β-呋喃果糖苷酶的固定化及其在低聚乳果糖合成中的应用

【目的】探索适宜的树脂作为载体固定β-呋喃果糖苷酶,并研究该固定化酶催化合成低聚乳果糖。【方法】选择9种大孔吸附树脂和碱性离子交换树脂固定β-呋喃果糖苷酶,筛选固定化效果较好的树脂作为载体。用聚乙烯亚胺(PEI)修饰得到PEI-树脂,采用吸附法将酶固定于PEI-树脂上,并对固定化条件进行优化。考察固定化酶的重复使用稳定性及其催化合成低聚乳果糖的能力。【结果】通过筛选发现大孔阴离子交换树脂D311固定化效果较好,经过PEI修饰后,D311固定化效果显著提高。用PEI修饰的载体PEI-D311固定果糖苷酶,最优固定化条件为:PEI浓度2%,加酶量103 U/g,吸附温度25°C,吸附p H 6.0-8.0,吸附时间8 h。最优条件下固定化酶活达57 U/g,酶活回收率达55.3%。用固定化酶催化水解1 mol/L蔗糖,重复利用15批载体酶活没有明显降低。用固定化酶催化合成低聚乳果糖,8 h内低聚乳果糖产量最高达到137 g/L。【结论】PEI-D311固定的果糖苷酶具有较好的重复使用稳定性及较高的低聚乳果糖合成能力,这为固定化酶法生产低聚乳果糖研究奠定了基础。     

产β-呋喃果糖苷酶节杆菌发酵条件的优化

通过单因素和响应面分析获得节杆菌产生β -呋喃果糖苷酶的最佳培养基配方为 :蔗糖 4 .4 5 % ,酵母膏 1.4 0 % ,蛋白胨 0 .93% ,(NH4 ) 2 HPO4 0 .4 % ,MgSO4 ·7H2 O 0 .13% ,低聚乳果糖产量可以达到 16 .38%。     

低聚异麦芽糖生产工艺研究

摘要：低聚异麦芽糖是一种功能性低聚糖,因其具有多种独特生理功能,被广泛地应用于食品、医药、饲料等领域,市场前景十分广阔,但是目前低聚异麦芽糖生产工艺中存在许多不足之处。本文首先介绍了低聚异麦芽糖的性质和生理功能,其次阐述了低聚异麦芽糖的生产工艺,并对如何构建和利用基因工程菌生产低聚异麦芽糖提出展望。     

不同碳水化合物对孤独症患儿肠道菌群 体外发酵产生短链脂肪酸的影响

目的探究孤独症谱系障碍(autism spectrum disorder,ASD)患儿与健康儿童的肠道菌群利用不同碳水化合物产生短链脂肪酸(short-chain fatty acids,SCFAs)的差异。方法前瞻性病例对照研究。选择2016年12月至2017年10月解放军总医院儿科门诊的15例ASD患儿为研究对象,并匹配15名健康儿童为对照组;收集研究对象粪便,稀释成10%悬浊液,然后分别接种到以乳果糖(LAU)、乳糖(LAT)、棉籽糖(RAF)、低聚半乳糖(GOS)、低聚异麦芽糖(IMO)和低聚甘露糖(MOS)为单碳源的肠道微生态小型模拟发酵系统中批量发酵24 h,检测SCFAs浓度、底物降解率和产气气压。结果 ASD组患儿粪便SCFAs浓度与对照组差异无统计学意义,在YCFA(无碳源对照培养基,蛋白发酵为主)培养基发酵后总SCFAs、乙酸和丙酸均低于对照组,差异有统计学意义(Z=-2.509、-2.509、-3.007,P=0.011、0.011、0.002);在添加LAT、RAF、GOS、IMO和MOS的培养基发酵后总SCFAs浓度显著高于对照组,差异有统计学意义(Z=2.385、2.344、2.675、2.344、2.302,P=0.016、0.019、0.007、0.019、0.021),而含LAU的培养基发酵后,两组研究对象总SCFAs浓度比较差异无统计学意义。结论 ASD患儿肠道菌群利用蛋白发酵产SCFAs能力显著低于健康对照,利用碳水化合物产SCFAs能力显著高于健康对照。6种发酵底物中,乳果糖是最适合ASD患儿的碳水化合物,有改善ASD患儿肠道菌群产SCFAs的潜力。     

