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衡水学院
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学生姓名 : 赵金风 系 专 年 学 别 : 应用化学系 业 : 应用化学 级 : 2008 级 号 : 200840600224

指导教师 : 王淑琼

衡水学院教务处印制

原 文

题目: Concentration of ?avonoids and phenolic compounds in aqueous and ethanolicpropolis extracts through nano?ltration
Abstract Propolis has a variable and complex chemical composition with high concentration of ?avonoids andphenolic compounds present in the extract. The extract varies with the solvent used in extraction. Ethanol extracts more phenolic acid and polar compounds than water. Before their use in industry, extracts must be concentrated but the use of high temperatures can degrade some compounds. Membrane pro-cesses is an option that allows concentration at low temperatures. Nano?ltration was carried out withaqueous and ethanolic extracts and each extract results in two distinct fractions: permeate and retentate. The capacity of the membrane to retain the compounds was veri?ed by spectrophotometric analysis: for aqueous solution, the membrane retained around 94% of the phenolic compounds and 99% of the ?avonoids, while for the ethanolic solution these values were 53% and 90%, respectively. Ferulic acid retentionindex was 1.00 and 0.88 to aqueous and ethanolic solutions, respectively. Thus, the nano?ltration processshowed high ef?ciency in the concentration of propolis extracts. 1 Introduction Over the last few decades, interest in functional foods has beengrowing fast, leading to the discovery of new functional components or processes that can improve food processing, as well as products that may help to retard aging or avoid diseases. In this context, bee products have gained the attention of consumers and researchers, due to their chemical compositions and functional properties. Propolis is one of the bee products with functional properties, but it cannot be consumed as a food because it is a resinous substance. It is prepared from the buds and exudates of certain trees and plants. These substances are transformed by the addition of wax and the enzyme glucosidase present in the bee saliva in order to form propolis (Bankova et al., 2000; Park et al., 1998). The product obtained is used by honeybees to protect the hives against invaders and contamination, to seal holes and to maintain the temperature. Some important characteristics have been reported for this
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substance, such as anti-microbial and antioxidant effects, anesthetic properties and others. Due to these characteristics, which can bring health bene?ts, propolis is considered a functional ingredient and is used in food, beverages, cosmetics and medicine to improve health and prevent diseases (Burdock, 1998; IFIC, 2009). There are over 150 constituents in propolis, including polyphenols, terpenoids, steroids and amino acids. Flavonoids are one of the most important groups and can represent around 50% of the propolis contents,depending on the region where it is collected, since its characteristics is in?uenced by plants and weather (Krell, 1996; Park et al., 1998). Kumazawa et al. (2004) tested the antioxidant activity of propolis from various geographic origins and showed different activities for each sample. Other studies indicated that the propolis from Europe and China contained many kinds of ?avonoids and phenolic acids, whereas the Brazilian samples had more terpenoids and prenylated derivatives of p-cumaric acid (Bankova et al., 2000). Finally, each combination of compounds in the propolis of acertain origin can represent speci?c characteristics in the ?nal product. The most common propolis extracting process uses ethanol as the solvent. However, this has some disadvantages such as the strong residual ?avor, adverse reactions and intolerance to alcohol of some people (Konishi et al., 2004). Researchers and industry are interested in producing a new type of extract with the same compounds extracted by the ethanolic method, but without the disadvantages. Water has been tested as the solvent, but resulted in a product containing less extracted compounds (Park et al., 1998). Konishi et al. (2004) tested water with a combination of some tensoactive compounds to replace part of the alcohol used in propolis extraction and all the tests were ef?cient in extracting it, and the product showed good anti-microbial activity. Depending on the application, the solvent in propolis extracts must be reduced or eliminated. The processes that are used today, as lyophilization, vacuum distillation and evaporation, have some disadvantages like the use of high temperatures and high energy consumption. Lyophilization requires large amounts of energy, since the sample needs to be maintained at ?20 ° for at least 24 h, and energy is also required for the sublimation of the C solvent used during preparation of the extract. Moreover, the method often requires a previous stage of concentration, maintaining the product at 70 ° until part of the solvent is C evaporated.

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Vacuum distillation requires great amounts of energy to generate the vacuum, and can lead to loss of compounds of low molecular weight, which can be removed together with the solvent evaporated in the system. Evaporation maintains the extract underheating at 70 ° C, until all the solvent is removed. This process, in addition to the high demand for energy, can degrade the ?avonoid and phenolic compounds in propolis, due to the temperature used. However, it is the process that gives greater ease of implementation in companies due to low cost on the equipment required compared with the previous cases. The use of membrane concentration processes has been growing due to certain advantages, such as: low temperatures, absence of phase transition and low energy consumption (Matta et al., 2004). This procedure is based on the principle of selective permeation of the solute molecules through semi-permeable, polymeric or inorganic membranes. The driving force for mass transfer across the membrane in most membrane processes, such as a micro?ltration, ultra?ltration, nano?ltration and reverse osmosis is mechanical pressure (Maroulis and Saravacos, 2003). Nano?ltration is a unit operation that permits many applications, such as solvent recovery from ?ltered oil, exchange of solvents in the chemical industry (Geens et al., 2006), concentration and puri?cation of ethanolic extracts of xantophylls, which is important in both the pharmaceutical and food industries (Tsui and Cheryan, 2007) and in wine concentration (Banvolgyi et al., 2006), as well as in juice concentration (Vincze et al., 2006) in the food industry. The objective of this study was to investigate the membrane concentration of propolis extracts using water and ethanol as the solvents, exclusively, verifying the quality of the concentrated products in terms of the retention of ?avonoids and phenolic compounds during processing. The process was evaluated according to the permeate ?ux, in?uence of temperature and pressure and concentration factor. The results obtained for each solution were compared to verify the viability of developing a new propolis product, based on water as the solvent. 2. Materials and methods 2.1. Propolis Raw propolis was obtained from Apis mellifera beehives in the State of S? Paulo, o Brazil, and was acquired in a single batch, in order to minimize the variability associated with the vegetation used for its production and the weather conditions. It was stored under

