Growth and laccase production by Pleurotus ostreatus in submerged and solid-state fermentation

Pleurotus ostreatus showed atypical laccase production in submerged vs. solid-state fermentation. Cultures grown in submerged fermentation produced laccase at 13,000 U l⁻¹, with a biomass production of 5.6 g l⁻¹ and four laccase isoforms. However, cultures grown in solid-state fermentation had a much lower laccase activity of 2,430 U l⁻¹, biomass production of 4.5 g l⁻¹, and three laccase isoforms. These results show that P. ostreatus performs much better in submerged fermentation than in solid-state fermentation. This is the first report that shows such atypical behavior in the production of extracellular laccases by fungi.     

Laccase production in solid‐state and submerged fermentation by Pleurotus ostreatus

A solid‐state fermentation (SSF) system for production of an industrially important enzyme laccase by Pleurotus ostreatus was developed by using potato dextrose yeast extract medium and polyurethane foam as a supporting material. The maximum laccase production in the SSF system was as high as 3×10⁵ U/L. Addition of inducers, such as copper and ferulic acid, further enhanced the laccase production in SSF. Moreover, the time required for the maximum laccase production was reduced to 6 days compared to 10 days reported earlier. The improvement achieved by the SSF system was investigated by comparing it to a submerged fermentation system (SmF), both experimentally and by using a standard theoretical model along with a parameter sensitivity analysis. Laccase production in SSF was found to be twice of that in SmF. One of the main reasons for higher laccase production in SSF compared to SmF was possibly due to the presence of higher proteolytic activity in SmF. Strong proteolytic activity in SmF presumably caused subsequent laccase degradation, which lowered the ultimate laccase production in SmF compared to SSF.     

Production of laccases by Pleurotus ostreatus in submerged fermentation in co‐culture with Trichoderma viride

Aims: To evaluate the production and stability of laccases by Pleurotus ostreatus in liquid co‐cultures with Trichoderma viride as a function of infection time and agitation rate. Methods and Results: Pleurotus ostreatus cultures were infected with T. viride spores at 30 and 48 h. Maximal laccase volumetric activity was seen after 48 h (control cultures) or 72 h (co‐cultures) of cultivation time. Only the cultures infected at 30 h showed an increased laccase volumetric activity compared to control cultures. After maximal laccase volumetric activity value was reached, a sharp decrease in it was observed in control cultures. Co‐cultures exhibited a comparatively lower loss of activity. The influence of P. ostreatus and/or T. viride on the stability of laccase volumetric activity and isoenzyme pattern was evaluated. Trichoderma viride induced changes in the laccase isoenzyme pattern. Agitated cultures increased biomass growth and specific productivity threefold and sevenfold, respectively, to the static cultures. Conclusions: The laccase volumetric activity is very likely the result of the balance between biosynthesis and degradation/biotransformation rates occurring during the cultures. The individual presence of P. ostreatus or T. viride in the culture negatively affected the volumetric laccase activity. Significance and Impact of the Study: The evaluation of culture parameters that could influence Trichoderma–basidomycetes interaction and laccase production during submerged fermentation has not been reported. This study showed how laccase production in co‐cultures of P. ostreatus and T. viride was influenced by the infection time and agitation/oxygenation conditions.     

不同木质纤维素诱导对糙皮侧耳液体发酵产漆酶活性的影响

以2株糙皮侧耳Pleurotus ostreatus栽培菌株为材料,研究在添加木屑、玉米芯和棉籽壳3种农林废弃物后,对其液体发酵产漆酶活性的影响。结果表明,不同糙皮侧耳菌株的漆酶活性具有极显著的差异(P0.001),仅第3天和第9天差异显著(P0.05);而不同培养基条件对漆酶活性也产生了极显著的影响(P0.001)。此外,不同菌株在产漆酶能力上存在一定的差异,在不同培养基条件下所表现出的差异也不一致。相比之下,在含棉籽壳的完全培养基中漆酶活性高于含木屑或玉米芯的完全培养基,说明棉籽壳对不同糙皮侧耳菌株产漆酶能力的诱导作用更强。     

