Microbial inactivation and cytotoxicity evaluation of UV irradiated coconut water in a novel continuous flow spiral reactor

Highlights

• Coconut water was irradiated using a novel flow-through UV system.

• Microbial kinetics was investigated.

• UV doses were validated and verified using a bacteriophage.

• All microbes were inactivated by > 5-log₁₀.

• UV-C irradiation could be an effective alternative for production of beverages.

Abstract

A continuous-flow UV reactor operating at 254 nm wave-length was used to investigate inactivation of microorganisms including bacteriophage in coconut water, a highly opaque liquid food. UV-C inactivation kinetics of two surrogate viruses (MS2, T1UV) and three bacteria (E. coli ATCC 25922, Salmonella Typhimurium ATCC 13311, Listeria monocytogenes ATCC 19115) in buffer and coconut water were investigated (D₁₀ values ranging from 2.82 to 4.54 mJ·cm^− 2). A series of known UV-C doses were delivered to the samples. Inactivation levels of all organisms were linearly proportional to UV-C dose (r² > 0.97). At the highest dose of 30 mJ·cm^− 2, the three pathogenic organisms were inactivated by > 5 log₁₀ (p < 0.05). Results clearly demonstrated that UV-C irradiation effectively inactivated bacteriophage and pathogenic microbes in coconut water. The inactivation kinetics of microorganisms were best described by log linear model with a low root mean square error (RMSE) and high coefficient of determination (r² > 0.97). Models for predicting log reduction as a function of UV-C irradiation dose were found to be significant (p < 0.05) with low RMSE and high r². The irradiated coconut water showed no cytotoxic effects on normal human intestinal cells and normal mouse liver cells. Overall, these results indicated that UV-C treatment did not generate cytotoxic compounds in the coconut water. This study clearly demonstrated that high levels of inactivation of pathogens can be achieved in coconut water, and suggested potential method for UV-C treatment of other liquid foods.

Industrial relevance

This research paper provides scientific evidence of the potential benefits of UV-C irradiation in inactivating bacterial and viral surrogates at commercially relevant doses of 0–120 mJ·cm^− 2. The irradiated coconut water showed no cytotoxic effects on normal intestinal and healthy mice liver cells. UV-C irradiation is an attractive food preservation technology and offers opportunities for horticultural and food processing industries to meet the growing demand from consumers for healthier and safe food products. This study would provide technical support for commercialization of UV-C treatment of beverages.

Introduction

There has been an increased interest in coconut water beverages in many parts of world due to rising consumer demands for food products with potential health benefits. Coconut water (CW; classified as a juice), is rapidly gaining popularity, with sales escalating over 300% since 2005 worldwide (Burkitt, 2009). Although the liquid endosperm remains sterile in an undamaged coconut (Awua, Doe, & Agyare, 2011), the compositional and physico-chemical properties of coconut water (pH of 4.2–6.0 and a_w of 0.995) make it susceptible to microbial growth and contamination (Walter, Kabuki, Esper, Sant'Ana, & Kuaye, 2009). Unhygienic handling and processing may introduce spoilage and pathogenic microbes to the raw product, with contamination of microbes like Salmonella enterica, Bacillus cereus, Listeria monocytogenes and Staphylococcus aureus.

Although there have been no outbreaks reported in coconut water, there remains the probability of microbial growth and survival of disease-causing organisms in coconut water, with repercussions for human health. Recent occurrences of food borne illness traced to consumption of unpasteurized apple and other low and high acid fresh juices have resulted in declaration of regulations requiring further processing for reduction of pathogens. For example, the United States Food and Drug Administration (US-FDA) instituted the federal juice Hazard Analysis Critical Control Point (HACCP) to ensure food safety of all juice products (US-FDA, 2000). This requires that manufacturers use adequate processing techniques, capable of achieving a 5-log₁₀ reduction in the numbers of most resistant pathogens (Goodrich, Schneider, & Parish, 2005).

The US-FDA states that fruit juice processing is required to be subjected to regulations of HACCP (Federal Register [FR], 2001) and related regulation (21 CFR 110). At present, thermal pasteurization is the dominant technology used to achieve these goals, with an accessible and well-understood strategy for treatment. The US-FDA has approved thermal pasteurization as an established technology for rendering fruit juice products safe from pathogenic microbes and enhancing the shelf-life of refrigerated juice products (Donahue, Canitez, & Bushway, 2004, US-FDA, 2001). The High-Temperature Short-Time (HTST) pasteurization process is widely used in large-scale continuous mode juice production (Rupasinghe & Yu, 2012). Although they are widely used, thermal processing techniques may bring about considerable changes in nutritional content of the juices (Caminiti et al., 2012). Because of these drawbacks, various non-thermal pasteurization techniques for achieving significant microbial inactivation are being evaluated. One of these novel non-thermal technologies to control pathogens is UV-C light.