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refrigeration (4 ° until use in the preparation of extracts. C) The propolis produced in this region is characterized as group 12 (Brazil has 12 different groups of propolis, with distinct characteristics) and presents a great amount of soluble substances, antimicrobial activity against Staphylococcus aureus and Streptococcus mutans and greater anti-in?ammatory activity than samples from other parts of the country, which can be associated with the higher concentrations of ?avonoids and phenolic compounds found in this group (Park et al., 2002). The ethanolic propolis solution was prepared from crude propolis previously comminuted in a bench blender with a 500 W motor, homogenized, weighed on a semi-analytical balance and mixed with 80% ethanol. The mixture was kept at room temperature for 7 days and manually stirred once a day. After this period, the sample was centrifuged (Beckman – Allegra 25-R, Beckman Coulter, German) at 8800 g for 20 min. The supernatant was ?ltered and refrigerated for 3 h at 4 °C and then ?ltered again for wax removal. Finally, the esulting extract was stored at room temperature in the dark. Preparation of the aqueous solutions followed the same procedures, using deionized water. Each solution was prepared in a proportion of 20% propolis and 80% solvent. Both extracts were evaluated with respect to their ?avonoid and phenolic compounds contents, to be compared with the concentrated products. 2.2. Determination of total ?avonoids The total ?avonoid content of the propolis solutions was determined by the aluminum complexation method (Marcucci et al., 1998). In this procedure, the extracted solutions were diluted in the proportion of 1:10 (0.5 mL) and mixed with 0.1 mL of 10% aluminum nitrate, 0.1 mL of 1 mol/L potassium acetate and 4.3 mL of 80% ethyl alcohol. The samples were kept at room temperature for 40 min and the absorbance read at 415 nm. Quercetin was used as the standard to produce the calibration curve. The mean of three readings was used and the total ?avonoid content expressed in mg of quercetin equivalents (mg/g). 2.3. Determination of the phenolic compounds The polyphenols in the propolis solutions were determined by the Folin–Ciocalteau colorimetric method (Kumazawa et al., 2004). According to this procedure, the extracted solution was previously diluted in the proportion of 1:10 (0.5 mL) and then mixed with 0.5 mL of the Folin–Ciocalteau reagent and 0.5 mL of 10% Na2CO3. The absorbance was read

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at 760 nm after 1 h of incubation at room temperature. Gallic acid was used as the standard to produce the calibration curve. The mean of three readings was used and the total phenolic content expressed in mg of gallic acid equivalents (mg/g). 2.4. HPLC determination The compounds present in the initial extract, permeate and concentrated products, were determined by HPLC as described by Parket al. (1998). Three hundred microliters of each solution were injected into a liquid chromatograph (Shimadzu, Tokyo, Japan) connected to a diode-array detector at 260 nm. The mobile phase was water/acetic acid (19:1, v/v) (solvent A) and methanol (solvent B), with a constant ?ow rate of 1 mL/min. The gradient started at 30% solvent B, passing to 60% at 45 min, 75% at 85 min, 90% at 95 min and back to 30% at 105 min. The column was maintained at a constant temperature of 30 ° and the C chromatograms processed using the computer software Chromatography Workstation (Shimatzu Corporation, Tokyo, Japan). The initial and concentrated samples were diluted in 1.5 mL of distilled water and the permeate sample was injected without dilution. The following authentic standards of phenolic acids and ?avonoids (Extrasynthese, Genay, France) were examined: q-cumaric acid, ferulic acid, cinnamicacid, gallic acid, quercetin, kaempferol, kaempferide, apigenin, isorhamnetin, rhamnetin, sakuranetin, isosakuranetin, hesperidin, hesperetin, pinocembrin, chrysin, acacetin, galangin, myricetin, tectochrysin and artepillin C, as they correspond to the most usual compounds present in propolis. 2.5. Membrane concentration In this study, the propolis extracts were concentrated using a tangential ?ltration system on a pilot scale, with a nano?ltration membrane as seen in the schematic diagram shown in Fig. 1. The experiments were performed on pilot equipment that permits the batch circulation mode, which means that both permeate and concentrate could be carried back to the feed tank. The permeate was totally removed just in a single experiment, where it was necessaryto obtain the concentrated product of the process. The nano?ltra tion module is equipped with a NF90 membrane (Osmonics, Minnetonka, USA) which is composed of polyamide and polysulphone, with 0.6 m2 of ?ltration area and 98% rejection of MgSO4 in a test performed by manufacturing with a spiral module at 20 ° and 6.0 bar. Approximately C 5.0 L of each solution permeated through the membrane over 30 min, this being the time necessary to complete the concentration in an open system, which means that the permeate

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was removed from the process. In the trials the permeate was removed and the retentate re-circulated until a concentration factor of around four. The concentration factor is calculated according to Eq. (1):

Fig. 1. Schematic diagram of the nano ?ltration unit where Vf is the total volume used in the feed, Vc is the volume collected in the concentrate fraction and Fc is the concentration factor. Other experiments were carried out at different temperatures (20–45 ° and pressures (2.0–5.0 bar), in order to evaluate the C) in?uence of these parameters on the permeate ?ux and the concentrated product quality. In these experiments, both the permeate and retentate were maintained under re-circulation in closed systems. The permeate ?ux was calculated according to the following equation: J=Vp/t*Ap (2)

where Vp is the permeate volume collected during the time intervalt and Ap is the membrane surface area of permeation. The quality of the ?ltration process was measured according to the quantity of ?avonoids and phenolic compounds present in permeate, evaluated as described in Sections 2.2–2.4, and the ef?ciency was measured according to the ?ux permeate rate and retention index. This index measures the relation between the amounts of the compound of interest in permeate and in the concentrated solution, which demonstrates the ability of the membrane to retain this compound under the experimental
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conditions. The index is calculated according to Eq. (3), in which R is the retention index, Cp is the concentration of the compound of interest in the permeate, and Cr is the concentration of the same compound in the retentate: R=1-Cp/Cr (3)

