Consortia of lactic acid bacteria strains increase the antioxidant activity and bioactive compounds of quinoa sourdough - based biscuits.

The aim of this work was to use consortia (two or three strains) of lactic acid bacteria (LAB) [Lactiplantibacillus plantarum CRL 1964 and CRL 1973, and Leuconostoc mesenteroides subsp. mesenteroides CRL 2131] to obtain quinoa sourdoughs (QS) for further manufacturing of quinoa sourdough-based biscuits (QB). Microbial grow and acidification were evaluated in QS while antioxidant activity (AOA), total phenolic compounds (TPC) and total flavonoid compounds (TFC) were determined in QS and QB. QS inoculated with LAB consortia respect to monocultures showed higher growth and acidification, AOA (7.9?42.6%), TPC (19.9?35.0%) and TFC (6.1?31.6%). QB prepared with QS inoculated by LAB consortia showed higher AOA (5.0-81.1%), TPC (22.5?57.5%) and TFC (14.0-79.9%) than biscuits inoculated by monocultures sourdoughs. These results were attributed to a synergic effect from LAB consortia. Principal component analysis showed the highest scores of the evaluated characteristics for biscuits made with consortia sourdough of two (CRL1964?+?CRL2131) and three (CRL1964?+?CRL1973?+?CRL2131) strains.

Biodiversity and technological potential of lactic acid bacteria isolated from spontaneously fermented amaranth sourdough.

UNLABELLED: Spontaneous fermented sourdoughs prepared from amaranth flour were investigated for the presence of autochthonous lactic acid bacteria (LAB) predominating microbiota. The doughs were fermented with daily backslopping on a laboratory scale at 30°C for 10 days. LAB counts ranged from 2·60 to 8·54 log CFU g(-1) with a pH declined from 6·2 to 3·8 throughout fermentation. The combined use of randomly amplified polymorphic DNA (RAPD)-PCR analysis and sequence analysis of 16S rRNA was applied for LAB intraspecies differentiation and taxonomic identification, respectively. Enterococcus, Pediococcus and Lactobacillus species were present in amaranth sourdoughs (AS). After the first refreshment step, Lactobacillus plantarum dominated AS until the end of fermentation. In coincidence, when DGGE analysis was performed, the occurrence of a progressive change in bacterial communities allowed the selection of Lact. plantarum as a dominant species. Moreover, technological, functional and safety characteristics of representative RAPD-biotypes were investigated. Lact. plantarum CRL1898 was selected as a potential candidate for gluten-free amaranth sourdough starter. SIGNIFICANCE AND IMPACT OF THE STUDY: Nowadays, there is an increasing interest in ancient noncereal gluten-free (GF) crops such as amaranth, due to their reported nutritional and health benefits. However, the use of these grains is still limited to traditional foods and bread making processes that are not yet well standardized. Results on the dynamics of autochthonous lactic acid bacteria (LAB) microbiota during laboratory spontaneous amaranth sourdoughs (AS) fermentation will contribute to overcome challenges for GF-fermented products development. In addition, knowledge about LAB diversity involving Enterococcus, Pediococcus and Lactobacillus species, with Lactobacillus plantarum predominating during AS fermentation, and their technological and functional properties provides the basis for the selection of autochthonous strains as starters cultures for novel gluten-free bakery products with enhanced nutritional, sensory and/or safety quality.

Biodiversity and technological-functional potential of lactic acid bacteria isolated from spontaneously fermented quinoa sourdoughs.

