Investigation and characterization of Myroides Odoratimimus strain

Subscriber access provided by UNIV OF LOUISIANA

Food Safety and Toxicology

Investigation and characterization of Myroides Odoratimimus strain 3J2MO on Aflatoxin B1 degradation Silivano Mwakinyali, Zhang Ming, Huali Xie, Qi Zhang, and Peiwu Li J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 25 Mar 2019 Downloaded from http://pubs.acs.org on March 27, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 27

Journal of Agricultural and Food Chemistry

1

Investigation and characterization of Myroides Odoratimimus strain

2

3J2MO on Aflatoxin B1 degradation

3

Silivano E. Mwakinyali1,2,4,6,ω,*, Zhang Ming1,2,4,ω, Huali Xie1,2,4, α,Qi Zhang1,2,4,†, Peiwu Li1,2,3,4,5,*,†

4

1

5

2

6

3

7

4

8

5

9

6

Oil Crops Research Institute, Chinese Academy of Agricultural Sciences, Wuhan 430062, PR China Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture, Wuhan, 430062, P. R. China Laboratory of Quality & Safety Risk Assessment for Oilseeds Products, Wuhan, Ministry of Agriculture, Wuhan, 430062, P. R.China Key Laboratory of Detection for Mycotoxins, Ministry of Agriculture, Wuhan 430062, PR China Quality Inspection and Test Center for Oilseeds Products, Ministry of Agriculture, Wuhan 430062, PR China National Food Reserve Agency (NFRA), Ministry of Agriculture, P.O Box 1050, Dodoma, United Republic of Tanzania

10

Author’s contribution

11

ω These authors did Investigation, Methodology, Conceptualization, Data curation, Formal analysis, Writing original draft; †, Supervision,

12 13

Validation, Conceptualization, Review and editing; α Formal analysis

14

Tel.: +86 27 86812943; Fax: +86 27 86812862; E-mail address: [email protected] (P. Li); [email protected] (S. Mwakinyali)

15

Abstract

16

Aflatoxin B1 (AFB1), is a type I carcinogen and one of the strongest naturally occurring that may

17

have injurious to human and livestock upon ingestion, inhalation or skin contact like

18

carcinogenic and mutagenic effects. It causes significant hazardous effects to food and animal

19

production industry. We found 3J2MO bacteria strain degraded AFB1 ultra-well and here it

20

were tested its AFB1 degradation ability and characterized. The strain degraded AFB1 about

21

93.82% after incubated for 48h in Luria-Bertani (LB) medium at 37 ℃

22

concentration of 100ppb and inoculation quantity of 1x107CFU/ml. High performance liquid

23

chromatography-fluorescence detection (HPLC-FLD) was used to determine AFB1 amount.

24

Maximum degradation rate was 89.23% at pH 8.5, 55.78% at an inoculation quantity of

25

1x108CFU/ml and 71.50% and 71.21% at 34℃and 37℃ respectively. Treatment with sucrose

26

and soluble starch as a carbon nutrients and beef extract and ammonium acetate as nitrogen

27

nutrients stimulated the degradation rate. Mg2+ and Ca2+ ions were activators for AFB1

*Corresponding Authors

1

ACS Paragon Plus Environment

with a final


Page 2 of 27

28

degradation; however, Mn2+, Fe3+, Zn2+ and Cu2+ were strong inhibitors. This bacteria have

29

potential bioremediation and detoxification of aflatoxin contamination biocontrol strategy in

30

both agricultural products and food industry matrices.

31

Keywords; Myroides Odoratimimus strain 3J2MO, aflatoxins, biocontrol, contamination, degradation

32

1. Introduction

33

Aflatoxins are noxious secondary metabolites that are produced by filamentous fungal species

34

like Aspergillus flavus and Aspergillus parasiticus distributed worldwide in environment which

35

normally contaminate staple foods and feeds and may have injurious to human and livestock

36

upon ingestion, inhalation or skin contact like carcinogenic, mutagenic, cytotoxic and

37

endocrine disrupting effects

38

carcinogenic potent chemical in nature, that attack liver 8.

