Enhanced kefiran production by mixed culture of Lactobacillus kefiranofaciens and Saccharomyces cerevisiae

In a batch mixed culture of Lactobacillus kefiranofaciens and Saccharomyces cerevisiae, which could assimilate lactic acid, cell growth and kefiran production rates of L. kefiranofaciens significantly increased, compared with those in pure cultures. The kefiran production rate was 36 mg l(-1) h(-1) in the mixed culture under the anaerobic condition, which was greater than that in the pure culture (24 mg l(-1) h(-1)). Under the aerobic condition, a more intensive interaction between these two strains was observed and higher kefiran production rate (44 mg l(-1) h(-1)) was obtained compared with that under the anaerobic condition. Kefiran production was further enhanced by an addition of fresh medium in the fed-batch mixed culture. In the fed-batch mixed culture, a final kefiran concentration of 5.41 g l(-1) was achieved at 87 h, thereby attaining the highest productivity at 62 mg l(-1) h(-1). Simulation study considered the reduction of lactic acid in pure culture was performed to estimate the additional effect of coculture with S. cerevisiae. Slightly higher cell growth and kefiran production rates in the mixed culture than those expected from pure culture by simulation were observed. These results suggest that coculture of L. kefiranofaciens and S. cerevisiae not only reduces the lactic acid concentration by consumption but also stimulates cell growth and kefiran production of L. kefiranofaciens.     

马乳酒样乳杆菌(Lactobacillus kefiranofaciens )合成开菲尔多糖培养基组成的优化

研究了发酵培养基组成对Lactobacillus kefiranofaciens的菌体生长以及合成开菲尔多糖(Kefiran)的影响。通过比较L. kefiranofaciens利用各种糖类和麦芽汁的发酵情况,发现麦芽汁最利于菌体生长及发酵产糖;各种氮源对L. kefiranofaciens发酵影响不同,其中以酵母粉最好,采用一品鲜酵母粉发酵的最佳浓度为10%;正交实验和RSA方法确定对发酵影响显著的三个无机盐是MnSO4.4H2O、MgSO4.7H2O和KH2PO4,最佳用量为0.2g/L,1g/L,4g/L,此时发酵获得开菲尔多糖产量为2.6±0.05g/L。L. kefiranofaciens发酵生产开菲尔多糖96h时可达到最佳发酵结果。     

Modelling and optimization of environmental conditions for kefiran production by Lactobacillus kefiranofaciens

A mathematical model for kefiran production by Lactobacillus kefiranofaciens was established, in which the effects of pH, substrate and product on cell growth, exopolysaccharide formation and substrate assimilation were considered. The model gave a good representation both of the formation of exopolysaccharides (which are not only attached to cells but also released into the medium) and of the time courses of the production of galactose and glucose in the medium (which are produced and consumed by the cells). Since pH and both lactose and lactic acid concentrations differently affected production and growth activity, the model included the effects of pH and the concentrations of lactose and lactic acid. Based on the mathematical model, an optimal pH profile for the maximum production of kefiran in batch culture was obtained. In this study, a simplified optimization method was developed, in which the optimal pH profile was determined at a particular final fermentation time. This was based on the principle that, at a certain time, switching from the maximum specific growth rate to the critical one (which yields the maximum specific production rate) results in maximum production. Maximum kefiran production was obtained, which was 20% higher than that obtained in the constant-pH control fermentation. A genetic algorithm (GA) was also applied to obtain the optimal pH profile; and it was found that practically the same solution was obtained using the GA.     

Kinetic modeling of kefiran production in mixed culture of Lactobacillus kefiranofaciens and Saccharomyces cerevisiae

