酶膜耦合法制备高聚合度壳寡糖

为了制备高聚合度壳聚糖,本研究通过对比分批酶解法和酶膜耦合法制备所得壳寡糖产物的聚合度差异,探索了利用酶膜耦合技术高效富集制备高聚合度壳寡糖的可行性。研究结果表明,酶膜耦合方法所得壳寡糖产物中DP 4~8壳寡糖的总收率高达78.1%,DP 4~8壳寡糖所占比例分别为16.5%、35.8%、18.9%、7.81%和5.12%。同时,以卷式膜系统代替板式膜系统,通过错流过滤的方式,可以有效降低实验过程中的不可逆膜污染(R_if=1.56×106 m-1),提高了料液底物浓度(30 g/L)。综上所述,本研究建立了一种基于酶膜耦合技术的高聚合度壳寡糖连续制备工艺,为高聚合度壳寡糖的应用和功能研究提供了基础。     

枯草芽孢杆菌壳聚糖酶在毕赤酵母中的高效表达及其酶解特性

研究枯草芽孢杆菌壳聚糖酶基因（BsCsn46）在巴斯德毕赤酵母（Pichia pastoris）GS115中的高效表达、重组酶性质及其酶解特性。重组菌在5 L发酵罐高密度发酵后胞外酶活力高达50 370 U/mL,蛋白质量浓度15.7 mg/mL。粗酶经强阴离子交换层析纯化,纯酶比活力为4 065.7 U/mg,最适pH 6.0,最适温度55 ℃,在45 ℃以下保持稳定。该酶水解3 g/100 mL壳聚糖得到主产物为二糖、三糖和四糖的壳寡糖,水解率为92.8%,壳寡糖得率为90.9%。本研究的重组壳聚糖酶产酶水平和水解效率高,为工业化制备壳聚糖酶及大规模制备壳寡糖的应用提供了理论支持。     

Process Characteristics of Hydrolysis of Chitosan in a Continuous Enzymatic Membrane Reactor

ABSTRACT: Crude enzyme from Bacillus cereus NTU-FC-4 was used to hydrolyze chitosan of 66% deacetylation in a membrane reactor, operated at 45 °C and pH 5, to continuously produce chitooligosaccharides. Major oligomers in the product from the reactor were chitobiose, chitotriose, chitotetraose, chitopentaose, and chitohexaose. When the membrane reactor was operated at an enzyme/substrate ratio of 0.2 (unit/mg) and residence time of 100 min, it reached steady state in 2.5 h. The system could be operated for 15 h and still maintained a stable product composition. When the volume replacement exceeded 2.5, the productivity of the membrane reactor became higher than that of the batch reactor, and the difference between them became even greater when the volume replacement was further increased. The apparent Michaelis constant (K_m) for the enzyme in the membrane reactor was 18.8 mg/mL, but the apparent K_m was 5.4 mg/mL for the batch reactor, suggesting that the affinity of the enzyme for chitosan was lower in the membrane reactor compared with the enzyme in the batch reactor. The estimated values of apparent V_max were 0.18 and 0.20 mg reducing sugar/mL/min for the enzyme in the membrane reactor and in the batch reactor, respectively, indicating that the enzyme activity was not greatly altered when used in the membrane reactor.     

Antioxidant activities of citrus herbal product extracts

Total phenolic content, DPPH free radical-scavenging activity, hydrogen peroxide-scavenging activity, ferrous ion-chelating activity and ferric-reducing antioxidant power (FRAP) of four citrus herbal products, Citri Reticulatae Pericarpium (CRP), Citri Reticulatae Viride Pericarpium (CRVP), Aurantii Immaturus Fructus (AIF) and Aurantii Fructus (AF) extracts were determined. EC₅₀ values of DPPH radical-scavenging activities ranged from 0.1 mg/ml (AF) to 1.59 mg/ml (AIF). EC₅₀ values of hydrogen peroxide-scavenging activities ranged from 0.08 mg/ml (AF) to 0.9 mg/ml (CRP). EC₅₀ values of ferrous ion-chelating activities ranged from 0.8 mg/ml (AF) to 2.08 mg/ml (AIF). The differences in DPPH free radical-scavenging activity, hydrogen peroxide-scavenging activity, and ferrous ion-chelating activity of all citrus herbal product extracts were significant. AF had the highest antioxidant activity. In this study, citrus herbal product extracts did not have good reducing power.     

