Research Article Received: 27 February 2014
Revised: 18 April 2014
Accepted: 18 April 2014
Published online in Wiley Online Library
Rapid Commun. Mass Spectrom. 2014, 28, 1515–1520 (wileyonlinelibrary.com) DOI: 10.1002/rcm.6927
Distribution of lysophosphatidylcholine in the endosperm of Oryza sativa rice Nobuhiro Zaima1*,†, Yukihiro Yoshimura2†, Yukio Kawamura3 and Tatsuya Moriyama1 1
Department of Applied Biological Chemistry, Graduate School of Agriculture, Kinki University, Nara 631-8505, Japan Department of Welfare Systems and Health Science, Faculty of Health and Welfare Science, Okayama Prefectural University, 111 Kuboki, Soja-shi, Okayama 719-1197, Japan 3 Department of Food and Nutrition, Faculty of Home Economics, Kyoto Women’s University, Kyoto 605-8501, Japan 2
RATIONALE: Sake is made from fermented rice and has been drunk in Japan for more than 1000 years. The rice must be polished prior to fermentation to obtain high-quality sake. It is traditionally recognized that the quality of sake is improved as the rice polishing ratio (percentage removed in the polishing process) increases. However, the underlying chemistry of the rice polishing process is incompletely understood. Herein, we analyzed the distribution of lysophosphatidylcholine (LPC) molecular species with unsaturated fatty acids in rice, as their presence is thought to exert a negative effect on the flavor of sake. METHODS: The distribution of LPC molecular species in rice was visualized via matrix-assisted laser desorption/ ionization mass spectrometry imaging (MALDI-MSI). RESULTS: LPC (16:0) is ubiquitously present in the endosperm of rice while LPC (18:0) is localized in the core of the endosperm. In contrast, LPC (18:2) and LPC (18:1) are present in the outer region of the endosperm. CONCLUSIONS: The enhancement of the quality of sake as the polishing ratio of the rice increases might be explained in terms of the distribution of LPC with unsaturated fatty acids in the rice. Copyright © 2014 John Wiley & Sons, Ltd.
Sake is a traditional alcoholic beverage that has been consumed in Japan for more than 1000 years. Sake is made from fermented rice and, prior to fermentation, the rice must be polished to enhance the flavor of the sake. During this polishing process, the outer layer of rice is removed and the weight of the rice decreases to 50–70% of the original mass. Generally, the quality of sake improves as the polishing ratio (percentage removed) of the rice increases. This is because the outer layer components of rice, which cause coloring and impart an unfavorable taste to sake, are removed by the polishing process. Isoamyl acetate has a banana-like flavor and is recognized as one of the most important flavor components of sake. It is synthesized from isoamyl alcohol by alcohol acetyltransferase (AATase) in sake yeast.[1] Unsaturated fatty acids reduce the production of isoamyl acetate by inhibiting the expression of AATase, causing a decline in the quality of sake. The unsaturated fatty acids in rice are derived from phospholipids (PL) including lysophosphatidylcholine (LPC), and from triacylglycerol (TG). Much of the PL and the TG are removed during the rice polishing process because they are mainly present in the outer layer of rice
(pericarp, seed coat, and aleurone layer).[2,3] On the other hand, we have shown previously that LPC is mainly present in the endosperm.[2] Therefore, most of the LPC remains in the endosperm of polished rice as a potential source of unsaturated fatty acids and may thus exert a negative effect on the production of isoamyl acetate. However, the detailed distribution of LPC molecular species containing unsaturated fatty acids in rice endosperm remains unknown. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) represents an ideal approach to the elucidation of the distribution of LPC molecular species in rice endosperm. MALDI-MSI is a two-dimensional mass spectrometric technique that has been used to elucidate the distribution of several biomolecules in tissue.[4,5] We recently presented a visual representation of the distribution of metabolites in both rice and black rice using MALDI-MSI.[2,6] In this study, we analyze the detailed distribution of LPC molecular species in rice via MALDI-MSI.
EXPERIMENTAL * Correspondence to: N. Zaima, Department of Applied Biological Chemistry, Graduate School of Agriculture, Kinki University, Nara 631-8505, Japan. E-mail: [email protected] These authors contributed equally to this work.
Rapid Commun. Mass Spectrom. 2014, 28, 1515–1520
Adhesive film (Cryofilm type IIC) was purchased from Leica Microsystems (Tokyo, Japan). Glass slides (Fisherbrand Superfrost Plus) were purchased from Fisher Scientific (Waltham, MA, USA). Methanol and distilled water were
Copyright © 2014 John Wiley & Sons, Ltd.
