海藻酸铝固定化酵母在杨梅果酒发酵中提高花色苷稳定性研究_英文_

2005, Vol. 26, No. 11 食品科学 ※工艺研究94 Aluminium (III)-Alginate Immobilized Yeasts to Enhance An- thocyanin Stability during Bayberry (Myrica rubra) Red Wine Fermentation ZHONG Rui-min1，ZENG Qing-xiao2，LIU Feng2，ZHU Ding-he1，ZHANG Wei-guo1 (1.Yingdong College of Biotechnology, Shaoguan University, Shaoguan 512005, China； 2.College of Food and Biotechnology, South China University of Technology, Guangzhou 510640, China) Abstract ：Bayberry (Myrica rubra) wine usually loses its red color, because its naturally-existing anthocyanins (CA) would be faded to a large extent during its free yeast cells fermentation (FCF). Anthocyanins are metabolically degraded by β-D- glucosidases that are widely expressed in fungi, yeasts and plants. To overcome this problem, aluminum (III) alginate-immobilized yeasts, instead of free ones, were utilized in the wine fermentation tests. Four commercially available Saccharomyces cerevisiae strains, Angel, Lalvin D254, Lalvin BM45, and Lalvin L2323 were evaluated. The CA stability in ICF (immobilized cells fermentation) of four yeast strains after 20 days was dramatically enhanced with the retaining rates as 50.0% (L.D254), 46.4% (Angel), 45.5% (L.L2323), and 42.7% (L.BM45), respectively, whereas, those corresponding values in FCF were only 22.7%, 20.9%, 24.5%, and 26.3%, respectively. The enhanced property of ICF on anthocyanins stability was due to the lower diffusion effect occurring in the compact outer layers of Al-alginate beads, the inhibition of β-D-glucosidases as ethanol to accumulate in medium, as well as the stabilization of the aluminum (III) in complex with anthocyanins. Moreover, during the early stage of the tests, ICF provided more stable fermentation rate as well as more slowly releasing of sulfur dioxide in medium, very important to the final retaining rate of CA. Key words：Aluminum (Ⅲ)-alginate；anthocyanin stability；β-D-glucosidase；bayberry (Myrica rubra) wine 海藻酸铝固定化酵母在杨梅果酒发酵中提高花色苷稳定性研究 钟瑞敏1，曾庆孝2，刘 锋 2，朱定和1，张卫国1 (1.韶关大学英东生物 工程 路基工程安全技术交底工程项目施工成本控制工程量增项单年度零星工程技术标正投影法基本原理 学院，广东 韶关 512005； 2.华南理工大学食品与生物工程学院，广东 广州 510640) 摘 要：杨梅果酒采用游离酵母发酵时，由于微生物所产生的β-D-葡萄糖苷酶对果汁中的呈色花色苷有脱糖作用， 导致其失色或降解，从而使杨梅果酒失去鲜艳的红色。为了解决这一问题，本研究采用海藻酸铝固定化酿酒酵母 (商品名分别为安琪，Lalvin D254, Lalvin BM45, and Lalvin L2323)代替游离酵母发酵杨梅果酒，对四种固定化酿酒 酵母的花色苷稳定作用进行了试验。结果表明四种酵母的海藻酸铝固定化发酵均表现出明显的护色效果，经过20d 发酵后，呈色花色苷保存率分别为50%(D254)、46.4%(安琪)、45.5%(L2323)、42.7%(BM45)，而在相应品种的 酿酒酵母的游离细胞发酵时，保存率仅分别为22.7%、20.9%、24.5%、26.3%。这种保护作用主要来自于海藻 酸铝固定化细胞珠致密外层的物质低扩散性、后期酒精对的抑制作用以及形成铝离子对花色苷的稳定作用。另外， 在发酵前期，海藻酸铝固定化酵母的平稳发酵速度和慢速消耗酒醪中的二氧化硫对最后的呈色花色苷保存率也有重 要意义。 关键词：海藻酸铝；花色苷稳定性；β-D-葡萄糖苷酶；杨梅果酒；酿酒酵母 中图分类号：TS262.7 文献标识码：A 文章编号：1002-6630(2005)11-0094-06 收稿日期：2004-11-17 基金项目：广东省教育厅自然科学基金资助项目(312-080164) 作者简介：钟瑞敏(1967-)，男，副教授，在读博士生，主要从事食品科学方面的研究。 95※工艺研究 食品科学 2005, Vol. 26, No. 11 1 Introduction The semitropical bayberry (Myrica rubra) grows widely in China, Korea and Japan (Tao et al., 2002). The berry, which abounds in anthocyanins(CA) such as cyanidin, pelargonidin and delphinidin (Ye et al., 1994), has been mainly used for semi-dry and dry styles of fruit wines in China since the last decade. However, the bayberry wine is easy to lose its CA through the extraction treatment during its fermentation. For example, in our previous work, the bayberry wine lost its CA to a great extent during the primary stage of FCF (free yeast cells fermentation), and the color quality in the resultant wine was greatly affected (Zhong et al., 2003). CA degradation is due to complicated causes in red fruits and wines. One of the reasons is that some of β-D- glucosidases may act as anthocyanin-β-D-glucosidases (or anthocyanases) by breaking the linkage between the glu- cose and the anthocyanidin moieties, therefore, the corre- sponding anthocyanindins will spontaneously be converted to brown or colorless compounds (Blom, 1983; Paloma et al., 2000). But β-D-glucosidases are usually used for enhance- ment of wine flavor by hydrolyzing of