Preparation of Sterile Raw Material - Chicken Eggshells in the Process of their Transformation into Selected Calcium Salts.

BACKGROUND: The chicken eggshells and their subcrustal membranes are a valuable source of calcium, but they are not further processed but disposed of as waste from the food industry. Chicken eggshells have high content (>95%) of calcium carbonate. Some properties suggest that eggshells may be a promising alternative to the present calcium sources used in the pharmaceutical industry. METHODS: The effect of roasting chicken eggshells with a selected organic acid (citric or fumaric or lactic acid) on microbiological purity, including the presence of fungi and bacteria Salmonella spp., Staphylococcus aureus, Escherichia coli of obtained calcium salts, was investigated. In this study, chicken eggshells were subjected to chemical reactions with organic acids (citric, fumaric or lactic acid) at two different calcium-acid molar ratios (1:1 or 1:3) and the mixture was heat-treated for 1 or 3 hours at a temperature of 100°C or 120°C. RESULTS AND DISCUSSION: It was found that lactic acid was 100% effective against fungi, and the remaining citric and fumaric acids were -50% (regardless of the other examined conditions). The type of acid used has a significant effect on fungal growth inhibition (p<0.05). Fumaric acid and lactic acid will be nearly 100% effective against bacteria (100% fumaric acid and 97% lactic acid effectiveness), regardless of other factors. CONCLUSION: Lactic acid is the most effective against pathogenic flora - fungi and bacteria. The transformation of chicken eggshells into calcium lactate can provide us with sterile calcium salt, free of 100% fungi and 97% of all bacteria.

Recent Advances on Purification of Lactic Acid.

With increasing interest in developing biodegradable polymers to replace fossil-based products globally, lactic acid (LA) has been paid extensive attention due to the high environment-compatibility of its downstream products. The mainstream efforts have been put in developing energy-efficient conversion technologies through biological and chemical routes to synthesize LA. However, to our best knowledge, there is a lack of sufficient attention in developing effective separation technologies with high atom economics for purifying LA and derivatives. In this review, the most recent advances in purifying LA using precipitation, reactive extraction, emulsion liquid membrane, reactive distillation, molecular distillation, and membrane techniques will be discussed critically with respect to the fundamentals, flow scheme, energy efficiency, and equipment. The outcome of this article is to offer insights into implementing more atomic and energy-efficient technologies for upgrading LA.

Lactate concentration in breast cancer using advanced magnetic resonance spectroscopy.

BACKGROUND: Precision medicine in breast cancer demands markers sensitive to early treatment response. Aerobic glycolysis (AG) upregulates lactate dehydrogenase A (LDH-A) with elevated lactate production; however, existing approaches for lactate quantification are either invasive or impractical clinically. METHODS: Thirty female patients (age 39-78 years, 15 grade II and 15 grade III) with invasive ductal carcinoma were enrolled. Lactate concentration was quantified from freshly excised whole tumours with double quantum filtered (DQF) magnetic resonance spectroscopy (MRS), and Nottingham Prognostic Index (NPI), LDH-A and proliferative marker Ki-67 were assessed histologically. RESULTS: There was a significantly higher lactate concentration (t = 2.2224, p = 0.0349) in grade III (7.7 ± 2.9 mM) than in grade II (5.5 ± 2.4 mM). Lactate concentration was correlated with NPI (ρ = 0.3618, p = 0.0495), but not with Ki-67 (ρ = 0.3041, p = 0.1023) or tumour size (r = 0.1716, p = 0.3645). Lactate concentration was negatively correlated with LDH-A (ρ = -0.3734, p = 0.0421). CONCLUSION: Our results showed that lactate concentration in whole breast tumour from DQF MRS is sensitive to tumour grades and patient prognosis.

Rational engineering of Aerococcus viridansl-lactate oxidase for the mediator modification to achieve quasi-direct electron transfer type lactate sensor.

