Accepted Manuscript

Antifungal activity of Michelia alba oil in the vapor phase and the synergistic effect
of major essential oil components against Aspergillus flavus on brown rice

Sumethee Songsamoe, Narumol Matan, Nirundorn Matan

PII: S0956-7135(17)30056-7

DOI: 10.1016/j.foodcont.2017.02.010

Reference: JFCO 5445

To appear in: Food Control

Received Date: 14 June 2016

Revised Date: 07 January 2017

Accepted Date: 09 February 2017

Please cite this article as: Sumethee Songsamoe, Narumol Matan, Nirundorn Matan, Antifungal
activity of Michelia alba oil in the vapor phase and the synergistic effect of major essential oil
components against Aspergillus flavus on brown rice, Food Control (2017), doi: 10.1016/j.foodcont.
2017.02.010

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Highlights

-Michelia alba oil in vapor was able to inhibit the spore and mycelium of Aspergillus flavus.

- Michelia alba oil in the vapor phase extends the shelf-life of brown rice by 4-fold.

- The synergistic effect of linalool and caryophyllene at the ratio of 10:1 was found.

- Cooked brown rice with Michelia alba oil in vapor showed good qualities.

- Michelia alba oil in vapor could decrease the hardness of the cooked rice.
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1 Antifungal activity of Michelia alba oil in the vapor phase and the synergistic effect of

2 major essential oil components against Aspergillus flavus on brown rice

3 Sumethee Songsamoe1, Narumol Matan1*, Nirundorn Matan2

4 1Food Technology, School of Agricultural Technology,

5 2Materials Science and Engineering, School of Engineering and Resources,

6 Walailak University, Nakhon Si Thammarat, THAILAND 80160

7 *e-mail address: nnarumol@wu.ac.th

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24 Abstract

25 The objectives of this study were to investigate the effect of essential oil (Michelia alba) vapor

26 on the spore germination and mycelium growth of Aspergillus flavus on brown rice and to

27 perceive the shelf life of the brown rice could be extended to a longer storage time. Different

28 volumes (150, 300, 450 μl) of M. alba and a 300 μl linalool/caryophyllene combination at ratios

29 of 10:1, 1:1, and 1:10 were first absorbed into plant absorbent material (~20 mm) before being

30 put into a closed glass box (1L) containing A. flavus spore and the mycelium (~5 mm) in a

31 Malt Extract Agar (uncovered plate). Mold testing was also carried out on brown rice with A.

32 flavus spore suspension before being incubated at 25º C and 100% RH for 16 weeks. Quality

33 tests e.g. texture, a sensorial evaluation (hedonic scale) of brown rice were also conducted.

34 Results indicated that the vapor phase of M. alba at ≥ 300 μl L-1 air could inhibit both spore

35 germination and A. flavus mycelium. Antifungal activity of M. alba in air was strongly

36 correlated with the linalool/caryophyllene combination at the ratio of 10:1 in 300 μl L-1 air. In

37 addition, M. alba vapor at 300 μl L-1 air was found to extend the shelf-life of the brown rice by

38 four times (16 weeks) in comparison with the control treated without essential oil (4 weeks).

39 After being cooked, the hardness of brown rice with volatile essential oil was found to be

40 reduced by one third (compared to the control brown rice). The hedonic value (overall liking) of

41 cooked brown rice packed with M. alba vapor at 300 μl for 1 week and then stored for 16 weeks

42 was a 7, rated as “like moderately”. Therefore, this study has demonstrated the good potential of

43 M. alba vapor to control mold growth on the surface of brown rice.

44 Keywords: Michelia alba; vapor; brown rice; linalool; caryophyllene

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47 1. Introduction

48 Normally, brown rice is obtained by removing the outermost layer and hull from rice

49 kernels. Brown rice has a lot of nutritional value as it contains high protein, fiber, vitamins and

50 minerals (Lamberts et al., 2007). In addition, health promoting components such as tocopherols,