利用纤维二糖差向异构酶制备乳果糖的研究进展

乳果糖是由D-半乳糖和D-果糖两个基团通过β-1,4糖苷键连接而成的还原型二糖;乳果糖口服液具有治疗慢性便秘和肝性脑病的功效,在100多个国家作为常见非处方药(OTC)使用,需求量十分巨大;乳果糖还可以作为益生元改善人体肠道菌群关系。乳果糖的生产依赖化学法,其催化剂对人体有害,下游分离难度大。近年来,纤维二糖差向异构酶被发现能够高效催化乳糖制备乳果糖,该技术绿色环保、步骤简单,具有很强的产业化前景。本文结合自身研究经历对纤维二糖差向异构酶的研发情况进行总结,并综述了乳果糖酶法制备技术的现状。     

N-酰化低聚壳聚糖衍生物还原能力的研究

对低聚壳聚糖进行N-酰化改性,制得取代度相同的N-马来酰低聚壳聚糖(NMCOS),N-琥珀酰低聚壳聚糖(NSCOS),N-邻苯二甲酰低聚壳聚糖(NPCOS),其中NMCOS1、NSCOS1、NPCOS1的取代度均为0.25;NMCOS2、NSCOS2、NPCOS2的取代度均为0.49。考察了6种N-酰化低聚壳聚糖衍生物的还原能力。结果表明:当取代度相同时,N-邻苯二甲酰低聚壳聚糖的还原能力最强,其次是N-马来酰低聚壳聚糖,N-琥珀酰低聚壳聚糖的还原能力最差。这可能是由取代基的性质不同所致。     

生物活性物质—低聚半乳糖

本文介绍了生物活性物质 -低聚半乳糖的性质、种类及化学结构 ,综述了它在促进人体有益菌群、抑制有害菌群的生长 ,减少肠内腐败物质 ,促进钙吸收 ,改善血清脂质等方面的生理功能 ,从游离酶、固定化酶和完整细胞等角度详细介绍了低聚半乳糖的制备技术及其生产现状与前景     

益生元对人胃癌细胞株BGC-823细胞抑制作用的研究

目的 探讨益生元对胃癌细胞的作用效果,为开发安全、有效的预防和辅助治疗胃癌的药物奠定基础,并为益生元的有效应用打开新思路.方法 用MTT法研究不同浓度大豆低聚糖、低聚木糖、低聚果糖和低聚甘露糖在不同时间对BGC-823细胞的抑制作用.结果 大豆低聚糖、低聚木糖、低聚果糖和低聚甘露糖4种益生元对BGC-823细胞均有一定程度的抑制作用(P<0.05),抑制效果基本呈浓度时间依赖关系,但大豆低聚糖、低聚果糖的作用在48h和72h差别不大.在这4种益生元中,大豆低聚糖的抑制效果最强,低聚木糖次之,低聚果糖、低聚甘露糖的抑制效果较弱.作用48h、72h达到对BGC-823细胞半数抑制率的大豆低聚糖的浓度是100ms/ml左右;作用72h时达到对BGC-823细胞半数抑制率的低聚木糖浓度在100～125mg/ml;而125mg/ml低聚果糖和低聚甘露糖处理胃癌BGC-823细胞72h,均未达到50%的抑制率.结论 大豆低聚糖、低聚木糖、低聚果糖和低聚甘露糖对人胃癌细胞株BGC-823细胞有一定程度的抑制作用.     

化学脱色法在制备乳果糖中的应用

化学脱色法在制备乳果糖中的应用大连医科大学科达药业微生态研究所大连１１６０２４温朗聪袁杰利王红大连三株生态化妆品有限公司大连１１６０２４王崇霞乳果糖是一重要的双歧杆菌促生长因子,目前主要通过化学合成法获得。乳果糖广泛用于临床医药、保健品和食品添加剂等...     

