東京医療保健大学紀要

原 著著

第 13 巻第1号

2019. 3. 31

パツリンと糖の酵素存在下での反応挙動
Reaction of patulin with glucose in the presence of an enzyme

大道公秀

千葉瑞紀

Kimihide OHMICHI,

長谷川（谷内）友梨

Mizuki CHIBA,

五百藏良

Yuri TANIUCHI-HASEGAWA and Ryo IOROI

〈原

著〉

パツリンと糖の酵素存在下での反応挙動
Reaction of patulin with glucose in the presence of an enzyme

大道公秀，千葉瑞紀，長谷川（谷内）友梨，五百藏良東京医療保健大学

医療保健学部

医療栄養学科

Kimihide OHMICHI, Mizuki CHIBA, Yuri TANIUCHI-HASEGAWA and Ryo IOROIDivision of Medical Nutrition, Faculty of Healthcare, Tokyo Healthcare University, Tokyo, Japan要

旨：近年、マイコトキシンに係る研究分野では、マイコトキシンの配糖体や誘導体の存在が注目されている。パツリンについては、配糖体の存在がこれまでに報告されていない。仮にパツリンに配糖体があった場合には、リスクを過小評価することになる。そこで本研究では、酵素反応によりパツリンの配糖体化について、dextransucrase を用
い検討を行った。酵素反応液は LC-MS により精密質量分析を行った。その結果、パツリンの配糖体化は確認できなかった。しかし、パツリンと同様の組成式をもつが、276 nm に吸収がない、パツリンとは異なる構造の化合物が検出された。この化合物はdextransucrase の作用で生成した化合物である可能性があり、dextransucrase はパツ
リンを構造変換させる可能性を示唆した。
A b s t r a c t：In recent years, the presence of mycotoxin glycosides and mycotoxin derivatives

has emerged as a popular topic in the field of mycotoxin research. The presenceof glycosides in patulin has not thus far been reported, however, if glycosides doexist in patulin, the risks may be underestimated. Given these background, thisstudy examined whether patulin was glycosylated through an enzymatic reaction.
Dextransucrase was used for the enzymatic reaction, and the enzymatic reaction
solution was subjected to an accurate mass analysis by LC-MS to examine whether
mass ions and the mass spectrum corresponding to patulin glucoside could beobserved. In this study, the glycosylation of patulin was not be determined. However,a compound with the same formula, but different structure as patulin and showing
no absorption at 276 nm was detected. This compound may have been generated in areaction catalyzed by dextransucrase. Thus, this study indicated that dextransucrase
may catalyze the structural transformation of patulin.

キーワード：マイコトキシン、グルコシド、マスクドマイコトキシン、パツリン、dextransucrase、
精密質量分析
Keywords：mycotoxin, glucoside, masked mycotoxin, patulin, dextransucrase, accurate mass analysis

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東京医療保健大学 紀要

第1号

Kimihide OHMICHI2018 年

Mizuki CHIBA

Yuri TANIUCHI-HASEGAWA

Ryo IOROI

Introduction
Patulin, a mycotoxin produced by a variety of molds, particularly Penicillium expansum species, is toxic
to animals. Administration of patulin at large doses can cause gastrointestinal, hepatic, and/or lung impairment,
1-3）

including congestion, bleeding, and necrosis. The International Agency for Research on Cancer（IARC）has

classified patulin as a group 3 carcinogen（not classifiable for its carcinogenicity to humans））. Therefore, that4

hasn’t been investigated in detail. The Joint FAO（Food and Agriculture Organization of the United Nations）/
WHO Expert Committee on Food Additives（JECFA）reported the provisional maximum tolerable daily intake（PMTDI）of patulin as 0.4μg/kg bw/day ）. Although there have been no human cases or epidemiological5reports of patulin - poisoning, some cases of intoxication in bovines have been reported ）. The toxicity of patulin
6

in vivo is considered to result from its binding to the - SH groups of a physiological protein, which results in the
denaturation of the protein and the subsequent onset of toxicity ）.
7

Damaged apples that cannot be sold as raw fruit are sometimes used as a raw material for fruit juice.
Patulin - producing strains are known to enter and grow within damaged apples. Therefore, patulin may be foundin the juice of apples or other fruit juices. As apple juice consumption per body weight is higher in infants andchildren than in adults, the health hazards of patulin to infants and children are a major concern. To prevent thesehazards, the Japanese Ministry of Health, Labour and Welfare limited patulin content in apple juice to no morethan 0.050 ppm in accordance with CODEX STAN 193 - 1995

8, 9 ）.

