Introduction

Citrus fruits are the most commonly produced type of fruit in the world. They are used not only for foods and drinks but in perfumes and soaps, and many other aromatic products. Today it is possible to find many different types of citrus on the market, but in recent years there has been a growing interest in Japanese citrus, and many types of foods and drinks are derived from Japanese citrus. Some qualities embodied in the image of Japanese citrus are: elegant, invigorating, calming, and fondly familiar.
In this paper, I would like to introduce varieties of citrus that have an image of being "Japanese" and that are not limited to varieties indigenous to Japan, and then, to introduce an aroma analysis approachs aiming to develop more genuine flavors.

Leading Japanese Citrus Fruits and their Volatile Components

Currently more than 25 species of citrus are cultivated in Japan. Among these, one can cite the followings as being the most popular: Unshu mikan (Satsuma mandarin or Citrus unshiu Marcov., with the largest quantity of production in Japan), Iyokan (Citrus iyo Hort. ex Tanaka), Natsu mikan (Citrus natsudaidai Hayata), Hassaku (Citrus hassaku Hort. ex Tanaka), Yuzu (Citrus junos Sieb. ex T. Tanaka), Sudachi (Citrus sudachi Hort. ex Shirai), and Kabosu (Citrus sphaerocarpa Hort. ex Tanaka). The Western citrus that immediately come to mind are: navel oranges, Valencia oranges, mandarin oranges, grapefruits, lemons, and limes. First of all, I will introduce several leading Japanese citrus and show how their distinctive volatile components differ from those of Western citrus.

Unshu Mikan

The leading citrus produced in Japan is easily the Unshu mikan. Its aroma is comparatively weak, but the taste is sweet, the eating quality is good, and the peel is thin, making it easy to peel off. The place of origin is Kagoshima Prefecture, formerly called Satsuma. It is said that seeds came from Zhejiang Province in China approximately 500 years ago, from which chance seedlings grew. Many cultivated varieties exist, which are classified according to the time of harvest: early varieties, medium maturing, common Unshu, and late varieties.
Terpene hydrocarbons such as limonene and γ-terpinene account for approximately 98% of the peel oil volatile components. Among the oxygenated components, which in general produce the distinctive aroma of the citrus, linalool exists in the largest amounts, followed by α-terpineol and decanal. Compared to Mediterranean mandarins and tangerines, which are botanically identical species, it has a lot of sesquiterpene hydrocarbons, which give it a woody fragrance. The oxygenated components in the juice has a high content ratio of isoamyl alcohol and low-boiling aliphatic compounds such as trans-2-hexenal and hexanal, which possess a green note - quite different in composition from the peel oil.

Iyokan

With a fragrant odor similar to sweet orange and a good sour-sweet balance, this citrus is the second most frequently grown citrus in Japan next to the Unshu mikan. It was discovered as a chance seedling in Yamaguchi Prefecture in 1886, and was originally called the Minato mikan, but after being transplanted to Matsuyama City in Ehime Prefecture, formerly called Iyo, it was sold as Iyo mikan. Later, to avoid confusion with Ehime-produced Unshu mikan, it started being called the Iyokan. Today, approximately 80% of Iyokan are grown in Ehime Prefecture.
Limonene and α-terpinene account for about 95% of the peel oil, with high amount of linalool among the oxygenated components, yet it has few aldehydes.

Natsu Mikan

The next largest production among citrus in Japan after the Unshu mikan and Iyokan is Natsu mikan, which is a late variety that ripens between April and May. Its origin is said to be from a citrus seed that washed ashore on the coast of Nagato City, Yamaguchi Prefecture in the 1700s. It is recognized as a natural hybrid that originates with Buntan (Citrus grandis Osbeck). Amanatsu is a mutant species of Natsu mikan, which has little acidity in the fruit, and accounts for the majority of Natsu mikan grown today. The juice is ordinarily used for soft drinks or mixed with Unshu mikan juice, while the peel is made into marmalade.
Terpene hydrocarbons such as limonene and α-terpinene account for approximately 97% of the peel oil volatile components, while the content of α-terpineol and decanal is high among the oxygenated components.

Hassaku

This is the fourth most often cultivated citrus in Japan by production volume. It was discovered in Hiroshima Prefecture around 1860 as a chance seedling. In botanical classification, it is a species related to Buntan. The peel is thick and the fruit is slightly hard. It has little juice, but the acidity is low, so it is easy to eat.
Monoterpene hydrocarbons such as limonene account for approximately 99% of the peel oil volatile components, while the content ratio of oxygenated components is low compared to Western citrus.

Yuzu

Yuzu could be considered the sour citrus that Japanese people are most familiar with. Its cultivation in Japan has a long history dating back to around the Nara period (710-794), but originally it came from China's Yangtze River basin area. Aside from its uses in cooking for adding fragrance or sour flavor, it is also popular as an aroma for bathwater additives to make a "Yuzu bath" in Japan.
Because it has a unique flavor that is refreshing and adds panache, a great deal of research has been done in Japan on its volatile components. Trace amounts of the following compounds have been found in its peel oil: cis-9-dodecenolide,
2,6-dimethyl-2,7-octadien-1,6-diol, 6-methyloctanal, 8-methylnonanal, 8-methyldecanal,
1,3,5-undecatriene, 1,3,5,7-undecatetraene, dimethyl trisulfide, 1-p-menthen-8-thiol.
In comparison to lemon, the world's leading citrus used for cooking, Yuzu is similarly high in monoterpene hydrocarbons at approximately 96%, but unlike lemons, which have a lot of aldehydes and esters such as neral and geranial, it has been reported that Yuzu has a high content of alcohols such as linalool and thymol, which has a phenolic odor. Important components that give the lemon woody aroma are β-bisabolene and α-bergamotene, while Yuzu is known to have bicyclogermacrene and germacrene D.

