High-Speed Counter-Current Chromatography (HSCCC) Purification

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High-Speed Counter-Current Chromatography (HSCCC) Purification of Antifungal Fatty Acids from Plant Seed Oil and Lactobacillus Fermentation Nuan Yi Liang, Pengfei Cai, Datong Wu, Yuanjiang Pan, Jonathan M. Curtis, and Michael G. Gänzle J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05658 • Publication Date (Web): 10 Dec 2017 Downloaded from http://pubs.acs.org on December 12, 2017

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Journal of Agricultural and Food Chemistry

High-Speed Counter-Current Chromatography (HSCCC) Purification of Antifungal Hydroxy Unsaturated Fatty Acids from Plant Seed Oil and Lactobacillus Cultures

Nuanyi Liang a), Pengfei Cai b), Datong Wu b), Yuanjiang Pan b), Jonathan M. Curtis a) and Michael G. Gänzle a,)*

a)

University of Alberta, Department of Agricultural, Food and Nutritional Science,

Edmonton, Canada b)

Zhejiang University, Department of Chemistry, Zhejiang University, Hangzhou

310027, China

*

Correspondent footnote, phone: + 1-780-492-0774, E-mail: [email protected]

ACS Paragon Plus Environment

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Abstract:

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Hydroxy unsaturated fatty acids (HUFA) can function as antifungal agents. To

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investigate the antifungal spectrum, i.e. the scope of in vitro fungal inhibition activities

4

of HUFA and their potential applications, three HUFA were produced by microbial

5

transformation or extracted from plant seed oils; compounds included coriolic acid (13-

6

hydroxy-9,11-octadecadienoic acid) from Coriaria seed oil, 10-hydroxy-12-octadecenoic

7

acid from cultures of Lactobacillus hammesii and 13-hydroxy-9-octadecenoic acid from

8

cultures of Lactobacillus plantarum TMW1.460∆lah. HUFA were purified by high-speed

9

counter-current chromatography (HSCCC), characterized by LC-MS and MS/MS, and

10

their antifungal activity was evaluated with 15 indicator fungal strains. HUFA had a

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different antifungal spectrum when compared to unsaturated fatty acids with comparable

12

structure but without hydroxy groups. The inhibitory effect of HUFA specifically

13

targeted filamentous fungi including Aspergillus niger and Penicillium roquefortii, while

14

yeasts including Candida spp. and Saccharomyces spp. were resistant to HUFA. Findings

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here support development of food applications for antifungal HUFA.

16 17

Keywords: fungal food spoilage, antifungal, Aspergillus, Penicillium, hydroxy

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unsaturated fatty acids (HUFA), high-speed counter-current chromatography (HSCCC),

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antifungal activity


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Introduction

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Pathogenic and spoilage fungi present enormous challenges to agriculture.1,2 Spoilage

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fungi, e.g. Aspergillus clavatus and Mucor plumbeus, and phytopathogenic fungi, e.g.

23

Leptosphaeria maculans and Pyrenophora teres f. teres, affect the yield or quality of

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agricultural products. 3 , 4 Mycotoxin producing fungi, e.g. Aspergillus flavus and

25

Aspergillus parasiticus, are a safety concern in food and feed.3 Increased virulence or

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resistance of spoilage or pathogenic fungi towards common fungicides,2 poor hygienic

27

practices, 5 the abusive use of chemically synthesized fungicides 6 and climate change 7

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impede control of fungi in the food supply, and necessitate development of alternative or

29

complementary fungicides.

30

Unsaturated fatty acids (UFAs) and hydroxy unsaturated fatty acids (HUFA) include

31

compounds with antifungal activity.8,9 Their antifungal activities have been reported to

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relate to specific structural attributes9,10 but structure-function relationships have not been

33

sufficiently explored. Moreover, the antifungal activity of individual HUFA has been

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evaluated only with few indicator organisms. Coriolic acid [(R)-13-hydroxy-9(Z),11(E)-

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octadecadienoic acid, or 13-OH C18:2], was first extracted and characterized in its

36

methyl ester form. 11 In wheat dough, coriolic acid is generated through lipoxygenase

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activity, followed by reduction by low molecular weight thiols.10,12 Coriolic acid extends

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the mould-free shelf life of wheat bread;10 lipid oxidation and thiol exchange reactions

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are additionally linked to bread volume and flavor.12 In plants and mushrooms, coriolic

40

acid contributes to the defense against phytopathogens. 13,14,15 However, the inhibitory

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spectrum of coriolic acid is unknown, as is the range of HUFA structures showing

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comparable antifungal activity. In order to further understand the influence of the


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molecular structure on the antifungal activity and the other biological roles of HUFA, an

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effective method for the production of specific HUFA is required.

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Coriolic acid was previously obtained by extraction from hydrolyzed plant seed oil,16

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chemical synthesis,17 by enzymatic conversion from unsaturated fatty acids (UFA)18 and

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from recombinant bacteria.19 Lactobacillus spp. expressing linoleate hydratases convert

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linoleic acid to the antifungal 10-hydroxy-12-octadecenoic acid (10-OH C18:1) or 13-

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hydroxy-9-octadecenoic acid (13-OH C18:1).10, 20 Coriolic acid inhibited the growth of

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the phytopathogenic blast fungus Magnaporthe grisea,43 Phytophthora parasitica and

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Cladosporium herbarum at low effective dose15. However, the amounts of HUFA that

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were obtained in previous studies did not allow determination of the inhibitory spectrum

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and potential application.10,21

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High-speed counter-current chromatography (HSCCC) has been used to purify

55

antimicrobial compounds in sufficient quantities for determination of the inhibitory

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spectrum and mode of action of natural antimicrobial compounds.22 HSCCC employs the

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partition of analytes into two immiscible liquids,24 has a high sample loading capacity

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and high recoveries of the target analytes.23,24,25 HSCCC separated fatty acid esters from

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fish oil but separation methods for fatty acids differing in their degree of unsaturation

60

required different solvent systems 26 , 27 , 28 and HSCCC solvent systems have not been

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optimized to for separation of HUFA. It was therefore the aim of this study to develop

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HSCCC as purification method for HUFA, and to obtain purified HUFA in sufficient

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amounts to investigate their inhibitory spectrum with mycelial fungi, conidiospores, and

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yeasts. Coriolic acid, 10-OH C18:1 and 13-OH C18:1, were extracted from plants or

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microbial cultures, purified by HSCCC, and their purity and molecular structure were


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characterized by liquid chromatography coupled with tandem mass spectrometry (LC-

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MS/MS).

