also be considered. For example, Ramonaite et al. [81] identified the connection between children with campylobacteriosis and urban environmental sources in Lithuania, however it was deemed to be limited due to rather cold climatic conditions.

1.3. Campylobacter in broiler production chain

Broilers are infected in the first three weeks of life by a horizontal route and can carry up to 109_{CFU campylobacter per gram of cecal contents [82,} 83]. According to EFSA, the prevalence of campylobacters in broiler batches vary between 2 and 100% in different countries with the mean count reaching up to 70% of contaminated primary production [84]. Campylobacters may survive in broiler faeces as well as sheep, goose and hen at 10–20 °C for 2–

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risk factor for bacteria spreading from bird intestines [89]. Additionally, C. jejuni may survive for at least 21 day in poultry processing environment and pose a risk for contamination of previously free from campylobacter broiler flocks, entering the slaughter line [2]. As a result, carcasses are contaminated with campylobacters.

At the moment a few measures for the reduction of broiler flocks colonisation are considered: vaccination, the use of bacteriocins, probiotic bacteria, prebiotics, plant extracts and organic acids [83, 90–93]. However, further studies and high-strengh evidence of efficiency is required for the possible application of these measures.

Figure 1.3.1. The prevalence of campylobacters in fresh broiler meat in EU countries during 2010–2015 period

Poultry is considered to be the most important source of human campylobacteriosis with 50–80% of confirmed cases attributed to chicken reservoir [8, 16]. Additionally, broiler meat is considered to be the main single source of human campylobacteriosis [66]. The prevalence of campylobacter during 2010–2015 year period in fresh broiler meat samples at slaughter, processing and at retail is presented in Figure 1.3.1. [8, 41, 66, 94, 95]. The contamination of broiler meat varied considerably in different year, however, trend analysis in EU level is not possible due to the diversity of reporting countries providing monitoring results each year and different

15,0 20,0 25,0 30,0 35,0 40,0 45,0 50,0 2010 2011 2012 2013 2014 2015 %

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experimental techniques [66]. However, in comparison to the average of contaminated broiler meat samples in 2015 (46.8%), significantly lower numbers of positive samples were reported for fresh turkey meat (15.7%), while the number of campylobacter-positive single samples or batches of fresh bovine and fresh pig meat varied from 0.4% to 3.4% sampling units [66].

1.4. Control of Campylobacter in poultry processing 1.4.1. Natural bioactive compounds as antimicrobial agents

There is a growing need to use natural compounds for effective decontamination purposes [96–98]. The efficiency of various natural bioactive compounds is discussed in literature [99, 100]. Lactic acid, linalool and cinnamaldehyde are between the most effective natural antimicrobials against a variety of bacteria [101–104].

Lactic acid (sinonyms include 2-hydroxypropanoic acid, Ammonium Lactate, Sarcolactic acid) is an odourless and colorless to yellow liquid or crystals, completely soluble in water [105, 106]. It can be found as a normal intermediate in oxidation and metabolism of sugar [107]. Lactic acid could be used as alkalinizing and bactericidal agent in human medicine, for the treatment of skin warts and smallcutaneous tumors, tissue irrigation [108– 110]. The fermentation of whey, cornstarch, potatoes and molasses is used for commercial production of lactic acid [110]. Also, lactic acid could be prepared by fermentation of carbohydrates like glucose, sucrose, lactose with Bacillus acidilactici, Lactobacillus delbrueckii, Lactobacillus bulgaricus and other lactic acid producing bacteria [110]. According to USA Environmental Protection Agency, more than 1 million pounds of lactic acid is produced annualy [111]. Approximately, 85% of lactic acid is used for food, beverage and bakery, 10% represent textile and leather and 5% is dedicated for chemicals and miscellaneous [112]. Currently, no acceptable daily intake (ADI) is determined for lactic acid as a food additive and ingredient [113]. Lactic acid is permitted to use for human consumption as a food additive E 270 and may be used quantum satis in food preparations [114].

Lactic acid is effective against Campylobacter jejuni, Listeria monocytogenes, Listeria innocua, Escherichia coli O157:H7, Salmonella enteritidis [101–103]. However, researchers often find working concentrations of lactic acid to be very different. For example, Riedel et al. [115] determined that 2.5% lactic acid effectively reduced C. jejuni numbers on poultry product, while others found that 5% lactic acid did not show a

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Therefore, further research is needed to estimate which lactic acid concentration is effective against this pathogen.

Cinanmaldehyde (other names include Trans-Cinnamaldehyde; Cinnamic aldehyde, 3-phenylprop-2-enaldehyde etc.) is an aldehyde responsible for the flavour and odor of cinnamon [116]. Cinnamaldehyde is a yellowish oily liquid, slightly soluble in water [117, 118], often applied for antimicrobial purposes in the form of aqueous emulsion [119–122]. It can be isolated from naturally occurring cassia and cinnamon bark essential oils and artificially prepared by condensation of benzaldehyde and acetaldehyde, oxidation of cinnamyl alcohol [118, 123, 124]. Also, it is recognized as an antioxidant for food products [125].

