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Inactivation of Aspergillus sp. spores on whole black peppers by nonthermal plasma and quality evaluation of the treated peppers
Accepted Manuscript Inactivation of Aspergillus sp. spores on whole black peppers by nonthermal plasma and quality evaluation of the treated peppers Takanori Tanino, Takuya Arisaka, Yuka Iguchi, Masayoshi Matsui, Takayuki Ohshima PII:
S0956-7135(18)30524-3
DOI:
https://doi.org/10.1016/j.foodcont.2018.10.023
Reference:
JFCO 6363
To appear in:
Food Control
Received Date: 5 July 2018 Revised Date:
19 September 2018
Accepted Date: 17 October 2018
Please cite this article as: Takanori Tanino, Takuya Arisaka, Yuka Iguchi, Masayoshi Matsui, Takayuki Ohshima, Inactivation of Aspergillus sp. spores on whole black peppers by nonthermal plasma and quality evaluation of the treated peppers, Food Control (2018), doi: 10.1016/j.foodcont.2018.10.023 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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Highlights
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A rotating non-thermal plasma (NTP) reactor was constructed and applied to the inactivation of Aspergillus sp. spores.
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Rotating the NTP treatment reactor improved the inactivation efficiency of Aspergillus sp. spores.
The NTP treatment using humidified gases also enhanced the inactivation of Aspergillus sp. spores.
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treatment.
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The antioxidative activity in NTP-treated whole black peppers remained relatively unaffected by the
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Inactivation of Aspergillus sp. spores on whole black peppers by nonthermal plasma and quality evaluation
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of the treated peppers
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3 Takanori Tanino 1, 2, Takuya Arisaka 1, Yuka Iguchi 3, Masayoshi Matsui 1, Takayuki Ohshima 1, 2, *
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1: Division of Environmental Engineering Science, Graduate School of Science and Technology, Gunma
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University
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2: Gunma University Center for Food Science and Wellness
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3: Department of Environmental Engineering Science, School of Science and Technology, Gunma
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Phone
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E-mail
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+81-277-30-1470
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[email protected]
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Key words
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Nonthermal plasma, pepper, spore, inactivation
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Abstract In this study, a rotating nonthermal plasma (NTP) reactor was designed and its practical use for
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the inactivation of microorganisms on dried granular agricultural products such as black pepper was
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investigated. As the target microorganism to inactivate in the rotating NTP reactor, Aspergillus spores were
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artificially inoculated on whole black peppers. Inactivation treatments were carried out under several
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conditions. Rotating the NTP reactor facilitated the inactivation of the Aspergillus spores on the whole
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black peppers, and a decrease in the survival ratio of three orders (3 Log) was achieved after 4 min
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treatment. Using humidified gases for inactivation treatment in the rotating NTP reactor also facilitated the
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inactivation of the Aspergillus spores on the whole black peppers, and no viable spores of Aspergillus sp.
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were detected after 4 min treatment. The quality of the NTP- and dry-heat-treated whole black peppers was
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compared with that of untreated peppers in terms of the piperine content and antioxidative activity. The
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piperine contents of the NTP- and dry-heat-treated black peppers showed no significant difference, but the
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antioxidative activity in the NTP-treated peppers was higher than that in the dry-heat-treated peppers.
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1. Introduction Numerous species and microorganisms derived from the soil and atmosphere adhere to the
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surface of agricultural products. Granular agricultural products such as beans, cereals, and spices are dried
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to improve their shelf life by removing water, which is required for microorganism growth. However, even
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after drying, there are active microorganisms in spore form on the surface of granular agricultural products.
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Some of these microorganisms may cause food poisoning and food loss. For example, Bacillus cereus
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spores are not inactivated by heating at 100˚C for 30 min, and germinate and grow in cooked rice and
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noodles preserved at room temperature. B. cereus produces cereulide, which causes vomiting when taken
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orally (Stenfors et al. 2008). Clostridium perfringens is also a spore-forming bacterium. This bacterium is
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obligately anaerobic and grows in cooked foods that lose oxygen upon heating. Eating food containing C.
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perfringens causes diarrhea because C. perfringens produces several toxins that cause enteric diseases
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inside the gastrointestinal tract (Smedley et al. 2004). In addition to bacteria, fungus is also a
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food-contaminating microorganism. Various Aspergillus species, especially A. flavus, produce aflatoxins,
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which are potent carcinogens, in the seeds of a number of crops not only before harvesting but also during
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storage (Klich 2007). The mass mortality of turkeys in the U.K. in the 1960s that occurred as a result of
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feeding them peanuts contaminated with aflatoxins is a well-known case showing the danger of aflatoxins.
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The contamination of food with aflatoxins renders it inedible, increasing food loss.
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The inactivation of pathogenic microorganisms on dried granular agricultural products is
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important to prevent food poisoning and food loss. For the inactivation of microorganisms on granular
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agricultural products, heating and fumigation are the most common treatments. However, heat and
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fumigation treatments are associated with the deterioration of food quality (e.g., color, flavor, and texture)
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and health concerns due to residual chemicals, respectively. Irradiation, one of the most promising
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substitutes, almost overcomes these problems but is also associated with public mistrust and is prohibited
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from being applied to food in some countries. Another emerging and promising inactivation treatment for
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microorganisms on dried granular agricultural products is atmospheric-pressure nonequilibrium plasma.
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This is also known as cold or nonthermal plasma (NTP), which can be produced using several types of
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generation system including corona discharge, dielectric barrier discharge, and plasma jet systems (Pankaj
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and Keener 2017). NTP generates various bactericidal factors, such as UV photons, reactive oxygen and
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nitrogen species, free radicals, and free electrons, and the inactivation of various microorganisms has been
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reported (Mai-Prochnow et al. 2014; Scholtz et al. 2015; Liao et al. 2017). Recently, NTP has attracted
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considerable attention as a nonthermal process in food processing research (Moisan et al. 2001; Thirumdas
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et al. 2015; Ito et al. 2017; Misra and Jo 2017). Several studies on the inactivation of microorganisms on
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dried granular agricultural products have been reported. Devi et al. (2017) investigated the effect of NTP on
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A. parasiticus and A. flavus on groundnuts; reductions in the Aspergillus spp. and aflatoxin B1 contents of
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more than 90% were observed after the plasma treatment. Mitra et al. (2014) revealed that the number of
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natural microbiota attached to the seed surface of Cicer arietinum, commonly known as chickpeas or
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garbanzo beans, was reduced by one and two orders of magnitude after 2 and 5 min NTP treatment,
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respectively. Lee et al. (2016) reported that NTP treatment reduced the number of microorganisms
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including pathogens inoculated on brown rice and caused slight changes in the physicochemical quality,
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pH, and color of the brown rice. In this study, we attempted to inactivate spores of Aspergillus sp.
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inoculated on the surfaces of whole black peppers. Previously, a decontamination study of whole black
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peppers using cold plasma was reported (Hertwig et al. 2015); however, no examples of the practical use of
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NTP treatment for the inactivation of microorganisms on dried granular agricultural products have yet been
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reported. We designed a rotating NTP treatment reactor and used it for the inactivation of microorganisms
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on the surfaces of whole black peppers. The quality of the whole black peppers after NTP treatment was
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also evaluated in terms of the piperine content and antioxidant activity.