The reported cases of patulin content in apple juice include an investigation of 249 samples of domestic
apple juice by the Ministry of Agriculture, Forestry, and Fisheries of Japan. This report stated that althoughpatulin content was within the acceptable level in all samples, it exceeded quantification limit（0.010 ppm）in threesamples, with the maximum patulin level being 0.021 ppm ）. In addition to apples, patulin - contaminated grapes
10

have also been reported. However, overall, reports of patulin detection are rare in Japan. Ohmichi Laboratory ）11analyzed 20 samples of apple juice and grape juice between 2013 and 2014, but did not detect patulin levels higher
than 0.01 ppm in any sample.
In recent studies of mycotoxin assays, induced metabolites such as mycotoxin glucosides have attracted
attention

12 - 15 ）. Mycotoxins are metabolized in the detoxification processes of plants. Mycotoxin glucosides,

referred to as masked mycotoxins（or modified mycotoxins）, are especially well known. Masked mycotoxins areundetectable by routine analytical techniques, which increase their chances of remaining undetected, thereby
resulting in the underestimation of mycotoxin risk. As the ingested masked mycotoxins are decomposed by theendogenous enzymes of intestinal bacteria and the resultant “unmasked” mycotoxins exert toxicity, they are
regarded as important factors in risk evaluation. Glucoconjugates of Fusarium toxins, such as deoxynivalenol
glucoside, are known as naturally occurring masked mycotoxins. However, the identification of a patulin glucosidehas never been reported. The existence of a patulin glucoside would mean that the risk from patulin has been
underestimated.
Therefore, in this study, we investigated whether patulin is metabolized to glucoside by
enzymatic reactions. Dextransucrase （sucrose : （ 1 → 6 ）-α- D - glucan 6 -α- D - glucosyltransferase; EC.
2.4.1.5 ）is known as a glycosyltransferase supporting the construction of well - defined polysaccharides ）.
16

Dextransucrase（DS - ase）produced by Streptococcus bovis 148, isolated from bovine rumen, catalyzes
the cleavage of sucrose and polymerization of the resultant glucose molecules via α- 1,6 linkages
18 ）This enzyme is also reported to produce riboflavin glucoside . Considering these characteristics

17 ）

.

16 - 18 ）,

we examined whether DS - ase could catalyze the glycosidation of patulin.
The enzymatic reaction solution was subjected to an accurate mass analysis by LC - MS to examine
whether mass ions and mass spectral features corresponding to patulin glucoside could be observed.

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東京医療保健大学 紀要

パツリンと糖の酵素存在下での反応挙動第1号

2018 年

Materials and Methods
Experimental materials

Patulin was purchased from Sigma - Aldrich（MO, USA）.
Commercially available granulated sugar（MITSUI SUGAR）was used as the source of sucrose.

Streptococcus bovis 148, isolated from bovine rumen, was used as the test strain17 ） and cultured in glucose yeast extract - polypeptone（GYP）medium, with glucose serving as the carbon source. The cells were incubatedfor 20 h at 40ºC under anaerobic conditions, by carbon dioxide aeration, and serial neutralization of the medium
was performed using sterilized sodium bicarbonate powder. The culture solution was centrifuged（ 15,000×g, 15min）,and the obtained supernatant was dialyzed overnight against a jet of water. The resulting supernatant wasused as the enzyme solution for this study. DS - ase activity was 18.10 units/mL when glucose was the substrate.
Instruments

Accurate mass spectra and chromatograms were obtained on an LTQ Orbitrap Velos（Thermo Fisher
Scientific, Bremen, Germany）with a high resolution Orbitrap mass spectrometer（MS）connected to a high- performance lipid chromatography（HPLC）system comprising an UltimateTM Rapid Separation LC system witha photodiode array（PDA）detector（Thermo Fisher Scientific）.
General experimental procedures

