Growth characteristics of Bacillus subtilis (natto) in milk

In this paper, Bacillus subtilis (natto) was incubated to develop a possible functional ingredient in ice cream. A lab‐scale culture revealed that incubation in the sterilised milk without dilution and concentration at 37°C for 28 h could obtain ideal growth characteristics of Bacillus subtilis (natto), especially with continuous aeration. Following freezing operation of the cultured milk, survival content of Bacillus subtilis (natto) was at 49–92%, while nattokinase activity was conserved at 62–98% comparing with the initial contents, which indicating a potential for application of natto functional ingredient in frozen milk products.     

Mode of inactivation of probiotic bacteria affects interleukin 6 and interleukin 8 production in human intestinal epithelial-like Caco-2 cells

Five lactic acid bacteria and two bifidobacteria strains were heat or irradiation inactivated. Inactivated cultures were evaluated for their effects on cytokines interleukin (IL) 6 and IL-8 production in human intestinal-like Caco-2 cells. For both heat- and irradiation-inactivated cultures, production of IL-6 and IL-8 was dependent on the specific microorganism. However, with all of the cultures, both IL-6 and IL-8 production was significantly higher (P < 0.05) in Caco-2 cells that were treated with heat-inactivated probiotic bacteria compare to the irradiation-inactivated bacteria. In the majority of the cases, heat-inactivated bacteria induced IL-6 and IL-8 production, whereas irradiation-inactivated bacteria attenuated both cytokine production. Our results indicate that the same probiotic bacteria used in the same cell culture could provide opposite cytokine production and immune modulation results based on its mode of inactivation; therefore, it is important to describe inactivation methods and conditions in detail when characterizing probiotic effects.     

Effect of nonfat dry milk and major whey components on interleukin-6 and interleukin-8 production in human intestinal epithelial-like Caco-2 cells

Bovine nonfat dry milk (NDM) and major whey components (lactose, α-lactalbumin, and β-lactoglobulin) were evaluated for their effects on IL-6 and IL-8 production in human intestinal-like Caco-2 cells unstimulated or stimulated with IL-1β. All the whey components investigated and NDM induced IL-6 production by Caco-2 cells; the most significant increase was observed with β-lactoglobulin. In the case of IL-1β-stimulated cells, neither NDM nor the major whey components investigated contributed to the induction of IL-6 production after they were stimulated. Induction of IL-8 production by both α-lactalbumin and β-lactoglobulin was higher than that by lactose and NDM; α-lactalbumin was a more potent inducer of IL-8 than β-lactoglobulin and IL-1β alone in both unstimulated and stimulated cells. In Caco-2 cells that were stimulated with IL1-β, NDM and all the major whey components investigated had a synergistic effect on induction of IL-8 production, indicating that IL-8 induction was amplified by prior stimulation of cells by IL-1β. This synergistic effect was not observed with IL-6. Our results suggest that immunomodulatory properties of milk components may be affected by other complex events in the gut.     

Use of Bacillus subtilis to enrich isoflavone aglycones in fermented natto

BACKGROUND: Natto is a food made by fermenting cooked soybeans with Bacillus subtilis. Soybean isoflavones are reported to provide many health benefits, including oestrogenic effects. However, isoflavone aglycones may be absorbed faster and in higher amounts in the human intestine than their glucosides. This study aimed to investigate the content of isoflavone components in commercial natto products as well as the use of B. subtilis strains to ferment cooked soybeans to produce a high level of isoflavone aglycones in natto. RESULTS: The content and composition of isoflavones in commercial natto products were predominantly (>76%) isoflavone glucosides. Fermentation of cooked soybeans with B. subtilis BCRC 14718 at 37 °C for 48 h was more effective in converting glucosides to aglycones than with other strains of B. subtilis, increasing the proportion of isoflavone aglycones from 12 to 68% of the total isoflavones in the fermented natto. The proportions of the isoflavone aglycones daidzein and genistein in cooked soybeans fermented with B. subtilis BCRC 14718 for 48 h increased from 6 to 54% and from 5 to 13% respectively. CONCLUSION: Bacillus subtilis BCRC 14718 incubated with cooked soybeans produces higher levels of isoflavone aglycones, which may enhance health benefits over traditional fermented natto. Copyright © 2008 Society of Chemical Industry     