The l-lactate oxidase (LOx) based lactate sensors are widely used for clinical diagnostics, sports medicine, and food quality control. However, dissolved oxygen interference and electroactive interferent effects are inherent issues of current lactate sensors. In this paper, a quasi-direct electron transfer (quasi-DET) type lactate sensor was developed using rationally engineered Aerococcus viridans LOx (AvLOx) modified with amine-reactive phenazine ethosulfate (PES). Since the modification of wild type AvLOx by PES did not result quasi-DET, engineered AvLOx with additional Lys residue was designed. The additional Lys residue was introduced by substituting residue locating on the surface of AvLOx, and within 20 Šof the isoalloxazine ring of FMN. Among several constructed mutants, Ala96Leu/Asn212Lys double mutant showed the highest dye-mediated dehydrogenase activity with negligible oxidase activity, showing quasi-DET properties after PES modification, when the enzyme was immobilized on screen printed carbon electrode. The constructed electrode did not show oxygen interference in cyclic voltammetric analysis and distinct catalytic current with 20 mM l-lactate. The sensor performance of a chronoamperometric l-lactate sensor employing PES modified Ala96Leu/Asn212Lys AvLOx, marked with linear range between 0 and 1 mM, with sensitivity of 13 µA/mM∙cm2, and a limit of detection of 25 µM for l-lactate. By applying -200 mV vs. Ag/AgCl, l-lactate could be monitored with negligible interference from 170 µM ascorbic acid, 1.3 mM acetaminophen, 1.4 mM uric acid or 20 mM glucose. These results indicated that a quasi-DET type lactate sensor was developed that did not suffer from the interference of oxygen and representative electroactive ingredient compounds.

Scanning electrochemical microscopy imaging of poly (3,4-ethylendioxythiophene)/thionine electrodes for lactate detection via NADH electrocatalysis.

Herein we report the use of scanning electrochemical microscopy (SECM) together with electrochemical and spectroscopic techniques to develop and characterise a stable and uniformly reactive chemically modified platinum electrode for NADH electrocatalysis. In order to achieve this, a range of different approaches for thionine entrapment within an electropolymerised poly (3,4-ethylendioxythiophene) (PEDOT) film were evaluated using SECM imaging in the presence of NADH, demonstrating the uniformity of the reactive layer towards NADH oxidation. The effect of electrolyte type and time scale employed during PEDOT electropolymerisation was examined with respect to thionine loading and the resulting charge transport diffusion coefficient (DCT) estimated via chronoamperometry. These studies indicated a decrease in DCT as thionine loading increased within the PEDOT film, suggesting that charge transport was diffusion limited within the film. Additionally, thionine functionalised nanotubes were formed, providing a stable support for lactate dehydrogenase entrapment while lowering the rate of thionine leaching, determined via SECM imaging. This enabled lactate determination at Eapp = 0.0 V vs Ag/AgCl over the range 0.25-5 mM in the presence of 1 mM NAD+.

The Usefulness of Performing Biochemical Tests in the Saliva of Kickboxing Athletes in the Dynamic of Training.

The study aimed to determine the suitability of testing the saliva of kickboxing athletes to show changes in biochemical parameters in dynamic of training. 8 elite male athletes (mean age 17.29± 0.31 years, body mass 66.82± 3.46kg, with 5.62±0.96 years of training experience) participated in the study. Indicators of lipid peroxidation and glycolysis (the concentration of lactic acid and pyruvic acid) were defined before and after a training session. Significant increases in indicators of lipid peroxidation activity indicators and the concentration of lactic acid (4-fold) were observed; analysis of correlation matrices confirms the absence of expressed changes. At the same time, significant decreases in catalase (10-fold from 3.69 µkat/L to 0.39 µkat/L) and pyruvic acid (from 3.92 µl/l to 0.55 µl/l) were observed. Our results confirm the value of using saliva to determine training load in an individual. Moreover, the study provided information on the importance of indexes reflecting a correlation of various biochemical indicators to estimate the sufficiency of training loads. The ease of sampling and informational content of saliva are reasons to use such tests in monitoring athletes' functional state to prevent fatigue.

Positive Impact of Pulsed Electric Field on Lactic Acid Removal, Demineralization and Membrane Scaling during Acid Whey Electrodialysis.

