Development of lytic Lactococcus lactis bacteriophages in a Cheddar cheese plant

The mixed TK5 starter culture was used in a Danish factory as the only starter for production of Cheddar cheese for more than 11 years before the factory experienced serious bacteriophage attacks with inhibition of the acid production in the curd. The cheese whey contained some phages from the beginning, and gradually new phages appeared able to infect an increasing number of isolates. Three bacteriophages jw30, jw31 and jw32 were isolated from the factory whey collected in 1989–94 and compared with lytic bacteriophages isolated in the period 1982–86 (Josephsen et al., 1994) DNA hybridisation showed that the type phage P008 had high homology to the new phages jw30, jw31 and jw32 as had the phages isolated in 1982–84. The new phages had broader host ranges and higher burst sizes than the previously isolated phages, showing that the lytic phages had become more virulent with time.     

Growth energetics of Lactococcus lactis subsp. lactis biovar diacetylactis in cometabolism of citrate and glucose

Certain strains of lactic acid bacteria present in commercial cheese starters, characterized by faint transparent colonies on an agar plate containing 1 mg kg ⁻¹ crystal violet (CVT), were identified as Lactococcus lactis subsp. (ssp) lactis biovar diacetylactis. The effect of citrate on the growth of these strains (CVT strains) in the presence of glucose was studied, in comparison with L. lactis strains. Molar growth yield from glucose (Y_G, g dry weight/mole of glucose consumed) for CVT strains grown on glucose plus citrate was significantly higher than the control (i.e. without citrate), but not for other L. lactis strains tested. Enhanced Y_G was also observed at a pH-controlled experiment, indicating that enhanced Y_G did not result from a buffering effect of citrate. CVT strains, in contrast to other strains of the same species, were shown to obtain enough energy to enhance Y_G on glucose–citrate mixtures.     

High nisin-Z production during repeated-cycle batch cultures in supplemented whey permeate using immobilized Lactococcus lactis UL719

Nisin-Z production was studied during repeated-cycle pH-controlled batch (RCB) cultures using Lactococcus lactis subsp. lactis biovar. diacetylactis UL719 immobilized in κ-carrageenan/locust bean gum gel beads in supplemented whey permeate. After an initial colonization of gel beads during the first two cycles, nisin-Z production in bulk medium and gel beads was very similar for 1-h and 2-h cycle RCB cultures. A very high nisin-Z production (8200 IU mL⁻¹) was measured in the broth after the 1-h cycles, with a corresponding volumetric productivity of 5730 IU mL⁻¹ h⁻¹. This productivity is much higher than maximum nisin productivities reported in literature or maximum productivities obtained previously for free-cell batch cultures (850 IU mL⁻¹ h⁻¹), and free-cell (460 IU mL⁻¹ h⁻¹) or immobilized-cell (1760 IU mL⁻¹ h⁻¹) continuous cultures, using the same strain and fermentation conditions. The stability of RCB cultures was demonstrated for 24 and 36 1-h cycles carried out over 3 and 6-day periods, respectively. Changing environmental conditions during batch cultures resulted high nisin production.     

Kinetic modeling and sensitivity analysis of xylose metabolism in Lactococcus lactis IO-1

We proposed a kinetic simulation model of xylose metabolism in Lactococcus lactis IO-1 that describes the dynamic behavior of metabolites using the simulator WinBEST-KIT. This model was developed by comparing the experimental time-course data of metabolites in batch cultures grown in media with initial xylose concentrations of 20.3–57.8 g/l with corresponding calculated data. By introducing the terms of substrate activation, substrate inhibition, and product inhibition, the revised model showed a squared correlation coefficient (r²) of 0.929 between the experimental time-course of metabolites and the calculated data. Thus, the revised model is assumed to be one of the best candidates for kinetic simulation describing the dynamic behavior of metabolites. Sensitivity analysis revealed that pyruvate flux distribution is important for higher lactate production. To confirm the validity of our kinetic model, the results of the sensitivity analysis were compared with enzyme activities observed during increasing lactate production by adding natural rubber serum powder to the xylose medium. The experimental results on pyruvate flux distribution were consistent with the prediction by sensitivity analysis.     