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2. Materials and methods
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2.1 Preparation of whole black peppers with inoculated spores of Aspergillus sp. on their surfaces As the model of Aspergillus sp., we selected A. niger to safely carry out the experiments
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because it is widely used for brewing and is generally recognized as safe. A. niger was purchased from the
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American Type Culture Collection (ATCC number: 9142). A. niger was restored on a PDA solid medium
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[3.9% (w/v) potato dextrose agar] at 30˚C for 2 weeks, then the formed spores were suspended in saline.
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The suspension was filtered with a sterile gauze to remove the mycelia. An aliquot of the resulting spore
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suspension was plated onto a PDAT solid medium [3.9% (w/v) potato dextrose agar, 0.25% (v/v) Triton
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X-100] and incubated at 30˚C for 2 days. The living spore number in the suspension was determined by
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counting the number of formed colonies on the PDAT solid medium. The spore suspension was diluted to a
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cell concentration of approximately 5×106 cells/ml. The whole black peppers with A. niger spores
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inoculated on their surface were prepared as below. Commercially available whole black peppers were
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purchased at a domestic supermarket. 0.9 g of the whole black peppers was soaked in 6 ml of the diluted
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spore suspension, agitated briefly by a glass rod, then drawn from the suspension. They were placed on a
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clean bench and allowed to dry in air at ambient temperature for 16 h. Five hundred grams of whole black
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peppers with A. niger spores inoculated on their surface was prepared by repeating this preparation method,
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then the whole black peppers were gathered together and uniformized for use in the inactivation
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experiments. The initial living cell number (N0) on the whole black peppers was determined as follows. 0.9
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g of the whole black peppers inoculated with A. niger spores was soaked in 6 ml of saline and vigorously
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stirred, then an aliquot of appropriately diluted supernatant was plated onto a PDAT solid medium. In this
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study, a maximum of 1 ml of suspension was plated onto the PDAT medium, therefore the limit of
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detection was 1 living cell/ml. The number of colonies formed on the PDAT solid medium after incubation
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at 30˚C for 2 days was counted, from which N0 was determined. N0 was approximately 4×104 cells (/0.9 g
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whole black peppers).
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2.2 Rotating NTP treatment reactor and NTP inactivation treatment procedure
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In this study, we selected a surface dielectric barrier discharge as the NTP for use in the
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inactivation treatment. Figure 1 shows images and a schematic of the rotating NTP treatment reactor
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designed in this study. A quartz tube (I.D. 13 mm, O.D. 17 mm) and aluminum tape (1 mm width) were
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used as the dielectric material and electrode, respectively (Fig. 1a). The aluminum tape was attached
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alternately on the inner and outer surfaces of the quartz tube, and the inner and outer aluminum electrodes
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were connected to an AC high voltage (AC H.V.) power supply (AGF-B10, KASUGA DENKI Inc.,
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Japan) and the ground, respectively (Fig. 1b). The NTP was generated by applying an AC H.V. of 6.2 kV0-p
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with a frequency of 30 kHz to the reactor, and the length of the NTP generation area was 78 mm (Fig. 1c).
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The power consumed by the NTP was 21 W. A stepping motor controlled by an Arduino microcontroller
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was attached to the NTP treatment reactor to rotate it (Fig. 1d). Stainless-steel bearings were attached to the
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inside and outside edges of the NTP treatment reactor, and a copper tube was attached to the inside bearing.
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To avoid tangling of the electric wire tapes connected to the AC H.V. supply and the ground during the
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rotation of the NTP treatment reactor, they were connected to the copper tube and outer bearing,
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respectively. The copper tube was also used to supply gases to the NTP treatment reactor.
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The NTP inactivation treatment of the whole black peppers was carried out as follows. 0.9 g of
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the whole black peppers inoculated with A. niger spores was introduced into the NTP generation area of the
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reactor. The NTP treatment reactor containing the whole black peppers was rotated by the stepping motor
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and compressed air was introduced through the copper tube at a flow rate of 0.3 L/min. To modify the
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atmosphere inside the NTP treatment reactor, oxygen, humidified compressed air, or humidified oxygen
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was flowed at a rate of 0.3 L/min. Then NTP was generated by applying an AC H.V. to the reactor. After
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the NTP treatment, all the whole black peppers were removed from the reactor and soaked in 6 ml of saline.
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The living cell number (N) was determined by the same procedure as that used to determine N0 described
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above. The survival ratio was defined as N/N0. All inactivation experiments were performed in at least
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triplicate and the data in the figures show the mean of the experimental results.
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2.3 Evaluation of the black pepper quality
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The piperine content in the whole black peppers was evaluated as below. One gram of whole
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black peppers was well pounded in a mortar and the crushed pepper was soaked in 50 ml of 96% methanol.
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The suspension was refluxed for 3 h then the supernatant was filtered by filter paper. The resulting solution
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was placed in a measuring flask and made up to 100 ml by adding 96% ethanol. The absorbance of the
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solution at 343 nm, which is the absorption wavelength of piperine, was measured by a spectrophotometer. The
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evaluated
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2,2-diphenyl-1-picrylhydrazyl hydrate (DPPH) radical scavenging assay, reduction power assay, and
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thiobarbituric acid number assay. The DPPH radical scavenging assay was carried out in accordance with
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previous studies (Bandoniene et al. 2002; Suhaj et al. 2006). Five grams of whole black peppers was well
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pounded in a mortar and the crushed pepper was soaked in 50 ml of 80% methanol. The suspension was
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shaken at 130 rpm at room temperature for 1 h. The supernatant of the suspension was collected as a
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methanolic extract. 0.65 ml of the methanolic extract and 25 ml of a methanolic solution of DPPH (6 × 10-5
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M) were mixed. After 15 min incubation at room temperature, the absorbance at 515 nm (AA) was
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measured by a spectrophotometer. As a control experiment, 80% methanol was used instead of the
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methanolic extract to measure the absorbance after incubation (AC). The DPPH radical scavenging activity
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was calculated as
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% = [(AC – AA) / AC] × 100.
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The reduction power assay was carried out in accordance with Suhaj et al. (2006) with a slight
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modification. Two milliliters of the appropriately diluted methanolic extract, 2 ml of 0.2 M sodium
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phosphate buffer (pH 6.6), and 2 ml of 1% potassium ferricyanide were mixed, and the mixture was
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incubated at 50˚C for 20 min. After the incubation, 2 ml of 10% trichloroacetic acid was added, then the
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mixture was centrifuged at 200g for 10 min. One milliliter of the supernatant, 1 ml of distilled water, and
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0.2 ml of 0.1% ferric chloride were mixed. The absorbance of the mixture was measured at 700 nm.
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The thiobarbituric acid number assay was also performed in accordance with previous studies
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(Zin et al. 2002; Suhaj et al. 2006) with a slight modification. One milliliter of 20% trichloroacetic acid and
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1 ml of 0.36% thiobarbituric acid were added to 0.5 ml of the appropriately diluted methanolic extract. The
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mixture was reacted in a boiling water bath for 10 min. Then the absorbance of the mixture was measured
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at 532 nm.