The drying of acid whey is hindered by its high mineral and organic acid contents, and their removal is performed industrially through expensive and environmentally impacting serial processes. Previous works demonstrated the ability to remove these elements by electrodialysis alone but with a major concern-membrane scaling. In this study, two conditions of pulsed electric field (PEF) were tested and compared to conventional DC current condition to evaluate the potential of PEF to mitigate membrane scaling and to affect lactic acid and salt removals. The application of a PEF 25 s/25 s pulse/pause combination at an initial under-limiting current density allowed for decreasing the amount of scaling, the final system electrical resistance by 32%, and the relative energy consumption up to 33%. The use of pulsed current also enabled better lactic acid removal than the DC condition by 10% and 16% for PEF 50 s/10 s and 25 s/25 s, respectively. These results would be due to two mechanisms: (1) the mitigation of concentration polarization phenomenon and (2) the rinsing of the membranes during the pause periods. To the best of our knowledge, this was the first time that PEF current conditions were used on acid whey to both demineralize and deacidify it.

A novel semiconductor based wireless electrochemical sensing platform for chronic disease management.

Electrochemical sensors are very versatile and can be used for a diverse range of biomedical applications. In this paper, a novel fully-integrated wireless electrochemical sensing platform is presented. The platform uses standard semiconductor technology to create a miniaturized integrated bioelectronics system that consists of an electrochemical sensor, potentiostat, signal processing circuitry, wireless power harvesting circuitry, and wireless telemetry unit, all on a single microchip. The platform is orders of magnitude smaller than the state-of-the-art sensing systems and costs a fraction. At 1.4 mm × 1.4 mm size, the sensor costs less than $1 to manufacture. The presented design provides fundamental advantages in decreasing sensor noise and settling time, thus providing superior response compared to existing solutions. System design and implementation details are presented as well as examples for metabolic sensing (glucose, lactate, O2) applications. The system can have widespread applications in biosensing applications.

Microneedle-based biosensor for minimally-invasive lactate detection.

Here we report the first mediated microneedles-based biosensor for minimally invasive continuous sensing of lactate in the dermal interstitial fluid (ISF). To further demonstrate the capability of microneedle arrays as second generation biosensors we have functionalized gold microneedles with nanocarbons at which mediated electron transfer of lactate oxidase takes place. In particular the gold surface of the microneedles electrode has been modified in 3 subsequent steps: i) electrodeposition of Au-multiwalled carbon nanotubes (MWCNTs); ii) electropolymerization of the mediator, methylene blue (MB); iii) immobilization of the enzyme lactate oxidase (LOX) by drop-casting procedure. The resulting microneedle-based LOX biosensor displays an interference-free lactate detection without compromising its sensitivity, stability, selectivity and response time. The performance of the microneedle array, second generation biosensor for lactate detection was assessed in artificial interstitial fluid and in human serum, both spiked with lactate. The results reveal that the new microneedles lactate sensor holds interesting promise for the development of a real-time monitoring device to be used in sport medicine and clinical care.

Model-based design of an intermittent simulated moving bed process for recovering lactic acid from ternary mixture.

An intermittent simulated moving bed (3F-ISMB) operation scheme, the extension of the 3W-ISMB to the non-linear adsorption region, has been introduced for separation of glucose, lactic acid and acetic acid ternary-mixture. This work focuses on exploring the feasibility of the proposed process theoretically and experimentally. Firstly, the real 3F-ISMB model coupled with the transport dispersive model (TDM) and the Modified-Langmuir isotherm was established to build up the separation parameter plane. Subsequently, three operating conditions were selected from the plane to run the 3F-ISMB unit. The experimental results were used to verify the model. Afterwards, the influences of the various flow rates on the separation performances were investigated systematically by means of the validated 3F-ISMB model. The intermittent-retained component lactic acid was finally obtained with the purity of 98.5%, recovery of 95.5% and the average concentration of 38 g/L. The proposed 3F-ISMB process can efficiently separate the mixture with low selectivity into three fractions.

A Tube-Integrated Painted Biosensor for Glucose and Lactate.

Developing a simple and direct approach for sensitive, specific, and rapid detection of metabolic compounds is of great importance for a variety of biological, medical, and food applications. Tubes are a highly portable and accessible container shape which are widely used for scientific research in cell biology and chemical synthesis, and which are also of great use in domestic health care applications. Here, we show for the first time the development of a tube-based painted amperometric biosensor for the detection of glucose and lactate. The sensor was prepared by printing carbon graphite and silver/silver chloride inks on the interior wall of the tube and then immobilizing glucose oxidase or lactate oxidase on the sensor. The sensor showed a sensitive, rapid, and reliable detection of glucose and lactate. We anticipate that these results could open new avenues for the development of painted biosensors, and toward advanced biosensor applications.

