Fermentation by Lactobacillus fermentum Ogi E1 of different combinations of carbohydrates occurring naturally in cereals: consequences on growth energetics and alpha-amylase production

Glucose, fructose, sucrose and starch are naturally present in cereals. Fermentation of different combinations of these carbohydrates by Lactobacillus fermentum Ogi E1, a sourdough heterofermentative lactobacillus, was investigated to determine effects on fermentation kinetics, growth energetics and alpha-amylase production. Irrespective of the substrate combination, the strain was able to simultaneously produce alpha-amylase and consume starch, glucose, fructose and sucrose. In mixtures of starch with either sucrose or fructose or with both fructose and glucose, yields of alpha-amylase from biomass (Y(amy/x)) were similar to those observed for starch. However, for starch and glucose or starch, glucose, fructose and sucrose mixtures, both Y(amy/x) and the specific rate of alpha-amylase production decreased markedly. In fructose- or sucrose-containing mixtures, mannitol was formed stoichiometrically indicating that fructose served as electron acceptor, and acetate was produced at constant yield from biomass (Y(ac/x)) (1 g acetate g biomass(-1)). Acetate production was expected to confer to the strain a competitive advantage during natural fermentation by improving biomass formation and growth through an increase in the ATP gain. Y(ATP) varied depending on the carbohydrate mixture, indicating different effects of substrate mixtures on the efficiency in ATP coupling to biomass formation. Compared to starch fermentation, the highest value of Y(ATP) (29 g biomass mol ATP(-1)) was estimated for the starch/fructose mixture but no increase in mu(max) was observed. The lowest value (16 g biomass mol ATP(-1)) was obtained for the starch, glucose and fructose mixture, whereas for the mixture of all carbohydrates, Y(ATP) was similar to that obtained with starch alone (20 g biomass mol ATP(-1)) and it was intermediary for the starch and sucrose mixture (17 g biomass mol ATP(-1)). It is concluded that competitiveness of the strain cannot be based on expected energy gain in mixed substrate fermentation involving fructose and sucrose with glucose and starch, but rather on its ability to simultaneously use carbohydrates while producing alpha-amylase and to produce acetic acid. Acetic acid production could enhance the strain capacity to inhibit nonacid-tolerant, competitive microflora at the earlier stage of natural fermentation.     

碳源与罗伊氏乳杆菌LYS-1发酵上清液抑菌效果的关系

本研究考察了碳源对罗伊氏乳杆菌LYS-1发酵特性和发酵上清液抑菌效果的影响,以此探讨碳源与LYS-1代谢产生抑菌物质的关系。LYS-1接种于不同碳源配方的培养基中,测定发酵过程中酸度、活菌数的变化以及发酵上清液的抑菌效果。结果显示,LYS-1以蔗糖、麦芽糖和葡萄糖为碳源时抑菌圈直径依次为9.84 mm、8.55 mm和7.65 mm,以果糖、乳糖和甘油为碳源时则无抑菌圈。活菌数≥1.00×10^9CFU/mL时抑菌圈才会出现,且活菌数的增加有利于抑菌圈直径的增大。葡萄糖和蔗糖复配具有较优的抑菌效果,且蔗糖添加量在5%~10%范围内增加可以提高抑菌效果。以葡萄糖和果糖等当量替换蔗糖具有同等的抑菌效果,碳源为1.00%葡萄糖+10.00%蔗糖或6.26%葡萄糖+5.26%果糖时发酵上清液的抑菌效果最强。碳源影响LYS-1代谢产生抑菌物质,葡萄糖和果糖是影响发酵上清液抑菌效果的关键性因素。     

Influence of in-situ synthesized exopolysaccharides on the quality of gluten-free sorghum sourdough bread

