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A combined hurdle approach of slightly acidic electrolyzed water simultaneous with ultrasound to inactivate Bacillus cereus on potato
Accepted Manuscript A combined hurdle approach of slightly acidic electrolyzed water simultaneous with ultrasound to inactivate Bacillus cereus on potato Ke Luo, Seon Young Kim, Jun Wang, Deog-Hwan Oh PII:
S0023-6438(16)30200-6
DOI:
10.1016/j.lwt.2016.04.016
Reference:
YFSTL 5404
To appear in:
LWT - Food Science and Technology
Received Date: 16 January 2016 Revised Date:
7 March 2016
Accepted Date: 11 April 2016
Please cite this article as: Luo, K., Kim, S.Y., Wang, J., Oh, D.-H., A combined hurdle approach of slightly acidic electrolyzed water simultaneous with ultrasound to inactivate on potato, LWT - Food Science and Technology (2016), doi: 10.1016/j.lwt.2016.04.016. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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A combined hurdle approach of slightly acidic electrolyzed water simultaneous with
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ultrasound to inactivate Bacillus cereus on potato
3 Ke Luo1, Seon Young Kim1, Jun Wang2, and Deog-Hwan Oh1,*
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Kangwon National University, Chuncheon, Gangwon 200-701, Korea
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Department of Food Science and Biotechnology and Institute of Bioscience and Biotechnology,
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266-109, China
College of Food Science and Engineering, Qingdao Agricultural University, Qingdao, Shandong
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E-mail: [email protected] (Deog-Hwan Oh)
Author for correspondence. Tel: 82-33-250-6457; Fax: 82-33-241-0508;
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Abstract
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Slightly acid electrolyzed water (SAcEW) and ultrasound (US) treatment have emerged as an
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environmental-friendly antimicrobial agent. However, SAcEW treatment alone shows low
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antimicrobial efficiency. Therefore, the aim of this study was to develop a hurdle approach that
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combined SAcEW and ultrasound (US) to improve the reduction of Bacillus cereus as well as
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inhibition of the growth on potato. US treatment under different conditions of dip times, acoustic
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energy densities (AED) and temperatures were conducted to obtain the optimal condition. Our
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findings demonstrate that 3 min of US with 400 W/L of AED at 40 ºC treatment (US+40 ºC)
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significantly (p ≤ 0.05) reduced B. cereus population by 2.3±0.1 log CFU/g with minimal change
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in the color of potato. In addition, 3 min of SAcEW (pH, 5.3-5.5; ORP, 958-981 mV; ACC, 28-30
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mg/L) simultaneous with US+40 ºC treatment (SAcEW+US+40 ºC) caused a significant reduction
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in B. cereus by approximately 3.0 log CFU/g. Furthermore, SAcEW+US+40 ºC treatment
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efficiently extended lag time of B. cereus by 0.2-10.5 h, reduced that of specific growth rate by
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0.01-0.23 log CFU/h during storage at temperature from 5 to 35 ºC. Therefore, this combined
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hurdle technology can to improve microbial safety of potato during storage and distribution.
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Keywords: Hurdle technology, Ultrasound, Slightly acidic electrolyzed water, Predictive model,
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Bacillus cereus, Potato
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1. Introduction Bacillus cereus, a ubiquitous spore-forming Gram-positive bacterium, is widely recognized
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as one of the major foodborne pathogens (Rosenquis et al., 2005). This microorganism can survive
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against high temperatures due to formation of spore, and produce two distinct types of toxins
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according to symptoms: emetic type and diarrheal type. Emetic-type illness results from ingesting
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cereulide toxin that is secreted by B. cereus when its population approaches 6.0 CFU/g (King et al.,
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2007; Takeno et al., 2012), whereas diarrheal-type illness is generally induced by consumption of
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food that is contaminated by B. cereus-secreted diarrheal toxin (Kim et al., 2011). Cereulide toxin
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is very stable at the high temperatures and even can remain the toxic activity after heating at
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126 °C for 90 min (King et al., 2007). Similarly, emetic toxin can survive during the cooking
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procedure, such as frying, roasting, and microwave cooking (Agata et al., 2002).
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Potato is the most important vegetable crop and a common staple in the world. Due to its
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abundant starch and valuable nutrition, the consumer`s demand for potato products is rapidly
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increasing year by year. However, it has been reported that potatoes and the related products
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resulted in several foodborne diseases in the Unitized States, the Netherlands and the United
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Kingdom (Doan & Davidson, 2000). Moreover, a foodborne disease that poisoned 450 school
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children was occurred after consumption of mashed potatoes that was contaminated by the level of
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B. cereus ranging from 6.4 to 7.8 log CFU/g (MaEntaffer, 1978). In addition, potato is one of the
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most widely used ingredient in the kitchen, reflexing that potato is likely to be cross-contaminated
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by the contaminated cutting boards or kitchen tools as well as by self-contamination during the
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preparation for cooking.
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toxin capable of heat resistance on cut potato during the preparation for cooking can cause serious
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Thus, the formation of B. cereus spore and the secreted emetic-type
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foodborne illness, highlighting the importance of the presence of this microorganism on potato
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prior to consumption. Hurdle technology is a method to guarantee microbial safety, nutritional quality, and the
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economic viability of food product. Hurdle technology usually works by the simultaneous or
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sequential application of hurdle factors (e.g. heating, freezing, drying, increasing acidity, reducing
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redox potential, etc.). Slightly acidic electrolyzed water (SAcEW), as a hurdle factor, has emerged
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as an environmental-friendly antimicrobial agent offering high antimicrobial effect due to the
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present of a high concentration of hypochlorous acid (HOCl) (Cao et al., 2009). Huang et al. (2008)
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reported that HOCl produced by electrolyzed water can inactivate the microbial cell through
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inhibiting glucose oxidation by chlorine-oxidizing sulfhydryl groups, thus causing the disruption
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of protein synthesis, inhibiting oxygen uptake with formation of toxic N-chlorine derivatives of
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cytosine, thereby resulting in eventual cell death. Recently, the combinations of SAcEW and
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physical treatment methods have been studied in seek of higher efficiency in the reduction of
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bacterial pathogens (Rahman et al., 2010; Koide et al., 2011; Lee et al., 2012; Fereidoun et al.,
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2013a, 2013b; Luo & Oh, 2015, 2016). However, there is less study on the application of SAcEW
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or US or their combined treatments in potatoes. Furthermore, B. cereus was detected in raw potato
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with approximately 3.0 log CFU/g in our preliminary experiment. Therefore, this study presents a
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combined hurdle technology that combines SAcEW and ultrasound treatment to enhance the
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antimicrobial effect of SAcEW in B. cereus on potato, thereby improving microbial safety of
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potato.
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2. Materials and Methods
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2.1. Preparation of inoculum Three strains of Bacillus cereus (ATCC 11778, ATCC14579, and ATCC 21772) were obtained
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from the Department of Food Science; at the University of Georgia (Griffin, GA, USA). The strain
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was maintained at -80 °C in tryptic soy broth (TSB, Difco, Sparks, MD, USA) with 0.6% yeast
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extract (YE, Difco) and 20% glycerol until use. For preparation of the inoculum, the frozen
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suspension was transferred into TSB for overnight incubation at 35 ºC. The cultured strain was
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then streaked onto mannitol egg yolk polymyxin agar (MYP, Difco) with egg yolk enrichment 50%
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and antimicrobic vial P (Difco), and incubated at 37 °C for 24 h. After that, a single colony from
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the incubated plate was transferred to tube filled with 10 mL TSB and incubated at 35 °C for 24 h.
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When the cultures reached the late stationary phase, the cultured strain was centrifuged at 4000 g
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for 10 min at 4 ºC, and the supernatants were decanted. The cell pellets were washed twice with
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0.1% sterile peptone water (PW, Difco) and resuspended in 10 mL of the same solution to obtain a
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final cell concentration of approximately 8.0 log CFU/mL. The inoculum population was
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confirmed by plating an appropriately diluted culture (0.1mL) on the MYP plates and incubating
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at 35 ºC for 24-36 h.
