Unsaturated fatty acids liberated from VLDL cause apoptosis in endothelial cells

Certain unsaturated fatty acids (UFAs), cleaved from lipoproteins, are known to activate the serine/threonine protein phosphatase type 2C (PP2C) alpha- and beta-isoforms. To investigate the role of UFAs in apoptosis of endothelial cells, we cocultured human umbilical vein endothelial cells (HUVECs) with THP-1 monocytes. Phorbol-12-myristic-13-acetate (PMA)-treated THP-1 monocytes differentiated into macrophages and synthesized lipoprotein lipase (LPL), the major enzyme for hydrolysis of triglycerides. We demonstrated that LPL from THP-1 macrophages released UFAs from VLDL, which were capable of inducing apoptosis in HUVECs. Physiological concentrations of VLDL did not cause apoptosis in HUVECs, whereas the combination of VLDL with LPL-rich cell medium of THP-1 macrophages did. THP-1 macrophages and HUVECs in cocultivation did not interfere with each other. However, addition of VLDL to this coculture caused apoptosis in HUVECs. Furthermore, inhibition of LPL by adding orlistat to the culture medium and down-regulation of LPL by small interfering RNA (siRNA) reduced the extent of apoptosis of HUVECs. In conclusion, our results show that the amounts of UFAs liberated from lipoproteins are high enough to induce apoptosis in endothelial cells. This underlines the proatherogenic role of UFAs in hyperlipoproteinemias.     

Expression of CA-125 by progestational bovine endometrium: prospective regulation and function

Cancer antigen 125 (CA-125) is expressed by malignant human ovarian surface epithelial cells and derivatives of the Müllerian duct system. This study explored the expression, regulation, and function of CA-125 in the bovine uterus. CA-125 was localized by immunohistochemistry to the apical surfaces of epithelial cells lining the endometrium and proximal glands of the late luteal phase and early pregnancy; antigen was not detected during oestrus or the postpartum period. Production of CA-125 by bovine endometrial cells in vitro was upregulated by progesterone and interferon-tau. Immunopurified CA-125 from uterine flushes of dioestrous or pregnant cows was similar in biochemical composition (as determined by gel electrophoresis and amino acid content) to the human antigen isolated from incubation medium conditioned by the ovarian cancer cell line OVCAR-3. Bovine CA-125 inhibited complement-induced lysis of antibody-sensitized sheep erythrocytes. It is suggested that endometrial CA-125 exerts a progestational role in part by protecting maternal and embryonic cells from immune targeting and lysis.     

矢车菊素-3-O-葡萄糖苷进入巨噬细胞的荧光检测

利用荧光技术探索矢车菊素-3-O-葡萄糖苷（cyanidin 3-O-glucoside,C3G）在体外培养的巨噬细胞中的分布。首先采用荧光分光光度法扫描C3G的特异激发波长和发射波长,再利用激光共聚焦技术探讨C3G进入小鼠和人巨噬细胞的过程以及C3G在细胞中的定位,并分析C3G孵育时间对细胞内荧光强度变化的影响。结果表明：在488 nm和520 nm的激发波长下,C3G孵育15 min的细胞质内开始呈现绿色和红色荧光,并且随着孵育时间的变化,细胞内荧光强度逐渐增强,其中孵育60 min可观察到荧光布满细胞核,其荧光强度是孵育前的6.45 倍。研究表明采用特异波长的激光共聚焦断层扫描技术可示踪到花色苷在干预细胞内的分布,结果显示花色苷C3G可快速穿过巨噬细胞的细胞膜和核膜,直达细胞核。     