It is important to know the rate of fouling that occurs in the membrane process, and one way of measuring this is to compare the permeate ?ux of the solution under study with the permeate ?ux when water is used as feed solution, under different pressures. Usually a variation in system pressure will cause a change directly proportional to the permeate ?ux. The fouling in?uence was measured by comparison of the permeate ?ux of the aqueous propolis extract with the ?ux of distilled water only, increasing the pressure from 1.0 to 5.0 bar. 3. Results and discussion The membrane process was carried out with the aqueous and ethanolic solutions in a closed system, in which the retentate and permeate streams being conducted back and mixed in a feed tank isolated from the environment, to evaluate the variation in permeate ?ux with time. The temperature was maintained at 20 ° and the pressure at 5.0 bar. The results are C shown in Fig. 2.

After stabilization of the process, the permeate ?ux began to decrease, after around 15 min of processing. The rate of decrease was higher for the alcoholic extract than for the

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aqueous extract, evidencing a greater rate of fouling with the alcohol solution. After 20 min of processing, the permeate ?ux tended to stabilize, that is, concentration polarization already occurred and fouling did not increase with time. The permeate ?ux in the stable region was about 12.0 L/h m2 and 25.0 L/h m2 for alcoholic and aqueous solutions, respectively. The difference between the permeate ?ux of these solutions can be explained by their different compositions: the alcohol extract contains more compounds of low

molecular weight, thus its concentration is more dif?cult to achieve, and this reduces the ?ux rate. Some of these compounds form a kind of wax which can cause more fouling in membrane. Tsui and Cheryan (2007) used nano?ltration to purify alcoholic corn extracts in the production of xanthophylls, and obtained a permeate ?ux of around 10.0 L/h m2 when working at 27 bar and 50 ° Hossain (2003) studied the membrane concentration of C. anthocyanins from blackcurrant pomace extracts using ultra?ltration, obtaining a maximum permeate ?ux of 17.3 L/h m2 at 1.4 bar and 18 °C. Using nano?ltration a permeate ?ux of 20 L/h m2 was obtained at 20 bar and 50 ° in the concentration of red wine (Banvolgyi et C al., 2006). The red wine concentration process is important since it can be considered a similar process to the concentration of alcoholic propolis, considering that they have similar compounds in solution and use alcohol as the solvent. The similarities between the processes allow a comparison between results. Low pressures (around 6 bar) were used in the propolis concentration process, when compared to other processes cited in the literature, but even so the values obtained for the permeate ?ux were similar to those obtained in the other processes. Therefore this process can be assumed to be viable, mainly because of the reduced energy requirements necessary to generate the lower pressure.The pressure adopted was not characteristic of nano?ltration processes, but was suf?cient to carry out this concentration procedure. Fig. 3 shows the difference between the curve of the permeate ?ux for the aqueous propolis extract and the curve of the permeate ?ux for distilled water to measure the degree of fouling in the process with the aqueous propolis solution.The difference between the permeate ?uxes of water and the propolis solution shows the amount of fouling in the process, under the same conditions of temperature and pressure. This parameter increased, reaching 32% at 5.0 bar. The procedure also provided information on how the ?ux was

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affected by a pressure variation, showing that the ?ux changed linearly with the pressure in the region studied. In this pressure range of operation, the concentrated products did not present signi?cant variation among the experiments.

By increasing the temperature from 20 to 45 ° and maintaining the pressure at 6.0 bar it was C possible to determine the relation ship between the temperature and the permeability of the membrane. Permeate ?ux increased proportionally, by around 8% per degree of temperature, as shown in Fig. 4. This result may be attributed to the effect of temperature on the viscosity of the solution. Also, the composition of the concentrated products obtained at different temperatures showed no signi?cant difference among them, thus, a higher ?ux can be obtained by increasing the temperature. The initial solutions, permeates and concentrates obtained by nano?ltration in open system were all subjected to a spectrophotometric analysis as described in Sections 2.2 and 2.3. The results for the aqueous solution indicated that this permeate only contained small amounts of phenolic compounds and ?avonoids, while the permeate from the ethanolic solution showed greater amounts, mostly of low molar weight phenolic compounds. Considering the losses in the compounds of interest in the resulting permeate, as compared to the initial solution, it can be seen that the aqueous solution of propolis retained almost 99% of the ?avonoids and 84% of the phenolic compounds. However, for the ethanolic
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solution, these values were 90% and 53% for the ?avonoids and phenolic compounds, respectively,as shown in Table 1. The lower retention obtained can be explained by the occurrence of plasticization in the case of ethanolic solution. This phenomenon can cause a membrane swelling or dilation, which in turn can increase the membrane diffusivity and solubility, causing loss of compounds in process. The results for the determination of ?avonoids and phenolic compounds carried out by spectrophotometric methods were veri?ed by HPLC analysis, as described in Section 2.4. The substances were identi?ed by a comparison of their retention times and ultraviolet spectra with those of standards in the literature. Chromato grams were obtained from the initial aqueous extract, and from the concentrated and permeated products, which are represented in Fig. 5a–c, where the numbers 1–3 indicate the peaks identi?ed in the HPLC analysis.

