Cheap extraction of antibacterial compounds of berry pomace and their mode of action against the pathogen Campylobacter jejuni
Introduction
Campylobacter jejuni is one of the major enteric pathogens and is the source of much of the foodborne bacterial illnesses in the US. In 2012, Foodborne Diseases Active Surveillance Network (FoodNet) identified 14.3 campylobacteriosis cases for every 100,000 inhabitants of the twelve states of USA and this rate was 14% higher than the rates found in 2006–2008. This high prevalence rate may be attributed to the increased consumption of white meat specifically chicken and turkey. In recent years, poultry has been recognized to be the major source of campylobacteriosis in humans (Lin, 2009). Campylobacter colonizes in the poultry gut as normal flora. So far, the most widely used control measure against the colonization of Campylobacter in poultry gut is the use of antibiotics in feed and water, but resistant strains of C. jejuni to several antibiotics, including ciprofloxacin, nalidixic acid, erythromycin, tetracycline, and streptomycin, have been reported (Wieczorek, Szewczyk, & Osek, 2012). Consequently, the search for alternative natural and organic antimicrobials is now more essential than ever. Bioactive phytochemicals from berries, especially blackberry (Rubus fruticosus) and blueberry (Vaccinium corymbosum) pomace as feed or water supplement to reduce pre-harvest level of C. jejuni contamination in poultry, might be a feasible alternative.
Recently, the antimicrobial activity of phenolic compounds present in berry fruits and their pomaces against Campylobacter species and other intestinal pathogens was demonstrated (Biswas et al., 2012, Puupponen-Pimiä et al., 2005). Extraction of bioactive phytochemicals from fruit juice is too expensive for large scale application in the poultry industry. However, fruit pomace, the (semi) solid remains of crushed fruit after juice extraction, may provide a cheaper alternative. Fruit pomaces may make up as much of 30% of the original fruit weight and are produced in significant amounts in the US, where they are considered a waste product. Pomaces cannot be fed to animals directly since they are low in protein content and too acidic. Cheap extraction of the phenolics that are mainly responsible for the antibacterial action would provide a solution to both the fruit industry and the poultry industry. These phenolics are concentrated in the outer layers of fruit (e.g. skin) as their biosynthesis is dependent on UV-light (sunlight).
Several modes of actions of berry phenolics have been suggested for pathogen inhibition. These mechanisms include damaging the bacterial cell membrane (Lacombe, Tadepalli, Hwang, & Wu, 2013), inhibition of extracellular microbial enzymes (Scalbert, 1991), direct effect on microbial metabolism, and deprivation of substrates mandatory for microbial growth (Puupponen-Pimiä et al., 2004). These mechanisms would act indiscriminately against benign and pathogenic bacteria. Nevertheless, we have provided evidence that berry juice inhibited pathogenic bacteria while stimulating the growth of probiotics (Yang, Hewes, Salaheen, Federman, & Biswas, 2014). The aim of this work was two-fold: (1) Development of a cheap extraction method to concentrate the antibacterial ingredients of berry pomace; and (2) Investigation of the effects of these active ingredients on the expression of virulence genes of C. jejuni, in order to explain the noted sensitivity of this pathogen for the antibacterial activity of berry pomace. The findings from this research will provide significant insight into the effects of blackberry and blueberry pomace extracts on C. jejuni growth, alteration of physicochemical properties, virulence gene expression and its interactions with host cells.
Section snippets
Bacterial strain and growth condition
C. jejuni RM1221 (ATCC BAA-1062™) was used in the current study. The bacterium was grown in Blood agar (Himedia, India) with 5% defibrinated sheep blood (Ward's Science) at 37 °C under microaerophilic (10% CO2, 5% O2, and 85% N2) condition.
Preparation of pomace extracts
Blackberry and blueberry whole fruits were purchased from a local market (College park, MD) and brought into the lab. A kilogram of blackberry or blueberry was treated with 1 L boiling water for 3 min and the water was discarded. After cooling, a mixture of
Role of solvents on phenolic content extraction from pomaces
We have extracted the pomace phenolics using three different solvents (10% ethanol, 10% methanol in water, and water alone) and incubated at two different temperatures, 37 °C or 60 °C, (Table 2). We found that as a solvent, 10% ethanol in water was the most effective in extracting phenolic compounds from both blackberry (1.66 mg/mL GAE) and blueberry (1.43 mg/mL GAE) pomaces compared to other two solvents though the difference between ethanol and methanol in terms of extracting phenolic
Discussion
There are many natural components that are able to limit and inhibit the spread and virulence of foodborne bacterial pathogens. In our previous study, we found that both blueberry (Biswas et al., 2012) and blackberry (Yang et al., 2014) juice inhibited the growth of various foodborne bacterial pathogens including C. jejuni. Several literature also indicated that phenolic components of berry and berry products have antioxidant and offer many other health benefits (Biswas et al., 2012,
Acknowledgments
This work was supported by start-up package of Dr Debabrata Biswas (# 2-93108), Department of Animal and Avian Sciences, University of Maryland-College Park.
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