Growth and kefiran production rates of Lactobacillus kefiranofaciens were significantly enhanced in a mixed culture with Saccharomyces cerevisiae as compared with those in a pure culture. Because a positive effect on growth and kefiran production of L. kefiranofaciens in a mixed culture was observed, the elucidation of interaction between L. kefiranofaciens and S. cerevisiae may lead to higher productivity. Hence, microbial performance of each strain was investigated and analyzed by a mathematical model. The mathematical model for kefiran fermentation in a mixed culture of L. kefiranofaciens and S. cerevisiae was established, and the impact of S. cerevisiae on cell growth, kefiran formation, and substrate assimilation of L. kefiranofaciens were considered. The behavior of L. kefiranofaciens in a mixed culture was predicted using a developed mathematical model in this work, and the predictions were compared with the results from mixed culture experiments. The overall mathematical model is capable of describing the behavior of S. cerevisiae in a mixed culture as a lactic acid consumer, nitrogen source competitor and protective function inducer for L. kefiranofaciens. Furthermore, the constructed model described the phenomena in mixed cultures under aerobic and anaerobic conditions. Finally, the optimal inoculation ratios of S. cerevisiae to L. kefiranofaciens at 7-fold and 10-fold under aerobic and anaerobic conditions were obtained by applying the mixed culture model, respectively.     

Bioprocess development for kefiran production by Lactobacillus kefiranofaciens in semi industrial scale bioreactor

Lactobacillus kefiranofaciens is non-pathogenic gram positive bacteria isolated from kefir grains and able to produce extracellular exopolysaccharides named kefiran. This polysaccharide contains approximately equal amounts of glucose and galactose. Kefiran has wide applications in pharmaceutical industries. Therefore, an approach has been extensively studied to increase kefiran production for pharmaceutical application in industrial scale. The present work aims to maximize kefiran production through the optimization of medium composition and production in semi industrial scale bioreactor. The composition of the optimal medium for kefiran production contained sucrose, yeast extract and K₂HPO₄ at 20.0, 6.0, 0.25 g L⁻¹, respectively. The optimized medium significantly increased both cell growth and kefiran production by about 170.56% and 58.02%, respectively, in comparison with the unoptimized medium. Furthermore, the kinetics of cell growth and kefiran production in batch culture of L. kefiranofaciens was investigated under un-controlled pH conditions in 16-L scale bioreactor. The maximal cell mass in bioreactor culture reached 2.76 g L⁻¹ concomitant with kefiran production of 1.91 g L⁻¹.     

马乳酒样乳杆菌(Lactobacillus kefiranofaciens)产生的开菲尔多糖的分离及其结构特性

研究了Lactobacillus kefiranofaciens发酵产生的开菲尔多糖的初步纯化方法,并对开菲尔多糖的分子组成、分子量以及单糖之间的连接方式进行了研究.研究结果表明,采用酶解与Sevage试剂相结合的方法进行纯化,当蛋白去除率为93%时,多糖得率60%,样品中多糖的百分含量为96%.采用HPLC和红外光谱研究表明,培养基的组成会明显影响开菲尔多糖中葡萄糖与半乳糖的比例,由MRS培养基发酵产生的KEFⅠ的相对分子质量约为2.4×105Da,葡萄糖/半乳糖约为14;而由改良的MRS培养基发酵产生的KEFⅡ的相对分子质量约为1.5×105Da,葡萄糖/半乳糖约为110.Lactobacillus kefiranofaciens发酵的开菲尔多糖分子中均含有β糖苷键.     

马乳酒样乳杆菌(Lactobacillus kefiranofaciens)产生的开菲尔多糖的分离及其结构特性

研究了Lactobacillus kefiranofaciens发酵产生的开菲尔多糖的初步纯化方法,并对开菲尔多糖的分子组成、分子量以及单糖之间的连接方式进行了研究。研究结果表明,采用酶解与Sevage试剂相结合的方法进行纯化,当蛋白去除率为93%时,多糖得率60%,样品中多糖的百分含量为96%。采用HPLC和红外光谱研究表明,培养基的组成会明显影响开菲尔多糖中葡萄糖与半乳糖的比例,由MRS培养基发酵产生的KEFⅠ的相对分子质量约为2.4×105Da,葡萄糖/半乳糖约为1:4;而由改良的MRS培养基发酵产生的KEFⅡ的相对分子质量约为1.5×105Da,葡萄糖/半乳糖约为1:10。Lactobacillus kefiranofaciens发酵的开菲尔多糖分子中均含有β糖苷键。     

