固定枯草芽孢杆菌发酵生产类细菌素的研究

以海藻酸钙为材料,固定枯草芽孢杆菌(Bacillussubtilis)LL18-4,研究了不同条件下类细菌素效价的变化情况。结果表明,利用3%海藻酸钠在2%CaCl2条件下,得到固定化细胞颗粒的类细菌素效价和稳定性较好,可维持216h无破裂;对其固定化颗粒进行发酵条件的研究发现,培养温度37℃,pH值6.5,接种量1%,72h静止培养,固定菌所产类细菌素的抑菌活性高,最高效价为334.2u/mL。通过216h3批次循环的半连续培养后,固定菌活性仍能维持在260.9u/mL以上。     

Continuous Hydrolysis of Fructans in Jerusalem Artichoke Extracts using Immobilized Nonviable Cells of Kluyveromyces marxianus

Non-viable immobilized cells of Kluyveromyces marxianus in alginate beads having inulase (ß-2,1-fructan fructano-hydrolyase E.C.3.2.1.7) acitivity were used as biocatalyst in a packed bed reactor. Extracts of Jerusalem artichoke tubers were contacted with the biocatalyst for continuous conversion of the fructan component to fructose. In a bed reactor packed with 100 mL of beads, a volumetric productivity of 136 g/L/hr total reducing sugars was obtained with 98% substrate conversion. When operated continuously for 30 days, a 55% loss in the original activity was observed, giving a half life for the biocatalyst of 28 days.     

Technical note: Application of immobilized cells of Kluyveromyces marxianus for continuous hydrolysis to fructose of fructans in Jerusalem artichoke extracts

Dead cells of Kluyveromyces marxianus having inulase (β-D-fructo fructanohydrolase E.C.3.2.1.7) activity were immobilized in alginate beads and used as a biocatalyst in a packed bed reactor and a stirred batch reactor. Fructosans of Jerusalem artichoke tubers, after extraction, were utilized for continuous or semi-continuous production of fructose. In a bed reactor packed with 100 ml of beads, a volumetric productivity of 36 g/l/hr total reducing sugars was obtained with 98% substrate conversion. When operated continuously for 30 days, a half life of 28 days was observed for the biocatalyst. Using artichoke extract containing 20% fructan solution, 98% conversion could be achieved in a batch reactor in 20 hr. Repeated cycling of beads resulted in considerable loss of catalyst from the reactor and subsequent loss in catalytic activity, thus giving a half life of only 14 days.     

Configuration of a bioreactor for milk lactose hydrolysis

Permeabilized microbial cells can be used as a crude enzyme preparation for industrial applications. Immobilization and process recycling can compensate for the low specific activity of this preparation. For biomass immobilization, the common support is alginate beads; however, its low surface area and the low biomass concentration limit the activity. We here describe a biocatalyst consisting of a paste of permeabilized Kluyveromyces lactis cells gelled with manganese alginate over a semicircular stainless steel screen. A ratio of wet permeabilized biomass to alginate of 50:4 (wt/wt) resulted in a paste with maximum immobilized beta-galactosidase activity and maximum gel biomass retention. The biocatalysts retained activity better when stored in milk at 4 degrees C than in 50% glycerol. The unused biocatalysts stored in milk did not lose activity after 50 d. However, repeated use of the same biocatalyst 40 times resulted in almost 50% loss of activity. A bioreactor design with two different conditions of operation were tested for milk lactose hydrolysis using this biocatalyst. The bioreactor was operated at 40 degrees C as packed bed or with recirculation, similar to a continuous stirred tank reactor. The continuous system with recirculation resulted in 82.9% lactose hydrolysis at a residence time of 285.5 min (flow of 2.0 ml/min), indicating the potential of this system for processing low lactose milk, or even in processing other substrates, using an appropriate biocatalyst.     

Release of embryogenic carrot cells with high regeneration potency from immobilized alginate beads

For the mass-production of regenerated carrot plantlets, embryogenic carrot callus immobilized in calcium alginate gel beads was cultivated in a growth medium and the regeneration frequency of cells released from alginate gel beads was compared with that in a suspension culture. Cells released in the immobilized culture were regenerated at a frequency which was about 1.5 times higher than that obtained in the suspension culture. When CaCl2 was added to the growth medium at 5 mM, repeated batch culture for plantlet production continued for 245 d with no significant decrease in the productivity (1.6 x 10(5) plantlets/l-medium/d).     

The potential of immobilized cell technology to produce freeze-dried, phage-protected cultures of Lactococcus lactis

Lactococcus lactis cells were immobilized in calcium alginate beads and added to growth media to enable biomass production in the gel. The immobilized population was then freeze-dried. Survival during freeze-drying (FD), stability upon high-temperature storage and residual humidity were evaluated. This culture was compared to a classical liquid starter and fresh beads of immobilized L. lactis in simulated quarg manufacture. Acidifying characteristics, proteolytic activity, sensitivity to bacteriophage and sensory properties of quarg products were evaluated.

