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D-Lactic Acid (D-Lactate) (Rapid) Assay Kit

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00:07  Introduction
00:58  Principle
01:52   Reagent Preparation
02:30  Procedure
05:28   Calculations

D-Lactic Acid D-Lactate Rapid Assay Kit K-DATE Scheme
Product code: K-DATE

50 assays (manual) / 500 assays (microplate) / 450 assays (auto-analyser)

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Content: 50 assays (manual) / 500 assays (microplate) / 450 assays (auto-analyser)
Shipping Temperature: Ambient
Storage Temperature: Short term stability: 2-8oC,
Long term stability: See individual component labels
Stability: > 2 years under recommended storage conditions
Analyte: D-Lactic Acid
Assay Format: Spectrophotometer, Microplate, Auto-analyser
Detection Method: Absorbance
Wavelength (nm): 340
Signal Response: Increase
Linear Range: 0.5 to 30 μg of D-lactic acid per assay
Limit of Detection: 0.21 mg/L
Reaction Time (min): ~ 5 min
Application examples: Wine, soft drinks, milk, dairy products (e.g. cream, milk / whey powder, cheese, condensed milk and yogurt), foods containing milk (e.g. dietetic foods, bakery products, baby food, chocolate, sweets and ice-cream), vinegar, fruit and vegetables, processed fruit and vegetables, meat products, food additives, paper (and cardboard), cosmetics, pharmaceuticals and other materials (e.g. biological cultures, samples, etc.).
Method recognition: Methods based on this principle have been accepted by DIN, GOST, IDF, EEC, EN, ISO, OIV, IFU, AIJN and MEBAK

The D-Lactic Acid (D-Lactate) (Rapid) test kit is suitable for the rapid, specific measurement and analysis of D-lactic acid in wine, beer, juice, milk, cheese, vinegar, meat and other food products.

Note for Content: The number of manual tests per kit can be doubled if all volumes are halved.  This can be readily accommodated using the MegaQuantTM  Wave Spectrophotometer (D-MQWAVE).

See our full list of lactic acid test kits and other organic acid assay kits.

Scheme-K-DATE DATE Megazyme

  • Extended cofactors stability. Dissolved cofactors stable for > 1 year at 4oC.
  • Very rapid reaction with most samples (~ 5 min) 
  • Very competitive price (cost per test) 
  • All reagents stable for > 2 years after preparation 
  • Mega-Calc™ software tool is available from our website for hassle-free raw data processing 
  • Standard included
  • Suitable for manual, microplate and auto-analyser formats
Megazyme publication

Megazyme “advanced” wine test kits general characteristics and validation.

Charnock, S. J., McCleary, B. V., Daverede, C. & Gallant, P. (2006). Reveue des Oenologues, 120, 1-5.

Many of the enzymatic test kits are official methods of prestigious organisations such as the Association of Official Analytical Chemicals (AOAC) and the American Association of Cereal Chemists (AACC) in response to the interest from oenologists. Megazyme decided to use its long history of enzymatic bio-analysis to make a significant contribution to the wine industry, by the development of a range of advanced enzymatic test kits. This task has now been successfully completed through the strategic and comprehensive process of identifying limitations of existing enzymatic bio-analysis test kits where they occurred, and then using advanced techniques, such as molecular biology (photo 1), to rapidly overcome them. Novel test kits have also been developed for analytes of emerging interest to the oenologist, such as yeast available nitrogen (YAN; see pages 2-3 of issue 117 article), or where previously enzymes were simply either not available, or were too expensive to employ, such as for D-mannitol analysis.

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Megazyme publication

Grape and wine analysis: Oenologists to exploit advanced test kits.

Charnock, S. C. & McCleary, B. V. (2005). Revue des Enology, 117, 1-5.

It is without doubt that testing plays a pivotal role throughout the whole of the vinification process. To produce the best possible quality wine and to minimise process problems such as “stuck” fermentation or troublesome infections, it is now recognised that if possible testing should begin prior to harvesting of the grapes and continue through to bottling. Traditional methods of wine analysis are often expensive, time consuming, require either elaborate equipment or specialist expertise and frequently lack accuracy. However, enzymatic bio-analysis enables the accurate measurement of the vast majority of analytes of interest to the wine maker, using just one piece of apparatus, the spectrophotometer (see previous issue No. 116 for a detailed technical review). Grape juice and wine are amenable to enzymatic testing as being liquids they are homogenous, easy to manipulate, and can generally be analysed without any sample preparation.