51 phytosterol, -oryzanol, -aminobutyric acid (Gani et al., 2012), phenolics, flavonoids and

52 proanthocyanidin (Zhou et al., 2014) have been reported to be found in brown rice and brown

53 rice products. By modulating lipid metabolism and oxidative stress (Imam et al., 2014), these

54 nutritional compounds may decrease the risk of cardiovascular diseases for humans. On the

55 other hand, brown rice has harder texture and lower palatability than white rice according to the

56 lower water absorption of the outer pericarp of brown rice that restricts water diffusion during

57 cooking (Billiris et al., 2012). Furthermore, brown rice and brown rice products are susceptible

58 to postharvest diseases caused by various molds. In particular, Aspergillus flavus is a major

59 mold of brown rice and brown rice products and causes postharvest losses and diseases at high

60 frequency (Suhem et al., 2013; Suhem et al., 2015). A. flavus can produce aflatoxins which pose

61 a risk to human health (Peraica et al., 1999) because of its nephrotoxic, immunotoxic, mutagenic,

62 teratogenic and carcinogenic effects. Numerous studies have found high levels of mold

63 contamination in rice due to the contamination of A. flavus during harvest, handling,

64 transportation, and storage (Lai et al., 2015; Reddy et al., 2009). Furthermore, A. flavus is one of

65 the most contaminant of stored agricultural commodities that have been reported around the

66 world (Dwivedy et al., 2016; Klich 2007). Therefore, the application of essential oils such as

67 Litsea cubeba vapor on brown rice snack bars (Suhem et al., 2015) and development of

68 absorbent material containing bergamot oil (Songsamoe et al., 2016) against the growth of A.

69 flavus in rice has been studied in recent years. Thanaboripat et al. (1997) also found that garlic at

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70 40,000 µg ml-1 and carrot at 60,000 µg ml-1 gave the best reduction of A. flavus and inhibited an

71 aflatoxin production in rice. The study agreed with the Dwivedy et al. (2016) findings where

72 Mentha spicata essential oil at 1.0 µl L-1 could protect chickpea food system from A. flavus.

73 Essential oil is normally obtained from vapor distillation of plants. The name of the

74 essential oil depends on its extracted ingredients (Burt, 2004). Michelia alba oil is an essential

75 oil and can be extracted from M. alba flowers. This essential oil is used in medicinal systems for

76 treating a number of diseases including inflammatory conditions (Parimi & Kolli, 2012) and

77 cancer (Noysang et al., 2014). Furthermore, M. alba is one of medicinal plants with a long

78 history of safe use in traditional medicine (Kumar et al., 2012; Wang et al., 2010). The volatile

79 constituents of M. alba flowers were found to contain α-myrcene, (S)-limonene, (R)-fenchone,

80 linalool, camphor and caryophyllene (Shang et al., 2002). Some of these, such as linalool

81 (Shimada et al., 2014) and limonene (Frankova et al., 2016), were reported to be used against

82 molds. However, using Michelia alba oil against A. flavus on brown rice has not been reported.

83 Therefore, the objectives of this study were to investigate the effects of Michelia alba oil

84 and its main components in the vapor phase on the growth of A. flavus on cooked brown rice and

85 to investigate the potential application of Michelia alba oil vapor to control mold spoilage on it

86 during long storage and its quality.

87 2. Materials and methods

88 2.1 Essential oil

89 Michelia alba was obtained from Thai China Flavors & Fragrances Co., Ltd of Thailand.

90 Linalool (73%) and caryophyllene (7%) were identified as major constituents by using gas

91 chromatography-mass spectrometry analysis (Hewlett-Packard, Model 7890A, USA). Both pure

92 components were purchased from Merck, Ltd. (Germany).