39

The global contamination of foods and feeds with mycotoxins is a significant agricultural and

40

medical problem

41

consumed cereals such as maize, rice and peanut and regrettably are also the greatest prone

42

crops to mycotoxins and results to massive economic losses each year, including health

43

endangerment and loss in human and animal life, decrease livestock productivity and so on

44

8,2,11,12,13.

45

contaminated with mycotoxins at various levels, and around 20%-50% of all cereal products

46

produced globally are contaminated with mycotoxins to various extend, which sometimes are

47

unpredictable and difficult to estimate making it a worldwide food safety and public health

48

problem 11.

49

Fungal growth and mycotoxin production may occur at various stages in the food chain from

9,10.

1,2,3,4,5,6.

AFB1 is the most poisonous aflatoxin

7

due to its

This is due to their presence and negative effects in a globally major

In a global perspective, about 25% of maize and its products were reported to be

2


Page 3 of 27


50

pre to post harvest14,13 like in the field, processing or during storage under desirable weather

51

such as temperature and humidity conditions11. Due to climate change and global warming

52

the world is undergoing nowadays, countries having temperate climate like warmer, tropical

53

and subtropical climatic conditions are also most likely to be impacted by aflatoxins producing

54

fungi and consequently aflatoxins

55

region and Sub Saharan Africa are at a high risk for chronic exposures to hazardous effects of

56

mycotoxins 8,12,20.

57

Reducing and removing both pre and postharvest aflatoxins contamination in crops is a huge

58

challenge facing health and agriculture researchers recently. Aflatoxins contamination can be

59

minimized by following proper crop field management including good agriculture practices.

60

This will contribute greatly to achieving the goal of devising novel strategies to eliminate pre-

61

and postharvest aflatoxins contamination, resulting to a safer food and feed supply21.

62

When considering aflatoxins mitigation strategy, one must bear in mind that, basic molecular

63

structure of all aflatoxins is carbon in nature, so microorganisms that could utilize it as their

64

carbon source might also be able to degrade it. Therefore, the metabolizing processes could

65

result in the degradation of these mycotoxins

66

molecular structure of all aflatoxins

67

source. Hence, biodegradation of aflatoxins using microorganisms is considered to be an

68

auspicious strategy for aflatoxins remediation because it is safe, selective, effective and

69

environmental friendly compared to other strategies.

70

In an efforts of mitigating the effects of aflatoxins, a number of bacteria isolates with

71

biodegradation ability have been characterized so far and seems to be better, from quality and

15,16,17,18,19.

22,23

Like East African countries which are in tropical

16.

Like difuran ring and coumarin is the basic

and some microorganism utilize it as their carbon

3



24,

such as Rhodococcus erythropolis

72

safety point

73

subtilis UTBSP1

74

licheniformis CFR1

75

and Pseudomonas fluorescens strain 3JW1

76

During decomposition reaction, unknown and noxious by products might be created which are

77

capable to react with other metabolites creating unmanageable and unwelcome mixture of

78

reagents34. Therefore, besides monitoring mycotoxin degradation by immunochemical or

79

other analytical methods, it is very essential to develop applicable methods to detect

80

hazardous metabolites produced during mycotoxin degradation like ELISA

81

SOS-Chromotest 36 so as to safeguard human and livestock health.

82

In an attempt to investigate new strain that will not change the characteristics of food during

83

AFB1 degradation, bacteria Myroides Odoratimimus strain 3J2MO was obtained from the

84

laboratory library after it displayed strong degradation activity on aflatoxins. Myroides

85

Odoratimimus is a gram-negative, non-fermentative, obligately aerobic, yellow pigmented,

86

and non-motile rod-shaped bacterium with a characteristic fruity odor and commonly found in

87

environment such as water bodies and soil. The genus Myroides comprises two species,

88

Myroides odoratus and Myroides odoratimimus 37,38. They are seldom clinical isolates and are

89

frequently

90

immunocompromised patients and rarely, immunocompetent host

91

M.Odoratimimus is an unusual pathogen, but from time to time can be life threatening 38 like

92

causing urinary tract infections and nosocomial outbreaks 41,42 ,pneumonia and meningitis 43.