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Fully automatic D-lactate assay using a modified commercially available method.

Rasmussen, R. W., Straarup, D., Thorlacius-Ussing, O., Handberg, A. & Christensen, P. A. (2021). Scandinavian Journal of Clinical and Laboratory Investigation, 1-6.

Intestinal infarction is the fast-evolving endpoint of impaired blood perfusion to an intestinal segment which may have fatal outcome. Early diagnosis and treatment within 6 h reduce mortality. Currently, d-lactate is a promising biomarker, however, not available in the acute clinical setting. The aim of this study is implementation of d-lactate analysis in a routine clinical setting. We used a spectrophotometric method, based on enzymatic oxidation of d-lactate by d-lactate dehydrogenase (D-LDH) coupled to the reduction of nicotinamide-adenine dinucleotide (NAD+). The amount of NADH formed in this reaction is equivalent to d-lactate. The primary concern in this method is interfering NADH formed by oxidation of l-lactate by l-lactate dehydrogenase (L-LDH). A commercially available kit for d-lactate measurement was implemented on our existing automated routine laboratory equipment including pH-inactivation of L-LDH. Our setup fulfilled clinical quality goals. We were able to measure d-lactate with an acceptable performance of the analysis and a short turn-around time. The method can be used to distinguish between the expected cut-off for intestinal ischemia around 0.3 mM and the upper reference limit of 0.05 mM. With a turnaround time of just 9 min, the analysis has potential as a readily available detection of circulating d-lactate for early diagnosis of intestinal ischemia.

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Differential cytokine and metabolite production by cervicovaginal epithelial cells infected with Lactobacillus crispatus and Ureaplasma urealyticum.

Cavanagh, M., Amabebe, E. & Anumba, D. O. (2020). Anaerobe, 62, 102101.

Introduction: We sought to quantify targeted metabolites (d-lactate, pyruvate, urea, ammonia) and the cytokine IL-8 produced by human cervicovaginal epithelial cells co-cultured with Ureaplasma urealyticum (a preterm birth-associated bacterium) or Lactobacillus crispatus (a healthy vaginal commensal associated with term birth). Methods: Concentrations of D-lactate, pyruvate, urea and ammonia measured by enzyme-based spectrophotometry and IL-8 by ELISA were determined and compared between monolayer-cultured HeLa cells (ATCC 35241) infected with strains of U. urealyticum (ATCC 27618, 0.5 mL = 3640 CFU/mL, U. urealyticum) or L. crispatus (ATCC 33820, MOI = 10,000, 1000 and 100, L. crispatus) and incubated in 5% CO2 at 37°C for 24 h. Uninfected HeLa cells (Hc) were used as controls and cytotoxicity was determined by the amount (optical density) of lactate dehydrogenase (LDH) released by the dead HeLa cells. Results: The amount of LDH released by untreated Hc (P = 0.002) and U. urealyticum-infected cells (P < 0.0001) was higher than those of L. crispatus-infected cells, with U. urealyticum-infected cells recording the highest % cytotoxicity and L. crispatus-infected cells MOI 10,000 (Lc10,000) the least (P < 0.0001). Though there was no significant difference in the concentration of urea between the samples, U. urealyticum-infected cells showed higher ammonia compared to other samples (p = 0.03). In contrast, all L. crispatus samples had higher D-lactate than untreated Hc (p = 0.01) and U. urealyticum-infected cells (P = 0.01). Also, Lc10,000 had the highest D-lactate (p = 0.001) and lowest pyruvate (P = 0.04, excluding UU) compared to other samples. Furthermore, U. urealyticum-infected cells produced the highest IL-8 (P = 0.01) compared to other samples, with Lc10,000 producing undetectable levels. Conclusion: Infection of cervicovaginal epithelial cells by U. urealyticum stimulates production of ammonia from urea and induces elevated IL-8 production possibly leading to significantly higher cytotoxicity. In contrast, L. crispatus appeared protective against HeLa cell inflammation and death, producing more D-lactate and less IL-8, consistent with a role for L. crispatus in promoting vaginal floral health and reducing infection/inflammation-associated preterm birth.