Table 2 shows the results of the quantitative analysis for all samples from the aqueous propolis solution. Comparing these data, it can be seen that there were no losses of ferulic acid to the permeate and only 20% of the caffeic acid present in the initial solution was lost to the permeate, this compound thus being the most abundant in the concentrated extract, of the compounds identi?ed. All the aqueous solutions showed peaks located in the region that

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represents a retention time of up to 20 min. This occurred since the water, being a polar material, only extracts polar compounds. The last peak identi?ed in the permeated solution, probably does not represent an isolated compound but interference in the system, since this compound was not present in the other chromatograms. Ferulic acid was not identi?ed in the permeated solution, indicating that no losses to the permeate occurred. The other peaks in the chromatograms represented compounds that could not be identi?ed according to the standards in the literature. The permeated solution showed a small amount of compounds in low concentration, allowing to verify the ef?cacy of the membrane concentration process.

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Park et al. (1998) analyzed an aqueous propolis solution prepared in the laboratory, using HPLC, and obtained similar results to those presented in Fig. 5a, reporting peaks with low retention times that represent polar substances, and identifying the compounds quercetin and pinocembrin. In their experiment, the proportions of water and alcohol in the solvents for propolis extraction were varied. Initial solutions contained 0–90% of alcohol, through which it was demonstrated that increasing the proportion of alcohol in the solution also increased the amount ofextracted ?avonoids and phenolic compounds in the propolis.The chromatograms obtained for the ethanolic solutions are presented in Fig. 5d–f. It was possible to identify peaks in the ethanolic solution throughout the process. Compounds with a retention time greater than 20 min are apolar and were extracted by ethanol, this being an advantage of the use of this solvent as compared to water, which does not extract apolars. On analyzing the chromatogram it can be seen that a considerable amount of cumaric acid was lost to the permeate (peak number 2 in Fig. 5f). This acid belongs to the phenolic acid class and has a low molar weight, which could explain the low retention capacity of the membrane for this compound. The results of the spectrophotometric analysis shown in Table 1 indicate a loss of phenolic compounds to the permeate, accounting for the cumaric acid and other compounds not being identi?ed by the HPLC method. Using the data given in Tables 2 and 3 the retention index could be calculated from Eq. (2). These results can be observed in Table 4. The retention indexes veri?ed that the membrane process retained the compounds of interest better when using aqueous solutions, since this resulted in smaller losses of the compounds studied to the permeate, as compared to such losses when working with the alcoholic solution. Cumaric acid was an exception, since only 56% of this compound was not lost to permeate. Despite the loss of compounds, the values obtained represent a high retention index and verify that the nano?ltration process is appropriate for propolis extracts.During their study on the concentration of red wine by nano?ltration, Banvolgyi et al. (2006) obtained a retention index of 88% for total acids, 50% for free sulfuric acids and 93% for the total extracts. Tsui and Cheryan (2007), working with the puri?cation of xantophylls by nano?ltration obtained a retention index of 90% for total solids, 88% for proteins and 98% for xanthophylls, the major compound of their study. In the present study, the values obtained for the retention indexes were very similar to

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those cited in the literature for similar processes. .4 Conclusions The results showed that nano?ltration can be considered as a good alternative for concentrating propolis extracts, since the membrane retained most of the ?avonoids and phenolic compounds, which are of major importance to propolis quality. Particularly in the case of the aqueous extract, it could be considered that there was no loss of compounds to the permeate solution, since almost 100% of the major compounds were retained. In the experiments with alcoholic propolis, the losses were considerable but this is a consequence of the higher amount of compounds extracted by alcohol. However, the method can be used for alcoholic solution since almost 90% of the ?avonoids were retained. Application of this technology could increase the use of propolis in many industrial applications, it being feasible to use the aqueous extract in new research projects and in the development of new products with functional properties. Furthermore, this process allows removal of the solvent from the extract, reducing the disadvantages associated with using alcoholic extractions. It should be noted that compared with other concentration methods, in the membrane process the product is not submitted to high temperatures and there is no change in the physical state of the solvent, which means that the functional properties of the compounds of interest are preserved and the process as a whole is energy saving.