纳豆芽孢杆菌生产维生素K2的连续发酵工艺优化

为优化维生素K2的高密度连续发酵生产工艺,通过对纳豆芽孢杆菌发酵培养,研究了培养基组成、转速、p H、溶氧量、后处理技术等因素对维生素K2产量的影响。实验结果表明以香菇菌渣复配蛋白胨为氮源(5%),甘油为碳源(5%),pH为7,通气比大于1.5 vvm,转速为600 r/min时,采用高密度连续发酵技术,5 L罐发酵产量最高达35.58 mg/L。发酵液萃取后,废液再通过纳滤膜截流,提高维生素K2回收率,并制成富含维生素K2的菌菇粉。维生素K2的结构经高效液相及质谱确证。大鼠急性毒性实验证明该菌菇粉产品安全无毒。      

纳豆枯草芽孢杆菌生物发酵川穹嗪初探

据资料显示,纳豆芽孢杆菌具有生物合成川穹嗪(TMP)等吡嗪类物质的特性,但并未深入展开研究。本文考察了液体发酵培养和固体发酵过程中底物乙偶姻(ACT)的加入时间、乙偶姻添加量及种子液的加入量对TMP生物合成的影响。实验结果表明,液体培养基组成为20%大豆浸泡物、0.5%乙偶姻时最有利于TMP的合成;固体发酵过程中,当前体在培养84h时加入TMP的合成量最高,TMP产率随底物乙偶姻加入量的增加而增加,与种子液的加入量呈负相关。      

Molecular mechanisms of the naringin low uptake by intestinal Caco-2 cells

Naringin, the main flavanone of grapefruit, was reported to display numerous biological effects: antioxidant, hypocholesteremic, anti-atherogenic and favoring drug absorption. Naringin absorption mechanisms were studied in Caco-2 cells (TC7 clone). We investigated the possible involvement of several membrane transporters implicated in polyphenolic compounds intestinal transport (sodium-dependent glucose transporter 1, monocarboxylate transporter, multidrug-associated resistance proteins 1 and 2, and P-glycoprotein). Naringin was poorly absorbed by Caco-2 cells, according to its low value of apparent permeability coefficient (P(app) = 8.1 +/- 0.9 x 10(-8) cm/s). In the presence of verapamil, a specific inhibitor of P-glycoprotein, cellular uptake was increased by almost threefold after 5 min, and P(app) was doubled after 30 min. Our results indicated the involvement of P-glycoprotein, an ATP-driven efflux pump, capable of transporting naringin from the Caco-2 cell to the apical side. This phenomenon could explain, at least in part, the low absorption of this flavanone at the upper intestinal level.     

培养条件及表面活性剂的添加对纳豆芽孢杆菌生物膜形成的影响

采用改良的微孔板法培养纳豆芽孢杆菌(Bacillus subtilis natto),通过结晶紫染色定量分析产生的生物膜。结果表明:培养条件为pH 7、温度37℃、培养72 h且在5 g/100 mL葡萄糖-5 g/100 mL NaCl条件下成膜能力最佳。柠檬酸二铵和聚乙二醇-200(polyethylene glycol-200,PEG-200)随着质量浓度的增加对生物膜的形成量呈现先下降后上升的趋势,十二烷基磺酸钠(sodium dodecyl sulfate,SDS)抑制生物膜的形成,十六烷基三甲基溴化铵(cetyl trimethyl ammonium bromide,CTAB)促进生物膜的形成。所以低温抑制生物膜的形成,一定浓度的NaCl和葡萄糖促进生物膜形成,不同的表面活性剂对其生物膜的影响机制不同,为纳豆芽孢杆菌生物膜的研究提供理论基础。     

Binding of the hop (Humulus lupulus L.) chalcone xanthohumol to cytosolic proteins in Caco-2 intestinal epithelial cells

Used in the brewing of beer, hops (Humulus lupulus L.) contain the prenylated chalcone xanthohumol, which is under investigation as a cancer chemoprevention agent and as a precursor for the estrogenic flavanones isoxanthohumol and 8-prenylnaringenin. The uptake, transport and accumulation of xanthohumol were studied using the human intestinal epithelial cell line Caco-2 to help understand the poor bioavailability of this chalcone. Studies were carried out using Caco-2 cell monolayers 18-21 days after seeding. The apparent K(m) and V(max) values of xanthohumol accumulation in Caco-2 cells were determined, and the protein binding of xanthohumol in sub-cellular fractions of Caco-2 cells was investigated. Approximately 70% of xanthohumol added to the apical side of Caco-2 cells accumulated inside the cells, while 93% of the intracellular xanthohumol was localized in the cytosol. Xanthohumol accumulation was temperature dependent and saturable with an apparent K(m )value of 26.5 +/- 4.66 muM and an apparent V(max) of 0.215 +/- 0.018 nmol/mg protein/min. Facilitated transport was not responsible for the uptake of xanthohumol, instead, accumulation inside the Caco-2 cells was apparently the result of specific binding to cytosolic proteins. These data suggest that specific binding of xanthohumol to cytosolic proteins in intestinal epithelial cells contributes to the poor oral bioavailability observed previously in vivo.     

