Continuous production of sialyllactose from colominic acid using a membrane reactor

Continuous production of sialyllactose, as a typical sialylsaccharide, was examined using a membrane reactor. The synthesis of sialyllactose through the transfer reaction catalyzed by neuraminidase, has been reported to be a suitable process for industrial production, but it still has drawbacks such as a low yield, high enzyme cost, and hydrolysis by the enzyme of sialyllactose formed. We attempted to solve these problems by utilizing an appropriate membrane reactor system. We first investigated the effects of various reaction conditions on sialyllactose productivity, and found that the productivity was independent of the enzyme concentration and reaction temperature but dependent on the substrate and buffer concentrations and the hydraulic retention time (HRT). We then selected a suitable membrane that allowed sialyllactose to permeate but rejected the substrate and enzyme. Finally, we constructed a membrane reactor system with a cut-off molecular weight of 3000 and applied it to continuous sialyllactose production from colominic acid at an HRT of 80 min. Using substrate concentration of 25 g/l the system performed with a high level of productivity and gave a good yield, while maintaining high transfer ratio of 4-5% over a 160-h test period.     

废蜜制取乳酸新菌种

回顾了利用糖蜜等有机化合物进行乳酸生产的技术和菌种研究进展。发酵工业上应用最多的生产乳酸的原料是葡萄糖、蔗糖、淀粉等糖类物质，虽然用纯糖物质发酵得到的乳酸产率高，提取也相对容易，但用高价的糖生产低价格的乳酸在经济上是不合算的，且还浪费了资源。利用含丰富碳水化合物的有机废弃物发酵生产乳酸，不但可以解决废弃物的资源化问题，还能降低乳酸的生产成本，具有广阔的开发前景，甜菜废蜜就是一种可以利用的底物。     

Continuous production of tempe-based bioactive peptides using an automated enzymatic membrane reactor

The utilization of bioactive peptides from food-grade raw materials has received increasing interest, especially for the production of functional foods. Within this study, a combination of fermentation and in vitro papain hydrolysis was devised to produce bioactive peptides exhibiting antioxidant property and angiotensin I-converting enzyme (AICE) inhibitory activity. The utilization of an automated membrane reactor system developed in this study could facilitate the continuous hydrolysis of tempe flour-rich in protein under constant flux, thus constant residence time operation. The optimum operating conditions were: [E]/[S] = 10% (w/v) and τ = 9 h. With a 10-kDa membrane filtration, the resulted antioxidant capacity and AICE inhibition were 0.033 mg AEAC/mL and 50.9%, respectively. Further separation of permeate with a 2-kDa membrane cut-off leveled off the antioxidant capacity but increased the AICE inhibitory activity (i.e., Reduction of IC₅₀ of ~40%). By this, the obtained IC₅₀ for AICE was 0.08 mg protein/mL, and this value was comparable with the IC₅₀ values reported in the literature.Industrial relevanceThe production of bioactive peptides from soybean through a combination of fermentation and in vitro enzymatic hydrolysis is limitedly reported. In this work, fermented soybean (tempe) was further hydrolyzed continuously using an automated membrane reactor. The hydrolysis under optimum operating conditions could increase the antioxidant activity and AICE inhibition of permeate. This work is expected to increase the commercialization of tempe-based food products by using safer technological approaches (i.e., fermentation and membrane technology).     

A mini-scale mass production and separation system for secretory heterologous proteins by perfusion culture of recombinant Pichia pastoris using a shaken ceramic membrane flask

The perfusion culture technique using a shaken ceramic membrane flask (SCM flask) was applied to the production of a secretory heterologous protein. A recombinant methylotrophic yeast strain, Pichia pastoris, was cultured aerobically on a reciprocal shaker using an SCM flask. High-level production of human serum albumin (HSA) was attempted by increasing both the cell concentration and the expression level of the recombinant gene. In the two-stage culture method, the cell concentration was first raised to 17 g/l by feeding glycerol, after which the expression of HSA was induced by feeding methanol. However, the concentration of HSA in the effluent filtrate was as low as 0.15 g/l, while the cell concentration continued to increase. In contrast, HSA was effectively produced by feeding methanol from an early stage of the culture. In this case, the HSA concentration reached 0.24 and 0.46 g/l, respectively, using the growth-associated production method without and with aeration into the head space of the SCM flask. The results showed that supplying sufficient oxygen together with the growth-associated induction method are effective for obtaining high-level expression of the methanol-inducible recombinant gene of P. pastoris. An HSA concentration in the filtrate of 1.5 g/l was finally achieved when the cell concentration was increased to 53 g/l by supplying oxygen-enriched gas to the SCM flask. The yield and productivity of HSA reached 2.6-fold and 10-fold those obtained in an ordinary fed-batch culture using a shake flask, and these levels were readily achieved by continuous replenishment of the culture supernatant. The achievements made in this study should contribute to the development of a handy bioreactor system for mini-scale mass production of target proteins with separation at high purity.     