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译 文

题目:黄酮类化合物和酚类化合物在水中和乙醇蜂胶中通过纳滤膜 的提取率
关键词:蜂胶 膜浓度 黄酮类化合物 酚类化合物 纳滤 摘要:蜂胶具有可变的、复杂的化学成分,且蜂胶中黄酮类化合物的浓度较高,酚 类化合物存在于蜂胶萃取物。提取溶剂不同,提取物也有所变化。乙醇胺比水更容易提 取酚酸和极性化合物。在他们应用于工业之前,提取物应被浓缩,与此同时高温会降解 一些化合物。 膜处理是一个能允许低温浓缩的一个选择。 纳滤膜应用于水和乙醇的提取, 但是每一种都会产生不同的结果:渗透、滞留物。用膜来保留化合物能力用分光光度法 进行了验证: 水溶法,膜保持94%左右的酚类化合物和99%的黄酮类化合物,而对乙醇溶解 这些数值分别为53%和90%。水和乙醇的相比酸的保留值为1.00和0.88。 因此,纳滤过程 对蜂胶提取物浓度有明显的较高的提取率。 1导语 在过去的几十年,对于功能食品的兴趣快速增长,使得改进食品加工新功 能成分或工艺有所发展,也产生了可能帮助延缓衰老或避免疾病的产品。在此 背景下,蜂产品由于其化学成分和功能特性吸引了消费者和研究人员的注意。 蜂胶是一种拥有功能特性的蜂产品,但它不能作为食品食用,因其是一种树脂 质物质。它由花蕾和某些植物的渗出物制成。这些物质通过添加蜂蜡和蜜蜂唾 液中存在的酶葡萄糖苷酶转化为蜂胶,得到的产物被蜜蜂用来保护蜂巢免遭入 侵者和污染物的侵害,用来封孔和保持温度。这种物质的一些重要特性已被报 告,例如抗微生物和抗氧化作用,麻醉特性及其他。由于这些能带来健康益处 的特性,蜂胶被认为是一种功能成分,应用于食品、饮料、化妆品和药物以改 善 健 康 及预防疾病。蜂胶中有 超过 150种成 分,包括多酚类、萜类、类固醇和氨 基酸。类黄酮是最重要的基团之一,能够代表大约 50%的蜂胶成分,这根据收集 蜂 蜜 的 地区而有所不同, 因为其 性质受植物和气候的影响。 Kumazawa等人 (2004) 检 测 了 来自不同地理区域的蜂胶的抗氧化活性,并展示了每个样本的不同活性 。 其他研究表明,来自欧洲和中国的蜂胶含有多种类黄酮和酚酸,而来自巴西的 样本含有更多的萜类和磷香豆酸异戊二烯衍生物。最后,不同来源的蜂胶中每 个 化 合 物的组合都能代表最终产品的特性。 最常见的蜂胶提取工艺使用乙醇作为溶剂。然而,这种方法有一些缺点,
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例如强烈的残留 味道,不良反应和某些人群的乙醇不耐受 (Konishi等 , 2004)。 研究者和企业对生产一种新型的提取物很感兴趣,它要有用乙醇方法提取出来 的相同化合物,但没有其缺点。将水作为溶剂测试,得到的产品中提取出的化 合物较少 (Park等, 1998)。 Konishi等人用含有某些化合物的组合测试水 取代蜂 胶 提取中使用的部分乙醇,所有试验在提取蜂胶上都有效,产品也显示出很好的 抗 微 生 物活性。 根据应用,蜂胶提取中的溶剂必须被分解或剔除。现在使用的工艺,如冻 干法、真空蒸馏和蒸发,都有一些像高温的使用和高能源消耗的缺点。冻干 法 需 要 大 量 的 能 量 , 因 为 样 本 需 要 在 零 下 20℃ 下 , 保 持 至 少 24小 时 , 提 取 准 备 过 程中溶剂升华时也需要能量。此外,此方法经常需要一个浓缩的前期阶段,将 产 品 保 持在70℃直到溶剂部分 蒸发。 真空蒸馏需要大量能量造成真空,导致低分子化合物的损失,此化合物可 以 与 系 统 中 蒸 发 的 溶 剂 一 起 被 移 除 。 蒸 发 将 提 取 物 保 持 在 70℃ 加 热 , 直 到 所 有 溶剂都被移除。这种工艺,除了高能源需求外,由于使用的高温,还会分解蜂 胶中的类黄酮和酚类化合物。然而,正是这种工艺给企业中的实施带来了极大 便 利 , 因其与以往案例相比在所需设备上花费较小。 膜浓缩工艺的使用由于某些优点而变得越来越多,优点有:低温、无需相 变 和 低 能 耗 (Matta等 ,2004)。 此 过 程 基 于 溶 质 分 子 通 过 半 渗 透 、 聚 合 或 无 机 膜 时的选择性渗透原则。在大多数膜工艺中,例如微滤、超滤、纳滤和反渗透, 物 质 穿 过膜的驱动力是机械压力 (Maroulis and Saravacos, 2003)。纳滤是一 种 允 许多种应用的单元操作,例如从过滤油中回收溶剂、化工企业中的溶剂交换 (Geens等, 2006),对 乙醇提取叶黄素的浓缩和纯化,这对制药和食品工业都很重 要 (Tsui and Cheryan, 2007),还有在酒类 浓 缩(Banvolgyi等, 2006),以及食品工业 的 果 汁 浓缩上 (Vincze等, 2006)。 此研究的目的是研究蜂胶提取物用水和乙醇作为溶剂进行的膜浓缩,且只 检验过程中浓缩产物关于类黄酮和酚类化合物保留方面的质量。此工艺根据渗 透熔剂及温度、压力和浓缩因素的影响而评价。每种溶液得出的结果相比较, 以 此 检 验用水做溶剂生产新的蜂胶产品的耐久性。 2. 材料和方法 2.1. 蜂胶 蜂胶原料从巴西圣保罗州的蜜蜂蜂巢中获得,并以单个批量的形式取得,