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Infection/inflammation-associated preterm delivery within 14 days of presentation with symptoms of preterm labour: A multivariate predictive model.

Amabebe, E., Reynolds, S., He, X., Wood, R., Stern, V. & Anumba, D. O. (2019). PLoS One, 14(9), e0222455.

Multi-marker tests hold promise for identifying symptomatic women at risk of imminent preterm delivery (PTD, <37 week’s gestation). This study sought to determine the relationship of inflammatory mediators and metabolites in cervicovaginal fluid (CVF) with spontaneous PTD (sPTD) and delivery within 14 days of presentation with symptoms of preterm labour (PTL). CVF samples from 94 (preterm = 19, term = 75) singleton women with symptoms of PTL studied between 19+0-36+6 weeks’ gestation were analysed for cytokines/chemokines by multiplexed bead-based immunoassay, while metabolites were quantified by enzyme-based spectrophotometry in a subset of 61 women (preterm = 16, term = 45). Prevalence of targeted vaginal bacterial species was determined for 70 women (preterm = 14, term = 66) by PCR. Overall, 10 women delivered within 14 days of sampling. Predictive capacities of individual biomarkers and cytokine-metabolite combinations for sPTD and delivery within 14 days of sampling were analysed by logistic regression models and area under the receiver operating characteristic curve. Fusobacterium sp., Mubiluncus mulieris and Mycoplasma hominis were detected in more preterm-delivered than term women (P<0.0001), while, Mcurtisii was found in more term-delivered than preterm women (P<0.0001). RANTES (0.91, 0.65-1.0), IL-6 (0.79, 0.67-0.88), and Acetate/Glutamate ratio (0.74, 0.61-0.85) were associated with delivery within 14 days of sampling (AUC, 95% CI). There were significant correlations between cytokines and metabolites, and several cytokine-metabolite combinations were associated with sPTD or delivery within 14 days of sampling (e.g. L/D-lactate ratio+Acetate/Glutamate ratio+IL-6: 0.84, 0.67-0.94). Symptomatic women destined to deliver preterm and within 14 days of sampling express significantly higher pro-inflammatory mediators at mid to late gestation. In this cohort, IL-6, Acetate/Glutamate ratio and RANTES were associated with delivery within 14 days of sampling, consistent with their roles in modulating infection-inflammation-associated preterm labour in women presenting with symptoms of preterm birth. Replication of these observations in larger cohorts of women could show potential clinical utility.

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Reproducible, high-yielding, biological caproate production from food waste using a single-phase anaerobic reactor system.

Nzeteu, C. O., Trego, A. C., Abram, F. & O’Flaherty, V. (2018). Biotechnology for Biofuels, 11(1), 108.

Background: Nowadays, the vast majority of chemicals are either synthesised from fossil fuels or are extracted from agricultural commodities. However, these production approaches are not environmentally and economically sustainable, as they result in the emission of greenhouse gases and they may also compete with food production. Because of the global agreement to reduce greenhouse gas emissions, there is an urgent interest in developing alternative sustainable sources of chemicals. In recent years, organic waste streams have been investigated as attractive and sustainable feedstock alternatives. In particular, attention has recently focused on the production of caproate from mixed culture fermentation of low-grade organic residues. The current approaches for caproate synthesis from organic waste are not economically attractive, as they involve the use of two-stage anaerobic digestion systems and the supplementation of external electron donors, both of which increase its production costs. This study investigates the feasibility of producing caproate from food waste (FW) without the supplementation of external electron donors using a single-phase reactor system. Results: Replicate leach-bed reactors were operated on a semi-continuous mode at organic loading of 80 g VS FW l-1 and at solid retention times of 14 and 7 days. Fermentation, rather than hydrolysis, was the limiting step for caproate production. A higher caproate production yield 21.86 ± 0.57 g COD l-1 was achieved by diluting the inoculating leachate at the beginning of each run and by applying a leachate recirculation regime. The mixed culture batch fermentation of the FW leachate was able to generate 23 g caproate COD l-1 (10 g caproate l-1), at a maximum rate of 3 g caproate l-1 day-1 under high H2 pressure. Lactate served as the electron donor and carbon source for the synthesis of caproate. Microbial community analysis suggested that neither Clostridium kluyveri nor Megasphaera elsdenii, which are well-characterised caproate producers in bioreactors systems, were strongly implicated in the synthesis of caproate, but that rather Clostridium sp. with 99% similarity to Ruminococcaceae bacterium CPB6 and Clostridium sp. MT1 likely played key roles in the synthesis of caproate. This finding indicates that the microbial community capable of caproate synthesis could be diverse and may therefore help in maintaining a stable and robust process. Conclusions: These results indicate that future, full-scale, high-rate caproate production from carbohydrate-rich wastes, associated with biogas recovery, could be envisaged.

