番石榴揮發性香氣化合物分析之研究

Abstract

第一部分：台灣產番石榴果實香氣特性之研究 番石榴（Psidium guajava L.）為桃金孃科番石榴屬之長綠灌木或小喬木，原產於中美洲，目前廣泛栽種於熱帶與亞熱帶地區。由於番石榴品種甚多，台灣產之中山月拔具有甚佳的加工特性及特殊香氣，適合果汁加工為本省生產番石榴果汁之主要原料。本研究以固相微萃取法（solid-phase microextraction, SPME）或水蒸氣蒸餾溶劑萃取方法結合氣相層析儀（gas chromatography）或氣相層析香氣描述法（GC-Sniffing）及氣相層析質譜儀 （gas chromatograph-mass spectrometer, GC-MS）進行台灣產番石榴揮發性成分、色澤及特性之研究，主要研究內容為： 1. 不同成熟度中山月拔果實後熟時間其色澤及揮發性成分之變化。 2. 全果及切塊中山月拔揮發性成分之比較。 3. 不同成熟度中山月拔果實揮發性成分之差異。 4. 加熱溫度及時間對番石榴果汁揮發性成分之影響。 5. 中山月拔揮發性成分及香氣特性描述。 6. 不同品種番石榴未熟果之揮發性成分分析。 結果顯示，不同後熟時間之未熟果，Hunter’s之 L值與b值隨後熟時間的增加而提高；熟果與過熟果其Hunter’s之L值與b值有先上升後下降趨勢；Hunter’s 之a值部份，未熟果、熟果與過熟果隨不同後熟時間的增加，呈現先上升後下降再上升的趨勢。未熟果之主要揮發性酯類成分為cis-3-hexenyl acetate，cis-3-hexenyl acetate、hexyl acetate 及ethyl hexanoate，為熟果與過熟果主要酯類成分， cinnamyl acteate只在過熟果中檢測出。成熟時以cis-3-hexenyl acetate最先生成，methyl butanoate及ethyl acetate在後熟晚期生成。 切塊之中山月拔，未熟果含75.96% sesquiterpene，但成熟時含量劇減，過熟果只含7.73% sesquiterpene，未成熟番石榴主要香氣來自sesquiterpene、1,8-cineole及monoterpene。 六種不同成熟度之中山月拔果實，其酯類化合物之變化以ethyl hexanoate、cis-3-hexenyl acetate及hexyl acetate三種化合物含量變化最明顯。α-pinene、1,8-cineole及β-caryophyllene等成分則隨成熟度提高有下降趨勢。 在是否調整pH值及不同加熱溫度與時間之處理，對番石榴果汁揮發性影響試驗中，發現中山月拔果汁以原汁80oC加熱5 min後揮發性成分總量最高，調成pH 3.60果汁與原汁皆以80oC加熱10 min後揮發性成分總量最低。珍珠拔果汁以調成pH 3.60果汁經90oC加熱1 min揮發性成分總量最高，以80oC加熱10 min揮發性成分總量最低。 在中山月拔揮發性成分及香氣描述實驗中，乃利用水蒸氣蒸餾-溶劑萃取法，結合GC及GC-MS分析與鑑定中山月拔之揮發性分析。結果共鑑定出65種揮發性成分。主要成分為α-pinene、1,8-cineole、β-caryophyllene、nerolidol、globule、六碳醛、六碳醇及六碳酯。以氣相層析香氣描述法分析出六碳醛、六碳醇、六碳酯及1,8-cineole 為中山月拔獨特香氣。 分析六品種番石榴未熟果之揮發性成分的研究，乃利用SPME萃取法結合GC及GC-MS分析及鑑定六種不同品種番石榴之青果，共鑑定出35種揮發性成分，在六品種番石榴中，未熟果揮發性成分以β-caryophyllene（47.74-58.28%）及aromadendrene（7.11-14.58%）所佔百分比最高。而品種間以中山月拔含高百分比之cis-3-hexenyl acetate、1,8-cineole 及allo-ocimene等成分；珍珠拔含高百分比之limonene及α-copaene等成分。

第二部分：台灣產番石榴葉香氣特性之研究 番石榴葉（Psidium guajava L. fam. Myrtaceae），台灣常用來作為治療糖尿病之民間藥，番石榴葉在其他國家亦廣泛用來治療咳嗽、腹瀉、抗菌與胃腸等疾病。本研究針對台灣產不同品種番石榴葉之揮發性成分，萃取與區分物進行分析與鑑定，並對其揮發性成分進行氣相層析嗅聞（GC-sniffing）法分析，以期望獲得更多的有關番石榴葉所含揮發性成分之資訊，以利擴大其應用價值。其中主要研究內容為： 1. 台灣產六種番石榴葉揮發性成分之研究 2. 綠色及黃色番石榴葉色澤及揮發性成分之比較 3. 不同萃取方法對世紀拔葉揮發性成分之影響 4. 不同蒸餾時間對世紀拔葉蒸餾液區分物揮發性成分之影響 5. 世紀拔葉均質液調成不同pH值對其精油收率之影響 6. 中山月拔葉精油香氣特性描述之研究 7. 番石榴葉精油用於香料配方調配與其品評 六品種不同品種之番石榴葉製得精油其收率分別為：中山月拔1.83 ± 0.40 g/kg、世紀拔葉片1.25 ± 0.33 g/kg、梨仔拔1.38 ± 0.35 g/kg、紅拔1.50 ± 0.38 g/kg、珍珠拔1.03 ± 0.25 g/kg及水晶拔1.08 ± 0.28 g/kg。 六品種番石榴葉之精油以直接注射-氣相層析法（DI-GC）共鑑定出69種揮發性成分，包括了1種含硫化合物、3種醇類、 3種醛類、4種酯類、1種酮類、13種萜醇類、4種萜酯類、 2種萜類氧化物、1種萜酮類及36種萜類碳氫化合物及1種雜環類化合物。其中中山月拔與世紀拔主要揮發性成分皆為α-pinene、1,8-cineole及β-caryophyllene；梨仔拔與紅拔主要揮發性成分皆為1,8-cineole、β-caryophyllene及aromadendrene；珍珠拔主要揮發性成分為α-copaene、β-caryophyllene及caryophyllene oxide; 水晶拔主要揮發性成分為α-copaene、β-caryophyllene及aromadendrene。 六品種番石榴葉之精油以SPME-GC共鑑定出61種揮發性成分包括了1種含硫化合物、1種醇類、 3種醛類、3種酯類、1種酮類、10種萜醇類、4種萜酯類、 2種萜類氧化物、1種萜酮類及34種萜類碳氫化合物、1種雜環類化合物。其中中山月拔與世紀拔主要揮發性成分為1,8-cineole、α-pinene及β-caryophyllene；梨仔拔與紅拔主要揮發性成分皆為1,8-cineole、β-caryophyllene及aromadendrene；珍珠拔主要揮發性成分為limonene、α-copaene及β-caryophyllene; 水晶拔主要揮發性成分為1,8-cineole、α-copaene、β-caryophyllene。 不同成熟度番石榴葉揮發性成分之變化以黃葉揮發性總量較高，綠葉中hexanal成分比黃葉多約10倍以上，且單萜烯類含量比黃葉多，倍半萜烯類則黃葉含量較多。 世紀拔葉揮發性成分以水蒸氣蒸餾-溶劑法萃取比水蒸氣蒸餾法萃取總量較高。 