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93 2.2. Culture

94 A strain of mold (Aspergillus flavus WU 1511) isolated from the brown rice was obtained

95 by the Innovation of Essential Oil for Food Safety and Packaging laboratory of Walailak

96 University in Nakhon Si Thammarat, Thailand. Mycelium of A. flavus on malt extract agar

97 (MEA; Merck Ltd., Thailand) was prepared for 10 days at 25C. Spore suspensions of A. flavus

98 (10-1 - 10-8 spore ml-1) were collected by flooding the surface of the plates with ~9 ml of sterile

99 water.

100 2.3 The effect of Michelia alba oil in the vapor phase with its main components on the growth of

101 A. flavus on MEA

102 The effect of the vapor phase of essential oil with its main components on spore

103 germination and mycelium growth of A. flavus was investigated by using Malt Extract Agar

104 (MEA). Petri dishes with MEA agar were separated into two groups for spore and mycelium

105 tests. The first group of MEA plates was spread with 0.1 ml of A. flavus at concentrations from

106 101 CFU ml-1 to 108 CFU ml-1. The lowest concentration for microbial population detected was

107 101 CFU ml-1. For the second group, MEA was aseptically inoculated by placing mycelium of A.

108 flavus (diameter ~ 5 mm) to the center. Next, absorbent material made from water hyacinth root

109 (~20 mm) was prepared according to the method of Songsamoe et al. (2016). Different

110 volumes of M. alba oil (150 µl, 300 µl and 450 µl) were added into each absorbent disc. An

111 absorbent without essential oil was done for the control. Then, the lidless inoculated plates and

112 each adsorbent were separately added inside a glass box (1L). All boxes were incubated at 25C

113 for 10 days. Measurements of mold growth by colony count and diameter (mm) of the mycelium

114 growth on the plates were checked every day for 10 days. The vernier caliper (Winton,

115 Gammaco Thailand Co., Ltd.) was used for the determination of the mycelium growth.

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116 For the main components of Michelia alba, 300 µl of linalool, 300 µl of caryophyllene

117 and 300 µl of 10:1, 1:1 and 1:10 mixtures of linalool and caryophyllene were selected and used

118 for this study. Each adsorbent was placed into the glass box according to the same method above

119 and was incubated for 10 days.

120 2.4 Inhibition of A. flavus and natural mold on brown rice by volatile Michelia alba oil

121 Brown rice (moisture content  12%) was prepared from Khai Mod Rin (NSRC9500113)

122 unhusked rice, a local rice grown in the Nakhon Si Thammarat province of Thailand. The rice

123 was provided from the Nakhon Si Thammarat Rice Research Center, Nakhon Si Thammarat

124 province, Thailand. Brown rice (375 g) without and with 37.5 ml of A. flavus at concentration of

125 108 CFU ml-1 on the surface was added into a sterilization glass box (1L). Next, M. alba at 300

126 µl and at 300 µl with linalool and caryophyllene at the ratio of 10:1 was added into the plant

127 absorbent material (~20 mm) and was placed into the glass box. After the box was closed,

128 brown rice was stored at 25C for 16 weeks. For mold count, brown rice specimens (25 g) were

129 placed into sterile stomacher bags with 225 ml of buffered peptone water (BPW; Nissui

130 Pharmaceutical CO., LTD, Tokyo, Japan). Then, the counting of A. flavus was performed on

131 MEA by using a method proposed by Suhem et al., (2013). The control was done in the same

132 way but without essential oil in the absorbent material.

133 2.5 Cooked brown rice quality measurements

134 Brown rice containing M. alba vapor at 300 µl was investigated for quality measurements

135 after cooking as well as brown rice with 300 µl of linalool and caryophyllene at the 10:1 ratio.

136 The control (without essential oil) was also done for the following measurements.

137 2.5.1 Cooking time

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138 Two hundred grams of brown rice were soaked in a pot for 5 minutes with 300 ml of

139 deionized water. After the rice was cooked with an automatic rice cooker (CR-110, 1L, 400 W,

140 220 V, 50 Hz, Otto Kingglass Co., Ltd. Thailand), a thermostat coupled with a microswitch

141 automatically switched it off. Cooking time (in minutes) was recorded by using a digital clock

142 (Eppendorf Thailand Co., Ltd.).