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Sharpea and Kandleria are lactic acid producing rumen bacteria that do not change their fermentation products when co-cultured with a methanogen.

Kumar, S., Treloar, B. P., Teh, K. H., McKenzie, C. M., Henderson, G., Attwood, G. T., Waters, S. M., Patchett, M. L. & Janssen, P. H. (2018). Anaerobe, 54, 31-38.

Sharpea and Kandleria are associated with rumen samples from low-methane-emitting sheep. Four strains of each genus were studied in culture, and the genomes of nine strains were analysed, to understand the physiology of these bacteria. All eight cultures grew equally well with D-glucose, D-fructose, D-galactose, cellobiose, and sucrose supplementation. D-Lactate was the major end product, with small amounts of the mixed acid fermentation products formate, acetate and ethanol. Genes encoding the enzymes necessary for this fermentation pattern were found in the genomes of four strains of Sharpea and five of Kandleria. Strains of Sharpea produced traces of hydrogen gas in pure culture, but strains of Kandleria did not. This was consistent with finding that Sharpea, but not Kandleria, genomes contained genes coding for hydrogenases. It was speculated that, in co-culture with a methanogen, Sharpea and Kandleria might change their fermentation pattern from a predominately homolactic to a predominately mixed acid fermentation, which would result in a decrease in lactate production and an increase in formation of acetate and perhaps ethanol. However, Sharpea and Kandleria did not change their fermentation products when co-cultured with Methanobrevibacter olleyae, a methanogen that can use both hydrogen and formate, and lactate remained the major end product. The results of this study therefore support a hypothesis that explains the link between lower methane yields and larger populations of Sharpea and Kandleria in the rumens of sheep.

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Are serum amyloid A or D-lactate useful to diagnose synovial contamination or sepsis in horses?.

Robinson, C. S., Singer, E. R., Piviani, M. & Rubio-Martinez, L. M. (2017). Veterinary Record, vetrec-2017.

Synovial sepsis in horses is life threatening and accurate diagnosis allowing prompt treatment is warranted. This study assessed the diagnostic value of serum amyloid A (SAA) and D-lactate in blood and synovial fluid (SF) as diagnostic markers of synovial sepsis in horses and correlated them with total nucleated cell count (TNCC), percentage of neutrophils (%N) and total protein (TP) in SF. Blood and SF SAA and D-lactate concentrations were determined in a case–control observational study including 112 horses (38 with synovial contamination or sepsis (SCS), 66 with non-septic intra-synovial pathology (NSISP) and 8 controls). Blood and SF SAA were significantly higher in SCS than in NSISP and control horses. SAA values were similar in NSISP and control horses. SF SAA was moderately correlated with synovial TNCC, TP and blood SAA. Blood and SF SAA were 82.4 per cent and 80 per cent sensitive and 88.9 per cent and 73 per cent specific for diagnosis of SCS, with cut-off values of 60.7 and 1.14 µg/ml, respectively. Blood and SF D-lactate concentrations were not significantly different between groups. This study shows that blood and SF SAA concentrations can aid to distinguish SCS from non-septic synovial pathology; however, D-lactate was not useful.

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Long-term adaptive evolution of Leuconostoc mesenteroides for enhancement of lactic acid tolerance and production.

Ju, S. Y., Kim, J. H. & Lee, P. C. (2016). Biotechnology for Biofuels, 9(1), 240.