甘草多糖对小鼠腹腔巨噬细胞NO、iNOS及iNOS mRNA表达的影响

通过研究甘草多糖(GP)对小鼠腹腔巨噬细胞NO、iNOS 的生成及iNOS mRNA 表达的影响,结果表明：GP 能剂量依赖性地增加活化的巨噬细胞中NO 的生成量及iNOS 的活性,当GP 的添加浓度为400μg/ml 时,NO、iNOS 的分泌量和iNOS mRNA 表达达到最大。固定GP 添加量为100μg/ml,培养时间在48h 时,NO、iNOS 的分泌量和iNOS mRNA 表达量最大。这表明GP 刺激巨噬细胞NO 合成的调节可发生在iNOS 的转录水平,从而刺激巨噬细胞NO 大量合成与释放,GP 的免疫调节作用和抗肿瘤作用机制可能与GP 刺激巨噬细胞iNOS 从头合成从而增加NO 生成有关。     

Sodium and potassium content and viability of mouse mammary gland tissue and acini

Isolated acini from lactating mouse mammary glands were prepared by collagenase and hyaluronidase digestion of tissue. Mammary tissue or acini incubated in vitro in tissue culture medium or a similar Ringer's solution lost K and gained Na. Intracellular concentrations approached, but did not equal, the concentrations in the external solution. This ion shift was largely prevented by incubating in a solution with ionic composition resembling mouse milk. In paired experiments, incubation with ouabain (1 mM) caused further increases in Na and decrease in K, suggesting that a functional Na+-K+-ATPase was present. Viability of acini was indicated by normal ATP content and morphology. The ion shift in NaCl-based solutions was slower at 0 degrees C than at 37 degrees C, suggesting that the flux is a membrane-regulated process. Under identical procedures, ion shifts did not occur in thymocytes or a cultured mammary cell line but were seen in both lactating and nonlactating mammary tissue. Nonlactating mammary tissue had a high Na and low K concentration in vivo. As predicted by previous models for the mechanisms of milk secretion, intracellular electrolyte content in mammary epithelial cells appears to be responsive to the ion concentration in the extracellular environment.     

Intracellular accumulation, subcellular distribution, and efflux of tilmicosin in bovine mammary, blood, and lung cells.

Tilmicosin is a semisynthetic macrolide antibiotic currently approved for veterinary use in cattle and swine to combat respiratory disease. Because the concentrations of tilmicosin are generally low in bovine serum, the interaction of tilmicosin with three types of bovine phagocytes (monocyte-macrophages, macrophages, and neutrophils from blood, lungs, and mammary gland, respectively) and mammary gland epithelial cells was evaluated to provide an understanding of potential clinical efficacy. After incubation with radiolabeled tilmicosin, uptake was determined and expressed as the ratio of the intracellular to the extracellular drug concentration. Accumulation of tilmicosin at 4 h of incubation by the alveolar macrophages (Cc/Ce 193) was 4 to 13 times more than that observed in monocyte-macrophages (Cc/Ce 43), neutrophils, (Cc/Ce 13), or mammary epithelial cells (Cc/Ce 20). Subcellular distribution showed that 70 to 80% of tilmicosin was localized in the lysosomes. Uptake in mammary gland cells was dependent on cell viability, temperature, and pH, but was not influenced by metabolic inhibitors or anaerobiosis. However, lipopolysaccharide exposure increased tilmicosin uptake by the bovine mammary macrophages and epithelial cells. When neutrophils and epithelial cells were incubated in the presence of tilmicosin and extracellular tilmicosin was then removed, 40% of the intracellular tilmicosin remained cell associated after 4 h of incubation (i.e., 60% effluxed), but only 25% remained in macrophages. These in vitro interactions of tilmicosin with bovine phagocytes and epithelial cells suggest an integral role in effecting clinical efficacy.     