1515
†
Materials
N. Zaima et al. purchased from Nacalai Tesque (Kyoto, Japan). 2,5Dihydroxybenzoic acid (DHB) and carboxymethyl cellulose (CMC) were obtained from Bruker Daltonics (Bremen, Germany) and Wako Pure Chemical Industries (Osaka, Japan), respectively. All chemicals used in this study were of the highest purity available. Oryza sativa rice (Koshihikari) was purchased from a local market in Nara, Japan. Preparation of rice sections Rice seeds were freeze-embedded with 2% CMC at 20°C, and rice sections were then prepared according to the Kawamoto method with slight modifications.[7] Briefly, the rice sections were attached to adhesive film and sliced to 20-μm thickness using a cryostat (CM 1850; Leica Microsystems, Wetzler, Germany). Sections were attached to a glass slide with adhesive tape. Mass spectrometry imaging MALDI-MSI analysis was performed using a LTQ-XL linear ion trap mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA). Samples were prepared according to a previously published method.[2] Briefly, 50 mg/mL DHB in methanol/water (7:3, v/v) was used as a matrix, and the matrix solution (500 μL) was sprayed uniformly over the rice sections using an airbrush with a 0.2-mm nozzle (Procon Boy FWA Platinum; Mr. Hobby, Tokyo, Japan). Data were acquired with a 50-μm step size in positive ion mode. The laser energy was set to 30 μJ. Ions with m/z values in the range of 300–1000 were measured. For tandem mass spectrometric (MS/MS) analysis of the LPC in the sections, the collision energy was set to 35% of the maximum available energy for the LTQ-XL, and the laser energy was set to 30 μJ. ImageQuest software
(Thermo Fisher Scientific) was used to create twodimensional ion-density maps. To create the MS image for each m/z value, the ion intensity at each pixel was divided by the total ion current using the ImageQuest software. The distribution of the MS/MS product ions was visualized without division by the total ion current. Semi-quantitative analyses of the ion intensity in parts of the rice section were carried out using the ImageQuest software. The ion intensity obtained in region 2 (see Fig. 1(a)) of each m/z value was set to 100%, as the control region. Data were collected from three different Koshihikari rice grains. Statistical analysis Statistical analysis was performed with Stat View 5.0 (SAS Institute, Tokyo, Japan). The statistical differences in the ion intensity were determined using one-way analysis of variance (ANOVA) with Dunnett’s multiple comparison post hoc test. Differences were considered significant at p
Revised: 18 April 2014
Accepted: 18 April 2014
Published online in Wiley Online Library
Rapid Commun. Mass Spectrom. 2014, 28, 1515–1520 (wileyonlinelibrary.com) DOI: 10.1002/rcm.6927
Distribution of lysophosphatidylcholine in the endosperm of Oryza sativa rice Nobuhiro Zaima1*,†, Yukihiro Yoshimura2†, Yukio Kawamura3 and Tatsuya Moriyama1 1
Department of Applied Biological Chemistry, Graduate School of Agriculture, Kinki University, Nara 631-8505, Japan Department of Welfare Systems and Health Science, Faculty of Health and Welfare Science, Okayama Prefectural University, 111 Kuboki, Soja-shi, Okayama 719-1197, Japan 3 Department of Food and Nutrition, Faculty of Home Economics, Kyoto Women’s University, Kyoto 605-8501, Japan 2
RATIONALE: Sake is made from fermented rice and has been drunk in Japan for more than 1000 years. The rice must be polished prior to fermentation to obtain high-quality sake. It is traditionally recognized that the quality of sake is improved as the rice polishing ratio (percentage removed in the polishing process) increases. However, the underlying chemistry of the rice polishing process is incompletely understood. Herein, we analyzed the distribution of lysophosphatidylcholine (LPC) molecular species with unsaturated fatty acids in rice, as their presence is thought to exert a negative effect on the flavor of sake. METHODS: The distribution of LPC molecular species in rice was visualized via matrix-assisted laser desorption/ ionization mass spectrometry imaging (MALDI-MSI). RESULTS: LPC (16:0) is ubiquitously present in the endosperm of rice while LPC (18:0) is localized in the core of the endosperm. In contrast, LPC (18:2) and LPC (18:1) are present in the outer region of the endosperm. CONCLUSIONS: The enhancement of the quality of sake as the polishing ratio of the rice increases might be explained in terms of the distribution of LPC with unsaturated fatty acids in the rice. Copyright © 2014 John Wiley & Sons, Ltd.