不同诱导培养基对糙皮侧耳液体发酵产漆酶活性的影响

利用1株糙皮侧耳Pleurotus ostreatus栽培菌株为材料,研究添加碱性木质素或者配合简单碳源或氮源后对其液体发酵产漆酶活性的影响。结果表明,不同诱导培养基对糙皮侧耳漆酶活性具有极显著的影响（P<0.001）,而且不同诱导培养基对糙皮侧耳菌丝生物量也产生了极显著的影响（P<0.001）。此外,只利用碱性木质素或者是再添加碳源葡萄糖均有利于糙皮侧耳产漆酶,既包括产漆酶酶活性的提升,同时也包括产漆酶时间的提前,但只利用碱性木质素诱导不利于菌丝生物量的积累;而富含简单碳/氮源的诱导培养基,无论是否含碱性木质素,均有利于菌丝生物量的积累,其中,富含简单碳/氮源的培养基中再添加碱性木质素后的菌丝生物量和漆酶活性均高于不添加碱性木质素时的菌丝生物量和漆酶活性。相比而言,含碱性木质素的培养基中测得的漆酶活性大部分时间下都要高于不含木质素的简单碳/氮源培养基,含碱性木质素的培养基对糙皮侧耳菌株产漆酶的诱导作用更强。     

Biosynthesis of vitamins B by the fungus Pleurotus ostreatus in a submerged culture

The intra- and extracellular contents of vitamins were studied in the course of submerged cultivation of the higher basidial mushroom Pleurotus ostreatus (Jacq.: Fr.) Kummer st. IMBF-1300 on liquid nutrient media. This strain was found to be autotrophic in respect of thiamin (vitamin B1), riboflavin (vitamin B2), niacin (vitamin B5), pyridoxine (vitamin B6) and biotin (vitamin B7), but it failed to synthesize cyanocobalamin (vitamin B12). The composition and pH of the culture medium, containing such complex biostimulating supplements as maize extract and concentrated potato sap noticeably influence the contents of vitamins B1, B5 and B7 in the mycelium, and to a less degree they change the level of the intracellular biosynthesis of vitamins B2 and B6. Higher excretion of vitamins B5, B7 and especially B6 was observed on the semisynthetic media during the postexponential growth. Under experimental conditions vitamins B1 and B2 were accumulated only in the cells. The dry mycelium of P. ostreatus obtained by submerged cultivation on liquid media is a valuable source of B vitamins and, especially, of niacin. Thus the oyster mushroom and other edible mushrooms can be put at one of the top places among food-stuffs by the content of niacin.     

Production of biomass and ligninolytic enzymes by Pleurotus ostreatus in submerged culture.

The production of biomass and ligninolytic enzymes by Pleurotus ostreatus was analysed in synthetic medium with yeast extract and different glucose concentrations (0.5 - 20 g/l), at different pH (3.5-6.5) and incubation temperatures (23-32 degrees C). The best culture condition were: initial glucose concentration of 5 g/l, initial pH between 5.5-6.5 and incubation temperature between 26-29 degrees C. The saturation constant for glucose (Ks) was 1.75 g/l. The biomass concentration reached 8.6 g/l with a glucose addition of 20.0 g/l to the culture medium. The control of pH allowed an increment of 0.5 g/l of biomass concentration. The birreactor produced pellets with a homogeneous distribution of diameter size of 3.4 -/+ 0.2 mm. Approximately, 307 U/l of laccase and 0.41 U/l of manganese peroxidase were obtained in extracellular liquid medium and 0.015 U/g of laccase and 0.809 U/g of manganese peroxidase were obtained in solid substrate. Lignin peroxidase activity was not detected at any condition.     