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2.2. Preparation of samples and inoculation Potatoes purchased from a local supermarket (Chuncheon, Korea) were aseptically
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transported to the laboratory and used within 24 h following storage at 4 ºC. Potato samples were
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washed until the adherent dust on the surface cleans up and aseptically cut into pieces of 10 ± 0.2
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g with similar size (3×3 cm) using a sterile knife. The samples were then spot inoculated by 5
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to obtain an initial level of approximately 6.0 log CFU/g. Inoculated samples were air-dried in
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hood for 1 h at 22 ± 2ºC to adhere bacteria on the surface completely, and then immediately
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exposed to sanitizing treatments.
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2.3. Preparation of slightly acidic electrolyzed water (SAcEW)
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SAcEW was produced by electrolysis of a dilute hydrochloric acid solution (6%) in an
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electrolysis device (BIOCIDER, model BC-360, Rvd crop., Gyeonggi, Korea) at a setting of 22.8
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V and 2.5 A. SAcEW with an available chlorine concentration (ACC) of 28-30 mg/L was
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collected at which the stable amperage was reached. The pH, oxidation reduction potential (ORP),
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and ACC of the collected SAcEW were immediately measured before treatment, using a
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dual-scale pH meter (Accumet model 15, Fisher Scientific Corp., Fair Lawn, N.J.) bearing pH and
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ORP electrodes. The ACC was determined by using a digital chlorine test kit (RC-3F, Kasahara
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Chemical Instruments Corp., Saitama, Japan). The collected SAcEW (pH, 5.0-5.2; ORP, 930-950
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mV; ACC, 28-30 mg/L) was stored in polypropylene containers until use.
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2.4. Experimental procedure A complete factorial design (3×3×4) was carried out to evaluate the antimicrobial efficacy of
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ultrasound treatment under different conditions in B. cereus on 10 g of cut potato (3×3 cm). For
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example, the different conditions of dip times (1, 3, and 5 min), temperatures (25, 40, and 60 ºC),
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and acoustic energy densities (AED) (0, 100, 200, and 400 W/L) were carried out in US treatment.
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The group of B. cereus-inoculated potato that were treated at AED of 0 W/L were considered as 6
ACCEPTED MANUSCRIPT the control comparing with US treatment groups. A rectangular tank-type (721×451×297 mm)
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ultrasonic cleaner (JAC-4020, KODO Technical Research Co., Ltd., Hwaseong, Gyeonggi-do,
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South Korea) filled with 6 L of deionized water (DW) was used for US treatment. For factorial
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experiment, the inoculated samples were aseptically placed into the ultrasonic cleaner (KODO
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Technical Research Co., Ltd) fixed at a frequency of 40 kHz and different AED of 0, 100, 200, and
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400 W/L with setting at different temperatures (25, 40, and 60 ºC) prior to the experiment. Due to
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the increasing temperature during US, 4 ºC of sterilized DW was added to maintain at measured
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temperatures. After each factorial experiment, DW was removed and the tank was sterilized by
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ultraviolet light irradiation for 10 min, serially rinsed with 70% ethanol and left to dry. All
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independent factors were replicated three times.
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For combined treatments of SAcEW-US (SAcEW treatment followed by US treatment),
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US-SAcEW (US treatment followed by SAcEW treatment) SAcEW+US (SAcEW treatment
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simultaneous with US treatment), the ultrasonic cleaner (KODO Technical Research Co., Ltd)
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fixed was filled by 6 L of SAcEW with frequency of 40 kHz and AED of 400 W/L at 40 ºC prior to
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treatment. The samples were then aseptically placed into SAcEW-contained ultrasonic cleaner
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After each combined treatment, SAEW was removed and the tank was sterilized by following the
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above procedure in factorial experiment. All combined treatments were conducted in duplicates
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with 3 replicates of each treatment.
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2.5. Sampling B. cereus growth on untreated and treated potato
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10 ± 0.2 g of untreated and treated (SAcEW+US) potato samples (3×3 cm) were placed in a
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stomacher bag (Nasco Whirl-pak, Janesville, WI) with careful mark and isothermally stored in the 7
ACCEPTED MANUSCRIPT incubator at 5, 10, 15, 20, 25, 30, and 35 ºC . Longer sampling intervals were applied at lower
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temperatures, while shorter intervals were chosen for higher temperatures (Ding et al., 2010). For
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homogenization, stomacher bags containing the sample were taken out from the incubator at
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appropriate sampling time, added by 90 mL of 0.1% sterile PW, and homogenized for 2 min in a
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stomacher (Lab-blender 400, Seward, London, UK). After that, aliquots of the homogenate were
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serially diluted in sterile PW and aliquots (0.1 mL) were spread-plated onto the mannitol egg yolk
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polymyxin agar (MYP, Difco) with egg yolk enrichment 50% and antimicrobic vial P (Difco). All
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plates were incubated at 37 ºC for 24 h, and expressed as log CFU/g. All experiments were
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performed three times.
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2.6. Model development and validation
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Bacterial numbers in log CFU/g were used for statistical analysis. The growth parameters,
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specific growth rate (SGR [log CFU/h]) and lag time (LT [h]), were estimated by fitting the treated
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and untreated (control) growth data using the Baranyi model (Ep. 1) (Baranyi & Roberts, 1994).
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The growth curves were fitted using the DMFit Add-In software in Excel, Institute of Food
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Research, Norwich, UK.
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= + −
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1 +
− 1 −
Eq.1
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= + 1 − + −ℎ − − − ℎ
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where Y(t) is the microbial count in units of log CFU/g at time, t (h), Y0 is the logarithm of initial
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microbial count (log CFU/g), Ymax is the logarithm of maximum microbial count (log CFU/g), m
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characterizes the curvature before the stationary phase, A(t) is a rescaling of t, µ is the specific
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growth rate (log CFU/h), υ is the rate of increase of the limiting substrate, assumed to be equal to 8
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µ, λ is the lag phase duration (h), and h0 is the physiological state of the microorganism under
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consideration. The obtained SGR and LT values were analyzed in SPSS version 21.0 (Statistical Package for
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the Social Sciences, Chicago, IL, USA) to develop a square root model and natural logarithm
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model (Eqs. 2 and 3) using the following equation (Delignette-Muller et al., 1995):
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= −
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Eq. 2
Ln# = −
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where µ max is the specific growth rate, λ is the lag time, b is the slope of the regression line, T is
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the temperature (ºC), and T0 is a notional minimum temperature for growth.
Eq. 3
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In this study, the goodness-of-fit of the models was evaluated by the coefficients of
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determination (R2) and the adjusted determination coefficient (R2Adj). In addition, the bias factor
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(Bf) (Eq. 4), the accuracy factor (Af) (Eq. 5), and % standard error of prediction (%SEP) (Eq. 6)
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were used to compare the differences between the observed data and the predicted data obtained
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by the developed models in order to validate the preformation of the models.
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$% = 10
% = 10
∑ )*+,-./⁄*01 4 3
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∑|)*+,-./⁄*01| 4 3
Eq.5
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%789 = :;<=;>?@ A
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where n is the number of observations, obs is the observed value, pred is the predicted value, p is
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the number of model parameters.
∑>?@BC<;DE
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2.7. Statistical analysis Means of bacterial populations (log CFU/g) were calculated from three replications (each 9
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interval consisted of two plates) of each experiment. Data were tested by one factor analysis of
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variance (ANOVA) using the SPSS statistical package. Differences between sample means were
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analyzed according to Tukey test.
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3. Results
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3.1. Bacteriological analyses of potato
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In this study, the microbiological quality of certain potato samples with respect to aerobic,
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coliform, and pathogenic microorganisms were determined as background information. Table 1
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reveals that the mean of total bacterial count and coliforms in potato samples were 3.34 and 1.22
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CFU/g, respectively. Escherichia coli, Listeria monocytogenes, Samonella spp., and
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Staphylococcus aureus were not detected with 1.0 log CFU/g of detection limit. However, a mean
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of 3.0 log CFU/g B. cereus was isolated in potato samples.
