Fermentation of brined turnip roots using Lactobacillus plantarum and Leuconostoc mesenteroides starter cultures

The controlled fermentation of turnip slices using Lactobacillus plantarum or Leuconostoc mesenteroides as starter cultures led to earlier acid production and earlier and more pronounced inhibition of Enterobacteriaceae than with uninoculated (natural) fermentation. Unlike the natural fermentation, the controlled fermentations did not show a yeast secondary fermentation and also had a better colour. Due to its ability to produce higher amounts of acid, the use of Lact. plantarum is more desirable than of Leuc. mesenteroides.     

Production and properties of a dextransucrase from Leuconostoc mesenteroides IBT-PQ isolated from ‘pulque’, a traditional Aztec alcoholic beverage

Dextransucrase was produced from a Leuconostoc mesenteroides isolated from pulque, a traditional Aztec alcoholic beverage produced from agave juice containing sucrose as the main carbon source. Almost all the dextransucrase activity (87%) was associated with the cells, and was unusually high (1.04 U mg⁻¹ of cells). The culture medium composition was optimized through a Box-Behnken method resulting in a process yielding 2.2 U ml⁻¹ of insoluble glucosyltransferase activity. The enzyme had a molecular weight of 166 kDa. Optimal temperature was 35°C with a half-life of 137 min at the same temperature. As with dextransucrase from the industrial strain L. mesenteroides NRRL B-512F, the enzyme showed Michaelis–Menten kinetic behavior with excess substrate inhibition (K _m and K _i values of 0.026 M and 1.23 M respectively); produced soluble linear dextran with glucose molecules linked mainly in α(1–6) with branching in α(1–3) in a proportion of 4:1 as shown by NMR studies; and produced a high yield of isomalto-oligosaccharides in the presence of maltose. Received 4 February 1998/ Accepted in revised form 25 July 1998     

The production of the enzyme dextransucrase using nonaerated fermentation techniques

High yields of the enzyme dextransucrase have been produced repeatedly by fed-batch fermentation techniques. Activities in excess of 21.9 U/cm(3) have been obtained by culturing Leuconostoc mesenteroides NRRL B-512(F) under nonaerated fed-batch fermentation conditions. Aerobic fermentations carried out under identical conditions have consistently produced enzyme of less than 17 U/cm(3), but with no difference in the final cell concentration in the broth. Different types of yeast extract have been found to have significant effect on the final cell concentration and more especially on the enzyme activity with enzyme yields varying by as much as 50% when different types of yeast extracts were used.     

Investigation of production of dextran and dextransucrase by Leuconostoc mesenteroides immobilized within porous stainless steel

Cells of Leuconostoc mesenteroides were immobilized within porus, stainless-steel (SS) supports and used for dextransucrase (DS) and dextran production. The pore size of the support significantly affected the dextran yields, which were greatest with average pore sizes of 2-5 mum. All immobilized-cell biocatalysts in porous stainless steel produced higher yields than free cells, with the exception of cells confined in submicrometer pores (0.5 mum). Coating supports of larger pore size (40 and 100 mum) with calcium alginate enhanced the cell-loading capacity of the supports and increased dextran and fructose yields in the cell-free broth. Controlled, fed-batch, DS production (activation), as a step preliminary to dextran production, significantly improved the subsequent dextran and fructose yields and shortened the time required to attain the maximum such yields. Scanning electron microscopy (SEM) of immobilized L. mesenteroides in stainless steel shows an irregular pattern of the microorganism inside the pores of the solid supports. Coating the porous solid supports with a cell-free calcium alginate layer led to an increase in the cell density inside the support. Cell growth inside the coated, porous stainless steel had no distinct growth form. (c) 1992 John Wiley & Sons, Inc.     

Inactivation of Leuconostoc Mesenteroids NRRL B-512F Dextransucrase by Specific Modification of Lysine Residues with Pyridoxal-5′-Phosphate

Abstract

Dextransucrase from Leuconostoc mesentwoides NRRL B-512F was inactivated by pyridoxal-5′-phosphate (PLP). The inactivation was reversible in as much as the loss of enzyme activity was completely reversed by prolonged dialysis. PLP-modified dextransucrase after reduction with sodium borohydride showed a characteristic fluorescence emission maximum at 397 nm when excited at 325 nm. The stoichiometric results indicated that four lysine residues are modified by PLP under the experimental conditions. These results established for the first time that lysine residues are essential for the activity of dextransucrase.     

Synthesis, structural analysis and application of novel acarbose-fructoside using levansucrase

Acarbose-fructoside (acarbose-Fru) was newly synthesized via the acceptor reaction of a levansucrase from Leuconostoc mesenteroides B-512 FMC with acarbose and sucrose. The resultant product was separated with 10.5% purification yield via Bio-gel P-2 column chromatography and HPLC. Its structure was determined to be 1^I-β-d-fructofuranosyl α-acarbose, according to the results of ¹H, ¹³C, HSQC, and HMBC analyses. Acarbose-Fru was inhibited competitively on α-glucosidase (A. niger and baker's yeast) but mixed noncompetitively on α-amylases (A. oryzae and porcine pancreatic). Compared to acarbose, acarbose-Fru exhibited inhibition potency of 1.12 or 1.52 on A. niger α-glucosidase or A. oryzae α-amylase, respectively. Additionally, acarbose-Fru was identified as a novel substrate for dextransucrase with K_m and V_max values of 189.0 mM and 8.51 μmol/(mg min), respectively. Therefore, acarbose-Fru as a substrate might be synthesized novel acarbose derivatives by using dextransucrase.     