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All quality evaluation experiments were performed ten times, and the data shown in table are expressed as mean ± standard deviation.
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3. Results and discussion
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3.1 Effect of the reactor rotation on the NTP inactivation treatment
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We studied the inactivation effect of the rotating NTP treatment reactor designed in this study
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(Fig. 1) on the spores of Aspergillus sp. inoculated artificially on the surface of whole black peppers. The
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NTP inactivation treatment was carried out at various rotation rates (Fig. 2). The NTP treatment without
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rotation slightly but significantly decreased the survival ratio, and a decrease in the survival ratio of one
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order was detected after 4 min treatment. On the other hand, the NTP treatment with rotation markedly
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decreased the survival ratio. A decrease in the survival ratio of almost two orders was detected after 1 min
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treatment under all rotation conditions. The rotation of the reactor proved to be effective for decreasing the
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survival ratio but the rotation rate hardly affected the efficiency. For all rotation rates, the survival ratio
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gradually decreased with the treatment time and an almost three-order decrease in the survival ratio was
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achieved after 4 min treatment. The lowest survival ratio after 4 min treatment was 0.71×10-3 for the NTP
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treatment involving rotation at 50 rpm. In the case of simply rotating the reactor without NTP generation, it
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was confirmed that the survival ratio did not decrease significantly. Therefore, the decrease in the survival
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ratio in the NTP treatment with rotation is due to the inactivation of the Aspergillus sp. spores on the surface
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of whole black peppers rather than the elimination of the Aspergillus sp. spores from the surface of the
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peppers by the vibration induced by rotation. Rotating the NTP treatment reactor moved and mixed the
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peppers in the reactor, facilitating the contact and exposure of various surfaces of the peppers to the NTP.
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This allowed bactericidal factors produced by the NTP to act efficiently on the spores of the Aspergillus sp.
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on the surface of whole black peppers.
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3.2 Effect of the atmosphere inside the NTP treatment reactor on the NTP inactivation treatment
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The species and amounts of some bactericidal factors generated by NTP, such as reactive
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oxygen and nitrogen species and free radicals, are strongly affected by the atmosphere of the NTP
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generation field. For example, using oxygen instead of air facilitates the production of reactive oxygen
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species and ozone, and the use of a humid gas also facilitates the production of hydroxyl radicals, which are
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the most reactive species among the reactive oxygen species (Lu and Wu 2013; Mouele et al. 2015).
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Because employing oxygen and humidified gas in practical use is expected to be feasible, we studied the
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effect of these atmospheres inside the rotating NTP treatment reactor on the NTP inactivation treatment of
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whole black peppers. Oxygen, humidified compressed air, or humidified oxygen was introduced into the
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NTP reactor, which was rotated at 10 rpm, at a flow rate of 0.3 L/min. The experiment was carried out at
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20˚C and the relative humidity of the humidified gases was 45%. Figure 3 shows the results of the NTP
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inactivation treatment using each gas. Compared with the results of the NTP inactivation treatment using
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compressed air (Fig. 2), the NTP treatment using oxygen did not improve the inactivation efficiency. On
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the other hand, the NTP treatment using the humidified gases enhanced the decrease in the survival ratio,
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and no viable spores of Aspergillus sp. were detected after 4 min treatment (Fig. 3a). This result shows that
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it is advantageous to facilitate the production of hydroxyl radicals using a humidified gas to achieve high
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inactivation efficiency. However, increasing the gas flow rate to 0.9 L/min to supply more of the humidified
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gas only slightly affected the inactivation efficiency (Fig. 3b). The volume in which the NTP is generated is
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approximately 0.01 L; thus, the flow rate of 0.3 L/min supplies a volume of gas equal to 30 times that in
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which the NTP is generated per 1 min. This is considered to be almost sufficient to supply all the humidity
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required to facilitate the production of hydroxyl radicals. Therefore, it is concluded that the threefold
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increase in the amount of humidified gas did not significantly improve the inactivation efficiency.
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3.3 Effect of the filling rate in the NTP treatment reactor on the NTP inactivation treatment
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The filling rate is expected to be important when applying the rotating NTP treatment reactor
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for practical use because the whole black peppers in the NTP reactor must be moved and mixed to expose
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the entire surface of the peppers to the NTP. Filling rates were calculated by assuming the weight and
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diameter of a whole black pepper to be 0.05 g and 4.5 mm, respectively. Whole black peppers with weights
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of 0.9, 2.7, and 4.5 g were introduced into the NTP treatment reactor, for which the filling rates were 8.3, 25,
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and 41%, respectively (Fig. 4). For the filling rates of 8.3 and 25% (Figs. 4a, b), the whole black peppers
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were moved and mixed by the rotation of the NTP reactor, although some of the peppers lay on top of the
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other ones at the filling rate of 25%. For the filling rate of 41% (Fig. 4c), the peppers were almost fully
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packed in the NTP reactor and were hardly moved or mixed by the rotation of the NTP reactor. The NTP
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treatment with these filling rates was carried out with the NTP treatment reactor rotated at 10 rpm and
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humidified compressed air provided at a flow rate of 0.6 L/min. Increasing the filling rate decreased the
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inactivation efficiency (Fig. 5). For the filling rate of 25%, a decrease in the survival ratio of almost three
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orders was detected after 4 min treatment, whereas after the same treatment time, a slight decrease in the
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survival ratio of less than one order was detected for the filling rate of 41%. From these results, to achieve
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the efficient inactivation of spores of Aspergillus sp. on the surface of whole black peppers, the filling rate
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must be controlled so that the whole black peppers do not lie on top of each other.
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In this study, a more than four-order decrease in the survival ratio of spores of Aspergillus sp.
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was achieved after 4 min of 21 W NTP treatment. The inner surface area and volume of the NTP
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generation area were 3184 mm2 and 10348 mm3, respectively. Therefore, the area and volumetric energy
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densities were 6.5 mW/mm2 and 2.0 mW/mm3, respectively. In a previous study, Hertwig et al. (2015)
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showed that inactivations of 0.7 log10, 0.6 log10, 0.8 log10, and 2.7 log10 were achieved for the total
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mesophilic aerobic count, total spore count, B. subtilis spores, and Salmonella enterica on whole black
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peppers, respectively, after 15 min of direct radio-frequency plasma jet treatment. Devi et al. (2017)
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showed that the complete inactivation of Aspergillus spp. spores inoculated on groundnuts at a
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concentration of 1.45 × 103 cfu/g by 60 W NTP required 27 min treatment. Dasan et al. (2016) showed that
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a 655 W atmospheric-pressure fluidized-bed plasma reactor achieved a 4.5 log (cfu/g) reduction in the
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number of A. flavus spores on hazelnuts after 5 min treatment. Although a simple comparison cannot be
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made between these studies and this study because the microorganisms and the particle shapes used in
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these studies are considerably different, the inactivation treatment conditions using the rotating NTP
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treatment reactor developed in this study are considered to have a reasonably high bactericidal effect.