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3.2. Efficacy of ultrasound at different temperature Initial population of B. cereus was approximately 6.0 log CFU/g in the samples. The
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antimicrobial effect of DW and US treatment at different conditions against B. cereus on potato
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are listed in Table 2. In DW treatment, there was no significant difference (p > 0.05) in the
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bacterial reduction between 40 and 60 ºC. Regardless of temperatures, US treatment with 400 W/L
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of AED efficiently reduced (p ≤ 0.05) the number of B. cereus on potato by the reduction ranging
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from 0.19 to 1.45 log CFU/g, when compared to those of treatment without US and of US
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treatment with 100 W/L and 200 W/L of AED. Similarly, the bacterial reductions were increased
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by increasing temperature from 25 ºC to 60 ºC. At 40 and 60 ºC, the bacterial reductions at 1 min
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significantly different (p > 0.05) between 3 and 5 min regardless of the AED. The bacterial
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reductions caused by US treatment at 60 ºC were higher than those at 25 and 40 ºC. However, a
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significant change (p ≤ 0.05) in the color of potato was observed when the temperature was
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increased up to 60 ºC (data not shown). Therefore, the US treatment with the setting of at 400 W/L,
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40 °C, and 3 min was chosen as the optimal conditions and derived in subsequent experiment.
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The antimicrobial effect of DW (control), SAcEW, SAcEW followed by US (SAcEW-US),
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US followed by SAcEW (US-SAcEW), and SAcEW simultaneous with ultrasound (SAcEW+US)
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treatments against B. cereus on potato are shown in Fig. 2. The bacterial reductions achieved by
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SAcEW or ultrasound or their combination treatment were significantly greater (p ≤ 0.05) than
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those of control. The antimicrobial effects of combination treatments were significantly greater
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that (p ≤ 0.05) those of individual treatments. Fig. 2 shows that SAcEW+US treatment caused
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approximately 3.0 log CFU/g reduction in B. cereus which is significantly greater (p ≤ 0.05) than
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those of SAcEW-US and US-SAcEW treatments. Therefore, SAcEW+US treatment was derived
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in bacterial growth experiment.
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3.4. Effect of SAcEW+US treatment on growth of B. cereus on the potato
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The growth of B. cereus on untreated and treated (SAcEW+US) potatoes during storage at
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different temperatures (5, 10, 15, 20, 25, 30, and 35 ºC) are shown in Fig. 3. The initial population
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of B. cereus on untreated and treated potatoes were 5.8 and 3.4 log CFU/g, respectively. As shown 11
ACCEPTED MANUSCRIPT in Fig. 3, all the isothermal growth curves clearly exhibited the lag period, exponential growth and
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stationary phases. The populations of B. cereus on treated (SAcEW+US) potato at the end of
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storage were lower than that of control regardless of storage temperature (Fig. 3). All raw
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experimental data were fitted into the Baranyi model to calculate specific growth rate (SGR, log
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CFU/h) and lag time (LT, h) of B. cereus growth on untreated and treated (SAcEW+US) potato at
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different storage temperatures, and the estimated growth parameters are listed in Table 3. The
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Baranyi model provided a good statistical fitness based on a high correlation coefficient (R2
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0.97). As expected, an increased SGR and decreased LT were observed with increasing
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temperature. The results show that the SGR values of B. cereus growth on treated potatoes were
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lower than those of untreated potatoes, whereas the greater LT values were observed in treated
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group. Subsequently, the secondary models were developed to describe the variation of SGR and
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LT values that were obtained from untreated and treated potatoes as a function of temperature,
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using equations (2) and (3) (Table 4). Bias (Bf) and accuracy (Af) factors of developed SGR and
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LT models were at a range from 1.00 to 1.07 and from 1.02 to 1.08 (Table 4). The proportion of
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standard error of prediction (%SEP) for the corresponding SGR and LT models were from 2.04%
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to 8.24%, respectively.
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4. Discussion
In our preliminary experiment, B. cereus was detected in potato without E. coli, L.
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monocytogenes, Samonella spp., and S. aureus (Table 1). The previous study on the monitoring of
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foodborne pathogens in agricultural products reported that 4.6 log CFU/g of B. cereus was
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detected in potato (Thapa et al., 2008). The current study was, therefore, conducted to develop a 12
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potato. Table 2 shows that antimicrobial effect of US treatment was efficiently (p ≤ 0.05) increased
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in terms of increasing the temperature. The increased reductions might be due to the enhancement
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of heat transfer by cavitation bubble generated during ultrasound treatment (Kim et al., 2004; Liu
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et al., 2014), thus causing the increased sterility efficacy on the surface of products. Furthermore,
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Earnshaw et al. (1995) stated that ultrasound combined with heat treatment was more effective in
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the cell death when compared to ultrasound treatment only. However, the color of potatoes treated
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by US at 60 ºC were significantly changed (p ≤ 0.05) (data not shown). In addition, previous
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studies related to electrolyzed water suggest that 3 min is the best dip time while using as sanitizer
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(Ding et al., 2010; Forghani et al., 2013; Huang et al., 2006; Nan et al., 2010; Rahman et al., 2013).
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Therefore, US treatment at 40 ºC for 3 min was considered as optimal condition, and applied in
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subsequent experiment that evaluated the antimicrobial effect of different combined treatments
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(DW, SAcEW, SAcEW-US, US-SAcEW, SAcEW+US) (Fig. 2).
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Fig. 2 shows that SAcEW treatment alone caused approximately 2.3 log CFU/g bacterial
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reduction in B. cereus on potato. Mansur et al. (2015) reported that treatment with SAcEW (pH
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6.29, ACC 30 mg/l) at 40 ºC for 3 min reduced the number of E. coli O157:H7, L. monocytogenes,
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S. aureus, and S. Typhimurium on pork with the range of 1.2 to 1.8 log CFU/g. It has been
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reported that treatment with SAcEW (pH 6.0-6.5, ACC 20 m/l) for 3 min caused the bacterial
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reductions of 1.3 to 1.6 log CFU/g in Salmonella enteritidis on mung bean seeds and sprouts
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(Zhang et al., 2011). In comparison with the bacterial reduction reported by above studies, a
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higher antimicrobial effect of SAcEW was observed in this study. It might be mainly due to the
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differences in the structure of samples surface, therefore causing that chemical sanitizers are
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occasionally unsuccessful against bacteria which are tightly adhered on the corner in where the
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aqueous sanitizers are difficult to immerse (São José et al., 2014a). The SAcEW simultaneous with ultrasound treatment at 40 °C for 3 min achieved the
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approximately 3.0 log CFU/g reduction in B. cereus adherent on potato (Fig. 2). A number of
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studies have reported that ultrasound enables to enhance antimicrobial effect of chemical sanitizer
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(Mañas et al., 2000; Scouten & Beuchat, 2002; Baumann et al., 2005; Sagong et al., 2011; São
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José & Vanetti, 2012; Van Poucke et al., 2012; Luo & Oh, 2015, 2016). São José et al. (2014b)
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demonstrated that the combined treatment of US (40 kHz) and 1 % lactic acid at room temperature
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for 3 min resulted in 2.9 log CFU/g reduction in Salmonella enterica Enteritidis on green pepper.
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Furthermore, Forghani et al. (2013) reported that combined treatment of low concentration
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electrolyzed water (LcEW) and US (40 kHz) at 40 °C for 3 min caused a 2.4 log CFU/g reduction
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in E. coli O157:H7 on lettuce. This might be due to the generated high temperature and pressure
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during ultrasound treatment, which is able to damage the cell wall, promoting penetration of the
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sanitizers through the cell wall, thereby killing the microorganisms more quickly and thoroughly
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(Lillard et al., 1993; Piyasena et al., 2003; Sagong et al., 2011).
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The growth of B. cereus on potato was efficiently inhibited by SAcEW+US treatment at each
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measured temperature by not only decreasing LT but also increasing SGR (Table 3). Baranyi and
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Roberts (1994) stated that the cell stressed by treatment had lower metabolic activity compared to
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that of untreated cell, resulting in longer LT of treated cell. Also, the previously reported evidences
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demonstrated that chemical sanitizer combined with ultrasound was capable of extending the shelf
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life of food product by controlling the bacterial growth during the storage (Forghani et al., 2013;
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Yang et al., 2011).