Chromatographic separation of phenyllactic acid from crude broth using cryogels with dual functional groups.

Phenyllactic acid (PLA) is an important organic acid with wide antimicrobial activities against gram-positive and gram-negative bacteria and some fungi. This interesting compound can be synthesized by the microbial fermentation or the bioconversion using phenylpyruvic acid (PPA) as the key substrate and microorganisms as the whole-cell biocatalysts. However, the isolation of high-purity PLA with a high recovery from the crude fermentation or conversion broth is a challenging task. In this work, the separation of PLA from the crude conversion broth prepared by employing Lactobacillus buchneri cells as the whole-cell catalysts was achieved by the chromatography using the poly(hydroxyethyl methacrylate) (pHEMA)-based cryogel with a combination of anion-exchange and hydrophobic benzyl groups. The static adsorption behaviors of PLA under different salt concentrations and the adsorption capacities of PLA on the cryogel were measured experimentally. The chromatographic performance of PLA from the crude conversion broth was compared with that from the clarified broth. The results showed that the pHEMA-based cryogel has a high capacity of PLA, i.e., 14.64 mg mL-1 cryogel, and the adsorption of PLA was influenced by the salt concentration. By using deionized water as running buffer, PLA with a high purity of 97.6% was obtained with one step elution using 0.3 M NaCl as the elution solution with the recovery at the range of 80.2-90.8% from crude feedstock without any pretreatment at various flow velocities. These values were close to those obtained for the clarified broth, i.e., the purity of 98.4% and the recovery of 92.3% under the same chromatography conditions at 1 cm min-1. The cryogel was then applied to separate PLA from clarified feedstock, high purity (>96.7%) and recovery (>91.4%) of PLA were found with 20 cycles, which verified the selectivity and robustness of prepared pHEMA-VBTAC cryogel. Therefore, the chromatography using pHEMA-based cryogel with the dual functional groups is an effective approach for the isolation of PLA directly from the crude bioconversion broth and thus could be interesting in the separation and production of high-purity PLA in industry.

Minimizing the effects of oxygen interference on l-lactate sensors by a single amino acid mutation in Aerococcus viridansl-lactate oxidase.

l-lactate biosensors employing l-lactate oxidase (LOx) have been developed mainly to measure l-lactate concentration for clinical diagnostics, sports medicine, and the food industry. Some l-lactate biosensors employ artificial electron mediators, but these can negatively impact the detection of l-lactate by competing with the primary electron acceptor: molecular oxygen. In this paper, a strategic approach to engineering an AvLOx that minimizes the effects of oxygen interference on sensor strips was reported. First, we predicted an oxygen access pathway in Aerococcus viridans LOx (AvLOx) based on its crystal structure. This was subsequently blocked by a bulky amino acid substitution. The resulting Ala96Leu mutant showed a drastic reduction in oxidase activity using molecular oxygen as the electron acceptor and a small increase in dehydrogenase activity employing an artificial electron acceptor. Secondly, the Ala96Leu mutant was immobilized on a screen-printed carbon electrode using glutaraldehyde cross-linking method. Amperometric analysis was performed with potassium ferricyanide as an electron mediator under argon or atmospheric conditions. Under argon condition, the response current increased linearly from 0.05 to 0.5mM l-lactate for both wild-type and Ala96Leu. However, under atmospheric conditions, the response of wild-type AvLOx electrode was suppressed by 9-12% due to oxygen interference. The Ala96Leu mutant maintained 56-69% of the response current at the same l-lactate level and minimized the relative bias error to -19% from -49% of wild-type. This study provided significant insight into the enzymatic reaction mechanism of AvLOx and presented a novel approach to minimize oxygen interference in sensor applications, which will enable accurate detection of l-lactate concentrations.

Cascade Production of Lactic Acid from Universal Types of Sugars Catalyzed by Lanthanum Triflate.