乳酸乳球菌硫胺素转运蛋白ThiT提高酸耐受性的机制

碳代谢引起的酸积累严重影响了微生物细胞的代谢活性。为提高细胞在酸性环境下的耐受性,通过过量表达硫胺素转运蛋白ThiT,研究了重组菌株的酸胁迫耐受性,并在此基础上通过比较转录组学对照菌株和重组菌株在正常和酸胁迫条件下关键基因的转录水平进行了研究。结果表明,经过酸胁迫（pH 4.0） 处理后,重组菌株的最大存活率优势是对照菌株的16.2倍。转录组学的分析发现,过表达ThiT转运蛋白可提高细胞对碳水化合物的转运能力,从而为细胞抵御酸胁迫的耐受性提供更多的能量;此外寡肽、甜菜碱等相关转运基因的显著上调可帮助细胞抵御酸胁迫的损伤。作者探究了在乳酸乳球菌中过表达ThiT转运蛋白提高细胞酸胁迫耐受性的具体作用机制,为ThiT蛋白在其他工业微生物中的应用提供了理论基础。     

5种菠萝果实品质比较与评价

为利用干酪乳杆菌制备发酵菠萝果汁饮品,本研究对干酪乳杆菌LK-1（Lactobacillus casei LK-1,L. casei LK-1）的生长曲线、产酸、耐酸和耐糖等特性进行研究分析,并以活菌数和总酸为指标,通过单因素实验及响应面法考察L. casei LK-1发酵菠萝果汁的最佳条件。结果表明,L. casei LK-1在pH5~7和0%~10%质量分数葡萄糖环境下,具备良好的生长繁殖能力;在酸性（pH3）和高糖（40%质量分数葡萄糖）环境中培养3 h,其存活率分别为76%和71%;在MRS培养基中培养48 h,产酸量为5.35 g/kg,产酸能力良好,适用于果汁的发酵。L. casei LK-1发酵菠萝果汁的最佳条件为：初始pH6.8,发酵温度37 ℃,接种量1%（v/v）,发酵时间30 h,此条件下制备的发酵菠萝果汁饮品活菌数为8.99±0.04 lg CFU/mL,总酸含量为7.16±0.26 g/kg,且色泽鲜亮,酸甜适口,风味较好,为L. casei LK-1在发酵食品中的进一步应用提供了理论和技术依据。

     

The influence of oxygen on the differentiation of temperature-sensitive and temperature-insensitive Lactococcus lactis subsp. cremoris starter strains

The differentiation of temperature-insensitive and temperature-sensitive strains of Lactococcus lactis subsp. cremoris by using a modified sodium-β-glycerophosphate/milk medium is described (temperature-insensitive strains are defined as those that continue to grow at 38°C and temperature-sensitive strains as those that do not grow, or grow poorly, at 38°C). The physiological basis for the differentiation assay was examined by using L. lactis subsp. cremoris strains 2146 (temperature-insensitive) and 2182 (temperature-sensitive) as test strains. After aerobic incubation on the medium, strain 2146 formed uniform colonies, 0·5 mm in diameter, while strain 2182 formed larger colonies, 1·0–1·5 mm in diameter. The differential was dependent on the medium constituents, on an aerobic gas phase, and on the effects of H₂O₂ generated within the colonies. The addition of 0·5% (w/v) pyruvate to the medium facilitated the growth of colonies of strain 2146 to 3-mm diameter, while the colony size of strain 2182 remained at 1-mm diameter, and thus the colony-size differential between strains was reversed. The growth of both strains was inhibited at 0·4–0·8% air saturation during suspension culture in sodium-β-glycerophosphate/milk medium. The inclusion of catalase in the cultures overcame the growth inhibition. There was no observable difference between the two strains in their oxygen sensitivity or NADH oxidase/peroxidase enzymology.     

