低温胁迫下菠萝幼苗生长与生理特性变化

以国内外10个菠萝品种幼苗为材料,研究了它们在低温胁迫下的生长及其渗透调节物质、细胞膜透性的变化特征,并综合评价它们的耐寒性.结果表明:(1) 随低温处理时间的延续,各品种幼苗单株干重虽然均呈逐渐增加趋势,但不同品种生物量增加幅度存在显著差异.(2) 随着低温处理时间的延长,各品种叶片脯氨酸含量和细胞膜相对透性逐渐升高;可溶性蛋白含量总体上初期升高后期降低,而‘巴厘'、‘珍珠'、‘台农17号'、‘台农19号'和‘MD-2'的可溶性蛋白在处理末期又有所上升;可溶性糖含量变化趋势复杂.(3) 低温胁迫处理9 d后,各菠萝品种叶片中的脯氨酸和可溶性蛋白含量及细胞膜相对透性均增加;可溶性糖含量除‘珍珠'略有减少外,其余均增加.(4)经过各指标赋分分级综合评价后认为,‘MD-2'和‘巴厘'品种耐寒性最强;‘台农17号'、‘台农16号'、‘巴厘突变体'、‘台农13号'、‘台农19号'和‘无刺卡因'耐寒性中等;‘珍珠'和‘台农6号'耐寒性最弱.研究发现,菠萝耐寒性强弱可根据干物质积累结合其它生理指标来综合评判.     

施钙对‘台农17号’菠萝裂柄的生理影响及效果

了解菠萝中钙素营养与其裂柄的关系及其生理机制,本文以‘台农17号’菠萝为试验材料,研究施钙处理对其产量、裂柄率、D叶和果柄中Ca含量及果柄中相关酶活性的影响,同时分析裂柄率与其Ca含量及相关酶活性的相关关系。研究结果表明,施钙可以使菠萝增产,且降低菠萝裂柄率;催花期施钙的效果最好。相关分析表明,叶片和果柄的Ca含量与裂柄率均呈极显著负相关关系。钙肥施用后,果柄中原果胶(PP)含量显著增加;超氧化歧化酶(SOD)与过氧化氢酶(CAT)活性增强,且在催花10 d施钙处理活性表现最强;多酚氧化酶(PPO)和细胞壁水解酶(CX和PG)活性降低。菠萝裂柄率与果柄中的PP含量及CAT活性呈显著负相关关系,与其PPO活性成极显著正相关。以上结果表明,施用钙肥是减少‘台农17号’菠萝裂柄的重要措施,其适宜施用量为300kg·hm~(-2),适宜施用时期为快速生长Ⅱ期至催花后10 d之间。     

六种白腐菌预处理对毛白杨木材酶水解效果的影响

通过化学分析和酶水解试验,研究了不同的白腐菌对毛白杨的预处理效果及不同组分的降解对酶水解的影响。毛白杨木片经6种白腐菌预处理30d后,各组分都发生了降解,其中半纤维素的损失最为显著,Trametes ochracea C6888引起半纤维素降解率高达47.19%,其次是纤维素和酸不溶木素的降解。在后续酶水解过程中,6种白腐菌处理后的样品显示出不同的水解模式,菌株Trametes ochracea C6888、T. pubescens C7571和T. versicolor C6915预处理效果最为显著,还原糖得率在整个酶水解过程中一直高于对照,其中T. ochracea C6888在水解96h后还原糖得率达到15.93%,比未处理样品提高了25%。分析酸不溶木素降解率及半纤维素降解率与还原糖得率的关系发现,不同菌株在作用同一种基质时,预处理效果差异显著,木质素和半纤维素的脱除都会影响木质纤维素的酶水解。     