It was estimated that approximately 95% of cinnamaldehyde is consumed as flavour enhancer [123] and it corresponds to approximately 180,000 kg annual amount of cinnamaldehyde accessible from the use of cinnamon and cinnamaldehyde itself [126]. World Health Organization has established a temporary acceptable daily intake dose of 0.7 mg/kg body weight of humans in 1984 and has not increased this level since [126–128]. However, according to 2 year long oral chronic study with trans-cinnamaldehyde, NOAEL (no-adverse-effect-level) in rats was determined to be 200 mg/kg body weight per day and 550 mg/kg body weight per day in mice [129]. It was determined that cinnamaldehyde does not have a genotoxic potential in vivo and it did not show carcinogenic properties [130].

Linalool (sinonyms are 3,7-dimethyl-1,6-octadien-3-ol, 7-methyl-3-methyleneocta-4,6-dien-2-ol, allo-ocimenol) is a flavouring agent, naturally occurring in more than 200 essential oils, also, present in fruits, flowers and spice plants [131]. It is soluble in water, colorless liquid with floral odor similar to bergamot and French lavender [132–134]. The flavour profile of linalool consists of coriander, floral, lavender, lemon and rose [135]. Linalool is responsible for anti-imflammatory, anti-hyperalgesic, antinociceptic effects in animal models [136]. According to Environmental Protection Agency (EPA) approximately 10–50 million pounds of linalool are produced annually [111]. Consumers usually use this bioactive compound for products designated for air-care, cleaning and furnishing, laundry and dishwashing, personal care [137]. A NOAEL (no-adverse-effect level) for linalool was determined to be 365 mg/kg for body weigh per day [138].

Plant origin bioactive compounds like cinnamaldehyde and linalool have shown an antimicrobial effect against food pathogens like Campylobacter spp., S. aureus, Escherichia coli, Listeria monocytogenes, Helicobacter pylori, Arcobacter spp. [96, 97, 139]. Cinnamaldehyde is able to reduce the numbers of antibiotic susceptible and resistant C. jejuni strains in culture medium and on broiler breast fillets [104, 140]. Likewise, linalool found in

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many flowers and herbs, is effective against pathogenic bacteria like Campylobacter spp., Staphylococcus aureus, Listeria monocytogenes and Bacillus cereus in culture medium, but less efficient in food models [96]. However, there is insufficient amount of data about the effect of cinnamaldehyde and linalool against C. jejuni on poultry products and it needs further study.

The mechanism of action of bioactive compounds against bacteria The antimicrobial effect of bioactive compounds may be related to various mechanisms of action. For example, organic acids affect Gram-negative bacteria by difusion through the lipidic membrane and dissociation into anions and protons, therefore increasing cell pH; suppresion of bacteria growth through the inhibition of ATP (adenozintriphosphate) production and lack of energy for DNA synthesis, resulting in cell death [141]. Chaveerach et al. [142] determined by TEM (transmission electron microscopy) that after the application of organic acids part of campylobacter culture cells had lost their membranes. Additionally, the application of more than one organic acid was more effective in the reduction of campylobacter numbers.

On the other hand, plant extracts affect campylobacters through the catabolism of aminoacids like aspartate, alanine, glutamate, glutamine, metionine and serine [143, 144]. This mechanism of action reduce the ability of campylobacters to compete with other bacteria in the gastrointestinal tract [143]. Nonetheless, cell membrane disruption of campylobacteria is also considered as an important effect of plant extracts [144].

Interestingly, fruit extracts like orange oil were effective against ciprofloxacin-resistant campylobacters [98]. Additionally, Ravishankar et al. [140] found out that carvacrol and cinnamaldehyde showed similar efficiency against C. jejuni bacteria resistant to ciprofloxacin, erytromycin and gentamicin as against sensitive campylobacter strains. Therefore, further studies could explore the effect of plant derived antimicrobials as a solution for antimicrobial resistance in campylobacters.

1.4.2. The antimicrobial effect of spices

Natural spices and bioactive compounds are well known for their antimicrobial properties [104, 145–148]. While pure bioactive compounds like cinnamaldehyde, linalool and others are not approved for decontamination of poultry products, they could be used by consumers as marinating ingredients or for production of ready to cook broiler products in the form of spices [148]. Several spices including thyme, rosemary, basil and marjoram are considered to be effective antimicrobials against Gram-negative

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Thyme (Thymus vulgaris L., family Lamiaceae) is a well known aromatic plant, traditionally used as a flavor enhancer for various food preparations [156]. Also, thyme essential oil could be applied as herbal medicine due to antibacterial, antifulgal, anti-inflammatory properties [149]. Based on essential oil composition, thyme can belong to several chemotypes: linalool, borneol, geraniol, sabinene hydrate, thymol, carvacrol or a multi-component chemotype [157]. These chemotypes show which bioactive compound is responsible for specific properties of essential oil and it depends from ecological, geographical, physiological and other factors [157, 158]. While linalool, thymol and carvacrol are known for their antimicrobial properties against gram-positive and gram-negative bacteria [96, 151, 152], other constituents of thyme like borneol are able to induce analgesia in patients with postoperative pain and to increase the penetration of other medicine into the brain cells [159, 160]. Therefore, bioactive compounds as the constituents of thyme could be applied in a variety of scientific and medical fields.