UV light forms a part of the electromagnetic spectrum in between the wavelengths of X-rays and visible light. UV is a non-thermal, low temperature treatment, producing little or no known toxic or significant non-toxic by-products during treatment (Islam et al., 2016a, Islam et al., 2016b), with minimal loss of sensory attributes and low energy consumption. The wavelength of UV light ranges from 100 to 400 nm and is categorized as UV-A (320–400 nm), UV-B (280–320 nm), UV-C (200–280 nm) and vacuum UV (100–200 nm) (Koutchma, Forney, & Moraru, 2009). The UV wavelength of 253.7 nm is commonly used for disinfection of water, air and surfaces. UV-C light, in particular, has been shown to have lethality effects on bacteria, yeasts, molds and viruses. The ability of UV-C light to penetrate through the cell wall, blocking DNA transcription and replication results in restricting the microorganism's ability to grow and multiply (Azimi, Allen, & Farnood, 2012). For all these reasons, UV-C is a promising technology that could have advantages over thermal methods of pasteurization (Koutchma et al., 2009).

Currently, UV technology has been used to treat liquids foods including fresh juices and nectars to inactivate microorganisms such as E. coli, Salmonella, Shigella, Zygosaccharomyces bailli, and Saccharomyces cerevisiae (Donahue et al., 2004, Gabriel and Nakano, 2009, Lopez-Malo and Palau, 2005, Lu et al., 2010, Murakami et al., 2006), and protozoa such as Cryptosporidium parvum (Hanes et al., 2002); enzymes such as polyphenoloxidase, ATPase, acid phosphatase, carboxypeptidase A, and trypsin (Falguera et al., 2010, Guerrero-Beltrán and Barbosa-Cánovas, 2006, Ibarz et al., 2009).

In a recent study, we showed that using a collimated beam (Islam et al., 2016a, Islam et al., 2016b) and a flow-through UV system, treated apple juice resulted in little to no impact on the concentration of individual polyphenols and in-vitro-antioxidant activity. Though powerful in its proof-of-principle, the implementation of such a system in a food industry setting is challenging. Typical UV irradiation research studies utilize batch reactors (i.e., collimated beam devices); however, continuous-flow reactors are significantly more desirable for industrial food processes. The effect of UV irradiation on microbial and viral inactivation in coconut water using a flow-through system has not been reported to date.

Most of the UV irradiation studies in liquid foods do not consider the optical absorbance of the fluid, while using a batch or a continuous flow-through system (Unluturk et al., 2010, Caminiti et al., 2012). A simple analogy is that the UV Dose is the number of photons absorbed per surface area by an irradiated object during a particular exposure time. While UV dose delivered by UV system is often expressed as the product of the average UV intensity within the UV system and the theoretical treatment time, the experimental set-up gives intensity gradients within UV systems and gives rise to a distribution of delivered doses as opposed to a fixed value. Without proper mixing, fluid further from the lamp will receive a lower dose than that closer to the lamp. In this study, the optics (absorption coefficients) of the fluid are accounted for, and dose delivery is verified through bio-dosimetry, ensuring that target levels of disinfection are achieved, and allowing direct comparisons with other UV-C treatment studies. In this novel study, the UV fluence was quantified and verified using a MS2 (Single Stranded RNA virus). MS2 inactivation has a linear response to UV and hence can be used to quantify and confirm the UV fluence. This parameter is also known as RED (Reduction Equivalent Dose). If the RED for a UV system is 40 mJ·cm^− 2, it means that the UV system is delivering 40 mJ·cm^− 2 as measured by the validation organism.

Cytotoxicity of irradiated beverages is utmost important to make sure that a novel food processing technique such as UV irradiation does not produce toxic chemical compounds when treated at higher doses. In fact, none of the studies have evaluated the cytotoxicity of irradiated coconut water.

Through this study, using a novel continuous flow reactor the effectiveness of UV-C irradiation for the inactivation of Salmonella Typhimurium ATCC 13311, Escherichia coli ATCC 25922, Listeria monocytogenes ATCC 19115 and two bacteriophage (MS2 and T1UV) as model viruses in coconut water was investigated. In addition, this study also evaluated the cytotoxicity of UV-C irradiated coconut water on the mice liver cells and fibroblasts from normal colon cells (CCD-18Co).