不同番木瓜品种植株感染环斑花叶病毒后PAL、PPO、POD活性的变化

对不同品种番木瓜接种环斑花叶病毒后,测定植株苯丙氨酸解氨酶(PAL)、多酚氧化酶(PPO)、过氧化物酶(POD)的活性变化,比较各品种的抗病性。结果表明,未接种的5个品种间PAL、PPO、POD活性差异较小,其酶活性水平次序为马来10号> 蜜红3号> 马来6号> 马来2号> 台农2号。接种后,5个品种的PAL、PPO、POD活性明显高于各对照水平,其中马来10号变化最突出,台农2号变化最缓慢。     

中国石榴品种资源经济性状研究

本文测定了34个中国石榴主栽品种的单果重、可溶性固形物含量、糖酸比等9个经济性状,根据测定结果对各品种果实品质进行了模糊综合评判.结果表明,皖榴3号、皖榴2号、皖榴1号、大笨子等品种品质最佳;提出了评判结果等级划分的方法;各品种经济性状的相关与回归分析结果显示,果实百粒重与出汁率,可溶性固形物含量、可溶性糖含量与糖酸比,出汁率与糖酸比,糖酸比与可溶性糖含量均呈极显著正相关(α=0.01,F＞F0.01);出汁率与果皮光洁度呈极显著负相关(a=0.01,F＞0.01);主成分分析结果表明,前5个主成分累积方差贡献率达到87.67%,基本概括了全部9个性状的主要信息.     

聚乙二醇对菠萝蛋白酶的化学修饰

方法:用琥珀酸酐法活化的聚乙二醇对菠萝蛋白酶进行化学修饰,得到菠萝蛋白酶的修饰酶,对比研究三种菠萝蛋白酶:修饰酶、混合酶、天然酶的热稳定性及酸碱稳定性,考察金属离子对三种菠萝蛋白酶的影响。结果:当在55℃水浴保温100min后天然酶活力只保留20%,混合酶活力保留37%,修饰酶活力保留58%;在pH3.0-4.5及pH6.0-7.0的条件下,修饰酶活力高于天然酶活力。当Ca2 的浓度达到0.05mg/mL时,修饰酶的活力高达257.66%;当Mg2 的浓度达到0.035mg/mL时,修饰酶的活力高达147.25%。一价离子Na 对三种菠萝蛋白酶无明显影响。结论:修饰的菠萝蛋白酶对温度和pH值的稳定性均比天然酶有很大程度的提高。混合酶的活力介于天然酶和修饰酶之间说明聚乙二醇对菠萝蛋白酶有一定的保护作用。二价离子Ca2 、Mg2 对三种菠萝蛋白酶活力均有不同程度的激活作用。     

欧李果实发育期糖和酸组分及其含量的动态变化特性

以农大3号、农大4号、农大5号3个欧李品种为材料,测定果实发育过程中各组分糖、酸及总糖、总酸的含量,以明确欧李果实糖酸积累的动态变化特性.结果显示:(1)3个欧李品种果实成熟期糖含量、酸含量及糖酸比存在明显差异,其中农大3号品种的总糖含量最高,总酸含量最低,糖酸比值最高.(2)成熟期3品种各糖组分中均以果糖含量较高,葡萄糖和蔗糖含量较低,山梨醇含量微量;酸组分中均以苹果酸为主,柠檬酸少量.(3)3个品种果糖、蔗糖、葡萄糖、山梨醇含量在整个果实发育期均呈持续增加态势,并以果糖积累为主;农大3号果糖含量在8周后增幅明显高于另2个品种,且一直保持到果实成熟;3个品种蔗糖含量的变化趋势相近,在前期和中期增加缓慢,接近成熟的2～3周则迅速增加并占整个发育期积累量的70%以上.(4)3个品种酸含量的变化趋势较为相似,苹果酸和柠檬酸在果实发育的前期和中期含量均较低,在果实发育后期迅速增加,但接近果实成熟时又大幅下降.研究表明,果糖与苹果酸的含量及其动态变化是影响欧李果实糖酸比、决定果实风味的主要因素.     