143 2.5.2 Color measurement

144 The surface color of the cooked brown rice was measured (n=5) by a colorimeter

145 (MiniScan EZ, Hunter Associates Laboratory, USA). CIELab color coordinates were used to

146 determine the degree of lightness (L*), redness-greenness (+ or – a*) and yellowness-blueness (+

147 or – b*). The total color difference (E*) between cooked M. alba treated brown rice and cooked

148 control brown rice was calculated according to E*  (L*) 2  (a*) 2  (b*) 2

149 (Sirisoontaralak et al., 2015)

150 2.5.3 Texture measurement

151 Twenty grams of cooked brown rice were placed on a cylinder container for the texture

152 profile analysis (TPA) test by using a texture analyzer (LR 5K MK4, Lloyd Instrument Co., Ltd.,

153 England). A cylinder probe of 45 diameters was used to compress the sample to 75%

154 deformation at the test speed of 5 mm s-1. Hardness (N), springiness (mm), cohesiveness and

155 gumminess (N) were recorded.

156 2.5.4 Sensory evaluation

157 Brown rice specimens containing M. alba vapor at 300 µl and the control stored at 25C

158 for 1 week and 16 weeks were selected in this test. After being cooked, the brown rice specimens

159 were subjected to sensory analysis by an untrained panel (48 panelists, 29 females and 19 males)

160 ranging from 20 to 45 years of age. The cooked brown rice was randomly coded and presented to
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161 each panelist seated separately in a control booth. A 9-point hedonic scale ranging from “like

162 extremely” to “dislike extremely” was used to determine the degree of acceptance of the specimens

163 in terms of flavor, texture, color and overall liking (Meilgaard et al. 1999). The responses were

164 then converted to numerical values ranging from 1 for “dislike extremely” to 9 for “like

165 extremely”.

166 2.6 Statistical analysis

167 All results are expressed as the mean ± standard deviation. The data was statistically

168 treated by one-way ANOVA and Duncan's post hoc test, with p < 0.05 being considered to be

169 statistically significant. Statistical analysis was performed with Statistica software (StatSoft,

170 Tulsa, Oklahoma, USA).

171 3 Results and Discussions

172 3.1 Effects of M. alba with its main components vapor

173 Effects of M. alba vapor at different volumes of 150, 300, and 450 µl L-1air on the spore

174 germination of A. flavus and the growth of mycelium are presented in Fig 1(a-b). The highest

175 volume of the essential oil vapor (450 µl L-1 air) clearly showed a reduction of spore germination

176 from 8 log10 CFU ml-1 to zero within 3 days of storage at 25C, compared to 5 days when using

177 300 µl L-1 air. In addition, no growth of mycelium was also confirmed when the mycelium

178 plates took in the M. alba vapor at volumes of 300 and 450 µl L-1 air. On the other hand, lower

179 volume of M. alba at 150 µl L-1 air had little effect on both spore and mycelium reduction of A.

180 flavus. From this study, Michelia alba oil in vapor was able to inhibit the spore and mycelium of

181 A. flavus.

182 M. alba is recognized to contain aroma constituents and bioactivities that can be used as

183 anti-inflammatory agents to treat cramps, abdominal pain (Lee et al., 2005), fever, syphilis and

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184 malaria (Asaruddin et al., 2003). Furthermore, M. alba has been confirmed to work against

185 Salmonella enteritidis, Enterobacter cloacae and Bacillus subtilis (Rangasamy et al., 2007).

186 However, the results from this experiment confirmed that M. alba in the vapor phase (≥300 µl L-

187 1air) could completely inhibit spore germination and mycelium growth of A. flavus.

188 On the other hand, the effects of vapor phase with the main components (linalool and

189 caryophyllene) of M. alba against spore and mycelium of A. flavus are presented in Fig 2 (a-b).