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目的是最小化与用来生产的植物和气候条件有关的可变性。它储存在冷藏条件 下 (4 ° C),直到在提取制备中被使用。 在 此 地 区 生 产 的 蜂 胶 被 描 述 为 第 12组 ( 巴 西 有 12组 拥 有 区 别 特 征 的 蜂 胶 ) 并 呈 现 出大量的可溶性物质,抗金黄色葡萄球菌和变形链球菌的抗微生物活性 , 及比来自其他地区的样本更大的抗炎活性,而这可能与在此组中发现的高浓度 类 黄 酮 和酚类化合物有关 (Park等 , 2002)。 乙 醇 蜂胶溶液是由使用 500瓦马达的台架搅拌机粉碎、匀化、在半分析天 平 上称量过并混合 了 80%乙醇的天然 蜂胶制 成的。混合物在 室温 条件下存放 7天 并 每 天 人 工搅拌一次。此阶段后,样本以 8800转离心分离 20分钟)。过滤上层 清 液 并 将 其 在 4° C条 件 下 冷 藏 3小 时 , 然 后 再 次 过 滤 以 清 蜡 。 最 后 , 得 到 的 提 取 物 储 存 于 室 温条件下的黑暗中。 水 溶 剂 的 配 制 使 用 去 离 子 水 依 照 相 同 工 序 进 行 。 每 种 溶 液 都 以 20%蜂 胶 和 80%溶 剂 的 比 例 配 制 成 。 两 种 提 取 物 都 以 它 们 的 类 黄 酮 和 酚 类 化 合 物 含 量 来 评 价 , 用 来与浓缩产品比较。 2.2. 类黄酮总量测定 蜂 胶 溶 液 中 的 类 黄 酮 总 量 由 铝 络 合 方 法 测 定 (Marcucci等 ,1998)。 在 此 过 程 中 , 提 取出的溶液以 1:10(0.5 mL)的比例 稀释并混入 0.1mL浓 度为 10%的硝酸 铝 , 0.1mL浓度为 1 mol/L的乙酸钾和 4.3 mL浓 度为 80%的乙醇。样本在室温条件下放 置 40分钟,吸收率为 415nm。槲皮素作为标准产生校准曲线。三个读数的平均值 以 毫 克 槲皮素当量为单位使用,类黄酮总量也用同样的单位表述 (mg/g)。 2.3. 酚类化合物测定 蜂 胶 溶液中的多酚由比色 法测定 (Kumazawa等,2004)。按照此 过程,提取出 的 溶 液 先以1:10 (0.5 mL)的比例稀释 ,然 后混入 0.5mL 试剂和 0.5 mL浓度为 10% 的 Na 2 CO 3 。在室温条件下培养 1小时候吸收率为 760nm。没食子酸作为标准产生 校准曲线。三个读数的平均值以毫克没食子酸当量为单位使用,酚类化合物总 量 也 用 同样的单位表述 (mg/g)。 2.4. 高效液相色谱法测定 存 在 于最初提取物、渗透物和浓缩产品的化合物是由 Park等人(1998)描述 的 高效液相色谱法测定的。每种溶液取三百微升注入一台连接着二极管阵列检测 器 的 波 长260 nm的液相色谱仪。流动相为水 /乙酸(19:1, v/v) (溶 剂A)和甲醇 (溶 剂 B),恒定流动速率为 1 mL/min。梯度在开 始时为 30%浓度的溶剂 B,45分钟后 变

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成 60%,85分钟后变 成 75%,95分钟后变 成 90%,105分钟后回 到 30%。列保持恒 温 30 ° C,色谱使用计算机软件色谱工作站进行处理。初始和浓缩样本在 1.5 mL 蒸馏水中稀释,渗透样本不经稀释直接注入。以下酚酸和类黄酮的真实性标准 受 到 检 验 : Q键 香 豆 酸 , 阿 魏 酸 、 肉 桂 酸 、 没 食 子 酸 、 槲 皮 素 、 山 柰 酚 、 山 柰 素、芹菜素、 异鼠 李素、鼠李素、野樱 素、异野樱素、橙皮 苷、橙皮素、松属 素 、 白杨 素、 刺槐 素、 高良 姜素 、杨 梅黄 素 、杨 芽黄 素和 阿替 匹 林 C, 因为 它 们 与 蜂 胶中存在的最常见化合物对应。 2.5. 膜浓缩 在此研究中,蜂胶提取物使用中试切向过滤系统浓缩,使用示意图 1所示 的 纳滤膜。试验在适用批循环模式的中试设备上进行,这种模式意味着渗透物和 浓缩物都能够被送回给水箱。渗透物在一次试验中就被完全移除了,在此试验 中 需 要 获取浓缩产物。纳滤模块配备的是 NF90膜,这种膜由聚 砜和聚酰胺组成, 过 滤 面积 为 0.6m 2 ,在 一 次在 条件 为 20 ° C和 6.0巴使 用 螺旋 模块 进 行的 试验 中 硫 酸 镁 的 截留率为 98%。 大 约 5.0 L每种溶液 用了超过 30分钟时间渗透过膜,这段时间正是在开放 系 统 中 完 成浓缩过程所需的时间,也就意味着渗透物都在过程中被移除。试验 中 , 渗透物被移除,渗余物重新循环直到一个浓缩因素达到 4左右。这个浓缩因素根 据 方 程(1)计算:

式 中 Vf为给水箱的总 容积, Vc为浓缩馏分 的体积, Fc为浓缩因 素。 其 他 试验在不同温度 (20–45 ° C)和压力(2.0–5.0巴)下进行,目的是评估这 些 参数对渗透量和浓缩产物质量的影响。在这些试验中,渗透物和渗余物都维持 在 封 闭 系统的再循环下。渗透量根据以下公式计算: J J=Vp/t*Ap (2)

式 中 Vp为在间隔时间 t里收集的渗透物体积, Ap为渗透膜表面积。 过滤过程的质量根据渗透物中存在的类黄酮和酚类化合物的数量而衡量, 如 2.2–2.4 节中所描述的那 样评估,效率根据通量渗透速率和截留指数衡量。该 指 数 衡 量渗透物和浓缩液中化合物数量之间的关系,这展示了膜 在实验条件 下

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图 1 纳滤膜单元的原理图 截 留 该 化 合 物 的 能 力 。 该 指 数 根 据 公 式 (3)计 算 , 式 中 R为 截 留 指 数 , Cp为 渗 透 物 中化合物的浓度, Cr为 渗余物中相同化合物的浓度: R=1-Cp/Cr (3)

