Journal of Oleo Science Copyright ©2017 by Japan Oil Chemists’ Society J-STAGE Advance Publication date : November 13, 2017 doi : 10.5650/jos.ess17147 J. Oleo Sci. Quantification of Fraglide-1, a New Functional Ingredient, in Vinegars Aydan H. Yatmaz1, 2, Tetsuaki Kinoshita1, Akio Miyazato3, Masahiro Takagi3, Yoshio Tsujino3, Fumiaki Beppu1 and Naohiro Gotoh1＊ 1 Department of Food Science and Technology, Tokyo University of Marine Science and Technology, 4-5-7 Konan, Minato-ku, Tokyo 108-8477, JAPAN 2 Food Safety and Agricultural Research Center, Akdeniz University, 07058, Antalya, TURKEY 3 School of Materials Science, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Nomi, Ishikawa 923-1292, JAPAN Abstract: Vinegar is a widely used condiment in the world, and is produced from ethanol by acetic acid fermentation. Different fruits, vegetables, cereals, and wines can be used as ingredients for vinegar production. It is known that vinegar has many nutrient components such as organic acids, polyphenols, and aromatic compounds. Because of these bioactive components, it has many health benefits. China has a long history of producing vinegar and has been using it for health products and as medicine. Chinese aromatic Zhenjiang vinegar (Kozu) is produced from sticky rice. It is famous for its special flavor and health benefits. 5-Hydroxy-4-phenyl-butenolide (Fraglide-1) is a functional compound discovered in Kozu and has anti- fungal and anti-obesity effects. In this study, the Fraglide-1 content of different kinds of vinegars and ingredients, including Kozu samples and ingredients, was investigated. Fraglide-1 analysis was carried out via LC-MS/MS in multiple reaction monitoring mode. It was found that all the Kozu samples, as well as brown rice vinegar (Kurosu) samples, and the Chinese sticky rice husk used for the production of Kozu, contained Fraglide-1. Kozu production requires a 6-month- to 8-year-long aging process for its special flavor and aroma. Because of this long aging process, Fraglide-1 is thought to move from the sticky rice husk to Kozu. Key words: Fraglide-1, functional ingredient, Chinese sticky rice vinegar, quantification, vinegar 1 INTRODUCTION types of vinegar using different ingredients or fermentation Vinegar is the world’s oldest cooking ingredient and a processes. Grapes, apples, rice, sugarcane, wine（white, red common liquid food product consisting of acetic acid and and sherry wine）, malted barley, pure alcohol, etc. can be water. It is also used as an ingredient in salad dressings, used as raw material for vinegars1, 2）. ketchup, and other sauces. Vinegar is produced by fermen- Rice vinegar is an aged and filtered product obtained tation of raw plant materials containing sugar and starch. from the acetic fermentation of sugars derived from rice. It The production process consists of 2-stage fermentation. is very common in Asian countries and its excellent flavor The first stage is alcoholic fermentation by yeasts and the forms together with herbs, spices, and fruits due to its mild second stage is acetic fermentation by acetic acid bacteria1－3）. own flavor5, 6）. Commercial rice vinegar consists of water, There are two methods to produce vinegar. One is via a acetic acid, sugars, and other secondary constituents that traditional（slow, surface culture methods）process and the contribute to smell, taste and preserving qualities. The other is via a submerged（fast）process4）. In the traditional whole fermentation of rice vinegar is very complex and process, acetic acid bacteria grow abundantly on the media contains many physical and chemical reactions, which also surface because the oxygen concentration is high there. lead to color changes during the fermentation period6）. Submerged methods are usually used in the semi-continu- There are different kinds of rice vinegar depending on the ous process for industrial vinegar production and they are production method and the rice species. Komesu and characterized by a faster acidification process in which Abbreviations: N.Q., not quantified; PPARγ, peroxisome oxygen is supplied in the fermentation3）. There are many proliferation-activated receptor γ; s/n, signal/noise ＊Correspondence to: Naohiro Gotoh, Department of Food Science and Technology, Tokyo University of Marine Science and Technology, 4-5-7 Konan, Minato-ku, Tokyo 108-8477, JAPAN E-mail: [email protected] Accepted August 8, 2017 (received for review June 27, 2017) Journal of Oleo Science ISSN 1345-8957 print / ISSN 1347-3352 online http://www.jstage.jst.go.jp/browse/jos/ http://mc.manusriptcentral.com/jjocs 1 A. H. Yatmaz, T. Kinoshita, A. Miyazato et al. Kurosu vinegar are rice vinegars traditionally produced in Japan from unpolished and polished rice7－9）. The history of Chinese rice vinegar production is said to be more than 5000 years old10）. Chinese people have used vinegar as condiments and healthcare products, and even as medi- cines5）. In China, there are several kinds of vinegar such as aromatic