Lignocellulosic biomass conversion into value-added platform chemicals in the non-toxic, water-tolerant Lewis acid, and water solutions bears the hallmark of green chemistry. Lactic acid derived from biomass is an important chemical building block for biodegradable polymers such as polylactide. Herein, a universal method of converting lignocellulosic sugars into lactic acid using catalytic amount of water-stable Lewis acid La(OTf)3 is demonstrated. The lignocellulosic sugars studied in this work include 1) pyrolytic sugars from pyrolysis oil, and 2) sugars derived from ionic liquid (IL)-pretreated biomass. Under moderate conditions (250 °C, 1 h), levoglucosan (major pyrolytic sugar), glucose, and xylose were converted into lactic acid with carbon-based molar yields of 75, 74, and 61 %, respectively. Furthermore, roughly 49 mol % (based on levoglucosan) and 74 wt % (relative to pretreated biomass) of lactic acid were obtained from the conversion of pyrolytic sugars and sugar-rich fraction after lignin removal from switchgrass, respectively. To our knowledge, this is the first reported conversion of pyrolytic sugar into lactic acid by chemocatalysis and also lignocellulosic sugars are converted into lactic acid without hydrolysis. This approach could potentially be extended to other lignocellulosic sugars after simple removal of lignin from biomass pretreatment, rendering moderate to high yields of lactic acid.

Efficient in situ separation and production of L-lactic acid by Bacillus coagulans using weak basic anion-exchange resin.

To get rid of the dependence on lactic acid neutralizer, a simple and economical approach for efficient in situ separation and production of L-lactic acid was established by Bacillus coagulans using weak basic anion-exchange resin. During ten tested resins, the 335 weak basic anion-exchange resins demonstrated the highest adsorption capacity and selectivity for lactic acid recovery. The adsorption study of the 335 resins for lactic acid confirmed that it is an efficient adsorbent under fermentation condition. Langmuir models gave a good fit to the equilibrium data at 50 °C and the maximum adsorption capacity for lactic acid by 335 resins was about 402 mg/g. Adsorption kinetic experiments showed that pseudo-second-order kinetics model gave a good fit to the adsorption rate. When it was used for in situ fermentation, the yield of L-lactic acid by B. coagulans CC17 was close to traditional fermentation and still maintained at about 82% even after reuse by ten times. These results indicated that in situ separation and production of L-lactic acid using the 335 resins were efficient and feasible. This process could greatly reduce the dosage of neutralizing agent and potentially be used in industry.

Thallium stimulates ethanol production in immortalized hippocampal neurons.

Lactate and ethanol (EtOH) were determined in cell culture medium (CCM) of immortalized hippocampal neurons (HN9.10e cell line) before and after incubation with Thallium (Tl). This cell line is a reliable, in vitro model of one of the most vulnerable regions of central nervous system. Cells were incubated for 48 h with three different single Tl doses: 1, 10, 100 µg/L (corresponding to 4.9, 49 and 490 nM, respectively). After 48 h, neurons were "reperfused" with fresh CCM every 24/48 h until 7 days after the treatment and the removed CCM was collected and analysed. Confocal microscopy was employed to observe morphological changes. EtOH was determined by head space-solid phase microextraction -gas chromatography -mass spectrometry (HS-SPME-GCMS), lactate by RP-HPLC with UV detection. Tl exposure had significant effects on neuronal growth rate and morphology. The damage degree was dose-dependent. In not exposed cells, EtOH concentration was 0.18 ± 0.013 mM, which represents about 5% of lactate concentration (3.4 ± 0.10 mM). After Tl exposure lactate and EtOH increased. In CCM of 100 and 10 µg/L Tl-treated cells, lactate increased 24 h after reperfusion up to 2 and 3.3 times the control value, respectively. In CCM of 10 and 100 µg/L Tl-treated cells 24 h after reperfusion, EtOH increased up to 0.3 and 0.58 mmol/L. respectively. These results are consistent with significant alterations in energy metabolism, despite the low doses of Tl employed and the relatively short incubation time.

Longterm storage of post-packaged bread by controlling spoilage pathogens using Lactobacillus fermentum C14 isolated from homemade curd.