Rosemary (Rosmarinus officinalis L., family Lamiaceae) is a herbal plant, used for flavor enhancement [154]. Rosemary extract is also considered as an antioxidant mainly due to the effect of phenolic diterpenes carnosol and carnosic acid [161]. This property of aromatic plant is applied to prevent the oxidation of oils and off-flavors in food products [148]. Rosemary extracts have been approved for the use as food additive with no safety concern [148] and is listed in the section of Antioxidants by the number E392 [162].

Rosemary is effective in reducing bacteria numbers of Escherichia coli, Salmonella typhi, S. enteritidis, and Shigella sonei, Candida albicans due to bioactive compounds like 1,8-cineol and α-pinene [150, 154]. Likewise, Sienkiewicz et al. [155] determined that rosemary, basil and clary sage essential oils showed the greatest synergistic effect against Gram-negative and Gram-positive bacteria with antibiotics used for the treatment of wounds, while thyme, cinnamon, clove and lavender essential oils expressed great antimicrobial properties when applied separately. Alamprese et al. [153] recently also reported that the combination of rosemary extract and modified atmosphere packaging with nitrogen extended the shelf-life of whole wheat breadsticks by 42%. Interestingly, rosemary extract is able to inhibit cellular proliferation by altering the RNA Post-Transcriptional Modification, the Protein Synthesis and the Amino Acid Metabolism functions, also possibly inactivating the oncogene MYC in vivo [163]. Therefore, this bioactive compound is a promising agent in the treatment of tumours.

Similarly to thyme and rosemary, basil (Ocimum basilicum L., family Lamiaceae) is mostly used as a culinary herb for its organoleptic properties [164]. Basil essential oil was also determined to be effective in the reduction of Klebsiella pneumonieae when applied alone and even higher antimicrobial

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effect was achieved in combination with amoxicillin and clavulanic acid [165].Additionally, the numbers of Brochothrix thermosphacta, Escherichia coli, Listeria innocua, Listeria monocytogenes, Pseudomonas putida, Salmonella typhimurium, Shewanella putrefaciens, Micrococcus flavus could be efficienty reduced due to the involvement of 𝛾-Terpinene, methylchavicol [166, 167]. Also, basil essential oil is known for its antifungal activity, antioxidant and larvicidal properties [168–171]. The recommended daily dose depends from the amount of estragole in basil [172]. It was estimated that essential oil corresponds to 0.8% of basil plant, while 20–89% of essential oil is composed of estragole [173]. According to the Committee of Experts on Flavouring substances of the Council of Europe report in 2000, the limit of 0.05 mg/kg of estragole is recommended for daily intake. Therefore, 60 kg person could consume 3 mg of estragole as a constituent of basil and it would correspond to approximately 1–2 g o basil as a flavor enhancer on food products per day.

Marjoram (Origanum majorana L., family Lamiaceae) is an aromatic herb belonging to the genus Origanum. It is known for antimicrobial activity against various microorganisms including Escherichia coli, Salmonella enteritidis, Listeria ivanovii, Listeria inocula and Listeria monocytogenes with better efficiency in the reduction of E. coli and S. enteritidis [174]. It was assumed that major compounds like cis-sabinene hidrate, trans-sabinene hidrate, terpinene 4-ol and sabinene were responsible for the antibacterial activity of essential oil [174, 175]. Additionally, Waller et al. [176] reported that the essential oil of marjoram expressed antifungal activity against Sporothrix spp. due to 1,8-cineole in vitro. Interestingly, marjoram also expresses antioxidant properties and could suppress the proliferation of human hepatocellular carcinoma cells [177]. Currently, no acceptable daily intake dose is determined due to a lack of evidence of possible danger to human health.

1.4.3. Modified atmosphere as a control measure against C. jejuni

The shelf-life of meat and poultry may be extended by 50–400% when packaged under modified atmosphere, therefore, allowing long distance distribution, reduction of losses and maintaining high quality of meat [178, 179]. Modified atmosphere has a better retention of meat colour [179] and a superior appearance [180] in comparison to vacuum packaged meat.

Campylobacter jejuni numbers could be reduced by modified atmosphere packaging using different combinations of oxygen, carbon dioxide and nytrogen [103, 181–185]. High oxygen modified atmospheres are used in

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formation of oxymyoglobin [186, 187]. However, this feature of high oxygen MAP is not important when dealing with white meat like poultry [188]. Nonetheless, oxygen containing modified atmosphere is efficient in the reduction of microaerophilic bacteria like campylobacters [183, 184, 189]. Reactive oxygen intermediates (ROIs) are formed under oxygen enriched atmosphere conditions causing damage to nucleic acids, proteins and membranes and suggesting the reason for the reduction of C. jejuni numbers under oxygen containing modified atmosphere [103, 190].

On the contrary, modified atmosphere containing only carbon dioxide and nytrogen gas mixtures without additional oxygen are less efficient or does not have any effect in the reduction of campylobacters [103, 183, 184, 191]. However, lower carbon dioxide concentration (40%) may reduce the numbers of spoilage bacteria like total aerobic mesophilic bacterial count, total Enterobacteriaceae count and Pseudomonas, while higher concentrations (50–90%) are efficient in lactic acid bacteria caused poultry spoilage [184]. Carbon dioxide antimicrobial effect is explained by an affected functions of cell membrane, enzyme activity, the physicochemical properties proteins and an alteration of intracellular pH [192].