vinegar, mature vinegar, rice vinegar, and white vinegar11）. Chinese vinegar can have high volatility depend- ing on the ingredients and production procedures. Zhenji- ang vinegar is the most famous aromatic vinegar in South- Fig. 1 Structure of Fraglide-1（5-hydroxy-4-phenyl-2（5H） ern China. It is produced from sticky rice, koji（a specific furanone, 5-hydroxy-4-phenyl-2（5H）butenolide）. fermented cereal containing molds, yeasts, and bacteria） and sticky rice husk12, 13）. The characteristic aroma is tify Fraglide-1 in different kinds of vinegar and their ingre- formed during the fermentation process and the final dients to understand the origin of Fraglide-1. product contains many kinds of volatile compounds, such as esters, aldehydes, ketones, alcohols, and aromatic com- pounds10, 14, 15）. Furthermore, it was accepted as a Protected Geographical Indication by a European Union（No. 2 EXPERIMENTAL 501/2012）regulation16）. Vinegar has been used for health 2.1 Chemicals and Materials benefits since ancient times because of several bioactive Methanol and hexane were purchased from Kokusan compounds. Besides acetic acid, it contains different kinds Chemical Ltd.（Tokyo, Japan）. LC-MS/MS grade acetonitrile of organic acids such as tartaric, formic, lactic, citric, malic and ultrapure water were purchased from Wako Pure and succinic acid2, 17）and the type of ingredient affects the Chemical Industries, Ltd.（Osaka, Japan）. 5-Hydroxy- bioactive compounds in vinegar. 4-phenyl-2（5H）furanone（5-hydroxy-4-phenyl-2（5H）bu- Epidemiological studies have indicated that there is a tenolide（Fraglide-1）, CAS number: 78920-11-3）standard negative correlation between the intake of fruits and vege- was obtained from TCI Chemical Industry, Ltd.（Tokyo, tables and occurrence of cardiovascular disease. Fruits and Japan）. Vinegar and other food samples were purchased vegetables are a good source of phenolic compounds, vita- from a supermarket in Tokyo. Kozu samples were obtained mins E and C, carotenoids and phytosterols18）. It was from Kojun Japan Co. Ltd.（Osaka, Japan）. shown that vinegar has benefits on human health such as improving the digestive system, lowering lipid levels, regu- 2.2 Preparation of standard solution and calibration lation of blood pressure, stimulating appetite, and promot- curves ing recovery from exhaustion. Additionally, vinegar has an- The stock solution of Fraglide-1 standard was prepared tioxidant effects due to its high content of polyphenols, at 10 mg/ML in methanol. The calibration standards of and it prevents lipid peroxidation, hypertension, hyperlip- 1000, 100, 10, and 1 ng/mL were then prepared with ultra- idemia, inflammation, DNA（deoxyribonucleic acid）damage, pure water. and cancer5, 9）. It also has anti-glycemic effects on human health19）. 2.3 Sample preparation 5-Hydroxy-4-phenyl-butenolide（Fraglide-1）is a func- Ten mL of vinegar sample was diluted with 90 mL metha- tional compound found in Kozu（Chinese Zhenjiang fragrant nol and mixed homogenously, subsequently filtered and in- vinegar）. It is a butenolide compound, a class of lactone jected into the LC-MS/MS. For rice samples, a cleanup pro- with a four-carbon heterocyclic ring structure20（） Fig. 1）. cedure was needed first. For this purpose, 10 g sample was Butenolide compounds are known to have anti-cancer homogenized with 90 mL methanol on ice using blender at effects and bactericidal, fungicidal, antiviral, anti-inflam- 15000 rpm for 5 min. The homogenate was filtered through matory and antitumor properties21）. It was shown that Fra- filter paper and the methanol samples were washed 3 times glide-1 also has certain health benefits such as anti-fun- with hexane to eliminate any fat from the sample. After gal22）and anti-obesity effects23）. Tsujino found that that, the methanol extract was concentrated to 10 mL with Fraglide-1 from Kozu is an agonist for peroxisome prolifer- a rotary evaporator, the extract was filtered through a ation-activated receptor （γ PPARγ）at the cellular level23）. 0.45-μm hydrophilic nylon syringe filter（Filtstar Syringe Also, an in vivo study has shown that Fraglide-1 has an Filter, Starlab Scientific Co. Ltd., Shaanxi Province, China） anti-obesity effect in mice24）. Furthermore, the anti-oxidant and injected to the LC-MS/MS. activity of Fraglide-1 in cells was also reported25）. However, the content of Fraglide-1 in vinegars and their ingredients has not been investigated. In this study, we aimed to quan- 2 J. Oleo Sci. Quantification of FR-1 in Vinegars 2.4 Instrumentation and chromatographic condition for 3 RESULTS AND DISCUSSION LC-MS/MS analysis According to an WHO report more than 600 million The LC-MS/MS analysis was carried out using the Shi- adults are obese and a total of 1.9 billion adults are over- madzu LC-20AD（Shimadzu Co., Kyoto, Japan）equipped weight in the world27）. Obesity is the most important risk with an ABSciex Qtrap 4500-ESI（AB Sciex, Framingham, factor for metabolic diseases such as hyperlipidemia, hy- MA）with negative polarity.