Sequential treatment of hydrogen peroxide, vacuum packaging, and dry heat for inactivating Salmonella Typhimurium on alfalfa seeds without detrimental effect on seeds viability
Introduction
Since demand for healthful dietary foods has been increasing worldwide, sprouts have been attracting much consumer attention since they are highly nutritious. Among the various kinds of sprouts, alfalfa sprouts are one of the most common and popular, and their consumption around the world has also been increasing. Regardless of their nutritive value, paradoxically, alfalfa sprouts often pose a serious human health concern since they can carry foodborne pathogens when eaten raw or lightly cooked. A number of outbreaks associated with consumption of raw alfalfa sprouts support this view, and Salmonella spp. has been involved in many of these outbreaks. Since 1996, more than 30 cases of outbreaks linked to sprouts have been reported (CDC, 2011). An outbreak of Salmonella Saintpaul associated with alfalfa sprouts, which was the largest recorded case, occurred simultaneously in 14 states of the USA in 2009, resulting in 256 infections and 8 hospitalizations (CDC, 2009). In 2010, Salmonella Newport was the cause of a multistate outbreak which engendered 44 infections (CDC, 2010). In 2016, 36 people infected with the outbreak strains of Salmonella Reading or Salmonella Abony were reported from 9 states (Centers for Disease Control and Prevention (CDC), 2016a, Centers for Disease Control and Prevention (CDC), 2016b). Also, 26 people infected with the outbreak strains of Salmonella Muenchen (25 people) or Salmonella Kentucky (1 person) were reported from 12 states (Centers for Disease Control and Prevention (CDC), 2016a, Centers for Disease Control and Prevention (CDC), 2016b).
It is known that seeds are the most likely source of introducing pathogenic bacteria in sprout-associated outbreaks (National Advisory Committee on Microbiological Criteria for Foods (NACMCF), 1999a, National Advisory Committee on Microbiological Criteria for Foods (NACMCF), 1999b). Various potential routes that can contaminate seeds during sprouting exist, including contaminated irrigation water, unsanitary production equipment, misuse of animal fertilizers, and inadequate worker hygiene (National Advisory Committee on Microbiological Criteria for Foods (NACMCF), 1999a, National Advisory Committee on Microbiological Criteria for Foods (NACMCF), 1999b). Since the sprout-producing environment provides perfect conditions for pathogenic bacteria to proliferate and gives pathogens opportunity to internalize into sprouts, adequate interventions are needed for safe consumption of sprouts.
The U.S. Food and Drug Administration (FDA) and the National Advisory Committee on Microbiological Criteria for Foods (NACMCF) recommends a 5 log reduction of pathogen load on seeds and the FDA's guidance suggest seeds for sprouting should be sanitized with such as 20,000 ppm of calcium hypochlorite (US FDA, 1999). However, this method cannot guarantee elimination of pathogens on seeds. In addition, no study to date has reported both complete inactivation of pathogens while simultaneously preserving seed germination with a single treatment. Numerous studies have been conducted using various treatments, such as organic acids, hypochlorite, ozonated water, electrolyzed water, high hydrostatic pressure and gamma irradiation, to eliminate pathogens completely without affecting seed germination, but most were insufficient to achieve this goal (Lang et al., 2000, Rajkowski and Thayer, 2000; Sharma et al., 2002, Sharma and Demirci, 2003, Wuytack et al., 2003).
Thermal treatment has been evaluated and it was proved effective to reduce pathogen load on seeds to some degree (Jaquette et al., 1996, Neetoo and Chen, 2011, Weiss and Hammes, 2005). Especially, dry heat has been effectively applied alone or combined with other treatments to decontaminate seeds while simultaneously maintaining the germination rate under a suitable temperature range.
Hydrogen peroxide (H2O2) has a germicidal effect and it is designated as GRAS (Generally recognized as safe). It forms hydroxyl free radicals (OH−) which act as an oxidant, and attacks bacteria by destroying essential components such as lipids, proteins, and DNA (Mcdonnell and Russell, 1999). Hydrogen peroxide also has been studied extensively, but individual treatment did not reduce pathogen level sufficiently. Weissinger and Beuchat (2000) reported that immersing inoculated alfalfa seeds in solutions containing 2% hydrogen peroxide for 10 min reduced populations of Salmonella by 0.67 log CFU/g. Also, treatment of alfalfa seeds for 10 min with 8% hydrogen peroxide reduced populations of Salmonella by 3.27 log CFU/g (Holliday et al., 2001).
Normally, vacuum packaging has been used to extend shelf-life of fresh food or meat products by inhibiting growth of aerobic bacteria and preventing oxidative reactions (Church and Parsons, 1995). Accordingly, vacuum packaging has not been considered as an inactivation method, and no research has been done thus far combining heat treatment and vacuum packaging to inactivate pathogens.
The purpose of this study was to examine the efficacy of hydrogen peroxide, vacuum packaging, and dry heat when these treatments were applied alone or sequentially; and determine the impact of these treatments on seeds germinability. Furthermore, we aimed to demonstrate the impact of vacuum heating for improving dry heat efficiency.
Section snippets
Bacterial strains and preparation of inoculum
Three strains of Salmonella enterica subsp. enteric serovar Typhimurium (ATCC, 19585, ATCC 43971, and DT 104) used for inoculation were obtained from the School of Food Science bacteria culture collection of Seoul National University (Seoul, Korea). Stock cultures were stored at −80 °C combining 0.7 ml of culture with 0.3 ml of sterile 50% glycerol. Active cultures were produced by streaking stock cultures onto tryptic soy agar (TSA; Difco, BD) and incubating at 37 °C for 24 h. A single colony
Trend of moisture content of alfalfa seeds after hydrogen peroxide treatment
To compare hydrogen peroxide treated sample with untreated samples exposed to the same conditions and to prevent seeds from damage by wet heat, moisture content was measured at regular intervals following hydrogen peroxide treatment (data not shown). The moisture content of alfalfa seeds declined dramatically and reached stability after 12 h (6.56 ± 0.44%) which was similar to its original condition (6.47 ± 0.28%). The moisture content after 14 h of drying was 6.56 ± 0.32%, and this drying time
Discussion
The objective of this study was to demonstrate the effectiveness of vacuum packaging for enhancing the efficacy of dry heat to inactivate Salmonella Typhimurium on contaminated alfalfa seeds. We designed this study to follow a sequence of hydrogen peroxide treatment, drying, vacuum packaging, and dry heat so that packaged alfalfa seeds could be the final product of the procedure and easily distributed for commercial use. As a result, sequential treatment could yield a reduction of S.
Acknowledgments
This work was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry (IPET) through Agriculture, Food and Rural Affairs Research Center Support Program, funded by Ministry of Agriculture, Food and Rural Affairs (710012-03-1-HD220). This work was also supported by the National Research Foundation of Korea grant funded by the Korea government (NRF-2018R1A2B2008825).
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