Biotechnological conversion of spent coffee grounds into lactic acid.

This work investigates the potential bioconversion of spent coffee grounds (SCG) into lactic acid (LA). SCG were hydrolysed by a combination of dilute acid treatment and subsequent application of cellulase. The SCG hydrolysate contained a considerable amount of reducing sugars (9·02 ± 0·03 g l-1 , glucose; 26·49 ± 0·10 g l-1 galactose and 2·81 ± 0·07 g l-1 arabinose) and it was used as a substrate for culturing several lactic acid bacteria (LAB) and LA-producing Bacillus coagulans. Among the screened micro-organisms, Lactobacillus rhamnosus CCM 1825 was identified as the most promising producer of LA on a SCG hydrolysate. Despite the inhibitory effect exerted by furfural and phenolic compounds in the medium, reasonably high LA concentrations (25·69 ± 1·45 g l-1 ) and yields (98%) were gained. Therefore, it could be demonstrated that SCG is a promising raw material for the production of LA and could serve as a feedstock for the sustainable large-scale production of LA. SIGNIFICANCE AND IMPACT OF THE STUDY: Spent coffee grounds (SCG) represent solid waste generated in millions of tonnes by coffee-processing industries. Their disposal represents a serious environmental problem; however, SCG could be valorized within a biorefinery concept yielding various valuable products. Herein, we suggest that SCG can be used as a complex carbon source for the lactic acid production.

Chicken feather hydrolysate as an inexpensive complex nitrogen source for PHA production by Cupriavidus necator on waste frying oils.

The chicken feather hydrolysate (FH) has been tested as a potential complex nitrogen source for the production of polyhydroxyalkanoates by Cupriavidus necator H16 when waste frying oil was used as a carbon source. The addition of FH into the mineral salt media with decreased inorganic nitrogen source concentration improved the yields of biomass and polyhydrohyalkanoates. The highest yields were achieved when 10 vol.% of FH prepared by microwave-assisted alkaline hydrolysis of 60 g l-1 feather was added. In this case, the poly(3-hydroxybutyrate) (PHB) yields were improved by more than about 50% as compared with control cultivation. A positive impact of FH was also observed for accumulation of copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) when sodium propionate was used as a precursor. The copolymer has superior processing and mechanical properties in comparison with PHB homopolymer. The application of FH eliminated the inhibitory effect of propionate and resulted in altered content of 3-hydroxyvalerate (3HV) in copolymer. Therefore, the hydrolysed feather can serve as an excellent complex source of nitrogen for the polyhydroxyalkanoates (PHA) production. Moreover, by the combination of two inexpensive types of waste, such as waste frying oil and feather hydrolysate, it is possible to produce PHA with substantially improved efficiency and sustainability. SIGNIFICANCE AND IMPACT THE STUDY: Millions of tons of feathers, important waste product of poultry-processing industry, are disposed off annually without any further benefits. Thus, there is an inevitable need for new technologies that enable ecologically and economically sensible processing of this waste. Herein, we report that alkali-hydrolysed feathers can be used as a complex nitrogen source considerably improving polyhydroxyalkanoates production on waste frying oil employing Cupriavidus necator.

Use of several waste substrates for carotenoid-rich yeast biomass production.

Carotenoids are industrially significant pigments produced in many bacteria, fungi, and plants. Carotenoid biosynthesis in yeasts is involved in stress response mechanisms. Thus, controlled physiological and nutrition stress can be used for enhanced pigment production. Huge commercial demand for natural carotenoids has focused attention on developing of suitable biotechnological techniques including use of liquid waste substrates as carbon and/or nitrogen source. In this work several red yeast strains (Sporobolomyces roseus, Rhodotorula glutinis, Rhodotorula mucilaginosa) were enrolled into a comparative screening study. To increase the yield of these pigments at improved biomass production, several types of exogenous as well as nutrition stress were tested. Each strain was cultivated at optimal growth conditions and in medium with modified carbon and nitrogen sources. Synthetic media with addition of complex substrates (e.g. yeast extract) and vitamin mixtures as well as some waste materials (whey, potato extract) were used as nutrient sources. Peroxide and salt stress were applied too. The production of carotene enriched biomass was carried out in flasks as well as in laboratory fermentor. The best production of biomass was obtained in inorganic medium with yeast extract. In optimal conditions tested strains differ only slightly in biomass production. All strains were able to use most of waste substrates. Biomass and pigment production was more different according to substrate type. In laboratory fermentor better producers of enriched biomass were both Rhodotorula strains. The highest yields were obtained in R. glutinis CCY 20-2-26 cells cultivated on whey medium (cca 45 g per liter of biomass enriched by 46 mg/L of beta-carotene) and in R. mucilaginosa CCY 20-7-31 grown on potato medium and 5% salt (cca 30 g per liter of biomass enriched by 56 mg/L of beta-carotene). Such dried carotenoid-enriched red yeast biomass could be directly used in feed industry as nutrition supplement.