AIMS: To analyse lactic acid bacteria (LAB) diversity and technological-functional and safety properties of strains present during spontaneous fermented quinoa sourdoughs. METHODS AND RESULTS: Fermentation was performed by daily backslopping at 30°C for 10 days. Autochthonous LAB microbiota was monitored by a biphasic approach combining random amplified polymorphic DNA (RAPD)-PCR and rRNA gene sequencing with PCR-denaturing gradient gel electrophoresis (DGGE) analysis. Identification and intraspecies differentiation allowed to group isolates within nine LAB species belonging to four genera. A succession of LAB species occurred during 10-days backslopping; Lactobacillus plantarum and Lactobacillus brevis were detected as dominant species in the consortium. The characterization of 15 representative LAB strains was performed based on the acidifying capacity, starch and protein hydrolysis, γ-aminobutyric acid and exopolysaccharides production, antimicrobial activity and antibiotic resistance. CONCLUSION: Strains characterization led to the selection of Lact. plantarum CRL1905 and Leuconostoc mesenteroides CRL1907 as candidates to be assayed as functional starter culture for the gluten-free (GF) quinoa fermented products. SIGNIFICANCE AND IMPACT OF THE STUDY: Results on native LAB microbiota present during quinoa sourdough fermentation will allow the selection of strains with appropriate technological properties to be used as a novel functional starter culture for GF-fermented products.

Eficacia y seguridad del control de glucemia guiado por objetivo y dirigido por el personal de enfermería en una unidad de cuidados intensivos: un estudio prospectivo observacional / Effectiveness and safety of goal directed nurse-led blood glucose control in an intensive care unit: a prospective observational study

Objetivo valorar la eficacia y seguridad de un sistema de control de la glucemia guiada por objetivo en una UCI. Metodología estudio descriptivo prospectivo de 13 meses. Se recogieron diariamente los valores de todas las determinaciones de glucemia en los pacientes con perfusión continua de insulina. Resultados se recogieron 12.677 glucemias realizadas a 69 pacientes en tratamiento con insulina intravenosa; el 57,9% se encontraron en el rango objetivo (100-140mg/dl); no hubo ninguna glucemia menor de 40mg/dl y tan solo el 0,2% de ellas se encontraron entre 40-60mg/dl. Conclusiones para un adecuado control de las cifras de glucemia mediante el empleo de terapia con insulina intravenosa es esencial el manejo individualizado de la perfusión de insulina, ajustándose a las características de cada paciente individual. El método guiado por objetivos que planteamos ha permitido obtener mejores resultados que los de otros estudios (AU)

Objective to evaluate the effectiveness and safety of a nurse-led blood glucose control protocol in a medical ICU. Method a descriptive, prospective study was carried out for a period of 13 months. All blood glucose values from patients on insulin therapy for intensive glycemic control were recorded daily. Results A total of 12,677 blood glucose determinations were performed on the 69 patients under glycemic control; 57.9% of the determinations had predetermined study target values for blood glucose (100-140mg/dl) and 68.8% of the determinations had physiological blood glucose values (80-140mg/dl); no values under 40mg/dl were obtained, and only 0.2% were between 40-60mg/dl. Conclusions For an adequate blood glucose control using intensive insulin therapy, individual management of insulin infusion regimen is essential, adjusted to the characteristics of each patient. A nurse-led intervention has allowed better results to be obtained in comparison with other studies in which different protocols for insulin infusion are used (AU)

[Effectiveness and safety of goal directed nurse-led blood glucose control in an intensive care unit: a prospective observational study]. / Eficacia y seguridad del control de glucemia guiado por objetivo y dirigido por el personal de enfermería en una unidad de cuidados intensivos: un estudio prospectivo observacional.

OBJECTIVE: to evaluate the effectiveness and safety of a nurse-led blood glucose control protocol in a medical ICU. METHOD: a descriptive, prospective study was carried out for a period of 13 months. All blood glucose values from patients on insulin therapy for intensive glycemic control were recorded daily. RESULTS: A total of 12,677 blood glucose determinations were performed on the 69 patients under glycemic control; 57.9% of the determinations had predetermined study target values for blood glucose (100-140 mg/dl) and 68.8% of the determinations had physiological blood glucose values (80-140 mg/dl); no values under 40 mg/dl were obtained, and only 0.2% were between 40-60 mg/dl. CONCLUSIONS: For an adequate blood glucose control using intensive insulin therapy, individual management of insulin infusion regimen is essential, adjusted to the characteristics of each patient. A nurse-led intervention has allowed better results to be obtained in comparison with other studies in which different protocols for insulin infusion are used.