Bioactive dietary polyphenols inhibit heme iron absorption in a dose-dependent manner in human intestinal Caco-2 cells

Abstract: Although heme iron is an important form of dietary iron, its intestinal absorption mechanism remains elusive. Our previous study revealed that (‐)‐epigallocatechin‐3‐gallate (EGCG) and grape seed extract (GSE) markedly inhibited intestinal heme iron absorption by reducing the basolateral iron export in Caco‐2 cells. The aim of this study was to examine whether small amounts of EGCG, GSE, and green tea extract (GT) could inhibit heme iron absorption, and to test whether the inhibitory action of polyphenols could be offset by ascorbic acid. A heme‐⁵⁵Fe absorption study was conducted by adding various concentrations of EGCG, GSE, and GT to Caco‐2 cells in the absence and presence of ascorbic acid. Polyphenolic compounds significantly inhibited heme‐⁵⁵Fe absorption in a dose‐dependent manner. The addition of ascorbic acid did not modulate the inhibitory effect of dietary polyphenols on heme iron absorption when the cells were treated with polyphenols at a concentration of 46 mg/L. However, ascorbic acid was able to offset or reverse the inhibitory effects of polyphenolic compounds when lower concentrations of polyphenols were added (≤ 4.6 mg/L). Ascorbic acid modulated the heme iron absorption without changing the apical heme uptake, the expression of the proteins involved in heme metabolism and basolateral iron transport, and heme oxygenase activity, indicating that ascorbic acid may enhance heme iron absorption by modulating the intracellular distribution of ⁵⁵Fe. These results imply that the regular consumption of dietary ascorbic acid can easily counteract the inhibitory effects of low concentrations of dietary polyphenols on heme iron absorption but cannot counteract the inhibitory actions of high concentrations of polyphenols. Practical Application: Bioactive dietary polyphenols inhibit heme iron absorption in a dose‐dependent manner. The small amounts of polyphenolic compounds present in foods are capable of reducing heme iron transport across the intestinal enterocyte. However, the inhibitory effects of dietary polyphenolic compounds on heme iron absorption can be offset by ascorbic acid and can possibly be avoided by decreasing the consumption of polyphenols while simultaneously taking ascorbic acid.     

以豆粕为基质纳豆芽孢杆菌生长条件优化

研究纳豆芽孢杆菌在以豆粕为基质的发酵培养基中的生长条件。利用全自动生长曲线测定仪,以菌液浊度OD660nm为指标,对纳豆芽孢杆菌液体发酵培养基中的氮源(豆粕)和碳源(葡萄糖)配比进行研究,确定豆粕质量浓度50g/L,葡萄糖质量浓度10g/L为最佳配比,并通过单因素及正交试验筛选出影响纳豆芽孢杆菌生长的主要条件。再根据Box-Behnken试验优化,结果表明,采用功率80W超声波处理豆粕4.32min,且培养基初始pH6.16,发酵温度35.5℃时纳豆芽孢杆菌在发酵生长13h后达到最大生物量,此时OD660nm为1.635。     

Production of menaquinone (vitamin K2)-7 by Bacillus subtilis

Menaquinone-7 (MK-7) is a highly bioactive homologue of vitamin K. We obtained a diphenylamine-resistant mutant strain D200-41 from Bacillus subtilis strain MH-1 which was isolated from fermented soybeans, natto. The mutant strain exhibited decreased production of MK-6. Using strain D200-41, efficient production of MK-7 was achieved. We found that, compared with an agitated and aerated culture, production of MK-7 was increased by static culture. The sporulation of the cells progressed more slowly in a static culture than in an agitated culture. The maximum concentration of MK reached about 60 mg/l in a medium containing 10% soybean extract, 5% glycerol, 0.5% yeast extract and 0.05% K2HPO4 (pH 7.3) when D200-41 cells as well as MH-1 cells were statically cultured at 45 degrees C for 5 d after being cultured with shaking at 37 degrees C for 1 d.     