93

The objectives of the present study were to (1) evaluate degradation efficiency of Myroides

27, 30

25,

Page 4 of 27

Myxococcus fulvus ANSM068

Bacillus subtilis ANSB060

28,

Bacillus licheniformis

26, 29,

Bacillus Bacillus

Stenotrophomonas maltophilia 31, Mycobacterium fluoranthenivorans

regarded

low-grade

33.

32

Biodegradation strategies have risks as well.

opportunistic

pathogens

4


causing

35

and

infections 39,40.

in

Although

Page 5 of 27


94

Odoratimimus strain 3J2MO in LB liquid culture on AFB1 (2) examine factors influencing

95

degradation efficiency of LB liquid culture of Myroides Odoratimimus strain 3J2MO on AFB1

96

(Characterization).

97

2. Materials and Methods

98

2.1 Bacteria Strain, Growth Media, and Chemicals

99

Luria-Bertani (LB) media (10ml)

100

The sample AFB1 was purchased from Sigma-Aldrich (Jerusalem, Israel), the purity was 99.99%

101

and used as reference standard.

102

Dichloromethane, methyl alcohol and other chemicals mentioned above were analytical

103

reagents from Xilong Scientific Co. Ltd. (Guangdong, China).

104

Water was purified by a Milli-Q water system-RIOS16/Milli-QA10, Millipore, (Shanghai, China)

105

Strain. Myroides odoratimimus strain 3J2MO was provided by the Oil Crops Research Institute

106

of the Chinese Academy of Agricultural Sciences where it is stored in the refrigerator at -70℃

107

Media. Luria-Bertani (LB) medium prepared with the following composition (yeast extract 5

108

g/L, tryptone 10 g/L, NaCl 10 g/L. pH were adjusted to 7.0±0.2; Heated and boiled with

109

frequent agitation for few minutes (≈5 min) to completely dissolve the powder, then sterilized

110

by autoclaved for 30 min at 121℃)

111

Preparation of the bacteria strain. Myroides odoratimimus strain 3J2MO was inoculated in

112

the petri dish for 24 h at 37 ℃ , and then inoculated into liquid medium and shaking it

113

constantly (200 r/min) in an incubator for 24 h at 28℃ and Optical Density was determined to

114

get the suitable concentration.

115

2.2 Determination of AFB1 biodegradation by Bacteria strain 3J2MO 5



Page 6 of 27

116

AFB1, were mixed with strain 3J2MO in 10 ml LB medium. The final concentration of AFB1 was

117

100ppb. The final concentration of strain 3J2MO were 1*107CFU/ml determined by Thermo

118

Scientific Microplate Reader (Molecular Devices, USA). After 5 days (120 hours) of incubation

119

with constant shaking(200 r/min) at 28 ℃ in an incubator shaker from Tian Jin Honour

120

Instrument Co. Ltd (Type HNY-211B, power AC 220 ± 0%, frequency 50-60Hz). 1 ml were

121

collected, 2 ml dichloromethane were added, extracted, then removed 1ml upper solution,

122

the remained solution were evaporated and dried by using Termovap Sample Concentrator

123

called Trustworthy Instrument, type HX-NC12 from Science and Technology Co. Ltd (Wuhan,

124

China), added 1 ml methyl alcohol (methanol) to dissolve the AFB1 residual, then extracted for

125

30 minutes at 20 ℃ by using Ultrasonic Apparatus type DTX-27J with frequency 40KHz, power

126

220v/50Hz from Ding Tai Heng Sheng (Hubei, China), filtrated by 0.22µm filter membrane and

127

the amount of AFB1 were determined by using HPLC-FLD series 1100 from Agilent®

128

Technologies, USA and used Photochemical Derivative Instrument type HX-G from Wuhan

129

Century Trusty Technology Co, Ltd for post column derivatization system to improve the

130

sensitivity of fluorescence. This study was conducted three times and treatments were

131

replicated three times.