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3.4 Evaluation of the black pepper quality
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The quality of whole black peppers treated by the NTP reactor rotated at 10 rpm with
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humidified compressed air at a flow rate of 0.3 L/min for 4 min was analyzed. As the indices of the quality,
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we focused on the piperine content and antioxidant activity. Moreover, the antioxidant activity was
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evaluated by three assays; DPPH radical scavenging assay, reduction power assay, and thiobarbituric acid
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number assay. The quality of the NTP-treated whole black peppers was compared with those of untreated
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and dry-heat (180˚C, 1 h)-treated ones. Table 1 shows a summary of the quality of each sample. Compared
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with the quality of the untreated whole black peppers, those of the NTP- and dry-heat-treated ones showed
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various degrees of deterioration. The piperine contents of the NTP- and dry-heat-treated whole black
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peppers were decreased to approximately 60% of that of the untreated peppers and showed no significant
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difference. On the other hand, significant differences were detected in the antioxidant activity assay. The
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result of the DPPH radical scavenging assay showed that the dry-heat treatment decreased the radical
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scavenging activity of the whole black peppers by approximately 50%, whereas the NTP treatment
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decreased it by 30%. The reduction power assay showed a clearer difference; the dry-heat treatment
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decreased the reduction power of the whole black peppers by approximately 45%, whereas the NTP
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treatment decreased it by only 11%. According to these results, the degree of oxidation of the black peppers
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caused by the NTP treatment is low. This tendency was also clearly confirmed in the thiobarbituric acid
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number assay, which indicates the degree of lipid oxidation. The thiobarbituric acid numbers of the NTP-
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and dry-heat-treated whole black peppers were 1.6-fold and 3.4-fold higher than that of the untreated whole
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black peppers, respectively. This quality of the whole black peppers after the NTP treatment was relatively
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low compared with that after the γ-irradiation treatment reported by Suhaj et al. (2006). However, NTP
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treatment is a promising microorganism inactivation technology for dried granular agricultural products,
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especially in countries that prohibit the irradiation treatment of food, and the findings of this study are
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expected to be useful for realizing the practical use of NTP treatment as a nonthermal microorganism
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inactivation process.
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4. Conclusions
It was demonstrated that the rotating NTP reactor was effective for enhancing the inactivation of
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Aspergillus spores on whole black peppers. After 4 min treatment, the reduction of the survival ratio was
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one order in the NTP treatment without rotation, on the other hand, the survival ratio was 0.71×10-3 in the
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NTP treatment with rotation at 50 rpm. This enhanced inactivation was due to the effective contact and
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exposure of the entire surface of the whole black peppers to the NTP. Therefore, control of the filling rate to
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allow the peppers to move and mix in the NTP reactor is important. The use of humidified gases was also
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effective for enhancing the reduction in the survival ratio; no viable spores of Aspergillus sp. were detected
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after 4 min treatment. In the evaluation of the black pepper quality, the piperine content and antioxidant
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activity of NTP- and dry-heat-treated whole black peppers showed various degrees of deterioration
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compared with those of untreated peppers. Although NTP and dry-heat treatment reduced the piperine
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content to the same extent, the NTP treatment was more effective for retaining the antioxidant activity of
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the whole black peppers than the dry-heat treatment.
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This study was supported by JSPS KAKENHI Grant Number 15H02231.
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Lee K. H., Kim H.-J., Woo K. S., Jo C., Kim J.-K., Kim S. H., Park H. Y., Oh S.-K. & Kim W. H. (2016).
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Evaluation of cold plasma treatments for improved microbial and physicochemical qualities of brown rice.
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Liao X., Liu D., Xiang Q., Ahn J., Che S., Ye X. & Ding T. (2017). Inactivation mechanisms of
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non-thermal plasma on microbes: A review. Food Control, 75, 83-91.
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Lu X. & Wu S. (2013). On the active species concentrations of atmospheric pressure nonequilibrium
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plasma jets. IEEE Transactions on Plasma Science, 41(8), 2313-2326.
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Mai-Prochnow A., Murphy A. B., McLean K. M., Kong M. G. & Ostrikov K. (2014). Atmospheric
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pressure plasmas: Infection control and bacterial responses. International Journal of Antimicrobial Agents,
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43, 508-517.
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Misra N. N. & Jo C. (2017). Applications of cold plasma technology for microbiological safety in meat
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industry. Trends in Food Science and Technology, 64, 74-86.
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Mitra A., Li. Y.-F., Klämpfl T. G., Shimizu T., Jeon J., Morfill G. E. & Zimmermann J. L. (2014).
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Inactivation of surface-borne microorganisms and increased germination of seed specimen by cold
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atmospheric plasma. Food and Bioprocess Technology, 7, 645-653.
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Moisan M., Barbeau J., Moreau S., Pelletier J., Tabrizian M. & Yahia L. H. (2001). Low-temperature
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sterilization using gas plasmas: a review of the experiments and an analysis of the inactivation mechanisms.
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microorganisms from wastewater using different dielectric barrier discharge configurations—a critical
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review. Environmental Science and Pollution Research, 22, 18345-18362.
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Pankaj S. K. & Keener K. M. (2017). Cold plasma: background, applications and current trends. Current
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Opinions in Food Science, 16, 49-52.
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Scholtz V., Pazlarova J., Souskova H., Khun J. & Julak J. (2015). Nonthermal plasma — A tool for
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decontamination and disinfection. Biotechnology Advances, 33, 1108-1119.
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Smedley J. G. 3rd, Fisher D. J., Sayeed S., Chakrabarti G. & McClane B. A. (2004). The enteric toxins of
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Clostridium perfringens. Reviews of Physiology, Biochemistry and Pharmacology, 152, 183-204.
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Stenfors Arnesen L. P., Fagerlund A. & Granum P. E. (2008). From soil to gut: Bacillus cereus and its food
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poisoning toxins. FEMS Microbiology Reviews, 32, 579-606.
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Suhaj M., Rácová J., Polovka M. & Brezová V. (2006). Effect of γ-irradiation on antioxidant activity of
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black pepper (Piper nigrum L.). Food Chemistry, 97, 696-704.
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Thirumdas R., Sarangapani C. & Annapure U. S. (2015). Cold plasma: A novel non-thermal technology for
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food processing. Food Biophysics, 10, 1-11.
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Zin Z. M., Abdul-Hamid A. & Osman A. (2002). Antioxidative activity of extracts from Mengkudu
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(Morinda citrifolia L.) root, fruit and leaf. Food Chemistry, 78, 227-231.
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Figure 1
Images and schematic of the NTP treatment reactor. Photograph taken from above (a) and
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cross-section drawing (b) of the electrode structure. Dark-field image of NTP (c). Photograph of the
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rotating NTP treatment reactor (d).
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Time course of survival ratio in the NTP inactivation treatment for several rotation rates. The
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NTP reactor was rotated at 0 (squares), 10 (triangles), 30 (circles), and 50 (diamonds) rpm. Closed and
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open symbols represent the treatment with and without NTP generation, respectively.
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Figure 3
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Time course of survival ratio in the NTP inactivation treatment using several gases. Oxygen
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(circles), humidified compressed air (triangles), or humidified oxygen (squares) was introduced into the
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NTP reactor at flow rates of 0.3 (a) and 0.9 (b) l/min. Closed and open symbols represent the treatment with
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and without NTP generation, respectively. N.D. means that living cells were not detected.