乳果糖研究进展

乳果糖研究进展大连医科大学科达药业有限公司微生态研究所大连１１６０２４刘仁宽,温朗聪大连三株化妆品有限公司大连１１６０２３王崇霞乳果糖又称１一４β半乳糖苷果糖,或称乳酮糖。它是一种合成双糖,含有一分子半乳糖和一分子果糖。乳果糖是Ｍｏｎｔｇｏｍｅｒｙｃ...     

Current studies on physiological functions and biological production of lactosucrose

Lactosucrose (O-β-d-galactopyranosyl-(1,4)-O-α-d-glucopyranosyl-(1,2)-β-d-fructofuranoside) is a trisaccharide formed from lactose and sucrose by enzymatic transglycosylation. This rare trisaccharide is a kind of indigestible carbohydrate, has good prebiotic effect, and promotes intestinal mineral absorption. It has been used as a functional ingredient in a range of food products which are approved as foods for specified health uses in Japan. Using lactose and sucrose as substrates, lactosucrose can be produced through transfructosylation by β-fructofuranosidase from Arthrobacter sp. K-1 or a range of levansucrases, or through transgalactosylation by β-galactosidase from Bacillus circulans. This article presented a review of recent studies on the physiological functions of lactosucrose and the biological production from lactose and sucrose by different enzymes.     

Continuous production of lactosucrose by immobilized Sterigmatomyces elviae mutant

In this study, in order to develop a continuous production process of lactosucrose in a packed-bed reactor, Sterigmatomyces elviae ATCC 18894 was selected and mutated. The mutant strain of S. elviae showed 54.3% higher lactosucrose production than the wild type. Reaction conditions such as temperature, pH, substrate concentration and flow rate were also optimized. Under optimized reaction conditions (50 degrees C, pH 6.0, 25% sucrose and 25% lactose as substrate, flow rate 1.2 ml/min), the maximum concentration of lactosucrose (192 g/l) was obtained. In a packed-bed reactor, continuous production of lactosucrose was performed using S. elviae mutant immobilized in calcium alginate, and about 180 g/l of lactosucrose production was achieved for 48 days.     

Optimization of culture medium for lactosucrose ( G-beta-D-galactosylsucrose) Production by Sterigmatomyces elviae mutant using statistical analysis

In this study, the optimization of culture medium using a Sterigmatomyces elviae mutant was investigated using statistical analysis to increase the cell mass and lactosucrose ((4)G-beta-D-galactosylsucrose) production. In basal medium, the cell mass and lactosucrose production were 4.12 g/l and 140.91 g/l, respectively. However, because of the low cell mass and lactosucrose production, optimization of culture medium was carried out to increase the cell mass and lactosucrose production. Culture media were optimized by the S. elviae mutant using analysis of variance (ANOVA) and response surface methodology (RSM). Central composite designs using RSM were utilized in this investigation. Quadratic models were obtained for cell mass and lactosucrose production. In the case of cell mass, optimal components of the medium were as follows: sucrose 1.13%, yeast extract 0.99%, bactopeptone 2.96%, and ammonium sulfate 0.40%. The predicted maximum value of cell mass was about 5.20 g/l and its experimental value was 5.08 g/l. In the case of lactosucrose production, optimal components of the medium were as follows: sucrose 0.96%, yeast extract 1.2%, bactopeptone 3.0%, and ammonium sulfate 0.48%. Then, the predicted maximum value of lactosucrose production was about 194.12 g/l and the corresponding experimental value was about 183.78 g/l. Therefore, by culturing using predicted conditions, the real cell mass and lactosucrose production increased to 23.3% and 30.42%, respectively.     