132

2.3 Effect of time length and AFB1 concentration on degradation level by Myroides

133

Odoratimimus strain 3J2MO

134

AFB1, were mixed with bacteria strain 3J2MO in 10 ml LB medium. The final concentrations of

135

AFB1 were 50ppb, 100ppb, 200ppb, 500ppb, and 1ppm. The final concentration of strain

136

3J2MO were 1*107CFU/ml. Then were incubated at different time interval of 0 h, 6 h, 12 h, 24

137

h, 48 h, 72 h, 96 h and 120 h with constant shaking(200 r/min) at 28℃ in an incubator shaker. 6


Page 7 of 27


138

Other procedures were the same as described in section 2.2 above. This study was conducted

139

three times and treatments were replicated three times.

140

2.4 Effects of incubation time on AFB1 degradation by Myroides odoratimimus strain 3J2MO

141

AFB1, were mixed with bacteria strain 3J2MO in 10ml LB medium. The final concentration of

142

AFB1 was 100ppb. The final concentration of strain 3J2MO were 1*107CFU/ml determined as

143

described above. Then were incubated at different time interval of 0 h, 6 h, 12 h, 24 h, 48 h, 72

144

h, 96 h and 120 h with constant shaking (200 r/min) at 28℃ in an incubator shaker. Other

145

procedures were the same as described in section 2.2 above. This study was conducted three

146

times and treatments were replicated three times

147

2.5 Effects of pH on AFB1 degradation by Myroides odoratimimus strain 3J2MO

148

AFB1, were mixed with bacteria strain 3J2MO in 10ml LB medium. Final concentrations of AFB1

149

were 100ppb. The final concentration of strain 3J2MO were 1*107CFU/ml determined as

150

described above. In the pH tests, initial pH values of the reaction mixture were adjusted to 5.0,

151

5.5, 6.0, 6.5, 7.0, 7.5, 8.0 and 8.5 with relevant sodium phosphate buffers (Sodium hydroxide

152

and phosphoric acid). They were measured by pH-meter and incubated for 36 h with constant

153

shaking at (200 r/min) at 28℃ in an incubator shaker. Other procedures were the same as

154

described in section 2.2 above. This study was conducted three times and treatments were

155

replicated three times

156

2.6 Effects of temperature on AFB1 degradation by Myroides odoratimimus strain 3J2MO

157

AFB1, were mixed with bacteria strain 3J2MO in 10 ml LB medium. Final concentrations of

158

AFB1 were 100ppb. The final concentration of strain 3J2MO were 1*107CFU/ml. Then were

159

incubated at different temperature levels of 25℃, 28℃, 31℃, 34℃and 37℃ for 36 h with 7



Page 8 of 27

160

constant shaking (200 r/min) in an incubator shaker. Other procedures were the same as

161


162


163

2.7 Effects of bacteria concentrations on AFB1 degradation by Myroides odoratimimus strain

164

3J2MO

165

AFB1, were mixed with bacteria strain 3J2MO in 10ml LB medium. Final concentration of AFB1

166

was 100ppb. Bacterial were adjusted at different final concentrations levels of 1*105 CFU/ml,

167

1*106 CFU/ml, 1*107 CFU/ml, 1*108 CFU/ml and 1*109 CFU/ml determined by Thermo

168

Scientific Microplate Reader (Molecular Devices, USA) and incubated for 36h with constant

169

shaking (200 r/min) at 28 ℃ in an incubator shaker. Other procedures were the same as

170


171


172

2.8 Effects of nutrients and metal ions on the degradation performance of Myroides

173

odoratimimus strain 3J2MO

174

AFB1, were mixed with bacteria strain 3J2MO in 10ml LB medium. Final concentrations of AFB1

175

were 100ppb. The final concentration of strain 3J2MO were 1*107 CFU/ml. The effect of

176

different metal ions on degradation were determined by adding 0.2 mg/ml of Mg2+, Zn2+, Cu2+,

177

Mn2+, Fe2+, and Ca2+ in the form of (MgSO4, ZnS04.7H20, CuSO4.5H2O, MnSO4.H2O, FeSO4.7H2O,

178

CaCl2, respectively, and then were incubated for 36 h with constant shaking (200 r/min) at 28

179

℃ in an incubator shaker. Then other process for AFB1 determination by HPLC-FLD was

180

performed as described in section 2.2 above. This study was conducted three times and