1.4.4. The effect of antagonistic activity expressing bacteria against C. jejuni

The antimicrobial effect of modified atmosphere could be enhanced by other bacteria expressing antagonistic activity against C. jejuni. For example, lactic acid bacteria like Leuconostoc pseudomesenteroides PCK18, Lactobacillus curvatus DN317 are known for their reduction ability against Campylobacters [191, 193]. This effect is explained by the production of hydrogen peroxide, organic acids and bacteriocins [194]. Also, the efficiency of antimicrobial lactobacillus strains effect could be extended to gastrointestinal tract as they are tolerant to acid and bile and may prevent adhesion and invasion of C. jejuni [195].

Additionally, spoilage bacteria like Pseudomonas sp. are known for their antagonistic activity against Bacillus cereus and Pedobacter sp. in vitro [196, 197, 198]. Garbeva and Boer [197] and Chakraborty et al. [198] determined that Pseudomonas sp. were competing with another bacteria based on interference strategy as Pseudomonas sp. produced inhibitor and negatively influenced the growth of other bacteria. Additionally, an antagonistic metabolite against B. cereus was secreted by Pseudomonas fluorescens in a co-culture under low level of iron [196]. Nonetheless, P. fluorescens ATCC 13525 may be regarded as safe for humans, as P. fluorescens, isolated from

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environmental sources and having the optimal growth temperature of 25–30 °C are not virulent to human cells [199].

1.4.5. Antimicrobial surfaces as a control measure

Up to now, there are no effective measures for the elimination of C. jejuni on animal carcasses. As a result, bacteria spread from contaminated carcasses and slaughterhouse equipment to processed meat products [200]. One way to protect the final product from contamination with pathogenic bacteria is to use physical and chemical decontamination measures in slaughterhouse environment. Therefore, cleaning of slaughterhouse equipment with high-pressure water, sodium hydroxide and potassium hydroxide combination is usually followed by the appliance of various disinfectants like quaternary ammonium compounds combined with glutaraldehyde, poly (hexamethylene biguanide) hydrochloride and foam alkaline detergents [2, 182, 201, 202]. However, they are not always efficient in the reduction of Campylobacter jejuni due to bacteria resistance to disinfectants [76].

The appliance of antimicrobial surfaces with silver could express a prolonged effect against pathogenic bacteria and low cytotoxicity to the final product [203]. Silver is known for antimicrobial properties against Gram-negative and Gram-positive bacteria [204] and silver nanoparticles, ranging in size from 1 to 100 nm, are efficient in the reduction of pathogenic bacteria like Escherichia coli, Staphylococcus aureus, Vibrio cholera and Streptococcus sanguinis [205–208]. The reduction effect is based on an increased contact surface between silver nanoparticles and bacteria cell [204, 209, 210]. Additionally, nanoparticles in the diameter lower than 10 nm have a better reactivity as electronic effects are produced during the interaction with target bacteria [211]. On the other hand, the appliance of silver nanoparticles in colloidal form usually has a short antimicrobial effect as silver ions are released quickly [212]. However, the antimicrobial effect could be prolonged by the incorporation of Ag nanoparticles into firm materials as diamond like carbon (DLC), expressing mechanical resistance [213, 214]. Silver nanoparticles electron magnetron sputtered into DLC material release silver ions slowly and enhances the antimicrobial effect of this kind of surface [215].

1.5. Detection and enumeration of Campylobacters

Cultivation on culture media is widely used as a method for the detection and quantification of bacteria numbers in various samples. However, the

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(processed or unprocessed, fresh, refrigerated, etc.), bacteria life phase (lag phase, ability to divide, viable-but-not-culturable form, dead) and others [216]. Traditional cultivation of bacteria on culture media show only the culturable bacteria numbers, while subpopulations belonging to the other viability forms of bacteria remain undetected [217]. On the other hand, direct molecular methods like quantitative real-time PCR may be used to estimate the number of all kinds of bacteria life phases [216].

Quantitative real-time PCR (qPCR) – is an enhanced version of a standard polymerase chain reaction, when the amount of DNA is evaluated after each cycle with the help of fluorescentic dyes or probes, marked with fluorophores. The application of probes increases reaction specificity and reduces the incidence of mistakes [218]. Specific to DNA target forward and reverse primers are used for the amplification of the final product. They should be checked for the unwanted formation of primer-dimer complexes, unspecific hybridization and possibility of a mistakingly positive result [219]. No template control is mandatory in the experiment and internal amplification control is voluntarily added to show if negative result is due to the absence of target DNA and not to the amplification conditions.

The results are collected significantly faster in comparison to standard PCR due to the sensitivity of fluorescentic reagents and the absence of some steps, necessary for standard PCR, for example, electrophoresis. qPCR is useful for the identification and quantification of bacteria and viruses numbers and evaluation of gene expression [220–222].

During the qPCR process, the level of fluorescence depends from the number of newly formed DNA copies. The more DNA copies are amplified, the higher fluorescence level will be registered. Fluorescence occurs when optical thermocycler system enlights the fluorophores present in fluorescentic dyes or probes with a certain wavelength light, which is emitted in another wavelength. Therefore, the fluorescence is registered by termocycler’s data collecting system. After a few cycles the level of fluorescence reaches the threshold level, stated above the possible background fluorescence, not related to target DNA, and further increases exponentially. Afterall, fluorescence is suppressed, when high number of DNA is amplified, reaction intensity decreases and no further light emission is registered in thermocycler. Therefore, fluorescence is no longer connected to the number of initial DNA [223].