terpenyl-β-D-glu- cosides into terpenols (Marie-Paule et al., 1998). These en- zymes are widely expressed in plants, fungi (such as As- pergillus niger) and yeasts (Zeng et al., 1998; Marie-Paule et al., 1998; Paloma et al., 2000). However, in red fruits and wines, fungal and yeast (especially Candida and Hanseniaspora genera) β-D-glucosidases as extracellular enzymes have been described as being responsible for in- ducing loss of colour (Manzanares et al., 1999; Sánchez- Torres et al., 1998). Although Saccharomyces cerevisiae strains are not considered as a good producer of extracellu- lar β-D-glucosidases (Mateo et al., 1997; Paloma, 2000), they also cause most of the young bayberry wine CA discolora- tion at the first stage of free cell fermentation (FCF) due to the microbial metabolisms. It has been reported that the immobilized cell systems can influence yeast metabolism (Melzoch et al., 1994; Russell, 1992; Rhiel et al., 2002). Alginate has often been investigated as a suitable immobilizing material that can restrict the diffu- sion of various solutes, such as proteins, sugars and salts, in and out of the gel (Amsden, 1999; Eric et al., 2001). Algi- nate-immobilized yeast can reduce the diffusion coefficients of both yeast enzymes and large molecular colorants out and into of the gel. This suggests that anthocyanins may be protected more or less due to the restriction caused by immobilization. In the present study, Al-alginate beads in good strength, high density and low friability were used to immobilize yeast cells during the first stage of bayberry wine fermentation. The CA stability of young bayberry wine inoculated by four commercially available yeasts of Saccharomyces cerevisiae strains was evaluated both in FCF and ICF tests. 2 Materials and Methods 2.1Yeast strains A total four active dry yeasts belonging to Saccharomy- ces cerevisiae strains were used. Lalvin D254, Lalvin BM45, and Lalvin L2323 were purchased from LALLEMAND S.A. (Shanghai, China), and Angel from Angel Yeast Co. (Hubei province, China), all yeast strains were prepared according to manufactures' recommendation. 2.2Preparation of must 'Biji' bayberry must (pH3.5, containing 8.9mg/L free SO2) was from Yongchen Bayberry winery (Zhoujiang province, China) in 2002. The must was used after being adjusted with sugar to 26.0%(W/V) total solid concentration and then sterilized at 85℃ for 15min. 2.3Immobilization of cells Each of the four active dry yeast strains (3g) was suspended in 50ml of a 4% sterile sugar solution and incu- bated at 28℃ for 1h, then the suspension was mixed with 250ml of a 4% sterile alginic acid sodium salt solution. The mixture was injected with syringes (#7) drop by drop into a 1000ml sterile 2% CaCl2 solution for 2h while stirring continuously. The beads (2～3mm diameter) were washed with de-ionized water three times and then hardened in 1000ml sterile 1% Al2(SO4)3 solution at 4℃ for 24h, eventually washed three times with de-ionized water again. The Al-algi- nate beads without yeast cells also were prepared according to the above procedure as control. The two kinds of beads were both divided in three batches and directly used for wine making. 2.4Fermentation conditions 2.4.1Free cells fermentation The fermentation was carried out in triplicate with 4-litre hydroseal flasks for each yeast strain. Two litres of sterile bayberry juice and 80g Al-alginate beads without yeast cells were added into flask. The medium was inoculated with 1g dry yeast which was first activated at 28℃ for 1h in 30ml 4% sterile sugar solution. The flask was incubated at stationary 20℃. 2005, Vol. 26, No. 11 食品科学 ※工艺研究96 2.4.2Immobilized cells fermentation In the case of immobilized cells fermentation, the condi- tions were same as those of free cells fermentation except for inoculum form. Each batch medium was inoculated with 100g of Al-alginate beads with entrapped cells of the four yeast strains. 