Exaggerated response to mild stress in rats fed high-fat diet

It has been suggested that high-fat (HF) diet exaggerates the stress-induced release of glucocorticoids due to activation of the hypothalamic-pituitary-adrenal (HPA) axis. In an initial experiment, in which rats were fed HF diet for 4 days, we found that HF-fed controls stopped gaining weight, indicating that they were hyperresponsive to the mild stress of tail bleeding but responded the same as low-fat (LF)-fed rats to the more severe stress of restraint. A second experiment confirmed these results when rats fed a HF diet for 4 days showed an exaggerated corticosterone release in response to an intraperitoneal injection of saline and movement to a novel cage, compared with LF-fed rats. Experiment 3 tested the same parameters as experiment 2 but interchanged the diets. This allowed us to differentiate between the effects of the dietary fat and the novelty of the diet. Additionally, this experiment determined whether hyperresponsiveness to mild stress in HF-fed rats was sustained during a prolonged exposure to diet. The results confirmed that a HF diet, not novelty, exaggerated the endocrine stress response after 9 days on the diet but that the effect was no longer present after 23 days on the diet. The hyperresponsiveness of the HPA axis in HF-fed rats is similar to that observed in animals that have been exposed to a significant chronic or acute stress, suggesting that the HF diet may initially be perceived as a stressor.     

Endothelial and vascular dysfunctions and insulin resistance in rats fed a high-fat, high-sucrose diet

This study was designed to examine the effects of a high-fat, high-sucrose (HFHS) diet on vascular and metabolic actions of insulin. Male rats were randomized to receive an HFHS or regular chow diet for 4 wk. In a first series of experiments, the rats had pulsed Doppler flow probes and intravascular catheters implanted to measure blood pressure, heart rate, and regional blood flows. Insulin sensitivity and vascular responses to insulin were assessed during a euglycemic hyperinsulinemic clamp performed in conscious rats. In a second series of experiments, new groups of rats were used to examine skeletal muscle glucose transport activity and to determine in vitro vascular reactivity, endothelial nitric oxide synthase (eNOS) protein expression in muscle and vascular tissues and endothelin content, nitrotyrosine formation, and NAD(P)H oxidase protein expression in vascular tissues. The HFHS-fed rats displayed insulin resistance, hyperinsulinemia, hypertriglyceridemia, hyperlipidemia, elevated blood pressure, and impaired insulin-mediated renal and skeletal muscle vasodilator responses. A reduction in endothelium-dependent vasorelaxation, accompanied by a decreased eNOS protein expression in muscles and blood vessel endothelium, and increased vascular endothelin-1 protein content were also noted in HFHS-fed rats compared with control rats. Furthermore, the HFHS diet induced a reduced insulin-stimulated glucose transport activity in muscles and increased levels of NAD(P)H oxidase protein and nitrotyrosine formation in vascular tissues. These findings support the importance of eNOS protein in linking metabolic and vascular disease and indicate the ability of a Westernized diet to induce endothelial dysfunction and to alter metabolic and vascular homeostasis.     

Functioning of oxidative phosphorylation in liver mitochondria of high-fat diet fed rats

We proposed that inhibition of mitochondrial adenine nucleotide translocator (ANT) by long chain acyl-CoA (LCAC) underlies the mechanism associating obesity and type 2 diabetes. Here we test that after long-term exposure to a high-fat diet (HFD): (i) there is no adaptation of the mitochondrial compartment that would hinder such ANT inhibition, and (ii) ANT has significant control of the relevant aspects of oxidative phosphorylation. After 7 weeks, HFD induced a 24+/-6% increase in hepatic LCAC concentration and accumulation of the oxidative stress marker N(epsilon)-(carboxymethyl)lysine. HFD did not significantly affect mitochondrial copy number, oxygen uptake, membrane potential (Deltapsi), ADP/O ratio, and the content of coenzyme Q(9), cytochromes b and a+a(3). Modular kinetic analysis showed that the kinetics of substrate oxidation, phosphorylation, proton leak, ATP-production and ATP-consumption were not influenced significantly. After HFD-feeding ANT exerted considerable control over oxygen uptake (control coefficient C=0.14) and phosphorylation fluxes (C=0.15), extra- (C=0.23) and intramitochondrial (C=-0.56) ATP/ADP ratios, and Deltapsi (C=-0.11). We conclude that although HFD induces accumulation of LCAC and N(epsilon)-(carboxymethyl)lysine, oxidative phosphorylation does not adapt to these metabolic challenges. Furthermore, ANT retains control of fluxes and intermediates, making inhibition of this enzyme a more probable link between obesity and type 2 diabetes.     