One potent lactic acid bacterial strain C14 with strong antifungal activity was isolated from homemade curd. Based on morphological as well as biochemical characters and 16S rDNA sequence homology the strain was identified as Lactobacillus fermentum. It displayed a wide antimicrobial spectrum against both Gram-positive and Gram-negative pathogenic bacteria, and also against number of food spoilage, plant and human pathogenic fungi. The cell free supernatant (CFS) of the strain C14 was also effective against the fungi tested. Inhibition of radial growth of Penicillium digitatum, Trichophyton rubrum and Mucor sp. was noticed in the presence of CFS of C14 even at low concentration (1%). More than 94.3 ± 1.6% and 91.5 ± 2.2% inhibition of conidial germination of P. digitatum and Mucor sp. were noticed in the presence of 10-fold-concentrated CFS of C14. Massive deformation of the fungal mycelia was observed by SEM studies, and losses of cellular proteins and DNA are also evident upon its treatment with C14. HPLC analysis revealed the presence of phenyl lactic acid, lactic acid along with some unidentified compounds in the antifungal extract. Challenge experiment showed immense potential of the strain C14 in preventing the spoilage of bread samples caused by Mucor sp. and Bacillus subtilis. The bread samples remained fresh upto 25 days even after inoculation with Mucor sp. (3.7 × 104 spores /ml) and B. subtilis (4.6 × 104 CFU /ml). Along with the antifungal properties, the isolated lactic acid bacterial strain also showed very good antioxidant activities. Unchanged level of liver enzymes serum glutamic pyruvic transaminase and serum glutamic oxaloacetic transaminase in albino mice upon feeding with C14 also suggested non-toxic nature of the bacterial isolate.

Recombinant Lactococcus lactis for efficient conversion of cellodextrins into L-lactic acid.

Lactic acid bacteria (LAB) are among the most interesting organisms for industrial processes with a long history of application as food starters and biocontrol agents, and an underexploited potential for biorefineries converting biomass into high-value compounds. Lactic acid (LA), their main fermentation product, is among the most requested chemicals owing to its broad range of applications. Notably, LA polymers, that is, polylactides, have high potential as biodegradable substitutes of fossil-derived plastics. However, LA production by LAB fermentation is currently too expensive for polylactide to be cost-competitive with traditional plastics. LAB have complex nutritional requirements and cannot ferment inexpensive substrates such as cellulose. Metabolic engineering could help reduce such nutritional requirements and enable LAB to directly ferment low-cost polysaccharides. Here, we engineered a Lactococcus lactis strain which constitutively secretes a ß-glucosidase and an endoglucanase. The recombinant strain can grow on cellooligosaccharides up to at least cellooctaose and efficiently metabolizes them to L-LA in single-step fermentation. This is the first report of a LAB able to directly metabolize cellooligosaccharides longer that cellohexaose and a significant step toward cost-sustainable consolidated bioprocessing of cellulose into optically pure LA.

ROS mediated selection for increased NADPH availability in Escherichia coli.

The economical production of chemicals and fuels by microbial processes remains an intense area of interest in biotechnology. A key limitation in such efforts concerns the availability of key co-factors, in this case NADPH, required for target pathways. Many of the strategies pursued for increasing NADPH availability in Escherichia coli involve manipulations to the central metabolism, which can create redox imbalances and overall growth defects. In this study we used a reactive oxygen species based selection to search for novel methods of increasing NADPH availability. We report a loss of function mutation in the gene hdfR appears to increase NADPH availability in E. coli. Additionally, we show this excess NADPH can be used to improve the production of 3HP in E. coli.

Isolation and identification of l/d-lactate-conjugated bufadienolides from toad eggs revealing lactate racemization in amphibians.

Three pairs of bufadienolide l/d-lactate epimers (1-6) were isolated from the eggs of the toad Bufo bufo gargarizans. The structures were elucidated by using spectroscopic methods, X-ray diffraction analysis and a modified Mosher's method. Compounds 1-6 represent the first occurrence of lactate-conjugated bufadienolides in nature, and illustrate the existence of an enzyme-controlled epimerization from l- to d-lactate in amphibians. The biosynthetic pathways, in which two key enzymes might be involved (i.e., lactate racemase and acyltransferase), were proposed. In addition, the biological assays revealed that compounds 1-4 are potent cytotoxic agents against human gastric cancer cells BGC-823 and human lung cancer cells A549 with IC50 values in a range of 8.0 to 80.0 nM.