The population in the beads prior to FD was 7 × 10¹⁰ CFU/g. The best survival rate to FD (62–79%) was obtained when the beads were mixed with a milk-based protective solution. In the absence of protective ingredients (milk solids, sucrose and vitamin C) the alginate beads had high water content following FD. Survival of the immobilized cells during FD was as high for immobilized cells as that of free cells. Stability of the immobilized cells at high-temperature storage (45–55°C) was higher than for the free cells. Quarg cheese was succesfully produced in 5 h with the immobilized freeze-dried (IFD) culture, but sensory evaluation confirmed a significant texture difference between cheeses made with free or IFD cultures. Higher inoculation rates were required with the IFD culture to obtain the same acidifying activity as classical fresh liquid starters. The IFD culture performed well under phage contamination; following a 6-h fermentation, 30% of the cells remained viable and active in the phage-contaminated sample. Increase in non-protein amino compounds (0·2 g/100 g cheese) over a 30-day storage period at 4°C was similar in quarg cheeses made with fresh or IFD starters, despite the higher inoculation rate used with the IFD culture.     

海藻酸钠-壳聚糖-海藻酸钠微胶囊二步法固定化β-葡萄糖苷酶的制备

以二步法制备的ACA微胶囊为载体,对β-葡萄糖苷酶进行固定化,以固定化β-葡萄糖苷酶的酶比活力和酶的稳定性为考查指标,对影响二步法制备固定化β-葡萄糖苷酶的各因素及其性质进行了探讨。ACA微胶囊二步法固定β-葡萄糖苷酶的优化条件是:3.5%海藻酸钠溶解0.15g酶,逐滴滴入到2%的CaCl2溶液中引发25min,所形成微球先在0.4%壳聚糖(0.5%(v/v)醋酸溶解)溶液中进行成膜反应,再在0.2%海藻酸钠进行覆膜反应,然后用1%戊二醛交联4h(4℃)。用上述最适条件制备固定化酶,总酶活的回收率为68.3%。4℃下贮藏,固定化β-葡萄糖苷酶的酶活力在一个月内保持稳定,重复使用3次后其活力仍保持在原来的80%以上。固定化酶反应的最适温度是60℃,最适pH是4.6。     

固定化增殖细胞发酵半纤维素糖类的研究

采用海藻酸钠包埋及进一步增殖培养，将休哈塔假丝酵母（ＣａｎｄｉｄａｓｈｅｈａｔａｅＲ）的细胞密集固定在凝胶珠表层，大大减小了传递阻力，为该菌的“半好气”发酵创造了有利条件。试验结果表明，ＣａＣｌ２浓度以２％为宜，凝胶中掺入适量的Ａｌ２Ｏ３（１．２％）能明显增加凝胶珠的机械强度及使用寿命。固定化增殖细胞能够利用己糖和戊糖及８０ｇ／ｌ的混合糖（己糖与戊糖各占一半）。经１２ｈ发酵，糖的利用率达９０．５％（相同条件下的游离细胞发酵需４８ｈ）。混合糖发酵的适宜条件为：温度３４～３６℃；初始糖浓度８０ｇ／Ｌ；通气量３．３ｍｌ／ｍｌ发酵体积·ｈ。固定化休哈塔假丝酵母能成功地发酵玉米秆水解液、杨木水解液及亚硫酸盐制浆废液，酒精得率达理论值的９０％以上，具有广阔的实际应用前景。     

海藻酸钠固定灵芝细胞对胞外三萜类产量的影响

采用海藻酸钠和氯化钙对灵芝细胞进行固定,研究包埋法固定灵芝细胞的工艺条件,探讨固定化工艺对灵芝胞外三萜类产量的影响。结果表明:固定灵芝细胞的最佳工艺条件为:4.5%海藻酸钠、2.2%CaCl2、培养基pH值自然,接种40个固定化小球,28℃、110r/min培养12d,胞外三萜类产量为(49.53±1.37)×10-2mg/mL。固定化灵芝细胞较未固定细胞,具有更好的耐酸、耐碱性,长时间培养仍具有较高的产三萜类能力,回收利用次数可达5次。     

Immobilized bacterial α-amylase for effective hydrolysis of raw and soluble starch

The major concern in an enzymatic process is the instability of the enzyme under repetitive or prolonged use and inhibition by high substrate and product concentration. Immobilization is a very effective alternative in overcoming problems of instability and repetitive use of enzymes. Entrapment method of immobilization is advantageous over other methods as they do not involve chemical modification of the enzyme. α-Amylase produced by Bacillus amyloliquefaciens ATCC 23842 was immobilized in calcium alginate beads and used for the effective hydrolysis of soluble and raw potato starch which was comparable to the free enzyme. The levels of parameters (sodium alginate, calcium chloride and curing time) that significantly influence the immobilization of α-amylase in calcium alginate were analyzed and optimized using response surface methodology. Reactor studies were performed to study the reusability and operational stability of the beads. The alginate beads retained more than 60% of their initial efficiency after five batches of successive use and 40% of efficiency was exhibited in the 6th and 7th batch run of 6 h duration.     