The interpretation of results is based on the level of threshold cycle (Ct). It is measured, where fluorescence curve crosses threshold level. Threshold level should be stated in the optimal position that fluorescence level of the samples would cross it in exponential phase. Additionally, threshold level should be the same for all samples tested at the same time [223]. At the end

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of the process, reaction efficiency will be automatically counted by thermocycler system if the standard curve was prepared. Standard curve may be done with serial dilutions of the standard sample, which is purified PCR product or plasmid with target DNA [224].

DNA based methods like PCR and real-time PCR are fast, sensitive, accurate and permit the qualitative and quantitative evaluation of the amount of microorganisms in food products [225]. Moreover, subdominant populations of bacteria could be detected without the selective enhancement, even when there are a high amount of other bacteria [226]. Therefore, these methods may help to evaluate the contamination level of food products.

1.6. Total bacterial count and the shelf-life of poultry product The shelf-life of poultry meat product depends on various factors including initial bacterial load on broiler carcasses, cross-contamination from slaughterhouse environment, hygiene during the processing of meat, packaging, temperature control and marinating ingredients during storage [189, 227–231]. Total count of bacteria is considered the gold standard for the determination of the shelf-life of chicken meat [232]. It is a better approach for the shelf-life evaluation in comparison to sensory assessment due to a high cost of human sensory panel training and standardization issues of the method [233]. The most commonly used method for the enumeration of microorganisms under aerobic conditions cultivation at 30 °C for 72 h is defined in ISO 4833-1:2013 standard. However, some studies evaluating the shelf-life of poultry meat under modified atmosphere packaging or under cold storage require the enumeration of microorganisms, growing under anaerobic conditions, or evaluating the numbers of psychrotrophic microbial counts. In these cases total anaerobic mesophilic bacterial count, total anaerobic psychrotrophic bacterial count could be enumerated as well with longer cultivation period of 5–10 days [234–236].

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2. MATERIALS AND METHODS

2.1. Study design

The experiments were carried out between 2012–2017 at the Lithuanian University of Health Sciences, Veterinary Academy, Department of Food Safety and Quality. Part of the experiments was performed in a poultry processing facility. Antimicrobial surfaces with thin (5 nm) and thick (40 nm) silver layers and surfaces covered with diamond like carbon (DLC) nanocomposite with embedded silver nanoparticles (DLC:Ag) were prepared in Kaunas University of Technology, Institute of Materials Science by Dr. Šarūnas Meškinis and Dr. Andrius Vasiliauskas. Study was divided in four stages (Fig. 2.1.1.)

Fig. 2.1.1. Study design

Stage I: The antimicrobial effect of lactic acid, cinnamaldehyde and linalool against C. jejuni was evaluated in culture medium under various concentrations of bioactive compound. Afterwards, tested bioactive compounds were applied on broiler breast fillets to determine the working concentrations. In this case total aerobic mesophilic count was evaluated as a product shelf-life indicator.

Stage I. Investigation of the antimicrobial effect of selected bioactive compounds against C. jejuni

Stage II. Modeling the production technology of poultry product by the appliance of selected concentrations of bioactive compounds and natural spices

Stage III. Antagonistic activity expressing bacteria as antimicrobial agents against C. jejuni under modified atmosphere packaging

Stage IV. Evaluation of antibacterial surfaces with silver layers and Ag nanoparticles with classical and molecular methods

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Stage II: The technology of poultry product production was modeled by the enrichment of selected commercial marinade with tested bioactive compounds (1) and the replacement of commercial marinade with natural spice-based marinade (2). Afterwards, the most effective against C. jejuni natural marinade was evaluated under real poultry processing conditions for the ability to reduce the numbers of other bacteria present during poultry processing (3).

1) Commercial marinade containing spices and chemical additives (calcium lactate, sodium diacetate and monosodium glutamate) from poultry processing facility was selected for modeling of poultry production technology. It was enriched with various concentrations of lactic acid, linalool and cinnamaldehyde to determine if newly formed combination of commercial marinade and bioactive compounds had increased antimicrobial properties against C. jejuni. In paralel total aerobic mesophilic bacterial count was tested to show the efficiency of this marinade in the extension of the shelf life of poultry product.

2) Natural marinades were formed from spices often used in domestic kitchens (black pepper, sweet red pepper, etc.) and natural spices (thyme, rosmary, basil, marjoram) expressing antimicrobial activity against various bacteria. No artificial chemical additives were used in experimental spice-based marinades. Commercial marinade containing only original ingredients received from poultry processing facility was tested as control and the antimicrobial effect against C. jejuni and total mesophilic aerobic bacterial count was compared between natural and commercial marinades.

3) The antimicrobial properties against other microorganisms (E. coli, yeasts, total aerobic mesophilic bacterial count) were tested in poultry processing facility under real poultry processing conditions with the most effective against C. jejuni thyme-based marinade. Marinated broiler wings (20 kg) were packed under aerobic conditions, modified atmosphere and vacuum to evaluate how different atmospheric conditions affect the microbiological quality of the final product.