DS - ase was added in the presence of sucrose at a 10 - fold lower concentration than that of patulin in termsof the molar ratio, as shown below. The control for comparison consisted of the deactivated enzyme solution
prepared by autoclave sterilization.
The composition of the enzyme reaction solution was :32 mM Patulin（aqueous solution）
	400μl3.2 mM Sucrose（aqueous solution）	400μl
Enzyme solution

200μl

The enzymatic reaction mixture was conducted at 40℃ for 24 h.
LC-MS analysis

Each reaction solution was subjected to LC - MS analysis. The LC - MS parameters for the operation of  the electrospray interface（ESI）were optimized as follows : heater temperature 400℃ , negative ionizationmode, sheath gas 40 arbitrary units（au）, aux gas 5 au, capillary temperature 230℃ , I spray voltage 3.5 kV.
Chromatographic separation was performed on an ACQUITY UPLC HSS C18（ 100 × 2.11 mm I.D., 1.8 μm
particle size）column（Waters, MA, USA）at 40℃ with flow rate of 0.3 ml/min. The carrier phrase comprised  
water/formic acid（ 99.9 : 0.1, v/v; solvent A）and acetonitrile/formic acid（ 99.9 : 0.1, v/v; solvent B）. First, thesolvent proportion was 5% B for 7 min, which was then increased to 90% B within 20 min, and held for 5 min at
90% B. Thereafter, the proportion of B was decreased to 5% within 0.1 min, and held at 5% for 4.9 min prior to thenext sample injection. The LC - MS system was operated in full spectra acquisition mode, over a mass range of m/z70 - 600, at an ultra high - resolution power of 100,000（m/z 200）.An accurate mass/high resolution full scan, all ion
MS/MS spectrum acquisition with collision energy and PDA chromatography were simultaneously performed ina single run. Fragmentation was achieved in an optional collision induced dissociation（CID）cell, with collisionenergy of 35.0 eV. Each scan event was performed at negative polarity. Chromatograms obtained by PDA wereextracted at a UV wavelength of 276 nm.

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東京医療保健大学 紀要第1号

2018 年

Kimihide OHMICHI

Mizuki CHIBA

Yuri TANIUCHI-HASEGAWA

Ryo IOROI

Results and Discussion
The chromatogram of the enzyme reaction solution is shown in Fig. 1. Patulin was detected at a retention
time of 11.27 min. This was confirmed by the patulin - equivalent m/z 153［M - H］− in the mass chromatogram（second panel in Fig. 1 ）. The mass spectrum of patulin is shown in Fig. 2. The second panel of Fig. 2 shows thepresence of ions with m/z values of 81, 109, 125, and 135 as fragments from cleaved patulin.
−［M - H］（the second panel of Fig. 1）,a peak was seen at a retention time
In the patulin - equivalent m/z : 153of 6.75 min. Essentially, a compound with the same composition - formula as patulin was eluted at a retention timeof 6.75 min, which was different from the of patulin peak（ 11.27min）in the reaction mixture solution containing
400μl of 32 mM patulin aqueous solution, 400μl of 3.2mM sucrose aqueous solution, and 200μl of DS - ase. The
MS/MS spectrum of the compound, determined by CID, was similar to the cleavage pattern of patulin（Fig. 2 &3 ）.
Similar to Fig. 2, we obtained ions with m/z values of 81, 109, 125, and 135 in Fig. 3, thereby suggesting thatthe generated compound has a formula similar to that of patulin and eluted at a retention time of 6.75 min.
The fourth panel of Fig. 1 shows the PDA absorption at 276 nm. A strong peak derived from patulin wasobserved at 11.19 min（close to 11.27 min）, but no peak was found at 6.75 min. The compound at 6.75 min wasconsidered to have a structure that, for an unknown reason, lacked the relevant absorption.
This study showed that patulin may react with DS - ase to subsequently produce a compound similar topatulin in structure but lacking absorption at 276 nm.
−, a peak was
In the mass chromatogram of the patulin glucoside - equivalent m/z 315.0690–315.0754 ［M - H］