The majority of gluten-free breads on the market are of poor sensory and textural quality. Exopolysaccharides (EPS) formed from sucrose during sourdough fermentation can improve the technological properties of gluten-free breads and potentially replace hydrocolloids. In this study, the influence of in situ formed EPS on dough rheology and quality of gluten-free sorghum bread was investigated. Dextran forming Weissella cibaria MG1 was compared to reuteran producing Lactobacillus reuteri VIP and fructan forming L. reuteri Y2. EPS containing bread batters were prepared by adding 10% and 20% of sourdough. As control served batters and bread containing sourdoughs fermented without sucrose and batters and bread without sourdough addition. The amount of EPS formed in situ ranged from 0.6 to 8.0 g/kg sourdough. EPS formed during sourdough fermentation were responsible for the significant decrease in dough strength and elasticity, with in situ formed dextran exhibiting the strongest impact. Increased release of glucose and fructose from sucrose during fermentation enhanced CO? production of yeast. Organic acids in control sourdough breads induced hardening of the bread crumb. EPS formed during sourdough fermentation masked the effect of the organic acids and led to a softer crumb in the fresh and stored sorghum bread. Among EPS, dextran showed the best shelf life improvements. In addition to EPS, all three strains produced oligosaccharides during sorghum sourdough fermentation contributing to the nutritional benefits of gluten-free sorghum bread. Results of this study demonstrated that EPS formed during sourdough fermentation can be successfully applied in gluten-free sorghum flours to improve their bread-making potentials.     

Environmental influences on exopolysaccharide formation in Lactobacillus reuteri ATCC 55730

Lactobacillus reuteri is known to produce exopolysaccharides (EPS), which have the potential to be used as an alternative biothickener in the food industry. In this study, the effect of several environmental conditions on the growth and EPS production in the L. reuteri strain ATCC 55730 was determined. The expression of the corresponding reuteransucrase gene, gtfO, was investigated over time and the results indicated that the expression increased with growth during the exponential phase and subsequently decreased in the stationary phase. Fermentation with glucose and/or sucrose as carbon and energy source revealed that gtfO was constitutively expressed and that the activity profile was independent of the sugar source. In the applied ranges of parameter values, temperature and pH were the most important factors for EPS formation and only temperature for growth. The best EPS yield, 1.4 g g(-1) CDW, was obtained at the conditions 37 degrees C, pH 4.5 and 100 g l(-1) sucrose, which were close to the estimated optimal conditions: pH 4.56 and 100 g l(-1) sucrose. No EPS formation could be detected with glucose. In addition, no direct connection between the expression and the activity of reuteransucrase could be established. Finally, the strain ATCC 55730 was benchmarked against 14 other L. reuteri strains with respect to EPS production from sucrose and abilities to metabolise sucrose, glucose and fructose. Eight strains were able to produce glucan and a corresponding glucansucrase gene was confirmed for each of them.     

Headspace flavour compounds produced by yeasts and lactobacilli during fermentation of preferments and bread doughs.

Production of volatile flavour compounds during fermentation with pure cultures of Saccharomyces cerevisiae and Candida guilliermondii, Lactobacillus brevis and Lactobacillus plantarum have been investigated, using wheat doughs and several preferements as substrates. For yeast, preferments consisted of 10% (w/v) glucose, maltose and sucrose solutions, whereas for lactobacilli they consisted of supplemented and unsupplemented (3% and 10% (w/v)) glucose solutions, and a 10% (w/v) wheat flour slurry. Seven volatile compounds (acetaldehyde, acetone, ethyl acetate, ethanol, hexanal+isobutyl alcohol, and propanol) were detected when using yeasts. All these compounds, except propanol, appeared for all the substrates assayed, with ethanol as the predominant component. Generally, S. cerevisiae produced higher amounts of the different components than C. guilliermondii. Both yeasts produced larger amounts of volatile flavour compounds during fermentation in glucose and sucrose solutions than in maltose or wheat dough. In general the yeasts examined produced more flavour components than the lactobacilli. For the lactobacilli the highest number of volatile flavour compounds were observed for substrates containing flour.     