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Materials and methods

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Chemicals and reagents

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HPLC-grade solvents hexane, ethyl acetate, methanol, water, acetonitrile, chloroform

71

and OptimaTM grade acetic acid were purchased from Fisher Scientific (Ottawa, Canada).

72

Fatty acid standards including tetradecanoic (myristic) acid, hexadecanoic (palmitic)

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acid, 9-cis-octadecenoic (oleic) acid, 12-hydroy-9-cis-octadecenoic (ricinoleic) acid, 9-

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cis,12-cis octadecadienoic (linoleic) acid, octadecanoic (stearic) acid, 9-cis,12-cis,15-cis

75

octadecatrienoic (alpha linolenic) acid with purity >99% were purchased from Nu-Chek

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Prep, Inc (Elysian, MN, USA). Microbiological media were purchased from Fisher

77

Scientific.

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Microbial strains and culture conditions

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L. hammesii DSM16381 29 and L. plantarum TMW1.460∆lah21 were cultured on

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modified De Man Rogosa Sharpe (mMRS) medium for 24 h at 30 °C. Microbial

81

conversion of linoleic acid to HUFA was achieved by sub-culturing of the respective

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strains with a 5% (v/v) inoculum in mMRS broth containing 4 g/L linoleic acid, followed

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by incubation for 4 d at ambient temperature anaerobically.10

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Aspergillus niger FUA5001, Mucor plumbeus FUA5003, Penicillium roqueforti

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FUA5004, Aspergillus clavatus FUA5005 and Aspergillus brasiliensis ATCC16404 were

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cultivated on malt extract agar (MEA) at 25 °C for 7 d in darkness to harvest spores as

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described. 30 Briefly, spores were freshly harvested from agar plates by adding 10mL

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physiological solution (0.9% sodium chloride and 0.1% tween 80) and harvesting of


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fungal biomass with a sterile inoculum loop. The spore suspension was filtered to

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eliminate mycelial cells and spores were counted with a hemocytometer (Fein-Optik,

91

Jena, Germany). 31 Spore counts were adjusted to 104 cfu/mL by dilution with mMRS

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broth. A. niger FUA5001 and P. roqueforti FUA5005 were also cultured on MEA

93

respectively for 2 d and their mycelia were harvested as the white thread-like edge of the

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colonies picked by 1mL pipette tips prior to the MIC test.

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The following yeasts were used in the MIC assays: Zygosaccharomyces spp.

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FUA4034, Candida albicans ATCC10231, Candida humilis FUA4001, Saccharomyces

97

cerevisiae FUA4011, Torulospora delbrueckii FUA4023, Wickerhamomyces anomalus

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FUA4024, Saccharomyces unisporus FUA4027, Candida valida FUA4030, Pichia

99

membranefaciens FUA4031, and Pichia orientalis FUA4032. Zygosaccharomyces spp.

100

FUA4034 was cultivated on Potato Dextrose (PD) agar for 2 d; other yeasts were

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cultivated on yeast extract peptone dextrose (YPD) agar. To prepare inocula for the MIC

102

assays, single colonies were picked and grown to the stationary phase in PD or YDP

103

broth for 2 d at 30 °C. The optical density at 600 nm of yeast cultures was related to the

104

cell count by counting with hemocytometer and the inocula of yeasts were adjusted to 104

105

cell / mL by dilution to the corresponding optical density.

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Extraction of fatty acid mixtures from Coriaria nepalensis Wall seed oil and from

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cultures of L. plantarum TMW1.460∆lah and L. hammesii DSM16381

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Powdered seeds of Coriaria nepalensis Wall (XinTai Seed Production and Wholesale

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Co. Jiangsu, China) were extracted with hexane. For small-scale extraction,

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approximately 5 g of powered seeds were extracted with 200 mL boiling hexane in a

111

Soxhlet extractor. After extraction for 6 h, the solvent was evaporated under reduced


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pressure. For large-scale production, 300 g of powdered C. nepalensis seed were boiled

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in two-liter round-bottom bottle with 600 mL hexane, refluxed for 6 h, followed by

114

separation of the solvent from the seed material and evaporation of the solvent in the

115

extract. Oil was stored at -20 °C with nitrogen.

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Saponification of Coriaria seed oil was performed as described with minor

117

modifications.32 In brief, 5 g of C. nepalensis seed oil was dissolved in 50 mL 10% KOH

118

in ethanol, refluxed at 70 °C for 4 h at a stirring speed of 200 rpm. After the reaction,

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50mL water was added and methanol was mostly evaporated under reduced pressure.

120

Non-saponifiable lipids in the aqueous fraction were extracted by adding 100 mL of

121

hexane; this extraction was repeated 3 times. The remaining aqueous phase was then

122

acidified with 12% (v/v) HCl, and fatty acids were extracted 3 times with 100 mL of

123

hexane. The hexane fractions were combined and washed by distilled water until a pH

124

over 5 was achieved. The final hexane extract was dried in rotary evaporator under

125

reduced pressure.

126

The extraction of fatty acid mixtures from cultures of Lactobacillus spp. was

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performed as described.21,33 In brief, 250 mL of cultures were adjusted to a pH of 2 and

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extracted with 250 mL of chloroform: methanol (85:15% v/v); the extraction was

129

repeated three times. Extracts in lower phase were combined and evaporated to dryness

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under vacuum.

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LC-MS and MS/MS analysis of fatty acids

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Fatty acids were dissolved in methanol to a concentration of 100 mg/L and analyzed

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using reversed-phase liquid chromatography coupled with negative-ion electrospray

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ionization-tandem mass spectrometry (LC-ESI-MS/MS) and normal phase liquid


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chromatography coupled with negative-ion atmospheric pressure photoionization-tandem

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mass spectrometry (LC-APPI-MS/MS). Fatty acids were separated using an Ascentis

137

Express C8 column (15 cm×2.1 mm, 2.7 µm) (Sigma, St. Louis, MO) at 25 °C using

138

Agilent 1200 series LC system (Agilent Technologies, Palo Alto, CA, USA). Samples

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were injected (5 µL) at 15 °C and eluted at 0.25 mL/min with a gradient of water with 10

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mM ammonium acetate and 0.2% acetic acid (A) and 98% acetonitrile and 2% of water

141

with 10 mM ammonium acetate and 0.2% acetic acid (B). The gradient was set as follows:

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0-23 min 70% to 100% B, followed by re-equilibration to 70% B, with a total run time of

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30 min.