Stage III: The antibacterial effect of Pseudomonas fluorescens and Lactobacillus delbrueckii subsp. lactis against C. jejuni was evaluated on artificially inoculated fresh broiler wings packaged under modified atmosphere. Lactobacillus delbrueckii subsp. lactis (ATCC® 12315™) and Pseudomonas fluorescens (ATCC® 13525™), expressing antagonistic activity against pathogens, were chosen for this study. Two gaseous mixtures were prepared – one containing high oxygen concentration (60%CO2/40%O2), expected to reduce C. jejuni numbers on poultry product, and the second mixture without oxygen (70%CO2/30%N2), often used for

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bacteria P. fluorescens and anaerobic bacteria L. delbrueckii subsp. lactis will be effective in reduction of C. jejuni numbers, while high carbon dioxide modified atmosphere and modified atmosphere containing oxygen, respectively, will prevent the undesirable multiplication of artificially inoculated bacteria. Also, total aerobic mesophilic, total aerobic psychrotrophic and total anaerobic psychrotrophic bacterial count, pseudomonas numbers and lactic acid bacteria numbers were counted in order to evaluate the dynamics of naturally occuring and artificially inoculated bacteria numbers on poultry product.

Stage IV: The effect of thin (5 nm), thick (40 nm) silver layers and DLC based Ag nanocomposites (DLC:Ag) against two reference strains of Campylobacter jejuni NCTC 11168 and Listeria monocytogenes ATCC 7644 were evaluated. L. monocytogenes was tested to check if the antimicrobial surface efficient in the reduction of sensitive to environmental conditions C. jejuni was also effective against more resistant bacteria. C. jejuni and L. monocytogenes numbers were counted by culture-based enumeration on selective agars and quantitative real-time PCR (qPCR) including staining with propidium monoazide (PMA). PMA-qPCR was chosen as a method to accurately enumerate the numbers of viable and viable-but-not-culturable bacterial numbers by distinguishing them from dead bacterial cells.

2.2. Materials

Bacterial strains used in the experiments. The antimicrobial effect of lactic acid, cinnamaldehyde and linalool was tested with three Campylobacter jejuni strains representing 3 different MLST clonal complexes (1034, 464 and 443) chosen from C. jejuni isolates collected from poultry products during previous studies [77]. These C. jejuni strains were selected due to their prevalence in broiler products at Lithuanian retail market [237]. The effect of lactic acid against these strains was tested in culture medium. Similar growth abilities and vitality were observed with no statistically significant differences between them (data not shown). Therefore, the experiment was proceeded with one strain with sequence type and clonal complex 464 as it was also isolated from human clinical samples suggesting an important route of infection between broiler products and humans [237]. The same C. jejuni strain was also used for the experiment with spice-based marinades in stage II and modified atmosphere packed poultry product in stage III. Additionally, Pseudomonas fluorescens (ATCC® 13525™, American Type Culture Collection, Manassas, USA) and Lactobacillus delbrueckii subsp. lactis (ATCC® 12315™, American Type Culture Collection, Manassas, USA) were used for their possible competitive performance

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against C. jejuni. The antimicrobial effect of various surfaces was tested with Campylobacter jejuni subsp. jejuni NCTC 11168 (ATCC® 700819™, American Type Culture Collection, Manassas, USA) and Listeria monocytogenes ATCC® 7644™, American Type Culture Collection, Manassas, USA) reference strains. Listeria monocytogenes reference strain was chosen to represent a more resistant to environmental conditions bacteria in comparison to C. jejuni in order to evaluate the effect of antimicrobial surfaces.

Bioactive compounds and spices used in the experiments. Lactic acid ((L-(+)-Lactic acid, 50%, 69778, 1L, Sigma-Aldrich), linalool (Linalool, 97%, L2602, 500g, Sigma-Aldrich) and cinnamaldehyde (Cinnamaldehyde, ≥93%, W228613-1KG-K, Sigma-Aldrich) were selected due to antimicrobial activity against other bacteria described in literature. All spices were purchased in retail market. Country of origin and producer or packer of various spices used in this study are listed in Table 2.2.1.

Table 2.2.1. The list of spices used in experimental marinades

Name Country of origin Producer/Packer

Onion pellets China UAB Sauda, Kaunas r., Lithuania

Black pepper Vietnam UAB Sauda, Kaunas r., Lithuania

Aromatic pepper Mexico UAB Sveko, Vilnius, Lithuania

Curry India UAB Saldukas, Kalviškės v., Vilnius r.

Cumin Austria Kotanyi GmbH, Austria

Sweet pepper Spain UAB Sauda, Kaunas r., Lithuania

Curcuma India UAB Tavlinas, Širvintai r., Lithuania

Salt Austria Kotanyi GmbH, Austria

Thyme Austria Kotanyi GmbH, Austria

Rosemary Austria Kotanyi GmbH, Austria

Basil Austria Kotanyi GmbH, Austria

Marjoram Austria Kotanyi GmbH, Austria

Broiler meat. Broiler breast fillets, broiler wings and median parts of broiler wings represented poultry product in various study stages. In stage I experiment was conducted with breast fillets obtained from broilers originating from the same batch. They were stored at -80ºC and defrosted within 24 h at 4 °C before the experiment. Sample contamination was checked before the study and they were determined to be free from campylobacter. One sample consisted of 4x10 g of broiler breast fillet meat dedicated to four different testing periods. All pieces of broiler breast fillet meat were cut to be the same size and thickness.