190 Results found that only the 300 µl L-1 air combinations of linalool and caryophyllene at the 10:1

191 ratio totally showed inhibition of mycelium and spore germination growth of A. flavus. Using

192 other ratios (1:1, and 1:10) and caryophyllene alone had no affect on A. flavus. Nevertheless,

193 linalool alone at 300 µl L-1 air did reduce the mycelium and spore growth but it was not

194 complete; just the 300 µl L-1 air combination of linalool and caryophyllene at the ratio of 10:1

195 could completely inhibit A. flavus. Therefore, low amounts of caryophyllene in the air phase

196 could help boost the antifungal activity of linalool against both spore germination and mycelium

197 of mold. This might be the main reason why the air phase of M. alba inhibited growth of mold.

198 In addition, linalool (73%) and caryophyllene (7%) were found to be the main components of M.

199 alba for this test with the ratio of linalool to caryophyllene about 10:1.

200 Linalool was reported to be found in many essential oils and confirmed to be major

201 components in high concentrations greater than 60% (Duarte et al., 2016). High composition of

202 linalool in M. alba (73%) was also found in this research. In 1989, Vollmuth et al. reported that

203 no adverse effects were found when female mice were given 375 mg kg-1 of linalool via a

204 stomach tube for 5 days. In addition, Bickers et al. (2003) indicated that linalool has a low levels

205 of acute toxicity. Furthermore, caryophyllene is not only found in essential oil but can also be

206 isolated from the cultures of fungicolour fungus (Deyrup et al., 2006). Although, linalool

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207 (Bagamboula et al., 2004) or caryophyllene alone (Deyrup et al., 2006) in the liquid phase

208 showed lower antibacterial activity than essential oil alone, some reports indicated that

209 combinations between linalool and another component such as -terpineol exhibit strong

210 antibacterial activity against periodontopathic and cariogenic bacteria e.g. Porphyromonas

211 gingivalis and Aggregatibacter actinomycetemcomitans (Park et a., 2012). In addition, a

212 synergistic effect of the combination of caryophyllene with ascaridole or carvacrol against

213 promastigotes of Leishmania has been reported (Pastor et al., 2015). There are other reports

214 (Shimada et al., 2014; Deyrup et al., 2006) on the antifungal activity of linalool or caryophyllene

215 but for this research it can be noted that the combination of linalool and caryophyllene in the gas

216 phase at the ratio of 10:1 could inhibit mold spores and mycelium of A. flavus.

217 While, essential oils are very natural mixtures which are contained by many major

218 compounds and various minor components. However, when using essential oil in the air phase

219 only one or two components have shown effectiveness. Suhem et al. (2015) found that citral

220 (the main component of L. cubeba oil) was shown to be the strongest inhibitor on A. flavus,

221 better than the L. cubeba oil vapor. In 2016, Songsamoe et al. reported that while many main

222 components of bergamot oil were observed, only the vapor of limonene could affect enzymes

223 involved in spore germination by causing an extension of the lag phase after UV-C radiation. By

224 synergistically using two main components of an essential oil in a vapor phase, it is worth

225 considering that this could inhibit mold growth and could have potentially be used in food as a

226 natural antifungal preservative.

227 3.2 Effect of M. alba vapor on A. flavus and natural mold on brown rice

228 Growth of A. flavus spores on the brown rice stored from 4 to 16 weeks after being exposed

229 to 300 l L-1 air of M. alba and 300 l L-1 air of linalool and caryophyllene at the 10:1 ratio are

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230 shown in Fig 3. Spore counting on the brown rice was recorded every 4 weeks until the 16th week

231 of storage (4 fold). The results between the treated brown rice and the control were different after

232 the first 4 weeks of storage. Only treated brown rice with both M. alba and linalool- caryophyllene

233 vapors could extend the spore germination of A. flavus and natural mold. However, only vapor

234 with M. alba could completely inhibit natural mold from 4 weeks to 16 weeks and for at least 12

235 weeks if the brown rice contained A. flavus spores. Differences in the shelf-life observed in this