181

treatments were replicated three times. 8


Page 9 of 27


182

The effects of nitrogen were determined by adding 0.5 mg/ml respectively in the form of

183

Peptone (Total Nitrogen ≥ 14.0% Amino nitrogen ≥ 3.5%, Burning residue ≤ 10.0%, Dry weight

184

loss ≤ 6.0%), a soluble protein formed by partial hydrolysis, sometimes used as a liquid

185

medium for growing bacteria),

186

≤ 4.5%, Burning residue ≤ 15%, Alcohol soluble nitrogen ≥ 6%, Ethanol insoluble ≤ 10%,

187

Standard nitrate present), Tryptone (%w/w), pH 2% solution, 7.3±0.2 at 25℃, Total Nitrogen ≥

188

12.7%, Amino Nitrogen ≥ 3.7%; Sodium chloride ≤ 0.4%; Tryptophan present, provide source

189

of amino acids for the growing of bacteria), Ammonia acetate and Yeast Extract (%w/w), pH

190

0.5% solution, 7.0±0.2 at 25℃, Total Nitrogen ≥ 10.0-12.5%, Amino nitrogen ≥ 5.1%, Sodium

191

chloride ≤ 0.3%).

192

The effects of carbon were determined by adding 0.5mg/ml respectively in the form of soluble

193

starch, D-glucose, Sucrose, α-lactose and D-Fructose. Then were incubated for 36h with

194

constant shaking at (200 r/min) at 28 ℃ in an incubator. Then other process for AFB1

195

determination by HPLC-FLD was performed as described in section 2.2 above. This study was

196

conducted three times and treatments were replicated three times.

197

2.9 Determination whether pH or bacteria strain 3J2MO are responsible for AFB1

198

degradation

199

It is known from literature that when pH reaches 9.0 and above

200

degrade itself, so we performed another experiment to determine and confirm which among

201

the two are responsible in the AFB1 degradation process. Therefore AFB1, were mixed with

202

bacteria strain 3J2MO in 10 ml LB medium. Final concentrations of AFB1 were 100ppb. The

203

final concentration of strain 3J2MO was 1*107 CFU/ml determined by Thermo Scientific

Beef Extract (Solid matter ≥ 75%, Total Nitrogen ≥ 13%, NaCl

9


44,

aflatoxins begins to


Page 10 of 27

204

Microplate Reader (Molecular Devices, USA) and the pH of a mixture was adjusted with initial

205

pH of 9.0 and incubated for 120 h with constant shaking at (200 r/min) at 28℃ in an incubator.

206

Then other process of AFB1 determination was followed as described above. This study was

207

conducted three times and treatments were replicated three times.

208

Another experiment was conducted to determine the responsiveness of bacteria strain on

209

AFB1 degradation whereby three set of experiments were performed first, only LB medium +

210

AFB1, second LB medium + AFB1 + bacteria strain 3J2MO (1*107 CFU/ml) and third LB medium

211

+AFB1 + E-Coli (1*107 CFU/ml) and the initial pH was adjusted to 9.0, Final concentration of

212

AFB1 (100ppb), incubated for 12 h at 28 ℃ .Then other process for AFB1 determination by

213

HPLC-FLD was performed as described in section 2.2 above. This study was conducted three

214

times and treatments were replicated three times

215

2.10 Data collection and analysis

216

All above experiment was performed on HPLC-FLD series 1100 from Agilent with a 4.6mm ×

217

250mm 5μm Shim-pack VP-ODS C18, Column with a fluorescence detector, The injection

218

volume was 20 µL, The mobile phase was methanol: water (4:6, v/v), The total run time was

219

25 min, with flow rate of 0.8 mL/min. Temperature 35 ℃ and excitation wavelength 360nm,

220

emission wavelength 440nm.

221

Data were analyzed by Microsoft Excel 2010 and Statistical Analysis Software (SAS 9.2). Results

222

were statistically compared and expressed as means with standard deviation (SD). An ANOVA

223

(Analysis of variance) test was used to compare the mean value. Average values and standard

224

errors were shown in figures. Significant difference were considered when a p-value

Investigation and characterization of Myroides Odoratimimus strain

Recommend Documents