Potential of bacteriocinogenic Lactococcus lactis subsp. lactis inhabiting low pH vegetables to produce nisin variants

Antimicrobial behavior of lactic acid bacteria (LAB) has been explored since many years to assess their ability to produce bacteriocin, a natural preservative, to increase the shelf life of food. This study aims to characterize bacteriocin producing strains of lactic acid bacteria isolated from acidic to slightly acidic raw vegetables including tomato, bell pepper and green chili and to investigate their potential to inhibit food related bacteria. Among twenty nine LAB screened for antimicrobial activity, three exhibited antagonism against closely related bacterial isolates which was influenced by varying temperature and pH. They were identified up to strain level as Lactococcus lactis subsp. lactis TI-4, L. lactis subsp. lactis CE-2 and L. lactis subsp. lactis PI-2 based on 16S rRNA gene sequence. Their spectrum of inhibition was observed against food associated strains of Bacillus subtilis and Staphylococcus aureus. Moreover, L. lactis subsp. lactis PI-2 selected on the basis of higher antimicrobial activity was further evaluated for bacteriocin production which was detected as nisin A and nisin Z. These findings suggest the possible use of L. lactis strains of vegetable origin as protective cultures in slightly acidic as well as slightly alkaline food by the bio-preservative action of bacteriocins.     

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.     

Effect of aeration and dilution rate on nisin Z production during continuous fermentation with free and immobilized Lactococcus lactis UL719 in supplemented whey permeate

The influence of dilution rate (D) and aeration on soluble and cell-bound nisin Z production was investigated during continuous free (FC) and immobilized cell (IC) cultures with Lactococcus lactis subsp. lactis biovar diacetylactis UL719 in supplemented whey permeate. Maximum total bacteriocin titres during non-aerated continuous FC and IC cultures were obtained for low D, with 1490 and 1090 IU mL⁻¹ for 0.15 h⁻¹ or 0.25 and 0.5 h⁻¹, respectively. For both systems, aeration increased nisin total production with maximum titres of 2560 and 2430 IU mL⁻¹ for low D, respectively, as well as specific production. Volumetric productivity was the highest for an intermediate D of 0.4 h⁻¹ during FC cultures (460 IU mL⁻¹ h⁻¹ for both aerated and non-aerated cultures), while it increased continuously with D during IC cultures, reaching high values of 1090 and 1760 IU mL⁻¹ h⁻¹ at 2.0 h⁻¹ without and with aeration, respectively. In comparison with previous data for FC batch cultures, data from this study may indicate that during continuous fermentations at steady state, some steps in nisin biosynthesis are limiting. In these conditions, nisin production by immobilized cells is reduced.     

Functionality of freeze-dried L. casei cells immobilized on wheat grains

Lactobacillus casei cells were immobilized on wheat grains and the effect of nine cryoprotectants during freeze-drying was investigated. Survival and fermentative activity of the freeze-dried immobilized biocatalysts was studied by monitoring pH, lactic acid and lactose content in successive fermentations batches of both synthetic lactose medium and milk. Freeze-dried L. casei cells immobilized on wheat grains without using cryoprotectants resulted in high cell survival and metabolic activity. The same biocatalysts were stored at room temperature for 9 months and at 4 °C and −18 °C for 12 months. Reactivation of the stored biocatalysts was carried out in synthetic lactose medium. Storage at room and low temperatures (4 °C and −18 °C) resulted in about 5.11, 4.9 and 4.3 final pH respectively during fermentations, indicating the suitability of the immobilized biocatalysts for the production of mild and low pH dairy products. The immobilization of a probiotic microorganism, such as L. casei, on boiled wheat which contains prebiotic compounds might provide a potential synbiotic preparation.     

Fe3O4磁性纳米粒子固定化微泡菌褐藻胶裂解酶AlgL17的研究

本论文旨在优化微泡菌褐藻胶裂解酶AlgL17固定化条件,以期探索出高效的固定化条件。以单宁酸功能化的四氧化三铁磁性纳米粒子作为载体,对褐藻胶裂解酶AlgL17进行固定化条件优化。测定固定化酶的最适反应温度和最适反应pH,并对固定化酶进行傅里叶变换红外光谱(FTIR)和扫描电镜(SEM)检测。结果表明,褐藻胶裂解酶AlgL17最佳固定化条件为:加酶量2.7 U、载体量25 mg、固定时间5 h,固定化酶的最适反应条件为:温度35℃、pH8.5。FTIR和SEM结果显示,褐藻胶裂解酶AlgL17固定在载体表面。四氧化三铁磁性纳米粒子可以成功固定褐藻胶裂解酶AlgL17,固定化后的褐藻胶裂解酶具有工业应用的前景。     