Valorization of Cheese and Tofu Whey through Enzymatic Synthesis of Lactosucrose

This work deals with the development of a new bioprocess for the efficient synthesis of lactosucrose, a potential prebiotic oligosaccharide with a high value-added, from two important and inexpensive agro-industrial by-products such as tofu whey and cheese whey permeate. The bioconversion is driven by the ability of the enzyme levansucrase SacB from Bacillus subtilis CECT 39 to transfructosylate lactose contained in the cheese whey permeate by using not only sucrose but also raffinose and stachyose, which are present in considerable amounts in the tofu whey, as suitable donors of fructosyl moieties. The maximum lactosucrose concentration obtained from both by-products was 80.1 g L^-1 after a short reaction time 120 min at 37°C, leading to productivity and specific productivity values of 40.1 g lactosucrose L^-1 h^-1 and 80.1 mg lactosucrose U enzyme⁻¹ h⁻¹, respectively. Findings contained in this work could provide a new strategy to valorize agro-industrial by-products as cheese whey permeate and, specially, tofu whey by means of their use as renewable resources in the enzymatic synthesis of bioactive oligosaccharides.     

Production of short-chain fatty acids and gas from various oligosaccharides by gut microbes of carp (Cyprinus carpio L.) in micro-scale batch culture

We studied the metabolism of various oligosaccharides by carp (Cyprinus carpio) hindgut microbes by measuring gas productivity and organic acid production in gut contents using a 50-microl-scale batch culture system. Carp hindgut contents were incubated with 500 microg each of raffinose, lactosucrose, kestose, lactulose, gentiobiose, 4'-galactosyllactose and 6'-galactosyllactose and soybean-, xylo-, and isomalto-oligosaccharides or none (blank culture) at 25 degrees C for 6 h. The time-course of gas release from the culture (Y microl/culture) was expressed as an exponential function of incubation time (t) [Y=A+Bx(1-e(-kt))]; A, B and k are constants). Potential production of gas (A+B) from soybean-oligosaccharide and raffinose was larger than for the other saccharides except for kestose, and blank culture. The rate constant of gas (k) for lactosucrose was larger than that for isomalto- and xylo-oligosaccharide, lactulose, kestose or blank culture. Net production of total SCFA (sum of acetic, propionic and n-butyric acid weights) from cultures with soybean- and isomalto-oligosaccharides, raffinose, gentiobiose and lactosucrose was greater than that from blank culture. These results suggested that soybean-oligosaccharide and raffinose were potentially highly fermentable oligosaccharides for carp hindgut microbes. Chemical structures of oligosaccharides seem to play an important role in the fermentability. It is also likely that oligosaccharide utilization differs between mammals and teleosts.     

Lactosucrose production by various microorganisms harboring levansucrase activity

Production of the artificial sweetener, lactosucrose, by various microorganisms containing levansucrase activity was investigated. Of the tested bacteria, Bacillus subtilis was the most effective producer using lactose as an acceptor and sucrose as a fructosyl donor. Lactosucrose production by this strain was optimal at pH 6.0 and 55 °C whereupon 181 g lactosucrose l^–1 was produced from 225 g lactose l^–1 and 225 g sucrose l^–1 in 10 h.     

Lactosucrose bioconversion from lactose and sucrose by whole cells of Paenibacillus polymyxa harboring levansucrase activity

Lactosucrose, a functional trisaccharide, was produced from lactose as an acceptor and sucrose as a fructosyl donor by whole cells harboring transfructosylation activity of levansucrase. Levansucrase-induced cells of Paenibacillus polymyxa were obtained in the medium containing sucrose, and the transfructosylation activity in the whole cell was optimized for lactosucrose production. The optimal cell concentration, substrates ratio, temperature, and pH were 2.0% (w/v), 22.5% (w/v) lactose and 22.5% (w/v) sucrose, 55 degrees C, and 6.0, respectively. Under these conditions, the whole cells produced approximately 17.0% (w/v) lactosucrose in 6 h of reaction time with a productivity of 2.8% (w/v)/h.     