A new assay of chitosan in the presence of protein by hydrolysis with commercial α-amylase

A simple and rapid method for the assay of chitosan concentration was developed employing hydrolysis of chitosan by α-amylase. The hydrolyzed products were mainly glucosamine, N-acetylated glucosamine, and chitooligosaccharides and were determined colorimetrically using 3,5-dimethylsalicylic acid. The result observed that the absorbance at 540 nm was linear (regression coefficient: 0.9967) with respect to the concentration of chitosan. The content of chitosan determined by this method was comparable with that determined by either colorimetrical method or HPLC method. The new method is specific for estimation of chitosan, particularly in the sample containing protein with high accuracy, and can assay chitosan content up to 3.2 mg/mL.     

Preparation of water soluble chitosan by hydrolysis with commercial α-amylase containing chitosanase activity

Water soluble chitosan (WSC) was prepared by hydrolysing chitosan using α-amylase. The degradation was monitored by WSC recovery and the WSC structure was characterised by Fourier transform infrared spectroscopy. The optimal hydrolysis conditions were found to be at pH 5, 50 °C, 20 mg α-amylase in the reaction solution, with a reaction time of 4 h. Under these conditions, the average molecular weight of chitosan decreased to 730 Da. The resulting products were composed of chitooligosaccharides of DP 3–7. The hydrolysate was neutralised with 1 M NaOH, filtered, concentrated to 16% (w/v), precipitated with five volumes of ethanol to one volume of the concentrated hydrolysate solution, and dried at 60 °C for 3 h to yield a white powder. The WSC content of the product and the yield were 96.2% and 91.2% (w/w), respectively.     

Process optimisation and physicochemical characterisation of enzymatic hydrolysates of proteins from co‐products of Atlantic Salmon (Salmo salar) and Yellowtail Kingfish (Seriola lalandi)

The aim of this study was to develop an enzymatic hydrolysis process of protein co‐products for two major commercial fish species in Australia: Atlantic salmon (AS) and Yellowtail kingfish (YTK). The outcomes are to produce high protein recovery of fish protein hydrolysates within controlled molecular weight ranges that display enhanced physicochemical properties of oil binding and emulsification. Three enzymes (Flavourzyme, Neutrase and Alcalase) were applied to processing co‐products. Protein recovery and physicochemical properties were evaluated with increasing hydrolysis time from 30 min to 180 min and ratio of enzyme to substrate (E/S) from 0.5% to 3.0%. In order to achieve a product with optimum emulsifying capacity (50 ± 0.6 m² g^?1), an E/S ratio of 0.6–1.3% Flavourzyme was applied for 30–111 min with a protein recovery of 55%; in order to achieve a product with optimum oil‐binding capacity (8.3 ± 0.3 g oil g hydrolysates^?1), an E/S ratio of 2.3–3.0% Flavourzyme was applied for 25–64 min with a protein recovery of 70%. YTK protein hydrolysates were further membrane‐fractionated into five fractions (>100 kDa, 50–100 kDa, 30–50 kDa, 10–30 kDa and <10 kDa), and of these, the 10–30 kDa exhibited the best properties of oil binding (19 ± 0.3 g oil g hydrolysates^?1) and emulsification (57 ± 0.7 m² g^?1). These results demonstrate the importance of enzymatic hydrolysis of seafood co‐products into high‐value ingredients for food products and processing.     