Background: Lactic acid has been approved by the United States Food and Drug Administration as Generally Regarded As Safe (GRAS) and is commonly used in the cosmetics, pharmaceutical, and food industries. Applications of lactic acid have also emerged in the plastics industry. Lactic acid bacteria (LAB), such as Leuconostoc and Lactobacillus, are widely used as lactic acid producers for food-related and biotechnological applications. Nonetheless, industrial mass production of lactic acid in LAB is a challenge mainly because of growth inhibition caused by the end product, lactic acid. Thus, it is important to improve acid tolerance of LAB to achieve balanced cell growth and a high titer of lactic acid. Recently, adaptive evolution has been employed as one of the strategies to improve the fitness and to induce adaptive changes in bacteria under specific growth conditions, such as acid stress. Results: Wild-type Leuconostoc mesenteroides was challenged long term with exogenously supplied lactic acid, whose concentration was increased stepwise (for enhancement of lactic acid tolerance) during 1 year. In the course of the adaptive evolution at 70 g/L lactic acid, three mutants (LMS50, LMS60, and LMS70) showing high specific growth rates and lactic acid production were isolated and characterized. Mutant LMS70, isolated at 70 g/L lactic acid, increased D-lactic acid production up to 76.8 g/L, which was twice that in the wild type (37.8 g/L). Proteomic, genomic, and physiological analyses revealed that several possible factors affected acid tolerance, among which a mutation of ATPase ε subunit (involved in the regulation of intracellular pH) and upregulation of intracellular ammonia, as a buffering system, were confirmed to contribute to the observed enhancement of tolerance and production of D-lactic acid. Conclusions: During adaptive evolution under lethal stress conditions, the fitness of L. mesenteroides gradually increased to accumulate beneficial mutations according to the stress level. The enhancement of acid tolerance in the mutants contributed to increased production of D-lactic acid. The observed genetic and physiological changes may systemically help remove protons and retain viability at high lactic acid concentrations.

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Solid state fermentation with lactic acid bacteria to improve the nutritional quality of lupin and soybean.

Bartkiene, E., Krungleviciute, V., Juodeikiene, G., Vidmantiene, D. & Maknickiene, Z. (2015). Journal of the Science of Food and Agriculture, 95(6), 1336-1342.

BACKGROUND: The ability of bacteriocin-like inhibitory substance (BLIS)-producing lactic acid bacteria (LAB) to degrade biogenic amines as well as to produce L(+) and D(−)-lactic acid during solid state fermentation (SSF) of lupin and soya bean was investigated. In addition, the protein digestibility and formation of organic acids during SSF of legume were investigated. RESULTS: Protein digestibility of fermented lupin and soya bean was found higher on average by 18.3% and 15.9%, respectively, compared to untreated samples. Tested LAB produced mainly L-lactic acid in soya bean and lupin (D/L ratio 0.38–0.42 and 0.35–0.54, respectively), while spontaneous fermentation gave almost equal amounts of both lactic acid isomers (D/L ratio 0.82–0.98 and 0.92, respectively). Tested LAB strains were able to degrade phenylethylamine, spermine and spermidine, whereas they were able to produce putrescine, histamine and tyramine. CONCLUSIONS: SSF improved lupin and soya bean protein digestibility. BLIS-producing LAB in lupin and soya bean medium produced a mixture of D- and L-lactic acid with a major excess of the latter isomer. Most toxic histamine and tyramine in fermented lupin and soya bean were found at levels lower those causing adverse health effects. Selection of biogenic amines non-producing bacteria is essential in the food industry to avoid the risk of amine formation.

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Markers of perioperative bowel complications in colorectal surgery patients.

Hyšpler, R., Tichá, A., Kaška, M., Žaloudková, L., Plíšková, L., Havel, E. & Zadák, Z. (2015). Disease Markers, 2015, Article ID 428535.

Colorectal cancer is a clinical condition whose treatment often involves intestinal resection. Such treatment frequently results in two major gastrointestinal complications after surgery: anastomotic leakage and prolonged ileus. Anastomotic leakage is a serious complication which, more often than not, is diagnosed late; to date, C-reactive protein is the only available diagnostic marker. A monocentric, prospective, open case-control study was performed in patients undergoing colorectal surgery. Intestinal fatty acid binding protein (i-FABP), citrulline, D-lactate, exhaled hydrogen, Escherichia coli genomic DNA, and ischemia modified albumin (IMA) were determined preoperatively, postoperatively, and on the following four consecutive days. Bacterial DNA was not detected in any sample, and i-FABP and D-lactate lacked any distinct potential to detect postoperative bowel complications. Exhaled breath hydrogen content showed unacceptably low sensitivity. However, citrulline turned out to be a specific marker for prolonged ileus on postoperative days 3-4. Using a cut-off value of 20  µmol/L, a sensitivity and specificity of ~75% was achieved on postoperative day 4. IMA was found to be an efficient predictor of anastomosis leak by calculating the difference between preoperative and postoperative values. This test had 100% sensitivity and 80% specificity and 100% negative and 20% positive predictive value.