Sudachi

A species related to Yuzu, Sudachi has a soft and juicy fruit, and the fragrance is similar to that of Yuzu. The main growing region is Tokushima Prefecture today, but the origins are unknown. It is used to add aroma to broiled items or is processed into table vinegar.
The quality of the aroma is different in the peel and the juice. Among the oxygenated components that produce the distinctive aroma, in the peel oil there is high content of linalool and citronellol, which has a rosy note, while in the juice these exist in low concentration. Instead, a high content of α-terpineol, decanal and dodecanal produces its distinctive character.

Kabosu

This is a sour citrus that Japanese are very familiar with, much like Yuzu and Sudachi. It has a thin, smooth peel. The fruit is soft, juicy and very sour, with a distinctive flavor. The peel is used in spices and the juice is used to add aroma to broiled fish, for example. Kabosu are grown in Oita Prefecture, but the origins are unknown.
Compared to the volatile components of lemon or lime or Yuzu or Sudachi, Kabosu has a high content ratio of normal chain aldehydes such as octanal, decanal and dodecanal in its peel oil among oxygenated components, and a low concentration of linalool and α-terpineol. It has also been reported that (R)-(+)-citronellal is the characteristic aroma compound of the Kabosu.

Aroma Analysis of Japanese Citrus Juice

As noted above, many types of research related to the aroma analysis of Japanese citrus has been taking place. Here I would like to introduce the aroma analysis procedures for fruit juice used at Takasago aiming to reproduce more genuine flavors based on clarifying the characteristic components of each variety.

Analysis of the Oxygenated Fraction

Generally, a large portion of the volatile components of citrus peel oil and juice are terpene hydrocarbons such as limonene but the characteristic aroma compounds exist in the remaining oxygenated fraction.
For six varieties of Japanese citrus - Iyokan, Natsu mikan, Hassaku, Kiyomi, Shiranui and Sanbokan - the straight juices were applied to solvent extraction process and Solvent Assisted Flavor Evaporation (SAFE) to obtain aroma extracts. Then, using a silica gel column, the hydrocarbon and oxygenated fractions were fractionated and analyzed by means of GC/MS. Table 1 shows the content ratios of the main compounds of the oxygenated fraction. This result shows that there is the large variance in the content ratio of each component by variety. Natsu mikan and Hassaku have a high content ratio of nootkatone, the characteristic aroma compound of grapefruit. More detailed component analysis reveals a clear variance in the component balance from variety to variety, and the correlation between the odor characteristics and component balance of the citrus juice.

Investigating Odor Active Compounds Using GC/Olfactometry

GC/Olfactometry (GC/O) is a vital method for determining odor active compounds. Aroma Extract Dilution Analysis (AEDA) is often used to screen odor active compounds. With this method, GC/O is performed as the aroma extracts are diluted in stages. The process is repeated until finally no aroma compounds can be detected. The highest dilution ratio for each detected compound is called the FD (Flavor Dilution) factor. The FD factor is regarded as the measure of odor contribution of each compound.
After applying AEDA to the oxygenated fraction of the six varieties of Japanese citrus juice mentioned above, we found linalool to be the compound with the highest FD factor in all varieties. At the same time it became clear that the odor characteristics of linalool differed according to the variety. This suggests the possibility of variance in the enantiomeric ratios of linalool by variety.

Studying Enantiomeric Distribution of Odor Active Compounds

Within the aroma compounds in natural fruit peel oil and fruit juice there are many compounds that have enantiomers, and it is known that the enantiomeric ratios of these chiral compounds differ according to the variety. The odor characteristics and thresholds are different for each enantiomer, so by blending according to the natural enantiomeric ratios, it becomes possible to develop flavors closely approximates the natural aroma.
Using multidimensional (MD) GC to analyze the enantiomeric ratios of linalool in Japanese citrus peel oil, we found an excess of (3R)-(-)-form, which have a petitgrain-like woody note, in Unshu mikan and Yuzu, and an excess of (3S)-(+)-form, which have a lavender-like fruity note, in Iyokan, Shiranui and Sudachi (See Table 2). In the future, in regard to fruit juices as well, we expect to be able to use the data from analyzing the enantiomeric ratios of odor active compounds such as linalool in order to develop new flavors.

Conclusion

Japanese citrus are well-suited to the sensitive tastes of the Japanese people, and are unique among citrus fruits in the world. Many varieties of citrus are produced in Japan and new varieties are being developed every year. There are also some popular varieties such as Kiyomi and Shiranui that have a distinctive flavor. With the technological level of analytical equipments constantly improving, the interest for the aroma analysis of Japanese citrus is endless.
Citrus aromas will still be primarily blended from natural materials derived from fruits for some time to come, but by utilizing the knowledge gained from the analytical approach described above, and using a combination of sensory evaluation methods and statistical analysis it is certainly possible to develop flavors that more closely approximate the natural fruit.

Ken'ichi Tomiyama (2008)