2.5Analytical assays At 24h intervals, proper quantities of fermentation fluid samples were removed from the flasks and analyzed. Ab- sorption spectra were recorded with an UV- spectrophotom- eter (916, GBC Co., Australia) fitted with quartz cells. Every sample was filtered with an injector microfilter (0.22μm pore- size cellulose acetate membrane) prior to absorption spectra assay. The CA measurements imitated the well-established spectrophotometric methodology for making wine as de- scribed by Arnous (Arnous et al., 2002). The bayberry wine sample was placed in a 0.2cm path-length quartz cuvette, and the absorbance was measured at 520nm (A520). Follow- ing this, 0.02ml of a 20% sodium metabisulphite solution was added, the sample was mixed well and after 1min the absor- bance was read at 520nm (A520 SO2). Wine (0.02ml) was mixed with 0.98ml 1 N HCl solution (dilution 1:50) in a 1.5ml Eppendorf tube, vortexed and allowed to stand for 180 min at room temperature. The absorbance was read at 520nm (A520 HCl), using a 1.0cm path-length cuvette. For the blank, 0.02ml of a 12% ethanolic solution was used instead of wine. Absorbance readings were corrected according to the dilu- tion factor. The total anthocyanins and colored anthocya- nins were calculated according to the following formula: Total anthocyanins (TA) (mg/L) = 20 × [A520 HCl － (5/ 3)×A520 SO2] Coloured (ionised) anthocyanins (CA) (mg/L) = 20 × (A520－A520 SO2). Acidity was determined by using acid-base titration method (as citric acid). Alcohol concentrations were deter- mined by using a Gay-Lussac Alcoholmeter. Because samples ethanol was not extracted through distillation, apparent re- sidual soluble solid concentration (SSC) was determined by a hand-refractometer (ATAGO N-1α, Japan). All analyses were run in duplicate, unless specified, and values averaged. 3 Results and Discussion 3.1Fermentation parameters of young bayberry wine In ICF of four yeast strains, Al-alginate beads would decrease SSC of bayberry must by 2% due to the dilution effect of beads free water. In order to compensate for the free water effect between FCF and ICF of each yeast strain, proper amount of Al-alginate beads without yeast cells was added into each FCF medium. For both FCF and ICF processes, the fermentation time lasted for 20 days in order to investigate the changes of CA efficiently. The results were summarized in Table 1. Generally, the total acidity of bayberry must was higher than that of grape must, so this resulted in decreasing the fermentation rate of young bayberry wine. There was no obvious change be- tween the total acidities of FCF and ICF samples in both processes, but the ethanol concentrations of ICF were slightly lower than those of FCF for all strains. The λmax of all samples brought hypsochromic effect from 4 to 15nm and the shift number of FCF samples was larger than that of ICF (as also shown in figure 1). The hypsochromic shift of λmax indicated that anthocyanins with longerλmax were faded a lot, or anthocyanin-anthocyanin complexes were destructed. Furthermore, the absorbance values at λmax decreased dra- matically by more than 70% for all young bayberry wine of FCF. Because the TA and CA of bayberry must were just about 20mg/L and 12 mg/L, respectively, which is much less than those of aged grape wine (TA≈165.7mg/L and CA≈ 13.4 mg/L, on average respectively) (Arnous et al., 2002), the large loss of CA in young bayberry wine directly led to low color quality of end products. Due to their high reactivity, anthocyanins readily de- grade and form colourless or undesirable brown-coloured compounds. Many exterior factors affected the stability of anthocyanins during wine making, including temperature, light, pH, oxygen, and enzymes (Mazza & Miniati, 1993; Bol ǐvar et al., 2004). The effect caused by the former two factors was negligible during bayberry must fermentation because of the incubation media located in low temperature and dark conditions. Oxidations on CA generally caused