Effect of compatible solutes on the respiratory activity and growth of Escherichia coli K-12 under NaCl stress

The respiratory activity of Escherichia coli K-12 was inhibited by high NaCl concentrations. The addition of compatible solutes such as proline and ectoine led to the recovery of the respiration of E. coli K-12 inhibited by 1 M NaCl to a similar extent as did the addition of glycine betaine. Glucose, an exogenous substrate for the stimulation of respiratory activity, was not taken up by the cells in the presence of 1 M NaCl, but active uptake of glucose was observed following the addition of compatible solutes. As a result, we obtained a good correlation between respiratory activities and glucose uptake rates, suggesting that the glucose uptake activity inhibited by high NaCl concentrations was facilitated by these solutes. The addition of proline did not lead to cell proliferation in the minimal medium containing 1 M NaCl, although the cells took up proline quite efficiently under high osmolarity. On the other hand, cell growth occurred after a lag time in the medium containing glycine betaine or ectoine in place of proline. Similar actions of the compatible solutes mentioned above were observed for E. coli ATCC 9637.     

Potential osmoprotectants for the lactic acid bacteria Pediococcus pentosaceus and Tetragenococcus halophila

The physiological responses of the lactic acid bacteria Pediococcus pentosaceus and Tetragenococcus halophila (formely known as P. halophila), subjected to osmotic stress in the presence of molecules known to act as osmoprotectants for other bacteria were studied. In a defined medium, glycine betaine, dimethylsulfonioacetate, choline, proline and L-carnitine were able to relieve inhibition of growth at 0.8 M NaCl. The five compounds were shown to efficiently compete with glycine betaine transport, suggesting the existence of common transporter(s) for these molecules. T. halophila, the most tolerant strain, exhibited a larger spectrum of compatible solutes including dimethylsulfonioacetate, dimethylsulfoniopropionate and ectoine. Preliminary data suggest that restoration of growth by ectoine under osmotic constraint seems specific to the genus Tetragenococcus.     

Effect of duration of osmotic downshock and coexisting glutamate on survival and uptake of ectoine in halotolerant Brevibacterium sp. JCM 6894

Halotolerant Brevibacterium sp. JCM 6894 that was subjected to an osmotic downshock (0.7 M NaCl to 0 M) was examined for its survival and uptake of ectoine in the presence of ectoine and/or carbon sources. In the presence of ectoine alone, the rates of ectoine uptake by the 1 h-downshocked cells were low and high in the absence and presence of 0.7 M NaCl, respectively, which were in parallel with the rates of cell growth. The presence of glutamate or amino acids together with ectoine exerted a stimulative effect on the survival of downshocked cells. The incubation time of the cells subjected to osmotic downshock strongly affected ectoine uptake as well as the cell growth of this strain, suggesting that the transporter of ectoine in the strain JCM 6894 was stimulated during the osmotic downshock for about 1 h. Different downshock strengths had marked effects on the rate of ectoine uptake when the downshocked cells were incubated in the presence of NaCl.     

Characterization of the induction of increased thermotolerance by high osmolarity inSalmonella

Factors limiting the growth ofSalmonellaat high temperature were analyzed in minimal medium. The growth ofS. typhimuriumwas curtailed at 44°C by high temperature-dependent block in methionine synthesis. At 45°C, the organism could not grow in glucose-mini- mal medium even with methionine, but growth could be restored at this temperature by the addition of NaCl in the final concentration range of 0.15–0.3m. Similar results were obtained with twoS. enteritidisstrains. The growth stimulatory effect of high osmolarity at 45°C was not altered by the osmoprotectant compound glycine betaine. Exposure to 0.3mNaCl also increased the resistance ofS. typhimuriumto thermal death at 50°C and to the oxidizing agent H₂O₂. However, the high osmolarity-dependent induction of enhanced resistance to 50°C and to H₂O₂was completely abolished by glycine betaine. The finding that glycine betaine blocks the induction of resistance to lethal high temperature by high osmolarity could provide a means for enhancing the bactericidal efficacy of heating, because it may be possible to increase the heat sensitivity of contaminating bacteria by supplementing food products with this naturally occurring plant metabolite, prior to thermal processing.     