Biochemical and virulence characterization of viable but nonculturable cells of Vibrio parahaemolyticus

Vibrio parahaemolyticus is a common foodborne pathogen frequently causing outbreaks in summer. Maintenance of virulence by the viable but nonculturable (VBNC) state of this pathogen would allow its threat to human health to persist. This study reports on the change in virulence and concomitant changes in activity of two enzymes and fatty acid profiles when V. parahaemolyticus ST550 entered the VBNC state in the modified Morita mineral salt-0.5% NaCl medium incubated at 4 degrees C. The major change in fatty acid composition occurred in the first week, with a rapid increase in C15:0 fatty acid and saturated/unsaturated ratio while a rapid decrease in C16:1 was observed. The activity level of the inducible protective enzyme superoxide dismutase became undetectable in the VBNC state, whereas that of constitutive glucose-6-phosphate dehydrogenase did not change in either the exponential phase or the VBNC state. Cytotoxicity against HEp-2 cells and a suckling mouse assay showed that virulence was lowered in the VBNC state compared with exponential-phase cells. Longer incubation times were required by the VBNC cells to achieve the same level of virulence as seen in exponential-phase cells. Culturable cells were recovered on selective agar medium from the VBNC cultures injected into suckling mice, probably as the result of in vivo resuscitation. Results of this study add to our understanding of the biochemical and physiological changes that have not been reported when V. parahaemolyticus enters into the VBNC state.     

Expression and adaptive regulation of amino acid transport system A in a placental cell line under amino acid restriction

Trans-placental transport of amino acids is vital for the developing fetus. Using the BeWo cell line as a placental model, we investigated the effect of restricting amino acid availability on amino acid transport system type A. BeWo cells were cultured either in amino acid-depleted (without non-essential amino acids) or control media for 1, 3, 5 or 6 h. System A function was analysed using alpha(methyl-amino)isobutyric acid (MeAIB) transcellular transport studies. Transporter (sodium coupled neutral amino acid transporter (SNAT1/2)) expression was analysed at mRNA and protein level by Northern and Western blotting respectively. Localisation was carried out using immunocytochemistry. MeAIB transcellular transport was significantly (P < 0.05) increased by incubation of the cells in amino acid-depleted medium for 1 h, and longer incubation times caused further increases in the rate of transfer. However, the initial response was not accompanied by an increase in SNAT2 mRNA; this occurred only after 3 h and further increased for the rest of the 6-h incubation. Similarly, it took several hours for a significant increase in SNAT2 protein expression. In contrast, relocalisation of existing SNAT2 transporters occurred within 30 min of amino acid restriction and continued throughout the 6-h incubation. When the cells were incubated in medium with even lower amino acid levels (without non-essential plus 0.5 x essential amino acids), SNAT2 mRNA levels showed further significant (P < 0.0001) up-regulation. However, incubation of cells in depleted medium for 6 h caused a significant (P = 0.014) decrease in the expression of SNAT1 mRNA. System L type amino acid transporter 2 (LAT2) expression was not changed by amino acid restriction, indicating that the responses seen in the system A transporters were not a general cell response. These data have shown that placental cells adapt in vitro to nutritional stress and have identified the physiological, biochemical and genomic mechanisms involved.     

嗜酸乳杆菌S-层蛋白对肠道细胞黏附及巨噬细胞增殖的影响

比较去除S-层蛋白前后嗜酸乳杆菌对肠道细胞黏附及菌体自我凝集率的变化,并探究S-层蛋白对巨噬细胞增殖及溶酶体分泌的影响。采用4M氯化锂提取嗜酸乳杆菌ATCC 4356的表层蛋白,经透析及微孔过滤等步骤得到S-层蛋白,SDS-PAGE确定其分子量。利用酶标仪测定嗜酸乳杆菌在37℃孵育过程中(0 h~5.5 h)自我凝集率的变化情况,光学显微镜观察去除S-层蛋白前后嗜酸乳杆菌对结肠癌细胞HT-29的黏附情况变化。将S-层蛋白与巨噬细胞RAW264.7孵育2 h后,MTS法测定细胞增殖情况,并采用溶酶体荧光探针研究溶酶体分泌量的变化。结果表明:所提蛋白为嗜酸乳杆菌S-层蛋白(分子量M≈46ku)。嗜酸乳杆菌自我凝集率随孵育时间的延长而呈现出上升趋势,去除S-层蛋白后,菌体自我凝集率降低,嗜酸乳杆菌对HT-29细胞的黏附量也明显减少,且S-层蛋白能促进巨噬细胞增殖及其溶酶体分泌。     