菠萝皮渣羧甲基纤维素/海藻酸钠复合水凝胶珠固定化菠萝蛋白酶的制备及稳定性研究

本实验以菠萝皮渣羧甲基纤维素、海藻酸钠为原料,制备了菠萝皮渣羧甲基纤维素/海藻酸钠复合水凝胶珠,用于固定化菠萝蛋白酶。采用单因素法分析菠萝皮渣羧甲基纤维素与海藻酸钠的质量比、氯化钙的浓度、菠萝蛋白酶浓度、戊二醛体积分数和交联时间对固定化酶活性的影响。结果表明,固定化酶的优化制备工艺为:菠萝皮渣羧甲基纤维素与海藻酸钠的质量比为2:3,氯化钙的浓度为1.0%,菠萝蛋白酶浓度为2.0 mg/mL,戊二醛体积分数为1.0%,交联时间为60 min。制备的固定化酶比游离酶具有更好的热稳定性,在80℃环境下放置2.0 h后,固定化酶的相对酶活性为35.1%,而游离菠萝蛋白酶在此条件下几乎失活;在pH为11条件下放置24 h后,游离酶的相对酶活性为43.2%,而固定化酶相对酶活性为85.1%,说明固定化酶比游离酶更耐受碱性环境。另外,固定化酶重复使用7次后,相对酶活性为60.5%,说明制备的固定化酶具有较好的重复使用性能。     

Alginate and Carrageenan Immobilization Effects on Schizosaccharomyces Pombe Activity and Stability

Calcium alginate and kappa-carrageenan were evaluated as entrapment matrices for whole cells of Schizosaccharomyces pombe. The fermentation activity of immobilized cells was enhanced by preincubation in nutrient medium. Alginate bead activity was significantly better than that with carrageenen beads (p<0.05). In grape must carrageenan beads disintegrated 10 times faster than alginate beads.     

LIPASE-CATALYZED ACYLATION OF MENTHOL WITH VINYL ACETATE IN ORGANIC MEDIA

Menthyl acetate was prepared using vinyl acetate with lipase PS-30 from Pseudomonas sp . as biocatalyst in organic media. Immobilization of the lipase by adsorption onto 0.5 mm diameter glass beads and the presence of molecular sieves improved the yield of menthyl acetate from 24 to 50%. Approximately 40% yield of menthyl acetate was achieved after 48 h incubation with the immobilized lipase. Equimolar ratio of substrates gave higher yields of product compared to higher mole ratios. Synthesis of menthyl acetate increased as the reaction temperature increased from 20 to 60C. An acceptable yield of menthyl acetate was achieved with 400 units of immobilized lipase PS. Less polar solvents appeared to be better than polar solvents for the enzymatic synthesis of menthyl acetate by transesterification.     

共固定化生产高含量低聚果糖的研究

生产低聚果糖过程中，副产物葡萄糖抑制酶反应，降低了产物中低聚果糖的含量。本文采用两种固定化方法，通过去除或转化葡萄糖，从而制备出高含量的低聚果糖。首先以２％戊二醛和０．１％丹宁于２０℃时处理固定化黑曲霉菌体与葡萄糖氧化酶，将菌体与酶共包埋得到固定化颗粒，所得到的共包埋产物于５０℃ｐＨ５．０条件下与５０％蔗糖溶液摇瓶反应２４ｈ，得到７１％的低聚果糖；又采用固定化黑曲霉增殖细胞与固定化葡萄糖异构酶协同作用方法，将５０％蔗糖溶液通入柱式反应器，连续生产得到高含量低聚果糖，产物中低聚果糖和果糖含量分别为６３％和１６％。     

Calcium alginate-immobilized cultures of lactic Streptococci are protected from bacteriophages

Calcium alginate-immobilized cultures of lactic streptococci were grown in milk and assessed for their sensitivity to homologous bacteriophage, proteolytic activity, and acid production. Immobilized cultures of Streptococcus lactis C2 and Streptococcus cremoris HP were protected from attack by bacteriophage due to the exclusion of phage particles from the gel matrix. These cultures were also functionally proteinase-deficient when immobilized in calcium alginate beads and grown in milk. Acid was produced by immobilized cultures at a lower rate than cells freely suspended in milk due in part to the inability of the immobilized cells to hydrolyze milk proteins. Agitation of immobilized cultures slightly increased acid production, suggesting that diffusional limitations of substrate into the beads contributed to decreased acid production. Use of immobilized cultures of lactic streptococci in certain dairy fermentations may be advantageous due to the protection of these cultures from attack by virulent bacteriophage.     