了解出现在膜过程中的污染速率是很重要的,测量改速率的一种方法是比 较 在 此 研究下的溶液的渗透量和用水作为液料、在不同压力下的渗透量。通 常 , 系统压力的变化会导致渗透量的正比例变化。污染影响根据水溶 蜂胶提取物的 渗 透 量 和仅用蒸馏水、压力从 1.0巴增加到 5.0巴的流量之间的比较而进行衡量 。 3. 结果与讨论 膜过程用水溶液和乙醇溶液在封闭系统中进行。在此系统中,渗余物和渗 透流被导回并混入一个与环境隔绝的给水箱,用来评估渗透量随时间的变化。 温 度 保 持在20 ° C,压力保持在 5.0巴。结果如图 2所示: 工 艺 稳 定 化 后 , 进 行 约 15分 钟 后 , 渗 透 量 开 始 下 降 。 乙 醇 提 取 物 的 下 降 率 高 于 水 溶 提 取 物 , 证 明 了 乙 醇 溶 液 的 污 染 率 更 高 。 进 行 20分 钟 后 , 渗 透 量 趋 于 稳定,即,浓差极化已经发生且污染并未随时间增加。乙醇和水溶液在稳定区 域 的 渗 透量分别为 12.0 L/h m 2 和25.0 L/h m 2 。溶液间不同的渗透量可根据它们的 不同成分解释:乙醇提取物包含更多的低分子化合物,因此更难浓缩,这也降 低 了 通 量率。其中某些化合物会形成某种蜡,可能造成更多的膜污染。 Tsui和Cheryan (2007)在生产叶黄素中 使 用纳滤来纯化乙醇玉 米提取物,得 出 工 作 压强为 27帕、 工作温度为 50 ° C时的渗透量约为 10.0 L/h m 2 。 Hossain(2003)

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时间 图 2 膜通量随时间的变化 研究了使用超滤从黑加仑果渣提取物中提取花青素的膜浓缩过程,在压强 为 1.4巴、温度为 18 ° C的条件下 得出的渗透量最大值为 17.3 L/h m 2 。使用纳 滤 , 在 压 强 为20巴、温度为 50 ° C的条件下浓缩红葡萄酒得出的渗透量为 20 L/h m 2 。 红葡萄酒浓缩过程很重要,因为从溶液的化合成分与都使用酒精做溶剂两方面 说,它可以视作与乙醇蜂胶浓缩类似的过程。过程的相似性允许结果间的比较。 在 蜂 胶 浓 缩 过 程 中 , 使 用 低 压 强 ( 约 6巴 ) 这 是 与 文 献 中 引 用 的 其 他 过 程 , 相比较而言的,但虽然如此,渗透量得出的值还是与其他过程得出的相似。因 此,此过程可认定为可行,主要由于设备产生低压强耗能较小。采用的压强不 是 纳 滤 过程的特征,但足以实施浓缩过程。 图 3显示了水溶蜂胶提取物渗透量曲线和蒸馏水渗透量曲线的差异,以 衡 量 水 溶 蜂 胶液浓缩过程的污垢度。 水和蜂胶溶液渗透量之间的差异显示了同等温度和压强条件下过程中的结 垢量。此参数在 压强 为 5巴的条件下 升至 32%。此过程 还提供 了通量如何受压强 变化影响的信息,显示了通量随着研究区域压强线性变化。在此操作压强范围 内 , 各 试验的浓缩产品并无显著区别。 将 温 度从 20° C上升至 45 ° C并保持压强为 6.0巴, 测定温度和膜的渗透性之间 的 关 系 是可能的。渗透量 成比例增长,大约每升高一度增长 8%,如图 4所示。此 结果可归因于温度对溶液黏度的影响。同样地,在不同温度获得的浓缩产品的 成 分 并 无显著差别,因此,高通量可通过增加温度获得。 初始溶液、在开放系统中通过纳滤获得的渗透物和浓缩物都受到 2.2和2.3节 中描述的光度分析。水溶液的结果表明,渗透物只含有少量的酚类化合物和类

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黄酮,而乙醇溶液的渗透物中的含量更大,主要为低分子酚类化合物。考虑到 所 得 渗 透物成分化合物与初始溶液相比的损失, 水溶蜂胶液保留了几乎 99%的 类 黄 酮 和84%的酚类化合物。然而,对乙醇溶液来说,这两个值分别为 90%和53%, 如 图1所示。得出的低截留率可由乙醇溶液样本发生增塑作用解释。此现象会导 致膜膨胀或扩张,这又反过来增加的膜的扩散系数和溶解度,引起过程中化合 物 的 损 失。 由分光光度法测出的类黄酮和酚类化合物的结果通过 高效液相色谱法分析 验 证 , 如2.4节所描 述 。这些物质 通过比 较 截留时间和 紫外线 光 谱,根据文 献中 的 标 准 鉴别。

压力 图3 压力对膜通量的影响 将 温 度从 20° C上升至 45 ° C并保持压强为 6.0巴, 测定温度和膜的渗透性之间 的 关 系 是可能的。渗透量成比例增长,大约每升高一度增长 8%,如图 4所示。此 结果可归因于温度对溶液黏度的影响。同样地,在不同温度获得的浓缩产品的 成 分 并 无显著差别,因此,高通量可通过增加温度获得。 初始溶液、在开放系统中通过纳滤获得的渗透物和浓缩物都受到 2.2和2.3节 中描述的光度分析。水溶液的结果表明,渗透物只含有少量的酚类化合物和类 黄酮,而乙醇溶液的渗透物中的含量更大,主要为低分子酚类化合物。考虑到 所 得 渗 透物成分化合物与初始溶液相比的损失, 水溶蜂胶液保留了几乎 99%的 类 黄 酮 和84%的酚类化合物。然而,对乙醇溶液来说,这两个值分别为 90%和53%, 如 图1所示。得出的低截留率可由乙醇溶液样本发 生增塑作用解释。此现象 会 导 致膜膨胀或扩张,这又反过来增加的膜的扩散系数和溶解度,引起过程中化合 物 的 损 失。

第 20 页

由分光光度法测出的类黄酮和酚类化合物的结果通过 高效液相色谱法分析 验 证 , 如2.4节所描 述 。这些物质 通过比 较 截留时间和 紫外线 光 谱,根据文 献中 的 标 准 鉴别。