Seasonal variation in amount of unabsorbed dietary carbohydrate from the intestine after breakfast in Japanese subjects

We previously showed that daytime dim-light exposure has a negative effect on the efficiency of dietary carbohydrate absorption in the evening, whereas evening-time dim-light exposure has a beneficial effect. These results suggest that seasonal changes in the environmental light may affect gastrointestinal activity, and that there might, therefore, be seasonal variation in the efficiency of dietary carbohydrate absorption from the intestine. In order to prove this hypothesis, we measured the amount of dietary carbohydrate unabsorbed from the intestine after a breakfast in healthy female Japanese subjects during the four seasons of the year. We estimated the amount of unabsorbed dietary carbohydrate by the breath hydrogen test, which measures the amount of hydrogen in exhaled air. A 6 g solution of lactosucrose, an indigestible trisaccharide, was used for comparison. Two groups of subjects, 12 subjects in Osaka and 14 subjects in Nagano, were studied in the winter (January to February), spring (April to May), summer (July to August), and autumn (October to November) of 2004. We found the following results: (1) In no season were there any significant differences between the two subgroups in the orocecal transit time of the breakfast and the lactosucrose solution. Nor were there any significant differences in the amount of unabsorbed dietary carbohydrate from the breakfast. (2) Using the pooled data of the total of 26 subjects, there was no significant seasonal variation in the orocecal transit time of the breakfast or the lactosucrose solution. (3) There was a significant seasonal variation in the amount of unabsorbed dietary carbohydrate from the breakfast. (4) The amount of unabsorbed dietary carbohydrate from the breakfast was largest in winter and smallest in autumn. Results in spring and in summer were similar and intermediate between those in winter and autumn. Post hoc multiple comparison tests showed that the amount of unabsorbed dietary carbohydrate in winter was significantly larger than in autumn. (5) In winter, the average ratio of the amount of unabsorbed dietary carbohydrate to the total amount of carbohydrate in the breakfast was about 12%; in autumn it was about 6%. These results clearly show that there is seasonal variation in the efficiency of intestinal dietary carbohydrate absorption among young female Japanese subjects.     

A new bioprocess for the production of prebiotic lactosucrose by an immobilized β-galactosidase

A new bioprocess for the synthesis of lactosucrose was studied using a covalently immobilized β-galactosidase on macrospheres of chitosan. The effects of temperature and pH on the production of lactosucrose and other oligosaccharides were evaluated. At 30 °C and pH 7.0, the maximum concentration of lactosucrose reached to 79 g L⁻¹. The change of the reaction conditions allowed to modify the qualitative profile of the final products without quantitative change in the total of oligosaccharides produced. At pH 7 and 30 °C, products profile was 79 g L⁻¹ of lactosucrose, 37 g L⁻¹ of galactooligosaccharides and 250 g L⁻¹ of total oligosaccharides, while at pH 5 and 64 °C the concentrations for the same compounds were 40, 62 and 250 g L⁻¹, respectively. The immobilization increased the thermal stability up to 260-fold. Using 300 g L⁻¹ of sucrose and 300 g L⁻¹ of lactose, and 8.5 mg of chitosan mL⁻¹, 30 cycles of reuse were performed and the biocatalyst kept the maximal lactosucrose synthesis. These results fulfill some important aspects for the enzyme immobilization and oligosaccharides synthesis: the simplicity of the protocols, the high operational stability of the enzyme and the possibility of driving the final products.     

Effect of post-prandial posture on orocecal transit time and digestion of milk lactose in humans

We examined the effect of post-prandial body posture on orocecal transit time and absorption of milk lactose using the breath hydrogen test. In this experiment, subjects ingested a cup of commercially available milk to which we had added a small amount of lactosucrose (an indigestible trisaccharide), and then they lay on their backs or sat on a chair for the first 4 hr (from 08:00 to 12:00). After four hours lying or sitting, they remained sedentary on a sofa for the second six hr (from 12:00 to 18:00). Participants' end alveolar breath samples were collected every 15 min from 08:00 to 12:30, then every 30 min from 13:00 to 18:00. The experiment was conducted on two consecutive days using a randomized, crossover study design. Examination showed that the orocecal transit time of the oligosaccharides (lactosucrose and milk lactose) under the post-prandial supine condition was significantly longer than that under the sitting condition. In addition, the amount of breath hydrogen excretion under the supine condition was significantly lower than under the sitting condition, indicating that the unabsorbed milk lactose moved into cecum under the supine condition is smaller than that under the sitting condition. These findings provide evidence that postprandial supine posture works more beneficially to digest and absorb milk lactose when compared to the sitting posture.