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Side views of NTP reactor with several amounts of black pepper. The filling rates were 8.3 (a),
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25 (b), and 40 (c) %.
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Time course of survival ratio in the NTP inactivation treatment for filling rate of whole black
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peppers in the reactor of 8.3% (circles), 25% (triangles), and 40% (squares). Closed and open symbols
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represent the treatment with and without NTP generation, respectively. N.D. means that living cells were
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not detected.
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Table 1 Comparison of piperine content and antioxidant activity in the black peppers treated for microbial inactivation.
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All the data are expressed as means ± standard deviations. Means with different numbers of stars in the same line differ significantly (p < 0.01).
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399 400 Table 1
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Comparison of piperine content and antioxidant activity in the black peppers treated for microbial
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inactivation. Piperine content
DPPH scavenging % [-]
Control (none)
25.3 ± 2.1 *
61.7 ± 2.6 *
NTP
15.9 ± 1.3 **
Dry-heat
15.2 ± 2.9 **
0.40 ± 0.09 *
43.3 ± 4.1 **
2.36 ± 0.23 **
0.62 ± 0.04 **
33.6 ± 2.5 ***
1.47 ± 0.26 ***
1.36 ± 0.03 ***
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2.68 ± 0.13 *
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OD532 [-]
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Thiobarbituric acid number
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Reduction power
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Quartz tube
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2 3 Treatment time [min]
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S0956-7135(18)30524-3
DOI:
https://doi.org/10.1016/j.foodcont.2018.10.023
Reference:
JFCO 6363
To appear in:
Food Control
Received Date: 5 July 2018 Revised Date:
19 September 2018
Accepted Date: 17 October 2018
Please cite this article as: Takanori Tanino, Takuya Arisaka, Yuka Iguchi, Masayoshi Matsui, Takayuki Ohshima, Inactivation of Aspergillus sp. spores on whole black peppers by nonthermal plasma and quality evaluation of the treated peppers, Food Control (2018), doi: 10.1016/j.foodcont.2018.10.023 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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Highlights
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A rotating non-thermal plasma (NTP) reactor was constructed and applied to the inactivation of Aspergillus sp. spores.
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Rotating the NTP treatment reactor improved the inactivation efficiency of Aspergillus sp. spores.
The NTP treatment using humidified gases also enhanced the inactivation of Aspergillus sp. spores.
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treatment.
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The antioxidative activity in NTP-treated whole black peppers remained relatively unaffected by the
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Inactivation of Aspergillus sp. spores on whole black peppers by nonthermal plasma and quality evaluation
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of the treated peppers
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3 Takanori Tanino 1, 2, Takuya Arisaka 1, Yuka Iguchi 3, Masayoshi Matsui 1, Takayuki Ohshima 1, 2, *
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1: Division of Environmental Engineering Science, Graduate School of Science and Technology, Gunma
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University
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2: Gunma University Center for Food Science and Wellness
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3: Department of Environmental Engineering Science, School of Science and Technology, Gunma
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Phone
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+81-277-30-1470
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[email protected]
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Key words
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Nonthermal plasma, pepper, spore, inactivation
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Abstract In this study, a rotating nonthermal plasma (NTP) reactor was designed and its practical use for
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the inactivation of microorganisms on dried granular agricultural products such as black pepper was
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investigated. As the target microorganism to inactivate in the rotating NTP reactor, Aspergillus spores were
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artificially inoculated on whole black peppers. Inactivation treatments were carried out under several
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conditions. Rotating the NTP reactor facilitated the inactivation of the Aspergillus spores on the whole
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black peppers, and a decrease in the survival ratio of three orders (3 Log) was achieved after 4 min
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treatment. Using humidified gases for inactivation treatment in the rotating NTP reactor also facilitated the
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inactivation of the Aspergillus spores on the whole black peppers, and no viable spores of Aspergillus sp.
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were detected after 4 min treatment. The quality of the NTP- and dry-heat-treated whole black peppers was
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compared with that of untreated peppers in terms of the piperine content and antioxidative activity. The
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piperine contents of the NTP- and dry-heat-treated black peppers showed no significant difference, but the
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antioxidative activity in the NTP-treated peppers was higher than that in the dry-heat-treated peppers.
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1. Introduction Numerous species and microorganisms derived from the soil and atmosphere adhere to the
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surface of agricultural products. Granular agricultural products such as beans, cereals, and spices are dried
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to improve their shelf life by removing water, which is required for microorganism growth. However, even
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after drying, there are active microorganisms in spore form on the surface of granular agricultural products.
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Some of these microorganisms may cause food poisoning and food loss. For example, Bacillus cereus
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spores are not inactivated by heating at 100˚C for 30 min, and germinate and grow in cooked rice and
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noodles preserved at room temperature. B. cereus produces cereulide, which causes vomiting when taken
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orally (Stenfors et al. 2008). Clostridium perfringens is also a spore-forming bacterium. This bacterium is
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obligately anaerobic and grows in cooked foods that lose oxygen upon heating. Eating food containing C.
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perfringens causes diarrhea because C. perfringens produces several toxins that cause enteric diseases
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inside the gastrointestinal tract (Smedley et al. 2004). In addition to bacteria, fungus is also a
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food-contaminating microorganism. Various Aspergillus species, especially A. flavus, produce aflatoxins,
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which are potent carcinogens, in the seeds of a number of crops not only before harvesting but also during
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storage (Klich 2007). The mass mortality of turkeys in the U.K. in the 1960s that occurred as a result of
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feeding them peanuts contaminated with aflatoxins is a well-known case showing the danger of aflatoxins.
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The contamination of food with aflatoxins renders it inedible, increasing food loss.
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The inactivation of pathogenic microorganisms on dried granular agricultural products is
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important to prevent food poisoning and food loss. For the inactivation of microorganisms on granular
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agricultural products, heating and fumigation are the most common treatments. However, heat and
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fumigation treatments are associated with the deterioration of food quality (e.g., color, flavor, and texture)
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and health concerns due to residual chemicals, respectively. Irradiation, one of the most promising
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substitutes, almost overcomes these problems but is also associated with public mistrust and is prohibited
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from being applied to food in some countries. Another emerging and promising inactivation treatment for
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microorganisms on dried granular agricultural products is atmospheric-pressure nonequilibrium plasma.