Efficient utilization of ectoine by halophilic Brevibacterium species and Escherichia coli subjected to osmotic downshock

Halophilic and non-halophilic bacteria subjected to osmotic downshock, from 0.7 M NaCl to deionized water, were examined for their survival, with the uptake and utilization of the cyclic amino acid ectoine, one of the representative compatible solutes, being taken into account. The uptake of ectoine added externally and survival of the cells were monitored as a function of incubation time in the presence and absence of NaCl. The halophilic Brevibacterium sp. JCM 6894 and B. epidermidis JCM 2593 actively accumulated ectoine regardless of the presence of NaCl, which led to cell survival. Brevibacterium casei JCM 2594 belonging to the same Brevibacterium species, however, revealed Na+-dependence of its uptake activity of ectoine. Non-halophilic Escherichia coli K-12 did not accumulate ectoine, and thereby this strain failed to survive irrespective of whether NaCl was present. The physiological meanings of the downshock procedure are discussed in connection with the uptake and the subsequent utilization of ectoine.     

Biosynthesis of exopolysaccharide by a Bacillus licheniformis strain isolated from ropy cider

Listeria monocytogenes accumulates low molecular weight compounds (osmolytes, or compatible solutes) in response to chill stress. This response has been shown to be responsible, in part, for the chill tolerance of the species. Among the osmolytes tested to date, glycine betaine, gamma-butyrobetaine and carnitine display the strongest cryoprotective effect. These osmolytes are not synthesized in the cell and must be transported from the medium. In this study, the compatible solute accumulation profile of the food-borne pathogen L. monocytogenes was determined in balanced growth and stationary phase cultures grown in milk whey at 7 and 30 degrees C. In balanced growth cultures at 7 degrees C, glycine betaine (720 nmol/10(10) cfu) and carnitine (130 nmol/10(10) cfu) were the major osmolytes accumulated by wild-type L. monocytogenes 10403S, whereas carnitine (490 nmol/10(10) cfu) was the dominant osmolyte and glycine betaine was present in smaller amounts (270 nmol/10(10) cfu) in a mutant (L. monocytogenes LTG59) blocked in the major glycine betaine uptake system, glycine betaine porter II. In strain 10403S, glycine betaine and carnitine were present in eightfold and twofold excess at 7 degrees C compared to 30 degrees C; the respective ratios for strain LTG59 were 6 and 8. The intracellular concentration of osmolytes in stationary phase cultures at 7 degrees C was markedly reduced compared to that during balanced growth. Furthermore, at 4 degrees C, small but highly significant differences in growth were observed between strains. Strain LTG59 grew with a lag phase that was significantly longer, a generation time that was significantly greater and reached a final cell yield that was significantly lower than that of strain 10403S. The elevated accumulation of carnitine in the absence of glycine betaine porter II was insufficient to confer the magnitude of the cryoprotective effect displayed by the wild type.     

Humectant permeability influences growth and compatible solute uptake by Staphylococcus aureus subjected to osmotic stress

The effects of different humectants (sodium chloride, sucrose, and glycerol) on the growth of and compatible solute (glycine betaine, proline, and carnitine) uptake by the osmotolerant foodborne pathogen Staphylococcus aureus were investigated. While growth in the presence of the impermeant humectants sodium chloride and sucrose induced the accumulation of proline and glycine betaine by cells, growth in the presence of the permeant humectant glycerol did not. When compatible solutes were omitted from low-water-activity media, growth was very poor in the presence of impermeant humectants. In contrast, the addition of compatible solutes had essentially no effect on growth when cells were grown in low-water-activity media containing glycerol as the humectant. Carnitine was found to accumulate to high intracellular levels in osmotically stressed cells when proline and glycine betaine were absent, making it a potentially important compatible solute for this organism.     