236 study between M. alba and linalool-caryophyllene vapors at 300 l L-1 air could possibly be caused

237 by the effect of various minor component gas in M. alba. While the combination of linalool and

238 caryophyllene at the ratio of 10:1 in air was effective in reducing mold spore spoilage in this

239 experiment, it could extend the shelf-life of brown rice by up to 12 weeks. Therefore, this work

240 has shown the potential for using a vapor phase with this combination at the ratio to extend the

241 shelf-life of brown rice under severe conditions (25C, 100%RH). Normally, the concentration of

242 essential oils or their main components is necessary to inhibit microbial growth. It is higher in

243 foods than in the medium according to the interactions between phenolic compounds in essential

244 oil and the food matrix (Nychas and Tassou, 2000). Higher concentrations of essential oil should

245 be used if food contains high fat content (Gill et al., 2002)

246 In this study, however, 300 l L-1 air of M. alba against mold in both brown rice (high in

247 carbohydrates) and MEA was used for this study. In addition, vapor of M. alba might be considered

248 as a potential use for rice storage because direct addition of essential oil into the surface of brown

249 rice has not been fully developed yet. Therefore, the benefits for using essential oil vapor at a low

250 volume are (1) it can increase food safety without chemical preservatives (2) it makes food safe to

251 consume and (3) the mild flavors of essential oil in the vapor phase don’t greatly affect the taste

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252 of foods. The technique could be extended to inhibit mold growth on various stored agricultural

253 products.

254 3.3 Quality of cooked brown rice and sensory evaluation

255 The cooking time, color and texture of the cooked brown rice specimens with and

256 without 300 µl L-1 air of M. alba and 300 µl L-1 air of linalool and caryophyllene at 10:1 in vapor

257 is shown in Table 1. From these results, the cooking time of the treated brown rice and the

258 control was not significantly different (p>0.05). The M. alba vapor treatment decreased the

259 lightness (L*) from 75.0 ± 1.0 to 71.9 ± 1.9. The L* value of the cooked M. alba treated brown

260 rice are higher than the cooked brown aromatic rice of 59±2.8 (Sirisoontaralak et al., 2015), the

261 cooked normal rice of 68.7±0.9 and the cooked waxy rice of 69.2±1.3 (Tian et al., 2014). In

262 addition, the total color difference (E*) caused by the M. alba vapor treatment of 5.23 ± 2.79 is

263 much smaller than those caused by various cooking methods and chilled storing conditions of

264 12-15 observed in the germinated brown aromatic rice (Sirisoontaralak et al., 2015).

265 For texture analysis, it was observed that M. alba vapor reduced the hardness of the

266 cooked brown rice in about one third time (from 351.96 ± 7.63 N to 243.13 ± 21.96 N). The

267 cooked brown rice with the 10:1 linalool-caryophyllene combination vapor also showed lower

268 hardness (275.51±13.95 N). According to the effect of essential oil vapor and combination of its

269 main components, it is clear that the hardness of the cooked brown rice changed substantially

270 after cooking. This decrease in hardness might be due to essential oil adsorption from the air to

271 the brown rice grains. After absorption, it can be responsible for a decrease in hardness and

272 gumminess of cooked rice specimens. In addition, it was noted that after vapor exposure the

273 cooking time was reduced with respect to the effect from the vapor.

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274 The results of the sensory flavor, texture, color and overall liking attributes of the cooked

275 brown rice are summarized in Fig. 4. Panelists gave higher flavor, texture and overall liking

276 marks for the cooked M. alba vapor treated brown rice than those of the untreated control. There

277 was no difference in color liking. Furthermore, overall liking of the cooked brown rice after 16

278 weeks of storage was not different from that of 1 week.

279 4.Conclusions

280 The results from this experiment indicated that the Michelia alba in vapor at ≥ 300 µl L-1

281 air was able to inhibit the spore and mycelium of A. flavus. It is confirmed that the synergistic

282 effect of linalool (the 1st main component) and caryophyllene (the 2nd main component) at the

283 10:1 ratio was the key factor in enhancing the antifungal activity of M. alba against mold.