四棱豆不同生育期叶片中过氧化物酶活力测定

比较了四棱豆不同生育期叶片中过氧化物酶活性,结果表明：该酶活力和比活力在不同生育期存在着明显差异,蛋白质含量变化不大。在成熟期,随着植株节位升高,酶活力及比活力均呈下降趋势,蛋白质含量则呈上升趋势。     

台湾菠萝品种在福建泉州的引种比较试验

以泉州地区主栽品种‘沙捞越’为对照,对引进的菠萝品种‘台农16号’和‘台农17号’进行种植试验,观察其植物学性状、农艺性状,测定果实品质性状,并调查抗寒性能。结果表明,‘台农16号’和‘台农17号’的单果重、可溶性固形物、维生素C含量均高于对照,且酸度较低,属优质鲜食品种;‘台农16号’抗寒力较强,可在该地区扩大示范面积;‘台农17号’抗寒力弱,可作杂交种质。     

蛋白鲜味肽调味品生产工艺初探

蛋白二次酶解技术生产蛋白鲜味肽能够在保有传统鲜味的同时,有效提高蛋白质得率,利于人体吸收。试验根据不同水产品的呈味特点,选择沙丁鱼和对虾为实验原料,利用复合蛋白酶对原料进行初步定向酶解并超滤,制备具有独特风味的短肽。采用响应面法优化酶解水产蛋白工艺,水解度作响应值,以探究酶添加量、酶解温度、时间及pH值对鲜味肽得率的影响,得到初步酶解制备鲜味肽最优工艺条件：复合蛋白酶加酶量3.3%、温度57℃、酶解时间3.5h、pH为7.1。     

Heterologous expression of the plant cysteine protease bromelain and its inhibitor in Pichia pastoris

Expression of proteases in heterologous hosts remains an ambitious challenge due to severe problems associated with digestion of host proteins. On the other hand, proteases are broadly used in industrial applications and resemble promising drug candidates. Bromelain is an herbal drug that is medicinally used for treatment of oedematous swellings and inflammatory conditions and consists in large part of proteolytic enzymes. Even though various experiments underline the requirement of active cysteine proteases for biological activity, so far no investigation succeeded to clearly clarify the pharmacological mode of action of bromelain. The potential role of proteases themselves and other molecules of this multi‐component extract currently remain largely unknown or ill defined. Here, we set out to express several bromelain cysteine proteases as well as a bromelain inhibitor molecule in order to gain defined molecular entities for subsequent studies. After cloning the genes from its natural source Ananas comosus (pineapple plant) into Pichia pastoris and subsequent fermentation and purification, we obtained active protease and inhibitor molecules which were subsequently biochemically characterized. Employing purified bromelain fractions paves the way for further elucidation of pharmacological activities of this natural product. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:54–65, 2017     

Application of an aqueous two‐phase micellar system to extract bromelain from pineapple (Ananas comosus) peel waste and analysis of bromelain stability in cosmetic formulations

Bromelain is a set of proteolytic enzymes found in pineapple (Ananas comosus) tissues such as stem, fruit and leaves. Because of its proteolytic activity, bromelain has potential applications in the cosmetic, pharmaceutical, and food industries. The present study focused on the recovery of bromelain from pineapple peel by liquid–liquid extraction in aqueous two‐phase micellar systems (ATPMS), using Triton X‐114 (TX‐114) and McIlvaine buffer, in the absence and presence of electrolytes CaCl₂ and KI; the cloud points of the generated extraction systems were studied by plotting binodal curves. Based on the cloud points, three temperatures were selected for extraction: 30, 33, and 36°C for systems in the absence of salts; 40, 43, and 46°C in the presence of KI; 24, 27, and 30°C in the presence of CaCl₂. Total protein and enzymatic activities were analyzed to monitor bromelain. Employing the ATPMS chosen for extraction (0.5 M KI with 3% TX‐114, at pH 6.0, at 40°C), the bromelain extract stability was assessed after incorporation into three cosmetic bases: an anhydrous gel, a cream, and a cream‐gel formulation. The cream‐gel formulation presented as the most appropriate base to convey bromelain, and its optimal storage conditions were found to be 4.0 ± 0.5°C. The selected ATPMS enabled the extraction of a biomolecule with high added value from waste lined‐up in a cosmetic formulation, allowing for exploration of further cosmetic potential. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:937–945, 2015     