brown poly- meric colorants formation (usually showed at absorbance 420nm) in wine aging stage (Gómez, 1995). However, as shown in table 1, most of CA were authentically faded ac- cording to the λmax and absorbance values of young bay- berry wine, which indicated that oxidation was also not the main destructive factor. pH was an important factor which affected the color change of anthocyanins (Brouillard et al., 1997), but the pH of bayberry must during fermentation steadily fixed in 2.9～3.1, a range which kept the light red color of bayberry juice as control. Therefore, the most important reason that the bayberry CA was faded greatly within several days of incubation was 97※工艺研究 食品科学 2005, Vol. 26, No. 11 Sample Total acidity (g of citric acid L-1) Ethanol concen. (% V/V)λ max(nm) Absorbance(λmax) Absorbance(420nm) Bayberry must 9.3 -- 526 2.86 1.10 Angel (FCF) 9.4 11.9 511 0.63 0.37 Angel (ICF) 9.4 11.0 521 1.20 0.58 L. D254 (FCF) 9.6 12.4 512 0.68 0.43 L. D254 (ICF) 9.4 11.6 522 1.23 0.58 L. BM45 (FCF) 9.8 12.6 512 0.82 0.47 L. BM45 (ICF) 9.3 11.6 520 1.08 0.51 L. L2323 (FCF) 9.5 11.4 513 0.74 0.41 L. L2323 (ICF) 9.3 11.1 521 1.12 0.50 Table 1 Fermentation parameters obtained in young bayberry wine fermentations with four yeast strains likely related to the metabolism of yeasts. Paloma et al. (2000) reported that many wine yeasts expressed anthocyanin-β- D-glucosidase activity during fermentation, and only yeast species belonging to the genera Dekkera, Rhodotorula and Schizosaccharomyces did not produce the enzyme. Al- though Saccharomyces cerevisiae strains were not recog- nized as being a good producer of extracellular β-D-glucosi- dases (Paloma et al., 2000), the obvious force of destruction of CA from yeast metabolism at the first stage of bayberry wine making was very strong. 3.2The effect of color preservation in ICF The change patterns of fermentation rate of CA both in FCF and ICF for the four yeast strains were shown in Figure 2. In ICF processes, all four yeast strains except for L.BM45 started fermentation on the sixth day, about two to four days later than FCF processes. This suggested that the restriction effect on metabolism of yeasts caused by lower diffusion coefficients of solutes in Al-alginate beads was also strong. It was well known that sulfur dioxide in must would depress the activity of yeasts in the first several days of brewing (Iconomopoulou et al., 2002; Kourkoutas et al., 2003). When compared with the changed amplitude of CA both in ICF and FCF media, the immobilized yeast cells slowly depleted sulfur dioxide but efficiently reproduced CA from colorless bisulfite- adducts. In the case of FCF, both sulfur dioxide and released CA were demolished violently within the next six days. As fermentation promoting, the released CA in ICF media also faded sharply at the accelerated stage of fermentation. Along with ethanol accumulation, metabolism of yeast was de- pressed gradually and the CA increased slightly due to trans- formation from colorless bisulfite-adducts. McMahon et al (1999) stated that grape β-glucosidases could exhibit a 60% loss of activity at ethanol concentrations of 3.5%. Mateo and Stefano (1997) also reported that Saccharomycesβ-glu- cosidase was inhibited by about 50% with 5% ethanol in the medium. The CA in bayberry wine stopped to be faded at final stage of fermentation should be partly benefited from this inhibition. The CA stability in the four Al-alginate immobi- lized yeast strains fermentation after 20 days was dramati- cally enhanced with the residual rates of 50.0% (D254), 46. 