not found at the aforementioned retention time of 6.75 min（third panel of Fig. 1）.In addition, although we could
see certain strong peaks in this mass chromatogram extracted by patulin glucoside - equivalent m/z（third panel ofFig. 1）,each MS/MS spectrum of detected compounds at patulin glucoside - equivalent m/z was not correspondedwith the cleavage pattern of patulin. The results of this accurate mass analysis did not show the production of
patulin glucoside in the enzyme reaction solution. Therefore, the generated compound was not a glucoside. The
glycosylation of patulin was not be determined in this study. In addition, compared with the deactivated enzyme
solution used as a control, peaks that were suggestive of the possible production of patulin glucoside were not
identified.
This study demonstrated the possible conversion of patulin to an unknown compound that resembled
patulin but lacked absorption at 276 nm. However there is a report that Lactobacillus plantarum can convert
patulin to E - /Z - ascladiol or hydroascladiol through ring - opening ）. Patulin biosynthesis involves a series of19

condensation and redox reactions, many, if not all, of which are enzyme catalyzed ）. Structural changes in the20

presence of DS - ase may occur depending on the reaction conditions.
Although patulin glucoside has not been reported, there was a report of patulin linked amino acids orprotein in cloudy apple juice and the compounds were named bound patulins ）. Therefore, we suggest the21

consideration of a decontamination strategy for patulin or bound patulin using enzymes such as DS - ase.
In this study, using accurate mass analysis, we first examined the possible production of patulin. However,its presence was elucidated as an absorption peak at 276 nm was not detected by PDA. This was analyticallysignificant because erroneous qualitative judgment can be overruled by accurate mass analysis combined with
PDA detection. In this study, we were able to establish the importance of complete analysis using multiple
detectors such as MS and PDA. The complementary use of these technologies should be incorporated into routine
analysis.
It should be noted that this generated compound（retention time : 6.75 min）could not be reproduced; thus,
it is possible that this may have been a contaminant in the detection. This aspect is still under examination.
In addition, it is important to note that in this study, it was shown that a false - positive peak for patulin
glucoside or patulin itself can be identified through the combined use of mass chromatogram, MS/MS spectrum
or chromatogram from the PDA data. The results of this study are indication of the importance of combinatorial̶ 18 ̶

̶ 19 ̶Third panel: mass chromatogram at m/z: 315.0880-315.0754 (patulin glucoside-equivalent mass).
First panel: Total ion current
chromatogram. Second panel: mass chromatogram at m/z: 153.0178-153.0208 (Patulin-equivalentmass).
Fourth panel: Chromatogram extracted from the PDA data at 276 nm.
Third panel: mass chromatogram at m/z: 315.0880-315.0754 (patulin glucoside-equivalent mass).
Fourth panel: Chromatogram extracted from the PDA data at 276 nm.

Fig. 1. Chromatogram of enzyme reaction solution

Fig. 1. Chromatogramof ionenzyme
reactionsolutionFirst panel: Total
current chromatogram.
Second panel:mass chromatogram at m/z: 153.0178-153.0208 (Patulin-equivalentmass).

パツリンと糖の酵素存在下での反応挙動
東京医療保健大学 紀要第1号

2018 年̶ 20 ̶

0

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109.0305

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139.0048200

199.0262

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199.0262

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53.32
70 55.38 59.16
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71.30 74.07

65

65.378070

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71.30 74.07
85

81.39
79.36 82.16

80

85

82.16
79.36 90

81.39
90

300

400

134.96123.03 127.07

124.99450452.3239

423.0199

134.96563.5869550

534.5518

165

150

155

165

162.82600

592.1208160

563.5869550

534.5518

150.06 152.99

First panel: full scan results. Second panel: MS/MS spectrum of precursor ion m/z : 153 with collision energy.

160

162.82145

600

592.1208500

495.9995

133.08 135.75 141.04

500495.9995

450

452.3239

97.40 99.34 105
107.37 109.81
127.07133.08150.06 152.99140
100
110 115.12 115 123.03 120
125 135.75 141.04
130
135
95
100
105 m/z110
115
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125
130
135
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m/z

400

423.0199

124.99371.0186 397.0179

107.37 109.81 115.12

109.07m/z

350

109.07309.0639 325.0589

97.40 99.34

89.29
9592.1089.29 92.10

262.9973250

234.0274m/z

350371.0186 397.0179

2. Mass spectrum obtained at 11.27 min
Fig. 2. Mass Fig.
spectrumobtained
11.27
First
panel: full scan results.
Second panel:at
MS/MS
spectrumminof precursor ion m/z : 153 with collision energy.

