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研究生:林鵬翔
研究生(外文):Peng-Hsiang Lin
論文名稱:具抗氧化特性新式香菇柄酒之開發研究
論文名稱(外文):Development of a Novel Alcoholic Beverage with Antioxidant Properties from Shiitake Stipe
指導教授:方繼方繼引用關係
學位類別:博士
校院名稱:國立中興大學
系所名稱:食品暨應用生物科技學系所
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2010
畢業學年度:98
語文別:英文
論文頁數:177
中文關鍵詞:香菇柄含酒精性飲料酒精發酵酒精耐度自由選擇法抗氧化
外文關鍵詞:Shiitake stipeAlcoholic beverageAlcoholic fermentationEthanol toleranceFree-choice profileAntioxidant activity
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香菇為臺灣菇類中產量最高者,其產區主要落於中部地區,如台中縣及南投縣。消費者食用香菇大抵以柔軟之菌傘為主,而強韌之菇柄部位大都先剪除,乾燥後視為廉價副產品販售或丟棄。因此,菇柄之再利用可增加菇農收益,並能降低廢棄物量,進而減少環境污染。利用菇柄為材質開發具特色及功能性新產品(如酒飲料)可作為地方特產。因此,本研究在於利用菇柄萃取液為氮源,研發具特色及抗氧化功能之新式菇柄酒。
本研究首先評估四株酵母菌在含菇柄萃取液的發酵液之發酵能力及其酒精耐度。此四株菌株分別源自麵包廠、清酒廠、啤酒廠及酒精製造廠。初始發酵液成分為菇柄液(6 g/100 mL)及蔗糖(25 g/100 mL)。結果顯示,菇柄萃取液之氮量足以提供酵母菌株增殖,並促使菌株進行糖發酵及產生酒精,然而其菌株增殖率、糖利用率及酒精產量依菌株不同而異。於發酵終點,其酒精產量(g/100 mL)分別為11.0 ± 0.7(菌株BCRC 20262)、11.5 ± 0.4(菌株BCRC 22236)、9.5 ± 0.1(菌株BCRC 21686)及8.8 ± 0.3(菌株BCRC 21992)。發酵過程中,菌株對蔗糖之分解速率以BCRC 21992最快,其次為BCRC 21686,再依序為BCRC 22236及BCRC 20262。相異於蔗糖分解速率,BCRC 21992對葡萄糖之利用率最弱,其它三株菌株則對葡萄糖均有較高之利用率。另者,對果糖利用率以BCRC 20262及BCRC 22236最佳,再依序為BCRC 21686與BCRC 21992;依此結果,四株菌株中以BCRC 22236最適於作為產製菇柄酒之菌株。在酒精耐度方面,菌株之葡萄糖利用率與發酵過程之酒精產量的相關性高於其菌數增殖率;因此,評估菌株之酒精耐度或可建立一快速且簡單方法以應用於高酒精產量菌株之初步篩選。喜好度官評試驗結果顯示,菌株BCRC 22236所發酵之菇柄酒具有與台灣產白葡萄酒之相近接受度,且優於法國產製之白葡萄酒。
本研究結果亦顯示菇柄量對酒精發酵效率具有顯著性影響。發酵過程中,酵母菌增殖率不因菇柄萃取液中氮量(酵母可利用氮)高低而異。然而,酒精產量、蔗糖、果糖及葡萄糖利用率則隨菇柄用量增加而提升。達發酵終點(12天)時,添加2、4及6 (g/100 mL)菇柄用量之酒精產量(g/100 mL)分別為6.1 ± 0.3、9.1 ± 0.5 及11.5 ± 0.3。初始發酵液之色澤可能主要來自蔗糖,而其色澤亦依菇柄用量及發酵天數呈現顯著差異性。菇柄用量高者之發酵液色澤具有較低白色度及較高紅度與較高黃度。然而,發酵初期(二天後),其白色度快速增加而紅色度則明顯降低,但黃色度僅些微降低,此色澤變化與菌數增長及蔗糖快速消耗有直接關聯,推測其可能肇於蔗糖之色澤為酵母菌分解或吸收所致。本研究亦評估菇柄用量對菌株之酒精耐度影響,結果顯示,高菇柄用量可顯著增加菌株之酒精耐度(以糖利用率而言),此可能為萃取液中之酵母可利用氮量較高所致。
本研究亦進一步分析大規模製備之菇柄酒於發酵前後之成分變化及成品之官能特性,並與二種市售白葡萄酒作比較。結果顯示,相較於二種市售白葡萄酒,菇柄酒具有低酸度、高糖度、高黏度及高酒精度等特質。在色澤方面、菇柄酒之色澤較白葡萄酒深,且伴隨高紅色度及高黃色度。再者,經由發酵後,菇柄液之精氨酸及胍氨酸為菌株利用殆盡,並可能因之產生大量脯氨酸。喜好度分析結果顯示,除香氣略遜之外,菇柄酒之色澤、口感、香味及總接受度均與台灣產白葡萄酒相近,且較法國白葡萄酒佳。以自由選擇法分析菇柄酒之官評特徵,得知其較白葡萄酒具有稠度、苦味、啤酒味及香菇味。由此顯示,本研究所開發之香菇柄酒具備菇味及啤酒味等特殊風味。
本研究亦探討發酵過程及菇柄量對菇柄酒之抗氧化性變化。結果顯示,菇柄量遞增可強化菇柄酒之抗氧化成分含量及其抗氧化活性。然而,發酵初期之DPPH自由基清除力略為降低。另者,發酵作用可增強還原力及總酚量,而亞鐵螯合力及總類黃酮量於發酵初期即為菌株所降解而顯現微量。比較菇柄液與酵母萃取物為氮源於發酵前後之抗氧性,發現使用菇柄之發酵液的初始抗氧化力顯著高於使用酵母萃取物者。依同等初始氮量(酵母可利用氮)而言,發酵後菇柄酒之DPPH自由基清除力、還原力及總酚量分別為酵母萃取物者之1.76~2.08倍、1.5~1.7倍及1.25~1.40倍。由此可預期,香菇柄萃取液可作為酵母萃物之替代氮源,以提昇發酵酒之抗氧化力,且或許可因此增加飲用者體內之抗癌性及降低心血管疾病發生率。綜合言之,香菇柄萃取液能取代酵母萃取物為酒精發酵之氮源,且可開發出一種具特殊風味及功能性之酒精性飲料。