利用甘蔗糖蜜厌氧发酵产丁二酸的研究

对甘蔗糖蜜作为廉价碳源厌氧发酵制备丁二酸进行初步研究.实验证明,Actinobacillus succinogenes NJ113能够利用葡萄糖、果糖、蔗糖等碳源,制备丁二酸为菌体利用廉价甘蔗糖蜜作碳源进行丁二酸发酵提供可行性依据.通过比较不同的糖蜜预处理方法,得出经硫酸处理的糖蜜发酵效果最好,丁二酸浓度37.73g/L,比未经处理的糖蜜所产丁二酸浓度高12.6%.考察不同的糖蜜添加量对发酵结果的影响表明,初始总糖浓度为65g/L时,丁二酸的产量最高为49.63g/L;在3L罐中进行放大实验,丁二酸产量46.91g/L,质量收率为72.2%,分别比混合糖(含蔗糖、果糖、葡萄糖)的发酵结果高9.8%、10%.     

利用CSTR生物制氢系统厌氧发酵的研究现状

生物制氢技术在开发能源方面具有重要的现实地位,连续流搅拌槽式反应系统（CSTR）采用机械搅拌,传质效率高,可有效提高产氢效率,同时此设备技术要求较为简单,能大幅降低生物制氢的成本,易于实现氢气的工业化生产.本文阐述了连续流搅拌槽式反应器（CSTR）生物制氢系统的研究进展,着重剖析了CSTR生物制氢系统的影响因素、底物范围和相关的技术研究,并对其发展和实际应用进行了简要论述.     

Preventing phage lysis of Lactococcus lactis in cheese production using a neutralizing heavy-chain antibody fragment from llama

Bacteriophage infection is still a persistent problem in large dairy processes despite extensive studies over the last decades. Consequently, new methods are constantly sought to prevent phage infection. In this paper, we show that phage neutralizing heavy-chain antibody fragments, obtained from Camelidae and produced at a large scale in the generally regarded as safe microorganism Saccharomyces cerevisiae, can effectively be used to impede phage induced lysis during a cheese process. The growth inhibition of the cheese starter culture by 10(5) pfu/ml cheese-milk of the small isometric-headed 936-type phage p2 was prevented by the addition of only 0.1 microg/ml (7 nM) of the neutralizing antibody fragment. The use of such antibody fragments in cheese manufacturing are a realistic and interesting option because of the small amount of antibody fragments that are needed. Moreover the antibodies are produced in a food grade microorganism and can easily be isolated from the fermentation liquid in a pure and DNA free form.     

Growth stimulation of a proteinase positive Lactococcus lactis strain by a proteinase negative Lactococcus lactis strain

Lactococcus lactis AMP15/pAMP31(D471R) is a proteinase negative, lactose negative strain with a modified oligopeptide transport system, and potential as a debittering agent due to its efficient utilization of hydrophobic peptides. Five wild L. lactis strains of dairy origin, which produced cheeses of high flavour quality, were cocultured with L. lactis AMP15/pAMP31(D471R) in an attempt to select adequate combinations of strains for use as defined cheese starters with potential debittering ability. Four of these strains, L. lactis B6, K16, M21 and P21, inhibited growth of L. lactis AMP15/pAMP31(D471R) at a level of 10(6) to 10(7) cfu mL(-1) after 24 h of incubation, even though production of bacteriocin-like compounds could only be proven for L. lactis M21. When L. lactis AMP15/pAMP31(D471R) was cocultured with the fifth strain, L. lactis N22, its growth was significantly (P<0.001) inhibited whereas growth of L. lactis N22 was significantly stimulated. The nature of the interaction was studied and it was established that L. lactis N22 is auxotrophic for folate, a compound produced and excreted by L. lactis AMP15/pAMP31(D471R).     