Stability,purification, and applications of bromelain: A review

Bromelain is a cysteine protease found in pineapple tissue. Because of its anti‐inflammatory and anti‐cancer activities, as well as its ability to induce apoptotic cell death, bromelain has proved useful in several therapeutic areas. The market for this protease is growing, and several studies exploring various properties of this molecule have been reported. This review aims to compile this data, and summarize the main findings on bromelain in the literature to date. The physicochemical properties and stability of bromelain under different conditions are discussed. Several studies on the purification of bromelain from crude extracts using a wide range of techniques such as liquid–liquid extractions by aqueous two‐phase system, ultrafiltration, precipitation, and chromatography, have been reported. Finally, the various applications of bromelain are presented. This review therefore covers the main properties of bromelain, aiming to provide an up‐to‐date compilation of the data reported on this enzyme. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:5–13, 2016     

Oriented immobilization of stem bromelain via the lone histidine on a metal affinity support

Bromelain is a basic, 23.8 kDa thiol proteinase obtained from the stem of the pineapple plant (Ananas comosus) and is unique for it contains a single histidine residue (His-158) in the polypeptide. Based on the technology of protein separation with immobilized metal ion affinity chromatography (IMAC), a method for oriented immobilization of bromelain was selected. Bromelain was successfully immobilized on iminodiacetic acid carrier Sepharose 6B. Cu²⁺ complexed with iminodiacetate (IDA) was used as the chelating ligand to bind the lone histidine on bromelain. Simultaneously, preparation of a high affinity immobilized preparation was attempted using a soluble cross-linked preparation of bromelain on Cu-IDA-Sepharose. However this second method proved unsuccessful, possibly due to poor histidine accessibility in the cross-linked preparation. The immobilized preparation obtained using uncrosslinked bromelain was more resistant to thermal inactivation, as evidenced by retention of over enzyme 50% activity after incubation at 60 °C, as compared to 20% retained by the native enzyme. The immobilized preparation also exhibited a broader pH-activity profile in acidic range. The native, immobilized and soluble cross-linked bromelain showed apparent Michaelis constant (K_m) values of 1.08, 0.42, 1.56 mg/ml, respectively, using casein as the substrate. While the maximum velocity (V_max) values of the soluble and immobilized preparations were comparable, cross-linked preparation showed a 20% decrease, suggesting inactivation. The mild conditions used for predominantly oriented immobilization exploiting the unique property of single histidine, the high recovery of immobilized preparations, the stability, reusability and the regenerability of the matrix are the main features of the method reported here.     

Inhibitory effects of fruit juices on cytochrome P450 2C9 activity in vitro

There is limited information on the effect of fruits on human cytochrome P450 (CYP) 2C9 activity. The objective of this study was to determine the effect of fruit juice on CYP2C9-mediated drug metabolism. Nine citrus fruits and eight tropical fruits were chosen. We investigated effects of the fruits on diclofenac 4'-hydroxylation and tolbutamide hydroxylation by human liver microsomes. Among the fruits, pineapple juice showed potent inhibition of CYP2C9 activity. The addition of 25 microl (5.0% v/v) of pineapple juice resulted in almost complete inhibition. Next we examined the inhibitory effect of bromelain, a cysteine protease in pineapple. Bromelain also strongly inhibited CYP2C9 activity. In addition, E-64, a cysteine protease inhibitor, almost entirely blocked inhibition by pineapple juice and bromelain. Thus we found that pineapple juice was a potent inhibitor of CYP2C9, and that the inhibitory effect might be due to the bromelain contained in pineapple.     

