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.
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.
In vitro and in vivo protective effects of fermented preparations of dietary herbs against lipopolysaccharide insult.
Bose, S., Song, M. Y., Nam, J. K., Lee, M. J. & Kim, H. (2012). Food Chemistry, 134(2), 758-765.
Lipopolysaccharide (LPS) is known to produce endotoxic shock by triggering systemic inflammatory responses. Here, we evaluated the protective effects of three fermented/re-fermented herbs, Rhizoma Atractylodis Macrocephalae, Massa Medicata Fermentata, and Dolichoris Semen, in an LPS-mediated inflammatory insult, either individually in vitro using RAW264.7 cells or in combination in in vivo using rats. In general, each of the fermented herbs showed appreciable in vitro anti-inflammatory activity, although the degree of this activity varied with the herb used. Moreover, a mixture of fermented herbal extracts in combination with probiotics significantly attenuated the blood endotoxin and CRP levels, as well as the gut permeability, and significantly augmented the intestinal Lactobacillus spp. colonisation in LPS-treated rats. However, these effects were not observed following the administration of the corresponding mixture of unfermented herbal extracts. Thus, our results highlight the beneficial impacts of the use of fermented herb products with probiotics to combat LPS-mediated inflammatory insults.
Botrytis cinerea isolates collected from grapes present different requirements for conidia germination.
Cotoras, M., Garcia, C. & Mendoza, L. (2009). Mycologia, 101(3), 287-295.
Botrytis cinerea presents high variability in several biological traits, which can be explained by the high degree of genotypic diversity among isolates. Because this genetic variability might be related to phenotypic differences the requirements for conidia germination of three natural isolates (G1, G5 and G11) obtained from grapes and belonging to the same genetic group were analyzed. The results showed that contact with a solid surface was a common requisite for conidia germination of the isolates but they differed in their nutritional requirements to germinate. Isolate G11 was able to germinate in the absence of a carbon or nitrogen source. G1 and G5 required the presence of a carbon source such as glucose, fructose or sucrose. In G11 and G5 isolates a much higher rate of germination was obtained in the presence of sucrose. It was shown with a pharmacological approach that the cAMP stimulated the germination only in those isolates requiring a carbon source. Conidia germination of G1 and G5 was inhibited by EGTA, a calcium chelator. Isolate G11 germinated in the presence of this compound. On the other hand the germination of three B. cinerea isolates required protein synthesis and did not require RNA synthesis. To explain the ability of isolate G11 to germinate in water the content of total and reducing sugars, mannitol/L-arabitol, trehalose, and proteins in the nongerminated conidia of the three isolates was compared. The isolates presented similar amounts of total and reducing sugars. In the three isolates the amount of mannitol/L-arabitol was higher than that of trehalose. In isolate G11 total protein content was twice higher than in the other isolates.
The effects of co-administration of probiotics with herbal medicine on obesity, metabolic endotoxemia and dysbiosis: A randomized double-blind controlled clinical trial.
Lee, S. J., Bose, S., Seo, J. G., Chung, W. S., Lim, C. Y. & Kim, H. (2013). Clinical Nutrition, 33(6), 973-981.
Backgrounds & aims: Probiotics help maintain balance in composition of the gut microbiota, and have been considered as a potential treatment for obesity. This study was conducted in order to assess the effects of probiotics when combined with herbal medicine in treatment of obesity. Probiotics were tested for the ability to modulate gut microbiota, gut permeability, and endotoxin level, which may have correlation with factors involved in obesity. Methods: A randomized, double-blind, placebo controlled study was conducted, in which patients with higher BMI (>25 kg/m2) and waist circumference (>85 cm) were enrolled and randomly assigned to receive Bofutsushosan with either probiotics or placebo capsules for a period of eight weeks. Assessment of body composition parameters, metabolic biomarkers, endotoxin level, gut permeability, and fecal bacteria in stool was performed at baseline and at week 8. The study was registered at the Clinical Research Information Service, approved by the Korea National Institute of Health (KCT0000386). Results: Although both groups showed a significant reduction in weight and waist circumference (p= 0.000), no significant differences in body composition and metabolic markers were observed. In correlation analysis, change in body composition showed positive correlation with endotoxin level (r= 0.441, p< 0.05 for BW; and r= 0.350, p< 0.05 for fat mass) and the population of gut Lactobacillus plantarum (r= 0.425, p< 0.05 for BW; and r= 0.407, p< 0.05 for BMI). The Gram negative bacterial population in gut also exhibited positive correlation with changes in body composition (WC) and total cholesterol level (r= 0.359, and 0.393, for the former and later parameters, respectively, p< 0.05 for both). While, the profile of gut Bifidobacterium breve population showed negative correlation with endotoxin level (r= −0.350, p< 0.05). Conclusions: Correlations between gut microbiota and change in body composition indicate that probiotics may influence energy metabolism in obesity. Correlation between endotoxin level and weight reduction indicates that probiotics may play an important role in prevention of endotoxin production, which can lead to gut microbiota dysbiosis associated with obesity.
Engineering of photosynthetic mannitol biosynthesis from CO2 in a cyanobacterium.
Jacobsen, J. H. & Frigaard, N. U. (2014). Metabolic Engineering, 21, 60-70.