在不同加熱時間對世紀拔揮發性成分影響試驗中以α-pinene、1,8-cineole及β-caryophyllene等成分變化最明顯，α-pinene與β-caryophyllene隨蒸餾時間的增加其含量隨之上升；1,8-cineole則隨蒸餾時間的增加其含量下降。 中山月拔葉之精油共鑑定出60種揮發性成分，揮發性總量為 1752.24 ± 235.48 mg/kg，主要揮發性成分為α-pinene、1,8-cineole、β-caryophyllene、aromadendrene。以GC-O分析，結果指出六碳醛及醇、α-pinene、1,8-cineole及萜烯類提供了的番石榴葉精油之主要香氣（主要為青香與木香）。 經水蒸氣蒸餾萃取之精油與香料單體混合成之天然香料添加於芭樂果汁，經品評後，喜好性與接受性都比未添加精油者喜好性高。 第三部份：番石榴及羅勒葉之組織培養 中文摘要 本研究以番石榴（Psidium guajava L.）及羅勒（Ocimun basilicum L.）之嫩葉為培植體，誘導癒合組織及建立懸浮細胞，並利用固相微萃取法（Solid-phase microextraction, SPME）結合GC及GC-MS分析，鑑定其懸浮細胞內揮發性之二次代謝物成分。 結果得知，在誘導癒合組織之部份，番石榴葉之嫩葉在黑暗下以含有0.5 mg/L BA 及1 mg/L IBA之1/2 MS固體培養基的誘導效果最佳，其誘導率可達70%；羅勒葉之嫩葉在黑暗下以含有0.1 mg/L Kinetin及1 mg/L 2,4-D之MS固體培養基的誘導效果最佳，其誘導率高達95%。 分別將兩種樣品誘導所得之癒合組織進行懸浮細胞培養，觀察細胞生長情形，經震盪培養30天後，再經靜置培養30天後，以SPME萃取其揮發性成分，並配合GC及GC-MS分析及鑑定其揮發性成分，結果顯示：番石榴葉之懸浮細胞未檢出揮發性成分；而羅勒葉細胞則可檢測出8種微量揮發性成分，其主要成分為limonene。

Abstract (Part1) Guava, Psidium guajava L. (Myrtaceae), which has an unique quince and banana-like odor, is a native to Central America. It is frequently cultivated as a food for its pleasant fruit that also was used in juice processing. Today, the trees can be found cultivated or growing wild in nearly Mesoamerican geographical area, all the countries of the Tropical World Belt, from the West Coast of Africa to the Pacific Region, including India, China and Taiwan. SPME or steam distillation-solvent extraction method was used for the isolation of volatile compounds of guava samples. The volatile compounds were then identified qualitatively and quantitatively by GC and GC-MS analyses. GC-sniffing was used to describe guava fruits aroma characterizations. Six research items were carried out in this study as the followings: 1. Changes of volatile components and colors of Chung-Shan guava fruits with different maturities during ripening. 2. Comparison of volatile compounds of Chung-Shan whole guava (unripe, ripe and overripe) fruits and their cubes. 3. Comparisons of volatile components of Chung-Shan guava fruits with different maturities. 4. Effect of heating temperature and time on the volatile components in guava juice. 5. Volatile constituents of guava fruit and its odor descriptors. 6. Volatile compounds in six cultivars of Taiwan unripe guava. Changes of Hunter’s L, a, b values of the fruits were studied: the unripe fruit’s L and b values were increased during maturation. The ripe and overripe fruits’ L and b values were increased first and then decreased during maturation. The main volatile compounds identified in the unripe fruit was cis-3-hexenyl acetate. The main volatile compounds identified in the ripe and overripe fruits were cis-3-hexenyl acetate, hexyl acetate and ethyl hexanoate. Changes of esters in fruits during ripening: cis-3-hexenyl acetate was produced first, methyl butanoate and ethyl acetate produced in later period. Volatile compounds of six maturities of Chung-Shan guava fruits were compared. The results showed that esters compounds such as ethyl hexanoate, cis-3-hexenyl acetate and hexyl acetate were changed significantly among six maturities of Chung-Shan guava fruits. The contents of α-pinene, 1,8-cineole and