Growth and metabolism of selected strains of probiotic bacteria, in maize porridge with added malted barley

A fermented probiotic maize porridge with high energy density and low viscosity was prepared, using maize flour and barley malt. The porridge was fermented with four probiotic strains (grown separately): Lactobacillus reuteri, Lb. acidophilus (LA5 and 1748) and Lb. rhamnosus GG. These strains were inoculated at two levels; to obtain approx. 7 or 6 log cfu g(-1) in the porridge at 0 h. The porridge was fermented for 24 h at 37 degrees C, and analysed for viable cell count, pH, organic acids, volatile aromatic compounds and sugar content. The inoculated cell concentration was shown to be particularly important during the first hours of the fermentation period, showing a delayed production of most metabolites in porridge inoculated with approx. 6 log cfu g(-1). Most strains reached maximum cell count after 12-h fermentation (7.2-8.2 log cfu g(-1)), with a pH below 4.0. Depending on the strain, lactic acid was produced in amounts ranging from 1360 to 4000 mg kg(-1). Lb. reuteri metabolised succinate, while pyruvate and small amounts of diacetyl were detected in porridge inoculated with Lb. acidophilus LA5 and Lb. acidophilus 1748. High amounts of diacetyl (6 mg kg(-1)) and acetoin (27 mg kg(-1)) were detected in porridge inoculated with Lb. rhamnosus GG. Porridge inoculated with Lb. acidophilus LA5 and Lb. acidophilus 1748, contained acetaldehyde, while both Lb. reuteri and Lb. rhamnosus GG reduced the acetaldehyde to ethanol. Lb. reuteri utilised both maltose and glucose as carbohydrate sources, while Lb. acidophilus LA5, Lb. acidophilus 1748 and Lb. rhamnosus GG utilised only glucose.     

罗伊氏乳杆菌培养基优化及其生长代谢研究

罗伊氏乳杆菌的MRS 培养基是富培养基,简化培养基组分,优化培养基组成和培养条件是微生物工业化培养的重要基础。本实验在传统MRS 培养基基础上,首先对氮源进行单因素优化,然后采用Plackett-Burman 和中心组合试验设计对影响罗伊氏乳杆菌CG001 生长的MRS 培养基和培养条件进行筛选优化,并在最优条件下研究该菌的生长代谢情况。结果表明：酵母膏质量浓度、葡萄糖质量浓度、硫酸锰质量浓度和温度是影响菌体质量浓度的4 个关键因素;经响应面法分析确定该菌的最优培养条件为：酵母膏质量浓度20g/L,葡萄糖质量浓度20g/L,醋酸钠质量浓度7g/L,柠檬酸铵质量浓度1g/L,磷酸氢二钾质量浓度3g/L,硫酸镁质量浓度0.2g/L,硫酸锰质量浓度为0.23g/L,吐温-80 质量浓度1g/L,初始pH6.2,培养温度38.6℃,最终菌体质量浓度达0.984g/L,相对优化前提高1.102 倍。发酵罐中菌体生长曲线呈S 型,4～12h 菌体处于对数生长期,之后趋于平衡,葡萄糖的代谢与菌体的生长速率相对应。     

Study of the behaviour of Lactobacillus plantarum and Leuconostoc starters during a complete wheat sourdough breadmaking process

The acidification properties, metabolic activity and technological performance of four individual Lactobacillus plantarum or Leuconostoc freeze-dried starters were investigated during a complete wheat sourdough breadmaking process including 0.2 g/100 g baker's yeast. Microbiological contents (lactic acid bacteria and yeasts), acidification characteristics (pH and total titratable acidity), soluble carbohydrates (maltose, glucose and fructose) and fermentative end-products (lactic and acetic acids, ethanol) contents were evaluated during both sourdough and corresponding bread dough fermentation. Biochemical and technological analysis of the resulting bread products are also presented. Some differences among strains in acidification properties and soluble carbohydrates availability were outlined both in sourdough and bread dough. Each individual Leuconostoc or Lb. plantarum starter was able to produce a characteristic fermentation and was found to ensure the production of breads with overall satisfactory acceptance.     