Lactobacillus kefiranofaciens流加发酵法生产开菲尔多糖

研究了开菲尔基质乳杆菌(Lactobacillus kefiranofaciens)在不同初始蔗糖浓度下的开菲尔多糖(Kefiran)分批发酵过程.结果表明,分批发酵过程不能实现Kefiran高产量、高底物转化率和高生产强度的相对统一.在此基础上,进一步考察分批补料、恒速流加和指数速率流加等不同培养方式对Kefiran发酵的影响.这几种培养方式都可以实现乳酸菌细胞和Kefiran的高产.综合比较,4g/(L·h)的蔗糖恒速流加为Kefiran生产较适宜的流加方式,细胞干质量浓度为63.6 g/L,Kefiran产量达到4.95 g/L.     

Batch propagation of Lactobacillus helveticus for production of lactic acid from lactose concentrated cheese whey with microaeration and nutrient supplementation

Continuous mix batch bioreactors were used to study the kinetic parameters of lactic acid fermentation in microaerated-nutrient supplemented, lactose concentrated cheese whey using Lactobacillus helveticus. Four initial lactose concentrations ranging from 50 to 150 g l^–1 were first used with no microaeration and no yeast extract added to establish the substrate concentration above which inhibition will occur and then the effects of microaeration and yeast extract on the process kinetic parameters were investigated. The experiments were conducted under controlled pH (5.5) and temperature (42 °C) conditions. The results indicated that higher concentrations of lactose had an inhibitory effect as they increased the lag period and the fermentation time; and decreased the specific growth rate, the maximum cell number, the lactose utilization rate, and the lactic acid production rate. The maximum lactic acid conversion efficiency (75.8%) was achieved with the 75 g l^–1 initial lactose concentration. The optimum lactose concentration for lactic acid production was 75 g l^–1 although Lactobacillus helveticus appeared to tolerate up to 100 g l^–1 lactose concentration. Since the lactic acid productivity is of a minor importance compared to lactic acid concentration when considering the economic feasibility of lactic acid production from cheese whey using Lactobacillus helveticus, a lactose concentration of up to 100 g l^–1 is recommended. Using yeast extract and/or microaeration increased the cell number, specific growth rate, cell yield, lactose consumption, lactic acid utilization rate, lactic acid concentration and lactic acid yield; and reduced the lag period, fermentation time and residual lactose. Combined yeast extract and microaeration produced better results than each one alone. From the results it appears that the energy uncoupling of anabolism and catabolism is the major bottleneck of the process. Besides lactic acid production, lactose may also be hydrolysed into glucose and galactose. The -galactosidase activity in the medium is caused by cell lysis during the exponential growth phase. The metabolic activities of Lactobacillus helveticus in the presence of these three sugars need further investigation.     

Lactic acid production from corn stover using mixed cultures of Lactobacillus rhamnosus and Lactobacillus brevis

Mixed cultures of Lactobacillus rhamnosus and Lactobacillus brevis was studied for improving utilization of both cellulose- and hemicellulose-derived sugars from corn stover for lactic acid production. During simultaneous saccharification and fermentation (SSF) of NaOH-treated corn stover by the mixed cultures, a lactic acid yield of 0.70 g/g was obtained, which was about 18.6% and 29.6% higher than that by single cultures of L. rhamnosus and L. brevis, respectively. Our results indicated that lactic acid yield from NaOH-pretreated corn stover by mixed cultures of L. rhamnosus and L. brevis was comparable to that from pure sugar mixtures (0.73 g/g of glucose/xylose mixture at 3:1 w/w).     

Sago starch as a low-cost carbon source for exopolysaccharide production by Lactobacillus kefiranofaciens

Low-cost sago starch was used as a carbon source for production of the exopolysaccharide kefiran by Lactobacillus kefiranofaciens. A simultaneous saccharification and fermentation process of sago starch for kefiran production was evaluated. Factors affecting the process such as an initial pH, temperature, starch concentration, including a mixture of α-amylase and glucoamylase were determined. The highest kefiran concentration of 0.85 g/l was obtained at the initial pH of 5.5, temperature of 30 °C, starch concentration of 4% and mixed-enzymes with activity of 100 U/g-starch. The use of a mixture of α-amylase and glucoamylase could enhance the productivity compared to the use of α-amylase alone. The optimal ratio of α-amylase to glucoamylase of 60:40 gave the highest kefiran production rate of 11.83 mg/l/h. This study showed that sago starch could serve as a low-cost substrate for kefiran production.     