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40 °C for 3 min caused synergic effects against B. ceruse on potato, as well as inhibition of the
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growth of B. ceruse during storage at different temperatures from 5 to 35 °C. Therefore, this
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hurdle approach can offer practical application in food industry to improve microbial safety of
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potato during storage or distribution. In addition, the obtained parameters and developed models
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can be used to set up the critical control points (CCP) in Hazard Analysis and Critical Control
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Point (HACCP) or the Quantitative Microbial Risk Assessment (QMRA) related to B. ceruse
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growth on potato.
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References
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São José, J. F. B. D., Andrade, N. J. D., Ramos, A. M., Vanetti, M. C. D., Stringheta, P. C., Chaves,
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ultrasound and organic acids. Int. J. Food Microbiol. 190, 9-13.
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Scouten, A. J., Beuchat, L. R., 2002. Combined effects of chemical, heat and ultrasound
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treatments to kill Salmonella and Escherichia coli O157:H7 on alfalfa seeds. J. Appl.
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Microbiol. 92, 668-674. 19
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Ogasawara, N., Hayashi, T., Shimizu, T., Kuhara, S., Hattori, M., Ohta, M., 2012. Complete
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Genome Sequence of Bacillus cereus NC7401, which produces high levels of the emetic
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toxin cereulide. J.Bacteriol. 194, 4767-4768.
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Thapa, S. P., Kim S. S., Hong S. S., Park D. S., Lim, C. K., Hur, J. H., 2008. Monitoring of
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bacterial pathogens in agricultural products and environments at farms in Korea. J. Appl. Biol.
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Chem. 51, 128-135.
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Van Poucke, K., Monbaliu, S., Munaut, F., Heungens, K., De Saeger, S., Van Hove, F., 2012.
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Genetic diversity and mycotoxin production of Fusarium lactis species complex isolates
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from sweet pepper. Int. J. Food Microbiol. 153, 28-37.
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Yang, Z., Cao, S., Cai, Y., Zheng, Y., 2011. Combination of salicylic acid and ultrasound to control
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postharvest blue mould caused by Penicillium expansum in peach fruit. Innov. food sci.
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emerg. technol. 12, 310-314.
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Zhang, C., Lu, Z., Li, Y., Shang, Y., Zhang, G., Cao, W., 2011. Reduction of Escherichia coli
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O157:H7 and Salmonella Enteritidis on mung bean seeds and sprouts by slightly acidic
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electrolyzed water. Food control. 22, 792-796.
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Figure Legends
424 Fig. 1. Hurdle technology procedure. (1) DW, deionized water; (2) US, ultrasound; (3) SAcEW, slightly
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acidic electrolyzed water; (4) SAcEW-US: SAcEW treatment followed by ultrasound treatment; (5)
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US-SAcEW, ultrasound treatment followed by SAcEW treatment; (6) SAcEW+US: ultrasound treatment
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combined with SAcEW treatment.
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Fig. 2. Inactivation effect of various decontamination treatments on Bacillus cereus in potato: DW,
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sterilized sistilled water (40 ºC, 3min). US, ultrasound (40 ºC, 3min); SAcEW, slightly acidic electrolyzed
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water (40 ºC, 3min); US-SAcEW, ultrasound treatment followed by SAEW treatment (40 ºC, 3min);
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SAcEW-US, SAEW treatment followed by ultrasound treatment (40 ºC, 3min); SAcEW+US, ultrasound
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treatment combined with SAEW treatment (40 ºC, 3min).
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Fig. 3. Effect of treatment with SAcEW+US on the growth of B. cereus on potato during storage at
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different temperatures. The following symbols are recommended: (○) the growth of B. cereus on untreated
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potato (control), (▲) the growth of B. cereus on potato by SAcEW+US.
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ACCEPTED MANUSCRIPT Table 1. Bacteriological analyses of potato
Micro-organisms
bulbs and tubers Potato (log CFU/g) 3.4±0.03 1.2±0.02 N.D* 3.1±0.03 N.D N.D N.D
Aerobic plate counts Total coliforms Escherichia coli Bacillus cereus Listeria monocytogenes Samonella spp. Staphylococcus aureus *
No detected
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ACCEPTED MANUSCRIPT Table 2. Effect of ultrasound on the reduction of B. cereus (means ± standard deviation; log CFU/g) inoculated on potato.
DW
US (100 W/L)
US (200 W/L)
*
1 3 5 1 3 5 1 3 5 1 3 5
40 ºC f‡
B 0.55 ± 0.03 B0.59 ± 0.04ef B0.60 ± 0.04ef C0.61 ± 0.03def C0.62 ± 0.03def C0.64 ± 0.08cdef C0.65 ± 0.05cde C0.66 ± 0.03bcde C0.70 ± 0.06abcd C0.72 ± 0.03abc C0.74 ± 0.09ab C0.79 ± 0.07a
*
60 ºC h
A0.69 ± 0.07 A1.49 ± 0.13d A1.51 ± 0.09d B0.85 ± 0.06g B1.11 ± 0.08f B1.31 ± 0.11e B1.35 ± 0.13e B1.61 ± 0.18c B1.74 ± 0.14c B1.90 ± 0.09b B2.32 ± 0.11a B2.45 ± 0.15a
A0.71 ± 0.06g A1.51 ± 0.09ef A1.53 ± 0.11ef A1.40 ± 0.15f A1.53 ± 0.14ef A1.60 ± 0.11e A1.75 ± 0.13d A1.95 ± 0.16c A2.01 ± 0.09c A2.28 ± 0.15b A2.91 ± 0.24a A2.98 ± 0.17a
Numbers within each row followed by different capital letters are significantly different (P ≤ 0.05). Numbers within each column followed by different small letters are significantly different (P ≤ 0.05). ‡ Treatment with sterilized deionized water (DW), ultrasound (US) at the AED of 100, 200, and 400 W/L.
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Dipping time (min)
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ACCEPTED MANUSCRIPT Table 3. Estimation of the specific growth rate (SGR) and lag time (LT) of B. cereus on untreated and
Temperature (ºC)
Control
SAcEW+US
R2
4 10
0.02e* 0.03e
0.01e 0.02e
82.1a 32.0b
91.6a 39.5b
0.991 0.973
15 20
0.12d 0.22c
0.07d
12.3c
15.6c
0.986
c
11.9
d
d
25 30
b
0.23 0.45a
b
0.12 0.23a
e
5.1 4.4e
0.978 0.992
35
a
a
e
0.46
0.11
0.24
treated potato at different storage temperatures. *
4.2
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13.3 d
6.0 4.8d 4.4
d
0.991 0.996
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Control
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LT (h)
SGR (log CFU/h)
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ACCEPTED MANUSCRIPT Table 4. Secondary models for B. cereus growth on water washed (control) and treated (SAcEW+US) potato.
(400W/L, 40° C, 3min)
†
ඥܵ = §ܴܩ0.021ܶ − 0.011
1.01
1.03
2.04
݊ܮሺ ¶ ܶܮሻ = −0.097ܶ + 4.407
1.07
1.08
7.13
ඥܵ = ܴܩ0.013ܶ + 0.045
1.00
1.02
3.11
݊ܮሺܶܮሻ = −0.131ܶ + 4.554
1.02
1.05
8.24
Bf
*
Bias factor. Accuracy factor. ‡ Proportion of standard error of prediction § Specific growth rate, SGR (log CFU/h). ¶ Lag time, LT (h).
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Af
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%SEP
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*
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Control
Equations
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S
S
W
U
cE
-U
1
0
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A
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W
c
S
-S
cE
cE
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S
A
A
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U
S
S
S
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M e a n re d u c tio n (lo g C F U /g )
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c d
b
2 a
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9
9
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7
L o g C F U /g
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6 5
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3 0
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20
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Fig. 3.
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L o g C F U /g
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ACCEPTED MANUSCRIPT 1. Bacillus cereus was detected in raw potato with approximately 3.0 log CFU/g。 2. SAcEW combined with US treatment caused synergistic effect against B. cereus. 3. Antimicrobial effect was enhanced by increasing acoustic energy density in ultrasound treatment.