Characterization of the acidic and basic limbs of a bell-shaped pH profile in the inhibitory activity of bromelain inhibitor VI

Bromelain inhibitor VI (BI-VI) is a cysteine proteinase inhibitor from pineapple stem and a unique two-chain inhibitor composed of two distinct domains. BI-VI's inhibitory activity toward the target enzyme bromelain is maximal at pH 4 and shows a bell-shaped pH profile with pKa values of about 2.5 and 5.3. This pH profile is quite different from that of bromelain, which is optimally active around pH 7. In the present article, to characterize the acidic limb, we first expressed the recombinant inhibitors designed to lose two putative hydrogen bonds of Ser7(NH)-Asp28(beta-CO2H) and Lys38(NH)-Asp51(beta-CO2H) and confirmed the existence of the hydrogen bonds by two-dimensional nuclear magnetic resonance (NMR). Moreover, it was revealed that these hydrogen bonds are not the essential electrostatic factor and some ionizable groups would be responsible for the acidic limb in the pH-inhibition profile. On the other hand, to characterize the basic limb, we examined the pH-dependent inhibition using the cysteine proteinase papain, some of whose properties differ from those of bromelain, and compared the data with the corresponding data for bromelain. The result suggests that the basic limb would be affected by some electrostatic factors, probably some carboxyl groups in the target proteinase.     

Pineapple stem bromelain immobilized on different supports: Catalytic properties in model wine

Bromelain from pineapple stem has been covalently immobilized on different supports to select the more efficient biocatalyst that should be applied toward unstable proteins in real white wine. In this preliminary study, catalytic properties of different immobilized bromelain forms were compared under wine‐like conditions, against a synthetic substrate (Bz‐Phe‐Val‐Arg‐pNA).Covalent immobilization affected protease kinetic properties, even if all immobilized forms presented both a better substrate affinity and higher half‐life (with the exception of a few procedures) with respect to the free enzyme. Stem bromelain was successfully immobilized on chitosan beads without glutaraldehyde thus yielding a food‐safe and promising biocatalyst for unstable real wine future application. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Bromelain, a cysteine protease from pineapple (Ananas comosus) stem, is an inhibitor of fungal plant pathogens

Aim: This study aimed to evaluate the effect of bromelain, a cysteine protease isolated from pineapple (Ananas comosus), on growth of several agronomically important fungal pathogens. Methods and Results: Purification of bromelain from pineapple stems was carried out by chromatography techniques, and its antimicrobial activity was tested against the fungal pathogens Fusarium verticillioides, Fusarium oxysporum and Fusarium proliferatum by broth microdilution assay. A concentration of 0·3 μmol l^?1 of bromelain was sufficient for 90% growth inhibition of F. verticillioides. The capability of bromelain to inhibit fungal growth is related to its proteolytic activity. Conclusions: The study demonstrates that stem bromelain exhibits a potent antifungal activity against phytopathogens and suggests its potential use as an effective agent for crop protection. Significance and Impact of the Study: The results support the use of a natural protease that accumulates at high levels in pineapple stems as alternative to the use of chemical fungicides for crop protection.     

Two phase partitioning and collagen hydrolysis of bromelain from pineapple peel Nang Lae cultivar

Extraction of bromelain from pineapple peel (Nang Lae cultv.) using aqueous two phase system (ATPS) was optimized. Some biochemical properties including collagen hydrolysis were also investigated. Bromelain predominantly partitioned to the polyethylene glycol (PEG)-rich phase. The highest enzyme activity recovery (113.54%) and purification fold (2.23) were presented in the top phase of 15% PEG2000–14% MgSO₄. Protein pattern and activity staining showed the molecular weight (MW) of bromelain to be about 29 kDa. The extracted bromelain showed the highest relative activity at pH 7.0 and 55 °C. Its activity was decreased continuously by increasing NaCl concentration (up to 1.5% (w/v)). The bromelain extract was applied to hydrolyze the skin collagen of beef and giant catfish (0–0.3 units). The β, α₁, α₂ of giant catfish skin collagen extensively degraded into small peptides when treated with 0.02 units of the bromelain extract. Bovine collagen was hydrolyzed using higher bromelain up to 0.18 units. This study showed the ATPS can be employed to partially purify bromelain from Nang Lae pineapple peel and the enzyme effectively hydrolyzed the collagens.