284 However, the underlining mechanisms warrant further investigation. Vapor of M. alba could

285 extend the shelf-life of brown rice for up to 16 weeks compared to just 4 weeks for the control.

286 Furthermore, the treatment of M. alba vapor improved qualities of the cooked brown rice by

287 lowering its hardness. Sensory panelists significantly preferred the flavor and texture of the

288 cooked brown rice treated with M. alba vapor stored for 1 week and 16 weeks more than the

289 untreated rice. These results indicate that the vapor phase of M. alba can be used for the

290 preservation of brown rice, achieving long term storage with good properties after cooking.

291 Acknowledgements

292 This study was supported by the Thailand Research Fund (TRF) through the Royal

293 Golden Jubilee Ph.D. Program (Grant. No. PHD/0090/2014), the Walailak University Fund and

294 the Office of the Higher Education Commission’s Higher Education Research Promotion

295 (HERP). The authors would like to thank Ms. Athiya Nonthakaew for her help on the sensory

296 experimental setup.

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8 150 ul/L air

7 300 ul/L air
6 450 ul/L air
Log10 cfu ml-1

Control
5

0
1 2 3 4 5 6 7 8 9 10
Exposure time (day)

(a)

30

150 ul/L air

25 300 ul/L air
450 ul/L air
Colony diameter (mm)

20 Control

15

10

0
1 2 3 4 5 6 7 8 9 10
Exposure time (day)

(b)

Fig.1 Effects of the M. alba vapor in different volume at 150, 300, and 450 µl L-1air on the

spore germination (a) and mycelium growth (b) of A. flavus in a closed box
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9 Linalool

8 Caryophyllene

Linalool :
7 Caryophyllene 10:1
Linalool :
6 Caryophyllene 1:1
Log10 cfu ml-1

Linalool :
5 Caryophyllene 1:10

0
1 2 3 4 5 6 7 8 9 10
Exposure time (day)

(a)

30 Linalool

Caryophyllene
25
Linalool :
Caryophyllene 10:1
Colony diameter (mm)

20 Linalool :
Caryophyllene 1:1
Linalool :
15 Caryophyllene 1:10

10

0
1 2 3 4 5 6 7 8 9 10
Exposure time (day)

(b)

Fig.2 Effect of vapor phase of linalool and caryophyllene and their mixtures against spore (a)

and mycelium of A. flavus (b) at 300 µl L-1air in a closed box

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5
Log10 cfu g-1

Time (week)
1 16
12
0 8
Control M. alba 4
Li:Ca (10:1) Control M. alba Li:Ca (10:1)

Natural mold A. flavus

Fig.3 Natural mold and A. flavus count on brown rice during storage for 16 weeks

9
a a Control
a a a a
8 a a a 300 ul/L air (1 week)
300 ul/L air (16 weeks)
7
b b
6 b
Scale (1-9)

0
Flavor Texture Color Overall
Attributes

Fig. 4 Sensory evaluation of cooked brown rice

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Table 1 Quality of cooked brown rice

Quality Factor Brown rice

Control 300 µl L-1 air of M. alba

Cooking time (min) 31.2 ± 0.4a 30.5 ± 0.5a

Color

L* 75.04 ± 1.03 a 71.92 ± 1.94b

a* 1.94 ± 0.11a 2.06 ± 0.48a

b* 15.76 ± 1.98b 19.35 ± 3.49 a

E* 5.23 ± 2.79

Hardness (N) 325.12 ± 21.37a 188.67 ± 14.06b

Springiness (mm) 4.05 ± 1.91a 4.77 ± 0.82a

Adhesiveness (N) 8.66 ± 3.00a 6.94 ± 3.42b

Cohesiveness 0.19 ± 0.08a 0.22 ± 0.02a

Gumminess (N) 59.35 ± 24.63a 40.43 ± 1.54a