Susceptibility of stored product insects to high concentrations of ozone at different exposure intervals

Ozone is a highly reactive gas with insecticidal activity. Past studies have indicated that ozone technology has potential as a management tool to control insect pests in bulk grain storage facilities. The objective of this study was to determine the efficacy of short periods of exposure to high ozone concentrations to kill all life stages of red flour beetle (Tribolium castaneum (Herbst)) (Coleoptera: Tenebrionidae), and Indianmeal moth (Plodia interpunctella (Hübner)) (Lepidoptera: Pyralidae), adult maize weevil (Sitophilus zeamais (Motsch.)) (Coleoptera: Curculionidae) and adult rice weevil (S. oryzae (L)) (Coleoptera: Curculionidae). Insects were treated with six ozone concentrations between 50 and 1800 ppm. The specific objective was to determine minimal time needed to attain 100% mortality. The most ozone-tolerant stages of T. castaneum were pupae and eggs, which required a treatment of 180 min at 1800 ppm ozone to reach 100% mortality. Eggs of P. interpunctella also required 180 min at 1800 ppm ozone to reach 100% mortality. Ozone treatments of 1800 ppm for 120 min and 1800 ppm for 60 min were required to kill all adult S. zeamais and adult S. oryzae, respectively. The results indicate that high ozone concentrations reduce the treatment times significantly over previously described results. Our results also provide new baseline information about insect tolerance to ozone treatment.     

Enzymatic preparation of chitooligosaccharides by commercial lipase

The effect of a commercial lipase on chitosan degradation was investigated. When four chitosans with various degrees of deacetylation were used as substrates, the lipase showed higher optimal pH toward chitosan with higher DD (degree of deacetylation). The optimal temperature of the lipase was 55 °C for all chitosans. The enzyme exhibited higher activity to chitosans which were 82.8% and 73.2% deacetylated. Kinetics experiments show that chitosans with DD of 82.8% and 73.2% which resulted in lower K_m values had stronger affinity for the lipase. The chitosan hydrolysis carried out at 37 °C produced larger quantity of COS (chitooligosaccharides) than that at 55 °C when the reaction time was longer than 6 h, and COS yield of 24 h hydrolysis at 37 °C was 93.8%. Products analysis results demonstrate that the enzyme produced glucosamine and chitooligosaccharides with DP (degree of polymerization) of 2–6 and above, and it acted on chitosan in both exo- and endo-hydrolytic manner.     

Highly efficient preparation of free all-trans-astaxanthin from Haematococcus pluvialis extract by a rapid biocatalytic method based on crude extracellular enzyme extract

A highly efficient, rapid, green and safe procedure for the preparation of free all-trans-astaxanthin from Haematococcus pluvialis algal extract, by a crude extracellular enzyme extract, was reported. The free all-trans-astaxanthin obtained by the biocatalysed method had fewer side products compared to the saponification procedure. Through single-factor experiments and a Box–Behnken design, it was possible to find the optimal biocatalytic conditions for the hydrolysis of 2 mg of H. pluvialis oil with 14.7 mg (protein content) of lyophilised crude extracellular enzyme extract obtained from Pseudomonas aeruginosa. The reaction was carried out in 30 min at pH 9.16 and 36 °C, in 5.5 mL total reaction volume, under nitrogen atmosphere and dark conditions. The hydrolysis ratio of the astaxanthin esters was 98.72%, and the production of free all-trans-astaxanthin was 82.83 μg per mg of H. pluvialis oil. The method herein reported was simpler than other enzymatic methods previously described and allowed saving of time and costs.     