Shiitake is a major cultivated mushroom in Taiwan, especially in the central district area such as Nantou County and Taichung County. However, the cap of shiitake is highly preferred by consumers compared to the stipe, which are commonly cut off, dried and sold as cheap by-products, or even discarded due to its tough structure. Utilization of the stipe may help increase farmers’ profits, and reduce the waste handling and pollution problems associated with the waste. In addition, using shiitake stipe as a substrate to develop a novel product with unique characteristics and functionality would lead to a special product of the area. Therefore, the present study was undertaken to develop a novel alcoholic beverage from shiitake stipes extract (SSE) as a nitrogen source with unique characteristics and antioxidant activities.
Four yeast strains, isolated from bakery, sake, beer and alcohol wineries, were firstly evaluated by screening their fermentative efficiency and ethanol tolerance in must with stipe extract. The initial must was composed of SSE with a quantity of 6 g/100 mL stipe supplemented and cane sugar (25 g/100 mL). The results demonstrated that the nitrogen quantity in SSE could enable yeast cell proliferation and promote good performance of sugar utilization and ethanol production. The rates of cell growth, sugar consumption and ethanol production during fermentation was significantly dependent on strains utilized. Ethanol production (g/100 mL) of 11.0 ± 0.7, 11.5 ± 0.4, 9.5 ± 0.1 and 8.8 ± 0.3 was obtained from strain BCRC 20262, BCRC 22236, BCRC 21686 and BCRC 21992, respectively, by the end of fermentation. The sucrose depletion rate for strains was in this descending order: BCRC 21992 > BCRC 21686 > BCRC 22236 > BCRC 20262. In contrast to sucrose utilization, three strains, BCRC 20262, BCRC 22236 and BCRC 21686, possessed higher glucose utilization ability than BCRC 21992. On the other hand, strains BCRC 20262 and BCRC 22236 showed the highest fructose utilization ability, followed by BCRC 21686 and BCRC 21992. Based on the above results, BCRC 22236 among the four subjected strains might be more available to develop the shiitake stipe alcoholic beverage (SSAB). Ethanol tolerance of strains in terms of glucose utilization rate was more consistent with the extent of ethanol production during fermentation than that observed for survival rate. Therefore, the evaluation of the glucose utilization rate in external ethanol conditions might help establish a simple and quick method to screen the strains capable of high ethanol production. By a hedonic test, the SSAB fermented from strain BCRC 22236 may provide comparable consumer acceptances with a commercial Taiwanese white wine, and have a significantly higher acceptability rating than the white wine vintaged from France.