Salt-free Miso Fermentation Using Ethanol, Sugars, and Polyols

Steam-cooked soybeans mixed with rice koji (2:1, w/w) were ground into a fine paste, combined with NaCl or sucrose, and supplemented with ethanol for miso fermentation at 28 °C for 8 wk. In products containing the same concentrations of NaCl and sucrose, proteolytic activities decreased with an increase in ethanol concentration. At the same level of ethanol (7.5% or 15%), protein hydrolysis was highest in products containing 12.5% sucrose and 0% NaCl and lowest in products containing 0% sucrose and 12.5% NaCl. NaCl enhanced and sucrose reduced the effects of ethanol on inhibition of koji proteases. A salt-free fermentation was achieved using 15% ethanol and 12.5% sucrose. When sucrose (12.5%) was substituted by glucose, fructose, sorbitol, or glycerol and supplemented with 15% ethanol, prevention of protease inactivation by ethanol was similar to that with sucrose.     

CHEMICAL AND SENSORY CHARACTERIZATION OF COMMERCIAL SAUERKRAUT1

Canned sauerkraut from eight U.S. companies was analyzed for salt, titra-table acidity (TA), fermentation substrates and end products, volatile sulfur compounds and sensory characteristics. The TA ranged from 0.9–1.5%, while salt content ranged from 1.4–2.0%, which was lower than in previous surveys. High performance liquid chromatography (HPLC) was used to monitor lactic, acetic, malic, succinic, propionic and butyric acids; mannitol, ethanol, propanol, glycerol, glucose, fructose and sucrose. Low concentrations of propionic acid, propanol and glycerol were found. These three compounds are not characteristic of lactic acid fermentations. No butyric acid was detected. GC analysis revealed seven main sulfur compounds (hydrogen sulfide, methanethiol, dimethyl sulfide, carbon disulfide, dimethyl disulfide, allyl isothiocyanate (AITC) and dimethyl trisulfide) and six other organic compounds (methanol, ethanol, n-propanol, 2propanol, acetaldehyde and ethyl acetate) in the headspace of sauerkraut juice. A profile panel characterized aroma, flavor and after-taste of sauerkraut with ten distinct notes. The sour, sulfur and salt notes had the greatest impact on sauerkraut flavor.     

Utilization of electron acceptors by lactobacilli isolated from sourdough

Lactobacillus sanfrancisco is frequently a prevalent organism in wheat and rye sourdoughs. Its growth and metabolism are strongly affected by agitation and the availability of maltose and electron acceptors such as oxygen and fructose. Upon agitation of a culture the length of the lag phase was reduced. The growth rate and final cell yield increased in the presence of oxygen and electron acceptors. Growing cells formed lactate and ethanol from maltose under anaerobic conditions. Intermediately excreted glucose did not repress maltose utilization. Upon aeration or addition of fructose to cultures growing on maltose, acetate was formed instead of ethanol. Fructose was reduced to mannitol. Added fumarate and malate were converted to lactate and did not serve as electron acceptors. The following observations are suggested to contribute to the competitiveness ofL. sanfrancisco in sourdough. Glucose is excreted in abundance of maltose and represses the maltose metabolism of competitors, maltose is split by maltose phosphorylase without the expenditure of adenosine 5-triphosphate (ATP), and additional metabolic energy is generated by the activity of acetate kinase in the presence of fructose which is abundant in the polyfructosanes of flour. The metabolic features ofL. pontis, L. reuteri, L. amylovorus andL. fermentum are to be described in a following communication.     

Controlled Fermentation of Spanish-type Green Olives

Pure culture fermentation of Spanish-type green olives was developed. The method used no heat treatment, included chlorination of both fermentor and olives, used sterile lye, water and brine, and acidification with lactic acid before inoculation. Lactobacillus pluntarum was used as test species. After 34 days fermentation, citric acid, mannitol and malic acid were completely degraded and ～ 90% of available glucose and fructose, but <30% sucrose, were utilized. Fermentation products were D- and L-lactic acid, ethanol, succinic, and acetic acid with a calculated carbon recovety of 107.5%. D-lactic predominaied over L-lactic acid. No differences were found between flavor of pure culture and naturally fermented olives, but there was a tendency towards preference of the latter.     