Use of controlled exogenous stress for improvement of poly(3-hydroxybutyrate) production in Cupriavidus necator.

The PHB production by Cupriavidus necator H16 depends on the type and concentration of stress factors and on the time of stress application. Hydrogen peroxide and ethanol significantly enhanced PHB accumulation in C. necator cells. Improved yields (10.9 g/L PHB) were observed after exposure of bacterial culture to 0.5 mmol/L H2O2 at the beginning of cultivation and to additional peroxide stress (5 mmol/L H2O2) after 60 h of cultivation (beginning of the stationary phase). Production was then approximately 28 % higher than in control (8.50 g/L PHB). The highest yields (11.2 g/L PHB) were observed when ethanol (0.5 %) was applied at the beginning of stationary phase. An application of exogenous stress could thus be used as a simple strategy for a significant improvement of PHB production in C. necator.

Antimutagenic properties of green tea.

In this work biological effects of two common kinds of green tea (Chinese Gunpowder and Japanese Sencha) were analyzed using three independent tests of antimutagenicity: 1) the Ames test with Salmonella typhimurium TA98, 2) cytogenetic analysis of peripheral blood lymphocytes (CAPL), and 3) test with Saccharomyces cerevisiae D7. Tea extracts were allowed to be antimutagenic based on their ability to inhibit the mutagenic effect of standard mutagens. Amounts of (-)catechin and (-)catechin gallate in tea extracts were determined by high performance liquid chromatography on reversed phase (RP-HPLC). Antioxidant capacity was found using total radical-trapping antioxidant parameter (TRAP) method. Extracts from Gunpowder and Sencha exhibited high antimutagenic activity in the Ames test (24.7+/-3.7% and 34.1+/-2, 1% of inhibition without metabolic activation; 74.9+/-1.7% and 62.7+/-4.3% of inhibition with metabolic activation, respectively) as well as in S. cerevisiae D7 test (Gunpowder: 62.7+/-5.7% of Trp convertants inhibition and 52.6+/-5.3% of Ilv revertants inhibition; Sencha: 45.6+/-4.2% of Trp convertants inhibition, 50.0+/-4.8% of Ilv revertants inhibition). In the CAPL method reduced number of abberant cells as well as decreased number of chromosome breaks was observed using both green tea extracts. Antioxidant capacity and antimutagenicity of green tea extracts was higher than activity of tea catechins and flavonoids.

Polymorphism Ncol in tumor necrosis factor B is associated with fasting glycemia and lipid parameters in healthy non-obese caucasian subjects.

BACKGROUND: The study was designed to investigate the associations among polymorphisms TNF-B Ncol and TNF-alpha -308G/A, plasma TNF-alpha levels and metabolic and anthropometric parameters related to insulin sensitivity in a set of 113 Caucasian subjects undergoing oral glucose tolerance test (oGTT). METHODS: Genotypes were detected by PCR; BMI, WHR, glycemia during oGTT, fasting immunoreactive insulin, fasting C-peptide, HbA(1c), total cholesterol, triglycerides, HDL, LDL and plasma TNF-alpha levels were measured in each subject. RESULTS: Type 2 diabetes was diagnosed in 10 subjects, impaired glucose tolerance (IGT) in 41, normal glucose tolerance (NGT) in 62. Significant differences among genotypes of the TNF-B Ncol were observed for FPG (P=0.0063), LDL (P=0.0179) and marginally for total cholesterol (P=0.0763) in NGT group. After the classification of NGT subjects into obese and non-obese according to BMI, associations of TNF-B Ncol with FPG, LDL and cholesterol were proved in non-obese subgroup only. TNF-alpha -308G/A polymorphism was not associated with any of the parameters studied. TNF-alpha levels did not revealed difference among NGT, IGT and DM groups or genotype-dependent differences. CONCLUSIONS: Our results indicate significant association of the TNF-B Ncol polymorphism with FPG, LDL and total cholesterol in normoglycemic non-obese Caucasian subjects. This polymorphism could be involved in genetic modulation of glucose and lipid homeostasis and regulation of insulin sensitivity already in healthy state. Disturbances of this regulation could be component of pathogenesis of type 2 diabetes mellitus.