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The Use of Lactic Acid Bacteria in the Fermentation of Fruits and Vegetables - Technological and Functional Properties.

Urbonaviciene, D., Viskelis, P., Bartkiene, E., Juodeikiene, G. & Daiva Vidmantiene, D. (2015). Biochemistry, Genetics and Molecular Biology, Biotechnology, Chapter 7.

The relationship between food and health has been investigated for many years, and therefore, the development of foods that promote health and well-being is a key research priority of the food industry. Fruits and vegetables are an essential part of human nutrition. Unfortunately, the daily intake of fruits and vegetables is estimated to be lower than the recommendation of the World Health Organization (WHO), who suggest a dietary intake of 450 and 500 g of fruits and vegetables, respectively. Vegetables are strongly recommended in the human diet because they are rich in antioxidants, vitamins, dietary fibres and minerals. The majority of vegetables consumed in the human diet are fresh, minimally processed, pasteurised or cooked by boiling in water or microwaving, and vegetables can be canned, dried, or juiced or made into pastes, salads, sauces, or soups. Fresh vegetables or those that have been minimally processed have a particularly short shelf-life because they are subjected to rapid microbial spoilage. In addition, the above cooking processes can cause a number of potentially undesirable changes in physical characteristics and chemical composition.

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Homo-fermentative production of D-lactic acid by Lactobacillus sp. employing casein whey permeate as a raw feed-stock.

Prasad, S., Srikanth, K., Limaye, A. M. & Sivaprakasam, S. (2014). Biotechnology Letters, 36(6) 1303-1307.

Casein whey permeate (CWP), a lactose-enriched dairy waste effluent, is a viable feed stock for the production of value-added products. Two lactic acid bacteria were cultivated in a synthetic casein whey permeate medium with or without pH control. Lactobacillus lactis ATCC 4797 produced D-lactic acid (DLA) at 12.5 g l-1 in a bioreactor. The values of Leudking–Piret model parameters suggested that lactate was a growth-associated product. Batch fermentation was also performed employing CWP (35 g lactose l-1) with casein hydrolysate as a nitrogen supplement in a bioreactor. After 40 h, L. lactis produced 24.3 g lactic acid l-1 with an optical purity >98%. Thus CWP may be regarded as a potential feed-stock for DLA production.

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The effect of ciliate fauna composition on murein content and mureinolytic activity in the rumen of sheep.

Bełżecki, G., Miltko, R., Kwiatkowska, E., Kowalik, B. & Michałowski, T. (2012). Journal of Animal and Feed Sciences, 21(1), 65-76.

The effect of the ciliates, Eudiplodinium maggii, Diploplastron affine and Entodinium caudatum, and natural protozoal fauna on the ruminal murein concentration and mureinolytic activity was examined on three rams, repeatedly defaunated and refaunated with Eudiplodinium maggii, Diploplastron affine, Entodinium caudatum and natural protozoal fauna. The number of ciliates varied from 18 (E. maggii) to 334 x 103/g rumen content (natural fauna). The murein concentration fluctuated between 180 and 277 mg/g dry matter (DM). The establishment of ciliates in the rumen of defaunated sheep decreased the murein content by 28-35% (P<0.05). Mureinolytic activity varied from 2.2 and 5.7 µg/g DM of rumen fluid/min and was the lowest in defaunated sheep and the highest in animals faunated with E. caudatum. The protozoa increased this activity from 32 (E. maggii) to 159% (E. caudatum). All examined parameters showed diurnal variations. The ciliate number was the greatest just before feeding and the smallest 4 h thereafter. The fluctuation pattern in murein content was inverse to that of protozoa concentration and mureinolytic activity.

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Engineering a cyanobacterial cell factory for production of lactic acid.

Angermayr, S. A., Paszota, M. & Hellingwerf, K. J. (2012). Applied and Environmental Microbiology, 78(19), 7098-7106.