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4. SAcEW+US+40 ºC treatment not only extended the lag time but also reduced specific growth
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rate of B. cereus.
S0023-6438(16)30200-6
DOI:
10.1016/j.lwt.2016.04.016
Reference:
YFSTL 5404
To appear in:
LWT - Food Science and Technology
Received Date: 16 January 2016 Revised Date:
7 March 2016
Accepted Date: 11 April 2016
Please cite this article as: Luo, K., Kim, S.Y., Wang, J., Oh, D.-H., A combined hurdle approach of slightly acidic electrolyzed water simultaneous with ultrasound to inactivate on potato, LWT - Food Science and Technology (2016), doi: 10.1016/j.lwt.2016.04.016. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1
A combined hurdle approach of slightly acidic electrolyzed water simultaneous with
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ultrasound to inactivate Bacillus cereus on potato
3 Ke Luo1, Seon Young Kim1, Jun Wang2, and Deog-Hwan Oh1,*
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Kangwon National University, Chuncheon, Gangwon 200-701, Korea
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Department of Food Science and Biotechnology and Institute of Bioscience and Biotechnology,
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266-109, China
College of Food Science and Engineering, Qingdao Agricultural University, Qingdao, Shandong
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*
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E-mail: [email protected] (Deog-Hwan Oh)
Author for correspondence. Tel: 82-33-250-6457; Fax: 82-33-241-0508;
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Abstract
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Slightly acid electrolyzed water (SAcEW) and ultrasound (US) treatment have emerged as an
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environmental-friendly antimicrobial agent. However, SAcEW treatment alone shows low
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antimicrobial efficiency. Therefore, the aim of this study was to develop a hurdle approach that
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combined SAcEW and ultrasound (US) to improve the reduction of Bacillus cereus as well as
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inhibition of the growth on potato. US treatment under different conditions of dip times, acoustic
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energy densities (AED) and temperatures were conducted to obtain the optimal condition. Our
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findings demonstrate that 3 min of US with 400 W/L of AED at 40 ºC treatment (US+40 ºC)
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significantly (p ≤ 0.05) reduced B. cereus population by 2.3±0.1 log CFU/g with minimal change
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in the color of potato. In addition, 3 min of SAcEW (pH, 5.3-5.5; ORP, 958-981 mV; ACC, 28-30
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mg/L) simultaneous with US+40 ºC treatment (SAcEW+US+40 ºC) caused a significant reduction
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in B. cereus by approximately 3.0 log CFU/g. Furthermore, SAcEW+US+40 ºC treatment
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efficiently extended lag time of B. cereus by 0.2-10.5 h, reduced that of specific growth rate by
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0.01-0.23 log CFU/h during storage at temperature from 5 to 35 ºC. Therefore, this combined
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hurdle technology can to improve microbial safety of potato during storage and distribution.
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Keywords: Hurdle technology, Ultrasound, Slightly acidic electrolyzed water, Predictive model,
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Bacillus cereus, Potato
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1. Introduction Bacillus cereus, a ubiquitous spore-forming Gram-positive bacterium, is widely recognized
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as one of the major foodborne pathogens (Rosenquis et al., 2005). This microorganism can survive
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against high temperatures due to formation of spore, and produce two distinct types of toxins
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according to symptoms: emetic type and diarrheal type. Emetic-type illness results from ingesting
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cereulide toxin that is secreted by B. cereus when its population approaches 6.0 CFU/g (King et al.,
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2007; Takeno et al., 2012), whereas diarrheal-type illness is generally induced by consumption of
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food that is contaminated by B. cereus-secreted diarrheal toxin (Kim et al., 2011). Cereulide toxin
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is very stable at the high temperatures and even can remain the toxic activity after heating at
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126 °C for 90 min (King et al., 2007). Similarly, emetic toxin can survive during the cooking
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procedure, such as frying, roasting, and microwave cooking (Agata et al., 2002).
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Potato is the most important vegetable crop and a common staple in the world. Due to its
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abundant starch and valuable nutrition, the consumer`s demand for potato products is rapidly
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increasing year by year. However, it has been reported that potatoes and the related products
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resulted in several foodborne diseases in the Unitized States, the Netherlands and the United
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Kingdom (Doan & Davidson, 2000). Moreover, a foodborne disease that poisoned 450 school
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children was occurred after consumption of mashed potatoes that was contaminated by the level of
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B. cereus ranging from 6.4 to 7.8 log CFU/g (MaEntaffer, 1978). In addition, potato is one of the
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most widely used ingredient in the kitchen, reflexing that potato is likely to be cross-contaminated
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by the contaminated cutting boards or kitchen tools as well as by self-contamination during the
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preparation for cooking.
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toxin capable of heat resistance on cut potato during the preparation for cooking can cause serious
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Thus, the formation of B. cereus spore and the secreted emetic-type
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foodborne illness, highlighting the importance of the presence of this microorganism on potato
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prior to consumption. Hurdle technology is a method to guarantee microbial safety, nutritional quality, and the
71
economic viability of food product. Hurdle technology usually works by the simultaneous or
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sequential application of hurdle factors (e.g. heating, freezing, drying, increasing acidity, reducing
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redox potential, etc.). Slightly acidic electrolyzed water (SAcEW), as a hurdle factor, has emerged
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as an environmental-friendly antimicrobial agent offering high antimicrobial effect due to the
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present of a high concentration of hypochlorous acid (HOCl) (Cao et al., 2009). Huang et al. (2008)
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reported that HOCl produced by electrolyzed water can inactivate the microbial cell through
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inhibiting glucose oxidation by chlorine-oxidizing sulfhydryl groups, thus causing the disruption
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of protein synthesis, inhibiting oxygen uptake with formation of toxic N-chlorine derivatives of
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cytosine, thereby resulting in eventual cell death. Recently, the combinations of SAcEW and
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physical treatment methods have been studied in seek of higher efficiency in the reduction of
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bacterial pathogens (Rahman et al., 2010; Koide et al., 2011; Lee et al., 2012; Fereidoun et al.,
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2013a, 2013b; Luo & Oh, 2015, 2016). However, there is less study on the application of SAcEW
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or US or their combined treatments in potatoes. Furthermore, B. cereus was detected in raw potato
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with approximately 3.0 log CFU/g in our preliminary experiment. Therefore, this study presents a
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combined hurdle technology that combines SAcEW and ultrasound treatment to enhance the
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antimicrobial effect of SAcEW in B. cereus on potato, thereby improving microbial safety of
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potato.
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2. Materials and Methods
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2.1. Preparation of inoculum Three strains of Bacillus cereus (ATCC 11778, ATCC14579, and ATCC 21772) were obtained
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from the Department of Food Science; at the University of Georgia (Griffin, GA, USA). The strain
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was maintained at -80 °C in tryptic soy broth (TSB, Difco, Sparks, MD, USA) with 0.6% yeast
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extract (YE, Difco) and 20% glycerol until use. For preparation of the inoculum, the frozen
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suspension was transferred into TSB for overnight incubation at 35 ºC. The cultured strain was
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then streaked onto mannitol egg yolk polymyxin agar (MYP, Difco) with egg yolk enrichment 50%
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and antimicrobic vial P (Difco), and incubated at 37 °C for 24 h. After that, a single colony from
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the incubated plate was transferred to tube filled with 10 mL TSB and incubated at 35 °C for 24 h.
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When the cultures reached the late stationary phase, the cultured strain was centrifuged at 4000 g
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for 10 min at 4 ºC, and the supernatants were decanted. The cell pellets were washed twice with
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0.1% sterile peptone water (PW, Difco) and resuspended in 10 mL of the same solution to obtain a
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final cell concentration of approximately 8.0 log CFU/mL. The inoculum population was
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confirmed by plating an appropriately diluted culture (0.1mL) on the MYP plates and incubating
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at 35 ºC for 24-36 h.