Ethanol yield and volatile compound content in fermentation of agave must by Kluyveromyces marxianus UMPe-1 comparing with Saccharomyces cerevisiae baker's yeast used in tequila production

In tequila production, fermentation is an important step. Fermentation determines the ethanol productivity and organoleptic properties of the beverage. In this study, a yeast isolated from native residual agave must was identified as Kluyveromyces marxianus UMPe-1 by 26S rRNA sequencing. This yeast was compared with the baker's yeast Saccharomyces cerevisiae Pan1. Our findings demonstrate that the UMPe-1 yeast was able to support the sugar content of agave must and glucose up to 22% (w/v) and tolerated 10% (v/v) ethanol concentration in the medium with 50% cells survival. Pilot and industrial fermentation of agave must tests showed that the K. marxianus UMPe-1 yeast produced ethanol with yields of 94% and 96% with respect to fermentable sugar content (glucose and fructose, constituting 98%). The S. cerevisiae Pan1 baker's yeast, however, which is commonly used in some tequila factories, showed 76% and 70% yield. At the industrial level, UMPe-1 yeast shows a maximum velocity of fermentable sugar consumption of 2.27g·L(-1)·h(-1) and ethanol production of 1.38g·L(-1)·h(-1), providing 58.78g ethanol·L(-1) at 72h fermentation, which corresponds to 96% yield. In addition, the major and minor volatile compounds in the tequila beverage obtained from UMPe-1 yeast were increased. Importantly, 29 volatile compounds were identified, while the beverage obtained from Pan1-yeast contained fewer compounds and in lower concentrations. The results suggest that the K. marxianus UMPe-1 is a suitable yeast for agave must fermentation, showing high ethanol productivity and increased volatile compound content comparing with a S. cerevisiae baker's yeast used in tequila production.     

Differential effects of glucose and fructose on hexose metabolism in dog spermatozoa

Incubation of dog spermatozoa with 10 mmol l(-1) glucose or fructose rapidly increased the intracellular content of glucose 6-phosphate and fructose 6-phosphate, although the effect of fructose was greater. These effects were correlated with increases in ATP, ribose 5-phosphate and glycogen contents, and in the rates of formation of L-lactate and CO2. In all cases, except for ATP and glycogen, the effect of fructose was greater than that of glucose. The total hexokinase activity of the crude extracts of dog spermatozoa was more sensitive to fructose than to glucose at lower concentrations (0.1-3.0 mmol l(-1)). Both monosaccharides induced a fast and intense increase in the overall tyrosine phosphorylation of dog spermatozoa, although their specific induced-phosphorylation patterns differed slightly. Glut 3 and Glut 5 hexose transporters were the main hexose transporters in dog spermatozoa; however, other possible SGLT family-related hexose transporters were also localized. These data indicate that, at concentrations from 1 mmol l(-1) to 10 mmol l(-1), fructose has a stronger effect than glucose on hexose metabolism of dog spermatozoa. These differences appear to be related to variations in the sensitivity of hexokinase activity. Moreover, the differential hexose metabolism induced by the two sugars had distinct effects on the function of dog spermatozoa, as revealed by the diverse patterns of tyrosine phosphorylation.     

Cultivation of the carotenoid-hyperproducing mutant 2A2N of the red yeast Xanthophyllomyces dendrorhous (Phaffia rhodozyma) with molasses

The carotenoid-hyperproducing mutant 2A2N of the yeast Xanthophyllomyces dendrorhous (Phaffia rhodozyma) was cultivated using sugar beet blackstrap molasses. This molasses was composed of 70% (w/v) total solid and 50% (w/v) total sugar. Biomass yield (biomass/carbohydrate) significantly decreased at >5% (v/v) molasses. Atomic emission spectrometry revealed that Na and P were the limiting nutrients when molasses was used. Molasses (5%, v/v) containing urea (30 g/l molasses) and sodium phosphate (NaH2PO4, 5 g/l molasses) was formulated for biomass production by the mutant. The optimal pH for carotenoid production was 4.9 during the growth phase and 2.6-3.5 during the stationary phase. The three main sugars in molasses (sucrose, glucose, and fructose) were assimilated by the mutant but fructose was consumed slowly. When the formulated medium with pH 4.5-5.5 was used, the maximal biomass yield was 36 g/l (0.18 g of yeast l(-1)h(-1) and 40 mg of carotenoid l(-1)) in fed-batch pilot-scale 100-l cultivation.     