Uptake and utilization of ectoine by halotolerant Brevibacterium sp. JCM 6894 subjected to osmotic downshock

The concentration changes of the cyclic amino acid ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyridine carboxylic acid) in Brevibacterium sp. JCM 6894 cells subjected to an osmotic downshock were investigated. When the cells grown in the presence of 2 M NaCl were suspended in deionized water, they immediately released about 60% of the ectoine synthesized intracellularly. During the subsequent incubation, we observed that both the extra- and intracellular concentrations of ectoine were reduced almost linearly with the incubation time. When ectoine was provided externally to the downshocked cells, a similar reduction in both intra- and extracellular ectoine concentrations was recognized. In addition, we observed an increase in ectoine accumulation at about 10 h of incubation, which indicates that ectoine was taken up by such downshocked cells in the absence of external Na+. Furthermore, the downshocked cells showed higher levels of survival, respiration, and growth in the presence of ectoine than in its absence. The ability to take ectoine up was induced in the cells grown in the presence of >0.25 M NaCl for >12 h. Thus, we conclude that even under the lower osmotic condition ectoine might be taken up and subsequently utilized by strain JCM 6894 subjected to the osmotic downshock, indicating that the uptake of ectoine by such cells occurred for the survival and growth of the cell itself rather than for cellular osmoregulation.     

Effects of exogenous compatible solutes on growth of the hyperthermophilic archaeon Sulfolobus solfataricus

Six known compatible solutes as well as twenty L-amino acids were individually added to a glucose minimal medium and their effects on the growth of Sulfolobus solfataricus (DSM 1617) were examined. Among the compatible solutes tested, putrescine, trehalose, and l-glutamate enhanced the growth of S. solfataricus. On the other hand, glycine betaine, choline, and L-proline showed little or no influence on cell growth. When cells were grown in the glucose medium supplemented with trehalose or L-glutamate, S. solfataricus preferentially utilized the compatible solute over glucose. The growth-enhancement effect of L-glutamate was also observed to be dependent on the glucose concentration in the medium: growth enhancement was higher when the concentration of glucose was low and gradually decreased with increasing glucose concentration. Interestingly, the effects of amino acids on cell growth differed markedly depending on the chemical nature of the amino acid added. While acidic amino acids-L-glutamate and L-aspartate-enhanced the growth rate, almost no growth was observed in the presence of glycine, L-leucine, L-valine, L-phenylalanine, L-threonine, L-methionine, or L-cysteine. Among all the low-molecular-weight solutes tested in this study, the growth-stimulation effect was most profound in the presence of L-glutamate. When S. solfataricus cells were grown in a glucose (1.0 g/l) medium supplemented with 3.0 g/l L-glutamate, the maximal cell density and growth rate were about 3.2- and 2.3-fold higher than those obtained without L-glutamate.     

甘氨酸甜菜碱及复合纤维素酶对酱油发酵的影响

将甘氨酸甜菜碱作为渗透压保护剂,于酱醪上罐时(0d)或酱醪发酵30d后添加到高盐稀态酱油发酵的酱醪中,同时添加外源复合纤维素酶,观察其对酱醪发酵的影响。结果表明：在酱醪上罐时(0d)或酱醪发酵30d后同时添加不同质量分数的甘氨酸甜菜碱(0.20%、0.30%)及质量分数0.15%的复合纤维素酶(105EGu/100g酱醪),均能有效提高发酵酱油中蛋白质转化率,达2.26%～7.92%。添加甘氨酸甜菜碱能显著改善高盐稀态发酵酱油头油品质,平均缩短发酵时间15d左右;另外,在酱醪发酵30d后再添加甘氨酸甜菜碱和外源复合纤维素酶其效果更佳,同0d时添加相比,蛋白质转化率平均提高了3.17%。     