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This is also known as cold or nonthermal plasma (NTP), which can be produced using several types of
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generation system including corona discharge, dielectric barrier discharge, and plasma jet systems (Pankaj
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and Keener 2017). NTP generates various bactericidal factors, such as UV photons, reactive oxygen and
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nitrogen species, free radicals, and free electrons, and the inactivation of various microorganisms has been
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reported (Mai-Prochnow et al. 2014; Scholtz et al. 2015; Liao et al. 2017). Recently, NTP has attracted
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considerable attention as a nonthermal process in food processing research (Moisan et al. 2001; Thirumdas
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et al. 2015; Ito et al. 2017; Misra and Jo 2017). Several studies on the inactivation of microorganisms on
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dried granular agricultural products have been reported. Devi et al. (2017) investigated the effect of NTP on
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A. parasiticus and A. flavus on groundnuts; reductions in the Aspergillus spp. and aflatoxin B1 contents of
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more than 90% were observed after the plasma treatment. Mitra et al. (2014) revealed that the number of
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natural microbiota attached to the seed surface of Cicer arietinum, commonly known as chickpeas or
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garbanzo beans, was reduced by one and two orders of magnitude after 2 and 5 min NTP treatment,
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respectively. Lee et al. (2016) reported that NTP treatment reduced the number of microorganisms
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including pathogens inoculated on brown rice and caused slight changes in the physicochemical quality,
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pH, and color of the brown rice. In this study, we attempted to inactivate spores of Aspergillus sp.
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inoculated on the surfaces of whole black peppers. Previously, a decontamination study of whole black
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peppers using cold plasma was reported (Hertwig et al. 2015); however, no examples of the practical use of
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NTP treatment for the inactivation of microorganisms on dried granular agricultural products have yet been
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reported. We designed a rotating NTP treatment reactor and used it for the inactivation of microorganisms
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on the surfaces of whole black peppers. The quality of the whole black peppers after NTP treatment was
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also evaluated in terms of the piperine content and antioxidant activity.
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2. Materials and methods
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2.1 Preparation of whole black peppers with inoculated spores of Aspergillus sp. on their surfaces As the model of Aspergillus sp., we selected A. niger to safely carry out the experiments
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because it is widely used for brewing and is generally recognized as safe. A. niger was purchased from the
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American Type Culture Collection (ATCC number: 9142). A. niger was restored on a PDA solid medium
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[3.9% (w/v) potato dextrose agar] at 30˚C for 2 weeks, then the formed spores were suspended in saline.
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The suspension was filtered with a sterile gauze to remove the mycelia. An aliquot of the resulting spore
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suspension was plated onto a PDAT solid medium [3.9% (w/v) potato dextrose agar, 0.25% (v/v) Triton
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X-100] and incubated at 30˚C for 2 days. The living spore number in the suspension was determined by
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counting the number of formed colonies on the PDAT solid medium. The spore suspension was diluted to a
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cell concentration of approximately 5×106 cells/ml. The whole black peppers with A. niger spores
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inoculated on their surface were prepared as below. Commercially available whole black peppers were
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purchased at a domestic supermarket. 0.9 g of the whole black peppers was soaked in 6 ml of the diluted
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spore suspension, agitated briefly by a glass rod, then drawn from the suspension. They were placed on a
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clean bench and allowed to dry in air at ambient temperature for 16 h. Five hundred grams of whole black
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peppers with A. niger spores inoculated on their surface was prepared by repeating this preparation method,
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then the whole black peppers were gathered together and uniformized for use in the inactivation
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experiments. The initial living cell number (N0) on the whole black peppers was determined as follows. 0.9
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g of the whole black peppers inoculated with A. niger spores was soaked in 6 ml of saline and vigorously
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stirred, then an aliquot of appropriately diluted supernatant was plated onto a PDAT solid medium. In this
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study, a maximum of 1 ml of suspension was plated onto the PDAT medium, therefore the limit of
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detection was 1 living cell/ml. The number of colonies formed on the PDAT solid medium after incubation
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at 30˚C for 2 days was counted, from which N0 was determined. N0 was approximately 4×104 cells (/0.9 g
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whole black peppers).
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2.2 Rotating NTP treatment reactor and NTP inactivation treatment procedure
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In this study, we selected a surface dielectric barrier discharge as the NTP for use in the
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inactivation treatment. Figure 1 shows images and a schematic of the rotating NTP treatment reactor
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designed in this study. A quartz tube (I.D. 13 mm, O.D. 17 mm) and aluminum tape (1 mm width) were
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used as the dielectric material and electrode, respectively (Fig. 1a). The aluminum tape was attached
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alternately on the inner and outer surfaces of the quartz tube, and the inner and outer aluminum electrodes
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were connected to an AC high voltage (AC H.V.) power supply (AGF-B10, KASUGA DENKI Inc.,
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Japan) and the ground, respectively (Fig. 1b). The NTP was generated by applying an AC H.V. of 6.2 kV0-p
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with a frequency of 30 kHz to the reactor, and the length of the NTP generation area was 78 mm (Fig. 1c).
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The power consumed by the NTP was 21 W. A stepping motor controlled by an Arduino microcontroller
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was attached to the NTP treatment reactor to rotate it (Fig. 1d). Stainless-steel bearings were attached to the
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inside and outside edges of the NTP treatment reactor, and a copper tube was attached to the inside bearing.
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To avoid tangling of the electric wire tapes connected to the AC H.V. supply and the ground during the
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rotation of the NTP treatment reactor, they were connected to the copper tube and outer bearing,
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respectively. The copper tube was also used to supply gases to the NTP treatment reactor.
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The NTP inactivation treatment of the whole black peppers was carried out as follows. 0.9 g of
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the whole black peppers inoculated with A. niger spores was introduced into the NTP generation area of the
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reactor. The NTP treatment reactor containing the whole black peppers was rotated by the stepping motor
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and compressed air was introduced through the copper tube at a flow rate of 0.3 L/min. To modify the
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atmosphere inside the NTP treatment reactor, oxygen, humidified compressed air, or humidified oxygen
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was flowed at a rate of 0.3 L/min. Then NTP was generated by applying an AC H.V. to the reactor. After
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the NTP treatment, all the whole black peppers were removed from the reactor and soaked in 6 ml of saline.
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The living cell number (N) was determined by the same procedure as that used to determine N0 described
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above. The survival ratio was defined as N/N0. All inactivation experiments were performed in at least
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triplicate and the data in the figures show the mean of the experimental results.
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2.3 Evaluation of the black pepper quality
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The piperine content in the whole black peppers was evaluated as below. One gram of whole
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black peppers was well pounded in a mortar and the crushed pepper was soaked in 50 ml of 96% methanol.
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The suspension was refluxed for 3 h then the supernatant was filtered by filter paper. The resulting solution
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was placed in a measuring flask and made up to 100 ml by adding 96% ethanol. The absorbance of the
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solution at 343 nm, which is the absorption wavelength of piperine, was measured by a spectrophotometer. The
antioxidant
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evaluated
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2,2-diphenyl-1-picrylhydrazyl hydrate (DPPH) radical scavenging assay, reduction power assay, and
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thiobarbituric acid number assay. The DPPH radical scavenging assay was carried out in accordance with
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previous studies (Bandoniene et al. 2002; Suhaj et al. 2006). Five grams of whole black peppers was well
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pounded in a mortar and the crushed pepper was soaked in 50 ml of 80% methanol. The suspension was
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shaken at 130 rpm at room temperature for 1 h. The supernatant of the suspension was collected as a
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methanolic extract. 0.65 ml of the methanolic extract and 25 ml of a methanolic solution of DPPH (6 × 10-5
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M) were mixed. After 15 min incubation at room temperature, the absorbance at 515 nm (AA) was
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measured by a spectrophotometer. As a control experiment, 80% methanol was used instead of the
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methanolic extract to measure the absorbance after incubation (AC). The DPPH radical scavenging activity
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was calculated as
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% = [(AC – AA) / AC] × 100.