Chemically fixed nurse cells for culturing murine or primate embryonic stem cells

In current and past practice, murine or primate embryonic stem (ES) cells are usually cultured on live nurse cells for growth that keeps the cells in an undifferentiated state. It is troublesome, however, to prepare nurse cells for each cell culture and it is difficult to completely remove the nurse cells when they are transferred. In this study, mouse and monkey ES cells were therefore grown on chemically fixed mouse embryonic fibroblast (MEF) or human amniotic epithelial (HAE) cells. MEF cells were fixed by incubation in a glutaraldehyde or formaldehyde solution. HAE cells were immortalized by transfection of hTERT and chemically fixed with the same reagents. When mouse ES cells were cultured on these chemically fixed cells, the mouse ES cells grew well and expressed alkaline phosphatase, SSEA-1, and Oct-3/4 as their markers, indicating their undifferentiated state. The monkey ES cells also grew well and expressed alkaline phosphatase, SSEA-4, and Oct-4 as their markers, indicating their undifferentiated state. Freeze-drying HAE or MEF cells did not change their ability to support the undifferentiated growth of ES cells. Additionally, the chemically fixed cells could be utilized repeatedly in the culture of ES cells. These results demonstrate that chemically fixed nurse cells are useful for the maintenance of ES cells in an undifferentiated state in culture.     

Impairment of Peroxisome Degradation in Pichia methanolica Mutants Defective in Acetyl-CoA Synthetase or Isocitrate Lyase

Single recessive mutations of the methylotrophic yeast Pichia methanolica acs1, acs2, acs3 and icl1 affecting acetyl-CoA synthetase and isocitrate lyase, and growth on ethanol as sole carbon and energy source, caused a defect in autophagic peroxisome degradation during exposure of methanol-grown cells to ethanol. As a control, a mutation in mdd1, which resulted in a defect of the ‘malic’ enzyme and also prevented ethanol utilization, did not prevent peroxisome degradation. Peroxisome degradation in glucose medium was unimpaired in all strains tested. Addition of ethanol to methanol-grown cells of acs1, acs2, acs3 and icl1 mutants led to an increase in average vacuole size. Thickening of peroxisomal membranes and tight contacts between groups of peroxisomes and vacuoles were rarely observed. These processes proceeded much more slowly than in wild-type or mdd1 mutant cells incubated under similar conditions. No peroxisomal remnants were observed inside vacuoles in the cells of acs1, acs2, acs3 and icl1 mutants after prolonged cultivation in ethanol medium. We hypothesize that the acs and icl mutants are defective in synthesis of the true effector—presumably glyoxylate—of peroxisome degradation in ethanol medium. Lack of the effector suspends peroxisome degradation at an early stage, namely signal transduction or peroxisome/vacuole recognition. Finally, these defects in peroxisome degradation resulted in mutant cells retaining high levels of alcohol oxidase which further led to increased levels of acetaldehyde accumulation upon incubation of mutant cells with ethanol. © 1997 by John Wiley & Sons, Ltd.     

莲原花青素抗黑色素瘤的研究

目的：探讨莲房和莲子壳中原花青素对黑色素瘤B16 生长的影响。方法：采用MTT 法测定莲房和莲子壳原花青素对B16 细胞的体外抑制作用;并借助显微技术观察B16 细胞形态;以荷黑色素瘤的C57BL/6J 小鼠为体内研究对象,通过瘤体积和瘤重测定,探讨体内抑瘤作用,采用黄嘌呤氧化酶法和硫代巴比妥酸法测定荷瘤鼠血清中超氧化物歧化酶(SOD)和丙二醛(MDA)含量。结果：莲房和莲子壳中原花青素体外对黑色素瘤B16 细胞均有较强的抑制作用,抑制率分别为87.4% 和78.2%,均使细胞的膜破损,细胞形态改变;体内能显著减小瘤体积和减轻瘤重,抑瘤率分别为55.3% 和46.7%,并能有效降低荷瘤小鼠血清中MDA 含量,提高SOD 活力。结论：莲房和莲子壳中原花青素对黑色素瘤均具有较强的抑制作用。     