144

Negative-ion ESI-MS was performed on a 3200 QTRAP triple-quadrupole mass

145

spectrometer (AB SCIEX, Concord, ON, Canada) coupled with a turbospray electrospray

146

ionization ion source. AB SCIEX Analyst 1.4.2 software was used for data acquisition

147

and processing. Full scan mode was to identify fatty acids and multiple reaction

148

monitoring (MRM) mode was used for the quantitation of fatty acids. Nitrogen was used

149

as the curtain gas, nebulizing gas and drying gas. The conditions of ion source and mass

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spectrometer were used as follow: full scan mass range was from m/z 100-1000 at a

151

frequency of 1 scan/sec, GS1, GS2 and curtain gas was set at 40, 60 and 25 arbitrary units

152

respectively; declustering potential (DP), ion spray voltage and ion source temperature

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was set at -60 V, -4500 V and 450 °C; the collision energy (CE), collision cell entrance

154

potential (CEP) and collision cell exit potential (CXP) were -36 V, -20 V and -1 V. Dwell

155

time was 40 ms. MS transitions and retention times of fatty acids analyzed by LC/ESI–

156

MS in the multiple reaction monitoring (MRM) mode are listed in Table 1. Calibration

157

curves were generated from dilution series of pure standards. Because no commercial


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external standards were available for coriolic acid, 13-OH C18:1 and 10-OH C18:1, the

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purity of HSCCC purified coriolic acid was verified by LC-MS and LC-UV, and by

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NMR analysis provided as service of the NMR facility of the Dept. of Chemistry at the

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University of Alberta (supplementary material Figure S2-S4). This purified fraction was

162

used as an external standard for quantitation of coriolic acid. The calibration curve

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established with ricinoleic acid was used to quantify its isomers 13-OH C18:1 and 10-OH

164

C18:1 in the following LC-APPI-MS/MS analysis.

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Normal phase LC-APPI-MS/MS was performed to confirm the molecular structure

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and concentration of fatty acids as described in a previous study with minor

167

modifications.20,21 Briefly, 2µL of 50 ppm fragmented fatty acids dissolved in hexane

168

were separated on a PVA-Sil column (Waters Ltd, Mississauga, Canada) at 25 oC with

169

mobile phase comprised of (A) 0.2% acetic acid in hexane and (B) 0.2% acetic acid in

170

isopropanol. The gradient was as follow: 0 min 99% A, 20 min 70% A, 20.1min 99% A,

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with total run time of 27min. Fatty acids were detected by APPI-MS/MS on a QStar Elite

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hybrid orthogonal Q-TOF mass spectrometer equipped with a PhotoSpray source

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(Applied Biosystems/MDS Sciex, Concord, Canada). The following parameters were

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used: nebulizer gas 65, auxiliary gas 10, curtain gas 25, ionspray voltage -1300 V, source

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temperature 325 oC, declustering potential (DP) -35 V, focusing potential (FP) -130 V

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and DP2 -13V. In addition to a total ion scan of m/z 50-1000, a product ion scan (m/z 50-

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700) of m/z 295.2 was also performed in fragmented Coriaria sample, and product ion

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scan (m/z 50-700) of m/z 297.2 was done for the two fragmented samples from

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Lactobacillus fermentation respectively. Data were analyzed using Analyst QS 2.0.

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Measurement of partition coefficient (K) values


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The solvent system for HSCCC separation was optimized by measuring the partition

182

coefficient (K) as described by Ito with modifications.24 The sample or standard was

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dissolved in approximately 10 mL of the respective solvent system, mixed and the upper

184

and lower phases were separated. After the addition of internal standard, fatty acids in the

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upper and lower phases were quantified by MRM mode of LC-ESI-MS/MS analysis.

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Results were recorded as the average of duplicate measurements. The partition

187

coefficients K of compounds of interest was calculated as:

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=

.

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Purification of fatty acids by high-speed counter-current chromatography (HSCCC)

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A TBE-300B HSCCC system was used to purify mono-HUFA. The system was

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configured with a Model 501 PrimeLine solvent delivery module (Analytical Scientific

192

Instruments, El Sobrante, CA), a multi-layer coiled column with 260 mL column capacity

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(β=0.5-0.8) and 0.019 mm i.d. tubing (Tauto Biotech, Shanghai, China), a VUV-24

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Visacon UV−Vis detector (Reflect Scientific Inc., Orem, UT) and a CHF 122SC fraction

195

collector (Avantec Toyo Kaisha Ltd., Tokyo, Japan).20,22 The 2 L solvent system was

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equilibrated after mixing and settling, and the upper phase was used as the stationary

197

phase and the lower phase as the mobile phase. The HSCCC column was filled with the

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stationary phase, rotated at 1000 rpm, followed by introduction of the mobile phase at a

199

constant flow rate of 3 mL/min. The column was equilibrated until the elution of the

200

mobile phase was constant. Samples (200 mg of Coriaria oil extract or 250 mg of extract

201

from Lactobacillus cultures) were dissolved in 5 mL upper phase and 5 mL lower phase

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and injected on the column. Elution of coriolic acid from Coriaria seed oil, or the mono-

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OH C18:2 fatty acids eluted before mono-OH C18:1 from fermentation was monitored at


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wavelength of 242 nm. Fractions were collected every 3 minutes, and the composition of

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each fraction was analyzed by ESI-MS/MS flow injection. Fractions containing mono-

206

HUFA of interest without interference compounds were combined, evaporated to dryness

207

under vacuum, and then freeze-dried to eliminate water. The identity of purified mono-

208

HUFA was confirmed by LC-MS/MS as described above.