Relations among serum ferritin, C282Y and H63D mutations in the HFE gene and type 2 diabetes mellitus in the Czech population.

Aims of the study were: (i) to determine the prevalence of mutations C282Y and H63D in the HFE gene causing hereditary hemochromatosis in patients with type 2 diabetes mellitus and non-diabetics, (ii) to investigate the relationship among HFE genotypes, serum ferritin and glucose intolerance and (iii) to assess possible association of HFE mutations with the susceptibility to develop late diabetic complications in the Czech population. Two approaches were employed - the case-control study comprising diabetics and non-diabetic controls (n = 326) and the cross-sectional study comprising subjects with a previously unknown defect of glucose tolerance (n = 113, oral glucose tolerance test performed in each subject). Allele frequencies of C282Y and H63D did not differ between diabetic and control groups nor among subjects with normal glucose tolerance (NGT), impaired glucose tolerance (IGT) and diabetes. Ferritin levels significantly differed between diabetic and non-diabetic women (P<1.10 (-3)) and among subjects with NGT, IGT and diabetes (P<0.05). Differences in ferritin levels related to particular genotypes of C282Y and H63D were not detected. Prevalence of diabetes in the first and second quartiles of ferritin distribution differed highly significantly from the prevalence in the third and fourth quartiles in women (P = 0.000037), OR = 3.50 (95% CI, 1.89-6.48). The extent of diabetic late complications did not correlate with ferritin plasma levels.

Polymorphisms 1704G/T and 2184A/G in the RAGE gene are associated with antioxidant status.

The formation of advanced glycation end products (AGEs) and oxidative stress are supposed to play an important role in the development of diabetic late complications. AGEs can bind to several binding sites including receptor of advanced glycation end products (RAGE). AGE-RAGE interaction results in free radical generation. The aim of the present study was to investigate the impact of previously described polymorphisms in the RAGE gene (G82S, 1704G/T, 2184A/G, and 2245G/A) on the glycoxidation status in non-insulin-dependent diabetes mellitus (NIDDM). A total of 371 unrelated caucasian subjects were enrolled in the study. The NIDDM group consisted of 202 subjects, and the presence of late diabetic complications in 5 particular localizations was expressed as an index (I(compl)). The nondiabetic group included 169 subjects. Glycated hemoglobin (HbA(1c)), glycated stratum corneum proteins (Amadori, AGE), total carotenoids, alpha- and beta -carotene, gamma-tocopherol, lutein, lycopene, and alpha-tocopherol were measured in each subject. Statistically significant differences in allele frequencies between the NIDDM and the nondiabetic groups were observed for the G82S and 2245G/A polymorphisms (P =.047 and .032, respectively). HbA(1c), Amadori, and AGE did not reveal any significant association with any of the polymorphisms analyzed. However, significant differences between subjects bearing "wild-type majority" genotypes 1704GG+2184AA and subjects with "mutated" genotypes were found for total carotenoids (P =.001), alpha-carotene (P =.046), beta-carotene (P =.028), lutein (P =.001), lycopene (P =.006), and alpha-tocopherol (P =.047). I(compl) significantly correlated with the plasma levels of all antioxidants (all P <.01), while no correlation of I(compl) with glycation variables was observed. The newly identified intron polymorphisms in the RAGE gene were proved to be associated with the antioxidant status in NIDDM subjects. The extent of diabetic vascular disease is related to the plasma levels of antioxidants.

Polymorphisms in the RAGE gene influence susceptibility to diabetes-associated microvascular dermatoses in NIDDM.