Antihypertensive effect of quercetin in rats fed with a high-fat high-sucrose diet

The effects of different levels of quercetin on the blood pressure were studied in 6-week-old male Sprague-Dawley rats. The rats were fed with a control diet or a high-fat high-sucrose (HFS) diet containing 0, 0.02, 0.07, 0.2, or 0.5% quercetin for 4 weeks. The systolic blood pressure and the lipid peroxides in the plasma were both higher in the rats fed with the HFS diet without quercetin than in the rats fed with the control diet. The nitric oxide synthase (NOS) activity in the vascular tissues and nitric oxide (NO) metabolites in the plasma and urine were both lower in these rats. A distinct depression of the increase in blood pressure was found in the rats fed with the HFS diets containing quercetin. Each level of quercetin examined was effective, the 0.5% level being much more effective than other levels. Dietary quercetin decreased lipid peroxidation in the plasma of the rats fed with the HFS diets. Quercetin also suppressed the decrease in NO metabolites in the plasma and urine, and the NOS activity in the vascular tissues of these rats. These results suggest that the increased NO availability caused by the elevated NOS activity, and the antioxidative activity in these rats fed with quercetin may be sources of the antihypertensive effect of quercetin.     

A krill oil supplemented diet suppresses hepatic steatosis in high-fat fed rats

Krill oil (KO) is a dietary source of n-3 polyunsaturated fatty acids, mainly represented by eicosapentaenoic acid and docosahexaenoic acid bound to phospholipids. The supplementation of a high-fat diet with 2.5% KO efficiently prevented triglyceride and cholesterol accumulation in liver of treated rats. This effect was accompanied by a parallel reduction of the plasma levels of triglycerides and glucose and by the prevention of a plasma insulin increase. The investigation of the molecular mechanisms of KO action in high-fat fed animals revealed a strong decrease in the activities of the mitochondrial citrate carrier and of the cytosolic acetyl-CoA carboxylase and fatty acid synthetase, which are both involved in hepatic de novo lipogenesis. In these animals a significant increase in the activity of carnitine palmitoyl-transferase I and in the levels of carnitine was also observed, suggesting a concomitant stimulation of hepatic fatty acid oxidation. The KO supplemented animals also retained an efficient mitochondrial oxidative phosphorylation, most probably as a consequence of a KO-induced arrest of the uncoupling effects of a high-fat diet. Lastly, the KO supplementation prevented an increase in body weight, as well as oxidative damage of lipids and proteins, which is often found in high-fat fed animals.     

Exponential growth kinetics for Polyporus versicolor and Pleurotus ostreatus in submerged culture

Simple mathematical models for a batch culture of pellet-forming fungi in submerged culture were tested on growth data for Polyporus versicolor (ATCC 12679) and Pleurotus ostreatus (ATCC 9415). A kinetic model based on a growth rate proportional to the two-thirds power of the cell mass was shown to be satisfactory. A model based on a growth rate directly proportional to the cell mass fitted the data equally well, however, and may be preferable because of mathematical simplicity.     

Dietary creatine supplementation lowers hepatic triacylglycerol by increasing lipoprotein secretion in rats fed high-fat diet

Recent studies have shown that dietary creatine supplementation can prevent lipid accumulation in the liver. Creatine is a small molecule that plays a large role in energy metabolism, but since the enzyme creatine kinase is not present in the liver, the classical role in energy metabolism does not hold in this tissue. Fat accumulation in the liver can lead to the development of nonalcoholic fatty liver disease (NAFLD), a progressive disease that is prevalent in humans. We have previously reported that creatine can directly influence lipid metabolism in cell culture to promote lipid secretion and oxidation. Our goal in the current study was to determine whether similar mechanisms that occur in cell culture were present in vivo. We also sought to determine whether dietary creatine supplementation could be effective in reversing steatosis. Sprague–Dawley rats were fed a high-fat diet or a high-fat diet supplemented with creatine for 5 weeks. We found that rats supplemented with creatine had significantly improved rates of lipoprotein secretion and alterations in mitochondrial function that were consistent with greater oxidative capacity. We also find that introducing creatine into a high-fat diet halted hepatic lipid accumulation in rats with fatty liver. Our results support our previous report that liver cells in culture with creatine secrete and oxidize more oleic acid, demonstrating that dietary creatine can effectively change hepatic lipid metabolism by increasing lipoprotein secretion and oxidation in vivo. Our data suggest that creatine might be an effective therapy for NAFLD.