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Minimum inhibitory concentration (MIC) assay

210

Antifungal activities of fatty acid standards and fatty acids after purification by

211

HSCCC were determined by a critical dilution assay.31 Fatty acids were dissolved in

212

100% ethanol to a concentration of 42.7 g/L for mono-HUFA and 85.3 g/L for oleic acid

213

and linoleic acid, respectively. A solution of calcium propionate (21.3 g/L in 50%

214

ethanol) was used for comparison. For each experiment, 100 µL of the fatty acid stock

215

solution was mixed with 100 µL mMRS broth (conidiospores and sporangiospores), YPD

216

or PD broth (yeasts) in 96-well microtiter plate, followed by a series of 2-fold dilutions

217

with the respective growth medium. Ethanol was evaporated by placing microtiter plates

218

without lid in a laminar flow hood until 50 µL ethanol placed in an empty well of the

219

same plate were completely evaporated. Wells were inoculated with 104cfu/mL spores or

220

vegetative cells of the indicator organism, or by adding mycelia picked from the thread-

221

like edge of colonies to each well. Controls contained inoculum but ethanol was used

222

instead of the fatty acid stock solutions. Growth was observed visually; the minimum

223

inhibitor concentration (MIC) was recoded one day after growth was visible in absence of

224

inhibitors. The MIC was reported as the minimum concentration of fatty acid analytes

225

that inhibited the growth of fungal strains tested. The MIC values were determined in

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three independent experiments using replicate preparations of conidiospores.


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Results

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Identification and quantification of fatty acids in Coriaria seed oil and lipids

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extracted from Lactobacillus cultures

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To compare the abundance of mono-HUFA from different sources, hydroxy fatty

231

acids in seed oil and lipid extracts of microbial cultures were quantified by LC-MS/MS

232

(Table 2). The MS/MS fragmentation pattern of coriolic acid included molecular ion [M-

233

H]- at m/z 295.2259, and the characteristic fragmentation ions of m/z 277.2162 [M-H-

234

H2O]-, 195.1393 [C12H19O2]- and 113.0971 [C7H13O]-.34 These distinctive fragments

235

were monitored in quantitative LC-MS/MS-MRM experiments. The MS/MS

236

fragmentations of 10-OH C18:1 and 13-OH C18:1 were consistent with prior reports.21

237

Characteristic fragment ions of 10-OH C18:1 and 13-OH C18:1 were observed at m/z

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185.1196 [C10H17O3]- and at m/z 197.1539 [C12H21O2]-, respectively, resulting from the

239

α-cleavage of hydroxy group at carbon-10 and 13 position respectively (Figure 1).20

240

Exact stereoisomerism of coriolic acid from Coriaria seed oil was proposed as (R)-13-

241

hdyroxy-cis-9, trans-11-octadecadienoic acid;11 this configuration differs from coriolic

242

acid produced by soy lipoxygenase and reduction of the peroxide.44 We confirmed the

243

cis/trans configuration of this structure using NMR analysis. Structure features of coriolic

244

acid as revealed by the NMR spectra (online supplementary figures S2, S3, and S4)

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matched published NMR data on coriolic acid.34

246

Linoleate hydratases of lactobacilli were reported to produce (S)-13-OH-cis-9-

247

octadecenoic acid from linoleic acid.35 L. hammesii converted linoleic acid into a mixture

248

of cis and trans isomers of 10-OH C18:1,10 however, the S configuration of 10-OH C18:1

249

from L. hammesii has not been confirmed. Likewise, the configuration of the 13-OH


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250

C18:1 produced by L. plantarum TMW1.460 remains unknown. Because our data on the

251

antifungal activity of coriolic acid demonstrates that R- and S-isomers of HUFA have

252

equivalent antifungal activity, the configuration of HUFA for lactobacilli was not further

253

investigated.

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Coriolic acid accounted for 67% of the fatty acids in Coriaria seed oil, consistent with

255

prior reports.16,32 Lipids extracted from cultures of L. hammesii and L. plantarum

256

TMW1.460∆lah consisted of the fermentation products 10-OH C18:1 and 13-OH C18:1,

257

respectively, linoleic acid, and traces of oleic acid.21 In both cases, isomers of coriolic

258

acid were also present, likely resulting from the auto-oxidation of linoleic acid. The yield

259

of 13-OH C18:1 from L. plantarum TMW1.460∆lah was lower than the yield of 10-OH

260

C18:1 from L. hammesii. This observation indicated that the 13-linoleate hydratase in

261

L. plantarum TMW1.460∆lah is less active than the 10-OH hydratase in L. hammesii.

262

Optimization of HSCCC solvent systems and preparative separation of HUFA by

263

HSCCC

264

To purify antifungal mono-HUFA fatty acids by HSCCC, the solvent system was

265

optimized to achieve a partition coefficient (K) in the range of 0.5 ≤ K ≤ 2.0 for analytes

266

of interest. In addition, the separation factor α=K1/K2 was considered if two compounds

267

were present in the same sample. Solvent systems were chosen to obtain α=K1/K2 of >

268

1.5.24,36 Twelve solvent systems were evaluated (Supplementary Table 1) and hexane-

269

ethyl acetate-methanol-water (HEMWat, 5:1.5:3:2, v/v/v/v) was chosen based on the

270

partition coefficient and the separation factor. The partition coefficient Kcoriolic acid was 1.5

271

and the separation factor

272

time for coriolic acid of 147 min. A faster elution of coriolic acid of 112 min was

was 1.5. This solvent system resulted in an elution


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achieved with hexane-ethyl acetate-methanol-water (HEMWat, 3.5:1.5:3.5:2, v/v/v/v) but

274

the partition and separation coefficients were reduced to Kcoriolic

275

acid=0.9

and

=1.3. Using this solvent system, adequate separation between the HSCCC

276

peaks of coriolic acid and ricinoleic acid was achieved such that the majority of coriolic

277

acid could be collected in pure fractions (Figure 2). Both solvent systems resulted in

278

consistent retention times (data not shown) with retention of >70% of the stationary

279

phase after equilibrium of HSCCC system. Sample loading of 202.60±2.08 mg of

280

saponified fatty acids from Coriaria oil per HSCCC run yielded 115.32±6.04 mg of

281

purified corioloic acid; this corresponds to a purification yield of 56.9±2.5% and an

282

average recovery of 85% in pure fractions of coriolic acid. The molecular structure and

283

purity of fatty acids was confirmed by LC-APPI-MS/MS (Figure 1 and Supplementary

284

Figure S1). After purification, all fatty acids eluted as single peak without contamination

285

that were detected by LC-APPI-MS/MS analysis (Figure S1).