The results also indicated that the amounts of stipe supplemented significantly affected fermentation efficiency in alcoholic fermentation. Following fermentation, yeast cell proliferation did not significantly vary with the yeast assimilable nitrogen (YAN) level in SSE with various stipe quantities used. However, the ethanol production and sugars depletion including sucrose, glucose and fructose increased in proportion to the increasing quantity of shiitake stipe supplemented in SSE. The ethanol production (g/100 mL) was obtained at 6.1 ± 0.3, 9.1 ± 0.5 and 11.5 ± 0.3 at day 12 when 2, 4, and 6 g/100 mL of stipe were supplemented, respectively. The cane sugar as a sucrose source might predominantly contribute to the pigments of the must. Following fermentation, the color values of broth altered as a function of stipe quantity and fermentation time. The must with a greater quantity of stipe exhibited lower intensity of lightness, and higher intensities of redness and yellowness. However, the lightness increased and redness reduced sharply during the first 2 days fermentation. The sharp changes of lightness and redness coincided with yeast cell proliferations and sucrose depletions. It is assumed that the pigment of cane sugar was discolored by yeast through degradation and/or adsorption. In the current work, the ethanol tolerance of the subjected strain was also evaluated in broths with various quantities of stipe supplemented. The results suggested that higher quantity of stipe used in SSE might significantly enhance the strain’s ethanol tolerance regarded as glucose utilization due to the pronounced quantity of YAN. However, the ethanol tolerance as cell proliferation differed insignificantly with various amounts of stipe used, where coincided with that observed in fermentation.
The chemical properties in must and fermented broth were assessed. The sensory characteristics of SSAB in laboratory bulk-scale from strain LN1 were evaluated and compared with the two commercial white wines vintaged from Taiwan and France, respectively. The results demonstrated that the SSAB exhibited lower acidity, and higher contents of fructose, Brix degree, specific viscosity and ethanol content than those values observed for two commercial white wines. Regarding color intensity, the SSAB exhibited more darkness, redness and yellowness than the two commercial white wines. Furthermore, consumption of some amino acids such as arginine and ornithine might result in the largely increasing proline content via fermentation. The hedonic test showed that the SSAB from strain LN1 was accepted as well as the Taiwanese white grape wine, except for the aroma note, and superior to those of the white wine produced from France. By the free-choice profiling method, the characteristics of the SSAB were perceived as stickiness, bitterness, beer flavor/aftertaste, and shiitake flavor/aftertaste when compared with commercial white wine. The result indicated that SSAB exerted a combination of tastes including beer-like and mushroom.
The effect of fermentation on the antioxidant components and activities were carried out with various concentrations of stipe supplemented in must. The results indicated that the contents of total phenols and flavonoids, and the antioxidant activities significantly improved with an increasing supplemented stipe. However, a slow variation in scavenging ability with a temporal reduction during the early period fermentation was observed. In addition, the reducing power and total phenols content of the broth was enhanced significantly via fermentation. The ferrous ion chelating ability and total flavonoids content decreased rapidly to the trace levels following 2 days of fermentation. The antioxidant activities in must and fermented broth from SSE as alternative nitrogen source were also compared to those from YE. In must, SSE showed stronger activities including DPPH scavenging ability, reducing power and ferrous ion chelating ability, and higher contents of total phenols and total flavonoids than YE. Following fermentation, the scavenging abilities on DPPH radicals, reducing power and total phenols content for SSE was a 1.76-2.08-fold, 1.5-1.7-fold and 1.25-1.40-fold increasing, respectively, stronger than those measured for YE with comparable initial YAN level. We suggested that SSE might replace the conventional nitrogen source such as YE to enhance antioxidant activity in fermented alcoholic beverages, and subsequently could be capable of providing consumer with protection against cancer and cardiovascular diseases. Overall, shiitake stipe extract could be used as nitrogen source in alcoholic fermentation and develop a novel alcoholic beverage with unique characteristics and functionalities.