Hydrogen production by fermentation using acetic acid and lactic acid

Microbial hydrogen production from sho-chu post-distillation slurry solution (slurry solution) containing large amounts of organic acids was investigated. The highest hydrogen producer, Clostridium diolis JPCC H-3, was isolated from natural environment and produced hydrogen at 6.03+/-0.15 ml from 5 ml slurry solution in 30 h. Interestingly, the concentration of acetic acid and lactic acid in the slurry solution decreased during hydrogen production. The substrates for hydrogen production by C. diolis JPCC H-3, in particular organic acids, were investigated in an artificial medium. No hydrogen was produced from acetic acid, propionic acid, succinic acid, or citric acid on their own. Hydrogen and butyric acid were produced from a mixture of acetic acid and lactic acid, showing that C. diolis. JPCC H-3 could produce hydrogen from acetic acid and lactic acid. Furthermore, calculation of the Gibbs free energy strongly suggests that this reaction would proceed. In this paper, we describe for the first time microbial hydrogen production from acetic acid and lactic acid by fermentation.     

Optimisation of bacteriocin production of Lactococcus lactis subsp. lactis MA23, a strain isolated from Boza

Lactococcus lactis subsp. lactis MA23 produces a bacteriocin (6400 AU/mL) that inhibits the growth of many Gram‐positive bacteria but is not active against Gram‐negative bacteria. This bacteriocin inhibits growth of lactococcal strains that are producing nisin, lacticin or lactococcin suggesting it to be different from these bacteriocins. The nutritional requirements and optimal growth conditions for MA23 bacteriocin production were studied with fed‐batch fermentations. The optimal pH, carbon source and nitrogen source for bacteriocin production were pH 6.5, sucrose (0.5%) and yeast extract (1%), respectively.     

腐乳源乳酸乳球菌17M1高密度培养条件研究

为了获得乳酸乳球菌(Lactococcus lactis)17M1的高密度培养方法,以菌体密度和活菌数为评价指标,以豆腐乳清为基础培养基,采用响应面法对该菌株的培养基组成进行优化,并研究其培养条件。结果表明,菌株17M1的最佳培养基组成为：在豆腐乳清中加入2.00%大豆蛋白胨、胰酪蛋白胨1.70%、柠檬酸铵2.30%。在此优化培养基中初始pH值6.15,于37℃培养15 h,乳酸乳球菌17M1的活菌数达到了1.17×10¹⁰CFU/mL,高于M17肉汤培养基活菌数(1.04×10⁹CFU/mL)。该研究结果为乳酸乳球菌17M1的工业化应用提供了技术支持。     

蔗糖及废蜜发酵生产L-乳酸

介绍了L-乳酸的性质及用途，结合糖厂实际，提出用蔗糖及废蜜发酵生产L-乳酸，并简述了发酵法生产L-乳酸的技术发展概况。     

Expression of amino acid converting enzymes and production of volatile compounds by Lactococcus lactis IFPL953

Lactococcus lactis contains a large number of key enzymes responsible for the formation of volatile compounds characteristic of cheese flavour. In the present work we have investigated the expression of genes codifying amino acid converting enzymes (AACE) and the formation of volatile compounds by L. lactis IFPL953 and its mutant lacking the hydroxy acid dehydrogenase PanE (L. lactis IFPL953ΔpanE). The growth in absence of isoleucine was the main induction factor in the expression of araT, bcaT, panE, and kivD. The expression of the gdh gene of L. lactis IFPL953ΔpanE increased remarkably during the stationary growth phase, particularly under isoleucine and valine starvation conditions. L. lactis IFPL953ΔpanE showed an enhanced formation of 2- and 3-methylbutanal and their corresponding alcohols and an increase in the formation of ketones. These findings contribute to a better knowledge of L. lactis AACE regulation and its potential application in cheese flavour formation.     