营养盐在固定化酵母生产蜂蜜酒中影响的研究

对营养盐类物质在固定化酵母生产蜂蜜酒中的影响进行研究,通过L9(34)正交试验确定了复合营养盐添加的最适添加量。     

Effect of a bacteriocin-producing Lactococcus lactis strain and high-pressure treatment on the esterase activity and free fatty acids in Hispánico cheese

The effect of milk inoculation with a bacteriocin-producing (BP) culture and of high-pressure (HP) treatment of 15-day-old Hispánico cheeses (400 MPa, 5 min, 10 °C), separately or combined, on the release of intracellular esterases and cheese lipolysis was investigated. Esterase activity and free fatty acids (FFAs) content increased during ripening of Hispánico cheese and palmitic, oleic and stearic acids being the most abundant FFAs. On day 15, the highest esterase activity was recorded for HP-treated BP cheese. The activity for HP-untreated BP cheese was the next highest. No difference in the activities was found between HP-treated and untreated cheeses made without BP culture. Total FFAs on day 15 were at a lower concentration in BP cheeses than in cheeses made without BP culture, probably due to the lower pH values of the former. The rate of total FFA accumulation from day 15 to day 50 was higher in BP cheeses (31.1–32.1% increase) than in cheeses made without BP culture (19.3–21.7% increase). The highest total FFA concentration on day 50 (612 mg kg⁻¹) was found for HP-untreated cheese made without BP culture.     

Potential production and preservation of dahi by Lactococcus lactis W8, a nisin-producing strain

Lactococcus lactis W8 produced nisin concomitantly while fermenting milk to “dahi”, a traditional Indian fermented milk. The activity of nisin was detected at 3 h of fermentation, which increased in parallel to growth of the organism and reached its maximum at 6 h. The activity remained essentially stable thereafter. At 7 h of fermentation of milk with the strain L. lactis W8 the pH of the medium dropped to 4.2, when the milk became converted to dahi. The produced dahi displayed antibacterial property against spoilage and pathogenic bacteria including Listeria monocytogenes. When L. monocytogenes was mixed with dahi at 5.2 log CFU/ml and stored at 4 °C, the number of L. monocytogenes gradually decreased and became undetectable at 10 h. L. lactis W8 appeared to be a suitable starter culture for production of dahi from milk and preservation of the dahi.     

Stepwise ethanolysis of tuna oil using immobilized Candida antarctica lipase

Ethanolysis of fish oil under mild conditions has been strongly desired for preparing the starting materials for the purification of ethyl docosahexaenoate. Thus, we attempted ethanolysis of tuna oil using immobilized Candida antarctica lipase. The immobilized lipase was inactivated in the presence of 2/3 molar equivalent of ethanol against the total fatty acids in tuna oil. To avoid such inactivation, the first step of ethanolysis was conducted at 40°C in a mixture of tuna oil and 1/3 molar equivalent of ethanol using 4% immobilized lipase. After a 10-h reaction, ethanol was consumed and 33% of tuna oil was converted to its corresponding ethyl esters (E-FAs). The reactant is named Gly/E-FA33. The lipase was not inactivated in the presence of 2/3 molar equivalent of ethanol against the total fatty acids in Gly/E-FA33. These findings and the consideration of several factors affecting ethanolysis of tuna oil led to the development of the two- and three-step ethanolyses. The two-step reaction was performed as follows: the first step was carried out at 40°C for 12 h in a mixture of tuna oil and 1/3 molar equivalent of ethanol with 4% immobilized lipase; the second step was performed for 36 h (total reaction period, 48 h) after adding 2/3 molar equivalent of ethanol. On the other hand, the three-step reaction was conducted as follows: the first step was conducted under the same conditions as those in the two-step ethanolysis; in the second and third steps, 1/3 molar equivalent of ethanol was added after 12 and 24 h, respectively; and in the third step, the mixture was shaken for 24 h (total, 48 h). Both types of ethanolyses achieved the conversion of 95% or more of tuna oil to its corresponding E-FAs. To investigate the lipase stability, the two- and three-step ethanolyses were repeated by transferring the enzyme to a fresh substrate mixture of the first step after finishing one cycle of reaction. The two- and three-step reactions maintained over 95% of the conversion for 70 d and over 100 d, respectively.     

