Yasushi Ifuku’s research while affiliated with Marine Biological Research Institute of Japan Co., Ltd. and other places

The cultivation of Satsuma mandarin (Citrus unshiu Marcov.) was originated in Japan and is one of the most important parts of Japanese agriculture. We determined changes in the amounts of limonoid aglycones and glucosides in the Satsuma mandarin fruit tissues during fruit growth and maturation as well as the contents of limonoid glucosides in commercial juices and juice processing by-products. Mature Satsuma mandarin fruit contains relatively low concentrations of bitter limonoid aglycones and high concentrations of non-bitter 17β-D-glucoside derivatives, such as limonin 17β-D-glucopyranoside and nomilin 17β-D-glucopyranoside. The concentration of limonoid glucosides in the fruit tissues is about 150 times higher than that of aglycones. Therefore, with the exception of early ripening cultivars, limonoid bitterness is very infrequently in Satsuma mandarin juices and its other processed products. The Satsuma mandarin juice processing by-products are also rich sources of limonoid glucosides. Molasses, for example, contains over 2000 ppm of total limonoid glucosides.

A new technique of supercritical carbon dioxide (SC-CO2) treatment, micro bubble SC-CO2 method, was applied to extraction of limonoids. This method could increase the concentration of SC-CO2 in sample solutions rapidly, so that limonoids were extracted effectively compared with conventional technique. Juice (sample A) and seed extract (sample B) of Natsudaidai were treated at 25 MPa, 35°C. The flow rate of CO2 was 4 g/min. The yield of limonoids was significantly higher than those extracted by conventional SC-CO2 method. When the extraction was carried out for 30 min, the extraction yield of limonoids in sample A and sample B were 92.5% and 42.4%, respectively. When methanol was added as an entrainer, the yield of sample B was increased up to 73.2%.

We examined immunological methods for the detection of microbial contaminants in fruit juice. Polyclonal antibodies against native yeast of 7 species, including Candida, Cryptococcus, Rhodotorula and Saccharomyces, and the monoclonal antibody against native C. intermedia, which was named mAb44 (IgG2a subclass), were prepared for use as probes in enzyme-linked immunosorbent assay (ELISA). These antibodies reacted with yeast cell-surface antigens that are thought to consist of protein moieties. The cross-reactivities between the antibodies and yeast antigens of 12 species were investigated by immunofluorescence and immunoblotting methods. C. intermedia polyclonal antibody showed extensive cross-reactivities with yeast antigens of 11 species. ELISA was carried out using C. intermedia polyclonal antibody and mAb44. Yeast cells (104-107) suspended per 100μl of fruit juice or PBS could be quantitatively measured using polyclonal antibody, although quantitative values were not obtained in the case of mAb44. When more than 103 per 100μl yeast cells were present, they were detectable by using mAb44 as well as polyclonal antibody. These results show that ELISA is a valuable method to detect microbial contaminants in fruit juice.

温州ミカン搾汁粕からの効率的なヘスペリジン抽出,回収方法について検討し,ヘスペリジン抽出試料として酵素処理搾汁粕が優れていることを示した.酵素処理によるヘスペリジンの分解,損失はほとんどなく,ヘスペリジンの抽出時間および析出時間は大幅に短縮できた,特に析出時間はヘスペリジン抽出液に共存するセルロース性物質と密接な関係があることが示唆された.ヘスペリジンの抽出条件として,酵素処理搾汁粕100gに対し,加水量300～400g,水酸化カルシウム添加量0.50～0.75g,抽出初発pH 12.00～12.25を設定した.

In the course of the development of an industrial recovering system of limonoids from citrus processing waste, an efficient method to recover seeds from the citrus processing waste (peel residue) was developed. The residue was first passed on a vibrating sieve (φ14.5×14.5 mm) to remove peel fragments and other contaminants, and then the remaining material was degraded either chemically or enzymatically. The degraded fraction was sieved with mesh screen (φ4.5×50mm) and 85-90% of the seeds was recovered in optimal condition. The limonoid content in the seeds was not affected when the first fraction was treated with acid or enzyme. However, the content decreased when it was treated with alkali.

温州ミカン搾汁粕中のヘスペリジンを効率的に抽出,回収するため,搾汁粕中に含有している夾雑物を分解除去しヘスペリジン濃度を高めるのに有効な酵素の選択を行った.ペクチナーゼやセルラーゼ活性を主体とした酵素剤を用いた場合,夾雑物の除去率が高く,得られた固形物の歩留りす少なく,ヘスペリジン濃度の高いことが示された.中でもペクチナーゼ活性を主としセルラーゼ,ヘミセルラーゼ活性を有する酵素剤PCH(起源; A. niger)で処理したとき,酵素処理中のヘスペリジンに与える影響も少なく, 24時間後のヘスペリジン残存率は94.1%と良好であった.また,酵素剤PCHによる温州ミカン搾汁粕の酵素処理は,初発pH 3.0～4.0,反応温度40℃,反応時間24時間が最適条件として示され,酵素処理粕中のヘスペリジン含量は処理前に比べて2.2倍に高められた.

最近,その生理活性が注日されているリモノイドおよびそのグルコサイドのカンキツ種子からのより有効な抽出方法としてSC-CO2抽出法について検討した.その結果,供試試料として脱脂乾燥夏柑種子粉末を用いてSC-CO2抽出したときCO2単独では各リモノイドの抽出率は有機溶媒抽出法に比べ著しく低かった.そこで抽出率の向上を目的として抽出温度,圧力,エントレーナーの併用およびCO2量の条件について実験した.リモノイドの抽出率は抽出圧力とCO2量に大きく依存したが,抽出温度の影響はほとんど受けなかった.各リモノイドの抽出率は抽出圧力を高めるに従い増加し, 300kg/cm2で最大となった.また,エントレーナーとしてはアセトンを添加したとき最も効果的であった. CO2の使用量についてはその使用量が多いほど高い抽出率が得られ,試料重量に対し50倍量のCO2量になるまでの変化が大きかった.最適条件(抽出温度, 60℃;圧力,300kg/cm2;エントレーナー,アセトン3%; CO2量,100g-CO2/g)における各リモノイドの抽出率はノミリン82.1%,オバクノン38.9%,リモニン19.1%,デアセチルノミリン13.9%であった.リモノイドグルコサイドについてはリモニングルコサイドを除き高い抽出率が得られたが,各成分の分画抽出はできなかった.

Limonoids and their glucosides in the seeds and barks of Phellondendron amurense (Kihada) were analyzed. The seeds contained limonin (1950 ppm), obakunone (20 ppm), limonin 17-beta-D-glucopyranoside (820 ppm) and obakunone 17-beta-D-glucopyranoside (1360 ppm). The barks contained limonin (6760 ppm), obakunone (1240 ppm) and nomilin (270 ppm).