0

10

150

139.0048

169.0155300

309.0639 325.0589

Mizuki CHIBA

20

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100

109.0305

89.0253

PATG_2_2u_m2 #2898-3018 RT: 11.09-11.47 AV: 22 NL: 2.76E5
F: ITMS - c ESI d Full ms2 153.02@cid35.00 [50.00-165.00]

0

10

20

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262.9973250

234.0274

PATG_2_2u_m2 #2960 RT: 11.27 AV: 1 NL: 2.38E5
T: FTMS - p ESI Full ms [70.00-600.00]
153.0204100
169.0155

C:¥Users¥...¥PATG_2_2u_m2

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Relative Abundance

2018 年

PATG_2_2u_m2 #2898-3018 RT: 11.09-11.47
AV: 22 NL: 2.76E5
40
F: ITMS - c ESI d Full ms2 153.02@cid35.00 [50.00-165.00]

Relative Abundance

PATG_2_2u_m2 #2960 RT: 11.27 AV: 1 NL: 2.38E5
T: FTMS - p ESI Full ms [70.00-600.00]
153.0204100

C:¥Users¥...¥PATG_2_2u_m2

Relative Abundance

第1号

Relative Abundance

東京医療保健大学 紀要

Yuri TANIUCHI-HASEGAWA
Ryo IOROI

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579.5087

150.04 153.06 156.99

First panel: full scan results. Second panel: MS/MS spectrum of precursor ion m/z: 153 with collision energy.

140
145

138.00 140.94
140

135

550

550

513.1356 536.9687
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513.1356 536.9687
500

138.00 140.94

133.04133.04130134.96134.96450452.5722 471.1173

450

452.5722 471.1173

130

123.07 127.04

125.00125.00400355.1374 377.1527 401.1340

109.07m/z

111.07 115.20
110

94.31 97.34 99.28

107.34300

292.1421
325.0549350

355.1374 377.1527 401.1340

109.07m/z

325.0549

261.0634

94.3181.30
97.34 99.28

217.0368

234.0273300

292.1421

Fig. 3. Mass spectrum obtained at 6.75 min

069.33 71.21
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102.8062

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PATG_2_2u_m2 #1671-1770 RT: 6.60-6.89 AV: 15 NL: 7.21E4
F: ITMS - c ESI d Full ms2 153.02@cid35.00 [50.00-165.00]

0

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261.0634
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First panel: full scan results. Second panel: MS/MS spectrum of precursor ion m/z: 153 with collision energy.
Fig. 3. Mass spectrum
obtained at 6.75 min

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234.0273
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パツリンと糖の酵素存在下での反応挙動
東京医療保健大学 紀要第1号

2018 年

東京医療保健大学 紀要

第1号

Kimihide OHMICHI2018 年

Mizuki CHIBA

Yuri TANIUCHI-HASEGAWA

Ryo IOROI

analytical approaches.

Conclusion
A compound with structural resemblance to patulin, but without absorption at 276 nm, was producedby the reaction of patulin with DS - ase. This finding shows the possible conversion of patulin to an unknown
compound that resembles patulin. Naturally occurring patulin glucoside has never been reported. The productionof patulin glucoside through enzymatic reactions was not successful. This study also suggests the importance ofcomplete analysis using a combination of mass chromatogram, MS/MS spectrum or chromatogram from the PDA
data.

Acknowledgement
The authors are grateful to Dr. Hiroyuki Nakagawa and Dr. Yousuke Matsuo（Food Research Institute,
National Agriculture and Food Research Organization）for their technical support and suggestions for LC - MSanalysis.

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パツリンと糖の酵素存在下での反応挙動第1号

2018 年17 ）Ioroi R, Hayashi T, Ohara N et al . Oligosaccharide Production by Dextransucrase of Streptococcus bovis No.148

Isolated from Bovine Rumen. Nippon Shokuhin Kogyo Gakkaishi（Journal of Japanese Society for Food Science andTechnology）1990； 37 : 355-362（In Japanese with an English abstract）
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