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2.2. Preparation of samples and inoculation Potatoes purchased from a local supermarket (Chuncheon, Korea) were aseptically
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transported to the laboratory and used within 24 h following storage at 4 ºC. Potato samples were
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washed until the adherent dust on the surface cleans up and aseptically cut into pieces of 10 ± 0.2
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g with similar size (3×3 cm) using a sterile knife. The samples were then spot inoculated by 5
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to obtain an initial level of approximately 6.0 log CFU/g. Inoculated samples were air-dried in
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hood for 1 h at 22 ± 2ºC to adhere bacteria on the surface completely, and then immediately
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exposed to sanitizing treatments.
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2.3. Preparation of slightly acidic electrolyzed water (SAcEW)
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SAcEW was produced by electrolysis of a dilute hydrochloric acid solution (6%) in an
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electrolysis device (BIOCIDER, model BC-360, Rvd crop., Gyeonggi, Korea) at a setting of 22.8
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V and 2.5 A. SAcEW with an available chlorine concentration (ACC) of 28-30 mg/L was
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collected at which the stable amperage was reached. The pH, oxidation reduction potential (ORP),
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and ACC of the collected SAcEW were immediately measured before treatment, using a
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dual-scale pH meter (Accumet model 15, Fisher Scientific Corp., Fair Lawn, N.J.) bearing pH and
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ORP electrodes. The ACC was determined by using a digital chlorine test kit (RC-3F, Kasahara
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Chemical Instruments Corp., Saitama, Japan). The collected SAcEW (pH, 5.0-5.2; ORP, 930-950
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mV; ACC, 28-30 mg/L) was stored in polypropylene containers until use.
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2.4. Experimental procedure A complete factorial design (3×3×4) was carried out to evaluate the antimicrobial efficacy of
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ultrasound treatment under different conditions in B. cereus on 10 g of cut potato (3×3 cm). For
129
example, the different conditions of dip times (1, 3, and 5 min), temperatures (25, 40, and 60 ºC),
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and acoustic energy densities (AED) (0, 100, 200, and 400 W/L) were carried out in US treatment.
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The group of B. cereus-inoculated potato that were treated at AED of 0 W/L were considered as 6
ACCEPTED MANUSCRIPT the control comparing with US treatment groups. A rectangular tank-type (721×451×297 mm)
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ultrasonic cleaner (JAC-4020, KODO Technical Research Co., Ltd., Hwaseong, Gyeonggi-do,
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South Korea) filled with 6 L of deionized water (DW) was used for US treatment. For factorial
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experiment, the inoculated samples were aseptically placed into the ultrasonic cleaner (KODO
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Technical Research Co., Ltd) fixed at a frequency of 40 kHz and different AED of 0, 100, 200, and
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400 W/L with setting at different temperatures (25, 40, and 60 ºC) prior to the experiment. Due to
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the increasing temperature during US, 4 ºC of sterilized DW was added to maintain at measured
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temperatures. After each factorial experiment, DW was removed and the tank was sterilized by
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ultraviolet light irradiation for 10 min, serially rinsed with 70% ethanol and left to dry. All
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independent factors were replicated three times.
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For combined treatments of SAcEW-US (SAcEW treatment followed by US treatment),
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US-SAcEW (US treatment followed by SAcEW treatment) SAcEW+US (SAcEW treatment
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simultaneous with US treatment), the ultrasonic cleaner (KODO Technical Research Co., Ltd)
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fixed was filled by 6 L of SAcEW with frequency of 40 kHz and AED of 400 W/L at 40 ºC prior to
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treatment. The samples were then aseptically placed into SAcEW-contained ultrasonic cleaner
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After each combined treatment, SAEW was removed and the tank was sterilized by following the
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above procedure in factorial experiment. All combined treatments were conducted in duplicates
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with 3 replicates of each treatment.
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2.5. Sampling B. cereus growth on untreated and treated potato
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10 ± 0.2 g of untreated and treated (SAcEW+US) potato samples (3×3 cm) were placed in a
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stomacher bag (Nasco Whirl-pak, Janesville, WI) with careful mark and isothermally stored in the 7
ACCEPTED MANUSCRIPT incubator at 5, 10, 15, 20, 25, 30, and 35 ºC . Longer sampling intervals were applied at lower
155
temperatures, while shorter intervals were chosen for higher temperatures (Ding et al., 2010). For
156
homogenization, stomacher bags containing the sample were taken out from the incubator at
157
appropriate sampling time, added by 90 mL of 0.1% sterile PW, and homogenized for 2 min in a
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stomacher (Lab-blender 400, Seward, London, UK). After that, aliquots of the homogenate were
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serially diluted in sterile PW and aliquots (0.1 mL) were spread-plated onto the mannitol egg yolk
160
polymyxin agar (MYP, Difco) with egg yolk enrichment 50% and antimicrobic vial P (Difco). All
161
plates were incubated at 37 ºC for 24 h, and expressed as log CFU/g. All experiments were
162
performed three times.
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2.6. Model development and validation
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Bacterial numbers in log CFU/g were used for statistical analysis. The growth parameters,
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specific growth rate (SGR [log CFU/h]) and lag time (LT [h]), were estimated by fitting the treated
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and untreated (control) growth data using the Baranyi model (Ep. 1) (Baranyi & Roberts, 1994).
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The growth curves were fitted using the DMFit Add-In software in Excel, Institute of Food
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Research, Norwich, UK.
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= + −
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1 +
− 1 −
Eq.1
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= + 1 − + −ℎ − − − ℎ
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where Y(t) is the microbial count in units of log CFU/g at time, t (h), Y0 is the logarithm of initial
173
microbial count (log CFU/g), Ymax is the logarithm of maximum microbial count (log CFU/g), m
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characterizes the curvature before the stationary phase, A(t) is a rescaling of t, µ is the specific
175
growth rate (log CFU/h), υ is the rate of increase of the limiting substrate, assumed to be equal to 8
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µ, λ is the lag phase duration (h), and h0 is the physiological state of the microorganism under
177
consideration. The obtained SGR and LT values were analyzed in SPSS version 21.0 (Statistical Package for
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the Social Sciences, Chicago, IL, USA) to develop a square root model and natural logarithm
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model (Eqs. 2 and 3) using the following equation (Delignette-Muller et al., 1995):
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= −
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Eq. 2
Ln# = −
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where µ max is the specific growth rate, λ is the lag time, b is the slope of the regression line, T is
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the temperature (ºC), and T0 is a notional minimum temperature for growth.
Eq. 3
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In this study, the goodness-of-fit of the models was evaluated by the coefficients of
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determination (R2) and the adjusted determination coefficient (R2Adj). In addition, the bias factor
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(Bf) (Eq. 4), the accuracy factor (Af) (Eq. 5), and % standard error of prediction (%SEP) (Eq. 6)
188
were used to compare the differences between the observed data and the predicted data obtained
189
by the developed models in order to validate the preformation of the models.
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$% = 10
% = 10
∑ )*+,-./⁄*01 4 3
'
Eq.4
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∑|)*+,-./⁄*01| 4 3
Eq.5
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%789 = :;<=;>?@ A
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where n is the number of observations, obs is the observed value, pred is the predicted value, p is
194
the number of model parameters.
∑>?@BC<;DE
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Eq.8
195 196 197
2.7. Statistical analysis Means of bacterial populations (log CFU/g) were calculated from three replications (each 9
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interval consisted of two plates) of each experiment. Data were tested by one factor analysis of
199
variance (ANOVA) using the SPSS statistical package. Differences between sample means were
200
analyzed according to Tukey test.
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3. Results
203
3.1. Bacteriological analyses of potato
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In this study, the microbiological quality of certain potato samples with respect to aerobic,
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coliform, and pathogenic microorganisms were determined as background information. Table 1
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reveals that the mean of total bacterial count and coliforms in potato samples were 3.34 and 1.22
207
CFU/g, respectively. Escherichia coli, Listeria monocytogenes, Samonella spp., and
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Staphylococcus aureus were not detected with 1.0 log CFU/g of detection limit. However, a mean
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of 3.0 log CFU/g B. cereus was isolated in potato samples.