β-caryophyllene compounds were low in unripe fruits but increased the contents during maturation. The comparision of volatile compounds contents in guava juice after heating with different times and pH values were carried out contents. In Chung-Shan guava juice, originally pH value, 80oC and heating for 5 min had the highest volatile content, while Jen-Ju guava Juice, adjust pH 3.60, 90 oC and heating for 1 min was the highest. Volatile compounds were isolated from Chung-Shan fruit by simultaneous steam distillation and solvent extraction according to the Likens-Nickerson method. Compounds were identified by capillary GC-MS and sensorially characterized by GC-sniffing. A total of sixty-five compounds were identified. The major constituents identified in the guava fruit were: α-pinene, 1,8-cineole, β-caryophyllene, nerolidol, globule, C6 aldehydes, alcohols and esters. The presence of C6 aldehydes and esters, terpenes and 1,8-cineole is thought to contribute to the unique flavor of the guava fruit. The volatile composition of six different cultivars of unripe guavas grown in Taiwan have been studied by headspace solid-phase microextraction (HS-SPME) coupled with GC and GC-MS. Totally 35 compounds were identified. including 24 terpene hydrocarbons, 2 terpene alcohols and also minor constituents of 1 alcohol, 2 aldehydes, 3 esters, 1 terpene ester and 2 terpene oxides. Although the volatile compounds composition of six cultivars were similar, β-caryophyllene (47.74-58.28%) and aromadendrene (7.11-14.58%) as the major constituent in all cultivars. quantitative differences in the composition of some constituents were observed. Chan-Shan guava contented with higher percentages of cis-3-hexenyl acetate, 1,8-cineole and allo-ocimene than other species. Abstract (part 2) Psidium guajava L. (fam. Myrtaceae) is a tree native of the Mesoamerican geographical area. The tree grows as a large spreading shrub or a small tree up to 15 m high. Guava leaves have been used to treat many ailments including cough and pulmonary disease in Bolivia and Egypt (Batick, 1984). In Mexico, guava leaves are extensively used to stop diarrhea (Lozoya et al., 1994) and for the alleviation of gastrointestinal disorder is a common practice originally inherited from traditional Aztec medicine (Lozoya et al., 2002). In Taiwan, it’s also known that leaf can improve glucose level with type 2 diabetes. The aim of this study was using GC-injection or SPME method to evaluate volatile compounds in guava leaf samples. The volatile compounds were then identified by GC and GC-MS. GC-sniffing also was used to describe guava volatile compounds characterization. In order to expect to get more information about guava leaves and extends their application fields. The research items were as the followings: 1. Studies on volatile compounds in six Taiwan guava cultivars leaves. 2. Comparisons of color and volatile components of green and yellow guava leaves. 3. Comparisons of Shih-Chi guava leaf volatiles extracted by steam distillation and steam distillation-solvent extraction. 