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。     

Effect of oxygen concentration on nitrogen removal by Nitrosomonas europaea and Paracoccus denitrificans immobilized within tubular polymeric gel

Tubular gel reactors containing Nitrosomonas europaea and Paracoccus denitrificans, which remove nitrogen from solutions through a process of nitrification and denitrification, require oxygen for ammonia oxidation, the first and rate-limiting step in the process. To accelerate ammonia oxidation, high concentrations of oxygen were applied to the reactors instead of air. Although a 50% O₂:N₂ gas mixture and pure oxygen were both toxic to free N. europaea cells, they actually accelerated ammonia oxidation by N. europaea immobilized within the tubular gel. Indeed, the rate of ammonia oxidation by a tube exposed to pure oxygen was twice that of one exposed to 20% O₂. When the distribution of N. europaea cells within the tubes was investigated using a fluorescently-labeled antibody, colonies were found on the external surface of the tube exposed to 20% O₂, but were located at a depth of 120–300 μm from the external surface in the case of the tube exposed to pure oxygen. The region between the external surface of the gel and the colonies apparently acted as a barrier, reducing the diffusion of oxygen and thus protecting the cells from oxygen cytotoxicity.     

The accelerating effect of aLactococcus lactis subsp.lactis lactose-utilization/proteinase-deficient strain on proteolysis and flavour development

This study describes the accelerating effect exerted on proteolysis and flavour development in cheese-curd slurries by combined application ofLactococcus lactis subsp.lactis IFPL359 and high concentrations of its Lac^– Prt^– derivative, (i.e. that which has reduced capacity to metabolize lactose and reduced proteolytic activity, strain T1). Cells of strain T1 partially lysed by either sonication or incubation with lysozyme, were also used to ascertain how proteolysis was affected by release of intracellular enzymes in the initial stage of incubation of the strains in cheese-curd slurries. The presence of strain T1 produced higher levels of non-protein nitrogen (NPN) and amine nitrogen (AN) during the first weeks of incubation when partially lysed cells had been added. Addition of whole cells of strain T1 produced higher values of NPN and AN at the end of incubation of slurries, accelerating proteolysis by about 2 weeks with respect to the control, which only contained the parental strain IFPL359. At the end of the experimental period higher amino acid levels were detected by HPLC in the slurry containing whole T1 cells. Volatile fractions of the different cheese-curd slurries were also analysed. The higher level of proteolysis produced by addition of high levels of strain T1 appeared to be related to release of intracellular enzymes by this strain owing to its greater capacity for autolysis.     

Continuous production of very enriched fructose syrup by the conversion of glucose to ethanol from glucose-fructose mixtures in an immobilized cell reactor

The yeast Saccharomyces cerevisiae ATCC 36859, which preferentially utilizes glucose in glucose-fructose mixtures, was immobilized and used for the continuous production of very enriched fructose syrup. A syrup containing fructose as 99% of the reducing sugars was produced from a 10.1% w/v glucose and 9.8% w/v fructose mixture at a dilution rate of 0.106 h⁻¹. Later in this process when the dilution rate was increased to 0.366 h⁻¹ the ethanol productivity was 12.7 g 1⁻¹ h⁻¹. This is 16% less than the value attained in a similar medium containing only glucose as the carbohydrate. The fructose content was also increased from 55 to 95% of the reducing sugars in a food grade mixture of high fructose corn syrup and Jerusalem artichoke juice. Purification of the product with activated carbon and ion-exchange resin produced a stable, colourless and very enriched fructose syrup suitable for human consumption.