Lactic acid production from whey permeate by immobilized Lactobacillus casei

Two matrices have been assessed for their ability to immobilize Lactobacillus casei cells for lactic acid fermentation in whey permeate medium. Agar at 2% concentration was found to be a better gel than polyacrylamide in its effectiveness to entrap the bacterial cells to carry out batch fermentation up to three repeat runs. Of the various physiological parameters studied, temperature and pH were observed to have no significant influence on the fermentation ability of the immobilized organism. A temperature range of 40–50°C and a pH range of 4.5–6.0 rather than specific values, were found to be optimum when fermentation was carried out under stationary conditions. In batch fermentation ～90% conversion of the substrate (lactose) was achieved in 48 h using immobilized cell gel cubes of 4 × 2 × 2 mm size, containing 400 mg dry bacterial cells per flask and 4.5% w/v (initial) whey lactose content as substrate. However, further increase in substrate levels tested (>4.5% w/v) did not improve the process efficiency. Supplementation of Mg²⁺ (1 mM) and agricultural by-products (mustard oil cake, 6%) in the whey permeate medium further improved the acid production ability of the immobilized cells under study.     

Acetic acid production from whey lactose by the co-culture ofStreptococcus lactis andClostridium formicoaceticum

Summary Acetic acid was produced from anaerobic fermentation of lactose by the co-culture ofStreptococcus lactis andClostridium formicoaceticum at 35° C and pHs between 7.0 and 7.6. Lactose was converted to lactic acid, and then to acetic acid in this mixed culture fermentation. The overall acetic acid yield from lactose was about 95% at pH 7.6 and 90% at pH 7.0. The fermentation rate was also higher at pH 7.6 than at pH 7.0. In batch fermentation of whey permeate containing about 5% lactose at pH 7.6, the concentration of acetic acid reached 20 g/l within 20 h. The production rate then became very slow due to end-product inhibition and high Na⁺ concentration. About 30 g/l acetate and 20 g/l lactate were obtained at a fermentation time of 80 h. However, when diluted whey permeate containing 2.5% lactose was used, all the whey lactose was converted to acetic acid within 30 h by this mixed culture.     

Carotenoid production by lactoso-negative yeasts co-cultivated with lactic acid bacteria in whey ultrafiltrate

Two strains were selected--the lactoso-negative yeast Rhodotorula rubra GED2 and the homofermentative Lactobacillus casei subsp. casei Ha1 for co-cultivation in cheese whey ultrafiltrate (WU) and active synthesis of carotenoids. Under conditions of intensive aeration (1.0 l/l min, 220 rpm), a temperature of 30 degrees C, WU with 55.0 g lactose/l, initial pH = 5.5, the carotenoid content in the cells reached a maximum, when the growth of the cultures had come to an end, i.e. in the stationary phase of the yeast. The maxima for dry cell accumulation (27.0 g/l) and carotenoid formation (12.1 mg/l culture medium) did not coincide on the 5th and 6th day, respectively. A peculiarity of the carotenoid-synthesizing Rh. rubra GED2 strain, co-cultivated with L. casei Ha1, was the production of carotenoids with high beta-carotene content (46.6% of total carotenoids) and 10.7% and 36.9% for torulene and torularhodin, respectively.     

A novel recycle batch immobilized cell bioreactor for propionate production from whey lactose