Additionally, samples in the second and the third stage of the study contained full broiler wings (65–75 g) and median parts of broiler wings (35–

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40 g), respectively, which were obtained from poultry processing facility early in the morning, not more than 24 h after slaughter. They were free of natural campylobacter contamination as taken from campylobacter free broiler batch and checked for natural contamination at the beginning of the study.

2.3. Preparation of bacterial inocula

C. jejuni, P. fluorescens, L. delbrueckii subsp. lactis and L. monocytogenes were stored at -80 ºC in BHI broth (REF 610008, Liofilchem, Italy) containing 30% glycerol. C. jejuni was cultivated on Blood agar base No.2 (REF 610188, Liofilchem, Italy) containing 5% of horse blood at 37ºC for 48 h under microaerophilic conditions (5% O2, 10% CO2, 85% N2). After cultivation full 10 µl loop of grown bacteria was transferred to a flask containing 50 ml of BHI broth and grown for 24 h under microaerophilic conditions, 37ºC. The number of flasks used represented the number of experimental samples. For example, one flask was used for inoculation with C. jejuni in study stage I, while 10 flasks containing 500 ml bacterial suspension was used in stage III.

Optical density of all tested bacterial species was measured at 600 nm wave length with spectrophotometer. Bacterial suspension with 0.2 optical density was formed in order to achieve C. jejuni numbers of approximately 7.0–8.0 log10 CFU/ml depending on the different experiments. Pseudomonas fluorescens was grown in Nutrient broth (REF 610037, Liofilchem, Italy) for 24 h under aerobic conditions at 26ºC to achieve 0.05 optical density representing 6 log10 CFU/ml concentration. Lactobacillus delbrueckii subsp. lactis was grown in MRS (de Man, Rogosa and Sharpe) broth (REF 4017292, Biolife, Italy) for 24 hours under anaerobic conditions, 37ºC to achieve 0.1 optical density representing 6 log10 CFU/ml. Lower P. fluorescens and L. delbrueckii subsp. lactis concentrations were chosen in order to achieve similar competition conditions on meat as C. jejuni are more sensitive to environment conditions than the aforementioned bacteria. L. monocytogenes was grown on ALOA (Agar Listeria acc. to Ottaviani & Agosti, REF 4016052, Biolife, Italy) with ALOA Enrichment Selective Supplements (REF 423501, Biolife, Italy) at 37 °C for 48 h under aerobic conditions. In stage IV different diluent was used – full 10 μl loop of grown bacteria was transferred to 1 ml of PBS solution (Oxoid, Basingtoke, England). Subsequently, optical density was measured at 600 nm wavelength with spectrophotometer in order to achieve C. jejuni and L. monocytogenes numbers of approximately 8.0 log10 CFU/ml.

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2.4. Preparation of antimicrobial surfaces

Hydrogenated DLC:Ag films were deposited by DC unbalanced reactive magnetron sputtering of silver target. The diameter of magnetron was 3 in. Monocrystalline silicon substrates were used. Mixture of the hydrocarbons (acetylene) and argon gas was used in the reactive magnetron sputtering. Ar gas flux was 80 sccm and C2H2 gas flux was 7.8 sccm.

Ag films were deposited by the same DC unbalanced reactive magnetron sputtering of silver target. Ar gas flux was 80 sccm. In all experiments substrate–target gap was set at 10 cm, magnetron target current was 0.1 A, base pressure was 5 × 10−4_Pa _and _work _pressure _was (4 ± 1) × 10−1_{Pa. Substrates with no additional bias were grounded.}

2.5. Experimental design

2.5.1. Investigation of the antimicrobial effect of selected bioactive compounds against C. jejuni

Treatment in culture medium. The experiment was done according to Rajkovic et al. (2009) technique. Bolton broth (CM 0983, Oxoid, England) was inoculated with 107_{CFU/ml C. jejuni. Control sample was not treated} with bioactive compound wile experimental samples were affected by 0.125%, 0.25%, 0.5%, 2% concentrations of lactic acid, 0.05%, 0.1%, 0.2% linalool and 0.01%, 0.05%, 0.1%, 0.2% cinnamaldehyde for 10 min with successive centrifugation for 2 min at 10400 x g. After the removal of supernatant, decimal dilutions were prepared and C. jejuni numbers were determined by cultivating on Blood agar base No.2 (REF 610188, Liofilchem, Italy) plates for 48 h at 37 ºC under microaerophilic conditions.