Synthesis and characterization of hydroquinone fructoside using <Emphasis Type="Italic">Leuconostoc mesenteroides</Emphasis> levansucrase

Hydroquinone (HQ) functions as a skin-whitening agent, but it has the potential to cause dermatitis. We synthesized a HQ fructoside (HQ-Fru) as a potential skin-whitening agent by reacting levansucrase from Leuconostoc mesenteroides with HQ as an acceptor and sucrose as a fructofuranose donor. The product was purified using 1-butanol partition and silica-gel column chromatography. The structure of the purified HQ-Fru was determined by ¹H and ¹³C nuclear magnetic resonance, and the molecular ion of the product was observed at m/z 295 (C12 H16 O7 Na)⁺. The HQ-Fru was identified as 4-hydroxyphenyl-β-d-fructofuranoside. The optimum condition for HQ-Fru synthesis was determined using a response surface method (RSM), and the final optimum condition was 350 mM HQ, 115 mM sucrose, and 0.70 U/ml levansucrase, and the final HQ-Fru produced was 1.09 g/l. HQ-Fru showed anti-oxidation activities and inhibition against tyrosinase. The median inhibition concentration (IC₅₀) of 1,1-diphenyl-2-picrylhydrazyl scavenging activity was 5.83 mM, showing higher antioxidant activity compared to β-arbutin (IC₅₀ = 6.04 mM). The K _i value of HQ-Fru (1.53 mM) against tyrosinase was smaller than that of β-arbutin (K _i  = 2.8 mM), indicating that it was 1.8-times better as an inhibitor. The inhibition of lipid peroxidation by HQ-Fru was 105.3% that of HQ (100%) and 118.9 times higher than that of β-arbutin (0.89% of HQ).     

Leuconostoc mesenteroides glucansucrase synthesis of flavonoid glucosides by acceptor reactions in aqueous-organic solvents

The enzymatic glucosylation of luteolin was attempted using two glucansucrases: the dextransucrase from Leuconostoc mesenteroides NRRL B-512F and the alternansucrase from L. mesenteroides NRRL B-23192. Reactions were carried out in aqueous-organic solvents to improve luteolin solubility. A molar conversion of 44% was achieved after 24h of reaction catalysed by dextransucrase from L. mesenteroides NRRL B-512F in a mixture of acetate buffer (70%)/bis(2-methoxyethyl) ether (30%). Two products were characterised by nuclear magnetic resonance (NMR) spectroscopy: luteolin-3'-O-alpha-d-glucopyranoside and luteolin-4'-O-alpha-d-glucopyranoside. In the presence of alternansucrase from L. mesenteroides NRRL B-23192, three additional products were obtained with a luteolin conversion of 8%. Both enzymes were also able to glucosylate quercetin and myricetin with conversion of 4% and 49%, respectively.     

Studies on Cell Wall of Alkalophilic Bacillus

Cell walls of alkalophilic Bacillus No. C-125 and No. A-59 which grew in different pH conditions were prepared and analyzed. In the walls from cells grown at pH 10.3 (pH 10.3-cell wall) and the walls from cells grown at pH 7.5 (pH 7.5-cell wall) of the alkalophilic bacilli, the contents of neutral sugar and phosphorus were low as compared with those of Bacillus subtilis 6160, while uronic acid and amino acids were abundant. The uronic acid content of the pH 10.3-cell walls was higher than that of the pH 7.5-cell walls in both strains. The insoluble fraction (peptidoglycan) of cell walls of Bacillus No. C-125 consisted of muramic acid, glutamic acid, alanine, diaminopimelic acid and glucosamine as in neutrophilic bacilli. In the TCA soluble fraction of pH 10.3-cell walls of Bacillus No. C-125, uronic acid was a polymer of glucuronic acid containing a small amount of hexosamine, and 2/3 of the ninhydrin positive material was glutamic acid which was derived mainly from poly γ-L-glutamic acid.     

Continuous Production of Dextran from Immobilized Cells of <Emphasis Type="Italic">Leuconostoc mesenteroides</Emphasis> KIBGE HA1 Using Acrylamide as a Support

The cells of L. mesenteroides KIBGE HA1 were immobilized for the production of dextran on acrylamide gel and gel concentration was optimized for maximum entrapment. Sucrose at substrate concentration of 10% produced high yield of dextran at 25°C with a percent conversion of 5.82 while at 35°C it was 3.5. However, increasing levels of sucrose diminished dextran yields. The free cells stopped producing dextran after 144 h, while immobilized cells continued to produce dextran even after 480 h. Molecular mass distribution of dextran from free cells indicate that it is identical to that of blue dextran while the molecular mass of dextran from immobilized cells is lower than that of free cells.