286

Antifungal activity of mono-HUFA and other UFA

287

HSCCC purification provided sufficient amounts of coriolic acid to test its antifungal

288

activity against a broad spectrum of organisms. The antifungal spectrum of coriolic acid

289

was compared to the two other major fatty acids in Coriaria seed oil, i.e., linoleic acid

290

and oleic acid, to ascertain the role of the hydroxy group on antifungal activity (Table 3).

291

Conidiospores and sporangiospores are asexual propagules produced by filamentous

292

fungi.

293

sporangiospores form M. plumbeus were sensitive to coriolic acid, with MICs ≤1 g/L.

294

The MIC of linoleic acid and oleic acid against spores was higher when compared to

295

coriolic acid (Table 3). Similarly, fungal mycelia were sensitive to coriolic acid but not to

37

Conidiospores from Aspergillus spp. and P. roquefortii as well as


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296

oleic and linoleic acids. In contrast to mycelia fungi, all yeasts tested in this assay were

297

resistant to coriolic acid (Table 3). This implies that the presence of hydroxy groups

298

highly contributes to the specific structure-function relationship of fatty acid to inhibit

299

foodborne spoilage and/or pathogenic fungi.

300

To determine whether the structure of monohydroxy unsaturated fatty acids

301

determines their antifungal activity, the MIC of coriolic acid, ricinoleic acid, 10-OH

302

C18:1 and 13-OH C18:1 was determined with two indicator strains (Table 4). Regardless

303

of their degree of unsaturation or the position of carbon double bond and hydroxy groups,

304

HUFA had a similar inhibition effect against the two filamentous fungi tested.

305

Discussion

306

This work developed HSCCC as an efficient method for preparative purification of

307

antifungal mono-HUFA from Coriaria seed oil and Lactobacillus cultures. The amounts

308

of purified compounds obtained by HSCCC were sufficient to investigate the antifungal

309

spectrum of mono-HUFA with a panel of 15 indicator strains. Remarkably, mycelial

310

fungi were sensitive to HUFA but yeasts were highly resistant.

311

To purify the target HUFA and avoid interference from other compounds in the

312

original samples, HSCCC was used as an efficient purification method. HSCCC purified

313

fatty acids from diverse matrixes without derivatization or combination with other

314

separation technologies. The separation factors α between fatty acids of interest in this

315

study and their closest elution interferences indicated good separation efficiency.24

316

Analytes were obtained in sufficient amounts to allow screening of their inhibitory

317

spectrum against a wide range of indicator organisms.


15


Page 16 of 33

318

The antifungal activity of HUFA was suggested to be specific for target fungi.9,15 This

319

study therefore compared the activity of HUFA against mycelial fungi to their activity

320

against yeast. Yeast strains included baker’s yeasts, S. cerevisiae, yeasts involved in

321

traditional food fermentations, i.e. C. humilis, T. delbrueckii, W. anomalus and S.

322

unisporus; the spoilage yeasts C. valida, P. membranefaciens, P. orientalis and

323

Zygosaccharomyces, and the pathogenic C. albicans. Coriolic acid was active against

324

asexual spores and mycelia of filamentous fungi but inactive against yeasts. HUFA are

325

thus unlikely to inhibit food spoilage caused by yeasts but can be applied as effective

326

antifungal agents in fermented foods that require growth and activity of yeasts. 38 In

327

contrast, medium-chain hydroxy fatty acids from L. plantarum inhibited yeasts more

328

actively than filamentous fungi.39 This may be explained by the diversity in chemical

329

composition of membrane in different fungi 40 and differences in the interaction of

330

medium chain and long chain fatty acids with fungal membranes.

331

The comparison of different antifungal HUFA also allowed for inference of structural

332

requirements of their antifungal activity. The antifungal activity of HUFA depended on

333

the presence of a hydroxy group in the middle of the carbon chain,9,10,15 and varied with

334

their number 41 , 42 and position15 of hydroxy groups. Unsaturation was critical to the

335

antifungal activity of long-chain HUFA10,43 but not for the middle-chain HUFA.39 The

336

position of the double bond15 and the chirality13,17,43 were reported to have only a minor

337

impact on the antifungal activity. Our data also support the conclusion that HUFA with R

338

and S configuration have equivalent activity. The activity of (R)-13-OH-cis-9, trans-11-

339

coriolic acid extracted from Coriaria seed oil against A. niger and P. roqueforti (this

340

study) was comparable to the activity of coriolic acid generated from soybean


16

Page 17 of 33


341

lipoxygenase.10,21 Soy lipoxygenase generates the S-isomer of coriolic acid (98% of (S)-

342

13-OH-cis-9, trans-11).44 The MIC data are in general agreement with prior studies,10,21

343

however, we observed a higher activity for ricinoleic acid when compared to previous

344

reports.10 This discrepancy to prior reports warrants further comparison of the activity of

345

ricinoleic acid and coriolic acid with a larger panel of indicator strains. The present study

346

additionally demonstrates that hydroxy groups are essential for the antifungal activity of

347

fatty acids. Moreover, all HUFA with a hydroxy group in the center of the acyl chain

348

exhibited comparable antifungal activity.

349

The mechanism of antifungal HUFA has been explained by their detergent-like

350

interactions with cell membranes9,40 and hence differences in fungal resistance of HUFA

351

may relate to variations in the membrane composition. However, the antifungal activity

352

of HUFA may also relate to other aspects of fungal physiology. For example, (R)-8-

353

hydroxy-cis-9,cis-12-octadecadienoic acid (8-OH C18:2), an isomer of coriolic acid, is a

354

precocious sexual inducer (psi) Bα factor that regulates the sexual development of

355

Aspergillus spp..45 The same compound has antifungal activity against phycomycetous

356

fungi.46 This compound was also associated with the regulation of conidia formation and

357

production of fumonisins by Fusarium verticillioides.47 Those roles of HUFA in fungal

358

physiology and ecology indicate that their mode of action may be species-specific. The

359

HSCCC protocol as developed in this study to allow the mg to g scale purification of

360

HUFA will facilitate future research on their mode of action.