4. Changes of volatile components in Shih-Chi guava leaves distillates during steam distillation. 5. Effect of different pH values of Shih-Chi guava leaf macerates on their volatile components in the essential oils. 6. GC-sniffing of Chung-Shan guava leaves volatiles. 7. Guava leaf essential oil used as a raw material for flavor formulation. Leaf essential oils of six cultivars of guava were obtained by steam distillation, the yields of essential oils were: Chung-Shan guava, 1.83 ± 0.40 g/kg; Shih-Chi guava, 1.25 ± 0.33 g/kg; Li-Tzy guava, 1.38 ± 0.35 g/kg; Red guava, 1.50 ± 0.38 g/kg; Jen-Ju guava, 1.03 ± 0.25 g/kg and Shuei-Jing guava, 1.08 ± 0.28 g/kg, respectively. Using direct GC-injection to analysis six cultivars of guava leaf essential oils, totally 69 compounds were identified, including 36 terpene hydrocarbons, 13 terpene alcohols, and also minor constituents of 3 alcohols, 3 aldehydes, 4 esters, 4 terpene esters, 2 terpene oxides, 1 terpene ketone, 1 ketone, 1 sulfur compound and 1 miscellaneous compound. The main compounds identified in the essential oil of Chung-Shan guava and Shih Chi guava were α-pinene, 1,8-cineole and β-caryophyllene. The major compounds identified in the essential oil of Li-Tzy guava and Red guava were 1,8-cineole, β-caryophyllene and aromadendrene. The main compounds identified in the essential oil of Jen-Ju guava were α-copaene, β-caryophyllene. The main compounds identified in the essential oil of Shuei-Jing guava were α-copaene, β-caryophyllene. Using SPME methods to analyses six cultivars of guava leaf essential oils, totally 61 compounds were identified, including 35 terpene hydrocarbons, 10 terpene alcohols, and also minor constituents of 1 alcohol, 3 aldehydes, 3 esters, 4 terpene esters, 2 terpene oxides, 1 terpene ketone, 1 ketone, 1 sulfur compound and 1 miscellaneous compound. The main compounds identified in the essential oil of Chung-Shan guava and Shih Chi guava were α-pinene, 1,8-cineole and β-caryophyllene. The major compounds identified in the essential oil of Li-Tzy guava and Red guava were 1,8-cineole, β-caryophyllene and aromadendrene. The main compounds identified in the essential oil of Jen-Ju guava was limonene, α-copaene and β-caryophyllene. The main compounds identified in the essential oil of Shuei-Jing guava were 1,8-cineole, α-copaene and β-caryophyllene . Results in analysis of essential oils, SPME method had a higher monoterpene contents obtained than GC-injection method while which had higher sesquiterpene contents. In the analysis of six cultivars of guava leaves by using SPME methods, totally 51 compounds were identified, including 34 terpene hydrocarbons, 8 terpene alcohols, and also minor constituents of 1 alcohol, 2 aldehydes, 3 terpene esters, 2 terpene oxides and 1 terpene ketone. The main compounds identified in the leaf volatile components of Chung-Shan guava and Shih- Chi guava were α-pinene, β-caryophyllene and aromadendrene. The major compounds identified in the leaf volatile components of Li-Tzy guava were 1,8-cineole, β-caryophyllene and