The role of dietary oxidized cholesterol and oxidized fatty acids in the development of atherosclerosis

The etiology of atherosclerosis is complex and multifactorial but there is extensive evidence indicating that oxidized lipoproteins may play a key role. At present, the site and mechanism by which lipoproteins are oxidized are not resolved, and it is not clear if oxidized lipoproteins form locally in the artery wall and/or are sequestered in atherosclerotic lesions following the uptake of circulating oxidized lipoproteins. We have been focusing our studies on demonstrating that such potentially atherogenic oxidized lipoproteins in the circulation are at least partially derived from oxidized lipids in the diet. Thus, the purpose of our work has been to determine in humans whether oxidized dietary oxidized fats such as oxidized fatty acids and oxidized cholesterol are absorbed and contribute to the pool of oxidized lipids in circulating lipoproteins. When a meal containing oxidized linoleic acid was fed to normal subjects, oxidized fatty acids were found only in the postprandial chylomicron/chylomicron remnants (CM/RM) which were cleared from circulation within 8 h. No oxidized fatty acids were detected in low density lipoprotein (LDL) or high density lipoprotein (HDL) fractions at any time. However, when alpha-epoxy cholesterol was fed to human subjects, alpha-epoxy cholesterol in serum was found in CM/RM and also in endogenous very low density lipoprotein, LDL, and HDL and remained in the circulation for 72 h. In vitro incubation of the CM/RM fraction containing alpha-epoxy cholesterol with human LDL and HDL that did not contain alpha-epoxy cholesterol resulted in a rapid transfer of oxidized cholesterol from CM/RM to both LDL and HDL. We have suggested that cholesteryl ester transfer protein is mediating the transfer. Thus, alpha-epoxy cholesterol in the diet is incorporated into CM/RM fraction and then transferred to LDL and HDL contributing to lipoprotein oxidation. We hypothesize that diet-derived oxidized fatty acids in chylomicron remnants and oxidized cholesterol in remnants and LDL accelerate atherosclerosis by increasing oxidized lipid levels in circulating LDL and chylomicron remnants. This hypothesis is supported by our feeding experiments in animals. When rabbits were fed oxidized fatty acids or oxidized cholesterol, the fatty streak lesions in the aorta were increased by 100%. Moreover, dietary oxidized cholesterol significantly increased aortic lesions in apo-E and LDL receptor-deficient mice. A typical Western diet is rich in oxidized fats and therefore could contribute to the increased arterial atherosclerosis in our population.     

Coffee and Maillard products activate NF-kappaB in macrophages via H2O2 production

In this study, we investigated the immunomodulatory activity of coffee and Maillard reaction products on macrophages in vitro. Stimulation of macrophages with coffee, but not with raw coffee extract in PBS, led to a 13-fold increased nuclear NF-kappaB translocation. A Maillard reaction mixture (25 mM D-ribose/L-lysine, 30 min at 120 degrees C) increased NF-kappaB translocation 18-fold (in PBS) or six-fold (in medium). MRPs also induced a two-fold increased NF-kappaB translocation in untransfected human embryonic kidney (HEK) cells as well as in HEK cells stably transfected with the receptor for advanced glycation endproducts (RAGE), indicating that the effect was not RAGE mediated. On the other hand, catalase totally abolished coffee- and MRP-induced NF-kappaB translocation. Consequently, up to 366 microM hydrogen peroxide was measured in the coffee preparation and Maillard mixtures used for cell stimulation. Stimulation of macrophages with MRPs did not lead to significantly increased IL-6 or NO release. Thus, it can be concluded that coffee and MRPs induce NF-kappaB translocation in macrophages via the generation of hydrogen peroxide.     