361

Food-borne fungi may develop resistance to the commonly used antifungal agents

362

including organic acids and their salts.48 HUFA are present in foods with a tradition of

363

safe use10,20,49 and were used as antifungal agents in food. Their activity in preventing


17


Page 18 of 33

364

fungal spoilage of bread was comparable to conventional preservatives.10 The resistance

365

of yeasts restricts the use of HUFA to foods where spoilage yeasts such as

366

Zygosaccharomyces bailii, Pichia spp. and Candida spp. are not relevant.50 Conversely,

367

the specific inhibitory spectrum allows their use in fermented foods, e.g. bread, where

368

inhibition of CO2 and flavor formation by S. cerevisiae or sourdough yeasts would

369

compromise food quality.10 Their use may also prevent phytopathogenic fungi in plant

370

production. In plants, UFA and their oxygenated derivatives not only act as antifungal

371

agents but also are critical in signaling self-defense response.15,51,52 The present study

372

extended the range of HUFA with potential use in food preservation and plant protection.

373

In conclusion, an efficient HSCCC-based method of purifying useful quantities of

374

antifungal fatty acids has been established, which enabled investigations of biological

375

functions of HUFA. A comparison of species-specific antifungal abilities among coriolic

376

acid, linoleic acid, oleic acid and two mono-hydroxy C18:1 fatty acids provided insights

377

into the structure-function relationship of antifungal fatty acids. It confirmed the

378

contribution of hydroxy group to the antifungal abilities of fatty acids. In addition, it has

379

demonstrated the potential application of antifungal fatty acids should be further explored

380

for prolonging food shelf-life and controlling plant diseases, both of which directly relate

381

to significant economic loss and health concern worldwide.

382

Acknowledgements

383

Western Grains Foundation and the Alberta Wheat Commission are acknowledged for

384

the funding of this project. Nuanyi Liang acknowledges financial support from Mitacs.

385

Michael Gänzle acknowledges financial support from the Canada Research Chairs

386

Program.

387 ACS Paragon Plus Environment

18

Page 19 of 33

388


References

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Science against microbial pathogens: current research and technological advances 2011, 1, 61-71. 10. Black, B. A.; Zannini, E.; Curtis, J. M.; Gänzle, M. G. Antifungal hydroxy fatty acids produced during sourdough fermentation: microbial and enzymatic pathways, and antifungal activity in bread. Appl. Environ. Microbiol. 2013, 79, 1866-1873. 11. Tallent, W. H.; Harris, J.; Wolff, I. A.; Lundin, R. E. (R)-13-Hydroxy-cis-9, trans-11octadecadienoic acid, the principal fatty acid from Coriaria nepalensis Wall seed oil. Tetrahedron Letters 1966, 7, 4329-4334. 12. Tsen, C. C.; Hlynka, I. Flour lipids and oxidation of sulfhydryl groups in dough. Cereal Chem. 1963, 40, 145. 13. Kato, T.; Yamaguchi, Y.; Hirano, T.; Yokoyama, T.; Uyehara, T.; Namai, T.; Yamanaka, S.; Harada, N. Unsaturated hydroxy fatty acids, the self defensive substances in rice plant against rice blast disease. Chem. Lett. 1984, 13, 409-412. 14. Stadler, M.; Mayer, A.; Anke, H.; Sterner, O. Fatty acids and other compounds with nematicidal activity from cultures of Basidiomycetes. Planta medica 1994, 60, 128-132. 15. Prost, I.; Dhondt, S.; Rothe, G.; Vicente, J.; Rodriguez, M. J.; Kift, N.; Carbonne, F.; Griffiths, G.; Esquerre-Tugaye, M. T.; Rosahl, S.; Castresana, C.; Hamberg, M.; Fournier, J., Evaluation of the antimicrobial activities of plant oxylipins supports their involvement in defense against pathogens. Plant Physiol. 2005, 139, 1902-1913. 16. Tallent, W. H.; Harris, J.; Spencer, G. F.; Wolff, I. A. Structure and intraglyceride distribution of coriolic acid. Lipids 1968, 3, 425.


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17 Kobayashi, Y.; Okamoto, S.; Shimazaki, T.; Ochiai, Y.; Sato, F., Synthesis and physiological activities of both enantiomers of coriolic acid and their geometric isomers. Tetrahedron Lett. 1987, 28, 3959-3962. 18. Nanda, S.; Yadav, J. S., Lipoxygenase biocatalysis: a survey of asymmetric oxygenation. J Mol. Catal. B-Enzym. 2003, 26, 3-28. 19 Sim, D. H.; Shin, K. C.; Oh, D. K., 13-Hydroxy-9Z,11E-octadecadienoic acid production by recombinant cells expressing Burkholderia thailandensis 13-lipoxygenase. J. AOCS 2015, 92, 1259-1266. 20 Black, B. A.; Sun, C. X.; Zhao, Y. Y.; Gänzle, M. G.; Curtis, J. M., Antifungal lipids produced by Lactobacilli and their structural identification by normal phase LC/atmospheric pressure photoionization-MS/MS. J. Agric. Food Chem. 2013, 61, 53385346. 21. Chen, Y. Y.; Liang, N. Y.; Curtis, J. M.; Gänzle, M. G., Characterization of linoleate 10-hydratase of Lactobacillus plantarum and novel antifungal metabolites. Front. Microbiol. 2016, 7, 1561. 22. Engels, C.; Gänzle, M. G.; Schieber, A., Fractionation of gallotannins from mango (Mangifera indica L.) kernels by high-speed counter-current chromatography and determination of their antibacterial activity. J. Agric. Food Chem. 2010, 58, 775-780. 23. Sutherland, I. A., Recent progress on the industrial scale-up of counter-current chromatography. J. Chromatogr. A 2007, 1151, 6-13. 24. Ito, Y., Golden rules and pitfalls in selecting optimum conditions for high-speed counter-current chromatography. J. Chromatogr. A 2005, 1065, 145-168.