Recovery of lactic acid by repeated batch electrodialysis and lactic acid production using electrodialysis wastewater

Repeated batch electrodialysis for lactic acid recovery was investigated using lactic acid solution and fermentation broth. In both cases, lactate fluxes averaged more than 7.0 moles/m2.h, lactate recovery reached more than 99% for all the batch runs, and specific energy consumption per unit lactate transported was lower than 0.25 kWh/kg-lactate. When electrodialysis wastewater was used as a fermentation medium, supplemented with 100 g/l glucose, up to 92.4 g/l lactic acid was produced with a productivity of 0.67 g/l.h. In addition, when electrodialysis wastewater was supplemented with 150 g/l whole-corn flour hydrolyzate and 5 g/l corn steep liquor, 2.5-fold and 1.8-fold increases in lactic acid productivity and maximum cell growth, respectively, were achieved, as compared with lactic acid fermentation using electrodialysis wastewater supplemented with glucose only.     

Continuous ethanol fermentation from non-sulfuric acid-washed molasses using traditional stirred tank reactors and the flocculating yeast strain KF-7

Waste molasses is one of the most important feedstock for ethanol production in Brazil as well as in many Southeast Asian countries, including China. Sulfuric acid pretreatment is employed in most ethanol distilleries in China to control bacterial contamination, which results in difficulties in the treatment of wastewater containing high levels of sulfate ions. In this study, a high efficiency, non-sterilized, continuous ethanol fermentation process without sulfuric acid pretreatment was developed using the flocculating yeast strain KF-7 and the widely utilized, traditional, stirred tank reactors. An alternative molasses medium feeding method, which differs from traditional methods, is proposed that effectively controls bacterial contamination. Separate feeding of 1.2-fold diluted molasses and tap water into the reactor proved to be effective against bacterial contamination during long-term continuous fermentation. By feeding yeast cells with high metabolic activity to the second reactor, a two-stage continuous fermentation process that yielded a high ethanol concentration of 80 g/l as well as high ethanol productivity of 6.6 g/l/h was successfully operated for more than one month. This fermentation process can be applied to ethanol distilleries in which traditional tank reactors are used.     

Production of poly-beta-hydroxybutyric acid by microorganisms accumulated from river water using a two-stage perfusion culture system

The perfusion culture system using a shaken ceramic membrane flask (SCMF) was employed to accumulate microorganisms separated from river water and to produce poly-beta-hydroxybutyric acid (PHB). Using a two-step culture method with a single SCMF, river microorganisms were cultured by separately feeding four representative carbon sources, n-propanol, lactic acid, methanol, and formic acid. After 140 h culture, the cell concentration and PHB content respectively reached 43 g/l and 35% when a propanol medium was fed. Using a two-stage perfusion culture with twin SCMFs, the seed cell mass was increased in the first SCMF and then supplied to the second flask for PHB production. As a consequence, the cellular PHB content rose to 51% in the second SCMF, while the cell concentration gradually increased to 25 g/l after 175 h perfusion culture. These results demonstrated the utility of the two-stage perfusion culture system for developing a cheap means of producing PHB coincident with wastewater treatment.     

Continuous fermentative hydrogen production using a two-phase reactor system with recycle

The effects of effluent recycle were examined in a two-phase anaerobic system where the first phase was operated for fermentative hydrogen production and the second for methanogenesis. The hydrogen reactor was operated as a chemostat at 35 degrees C and pH 5.5 with a 10 h hydraulic retention time, and the methane reactor was operated as an up-flow reactor at 28 degrees C and pH between 6.9 and 7.2. Two recycle ratios were examined: 0 and 0.98. Effluent recycle reduced the required alkalinity for pH control by approximately 40%. The H2 productivity metric, with a basis in electrons and incorporating both gaseous and dissolved H2, was developed as a more fundamental reporting method than the molar H2 yield. Without recycle, the H2 productivity was 0.115 g of H2 COD/g of feed COD, but decreased to 0.015 q of H2 COD/g of feed COD with recycle (COD = chemical oxygen demand). Mass balances indicated the lower H2 productivity during recycle was due to electrons being partitioned to methane and less-oxidized soluble constituents such as propionic acid, ethanol, and butanol. The results indicated that achieving high H2 productivity with nonsterile wastewaters will be challenging and membrane filtration of the recycle liquid may be required to exclude the return of hydrogen-consuming organisms.