体外评估十株乳杆菌对免疫细胞活性的影响

针对传统的两种糖基化接枝方法和改进方法从糖基化反应程度进行系统比较,并对产物氨基酸组成进行分析,以期进一步揭示蛋白质糖基化方法对于糖基化反应过程及产物的影响机制。本文利用干热法、湿热法和加压辅助湿热三种糖基化方法,对大豆7S球蛋白和葡聚糖的糖基化产物从反应程度、氨基酸组成等方面进行研究发现,结果表明:三种制备方法的反应程度高低顺序依次为:湿热法>加压辅助湿热法>干热法,说明压力能够对Maillard反应的进行具有一定的抑制作用,可以控制反应向理想阶段进行。大豆7S球蛋白和葡聚糖的糖基化主要发生在蛋白质肽链上的赖氨酸和精氨酸侧链上的自由氨基,干热法与其它两种方法相比糖链更有易于和精氨酸侧链上的自由氨基发生共价交联,而湿热法和加压辅助湿热法相比糖链更易于和赖氨酸侧链上的自由氨基发生共价交联。     

虫草灵芝孢子粉复合物对小鼠免疫功能的影响

目的：探讨虫草灵芝孢子粉复合物对小鼠免疫功能的调节作用。方法：实验将虫草灵芝孢子粉分为低、中、高3个剂量组,并设置对照组,分别喂饲小鼠30d,观察小鼠体质量及免疫器官指数变化、迟发型变态反应(DTH)、ConA诱导的淋巴细胞转化能力、血清溶血素生成、碳廓清能力和巨噬细胞吞噬鸡红细胞能力6个项目。结果：与对照组比较,虫草灵芝孢子粉高剂量组显著提高小鼠胸腺指数,低、中剂量组显著提高小鼠迟发型变态反应,高剂量组显著提高小鼠淋巴细胞的增殖能力,提示虫草灵芝孢子粉能增强小鼠特异性免疫应答。低、中剂量组还能显著提高巨噬细胞碳廓清能力和吞噬鸡红细胞能力,提示虫草灵芝孢子粉可增强正常小鼠巨噬细胞吞噬能力,增强小鼠非特异性免疫应答。结论：虫草灵芝孢子粉复合物能增强小鼠特异性免疫作用和非特异性免疫作用,具有增强小鼠免疫功能的作用。     

Effects of follicular cells and FSH on the resumption of meiosis in equine oocytes matured in vitro

It has been suggested that preculturing immature oocytes in a manner that maintains them in meiotic arrest may improve cytoplasmic maturation and, thereby, the eventual developmental competence of oocytes matured in vitro. This study examined the ability of follicular cells to maintain meiotic arrest in equine oocytes. Cumulus-oocyte complexes (COCs) recovered from dead mares were cultured for 38 h in M199 either attached to, or together with, different follicle wall components, as follows: (1) attached to the follicle wall, (2) cocultured with separated follicle wall, (3) attached to membrana granulosa (COCG), (4) COCGs cocultured with sheets of theca cells, (5) COCGs cultured in theca-cell conditioned medium, and (6) control COCs without any follicle wall components. When oocytes were cultured attached to their follicle wall, 79% remained in the GV stage throughout the 38 h incubation. However, when oocytes were cocultured with separate pieces of follicle wall, meiosis resumed and a similar proportion of oocytes progressed to metaphase II (79%) as under control conditions (84%). Only 16% of oocytes cultured while still attached to the membrana granulosa (COCGs) maintained the GV stage, whereas when COCGs were cocultured with theca cells or in theca-cell conditioned medium, significantly more oocytes remained in the GV stage (64 and 52%, respectively), indicating that theca cells secrete a meiosis-inhibiting factor. The effect of FSH on the meiosis-inhibiting activity of follicular cells was investigated by culturing COCs attached to the follicle wall and COCGs in the presence or absence of theca cells in medium containing FSH. Addition of 0.05 iu recombinant human FSH ml(-1) to the culture medium did not affect nuclear maturation and failed to overcome the suppressive effect exerted by the follicle wall or by theca cells, despite the fact that mRNA for the FSH receptor was found using RT-PCR in both cumulus and granulosa cells. These results demonstrate that the maintenance of meiotic arrest in equine oocytes during culture can be promoted by theca cells, which appear to act via a secreted inhibitory factor that cannot be suppressed or counteracted by FSH.     