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25. Hu, R. L.; Pan, Y. J., Recent trends in counter-current chromatography. Trac-Trend. Anal. Chem. 2012, 40, 15-27. 26 Cao, X.; Ito, Y., Supercritical fluid extraction of grape seed oil and subsequent separation of free fatty acids by high-speed counter-current chromatography. J. Chromatogr. A 2003, 1021, 117-24. 27 Li, D.; Schröder, M.; Vetter, W., Isolation of 6, 9, 12, 15-hexadecatetraenoic fatty acid (16: 4n-1) methyl ester from transesterified fish oil by HSCCC. Chromatographia 2012, 75, 1-6. 28 Du, Q.; Shu, A.; Ito, Y., Purification of fish oil ethyl esters by high-speed countercurrent chromatography using non-aqueous solvent systems. J. Liquid Chromatogr. Related Technol. 1996, 19, 1451-1457. 29. Valcheva, R., Korakli, M., Onno, B., Prévost, H., Ivanona, I., Ehrmann, M.A., Dousset, X., Gänzle, M.G., Vogel, R.F. Lactobacillus hammesii sp. nov., isolated from French sourdough. Int. J. System. Evol. Microbiol. 2005, 55, 763-767. 30. Zhang, C.G.; Brandt, M. J.; Schwab, C.; Gänzle, M. G., Propionic acid production by cofermentation of Lactobacillus buchneri and Lactobacillus diolivorans in sourdough. Food Microbiol. 2010, 27, 390-395. 31. Magnusson, J.; Schnürer, J., Lactobacillus coryniformis subsp. coryniformis strain Si3 produces a broad-spectrum proteinaceous antifungal compound. Appl. Environ. Microbiol. 2001, 67, 1-5. 32. Jin, Q. D.; Yu, C. H., The component acids of seed fats of Coriaria sinica Maxim. Acta Bot. Yunnanica 1980, 2, 228-229. 33. Shahzadi, A. Bio-transformation of fatty acids. Ph.D. thesis, University of Alberta,


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2012. URL (https://era.library.ualberta.ca/files/8336h250t/Shahzadi_Asima_Spring2012.pdf). 34. Ding, L.; Peschel, G.; Hertweck, C., Biosynthesis of archetypal plant self-defensive oxylipins by an endophytic fungus residing in mangrove embryos. Chembiochem. 2012, 13, 2661-2664. 35 Park, J. Y.; Lee, S. H.; Kim, K. R.; Park, J. B.; Oh, D. K., Production of 13S-hydroxy9(Z)-octadecenoic acid from linoleic acid by whole recombinant cells expressing linoleate 13-hydratase from Lactobacillus acidophilus. J. Biotechnol. 2015, 208, 1-10. 36. Marston, A.; Hostettmann, K., Developments in the application of counter-current chromatography to plant analysis. J Chromatogr. A 2006, 1112, 181-194. 37. Cole, G. T., Basic Biology of Fungi. In Medical Microbiology, 4th ed.; Baron, S., Ed. Galveston (TX), 1996. 38. Schnürer, J.; Magnusson, J., Antifungal lactic acid bacteria as biopreservatives. Trends Food Sci. Tech. 2005, 16, 70-78. 39. Sjogren, J.; Magnusson, J.; Broberg, A.; Schnürer, J.; Kenne, L., Antifungal 3hydroxy fatty acids from Lactobacillus plantarum MiLAB 14. Appl. Environ. Microbiol. 2003, 69, 7554-7557. 40 Avis, T. J.; Belanger, R. R., Specificity and mode of action of the antifungal fatty acid cis-9-heptadecenoic acid produced by Pseudozyma flocculosa. Appl. Environ. Microbiol. 2001, 67, 956-960; 41. Graner, G.; Hamberg, M.; Meijer, J., Screening of oxylipins for control of oilseed rape (Brassica napus) fungal pathogens. Phytochemistry 2003, 63, 89-95.


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42. Hou, C. T.; Forman, R. J., Growth inhibition of plant pathogenic fungi by hydroxy fatty acids. J. Ind. Microbiol. Biotechnol. 2000, 24, 275-276. 43. Kato, T.; Nakai, T.; Ishikawa, R.; Karasawa, A.; Namai, T., Preparation of the enantiomers of hydroxy-C18 fatty acids and their anti-rice blast fungus activities. Tetrahedron-Asymmetr. 2001, 12, 2695-2701. 44 Nikolaev, V.; Reddanna, P.; Whelan, J.; Hildenbrandt, G.; Reddy, C. C., Stereochemical nature of the products of linoleic-acid oxidation catalyzed by lipoxygenases from potato and soybean. Biochem. Bioph. Res. Comm. 1990, 170, 491496. 45. Mazur, P.; Nakanishi, K.; Elzayat, A. A. E.; Champe, S. P., Structure and synthesis of sporogenic Psi factors from Aspergillus nidulans. J. Chem. Soc. Chem. Comm. 1991, 1486-1487. 46. Bowers, W. S.; Hoch, H. C.; Evans, P. H.; Katayama, M., Thallophytic Allelopathy isolation and identification of laetisaric acid. Science 1986, 232, 105-106. 47. Scala, V.; Giorni, P.; Cirlini, M.; Ludovici, M.; Visentin, I.; Cardinale, F.; Fabbri, A. A.; Fanelli, C.; Reverberi, M.; Battilani, P.; Galaverna, G.; Dall'Asta, C., LDS1-produced oxylipins are negative regulators of growth, conidiation and fumonisin synthesis in the fungal maize pathogen Fusarium verticillioides. Front. Microbiol. 2014, 5, R669. 48. Nielsen, P. V.; Boer, E. d.; Samson, R. A.; Hoekstra, E. S.; Frisvad, J. C., Food preservatives against fungi. Introduction to food-and airborne fungi 2004, (Ed. 7), 357363. 49 Biermann, U.; Wittmann, A.; Grosch, W., The occurrence of bitter hydroxy fatty acids in oats and wheat. Fette, Seifen, Anstrichmittel 1980, 82, 236-240.


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50 Loureiro, V., Querol, A. The prevalence and control of spoilage yeasts in foods and beverages. Trends Food Sci. Technol. 1999, 10, 356-365. 51 , Vellosillo, T.; Aguilera, V.; Marcos, R.; Bartsch, M.; Vicente, J.; Cascón, T.; Hamberg, M.; Castresana, C., Defense activated by 9-lipoxygenase-derived oxylipins requires specific mitochondrial proteins. Plant Physiol. 2013, 161, 617-627. 52. Marcos, R.; Izquierdo, Y.; Vellosillo, T.; Kulasekaran, S.; Cascón, T.; Hamberg, M.; Castresana, C., 9-Lipoxygenase-derived oxylipins activate brassinosteroid signaling to promote cell wall-based defense and limit pathogen infection. Plant Physiol. 2015, 169, 2324-2334.


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Page 26 of 33

389

Figure legends

390

Figure 1. Negative-mode LC-APPI- MS/MS fragmentation spectra of antifungal fatty

391

acids purified via HSCCC. Panel A. coriolic acid. Panel B. 10-hydroxy-12-

392

octadecenoic acid (10-OH C18:1). Panel C. 13-hydroxy-9-octadecenoic acid (10-OH

393

C18:1). Inserts indicate molecular structure and proposed fragmentation pattern.