In vitro digestive stability and uptake by Caco-2 human intestinal cells of nonfluorescent chlorophyll catabolites

Nonfluorescent chlorophyll catabolites (NCCs) are commonly considered to be the final tetrapyrrolic products of chlorophyll catabolism in higher plants. This study describes two different NCCs, Ca-NCC-1 and Ca-NCC-2, during the ripening of pepper fruits. Although NCCs are present in fruits and vegetables that are or were green, and are part of our daily diet, no information is available about their bioavailability. After in vitro digestion of an extract of pepper NCCs (which reproduces the gastric and intestinal phases of physiological digestion), both have shown resistance to the digestive process and are efficiently incorporated into the aqueous micellar fraction. Assays of absorption by Caco-2 cell monolayers were performed, showing that only Ca-NCC-1 is efficiently absorbed. This is the first time that NCCs have been shown to be absorbed by human intestinal cells, in the same way as other chlorophyll catabolites from plants and vegetables.     

鼓槌石斛毛兰素诱导人结肠癌Caco-2细胞凋亡

目的:研究毛兰素对人结肠癌Caco-2细胞增殖的抑制作用及其诱导细胞凋亡的分子机制。方法:采用SRB法检测毛兰素对Caco-2细胞增殖的抑制作用,用Hoechst33342染色法观察细胞的形态学改变,流式细胞术检测细胞的凋亡率和细胞周期;蛋白免疫印迹法（Western Blot）检测细胞凋亡相关蛋白Caspase-3的表达水平。结果:与对照相比,毛兰素能抑制结肠癌细胞Caco-2的增殖,且抑制率随着药物浓度与时间增加,呈剂量时间效应,48 h半数抑制浓度IC50为0.845μg/m L;毛兰素能诱导Caco-2细胞凋亡,并诱导细胞周期阻滞于G₂期;Caspase-3活性裂解片段表达增高。结论:毛兰素对肿瘤细胞的增殖具有一定抑制作用且通过线粒体途径诱导Caco-2细胞凋亡。      

Effect of Bacillus cereus exocellular factors on human intestinal epithelial cells

To gain insight on the biological effects of the exocellular factors produced by Bacillus cereus, culture filtrate supernatants of different strains were coincubated with differentiated Caco-2 cells. Exocellular factors were able to detach enterocyte-like cells from the substratum after 1 h of incubation. In addition, microvilli effacing and dramatic changes on the cellular surface of enterocytes were found after incubation periods as short as 20 min. Since cell detachment was not inhibited by fetal calf serum, thiol activated cholesterol-binding cytolysin, cereolysin O, does not seem to be involved. Also, translocation of phosphatidylserine from the inner to the outer leaflets of the plasma membrane was demonstrated by using fluorescein isothiocyanate (FITC)-Annexin V. In contrast to the high capability of detaching Caco-2 cells shown by all the strains under study, the mitochondrial dehydrogenase activity was lowered by culture filtrate supernatants in a strain-dependent manner. For strain M2, the decrease in dehydrogenase activity was already evident after 30 min of incubation. Production of biologically active factors depends on the growth phase, and maximal activity was found in late exponential-early stationary phases. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of concentrated exocellular factors showed a very complex scenery supporting the multifactorial character of the biological activity of B. cereus.     

Grapefruit juice enhance the uptake of coenzyme Q10 in the human intestinal cell-line Caco-2

Coenzyme Q10 (CoQ10) is very widely consumed by humans as a food supplement. However, CoQ10 is taken up from the intestine into the circulation at a low rate. The absorption of compounds from the gastrointestinal tract is one of the important determinants for oral bioavailability. Secretory transport limits the oral bioavailability of compounds. It has been reported that efflux transport of CoQ10 is mediated by P-glycoprotein (P-gp) in Caco-2 cells. We tried to improve intestinal absorption of CoQ10 by modulating P-gp. Since grapefruit juice (GFJ) is reported to inhibit P-gp function, we investigated the effect of GFJ on the transport of CoQ10 by Caco-2 cells. In the presence of GFJ, the basal-to-apical transport of CoQ10 was decreased and the uptake of CoQ10 was increased. These findings suggest that the combined administration of CoQ10 and GFJ could enhance CoQ10 absorption.