蛋清溶菌酶与渗透剂对大肠杆菌的协同抑菌作用

为了提高溶菌酶对革兰氏阴性菌的抑菌性能,研究蛋清溶菌酶和渗透剂甘氨酸、EDTA-Na2复配对大肠杆菌ATCC25922、DH5α的抑菌效果,及细胞外膜渗透性和细胞表面形态的变化。结果表明：大肠杆菌ATCC25922对溶菌酶的敏感性明显强于DH5α,其外膜渗透性也高于大肠杆菌DH5α。当溶菌酶分别和渗透剂甘氨酸或EDTA-Na2复配后,对大肠杆菌ATCC25922和DH5α的抑菌性能都显著提高;三者共同作用时,对大肠杆菌ATCC25922的抑菌能力从101.0提高到103.45,对大肠杆菌DH5α则从100.35提高到103.15,表现出协同抑菌作用。细胞外膜渗透性的N-苯基-1-萘胺(NPN)测定结果表明：溶菌酶与甘氨酸、EDTA-Na2复配后,两种大肠杆菌的外膜渗透性相应提高,TEM电镜结果也证实溶菌酶与渗透剂对大肠杆菌细胞表面有协同破坏作用。因此改善大肠杆菌的外膜渗透性有助于增强溶菌酶对其抑菌效果。     

Glycine Betaine-Mediated Protection of Peas (Pisum sativum L.) During Blanching and Frozen Storage

We used glycine betaine (5–20% w/v) for blanching green peas (100°C, 60 s), and their subsequent freezing and storage (–20°C, 90 days). Blanching after the addition of glycine betaine at ≥10% (w/v) followed by a 90 day storage period which resulted in the most desirable outcome: higher vitamin C levels, a superior green color, enhanced organoleptic quality and texture, and improved retention of peroxidase and lipoxygenase activity relative to control peas (no glycine betaine added). Microscopic characterizations of control and treated peas revealed that glycine betaine acts as a cryoprotectant which maintains cellular integrity. Glycine betaine (10% w/v) could be used commercially for production of frozen peas with better quality attributes.     

甜菜碱对单针藻Monoraphidium sp.QLY-1油脂积累的影响

为提高单针藻Monoraphidium sp.QLY-1的生物量和油脂产量,本文结合两阶段策略,即异养-光诱导培养方法,研究甜菜碱（GB）对单针藻QLY-1生长和油脂积累的影响。结果表明,异养、自养的微藻生物量分别为（5.54±0.22）、（0.878±0.12）g/L,且异养比自养提高了5.3倍。此外,在光胁迫下,添加5 mmol/L甜菜碱时可有效提高微藻的油脂含量,其油脂含量（47.37%±2.93%）相比对照（36.68%±1.34%）提高了0.29倍（p<0.05）。进一步的研究表明,与对照相比,添加5 mmol/L甜菜碱时,藻细胞中性脂增加了12.83%±0.75%,对藻细胞内脂肪酸组成无显著性影响。研究表明,甜菜碱作为一种外源诱导子可有效促进微藻细胞中油脂积累,甜菜碱结合两阶段法可作为促进微藻细胞油脂积累的另一策略。      

代谢工程改造谷氨酸棒杆菌合成四氢嘧啶

为了获得四氢嘧啶生产菌株并利用此菌株提高四氢嘧啶产量,以谷氨酸棒杆菌（Corynebacterium glutamicum）野生菌株ATCC13032为研究对象,以赖氨酸积累量为评价指标,强化了天冬氨酸-β-半醛合成代谢流;通过阻断赖氨酸输出通道LysE,表达不同来源的四氢嘧啶操纵子ectABC,获得了四氢嘧啶生产菌株;通过强化天冬氨酸转氨酶和还原型烟酰胺腺嘌呤二核苷酸磷酸（NADPH）再生途径,进一步提高四氢嘧啶产量。结果表明,构建了一株高产赖氨酸的谷氨酸棒杆菌工程菌株LYS-3,赖氨酸积累量为28.48 g/L;表达操纵子HEectABC的谷氨酸棒杆菌ECT-3四氢嘧啶产量达到17.21 g/L;过表达基因aspC和ECpntAB谷氨酸棒杆菌ECT-6,摇瓶发酵四氢嘧啶产量达到21.85 g/L。研究结果可为天冬氨酸-β-半醛衍生物的生物合成提供参考。     