394

Figure 2. ESI-MS/MS flow injection analysis of HSCCC purification fractions

395

collected during HSCCC purification using solvent system of HEMWat (3.5:1.5:3.5:2,

396

v/v/v/v). Panel A, fractionation of hydrolyzed Coriaria seed oil. Panel B, fractionation

397

of lipids extracted from cultures of L. hammesii. Panel C. Fractionation of lipids

398

extracted from L. plantarum TMW1.460∆lah. The compositions of peaks of analytes

399

of interest were monitored by different ion transition, i.e., coriolic acid and 13-OH

400

C18:2 isomer (Q1/Q3=295.2/197.2), ricinoleic acid (Q1/Q3=297.2/185.1), 10-OH

401

C18:1 (Q1/Q3=297.2/185.1) and 13-OH C18:1 (Q1/Q3=297.2/197.1).

402


26

Page 27 of 33


Table 1. MS transition and retention time of fatty acids in LC/ESI–MS/MS-MRM quantitative analysis Compound

MS transitions (amu) Q1/Q3

Retention time (min)

Coriolic acid

295.2/277.2

5.2

13-OH C18:1

297.2/197.1

6.2

10-OH C18:1

297.2/185.1

6.2

Ricinoleic acid

297.2/183.1

6.2

Linolenic acid

277.2/277.2

11.9

Myristic acid

227.2/227.2

12.3

Linoleic acid

279.2/279.2

14.9

Palmitic acid

255.2/255.2

17.2

Oleic acid

281.2/281.2

18.4

Stearic acid

283.2/283.2

22.2

Heptadecanoic acid

269.2/269.2

19.8


27


Page 28 of 33

Table 2. Fatty acid content (%, w/w) in lipid extracts from C. nepalensis seed oil and lipids extracted from L. hammesii and L. plantarum TMW1.460∆lah cultures. Fatty acids* Lipid samples Coriaria oil L. hammesii extract L. plantarum TMW1.460∆lah extract

RA

CA

LA

PA

SA

OA

0.89±0.02 NF

66.7±3.4 6.24±0.00

10.6±0.2 67.3±22.2

1.78±0.30 NF

2.68±0.04 NF

4.67±0.33 0.67±0.08

NF

1.71±0.08

72.5±16.6

NF

NF

0.64±0.10

10-OH 13-OH C18:1 C18:1 NF NF 12.3±2.2** NF NF

4.06±0.35 **

*RA=ricinoleic acid; CA=R-coriolic acid; LA=linoleic acid; PA=palmitic acid; SA=stearic acid; OA=oleic acid; 10-OH C18:1=10-hydroxy-12-octadecenoic acid; 13-OH C18:1=13-hydroxy-9octadecenoic acid; NF=not found; **: 10-OH C18:1 and 13-OH C18:1 were quantified during the isomer ricinoleic acid in full scan mode in LC/APPI-MS/MS as standard. ***: Values are presented as means (n=2) ± standard deviation.


28

Page 29 of 33


Table 3. Minimum inhibitory concentration (MIC) of coriolic acid against food spoilage and pathogenic fungi compared to linoleic acid and oleic acid. Data are shown as means ± standard deviation of triplicate independent experiments. Strains

Conidiospores

Sporangiospores Mycelia

Yeasts

A. niger P. roqueforti A. brasiliensis A. clavatus M. plumbeus A. niger P. roqueforti C. albicans C. humilis S. cerevisiae T. delbrueckii W. anomalus S. unisporus C. valida P. membranaefaciens P. orientalis Zygosaccharomyces

MIC of fatty acids (g/L) Coriolic acid Linoleic acid Oleic acid 0.29±0.07 3.67±1.53 ≥8 0.33±0.14 3.33±1.15 ≥8 0.42±0.14 3.33±1.15 ≥8 0.38±0.13 6.67±2.31 ≥8 0.67±0.29 3.33±1.15 ≥8 0.07±0.05 6.00±3.46 ≥8 0.27±0.22 6.67±2.31 ≥8 ≥8 ≥8 ≥8 ≥8 6.67±2.31 ≥8 ≥8 ≥8 ≥8 ≥8 ≥8 ≥8 4.00±0.00 ≥8 ≥8 ≥8 ≥8 ≥8 ≥8 4.00±0.00 ≥8 ≥8 6.67±2.31 ≥8 ≥8 ≥8 ≥8 5.33±2.31 ≥8 ≥8


29


Page 30 of 33

Table 4. Minimum inhibitory concentration (MIC) comparison of four mono-hydroxy fatty acids against A. niger and P. roquefortii. Data represent means ± standard deviation of triplicate independent experiments. MIC of HUFA and propionate (g/L) Strains

Coriolic acid

Ricinoleic acid

10-OH C18:1

13-OH C18:1

Propionate

A. niger

0.29±0.07

0.33±0.14

0.50±0.00

0.42±0.14

1.04±0.46

P. roquefortii

0.33±0.14

0.42±0.14

0.42±0.14

0.42±0.14

2.62±0.91


30

Page 31 of 33


195.1393

Relative Intensity (%)

100

295.2259

50 113.0971 59.0137

100

150

200 250 m/z (DA)

300

350

185.1196

100 Relative Intensity (%)

179.1439 259.2051

50

B

50

279.2313 50

100

150

200 250 m/z (DA)

297.2425 300

350

297.2406

100 Relative Intensity (%)

277.2162

A

C

50 113.0969 197.1539 279.2297 99.0804 179.1435 50

100

150

200 250 m/z (DA)

300

350

Figure 1.


31


Page 32 of 33

6 Peak Area (x10 counts)

2.0

1.5

Coriolic acid Ricinoleic acid

A

1.0

0.5

0.0 40

60

80

100

120

140

Collection Time (min)


4.0

3.0

mono-OH C18:2 isomer 10-OH C18:1

B

2.0

1.0

0.0 60

80

100

120

140



2.0

1.5

mono-OH C18:2 isomer 13-OH C18:1

C

1.0

0.5

0.0 80

100

120

140

160

180


Figure 2


32

Page 33 of 33


TOC Graph


33

High-Speed Counter-Current Chromatography (HSCCC) Purification

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