Treatment on broiler breast fillets. The effect of lactic acid, linalool and cinnamaldehyde against C. jejuni on poultry meat was determined using broiler breast fillets with skin. At the beginning of experiment, 4x10 g pieces were cut from one broiler breast fillet to form one sample. Different samples represented various decontamination treatments and control. Samples were inoculated with 50 ml bacterial suspension containing 108_{CFU/ml C. jejuni} bacteria for 2 min and left for 1 h at 4 ºC temperature for the attachment of bacteria. After 1 h samples were decontaminated for 2 min with 50 ml of lactic acid (3%, 5%), linalool (0.5%, 1%, 1.5%, 2%) and cinnamaldehyde (0.5%, 1%, 1.5%, 2%, 2.5%, 3%). One sample (control) was not treated to show the initial count of C. jejuni on broiler breast fillets after inoculation. One sample was treated with water to show if washing without bioactive

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were removed without rinsing. Decimal dilutions of each sample were done immediately after decontamination, after 4, 24, and 96 hours of storage at 4 ºC temperature to examine if prolonged residual treatment could reduce C. jejuni numbers. The counts of C. jejuni were determined on Campylobacter blood free medium base (REF 610130, Liofilchem) with Campylobacter Charcoal Cefoperazone Desoxycholate Agar (CCDA) supplement (REF 81037, Liofilchem) incubated at 37 °C for 48 h under microaerophilic conditions. In parallel total bacterial count was determined at 30 ºC incubation for 72 h with pour plate method using Plate count agar (REF 610040, Liofilchem, Italy).

2.5.2. Modeling the production technology of broiler product by the appliance of selected concentrations of bioactive compounds and natural spices

The enrichment of commercial marinade

Commercial marinade was enriched with different concentrations of bioactive compounds: 4–5% lactic acid, 1–2% linalool and 1.5–2% cinnamaldehyde with a purpose to increase the antimicrobial properties of marinade. Four samples were experimental, while control sample was represented by broiler wings contaminated with C. jejuni and marinated with commercial spices but not treated with bioactive compounds.

Each sample consisted of seven wings, altogether weighing approximately 500 g. They were inoculated in a sterile plastic bag with bacterial suspension (50 ml). Equal distribution of inoculum was ensured by manually mixing the content of the bag from external side for 2 min. Afterwards broiler wings were put into another sterile bag and left for 1 h at 4 ºC temperature for the attachment of bacteria. After 1 h marination mix containing 21 ml of lactic acid, linalool, cinnamaldehyde (only cinnamaldehyde and lactic acid in sample No.2) and spices used for commercial marinade preparation were applied to the samples spreading solutions manually under aseptic conditions. Samples were kept at 4 ºC temperature under aerobic conditions. One broiler wing was taken from each sample bag and decimal dilutions of each sample were done using Buffered peptone water (REF 611014, Liofilchem, Italy) immediately after the treatment (0 h), after 4, 24, 72, 120, and 168 h of storage. The counts of C. jejuni were enumerated by plating of appropriate tenfold serial dilution on Campylobacter blood free medium base (REF 610130, Liofilchem, Italy) with Campylobacter CCDA supplement (REF 81037, Liofilchem, Italy) incubated at 37 ºC for 48 h under microaerophilic conditions. In parallel total aerobic mesophilic bacterial counts were determined at 30 ºC incubation for 72 h with pour plate method using Plate

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count agar (REF 610040, Liofilchem, Italy), including testing of the number of aerobic bacteria before inoculation of C. jejuni (N/N-Not contaminated/Not treated, Table 3.2.2.1). Experiment was repeated three times and each time new inoculum and marinades were prepared. No significant differences (P0.05) were determined between C. jejuni numbers in inoculum and the effect of marinades on pathogen considering three separate repetitions.

The antimicrobial effect of commercial marinade enriched with different concentrations of bioactive compounds was evaluated by comparing the numbers of C. jejuni in control and experimental marinades at certain time periods (after 0 h, 4 h, 24 h, 72 h, 120 h and 168 h).

The preparation of natural marinades expressing antimicrobial properties

Experiments were carried out using six marinades and one control sample. Experimental marinades consisted of natural spices (thyme, rosemary, basil, marjoram and others) and bioactive compounds (cinnamaldehyde, linalool, lactic acid) chosen according to the results from previous experiments (detailed composition of marinades is described in Table 2.5.2.1).

One commercial marinade contained only commercially produced spices combined with chemical additives (monosodium glutamate-E621, sodium diacetate-E262, calcium lactate-E327) (No.6, Table 2.5.2.1) and the other one contained commercially produced spices with addition of above mentioned bioactive compounds (No.5, Table 2.5.2.1). For experiments, commercial marinade was made according to the recipe used in a poultry producing company for broiler wings marinating. Fresh broiler wings from the same batch were used as a control sample (No.7) and were inoculated only with C. jejuni.

Testing procedure remained the same as in previous experiment with commercial marinade enrichment, however, the concentrations of linalool were slightly changed from 1% and 2% to 1% and 2.5% with an expectation of stronger antimicrobial effect. Experiment contained three repetitions and each time new inoculum and marinades were prepared. No significant differences (P0.05) were determined between C. jejuni numbers in inoculum and the effect of marinades on pathogen considering three separate repetitions.

The antimicrobial effect of experimental and commercial marinades was evaluated by comparing the numbers of C. jejuni in marinated samples and inoculated non-marinated broiler wing samples (further as control sample No.7) at certain time periods (after 0 h, 4 h, 24 h, 72 h, 120 h and 168 h). Additionally, the effect of chemical additives (E262, E327 and E621) was

THE APPLICATION OF BIOLOGICALLY ACTIVE COMPOUNDS AND ANTIBACTERIAL PROPERTIES EXPRESSING SURFACES FOR THE CONTROL OF CAMPYLOBACTER JEJUNI IN POULTRY PROCESSING

Gintarė Zakarienė