Recycle batch fermentations using immobilized cells of Propionibacterium acidipropionici were studied for propionate production from whey permeate, de-lactose whey permeate, and acid whey. Cells were immobilized in a spirally wound fibrous sheet packed in a 0.5-L column reactor, which was connected to a 5-L stirred tank batch fermentor with recirculation. The immobilized cells bioreactor served as a breeder for these recycle batch fermentations. High fermentation rates and conversions were obtained with these whey media without nutrient supplementation. It took approximately 55 h to ferment whey permeate containing approximately 45 g/L lactose to approximately 20 g/L propionic acid. Higher propionate concentrations can be produced with various concentrated whey media containing more lactose. The highest propionic acid concentration obtained with the recycle batch reactor was 65 g/L, which is much higher than the normal maximum concentration of 35 to 45 g/L reported in the literature. The volumetric productivity ranged from 0.22 g/L . h to 0.47 g/L . h, depending on the propionate concentration and whey medium used. The corresponding specific cell productivity was 0.033 to 0.07 g/L . g cell. The productivity increased to 0.68 g/L . h when whey permeate was supplemented with 1% (w/v) yeast extract. Compared with conventional batch fermentation, the recycle batch fermentation with the immobilized cell bioreactor allows faster fermentation, produces a higher concentration of product, and can be run continually without significant downtime. The process also produced similar fermentation results with nonsterile whey media. (c) 1995 John Wiley & Sons, Inc.     

Lactic acid production by mixed cultures of Kluyveromyces marxianus, Lactobacillus delbrueckii ssp. bulgaricus and Lactobacillus helveticus

Lactic acid production using Kluyveromyces marxianus (IFO 288), Lactobacillus delbrueckii ssp. bulgaricus (ATCC 11842) and Lactobacillus helveticus (ATCC 15009) individually or as mixed culture on cheese whey in stirred or static fermentation conditions was evaluated. Lactic acid production, residual sugar and cell biomass were the main features examined. Increased lactic acid production was observed, when mixed cultures were used in comparison to individual ones. The highest lactic acid concentrations were achieved when K. marxianus yeast was combined with L. delbrueckii ssp. bulgaricus, and when all the strains were used revealing possible synergistic effects between the yeast and the two lactic acid bacteria. The same synergistic effects were further observed and verified when the mixed cultures were applied in sourdough fermentations, proving that the above microbiological system could be applied in the food fermentations where high lactic acid production is sought.     

Fermentation of lactose to bio-ethanol by yeasts as part of integrated solutions for the valorisation of cheese whey

Cheese whey, the main dairy by-product, is increasingly recognized as a source of many bioactive valuable compounds. Nevertheless, the most abundant component in whey is lactose (ca. 5% w/v), which represents a significant environmental problem. Due to the large lactose surplus generated, its conversion to bio-ethanol has long been considered as a possible solution for whey bioremediation. In this review, fermentation of lactose to ethanol is discussed, focusing on wild lactose-fermenting yeasts, particularly Kluyveromyces marxianus, and recombinant Saccharomyces cerevisiae strains. The early efforts in the screening and characterization of the fermentation properties of wild lactose-consuming yeasts are reviewed. Furthermore, emphasis is given on the latter advances in engineering S. cerevisiae strains for efficient whey-to-ethanol bioprocesses. Examples of industrial implementation are briefly discussed, illustrating the viability of whey-to-ethanol systems. Current developments on strain engineering together with the growing market for biofuels will likely boost the industrial interest in such processes.     

Batch fermentation of whey ultrafiltrate by Lactobacillus helveticus for lactic acid production

Summary Cheese whey ultrafiltrate (WU) was used as the carbon source for the production of lactic acid by batch fermentation with Lactobacillus helveticus strain milano. The fermentation was conducted in a 400 ml fermentor at an agitation rate of 200 rpm and under conditions of controlled temperature (42° C) and pH. In the whey ultrafiltrate-corn steep liquor (WU-CSL) medium, the optimal pH for fermentation was 5.9. Inoculum propagated in skim milk (SM) medium or in lactose synthetic (LS) medium resulted in the best performance in fermentation (in terms of growth, lactic acid production, lactic acid yield and maximum productivity of lactic acid), as compared to that propagated in glucose synthetic (GS) medium. The yeast extract ultrafiltrate (YEU) used as the nitrogen/growth factor source in the WU medium at 1.5% (w/v) gave the highest maximum productivity of lactic acid of 2.70 g/l-h, as compared to the CSL and the tryptone ultrafiltrate (TU). L. helveticus is more advantageous than Streptococcus thermophilus and Lactobacillus delbrueckii for the production of lactic acid from WU. The L. helveticus process will provide an alternative solution to the phage contamination in dairy industries using Lactobacillus bulgaricus.