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The reduction power assay was carried out in accordance with Suhaj et al. (2006) with a slight
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modification. Two milliliters of the appropriately diluted methanolic extract, 2 ml of 0.2 M sodium
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phosphate buffer (pH 6.6), and 2 ml of 1% potassium ferricyanide were mixed, and the mixture was
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incubated at 50˚C for 20 min. After the incubation, 2 ml of 10% trichloroacetic acid was added, then the
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mixture was centrifuged at 200g for 10 min. One milliliter of the supernatant, 1 ml of distilled water, and
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0.2 ml of 0.1% ferric chloride were mixed. The absorbance of the mixture was measured at 700 nm.
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The thiobarbituric acid number assay was also performed in accordance with previous studies
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(Zin et al. 2002; Suhaj et al. 2006) with a slight modification. One milliliter of 20% trichloroacetic acid and
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1 ml of 0.36% thiobarbituric acid were added to 0.5 ml of the appropriately diluted methanolic extract. The
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mixture was reacted in a boiling water bath for 10 min. Then the absorbance of the mixture was measured
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at 532 nm.
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All quality evaluation experiments were performed ten times, and the data shown in table are expressed as mean ± standard deviation.
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3. Results and discussion
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3.1 Effect of the reactor rotation on the NTP inactivation treatment
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We studied the inactivation effect of the rotating NTP treatment reactor designed in this study
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(Fig. 1) on the spores of Aspergillus sp. inoculated artificially on the surface of whole black peppers. The
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NTP inactivation treatment was carried out at various rotation rates (Fig. 2). The NTP treatment without
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rotation slightly but significantly decreased the survival ratio, and a decrease in the survival ratio of one
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order was detected after 4 min treatment. On the other hand, the NTP treatment with rotation markedly
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decreased the survival ratio. A decrease in the survival ratio of almost two orders was detected after 1 min
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treatment under all rotation conditions. The rotation of the reactor proved to be effective for decreasing the
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survival ratio but the rotation rate hardly affected the efficiency. For all rotation rates, the survival ratio
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gradually decreased with the treatment time and an almost three-order decrease in the survival ratio was
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achieved after 4 min treatment. The lowest survival ratio after 4 min treatment was 0.71×10-3 for the NTP
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treatment involving rotation at 50 rpm. In the case of simply rotating the reactor without NTP generation, it
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was confirmed that the survival ratio did not decrease significantly. Therefore, the decrease in the survival
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ratio in the NTP treatment with rotation is due to the inactivation of the Aspergillus sp. spores on the surface
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of whole black peppers rather than the elimination of the Aspergillus sp. spores from the surface of the
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peppers by the vibration induced by rotation. Rotating the NTP treatment reactor moved and mixed the
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peppers in the reactor, facilitating the contact and exposure of various surfaces of the peppers to the NTP.
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This allowed bactericidal factors produced by the NTP to act efficiently on the spores of the Aspergillus sp.
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on the surface of whole black peppers.
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3.2 Effect of the atmosphere inside the NTP treatment reactor on the NTP inactivation treatment
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The species and amounts of some bactericidal factors generated by NTP, such as reactive
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oxygen and nitrogen species and free radicals, are strongly affected by the atmosphere of the NTP
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generation field. For example, using oxygen instead of air facilitates the production of reactive oxygen
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species and ozone, and the use of a humid gas also facilitates the production of hydroxyl radicals, which are
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the most reactive species among the reactive oxygen species (Lu and Wu 2013; Mouele et al. 2015).
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Because employing oxygen and humidified gas in practical use is expected to be feasible, we studied the
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effect of these atmospheres inside the rotating NTP treatment reactor on the NTP inactivation treatment of
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whole black peppers. Oxygen, humidified compressed air, or humidified oxygen was introduced into the
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NTP reactor, which was rotated at 10 rpm, at a flow rate of 0.3 L/min. The experiment was carried out at
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20˚C and the relative humidity of the humidified gases was 45%. Figure 3 shows the results of the NTP
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inactivation treatment using each gas. Compared with the results of the NTP inactivation treatment using
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compressed air (Fig. 2), the NTP treatment using oxygen did not improve the inactivation efficiency. On
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the other hand, the NTP treatment using the humidified gases enhanced the decrease in the survival ratio,
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and no viable spores of Aspergillus sp. were detected after 4 min treatment (Fig. 3a). This result shows that
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it is advantageous to facilitate the production of hydroxyl radicals using a humidified gas to achieve high
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inactivation efficiency. However, increasing the gas flow rate to 0.9 L/min to supply more of the humidified
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gas only slightly affected the inactivation efficiency (Fig. 3b). The volume in which the NTP is generated is
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approximately 0.01 L; thus, the flow rate of 0.3 L/min supplies a volume of gas equal to 30 times that in
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which the NTP is generated per 1 min. This is considered to be almost sufficient to supply all the humidity
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required to facilitate the production of hydroxyl radicals. Therefore, it is concluded that the threefold
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increase in the amount of humidified gas did not significantly improve the inactivation efficiency.
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3.3 Effect of the filling rate in the NTP treatment reactor on the NTP inactivation treatment
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The filling rate is expected to be important when applying the rotating NTP treatment reactor
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for practical use because the whole black peppers in the NTP reactor must be moved and mixed to expose
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the entire surface of the peppers to the NTP. Filling rates were calculated by assuming the weight and
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diameter of a whole black pepper to be 0.05 g and 4.5 mm, respectively. Whole black peppers with weights
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of 0.9, 2.7, and 4.5 g were introduced into the NTP treatment reactor, for which the filling rates were 8.3, 25,
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and 41%, respectively (Fig. 4). For the filling rates of 8.3 and 25% (Figs. 4a, b), the whole black peppers
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were moved and mixed by the rotation of the NTP reactor, although some of the peppers lay on top of the
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other ones at the filling rate of 25%. For the filling rate of 41% (Fig. 4c), the peppers were almost fully
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packed in the NTP reactor and were hardly moved or mixed by the rotation of the NTP reactor. The NTP
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treatment with these filling rates was carried out with the NTP treatment reactor rotated at 10 rpm and
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humidified compressed air provided at a flow rate of 0.6 L/min. Increasing the filling rate decreased the
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inactivation efficiency (Fig. 5). For the filling rate of 25%, a decrease in the survival ratio of almost three
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orders was detected after 4 min treatment, whereas after the same treatment time, a slight decrease in the
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survival ratio of less than one order was detected for the filling rate of 41%. From these results, to achieve
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the efficient inactivation of spores of Aspergillus sp. on the surface of whole black peppers, the filling rate
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must be controlled so that the whole black peppers do not lie on top of each other.
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In this study, a more than four-order decrease in the survival ratio of spores of Aspergillus sp.