To examine genetic polymorphism in the complete sequence of the Receptor of Advanced Glycation End products (RAGE) gene and its possible associations with diabetes-associated microvascular dermatoses (DAMD). Further, to analyze the distribution of individual genotype combinations on the particular polymorphic loci in the RAGE gene. A part of the RAGE gene spanning a region from -4 to 3334 bp was analyzed on a set of 45 subjects with non-insulin dependent diabetes mellitus (NIDDM) and parallel DAMD by means of PCR with subsequent heteroduplex and single-strand conformation polymorphism (SSCP) analyses. Allele frequencies and genotype combinations of novel common polymorphisms were determined in an associations study comprising four groups of subjects (n=390). Fourteen novel polymorphisms (R77C, V89V, 718G/T, 1704G/T, 1727A1728ins, H305Q, S307C, 2117A/G, 2184A/G, 2245G/A, 2249A/G, 2741G/A, and 3089ACdel) and one described previously (G82S) were identified. Significant association with microvascular dermatoses (MD) irrespective of NIDDM were found for exon mutation 82S (P= .004, after a correction for the number of comparisons P(corr) < .05) and marginally significant for intron variant 1704T (P= .032, P(corr)> .05). Calculated odds ratios for 82S and 1704T were 4.73 (95% CI, 1.51 to 14.77) and 1.73 (95% CI, 0.93 to 3.22), respectively. Certain individual genotype combinations of G82S, 1704G/T, and 2184A/G were significantly associated with the presence of MD (P= .00647) both in diabetic and non-diabetic study populations. The two novel polymorphisms (1704G/T and 2184A/G) together with the G82S were shown to influence the susceptibility to MD independent of diabetes itself.

Non-enzymatic glycation of epidermal proteins of the stratum corneum in diabetic patients.

A selected group of diabetic patients showed a statistically significant increase in levels of glycated proteins in the stratum corneum compared with a control group. The values of glycated proteins correlated with those of glycohaemoglobin (GHb), and in diabetic patients also with serum glucose concentrations. The values of glycated proteins (and GHb) exhibited a positive correlation with age both in a control group and in diabetic patients. The average values of glycated proteins (and GHb) were slightly higher in women than in men. Determination of glycated proteins levels of the stratum corneum can serve as a stable parameter for long-term monitoring of the course of non-enzymatic glycation in structural and connective tissues and thus also for the prognosis of the development of dermatological complications related to diabetes mellitus. In vitro incubation of stratum corneum proteins and keratin with glucose resulted in an increase of their glycation. The values of glycated proteins and glycated keratin increased proportionally to the glucose concentration and duration of incubation. Glucose binding to keratin and proteins of the insoluble stratum corneum fraction appeared to occur at practically the same rate, and it is a first-order reaction with regard to the glucose concentration. Water-soluble proteins of the stratum corneum undergo non-enzymatic glycation preferentially (on average, 83.4% of the total amount of glycated proteins is present in the soluble fraction), regardless of the initial content of glycated proteins in the sample. The content of glycated soluble proteins of a higher molecular weight significantly increased after 4 weeks of incubation with glucose.

Modified simplified method for isolation of lysostaphin from the culture filtrate of Staphylococcus staphylolyticus.

The present paper reports a modified method for isolation of lysostaphin--a bacteriolytic agent with specific affinity for staphylococcal cell wall. The proposed purification scheme includes three steps. The first procedure is ultrafiltration through a membrane filter giving a yield of 75.6%. The result of ultrafiltration is a concentrated, 10-times purified preparation of lysostaphin with specific activity 0.62 U/mg which can be used for digestion of S. aureus cells. Further step, performed by ion-exchange chromatography on DEAE-cellulose, yields a 60-times purified preparation containing a mixture of enzyme components of lysostaphin. The yield of this step is 47.2%, the preparation contains 3.54 U/mg protein. Using gel filtration on Sephadex G-50 a component with hexosaminidase activity was separated from the endopeptidase component on the basis of molar mass difference. A 270-times purified preparation of lysostaphin-endopeptidase with minimum of contaminating substances was obtained in this step. The yield of gel filtration was 22.1%, specific activity increased up to 16.3 U/mg protein.

Spectrophotometric detection of bacteriolytic activity of diluted lysostaphin solutions.

Turbidimetric method with spectrophotometric detection of changes in density of test bacteria S. aureus strain SA 812 for determination of bacteriolytic activity of lysostaphin was employed. Results of two evaluations are compared: (1) calculation of the relative value of turbidity decrease on the basis of the difference of absolute values of A540 at the beginning of reaction and after the incubation period, (2) following of time changes in A540 by monitoring the course of reaction directly in the constant-temperature cuvette of the spectrophotometer at 37 degrees C. Both arrangements yielded identical results, within the significance level of 0.05. With concentrated samples both methods yield reliable results; with diluted samples the accuracy of the "absolute" method decreases together with decreasing lysostaphin concentration much faster than with the "registration" method. The registration method makes it possible to detect even minute amounts of the lytic enzyme and thus to distinguish the values of activity in dilute samples even when data obtained by means of the "absolute" method cannot be considered as reliable. A unit of bacteriolytic activity can be expressed from the kinetic curve as an amount of enzyme preparation causing delta A540/min = 0.01.