Characterisation of 2-Cys peroxiredoxin isozyme (Prx1) from Taiwanofungus camphorata (Niu-chang-chih): Expression and enzyme properties

Peroxiredoxins (Prxs) are a family of antioxidant peroxidases. The functions of Prxs comprises of cell protection against oxidative stress and regulation of cell proliferation. A putative 2-Cys Prx isozyme (Prx1) cDNA was cloned from Taiwanofungus camphorata (commonly known as Niu-chang-chih in Taiwan). The deduced amino acid sequence is conserved amongst the reported Prxs. A 3-D homology structure was created for this Prx1. To characterise the T. camphorata Prx1, the coding region was subcloned into a pAVD10 and transformed into Escherichia coli. The recombinant 6His-tagged Prx1 was expressed and purified by Ni²⁺-nitrilotriacetic acid sepharose. The purified enzyme showed two forms using a 15% SDS–PAGE. The enzyme retained 60% activity at 60 °C for 2.5 min. The enzyme was stable under a broad pH range from 5 to 11. The enzyme showed 57% activity after 40 min of incubation at 37 °C with trypsin. The ability of the enzyme to protect intact supercoiled plasmid DNA from ^·OH induced nicking was demonstrated.     

Structural modification and characterization of rice starch treated by Thermus aquaticus 4-α-glucanotransferase

Rice starch was modified using Thermus aquaticus 4-α-glucanotransferase (TAαGTase) in this study. The changes in the molecular structure and the effect on the starch retrogradation by TAαGTase treatment were investigated on isolated rice starch. By treating TAαGTase, molecular weight profile of amylopectins shifted to higher elution time from 1.0 × 10⁸ to 2.4 × 10⁷ or 0.8 × 10⁷, depending on the level of enzyme dosage. Meanwhile, there were huge increases in the proportions of content corresponding to amylose size and even smaller molecules. On treating with TAαGTase, short branch chains (DP 1–8) increased, and longer branch chains (>DP 19) increased significantly as well, with a broader distribution up to DP 46 compared to the control rice starch. Amylose content decreased from 30.0 to 21.8–23.7%. This indicated that the amylose could be transferred to the amylopectin branch chain by the disproportionation of TAαGTase, resulting in lowering the amylose content and the formation of amylopectin with a broader branch-chain length distribution. TAαGTase modified rice starch showed that X-ray diffraction pattern of the B-type crystalline even before cold storage, and that a variety of cyclic glucans (DP 5–19) were produced by enzymatic reaction. In particular, the accelerated rate of starch retrogradation was clearly observed compared to the control due to an overall increase in the number of elongated long-branch chains, decrease in the amount of amylose–lipid complex, and the possible synergistic effects of these factors.     

Composition and inhibitory activities towards digestive enzymes of polyphenolic-rich fractions of Davidson's plum and quandong

Davidson's plum (DP; Davidsonia pruriens, F. Muell, Cunoniaceae) and quandong (QD; Santalum acuminatum, A.D.C., Santalaceae) are two leading commercially grown native Australian fruits used in a variety of food products such as jam, chutney, sauces, wine or herbal teas. Within this study their inhibitory activities towards key enzymes relevant to metabolic syndrome: α-glucosidase, pancreatic lipase and angiotensin I-converting enzyme were evaluated and compared to these of reference samples: rabbit eye (REB, Vaccinium ashei J.M. Reade) and southern highbush (SHB, Vaccinium darrowii × Vaccinium corymbosum) blueberries. Polyphenolic-rich fraction obtained from DP comprised ellagic acid and ellagitannins accompanied by flavonoids: myricetin, quercetin, rutin, and anthocyanins. This fraction contained the highest level of total phenolics and exhibited superior antioxidant capacity (Ferric Reducing Antioxidant Power, FRAP and Oxygen Radical Absorbance Capacity, ORAC) to polyphenolic-rich fractions of QD, REB and SHB. DP closely followed REB and SHB in their inhibitory activities against isolated α-glucosidase (IC₅₀ of 0.13; 0.097 and 0.091, respectively) and was the most efficient inhibitor of angiotensin converting-enzyme (ACE; 91.2% inhibition at extract concentration of 1 mg/mL). Polyphenolic-rich fraction of QD comprising hydroxycinnamic acids, quercetin and cyanidin 3-glucoside was the most effective inhibitor of pancreatic lipase (IC₅₀: 0.60 mg/mL).     