蔗糖水解制取结晶果糖技术研究

为提升制糖产业的综合效益,以白砂糖为原料开发附加值更高的结晶果糖,探讨用蔗糖原料制取高纯度结晶果糖的最优工艺方法.实验以高浓度蔗糖液(55％w/w)为原料,食品酸味剂柠檬酸为水解剂,通过蔗糖水解、脱色、阴阳离子树脂除盐,以钙型树脂分离、纯化果葡糖液,在常温下得到结晶果糖和结晶葡萄糖.实验优化蔗糖最佳水解工艺条件:水解温...     

离子色谱-脉冲安培法同时测定蔗糖酶和果聚糖酶活力的方法建立

本文旨在建立一种采用离子色谱仪同时测定蔗糖酶、果聚糖酶活力的方法。以蔗糖为底物,先加入蔗糖酶酶解并以硼氢化钠还原酶解产物葡萄糖和果糖得到甘露醇和山梨醇,再将蔗糖酶高温失活后加入果聚糖酶,得到含甘露醇、山梨醇、葡萄糖和果糖四种化合物的混合溶液。以水-氢氧化钠溶液-乙酸钠溶液为流动相进行梯度洗脱,在流速为1.0 mL/min、柱温为30 ℃条件下经CarboPaCTM PA1 250 mm×2 mm分离以测定四种化合物的含量。最后以甘露醇和山梨醇总量计算蔗糖酶活力,以葡萄糖和果糖的总量计算果聚糖酶的活力。结果表明:甘露醇、山梨醇、葡萄糖和果糖四种化合物在20 min内完成测定,且线性相关系数均达到0.9995以上,分离度均在1.0以上;果聚糖酶活力的测定可以采用蔗糖为底物;加入1.2 mL 10 mg/mL的硼氢化钠溶液并60 ℃水浴30 min后,葡萄糖和果糖能够分别完全转化为甘露醇和山梨醇;本方法测定的蔗糖酶活力、果聚糖酶活力与3,5-二硝基水杨酸比色法（DNS法）测定的蔗糖酶活力、果聚糖酶活力结果无显著性差异（P>0.05）;且测定的蔗糖酶活力、果聚糖酶活力含量结果的RSD分别为2.0%和1.7%,精密度好,且本方法不涉及传统方法所用的有毒有害试剂。因此,所建方法可以同时用于测定蔗糖酶和果聚糖酶的活力,同时也为其它酶活力测定提供了一种新的思路。     

Microaeration enhances productivity of bioethanol from hydrolysate of waste house wood using ethanologenic Escherichia coli KO11

This is the first study showing the successful application of waste house wood (WHW) to the pilot-scale production of bioethanol by hydrolysis using diluted acid and fermentation using the ethanologenic recombinant Escherichia coli KO11. The major sugars in the WHW hydrolysate were glucose, mannose and xylose; the percentages were approximately 35%, 35% and 20% (w/w), respectively. In anaerobic fermentation using a 5-l reactor in which the oxygen transfer rate (OTR) was 0 mmol/(l x h), KO11 consumed only 25% of the xylose in the WHW hydrolysate over the examined fermentation time of 100 h; however, hexoses such as glucose and mannose were consumed completely. Microaeration at an OTR of 4 mmol/(l x h) enhanced the xylose utilization ratio of KO11 to 100%, at which the ethanol concentration was 35.4 g/l and the ethanol yield was 0.42, although the maximum ethanol concentrations were 28.8 and 26.6 g/l at OTRs of 0 mmol/(l x h) and 15 mmol/(l x h), respectively. Moreover, this microaerobic fermentation at OTR of 4 mmol/(l x h) was applied to 1000-l scale bioethanol production using the WHW hydrolysate. The xylose utilization ratio reached 100% and the ethanol yield was determined to be 0.45 for a 63-h fermentation, which were comparable to those obtained from the laboratory-scale fermentation.