热胁迫和培养温度对大肠杆菌抗热性的影响

研究大肠杆菌O157:H7 ATCC 43889经50、60 ℃和70 ℃反复多次热胁迫处理与在10、28、36 ℃和45 ℃的条件下生长培养后对其80 ℃抗热性的影响。分别对ATCC 43889进行50、60 ℃和70 ℃的热胁迫,研究在一定的热力致死温度条件下杀死某细菌数量90%所需要的时间（D值）的变化,观察ATCC 43889热胁迫前后菌落形态和个体形态的变化;将ATCC 43889置于10、28、36 ℃和45 ℃培养至稳定期,分别测定其在80 ℃的存活量,再利用Weibull模型拟合其在80 ℃的热致死曲线。结果表明,50、60 ℃和70 ℃热胁迫处理均可诱导ATCC 43889抗热性增加,经10 次热胁迫并传代培养后,其D值分别为第1次热胁迫处理后的1.88、2.38 倍和8.18 倍,D值随热处理次数的增加不断增大,说明胁迫温度越高,D值越大,其抗热性越强;经过60 ℃和70 ℃热胁迫后,ATCC 43889菌落形态和个体形态与对照组相比差异显著;在80 ℃,ATCC 43889的致死曲线表明,胁迫温度越高,其抗热性越强（P＜0.05）;在10～45 ℃培养,随培养温度的升高,ATCC 43889的抗热性显著增加（P＜0.05）。利用Weibull模型可以较好地拟合ATCC 43889经过50、60 ℃和70 ℃热胁迫处理10 次后与10、28、36 ℃和45 ℃培养后在80 ℃的抗热性曲线,随着胁迫温度和培养温度的升高,ATCC 43889的抗热性都呈增加趋势。综上,一定热处理与培养温度可胁迫诱导大肠杆菌ATCC 43889抗性热增强和形态的变化。     

Effect of glycine on 5-aminolevulinic acid biosynthesis in heterotrophic culture of Chlorella regularis YA-603

Chlorella regularis strain YA-603 was previously found to exhibit a comparatively high growth rate and improved 5-aminolevulinic acid (ALA) productivity when cultured heterotrophically. Although the universal tetrapyrrole precursor ALA is synthesized from glutamate in algae and higher plants, in this study the addition of glutamate to the medium did not enhance ALA production in the heterotrophic culture of C. regularis YA-603. On the other hand, the addition of glycine, which is a precursor of ALA biosynthesis via the Shemin pathway in animal cells and some bacteria, enhanced both the specific growth rate and ALA production. Moreover, when a second glycine addition was made during the culture, the ALA concentration increased by about 1.5 times compared to that obtained with one glycine addition. From these results, it is suggested that the Shemin pathway contributes to ALA production, and that addition of glycine in the heterotrophic culture of C. regularis YA-603 can significantly increase the amount of ALA produced.     

EFFECTS OF DIETARY GLYCINE BETAINE ON BLOOD CHARACTERISTICS AND PORK QUALITY

This study was conducted to determine the effects of dietary glycine betaine on pork quality and blood characteristics. A total of 80 female pigs (Landrace  ×  Yorkshire  ×  Duroc) were randomly allotted into one of four experimental diet groups. Each group of pigs fed with a commercial diet (control) added with 0.2 g glycine betaine (T1), 0.4 g glycine betaine (T2) and 0.6 g glycine betaine (T3)/kg diet during 40 days. Glycine betaine concentrations in plasma and loin muscle were significantly increased by dietary glycine betaine; however, triglyceride concentration in serum was decreased by dietary glycine betaine. Creatine phosphokinase concentrations in plasma had no significant difference among the dietary groups. Redness ( a* ) was significantly higher in T2 and T3; however, at the first and seventh days of storage, redness ( a* ) was not significantly different between dietary groups. Shear force was significantly higher in dietary glycine betaine groups at the first day of storage. Cholesterol content was significantly lower in T2 and T3, whereas T1 was not significantly different compared with the control group. In fatty acid composition, the ratio of saturated fatty acids was increased, whereas unsaturated fatty acids were decreased by dietary glycine betaine.

PRACTICAL APPLICATIONS

Glycine betaine is an amino acid (trimethylglycine) present in most organisms, and is an obligatory intermediate in the catabolism of choline. Glycine betaine has been reported to affect some aspects of pork qualities. As a result of this study, dietary glycine betaine should improve meat redness and reduce cholesterol. However, dietary glycine betaine did not influence creatine phosphokinase levels in plasma. These results will be helpful to the pork industry and meat scientists for improving meat qaulity.