Effect of biosynthetic intermediates and citrate on the phenyllactic and hydroxyphenyllactic acids production by Lactobacillus plantarum CRL 778.

AIM: To evaluate the influence of biosynthetic precursors, intermediates and electron acceptors on the production of antifungal compounds [phenyllactic acid (PLA) and hydroxyphenyllactic acid (OH-PLA)] by Lactobacillus plantarum CRL 778, a strain isolated from home-made sourdough. METHODS AND RESULTS: Growth of fermentative activity and antifungal compounds production by Lact. plantarum CRL 778 were evaluated in a chemically defined medium (CDM) supplemented with biosynthetic precursors [phenylalanine (Phe), tyrosine (Tyr)], intermediates [glutamate (Glu), alpha-ketoglutarate (α-KG)] and electron acceptors [citrate (Cit)]. Results showed that the highest PLA production (0.26 mmol l(-1)), the main antifungal compound produced by Lact. plantarum CRL 778, occurred when greater concentrations of Phe than Tyr were present. Both PLA and OH-PLA yields were increased 2-folds when Cit was combined with α-KG instead of Glu at similar Tyr/Phe molar ratio. Similarly, glutamate dehydrogenase (GDH) activity was significantly (P < 0.01) stimulated by α-KG and Cit in Glu-free medium. CONCLUSION: Phe was the major stimulant for PLA formation; however, Cit could increase both PLA and OH-PLA synthesis by Lact. plantarum CRL 778 probably due to an increase in oxidized NAD(+). This effect, as well as the GDH activity, was enhanced by α-KG and down regulated by Glu. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study where the role of Glu and GDH activity in the PLA and OH-PLA synthesis was evidenced in sourdough lactic acid bacteria (LAB) using a CDM. These results contribute to the knowledge on the antifungal compounds production by sourdough LAB with potential applications on the baked goods.

Inhibition of citrus fungal pathogens by using lactic acid bacteria.

The effect of lactic acid bacteria (LAB) on pathogenic fungi was evaluated and the metabolites involved in the antifungal effect were characterized. Penicillium digitatum (INTA 1 to INTA 7) and Geotrichum citri-aurantii (INTA 8) isolated from decayed lemon from commercial packinghouses were treated with imazalil and guazatine to obtain strains resistant to these fungicides. The most resistant strains (4 fungal strains) were selected for evaluating the antifungal activity of 33 LAB strains, among which only 8 strains gave positive results. The antifungal activity of these LAB strains was related to the production of lactic acid, acetic acid, and phenyllactic acid (PLA). A central composite design and the response surface methodology were used to evaluate the inhibitory effect of the organic acids produced by the LAB cultures. The antifungal activity of lactic acid was directly related to its concentration; however, acetic acid and PLA showed a peak of activity at 52.5 and 0.8 mM, respectively, with inhibition rates similar to those obtained with Serenade((R)) (3.0 ppm) imazalil (50 ppm) and guazatine (50 ppm). Beyond the peak of activity, a reduction in effectiveness of both acetic acid and PLA was observed. Comparing the inhibition rate of the organic acids, PLA was about 66- and 600-fold more effective than acetic acid and lactic acid, respectively. This study presents evidences on the antifungal effect of selected LAB strains and their end products. Studies are currently being undertaken to evaluate the effectiveness in preventing postharvest diseases on citrus fruits.

Lactobacillus reuteri CRL 1100 as starter culture for wheat dough fermentation.