aromadendrene. The main compounds identified in the leaf volatile components of Jen-Ju guava were limonene, α-copaene and β-caryophyllene. The main compounds identified in the leaf volatile components of Red guava and Shuei-Jing guava were α-copaene, β-caryophyllene and aromadendrene. Volatiles components in guava leaves isolated by SPME method was thought to represent the aroma in raw leaves more truly because of less heat suffered than by the method of distillation. Comparison of raw leaves analyzed by SPME method and leaf essential oils by direct-GC, the SPME data possessed sesquiterpene hydrocarbons at a percentage nearly as high as that in the leaf essential oils. However, monoterpene alcohol and monoterpene percentages were less than that in the essential oils. It was postulated that heating decomposed componts caused by distillation during the essential oils preparation enhances the leaf with high volatile composition. The essential oil contained 2-ethyl furan, while the SPME-data did not. We speculated this resulted in the heating processing. Yellow leaf was found to have higher amount of total volatiles than that of green leaf, but hexanal content is about 10 times less than the green leaf. The yields of volatile by steam distillation-solvent extraction is higher than that by steam distillation of Shih-Chi guava leaves. α-Pinene and β-caryophyllene contents in the distillates increased with increasing time of distillation, but 1,8-cineole content decreased during distillation. Volatile compounds were isolated from guava leaves by simultaneous steam distillation and solvent extraction according to the Likens-Nickerson method. Compounds were identified by capillary GC-MS and sensorially characterized by GC-sniffing. A total of sixty compounds were indentified, total amounts of 1752.24 ± 235.48 mg/kg. The major constituents identified in the guava fruit were: α-pinene, 1,8-cineole, β-caryophyllene and aromadendrene. The presence of C6 aldehydes, α-pinene, 1,8-cineole and terpenes are thought to contribute to the unique flavor of the guava leaf essential oil. Natural leaf essential oils were used to enhance the aroma quality of guava juice effectively.

Abstract (Part 3) Guava (Psidium guajava L.) and basil (Ocimun basilicum L.) leaves were used as explants in this research. When guava leaves were cultured in half strength MS medium containing 0.5 mg/L BA and 1mg/L IBA the average callus induction rate 70% was observed whereas, average callus induction rate of 95% was obtained from basil’s young leaves were cultured in MS medium containing 0.1 mg/L Kinetin and 1mg/L 2,4-D. Suspension cell cultured was then established from those leaf –derived callus. The volatile compounds was detected after suspension cell was cultured for 30 days and then keep it in quiescent condition for 30 days by headspace solid-phase microextraction and analyzed by GC and GC-MS. Eight compounds were identified in the basil suspension of which limonene was the major compound. The volatile compounds in guava suspension cell was not detectable.