4% (Angel), 45.5% (L2323), and 42.7% (BM45), respectively, whereas, those corresponding values in the free cells fer- mentation were only 22.7%, 20.9%, 24.5%, and 26.3%, respectively. Anyway, the metabolism of yeast cells immobi- lized in Al-alginate was different from that of free yeast cells. The enhanced property of Al-alginate immobilized yeast on anthocyanins stability also was related to the pro- tective effect from the Al3+ in gel beads. Elhabiri et al (1997) reported that the ability of natural anthocyanins to form stable complexes with small highly charged metal ions such as Al3+ and Fe3+, and these metalloanthocyanin complexes could strengthen the pigment-copigment interaction leading to bathochromic shift and higher stability. In order to investi- gate whether possible free Al3+ diffused from gel beads contributed to such stable effect on anthocyanins, all young 2005, Vol. 26, No. 11 食品科学 ※工艺研究98 bayberry wine samples were scanned with UV- spectropho- tometer from 380 to 650nm. Figure 2 showed that all λ max of samples did not bring bathochromic shifts. But the Al3+ cations bonded with the alginate gel likely relocated and yielded aluminum (Ⅲ)-anthocyanin complex because the beads discovered were being red dyed after inoculation. 3.3Conclusions Compared to ICF process, the residual CA of young bayberry wine samples inoculated with four Saccharomyces cerevisiae strains decreased dramatically by more than 70% in FCF processes, and that directly led to low color quality of the end products. Although Saccharomyces cerevisiae strains were not recognized as good producers of extracellu- larβ-D-glucosidases, many other fungal and yeastβ- glucosidases were not inhibited by the concentrations of ethanol in table wine (McMahon et al., 1999). Among the documented species showing ethanol-stable glycosidases were Hanseniaspora vineae (Vasserot et al., 1989), Dekkera intermedia (Blondin et al., 1983), and Candida molischiana (Gonde et al., 1985). In this research, the CA degradation rate from the yeast metabolism at the first stage of bayberry wine making in FCF was very rapid. However, In ICF processes, immobilized yeast cells slowly depleted sulfur dioxide, but efficiently reproduced CA, which once was bleached by sulfur dioxide. This indicated that the restriction effect caused by lower diffusion coeffi- cients of solutes in aluminum (Ⅲ) alginate beads affected the metabolism of yeast cells. The CA stability in the four alumi- num (Ⅲ) alginate-immobilized yeast strains fermentation af- ter 20 days was greatly enhanced with the residual rates of 50.0% (D254), 46.4% (Angel), 45.5% (L2323), and 42.7% (BM45), respectively, whereas, those corresponding values in the free cells fermentation were less than 30% for all strains. The enhanced property of Al-alginate immobilized yeast on anthocyanins stability was due to the lower diffu- sion of the compact outer layers of Al-alginate beads, the inhibition onβ-glucosidases as ethanol accumulation in medium, and the more stable complex of aluminum- anthocyanins. Moreover, compared to FCF, ICF could pro- vide more stable fermentation rate during the early stage of fermentation. It also retarded the releasing of sulfur dioxide in medium, which was very important for the final residual of CA. However, we hoped that this work served as a motivation for seeking for exploring the details of mechanism about rapid discoloration during the wine making for some sorts of spe- cific red fruits abounded in anthocyanins, and eventually for 99※工艺研究 食品科学 2005, Vol. 26, No. 11 improving process and sensory quality of products. The further work on ICF effect was undertaken in our lab. Referen ces: [1]Amsden, Diffusion characteristics of calcium alginate gels[J]. Biotechnology and Bioengineering, 1999, 65(5): 605-610. [2]Arnous A M, K Panagiotis. Anthocyanin composition and colour characteristics of selected aged wines produced in greece[J]. Journal of Wine Researck, 2002, 13(1): 23-34. [3]Blom H. Partial characterization of a thermostable anthocya- nin-b-glycosidase from Aspergillus niger[J]. Food Chemistry, 1983, 12: 197-204. [4]Blondin B, R Ratomahenina, A Arnaud, et al. Purification and properties of the β-glucosidase of a yeast capable of fer- menting cellobiose to ethanol[J]. Microbiol Biotechnol, 1983, 17: 1-6. [5]BolǐVar A C, C Luis. Stability