Contents
List of figures………………………………………………………………………………… v
List of tables……………………………………………………………………..………… viii
List of abbreviations and full names……………………………………...……………… ..xi
Abstract (in Chinese)………………………………………………………………………… I
Abstract (in English)……………………………………………………………………….. IV
1. Introduction……………………………………………………………………………... 1
2. Literature review 6
2.1. Shiitake……………………………………………………………………………….. 6
2.2. Factors influencing the quality of wine/alcoholic beverages………………………...12
2.3. Ethanol tolerance of yeast……………………………………………………………26
2.4. Antioxidant properties and measurements…………………………………………...30
2.5. Functionalities of wines……………………………………………………………...42
2.6. Aromas profile and tastes in wines……………………......…………………………46
2.7. Sensory evaluation…………………………………………………………………...48
3. Materials and methods 59
3.1. Materials……………………………………………………………………………..59
3.2. Reagents……………………………………………………………………………..59
3.3. Yeast strains………………………………………………………………………….60
3.4. Primary culture preparation………………………………………………………….60
3.5. Culture broth preparation and fermentation conditions……………………………...61
3.6. Measurement of viable yeast count………………………………………………….62
3.7. Measurement of sugar contents……………………………………………………...62
3.8. Measurement of ethanol production…………………………………………………63
3.9. Measurement of yeast assimilable nitrogen content…………………………………63
3.10. Measurement of free amino acids………………………………………………......64
3.11. Measurements of pH and titratable acidity…………………………………………65
3.12. Determination of hunter color value………..………………………………………65
3.13. Assessment of specific viscosity………………………………………………...…66
3.14. Evaluation of ethanol tolerance of yeast strains……………………………………66
3.15. Sensory evaluation (hedonic test and FCP)………………………………………...67
3.16. Total phenol content measurement…………………………………………………68
3.17. Measurement of DPPH radical scavenging ability………………………………....69
3.18. Measurement of reducing power…………………………………………………...69
3.19. Assessment of ferrous ion chelating ability………………………………………...70
3.20. Total flavonoid content measurement………………………………………………71
3.21. Statistical analysis…………………………………………………………………..71
4. Results and discussion 73
4.1. Saccharomyces strains on fermentative efficiencies 73
4.1.1. Growth profile of yeast strains during fermentation………………….............73
4.1.2. Ethanol production by yeast strains during fermentation…………….............74
4.1.3. Sugars consumption profile during fermentation…………………….............75
4.1.4. Ethanol tolerance of yeast strains…………………………………………….77
4.1.5. Consumer acceptance of SSAB by hedonic test……………………………...80
4.1.6. Conclusion of strains effect…………………………………………………...81
4.2. Effects of stipes levels on fermentative efficiencies 88
4.2.1. Yeast proliferations during fermentation……………………………………...88
4.2.2. Sugars consumption during fermentation…………………………………….90
4.2.3. Ethanol production during fermentation……………………………………...91
4.2.4. Color profiles during fermentation…………………………………………...92
4.2.5. Stipe quantities upon the ethanol tolerance…………………………………...94
4.2.6. Conclusion of stipe level effect……………………………………………….96
4.3. Quality and sensory characteristics of the SSAB 102
4.3.1. Proximal components………………………………………………………..102
4.3.2. Color properties……………………………………………………………...103
4.3.3. Free amino acids profile…………………………………………………..…104
4.3.4. Sensory characteristics………………………………………………………106
4.3.5. Conclusion of SSAB characteristics………………………………………...108
4.4. Antioxidant properties during fermentation 116
4.4.1. DPPH radicals scavenging ability…………………………………………...117
4.4.2. Reducing power….………………………………………………………….118
4.4.3. Ferrous ion chelating ability…………………………………………….…..120
4.4.4. Total phenols content………………………………………………………..120
4.4.5. Total flavonoids content……………………………………………………..121
4.4.6. Conclusion of the antioxidant properties……………………………………124
5. Future prospects…………………………………………………………………………135
6. References………………………………………………………………………………..137


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