海藻酸钠固定化果胶酶的研究

以海藻酸钠为载体,采用交联吸附法固定果胶酶。通过单因素实验和正交实验考察固定化主要因素对固定化酶活力的影响,优化固定条件。结果表明,在海藻酸钠浓度为2%、氯化钙为1%、戊二醛浓度为3%的条件下,采用酶浓度为0.2mg/mL、pH值3.0、温度为40℃、固定时间为45min。固定化果胶酶活力最高,酶活回收率达到84.4%。     

固定化酵母发酵产酒精载体选择的研究

以海藻酸钙和甘蔗块为载体固定酵母细胞,进行蔗汁和废糖蜜酒精发酵。结果表明,以甘蔗汁为发酵培养基时甘蔗块固定化酵母发酵液中平均残糖锤度(20℃)比海藻酸钙包埋酵母发酵低0.36,酒精平均体积分数比海藻酸钙包埋酵母发酵高0.20%;以废糖蜜为发酵培养基时甘蔗块固定化酵母发酵液中平均残糖锤度(20℃)比海藻酸钙包埋酵母发酵低0.43,酒精平均体积分数比海藻酸钙包埋酵母发酵高0.23%,显示出甘蔗块固定化法酵母发酵优于海藻酸钙包埋法固定化酵母。此外,甘蔗汁培养基与废糖蜜培养基对总体发酵效果的影响非常接近,但综合考虑甘蔗汁与废糖蜜的成本,废糖蜜是工业发酵生产乙醇用培养基的更优选择。     

Stability evaluation of an immobilized enzyme system for inulin hydrolysis

The stability of free and Amberlite-immobilized inulinase, aiming at inulin hydrolysis was evaluated. The apparent activation energy of the biotransformation decreased when the immobilized biocatalyst was used, suggesting diffusional limitations, despite a decrease in the optimal temperature for catalytic activity for the immobilized biocatalyst. Thermal deactivation, of both forms of the biocatalyst, was evaluated by the linear inverted model. Inulinase immobilization consistently enhanced half-life of the enzyme, which increased up to 6-fold, as compared to the free form. Mean enzymatic activity was computed for both forms of the biocatalyst, and evidenced a decrease of optimal temperature with increased incubation periods. The deactivation energies estimated by an Arrhenius plot, evidenced a decrease of roughly 20% when free inulinase was used. The immobilized biocatalyst was effectively reused in successive batch runs for the hydrolysis of a 5% inulin solution.     

Mead production: fermentative performance of yeasts entrapped in different concentrations of alginate

Mead is an alcoholic drink known since ancient times, produced by yeast fermenting diluted honey. However, the production of mead has suffered in recent years, partially owing to the lack of scientific progress in this field. In this study, two strains of Saccharomyces cerevisiae, QA23 and ICVD47, were immobilized in 2 or 4% (w/v) alginate beads to assess the most effective alginate concentration for yeast immobilization to produce mead. Neither of the alginate concentrations was able to prevent cell leakage from the beads. The fermentation length was 120 h for both yeast strains. In all cases, at the end of the fermentation, the number of cells entrapped in the beads was higher than the number of free cells, and the total 4% alginate bead wet weight was significantly higher than the 2% alginate bead wet weight. In addition, the evaluation of mead quality showed that the yeast strain had significantly more influence on the physicochemical characteristics than the alginate concentration. Although the yeasts immobilized in the two alginate concentrations were able to perform the fermentation, further research is needed in order to understand the evolution of the yeast population inside the beads throughout the fermentative process. Copyright © 2014 The Institute of Brewing & Distilling     

海藻酸锰固定化酵母在木薯淀粉酒精发酵工艺中的应用研究

利用Mn2+对Ca2+呈换后形成的海藻酸锰固定化酵母细胞对木薯淀粉双酶法水解后的糖化液进行发酵.正交试验对海藻酸锰固定化细胞的固化条件和发酵条件进行了优化,固定化最佳条件为CaCl2浓度0.2mol/L,海藻酸钠浓度2.0％,MnSO4浓度1.2％.发酵最佳条件为初还原糖浓度20％,颗粒填充率30％,发酵液pH值为5.0.强度实验表明,海藻酸锰固定化酵母细胞的耐磷酸盐能力是海藻酸钙固定化酵母细胞的3倍左右.对海藻酸锰、海藻酸钙固定化酵母细胞进行13批次发酵对比试验,表明第3批次后海藻酸锰固定化酵母细胞的糖利用率和酒精度都比海藻酸钙固定化酵母细胞要高.