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3.2. Efficacy of ultrasound at different temperature Initial population of B. cereus was approximately 6.0 log CFU/g in the samples. The
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antimicrobial effect of DW and US treatment at different conditions against B. cereus on potato
214
are listed in Table 2. In DW treatment, there was no significant difference (p > 0.05) in the
215
bacterial reduction between 40 and 60 ºC. Regardless of temperatures, US treatment with 400 W/L
216
of AED efficiently reduced (p ≤ 0.05) the number of B. cereus on potato by the reduction ranging
217
from 0.19 to 1.45 log CFU/g, when compared to those of treatment without US and of US
218
treatment with 100 W/L and 200 W/L of AED. Similarly, the bacterial reductions were increased
219
by increasing temperature from 25 ºC to 60 ºC. At 40 and 60 ºC, the bacterial reductions at 1 min
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221
significantly different (p > 0.05) between 3 and 5 min regardless of the AED. The bacterial
222
reductions caused by US treatment at 60 ºC were higher than those at 25 and 40 ºC. However, a
223
significant change (p ≤ 0.05) in the color of potato was observed when the temperature was
224
increased up to 60 ºC (data not shown). Therefore, the US treatment with the setting of at 400 W/L,
225
40 °C, and 3 min was chosen as the optimal conditions and derived in subsequent experiment.
226 3.3. Efficacy of different combination treatments
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The antimicrobial effect of DW (control), SAcEW, SAcEW followed by US (SAcEW-US),
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US followed by SAcEW (US-SAcEW), and SAcEW simultaneous with ultrasound (SAcEW+US)
230
treatments against B. cereus on potato are shown in Fig. 2. The bacterial reductions achieved by
231
SAcEW or ultrasound or their combination treatment were significantly greater (p ≤ 0.05) than
232
those of control. The antimicrobial effects of combination treatments were significantly greater
233
that (p ≤ 0.05) those of individual treatments. Fig. 2 shows that SAcEW+US treatment caused
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approximately 3.0 log CFU/g reduction in B. cereus which is significantly greater (p ≤ 0.05) than
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those of SAcEW-US and US-SAcEW treatments. Therefore, SAcEW+US treatment was derived
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in bacterial growth experiment.
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3.4. Effect of SAcEW+US treatment on growth of B. cereus on the potato
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The growth of B. cereus on untreated and treated (SAcEW+US) potatoes during storage at
240
different temperatures (5, 10, 15, 20, 25, 30, and 35 ºC) are shown in Fig. 3. The initial population
241
of B. cereus on untreated and treated potatoes were 5.8 and 3.4 log CFU/g, respectively. As shown 11
ACCEPTED MANUSCRIPT in Fig. 3, all the isothermal growth curves clearly exhibited the lag period, exponential growth and
243
stationary phases. The populations of B. cereus on treated (SAcEW+US) potato at the end of
244
storage were lower than that of control regardless of storage temperature (Fig. 3). All raw
245
experimental data were fitted into the Baranyi model to calculate specific growth rate (SGR, log
246
CFU/h) and lag time (LT, h) of B. cereus growth on untreated and treated (SAcEW+US) potato at
247
different storage temperatures, and the estimated growth parameters are listed in Table 3. The
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Baranyi model provided a good statistical fitness based on a high correlation coefficient (R2
249
0.97). As expected, an increased SGR and decreased LT were observed with increasing
250
temperature. The results show that the SGR values of B. cereus growth on treated potatoes were
251
lower than those of untreated potatoes, whereas the greater LT values were observed in treated
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group. Subsequently, the secondary models were developed to describe the variation of SGR and
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LT values that were obtained from untreated and treated potatoes as a function of temperature,
254
using equations (2) and (3) (Table 4). Bias (Bf) and accuracy (Af) factors of developed SGR and
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LT models were at a range from 1.00 to 1.07 and from 1.02 to 1.08 (Table 4). The proportion of
256
standard error of prediction (%SEP) for the corresponding SGR and LT models were from 2.04%
257
to 8.24%, respectively.
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4. Discussion
In our preliminary experiment, B. cereus was detected in potato without E. coli, L.
261
monocytogenes, Samonella spp., and S. aureus (Table 1). The previous study on the monitoring of
262
foodborne pathogens in agricultural products reported that 4.6 log CFU/g of B. cereus was
263
detected in potato (Thapa et al., 2008). The current study was, therefore, conducted to develop a 12
ACCEPTED MANUSCRIPT hurdle technology which combined SAcEW and ultrasound to improve the microbial safety of
265
potato. Table 2 shows that antimicrobial effect of US treatment was efficiently (p ≤ 0.05) increased
266
in terms of increasing the temperature. The increased reductions might be due to the enhancement
267
of heat transfer by cavitation bubble generated during ultrasound treatment (Kim et al., 2004; Liu
268
et al., 2014), thus causing the increased sterility efficacy on the surface of products. Furthermore,
269
Earnshaw et al. (1995) stated that ultrasound combined with heat treatment was more effective in
270
the cell death when compared to ultrasound treatment only. However, the color of potatoes treated
271
by US at 60 ºC were significantly changed (p ≤ 0.05) (data not shown). In addition, previous
272
studies related to electrolyzed water suggest that 3 min is the best dip time while using as sanitizer
273
(Ding et al., 2010; Forghani et al., 2013; Huang et al., 2006; Nan et al., 2010; Rahman et al., 2013).
274
Therefore, US treatment at 40 ºC for 3 min was considered as optimal condition, and applied in
275
subsequent experiment that evaluated the antimicrobial effect of different combined treatments
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(DW, SAcEW, SAcEW-US, US-SAcEW, SAcEW+US) (Fig. 2).
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Fig. 2 shows that SAcEW treatment alone caused approximately 2.3 log CFU/g bacterial
278
reduction in B. cereus on potato. Mansur et al. (2015) reported that treatment with SAcEW (pH
279
6.29, ACC 30 mg/l) at 40 ºC for 3 min reduced the number of E. coli O157:H7, L. monocytogenes,
280
S. aureus, and S. Typhimurium on pork with the range of 1.2 to 1.8 log CFU/g. It has been
281
reported that treatment with SAcEW (pH 6.0-6.5, ACC 20 m/l) for 3 min caused the bacterial
282
reductions of 1.3 to 1.6 log CFU/g in Salmonella enteritidis on mung bean seeds and sprouts
283
(Zhang et al., 2011). In comparison with the bacterial reduction reported by above studies, a
284
higher antimicrobial effect of SAcEW was observed in this study. It might be mainly due to the
285
differences in the structure of samples surface, therefore causing that chemical sanitizers are
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occasionally unsuccessful against bacteria which are tightly adhered on the corner in where the
287
aqueous sanitizers are difficult to immerse (São José et al., 2014a). The SAcEW simultaneous with ultrasound treatment at 40 °C for 3 min achieved the
289
approximately 3.0 log CFU/g reduction in B. cereus adherent on potato (Fig. 2). A number of
290
studies have reported that ultrasound enables to enhance antimicrobial effect of chemical sanitizer
291
(Mañas et al., 2000; Scouten & Beuchat, 2002; Baumann et al., 2005; Sagong et al., 2011; São
292
José & Vanetti, 2012; Van Poucke et al., 2012; Luo & Oh, 2015, 2016). São José et al. (2014b)
293
demonstrated that the combined treatment of US (40 kHz) and 1 % lactic acid at room temperature
294
for 3 min resulted in 2.9 log CFU/g reduction in Salmonella enterica Enteritidis on green pepper.
295
Furthermore, Forghani et al. (2013) reported that combined treatment of low concentration
296
electrolyzed water (LcEW) and US (40 kHz) at 40 °C for 3 min caused a 2.4 log CFU/g reduction
297
in E. coli O157:H7 on lettuce. This might be due to the generated high temperature and pressure
298
during ultrasound treatment, which is able to damage the cell wall, promoting penetration of the
299
sanitizers through the cell wall, thereby killing the microorganisms more quickly and thoroughly
300
(Lillard et al., 1993; Piyasena et al., 2003; Sagong et al., 2011).