ANTIGENOTOXIC, FIBRINOLYTIC AND IMMUNOMODULATING ACTIVITY OF TRADITIONALLY FERMENTED SOY PRODUCT, CHUNGKUKJANG

Fibrinolytic, antigenotoxic and immunomodulating properties of chungkukjang were evaluated. The purified fibrinolytic enzyme was characterized as an alkaline serine protease with molecular weight of 22 kDa determined by fibrin zymography and stable over pH range 6.0–10.0 below 50C. The comet assay showed that N-methyl-N ' -nitro-N-nitrosoguanidine (MNNG)-induced DNA damage level (190.8 AU) in 3T3 cells was considerably decreased to 121.2 AU with chungkukjang extracts (CB) showing 49.7% of protection. Incubation of the 3T3 cells with CB significantly reduced the cytotoxicity of MNNG. [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) assay revealed that the addition of CB enhanced the proliferation of RAW 264.7 (mouse leukemic monocyte macrophage cell line) macrophages up to about 150% of the control levels. RAW 264.7 macrophages incubated with CB induced a strong increase in nitric oxide (NO), tumor necrosis factor -α (TNF-α) and interleukin-1α (IL-1α) with a dose-dependent manner. After 24 h incubation with 1 mg/mL of CB, NO, TNF-α and IL-1α showed the highest production of 511, 2.46 and 0.258 ng/mL, respectively.     

Factors influencing leucine catabolism by a strain of Staphylococcus carnosus.

The metabolism of leucine by resting cells of Staphylococcus carnosus 833 was studied according to three physicochemical factors: preculture condition (defined medium; complex medium), nitrate concentration (0% and 0.03%) and stirring condition (static or shaking). A factorial design was set up to test the effects of these factors, each at two levels. The results showed that resting cells of S. carnosus 833 produced 3-methyl butanal, 3-methyl butanol and 3-methyl butanoic acid from leucine. Whatever the incubation conditions, there was greater quantity of 3-methyl butanoic acid than 3-methyl butanal and 3-methyl butanol. The preculture and incubation conditions influenced the level of production of the 3 metabolites. The highest overall production of the 3 metabolites was observed when cells were incubated without nitrate in the reaction mixture. 3-methylbutanoic acid production was enhanced when S. carnosus 833 was precultivated in complex medium. 3-methylbutanal was only detected when cells were precultivated in defined medium. Stirring condition had no effect on leucine catabolism of S. carnosus 833.     

Effect of Cultural Conditions and Media on the Antimicrobial Activity of Streptococcus Thermophilus

When Streptococcus thermophilus was grown in basal medium with or without fermentable carbohydrate (lactose, glucose or sucrose at 2% level), antimicrobial activity was detected only in the cell-free medium and cells were free of any antimicrobial activity. Peanut milk, soymilk and basal medium with a fermentable sugar supported the production of antimicrobial compound(s) by S. thermophilus. Milk-based medium did not appear to be a necessity for such activity. When tested independently, the optimum temperature for maximum production of antimocrobial activity by S. thermophilus in whole milk was 50°C while the optimum time of incubation was 48 hr. An initial pH of 6.0 resulted in maximum antimicrobial activity in basal medium with 2% lactose.