Metabolic engineering of microorganisms has become a versatile tool to facilitate production of bulk chemicals, fuels, etc. Accordingly, CO2 has been exploited via cyanobacterial metabolism as a sustainable carbon source of biofuel and bioplastic precursors. Here we extended these observations by showing that integration of an ldh gene from Bacillus subtilis (encoding an L-lactate dehydrogenase) into the genome of Synechocystis sp. strain PCC6803 leads to L-lactic acid production, a phenotype which is shown to be stable for prolonged batch culturing. Coexpression of a heterologous soluble transhydrogenase leads to an even higher lactate production rate and yield (lactic acid accumulating up to a several-millimolar concentration in the extracellular medium) than those for the single ldh mutant. The expression of a transhydrogenase alone, however, appears to be harmful to the cells, and a mutant carrying such a gene is rapidly outcompeted by a revertant(s) with a wild-type growth phenotype. Furthermore, our results indicate that the introduction of a lactate dehydrogenase rescues this phenotype by preventing the reversion.

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Production of L-lactic acid from a green microalga, Hydrodictyon reticulum, by Lactobacillus paracasei LA104 isolated from the traditional Korean food, makgeolli.

Nguyen, C. M., Kim, J. S., Hwang, H. J., Park, M. S., Choi, G. J., Choi, Y. H., Jang, K. S. & Kim, J. C. (2012). Bioresource Technology, 110, 552-559.

The freshwater microalga, Hydrodictyon reticulum, that contained 47.5% reducing sugars including 35% glucose was used as substrate for the production of L-lactic acid (LA) by LA-producing bacteria. Lactobacillus paracasei LA104 was selected for fermentation in a 5-l fermentor since it was able to grow at pH 3, 60 g LA/l, 200 g glucose/l, 125 g NaCl/l, and 45°C and produced over 97.3% optically pure L-lactic acid with glucose as a substrate. Simultaneous saccharification and cofermentation from H. reticulum to L-LA using LA104 was investigated in a jar fermentor. The yield reached 46 g/100 g H. reticulum dry material, with a final concentration of 37.11 g/l and a productivity of 1.03 g/l/h. This is the first report of the production of L-LA from a microalga, and H. reticulum could be a potential feedstock for large-scale production of L-LA by LA104.

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D-Lactic acid production from dry biomass of Hydrodictyon reticulatum by simultaneous saccharification and co-fermentation using Lactobacillus coryniformis subsp. torquens.

Nguyen, C. M., Kim, J. S., Song, J. K., Choi, G. J., Choi, Y. H., Jang, K. S. & Kim, J. C. (2012). Biotechnology Letters, 34(12), 2235-2240.

D-Lactic acid production from dry biomass of the microalga, Hydrodictyon reticulatum, was carried out in a 5-l jar fermentor (initial pH 6, 34°C using CaCO3 as a neutralizing agent) through simultaneous saccharification and co-fermentation using the Lactobacillus coryniformis subsp. torquens. After 36 h, 36.6 g lactic acid/l was produced from 80 g H. reticulatum/l in the medium containing 3 g yeast extract/l and 3 g peptone/l in the absence of mineral salts. The maximum productivity, average productivity and yield were 2.38 g/l h, 1.02 g/l h and 45.8%, respectively. The optical purity of D-Lactic acid ranged from 95.8–99.6%. H. reticulatum is thus a promising biomass material for the production of D-Lactic acid.

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Modelling the Effect of Different Substrates and Temperature on the Growth and Lactic Acid Production by Lactobacillus amylovorus DSM 20531T in Batch Process.

Trontel, A., Baršić, V., Slavica, A., Santek, B. & Novak, S. (2010). Food Technology & Biotechnology, 48(3), 352-361.