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was achieved after 4 min of 21 W NTP treatment. The inner surface area and volume of the NTP
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generation area were 3184 mm2 and 10348 mm3, respectively. Therefore, the area and volumetric energy
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densities were 6.5 mW/mm2 and 2.0 mW/mm3, respectively. In a previous study, Hertwig et al. (2015)
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showed that inactivations of 0.7 log10, 0.6 log10, 0.8 log10, and 2.7 log10 were achieved for the total
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mesophilic aerobic count, total spore count, B. subtilis spores, and Salmonella enterica on whole black
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peppers, respectively, after 15 min of direct radio-frequency plasma jet treatment. Devi et al. (2017)
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showed that the complete inactivation of Aspergillus spp. spores inoculated on groundnuts at a
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concentration of 1.45 × 103 cfu/g by 60 W NTP required 27 min treatment. Dasan et al. (2016) showed that
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a 655 W atmospheric-pressure fluidized-bed plasma reactor achieved a 4.5 log (cfu/g) reduction in the
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number of A. flavus spores on hazelnuts after 5 min treatment. Although a simple comparison cannot be
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made between these studies and this study because the microorganisms and the particle shapes used in
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these studies are considerably different, the inactivation treatment conditions using the rotating NTP
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treatment reactor developed in this study are considered to have a reasonably high bactericidal effect.
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3.4 Evaluation of the black pepper quality
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The quality of whole black peppers treated by the NTP reactor rotated at 10 rpm with
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humidified compressed air at a flow rate of 0.3 L/min for 4 min was analyzed. As the indices of the quality,
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we focused on the piperine content and antioxidant activity. Moreover, the antioxidant activity was
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evaluated by three assays; DPPH radical scavenging assay, reduction power assay, and thiobarbituric acid
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number assay. The quality of the NTP-treated whole black peppers was compared with those of untreated
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and dry-heat (180˚C, 1 h)-treated ones. Table 1 shows a summary of the quality of each sample. Compared
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with the quality of the untreated whole black peppers, those of the NTP- and dry-heat-treated ones showed
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various degrees of deterioration. The piperine contents of the NTP- and dry-heat-treated whole black
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peppers were decreased to approximately 60% of that of the untreated peppers and showed no significant
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difference. On the other hand, significant differences were detected in the antioxidant activity assay. The
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result of the DPPH radical scavenging assay showed that the dry-heat treatment decreased the radical
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scavenging activity of the whole black peppers by approximately 50%, whereas the NTP treatment
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decreased it by 30%. The reduction power assay showed a clearer difference; the dry-heat treatment
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decreased the reduction power of the whole black peppers by approximately 45%, whereas the NTP
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treatment decreased it by only 11%. According to these results, the degree of oxidation of the black peppers
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caused by the NTP treatment is low. This tendency was also clearly confirmed in the thiobarbituric acid
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number assay, which indicates the degree of lipid oxidation. The thiobarbituric acid numbers of the NTP-
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and dry-heat-treated whole black peppers were 1.6-fold and 3.4-fold higher than that of the untreated whole
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black peppers, respectively. This quality of the whole black peppers after the NTP treatment was relatively
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low compared with that after the γ-irradiation treatment reported by Suhaj et al. (2006). However, NTP
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treatment is a promising microorganism inactivation technology for dried granular agricultural products,
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especially in countries that prohibit the irradiation treatment of food, and the findings of this study are
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expected to be useful for realizing the practical use of NTP treatment as a nonthermal microorganism
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inactivation process.
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4. Conclusions
It was demonstrated that the rotating NTP reactor was effective for enhancing the inactivation of
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Aspergillus spores on whole black peppers. After 4 min treatment, the reduction of the survival ratio was
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one order in the NTP treatment without rotation, on the other hand, the survival ratio was 0.71×10-3 in the
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NTP treatment with rotation at 50 rpm. This enhanced inactivation was due to the effective contact and
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exposure of the entire surface of the whole black peppers to the NTP. Therefore, control of the filling rate to
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allow the peppers to move and mix in the NTP reactor is important. The use of humidified gases was also
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effective for enhancing the reduction in the survival ratio; no viable spores of Aspergillus sp. were detected
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after 4 min treatment. In the evaluation of the black pepper quality, the piperine content and antioxidant
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activity of NTP- and dry-heat-treated whole black peppers showed various degrees of deterioration
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compared with those of untreated peppers. Although NTP and dry-heat treatment reduced the piperine
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content to the same extent, the NTP treatment was more effective for retaining the antioxidant activity of
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the whole black peppers than the dry-heat treatment.
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This study was supported by JSPS KAKENHI Grant Number 15H02231.
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Thirumdas R., Sarangapani C. & Annapure U. S. (2015). Cold plasma: A novel non-thermal technology for
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Zin Z. M., Abdul-Hamid A. & Osman A. (2002). Antioxidative activity of extracts from Mengkudu
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(Morinda citrifolia L.) root, fruit and leaf. Food Chemistry, 78, 227-231.
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Figure 1
Images and schematic of the NTP treatment reactor. Photograph taken from above (a) and
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cross-section drawing (b) of the electrode structure. Dark-field image of NTP (c). Photograph of the
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rotating NTP treatment reactor (d).
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Time course of survival ratio in the NTP inactivation treatment for several rotation rates. The
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NTP reactor was rotated at 0 (squares), 10 (triangles), 30 (circles), and 50 (diamonds) rpm. Closed and
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open symbols represent the treatment with and without NTP generation, respectively.
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Figure 3
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Time course of survival ratio in the NTP inactivation treatment using several gases. Oxygen
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(circles), humidified compressed air (triangles), or humidified oxygen (squares) was introduced into the
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NTP reactor at flow rates of 0.3 (a) and 0.9 (b) l/min. Closed and open symbols represent the treatment with
380
and without NTP generation, respectively. N.D. means that living cells were not detected.
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Figure 4
Side views of NTP reactor with several amounts of black pepper. The filling rates were 8.3 (a),
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25 (b), and 40 (c) %.
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Time course of survival ratio in the NTP inactivation treatment for filling rate of whole black
388
peppers in the reactor of 8.3% (circles), 25% (triangles), and 40% (squares). Closed and open symbols
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represent the treatment with and without NTP generation, respectively. N.D. means that living cells were
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not detected.
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Table 1 Comparison of piperine content and antioxidant activity in the black peppers treated for microbial inactivation.
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395 396
All the data are expressed as means ± standard deviations. Means with different numbers of stars in the same line differ significantly (p < 0.01).
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399 400 Table 1
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Comparison of piperine content and antioxidant activity in the black peppers treated for microbial
403
inactivation. Piperine content
DPPH scavenging % [-]
Control (none)
25.3 ± 2.1 *
61.7 ± 2.6 *
NTP
15.9 ± 1.3 **
Dry-heat
15.2 ± 2.9 **
0.40 ± 0.09 *
43.3 ± 4.1 **
2.36 ± 0.23 **
0.62 ± 0.04 **
33.6 ± 2.5 ***
1.47 ± 0.26 ***
1.36 ± 0.03 ***
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2.68 ± 0.13 *
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OD532 [-]
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Thiobarbituric acid number
OD700 [-]
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Reduction power
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Quartz tube
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To ground NTP generation area
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10 0-1 10 0-2
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1 1
2 3 Treatment time [min]
Fig. 2
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0-1 10
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2 3 Treatment time [min]
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Fig. 5
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