D-Mannitol (hereafter denoted mannitol) is used in the medical and food industry and is currently produced commercially by chemical hydrogenation of fructose or by extraction from seaweed. Here, the marine cyanobacterium Synechococcus sp. PCC 7002 was genetically modified to photosynthetically produce mannitol from CO2 as the sole carbon source. Two codon-optimized genes, mannitol-1-phosphate dehydrogenase (mtlD) from Escherichia coli and mannitol-1-phosphatase (mlp) from the protozoan chicken parasite Eimeria tenella, in combination encoding a biosynthetic pathway from fructose-6-phosphate to mannitol, were expressed in the cyanobacterium resulting in accumulation of mannitol in the cells and in the culture medium. The mannitol biosynthetic genes were expressed from a single synthetic operon inserted into the cyanobacterial chromosome by homologous recombination. The mannitol biosynthesis operon was constructed using a novel uracil-specific excision reagent (USER)-based polycistronic expression system characterized by ligase-independent, directional cloning of the protein-encoding genes such that the insertion site was regenerated after each cloning step. Genetic inactivation of glycogen biosynthesis increased the yield of mannitol presumably by redirecting the metabolic flux to mannitol under conditions where glycogen normally accumulates. A total mannitol yield equivalent to 10% of cell dry weight was obtained in cell cultures synthesizing glycogen while the yield increased to 32% of cell dry weight in cell cultures deficient in glycogen synthesis; in both cases about 75% of the mannitol was released from the cells into the culture medium by an unknown mechanism. The highest productivity was obtained in a glycogen synthase deficient culture that after 12 days showed a mannitol concentration of 1.1 g mannitol L-1 and a production rate of 0.15 g mannitol L-1 day-1. This system may be useful for biosynthesis of valuable sugars and sugar derivatives from CO2 in cyanobacteria.
Hepatoprotective effect of Lentinus edodes mycelia fermented formulation against alcoholic liver injury in rats.
Chung, W. S., Wang, J. H., Bose, S., Park, J. M., Park, S. O., Lee, S. J., Jeon. S. & Kim, H. (2015). Journal of Food Biochemistry, 39(3), 251-262.
The hepatoprotective effects of fermented black rice bran extracts (FF1 and FF2: black rice bran fermented by Lentinus edodes derived from mycelium supplemented with soybean or Hovenia dulcis) and their associated mechanisms were evaluated. In an in vitro experiment, FFs caused significant amelioration of the metabolic function of rat hepatocytes treated with NH4Cl. In addition, administration of FFs to rats with chronic liver injury induced by 12-week continual alcohol consumption resulted in significant restoration of body weight shrinkage, notable attenuation of excessive aspartate aminotransferase, alkaline phosphatase and endotoxin in serum, malondialdehyde in liver and the lactulose/mannitol ratio in urine. Furthermore, FF1 or FF2 also caused significant downregulation of gene expression of several critical inflammatory mediators (interleukin-6, tumor necrosis factor-alpha, cyclooxygenase-2 and inducible nitric oxide synthase). Histopathological findings also indicated that FFs reduced inflammation, necrosis and fatty infiltration in liver tissue. Taken together, FFs exert hepatoprotective effects through anti-inflammatory and anti-lipid peroxidative properties and regulation of intestinal permeability.
Strategic optimization of xylanase–mannanase combi-CLEAs for synergistic and efficient hydrolysis of complex lignocellulosic substrates.
Bhattacharya, A. & Pletschke, B. I. (2015). Journal of Molecular Catalysis B: Enzymatic, 115, 140-150.
Cost-effective application of lignocellulolytic enzymes holds the key towards commercialization of enzymatic hydrolysis of lignocellulosic biomass. Carrier free immobilization of enzyme(s) offers a lucrative prospect. Combined-cross linked enzyme aggregates (combi-CLEAs) are a novel prospective and this present study addresses the preparation, characterization and application of xylanase–mannanase combi-CLEAS on lime-preteated sugarcane bagasse and milled corn stover. X6-CLEAs, X7-CLEAs, L1-CLEAs and L7-CLEAs were prepared after elaborative optimization of the precipitating agent and glutaraldehyde concentration. The highest activity after precipitation was observed with acetone but following cross-linking with glutaraldehyde less than 60% activity was retained, while more than 60% activity was retained after precipitation with ammonium sulphate and cross-linking with glutaraldehyde. Accessory enzyme activities including α-arabinofuranosidase, β-xylosidase, esterases, β-mannosidase, α-galactosidase and β-glucosidase were also determined. More than an 1.5 fold increase in thermostability compared to the free enzyme was observed over a broad temperature range (50–70°C). Tri-synergy studies and quad synergy studies were used to generate combi-CLEAs with different protein ratios. Hydrolysis of lime pre-treated bagasse with combi-CLEAs at protein ratios corresponding to X6 (33.0%):X7 (17.0%):L1 (17.0%):L7 (33.0%) resulted in a 1.68 fold higher sugar release compared to the quad synergy model using free enzymes. Similarly, hydrolysis of corn stover with combi-CLEAs at protein ratios corresponding to X6 (40.0%):X7 (10.0%):L1 (10.0%):L7 (40.0%) resulted in an 1.58 fold higher sugar release compared to the sugar release observed with the quad synergy model using free enzymes. Monomeric sugars constituted 70–75% of reducing sugars released during hydrolysis. The role of accessory enzymes in improving enzyme synergy was clearly shown. The efficiency of combi-CLEAs compared to free enzymes makes them ideal candidates for the prudent and cost-effective commercialization of lignocellulolytic enzymes.