Amylolytic lactic acid bacterium Lactobacillus amylovorus DSM 20531T utilised glucose, sucrose and starch as a sole carbon and energy source. The three substrates were completely depleted from MRS medium during batch cultivations carried out in a laboratory scale stirred tank bioreactor at constant temperature (40°C) and pH value (5.5). Under the tested conditions, the bacterium was capable of conducting simultaneously starch hydrolysis and fermentation. A mixture of two stereoisomers, D-(-)- and L-(+)-lactic acid, was produced in all cases by highly efficient homofermentative bioprocess with 0.93 to 1 g of lactate produced per g of total (consumed) substrate. The effect of temperature on the kinetics of cell growth and lactic acid production by the amylolytic strain in the starch-containing medium was also investigated. Efficient simultaneous saccharification and fermentation (SSF) was obtained at 35, 40 and 45°C with completely degraded complex carbohydrate in 8 to 12 h and the product yield coefficient in the range from 0.91 to 0.93 g/g. Maximum values for substrate consumption rate (0.89 h-1), maximum specific growth rate (0.87 h-1), product formation rate (2.01 h-1), and productivity of lactic acid (1.45 g/(Uh)) were obtained at 45°C, while maximum biomass concentration (4.38 g/L) was attained at 40°C. The ratio of the two stereoisomeric forms of produced lactic acid was strongly affected by the temperature. Unstructured kinetic model was used to describe the consumption of the three substrates, bacterial biomass formation and lactic acid production by L. amylovorus DSM 20531T. The dependence of biokinetic parameters on temperature was described by cardinal temperature model. The applied models successfully predicted all experimental data.

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Effect of thermal processing during yogurt production upon the detection of staphylococcal enterotoxin B.

Principato, M., Boyle, T., Njoroge, J., Jones, R. L. & O'Donnell, M. (2009). Journal of Food Protection®,72(10), 2212-2216.

This research was conducted to examine the inherent properties of yogurt contaminated with staphylococcal enterotoxin B (SEB). Two types of yogurts were produced for this study. Type I yogurts were produced by adding SEB at the start of yogurt production, and type II yogurts were produced by adding SEB after the milk base had been boiled. Biochemical characteristics inherent to yogurt, including pH, lactic acid and acetaldehyde concentrations, were analyzed weekly for each batch beginning at a time just after production and throughout a storage period of at least 4 weeks. The presence of toxin during yogurt production did not result in any significant biochemical or physical changes in yogurt. However, we were unable to detect SEB toxin in type I yogurt using a commercially available enzyme-linked immunosorbent assay (ELISA). In contrast, SEB was easily detectable by our ELISA in type II yogurt samples. Higher levels of SEB were recovered from type II yogurt that had been stored for 1 week than from type II yogurt that had been stored for any other length of time. These results indicate that the biochemical characteristics of yogurt did not change significantly (relative to control yogurt) in the presence of either thermally processed SEB or native SEB. However, the ability to detect SEB by ELISA was dependent on whether the toxin had been processed.

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Sourdough-leavened bread improves postprandial glucose and insulin plasma levels in subjects with impaired glucose tolerance.

Maioli, M., Pes, G. M., Sanna, M., Cherchi, S., Dettori, M., Manca, E. & Farris, G. A. (2008). Acta Diabetologica, 45(2), 91-96.

Sourdough bread has been reported to improve glucose metabolism in healthy subjects. In this study postprandial glycaemic and insulinaemic responses were evaluated in subjects with impaired glucose tolerance (IGT) who had a meal containing sourdough bread leavened with lactobacilli, in comparison to a reference meal containing bread leavened with baker yeast. Sixteen IGT subjects (age range 52–75, average BMI 29.9 ± 4.2 kg/m²) were randomly given a meal containing sourdough bread (A) and a meal containing the reference bread (B) in two separate occasions at the beginning of the study and after 7 days. Sourdough bread was leavened for 8 h using a starter containing autochthonous Saccharomyces cerevisiae and several bacilli able to produce a significant amount of D-and L-lactic acid, whereas the reference bread was leavened for 2 h with commercial baker yeast containing Saccharomyces cerevisiae. Plasma glucose and insulin levels were measured at time 0, 30, 60, 120, and 180 min. In IGT subjects sourdough bread induced a significantly lower plasma glucose response at 30 minutes (p = 0.048) and a smaller incremental area under curve (AUC) Δ 0–30 and Δ 0–60 min (p = 0.020 and 0.018 respectively) in comparison to the bread leavened with baker yeast. Plasma insulin response to this type of bread showed lower values at 30 min (p = 0.045) and a smaller AUC Δ 0–30 min (p = 0.018). This study shows that in subjects with IGT glycaemic and insulinaemic responses after the consumption of sourdough bread are lower than after the bread leavened with baker yeast. This effect is likely due to the lactic acid produced during dough leavening as well as the reduced availability of simple carbohydrates. Thus, sourdough bread may potentially be of benefit in subjects with impaired glucose metabolism.

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