The effect of sucrose on the fermentation balance of Lactobacillus reuteri CRL 1100 and the invertase activity of this strain in wheat dough and culture medium (MRSs) was evaluated. The enzyme activity was dependent on the environmental pH releasing glucose and fructose from sucrose hydrolysis. Glucose was used as carbon source, while fructose was mainly used as electron acceptor to produce mannitol up to 10h of fermentation. Thereafter, fructose seemed to be metabolized by the heterofermentative pathway, which determined an increase in the concentration of acetate (6 mmol l(-1)), lactate (2 mmol l(-1)) and ethanol (1 mmol l(-1)) and the lack of mannitol formation after glucose depletion. The fermentation balance of Lb. reuteri CRL 1100 during the dough fermentation resulted in lower (63%) ethanol, higher (75%) acetate production and soluble carbohydrates concentrations, like MRSs cultures. This fermentation profile would be important to obtain an optimal growth of yeast and the optimal bread flavor and taste.

Functionality of lactic acid bacteria peptidase activities in the hydrolysis of gliadin-like fragments.

AIMS: To evaluate the role of the peptidase activities from sourdough lactic acid bacteria (LAB) in the degradation of alpha-gliadin fragments. METHODS AND RESULTS: Different proline-containing substrates were hydrolysed by LAB indicating pro-specific peptidase activities. Lactobacillus plantarum CRL 775 and Pediococcus pentosaceus CRL 792 displayed the highest tri- and di-peptidase activities, respectively. Lactobacillus plantarum strains hydrolysed more than 60%alpha-gliadin fragments corresponding to the 31-43 and 62-75 amino acids in the protein after 2 h. None of the LAB strains alone could hydrolyse 57-89 alpha-gliadin peptide; however, the combination of L. plantarum CRL 775 and P. pentosaceus CRL 792 led to hydrolysis (57%) of this peptide in 8 h. CONCLUSIONS: The capacity of LAB strains to degrade alpha-gliadin fragments was not correlated to individual peptidase activities. Several strains separately degraded the 31-43 and 62-75 alpha-gliadin fragments, while the 57-89 peptide degradation was associated with the combination of peptidase profiles from pooled LAB strains. This is the first report on the peptide hydrolase system of sourdough pediococci and its ability to reduce alpha-gliadin fragments. SIGNIFICANCE AND IMPACT OF THE STUDY: This study contributes to a better knowledge of sourdough LAB proteolytic system and its role in the degradation of proline-rich alpha-gliadin peptides involved in celiac disease.

Gluten breakdown by lactobacilli and pediococci strains isolated from sourdough.

AIMS: To evaluate the growth and metabolic activity of lactobacilli and pediococci strains in a gluten base medium (GBM), formulated for a proper selection of proteolytic strains to be used in sourdough fermentation. METHODS AND RESULTS: Proteolytic activity by lactic acid bacteria (LAB) was evaluated by SDS-PAGE and by the amino acids released determined by reversed-phase high-performance liquid chromatography. Only 13 LAB (nine lactobacilli and four pediococci), among the 42 evaluated were able to utilize gluten as nitrogen source and to grow in GBM. Pediococcus pentosaceus CRL 797 showed a similar proteolytic activity to lactobacilli strains. In the majority of the cultures, basic amino acid group increased (c. 80% after 12 h incubation) mainly due to the release of ornithine, a flavour precursor of bread. Lysine, a limiting essential amino acid in wheat flour, increased by 150% in cultures of P. pentosaceus CRL 797. CONCLUSIONS: This study allows selecting P. pentosaceus CRL 797 and L. plantarum CRL 759 as potential starter culture for type III sourdough fermentation. It is shown for the first time that pediococci strains isolated from sourdough are proteolytically active on gluten. SIGNIFICANCE AND IMPACT OF THE STUDY: The physiological studies on gluten breakdown by LAB will contribute to the better selection of strains to produce breads with enhanced organoleptic characteristics.

Proteolytic activity and reduction of gliadin-like fractions by sourdough lactobacilli.