Enzyme membrane reactor in isolation of antioxidative peptides from oil industry waste: A comparison with non-peptidic antioxidants

Edible oil industries suffer from the problem of seed meal utilization, which is recognized as a byproduct of edible oil. Present work has investigated production of peptide antioxidants, from oil seed meals to meet the increasing crave for natural antioxidants in food and pharmaceutical industries. Metallo-endopeptidase ‘Protease A Amano 2G’ (Aspergillus oryzae) was used to hydrolyze seed protein isolate in enzyme membrane reactor (EMR) and protein hydrolysate was sequentially fractionated by ultrafiltration to obtain potential peptide fraction. Degree of hydrolysis was varied within enzyme to substrate ratio 0.1–2 g/100 g and hydrolysis time 10–60 min to maximize peptide yield and antioxidant activity of peptides in vitro. Controlled hydrolysis with enzyme dose of 2 g/100 g, exhibited peptide yield of 4.60 ± 0.08 mg/100 mg meal protein in membrane reactor (DH 30.7%) and 4.23 ± 0.22 mg/100 mg meal protein in batch mode of hydrolysis (DH 29.3%). Antioxidant potential of peptide fractions were compared with commercial non-peptidic antioxidants and major findings confirm superior activity for protein fragments (IC₅₀-0.062 mg/mL) as compared to protein isolate (IC₅₀-0.038 mg/mL), with enhanced product stability by preventing oxidative deterioration. Results substantiate the prospect of EMR in obtaining peptides from edible oil industry waste, having comparable antioxidative potential with commercial non-peptidic antioxidants.     

响应面法优化微波辅助果胶酶制备壳寡糖的工艺

为优化壳寡糖制备工艺,提高壳寡糖得率,以壳聚糖为原料,采用微波法辅助果胶酶酶解壳聚糖制备壳寡糖,以还原糖含量作为壳寡糖的产率依据,通过单因素实验和响应面实验确定最佳工艺条件为:果胶酶加酶量2100 U/g,微波功率510 W,pH4.4,反应温度50℃,在此工艺条件下获得的降解产物中还原糖浓度为1.964 mg/mL,与单一果胶酶酶解法(1.747 mg/mL)相比提高了12%,与单一微波法(1.671 mg/mL)相比提高了18%。     

Performance of enzymatic wheat gluten hydrolysis in batch and continuous processes using Flavourzyme

The aim of this study was to investigate the process performance of wheat gluten hydrolysis with Flavourzyme in the conventional batch and in an enzyme membrane reactor system. The release of amino acids and peptides from wheat gluten using Flavourzyme in a batch process (T 50 °C, pH 5) is limited due to product inhibition. Investigation of Flavourzyme inhibition kinetics by l-amino acids revealed that the strongest inhibition was observed by l-cysteine. Thus, application of an enzyme membrane reactor with continuous product removal and substrate and enzyme retention was assessed. The highest productivity in the enzyme membrane reactor system was found using a 10 kDa molecular weight cut-off polyethersulfone membrane. This resulted in an increased product enzyme ratio from 2.1 mg product/nkat in the conventional batch process to 4.9 mg product/nkat with the enzyme membrane reactor after 20 h. The enzyme membrane reactor retained 80% of the initial space-time yield (STY_init 5 g/L/h) after nine days.     

Enzymatic Processing of Rice Husk Autohydrolysis Products for Obtaining Low Molecular Weight Oligosaccharides

Rice husks were enzymatically treated in aqueous media to cause the hydrolytic degradation of xylan. The reaction liquors (raw or refined by nanofiltration and ion exchange) were treated with endoxylanases to reduce the degree of polymerisation (DP) of the reaction products. Operating at the optimal temperature and pH of each enzyme, the effects of enzyme loading and reaction time were assessed. The enzyme Shearzyme 2x led to the highest proportion of oligomers with DP in the range 2-3, whereas the other endoxylanases (Pentopan Mono BG and Pulpzyme HC) gave mainly oligomers with DP in the range 4-5 or 6-7.     