温度 图 4 温度对水性溶胶膜通量的影响 色 谱 图从初始水溶提取物 以及浓缩和渗透产物中获得,见图 5 a–c,其中数 字 1-3。 代表高效液相色谱法分析中鉴别的峰值。色谱图从初始水溶提取物以及浓 缩 和 渗 透产物中获得,见图 5 a–c,其中数字 1-3 代表 高效液相色谱法 分析中 鉴 别 的 峰 值。 表 2 显示了对水溶 蜂胶溶液中所有样本的定量分析结果。比较这些数据, 可以看出渗透物中的阿魏酸没有损失,且初始溶液中的咖啡酸也只在渗透物中 损 失 了 20%, 因此 该化合物是浓缩 提取 物里所有鉴定出 的化 合物中最丰富的一 种。 所 有 的 水 溶 液 都 显 示 出 区 域 内 的 峰 值 最 大 为 20分 钟 的 截 留 时 间 。 这 是 因 为 水作为一种极性物质,只提取极性化合物。渗透溶液中最后一个鉴定的峰值可 能并不代表一种孤立的化合物,而干扰整个系统,因为该化合物并不存在于其 他 色 谱 图中。

第 21 页

色 谱 图从初始水溶提取物 以及浓缩和渗透产物中 获得,见图 5 a–c,其中 数 字 1-3代表 高效液相色谱法 分析中鉴别的峰值。 表 2显示了对水溶蜂胶溶液中所有样本的定量分析结果。比较这些数据 ,可 以看出渗透物中的阿魏酸没有损失,且初始溶液中的咖啡酸也只在渗透物中损 失 了20%,因此该化合物是浓缩提取物里所有鉴定出的化合物中最丰富的一 种 。 所 有 的 水 溶 液 都 显 示 出 区 域 内 的 峰 值 最 大 为 20分 钟 的 截 留 时 间 。 这 是 因 为 水作为一种极性物质,只提取极性化合物。渗透溶液中最后一个鉴定的峰值可 能并不代表一种孤立的化合物,而干扰整个系统,因为该化合物并不存在于其 他 色 谱 图中。 阿 魏酸没有在 渗透溶液中被 鉴定出来,表明渗透物没有发生损失。色谱 图 中 的其他峰值代表了不能根据文献中的标准鉴定的化合物。渗透溶液显示了少量 的 低 浓 度化合物,容许对膜浓缩过程的效验进行检验。

第 22 页

Park等 人 (1998)分 析 了 一 种 在 实 验 室 使 用 高 效 液 相 色 谱 法 制 备 的 水 溶 蜂 胶 液 , 得 出 了 和 图 5a中 显 示 的 类 似 结 果 , 报 告 了 拥 有 代 表 极 性 物 质 的 低 截 留 时 间 的 峰 值 ,并将该化合物鉴定为槲皮素和松属素。 在他们的试验中,用于蜂胶提取的溶液中水和乙醇的比例各有不同。初始 溶 液 中 含有0-90%的 乙醇,通过此研究表明,增加溶液中乙醇的比例也会增加蜂 胶 中 提 取出的类 黄酮和酚类化 合物的比例。 从 乙 醇溶液中得到的色谱 图呈现为图 5d-f。 在 过 程 中 鉴 定 乙 醇 溶 液 的 峰 值 是 可 能 的 。 需 要 超 过 20分 钟 的 截 留 时 间 的 化 合物是非极性的,并用乙醇提取,这就是使用此溶剂和水相比的优势,水不能 提取非极性物质。分析色谱图时,可能看到,数量可观的香豆酸在渗透物中损 失(图 5f中的 2号峰值) 。该酸属于酚酸类,分子量低,这解释了膜对于此化合物 的低截留能力。表 1中光度分析的结果表明了渗透物中酚类化合物的损失,解 释 了 香 豆 酸和其他化合物没有被高效液相色谱法鉴定出来的原因。 用 表 2和 表 3给 出的 数据 , 可以 根据 公 式 (2)算 出 截留 指数 。 这些结 果 可以 在 表 4中 观察。 截留指数证明膜过程用水溶液截留成分中的化合物效果更好,因为和使用 乙 醇 溶 液造成的损失相比,这造成的渗透物化合物损失较小。香豆酸是个例 外 , 因 为 只 有56%的该化合物没有在渗透物中损失。尽管化合物有损失,得到的值 还 是 呈 现 出高截留指数并证明纳滤过程对蜂胶提取是适用的。 在 使 用 纳 滤 对 红 葡 萄 酒 进 行 浓 缩 的 研 究 中 , Banvolgyi等 人 (2006)得 出 总 酸 的 截 留 指数为 88%,游离硫酸为 50%,总 提取物为 93%。研究 使用纳滤纯化叶黄 素 的Tsui和Cheryan(2007)得出总固形 物的截留指数为 90%,蛋白质为 88%,研究 的 主 要 化合物 ——叶黄素则为 98%。 在 本 研究中,得出的截留 指数与文献中引用的类似过程的数值非常相似。 4. 结论 结果显示,纳滤可以被视为用于浓缩蜂胶提取物的良好替代品,因为膜截 留了大部分类黄酮和酚类化合物,这对蜂胶质量来说非常重要。尤其在水溶提 取物样本中,可 以看 作渗透溶液中没 有化 合物损失,因为 它截 留了几乎 100%的 主要化合物。在用乙醇蜂胶进行的试验中,损失相当可观,但这是用乙醇提取 化 合 物 的数量更大的后果。然而 ,此方法可用于乙醇溶液,因为截留了几乎 90% 的类黄酮。此技术的应用能够增加很多工业应用中蜂胶的使用,使得在新研究

第 23 页

项目和具有功能特性的新产品研发中使用水溶提取物成为可能。此外,该过程 允 许 将 溶剂从提取物中移除,减少了与使用乙醇提取相关的缺点。应该注意 到 , 和其他浓缩方法相比,在膜浓缩过程中,产品不需要置于高温下,溶剂的物理 状态也没有改变,这意味着成分化合物的功能特性保持了下来,且整个过程都 很节能。

第 24 页


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