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The growth of B. cereus on potato was efficiently inhibited by SAcEW+US treatment at each
302
measured temperature by not only decreasing LT but also increasing SGR (Table 3). Baranyi and
303
Roberts (1994) stated that the cell stressed by treatment had lower metabolic activity compared to
304
that of untreated cell, resulting in longer LT of treated cell. Also, the previously reported evidences
305
demonstrated that chemical sanitizer combined with ultrasound was capable of extending the shelf
306
life of food product by controlling the bacterial growth during the storage (Forghani et al., 2013;
307
Yang et al., 2011).
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309
40 °C for 3 min caused synergic effects against B. ceruse on potato, as well as inhibition of the
310
growth of B. ceruse during storage at different temperatures from 5 to 35 °C. Therefore, this
311
hurdle approach can offer practical application in food industry to improve microbial safety of
312
potato during storage or distribution. In addition, the obtained parameters and developed models
313
can be used to set up the critical control points (CCP) in Hazard Analysis and Critical Control
314
Point (HACCP) or the Quantitative Microbial Risk Assessment (QMRA) related to B. ceruse
315
growth on potato.
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Yang, Z., Cao, S., Cai, Y., Zheng, Y., 2011. Combination of salicylic acid and ultrasound to control
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postharvest blue mould caused by Penicillium expansum in peach fruit. Innov. food sci.
418
emerg. technol. 12, 310-314.
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Zhang, C., Lu, Z., Li, Y., Shang, Y., Zhang, G., Cao, W., 2011. Reduction of Escherichia coli
420
O157:H7 and Salmonella Enteritidis on mung bean seeds and sprouts by slightly acidic
421
electrolyzed water. Food control. 22, 792-796.
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419
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ACCEPTED MANUSCRIPT 422 423
Figure Legends
424 Fig. 1. Hurdle technology procedure. (1) DW, deionized water; (2) US, ultrasound; (3) SAcEW, slightly
426
acidic electrolyzed water; (4) SAcEW-US: SAcEW treatment followed by ultrasound treatment; (5)
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US-SAcEW, ultrasound treatment followed by SAcEW treatment; (6) SAcEW+US: ultrasound treatment
428
combined with SAcEW treatment.
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425
429
Fig. 2. Inactivation effect of various decontamination treatments on Bacillus cereus in potato: DW,
431
sterilized sistilled water (40 ºC, 3min). US, ultrasound (40 ºC, 3min); SAcEW, slightly acidic electrolyzed
432
water (40 ºC, 3min); US-SAcEW, ultrasound treatment followed by SAEW treatment (40 ºC, 3min);
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SAcEW-US, SAEW treatment followed by ultrasound treatment (40 ºC, 3min); SAcEW+US, ultrasound
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treatment combined with SAEW treatment (40 ºC, 3min).
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430
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Fig. 3. Effect of treatment with SAcEW+US on the growth of B. cereus on potato during storage at
437
different temperatures. The following symbols are recommended: (○) the growth of B. cereus on untreated
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potato (control), (▲) the growth of B. cereus on potato by SAcEW+US.
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24
ACCEPTED MANUSCRIPT Table 1. Bacteriological analyses of potato
Micro-organisms
bulbs and tubers Potato (log CFU/g) 3.4±0.03 1.2±0.02 N.D* 3.1±0.03 N.D N.D N.D
Aerobic plate counts Total coliforms Escherichia coli Bacillus cereus Listeria monocytogenes Samonella spp. Staphylococcus aureus *
No detected
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1
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ACCEPTED MANUSCRIPT Table 2. Effect of ultrasound on the reduction of B. cereus (means ± standard deviation; log CFU/g) inoculated on potato.
DW
US (100 W/L)
US (200 W/L)
*
1 3 5 1 3 5 1 3 5 1 3 5
40 ºC f‡
B 0.55 ± 0.03 B0.59 ± 0.04ef B0.60 ± 0.04ef C0.61 ± 0.03def C0.62 ± 0.03def C0.64 ± 0.08cdef C0.65 ± 0.05cde C0.66 ± 0.03bcde C0.70 ± 0.06abcd C0.72 ± 0.03abc C0.74 ± 0.09ab C0.79 ± 0.07a
*
60 ºC h
A0.69 ± 0.07 A1.49 ± 0.13d A1.51 ± 0.09d B0.85 ± 0.06g B1.11 ± 0.08f B1.31 ± 0.11e B1.35 ± 0.13e B1.61 ± 0.18c B1.74 ± 0.14c B1.90 ± 0.09b B2.32 ± 0.11a B2.45 ± 0.15a
A0.71 ± 0.06g A1.51 ± 0.09ef A1.53 ± 0.11ef A1.40 ± 0.15f A1.53 ± 0.14ef A1.60 ± 0.11e A1.75 ± 0.13d A1.95 ± 0.16c A2.01 ± 0.09c A2.28 ± 0.15b A2.91 ± 0.24a A2.98 ± 0.17a
Numbers within each row followed by different capital letters are significantly different (P ≤ 0.05). Numbers within each column followed by different small letters are significantly different (P ≤ 0.05). ‡ Treatment with sterilized deionized water (DW), ultrasound (US) at the AED of 100, 200, and 400 W/L.
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6 7 8
25 ºC
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Temperature
Dipping time (min)
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Treatments‡
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ACCEPTED MANUSCRIPT Table 3. Estimation of the specific growth rate (SGR) and lag time (LT) of B. cereus on untreated and
Temperature (ºC)
Control
SAcEW+US
R2
4 10
0.02e* 0.03e
0.01e 0.02e
82.1a 32.0b
91.6a 39.5b
0.991 0.973
15 20
0.12d 0.22c
0.07d
12.3c
15.6c
0.986
c
11.9
d
d
25 30
b
0.23 0.45a
b
0.12 0.23a
e
5.1 4.4e
0.978 0.992
35
a
a
e
0.46
0.11
0.24
treated potato at different storage temperatures. *
4.2
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SAcEW+US
13.3 d
6.0 4.8d 4.4
d
0.991 0.996
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Within the same column, values not followed by the same capital letter are significantly different (p ≤ 0.05). † Unwashed potatoes. ‡ Combination treatment of SAcEW and ultrasound.
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11 12 13 14
Control
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LT (h)
SGR (log CFU/h)
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ACCEPTED MANUSCRIPT Table 4. Secondary models for B. cereus growth on water washed (control) and treated (SAcEW+US) potato.
(400W/L, 40° C, 3min)
†
ඥܵ = §ܴܩ0.021ܶ − 0.011
1.01
1.03
2.04
݊ܮሺ ¶ ܶܮሻ = −0.097ܶ + 4.407
1.07
1.08
7.13
ඥܵ = ܴܩ0.013ܶ + 0.045
1.00
1.02
3.11
݊ܮሺܶܮሻ = −0.131ܶ + 4.554
1.02
1.05
8.24
Bf
*
Bias factor. Accuracy factor. ‡ Proportion of standard error of prediction § Specific growth rate, SGR (log CFU/h). ¶ Lag time, LT (h).
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Af
‡
%SEP
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SAcEW+ US
*
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Control
Equations
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Fig. 1.
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Fig. 2.
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T r e a tm e n ts
A
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+
S
S
W
U
cE
-U
1
0
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A
W
W
c
S
-S
cE
cE
b
S
A
A
3
U
S
S
S
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D
M e a n re d u c tio n (lo g C F U /g )
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c d
b
2 a
ACCEPTED MANUSCRIPT °C
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°C
9
9
8
8
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7
L o g C F U /g
6 5
6 5
4
4
3
3 0
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T im e ( h )
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L o g C F U /g
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25
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T im e ( h )
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3 0 °C
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35
T im e ( h )
°C 9 8
L o g C F U /g
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T im e ( h )
7 6 5 4 3 0
10
20
T im e ( h )
Fig. 3.
200
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°C
L o g C F U /g
L o g C F U /g
15
150
T im e ( h )
30
40
ACCEPTED MANUSCRIPT 1. Bacillus cereus was detected in raw potato with approximately 3.0 log CFU/g。 2. SAcEW combined with US treatment caused synergistic effect against B. cereus. 3. Antimicrobial effect was enhanced by increasing acoustic energy density in ultrasound treatment.
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4. SAcEW+US+40 ºC treatment not only extended the lag time but also reduced specific growth
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rate of B. cereus.