AIMS: To characterize the peptide hydrolase system of Lactobacillus plantarum CRL 759 and CRL 778 and evaluate their proteolytic activity in reducing gliadin-like fractions. METHODS AND RESULTS: The intracellular peptide hydrolase system of Lact. plantarum CRL 759 and CRL 778 involves amino-, di- (DP), tri- (TP) and endopeptidase activities. These peptidases are metalloenzymes inhibited by EDTA and 1,10-phenanthroline and stimulated by Co2+. DP and TP activities of Lact. plantarum CRL 759 and CRL 778, respectively, were completely inhibited by Cu2+. Lactobacillus plantarum CRL 778 showed the highest proteolytic activity and amino acids release in fermented dough. The synthetic 31-43 alpha-gliadin fragment was hydrolysed to 36% and 73% by Lact. plantarum CRL 778 and CRL 759 respectively. CONCLUSIONS: Lactobacillus plantarum CRL 759 and CRL 778 have an active proteolytic system, which is responsible for the high amino acid release during sourdough fermentation and the hydrolysis of the 31-43 alpha-gliadin-like fragment. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides new information of use when obtaining sourdough starters for bread making. Moreover, knowledge regarding lactobacilli capable of reducing the level of gliadin-like fractions, a toxic peptide for coeliac patients, has a beneficial health impact.

Effect of galactose and glucose on the exopolysaccharide production and the activities of biosynthetic enzymes in Lactobacillus casei CRL 87.

AIMS: The objective of this work was to study the influence of the sugar source on exopolysaccharide (EPS) production and the activities of the enzymes involved in the synthesis of sugar nucleotides in Lactobacillus casei CRL 87. The relationship between these enzymes and EPS formation was determined. METHODS AND RESULTS: The concentration of EPS was estimated by the phenol/sulphuric acid method while the chemical composition of purified EPS was investigated using gas-liquid chromatography. Biosynthetic enzyme activities were determined spectrophotometrically by measuring the formation or disappearance of NAD(P)H at 340 nm. Polysaccharide production by Lb. casei CRL 87 was 1.7 times greater on galactose than on glucose. The isolated polymer was composed of rhamnose, glucose and galactose. The activities of uridine-diphosphate (UDP)-glucose-pyrophosphorylase, thymidine-diphosphate (dTDP)-glucose-pyrophosphorylase and the dTDP-rhamnose-synthetic enzyme system were higher in galactose-grown than in glucose-grown cells. When an EPS- mutant strain was used, galactokinase activity was not detected on galactose, this sugar not being available for the formation of sugar nucleotides for further EPS production. dTDP-glucose-pyrophosphorylase and dTDP-rhamnose-synthetic enzyme system activities were lower than the values found for the wild type strain. CONCLUSION: The carbon source present in the culture medium affects EPS production by Lb. casei CRL 87. The greater polymer synthesis by galactose-grown cells is correlated with the higher UDP-glucose-pyrophosphorylase, dTDP-glucose-pyrophosphorylase and dTDP-rhamnose-synthetic enzyme system activities. Initial sugar metabolism is also an important step for the synthesis of EPS precursors by this strain. SIGNIFICANCE AND IMPACT OF THE STUDY: Knowledge of the effect of the sugar source on EPS production and the activities of biosynthetic enzymes provides information about the mechanisms of regulation of the synthesis of EPS which can contribute to improving polymer production.

The peptide hydrolase system of Lactobacillus reuteri.

Peptide hydrolase system of Lactobacillus reuteri CRL 1098, a lactic acid bacteria of sourdough origin, was investigated. This microorganism has a broad range of peptidases consisting of an active aminopeptidase, X-Prolyl-dipeptidylaminopeptidase, dipeptidase and tripeptidase. Aminopeptidase, iminopeptidase and endopeptidase are most likely located in the cytoplasmic fraction showing no detectable association with the cell membrane, while dipeptidase and tripeptidase are mainly associated with the latter fraction. The peptidases are metalloenzymes activated by Co2+ and inhibited by Cu2+, Hg2+, Cd2+ and by metal-complexing reagents. The aminopeptidase activity inhibited by EDTA can be restored by Mn2+ while that of di- and tripeptidase treated with 1,10-phenantroline can be restored by Zn2+ and Co2+, respectively.