Sake is a traditional alcoholic beverage that has been consumed in Japan for more than 1000 years. Sake is made from fermented rice and, prior to fermentation, the rice must be polished to enhance the flavor of the sake. During this polishing process, the outer layer of rice is removed and the weight of the rice decreases to 50–70% of the original mass. Generally, the quality of sake improves as the polishing ratio (percentage removed) of the rice increases. This is because the outer layer components of rice, which cause coloring and impart an unfavorable taste to sake, are removed by the polishing process. Isoamyl acetate has a banana-like flavor and is recognized as one of the most important flavor components of sake. It is synthesized from isoamyl alcohol by alcohol acetyltransferase (AATase) in sake yeast.[1] Unsaturated fatty acids reduce the production of isoamyl acetate by inhibiting the expression of AATase, causing a decline in the quality of sake. The unsaturated fatty acids in rice are derived from phospholipids (PL) including lysophosphatidylcholine (LPC), and from triacylglycerol (TG). Much of the PL and the TG are removed during the rice polishing process because they are mainly present in the outer layer of rice
(pericarp, seed coat, and aleurone layer).[2,3] On the other hand, we have shown previously that LPC is mainly present in the endosperm.[2] Therefore, most of the LPC remains in the endosperm of polished rice as a potential source of unsaturated fatty acids and may thus exert a negative effect on the production of isoamyl acetate. However, the detailed distribution of LPC molecular species containing unsaturated fatty acids in rice endosperm remains unknown. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) represents an ideal approach to the elucidation of the distribution of LPC molecular species in rice endosperm. MALDI-MSI is a two-dimensional mass spectrometric technique that has been used to elucidate the distribution of several biomolecules in tissue.[4,5] We recently presented a visual representation of the distribution of metabolites in both rice and black rice using MALDI-MSI.[2,6] In this study, we analyze the detailed distribution of LPC molecular species in rice via MALDI-MSI.
EXPERIMENTAL * Correspondence to: N. Zaima, Department of Applied Biological Chemistry, Graduate School of Agriculture, Kinki University, Nara 631-8505, Japan. E-mail: [email protected] These authors contributed equally to this work.
Rapid Commun. Mass Spectrom. 2014, 28, 1515–1520
Adhesive film (Cryofilm type IIC) was purchased from Leica Microsystems (Tokyo, Japan). Glass slides (Fisherbrand Superfrost Plus) were purchased from Fisher Scientific (Waltham, MA, USA). Methanol and distilled water were
Copyright © 2014 John Wiley & Sons, Ltd.
1515
†
Materials
N. Zaima et al. purchased from Nacalai Tesque (Kyoto, Japan). 2,5Dihydroxybenzoic acid (DHB) and carboxymethyl cellulose (CMC) were obtained from Bruker Daltonics (Bremen, Germany) and Wako Pure Chemical Industries (Osaka, Japan), respectively. All chemicals used in this study were of the highest purity available. Oryza sativa rice (Koshihikari) was purchased from a local market in Nara, Japan. Preparation of rice sections Rice seeds were freeze-embedded with 2% CMC at 20°C, and rice sections were then prepared according to the Kawamoto method with slight modifications.[7] Briefly, the rice sections were attached to adhesive film and sliced to 20-μm thickness using a cryostat (CM 1850; Leica Microsystems, Wetzler, Germany). Sections were attached to a glass slide with adhesive tape. Mass spectrometry imaging MALDI-MSI analysis was performed using a LTQ-XL linear ion trap mass spectrometer (Thermo Fisher Scientific, San Jose, CA, USA). Samples were prepared according to a previously published method.[2] Briefly, 50 mg/mL DHB in methanol/water (7:3, v/v) was used as a matrix, and the matrix solution (500 μL) was sprayed uniformly over the rice sections using an airbrush with a 0.2-mm nozzle (Procon Boy FWA Platinum; Mr. Hobby, Tokyo, Japan). Data were acquired with a 50-μm step size in positive ion mode. The laser energy was set to 30 μJ. Ions with m/z values in the range of 300–1000 were measured. For tandem mass spectrometric (MS/MS) analysis of the LPC in the sections, the collision energy was set to 35% of the maximum available energy for the LTQ-XL, and the laser energy was set to 30 μJ. ImageQuest software
(Thermo Fisher Scientific) was used to create twodimensional ion-density maps. To create the MS image for each m/z value, the ion intensity at each pixel was divided by the total ion current using the ImageQuest software. The distribution of the MS/MS product ions was visualized without division by the total ion current. Semi-quantitative analyses of the ion intensity in parts of the rice section were carried out using the ImageQuest software. The ion intensity obtained in region 2 (see Fig. 1(a)) of each m/z value was set to 100%, as the control region. Data were collected from three different Koshihikari rice grains. Statistical analysis Statistical analysis was performed with Stat View 5.0 (SAS Institute, Tokyo, Japan). The statistical differences in the ion intensity were determined using one-way analysis of variance (ANOVA) with Dunnett’s multiple comparison post hoc test. Differences were considered significant at p