蜡样芽孢杆菌D-11降解粉末壳聚糖制备水溶性低聚糖的研究

为了省去制备胶状壳聚糖的工艺,能有效降解粉末壳聚糖利用到低聚糖的生产,利用自然环境中筛选得到的产壳聚糖酶菌株蜡样芽孢杆菌D-11,进行了基质条件优化实验。结果表明,以筛孔分别为20、40、60、80目的粉末壳聚糖(3±0.5 cm;脱乙酰度98.1%)为基质,培养液含有1%的吐温60时,降解粉末壳聚糖的能力比对照提高12%,当筛孔60目的粉末壳聚糖为碳源(浓度0.6%)时,降解粉末壳聚糖的能力提高62.5%。随着基质每毫克中酶活性的提高,对粉末壳聚糖的分解率也随之增加。同时利用薄层色谱法分析低聚糖的分布,有效证明了该D-11菌株具有降解粉末壳聚糖的能力,且发现壳聚糖酶对基质粉末状态的降解率有选择性。     

Physico-chemical Properties, Vitamin C Content, and Antimicrobial Properties of Pomegranate Fruit (Punica granatum L.)

Pomegranate (Punica granatum L.) fruit is widely used in the food and process industries due to its excellent nutritional and health value and as a raw material for the manufacture of secondary products such as jellies, dyes, and cosmetics. The objectives of this research were to determine the vitamin C content and antimicrobial properties of fresh and dried fractions of fruit peel and arils of locally grown and imported pomegranate in Oman. A significant variation in vitamin C content was found among the five varieties of pomegranate studied, ranging from 52.8 to 72.0 mg/100 g fresh weight (fw) for arils and 76.8 to 118.4 mg/100 g fw for peels. Irrespective of the variety of pomegranate, vitamin C content in the peel was significantly higher than the aril, with differences ranging from 24.4% to 97.0% depending on variety. Fruit fractions showed antimicrobial effects (inhibition zone) on Staphylococcus aureus and Pseudomonas aeruginosa but not Escherichia coli. Sun drying of fruit peel significantly (p ≤ 0.05) enhanced vitamin C retention and antimicrobial effects in comparison with oven drying presumably due to lower rate of moisture removal associated with low temperature drying over longer duration in comparison with short-time high-temperature oven drying.     

Composition and biological activities of the essential oil from Schisandra chinensis obtained by solvent-free microwave extraction

Applicability of solvent-free microwave extraction (SFME) for extraction of the fruits of Schisandra chinensis essential oil was examined; the composition and antioxidant activities and antibacterial activities of the essential oil were assessed in vitro. An orthogonal experiment (L₉ (3)⁴) was applied to optimize the extraction process. The optimum conditions were: extraction time, 45 min; microwave power, 800 W; diameter of powder particles, 0.25 mm; and proportion of water pretreatment, 30%. Under these conditions, the extraction yield was 1.75%. Thirty-five compounds, representing 91.12% of the oil, were identified, of which the major ones, ylangene (50.11%), β-himachalene (10.76%)，α-bergamotene (9.52%) and β-Chamigrene (5.41%), accounted for of 75.80% the oil.Antioxidant activity, IC₅₀ value of the essential oil was determined as 3.87 mg/mL by DPPH assay, and the inhibition values of the essential oil at 1.8 mg/mL was 41.88% by β-Carotene–linoleic acid bleaching assay. The essential oil was screened for antibacterial activity against both Gram positive (Staphylococcus epidermidis, Staphylococcus aureus, Bacillus subtilis) and Gram negative (Escherichia coli, Pseudomonas aeruginosa, Proteus vulgaris) bacteria. The essential oil showed antibacterial effect against all the gram (+) bacteria and gram (−) bacteria tested. These results show that S. chinensis essential oil could be considered as a natural alternative to food antioxidants and preservatives.