Histidine carboxylase of Leuconostoc oenos 9204: purification, kinetic properties, cloning and nucleotide sequence of the hdc gene.

Histidine decarboxylase (HDC) was purified to homogeneity from Leuconostoc oenos 9204, a wine lactic acid bacterium. Histidine decarboxylase comprised two subunits, respectively alpha and beta. The hdc gene was cloned and sequenced. The gene encodes a single polypeptide of 315 amino acids, demonstrating that Leuc. oenos 9204 HDC was synthesized as a precursor proHDC pi 6 (Mr 205,000). A cleavage between Ser-81 and Ser-82 generated the alpha (Mr 25,380) and beta (Mr 8840) chains, which suggested that the holoenzyme exists as a hexameric structure (alpha beta)6. At the optimal pH of 4.8, the HDC activity exhibited a simple Michaelis-Menten kinetic (K(m) = 0.33 mmol l-1, Vmax = 17.8 mumol CO2 min-1 mg-1), while at pH 7.6 it was sigmoidal (cooperativity index of 2). Histamine acted as a competitive inhibitor (Ki = 32 mmol l-1). The similarities of these results with those described for other bacterial HDC support the assumption that the pyruvoyl enzymes evolved from a common ancestral protein and have similar catalytic mechanisms. These results also confirmed that the main lactic acid bacterial species responsible for malolactic fermentation in red wine is able to produce histamine. Bacteria carrying the HDC activity must be avoided during selection of strains for the production of malolactic starters.

Characterization of two extracellular proteases fromLeuconostoc oenos.

Two extracellular proteolytic activities were characterized fromLeuconostoc oenos isolated from Argentinian wines. Both activities were maximal with autoclaved grape juice as substrate. The temperature and pH optima for the two proteolytic activities were different (30 and 40°C, and pH 4.0 and 5.5, respectively). Both enzymes were thermostable and their activities were unaltered by heating at 70°C for 15 min. Metal ions were not required for the activities. Neither enzyme appeared to be a serine protease but both were strongly inhibited by cysteine and ß-mercaptoethanol, indicating the involvement of disulphide bridges.

Protease production by Leuconostoc oenos strains isolated from wine.

Two separate, extracellular proteolytic activities were demonstrated in four strains of Leuconostoc oenos isolated from Argentinian wines. The first took place in the early growth phase and the other had its maximum at the end of growth. The two proteolytic enzymes had different pH and temperature optima. Divalent metal ions had different effects not only on each L. oenos strain but also on each of the two proteases from each strain.

Aspartate aminotransferase of Lactobacillus murinus.

Aspartate aminotransferase from Lactobacillus murinus is thermostable, its activity being not changed for two months at temperatures between 4 and -70 degrees C. Maximum activity was observed at 40 degrees C and pH 7.3 in phosphate buffer (30 mmol/L). delta G* Value of 26.3 kJ/mol was calculated from the Arrhenius plot. The Km values for L-aspartate and 2-oxoglutarate at pH 7.3 were 25 and 100 mmol/L, respectively. Sodium maleate and glutamate acted as inhibitors of the enzyme activity. The Ki values for sodium maleate with L-aspartate of 2-oxoglutarate as variable substrates were 1.1 and 0.5 mmol/L, respectively. The Ki values for glutamate with L-aspartate or 2-oxoglutarate were 8.0 and 4.0 mmol/L, respectively. An inhibitory effect was observed with 1 mM Hg2+ ions (1 mmol/L). The activity of the enzyme was diminished by only 12% in the absence of pyridoxal 5'-phosphate.