Enhancement of asymmetric bioreduction of ethyl 4-chloro acetoacetate by the design of composition of culture medium and reaction conditions

This study investigated the feasibility of improving the stereospecificity of yeast by the adjustment of the culture medium composition and the reaction conditions. The investigation was performed systematically, using an approach that integrates the Taguchi's array method and the steepest ascent method. The reaction yield and the product's ee were the two indexes of reaction performance. A desirability function was applied to combine these two indexes as a single objective function. The removal of peptone and malt extract from the YM medium increased the yeast's stereoselectivity, without reducing the production of biomass. The medium composition and the reaction conditions were then simultaneously optimized. The resulting optimal conditions were 30 g/l glucose for cultivation, 12 g/l yeast extract, a cultivation time of 12 h, 15 g/l glucose for reaction, 150 g/l yeast for reaction, a reaction buffer concentration of 0.2 M and a buffer pH of 8.5. Compared to the one before this study, the product's ee was improved from 82.1 to 92.3%, and the reaction yield was enhanced from 77.3 to 82.3%. Furthermore, the biomass production was increased considerably from 5.94 to 10.14 g DCW/l.     

Inulinase overproduction by a mutant of the marine yeast Pichia guilliermondii using surface response methodology and inulin hydrolysis

In this study, in order to isolate inulinase overproducers from the marine yeast Pichia guilliermondii, its cells were treated by using UV light and LiCl. The mutant M-30 with enhanced inulinase production was obtained and was found to be stable after cultivation for 20 generations. Response surface methodology (RSM) was used to optimize the medium compositions and cultivation conditions for inulinase production by the mutant M-30 in liquid fermentation. Inulin, yeast extract, NaCl, temperature, pH for maximum inulinase production by the mutant M-30 were found to be 20.0 g/l, 5.0 g/l, 20.0 g/l, 28 °C and 6.5, respectively. Under the optimized conditions, 127.7 U/ml of inulinase activity was reached in the liquid culture of the mutant M-30 whereas the predicted maximum inulinase activity of 129.8 U/ml was derived from RSM regression. Under the same conditions, its parent strain only produced 48.1 U/ml of inulinase activity. This is the highest inulinase activity produced by the yeast strains reported so far. We also found that inulin could be actively converted into monosaccharides by the crude inulinase.     

Bioprocess development of the production of the mutant P-219-L human d-amino acid oxidase for high soluble fraction expression in recombinant Escherichia coli

In order to examine the structure–activity relationship and the substrate specificity of human d-amino acid oxidase (h.DAO), a single amino acid mutation had been established as proline-219-luecine (P-219-L). The gene encoding mutant h.DAO has been cloned and expressed in Escherichia coli BL21 (DE3). It was observed that the host cell was negatively affected by the expressed mutant h.DAO, resulting in a remarkable decrease in the cell growth and consequently the amount of the produced enzyme. To overcome this problem, we investigated several factors that may affect the cell growth rate and the mutant h.DAO production such as optimization of the glucose concentration as a main carbon source and the yeast extract concentration as a main nitrogen source, optimization of dissolved oxygen (DO%) concentration and the addition of benzyl alcohol (BA, which can artificially induce a strong heat shock response at low temperature), to enhance the production of natively folded soluble fraction of the recombinant protein. These parameters were tested on both shake flask level and fed-batch bioreactor level. The Western blot analysis and the enzyme activity assay indicated the higher level of the mutant expression towards enhancement of the conditions by using our designed approach.The specific activity (which was used as an indicator for the level of the desired protein produced = U/mg protein) and the OD_600 nm of the host cells (which was used as an indicator for the cell growth), reached to be 0.061 U/mg protein and 3.44, respectively upon using fed-batch culture system containing the optimized medium composition (15 g/l glucose and 5 g/l yeast extract). While upon using the shake flask level, these values were 0.032 and 1.1, respectively. Enhancement of the cell growth and the enzyme production was noticed after DO% optimization upon using 500 rpm agitation speed and 1.8 v.v.m. (volume volume minute) aeration. The specific activity for the mutant enzyme and the OD_600 nm of the host cells reached to be 0.14 U/mg protein and 7.1, respectively. Finally upon using the optimized culture composition (15 g/l glucose and 5 g/l yeast extract), optimized DO% (using 500 rpm agitation speed and 1.8 v.v.m.) and 0.1 mM BA at the fed-batch bioreactor level, the specific activity and the OD_600 nm of the host cells increased significantly to be 0.21 U/mg protein and 11.3, respectively at 24 h culture. These results indicate the importance of our approaches to overproducing mutant h.DAO in soluble form in E. coli.     

An economical approach for d-lactic acid production utilizing unpolished rice from aging paddy as major nutrient source

In order to reduce the raw material cost of d-lactic acid fermentation, the unpolished rice from aging paddy was used as major nutrient source in this study. The unpolished rice saccharificate, wheat bran powder and yeast extract were employed as carbon source, nitrogen source and growth factors, respectively. Response surface methodology (RSM) was applied to optimize the dosages of medium compositions. As a result, when the fermentation was carried out under the optimal conditions for wheat bran powder (29.10 g/l) and yeast extract (2.50 g/l), the d-lactic acid yield reached 731.50 g/kg unpolished rice with a volumetric production rate of 1.50 g/(l h). In comparison with fresh corn and polished rice, the d-lactic acid yield increased by 5.79% and 8.71%, and the raw material cost decreased by 65% and 52%, respectively, when the unpolished rice was used as a major nutrient source. These results might provide a reference for the industrial production of d-lactic acid.     

Effects of culture conditions on the mycelial growth and bioactive metabolite production in submerged culture of Cordyceps militaris

The influence of initial pH value, various nitrogen sources, plant oils, and modes of propagation (shake-flask and static culture) on the production of biomass, exopolysaccharide (EPS), adenosine and, in particular, cordycepin, by Cordyceps militaris CCRC 32219 were investigated. Optimal conditions for mycelial growth, EPS and cordycepin production were observed at relatively low pH. Amongst organic sources, yeast extract (YE) was favorable for EPS and cordycepin production, while corn steep powder (CSP) was favorable for adenosine production. A lower C/N ratio was favorable for adenosine and cordycepin production; however, too low a C/N ratio led to diminished production. All plant oils tested stimulate mycelial growth and EPS production of C. militaris, but they did not show much effect on the adenosine and cordycepin production. A two-stage fermentation process by combining shake-flask fermentation with static culture significantly enhanced cordycepin production. A Box–Behnken experimental design was employed to optimize the production of cordycepin, which showed that the optimum conditions to produce cordycepin by C. militaris CCRC 32219 were at pH 6, YE concentration of 45 g/l and 8.0 day of the shake culture followed by 16 days of the static culture. Under the optimized conditions, the maximum production (2214.5 mg/l) of cordycepin was obtained, which is much higher than those reported up to date.     

Characterization of the extracellular biodemulsifier of Bacillus mojavensis XH1 and the enhancement of demulsifying efficiency by optimization of the production medium composition

The demulsifying bacterium XH1 was identified as a Bacillus mojavensis by the 16S rDNA gene. The extracellular biodemulsifier produced by this species was purified by ethanol extraction and column chromatography through a sephadex and silicon gel column. Preliminary investigation using UV–vis and TLC indicated that the biodemulsifier had two components a protein and a lipopeptide. All major components of the medium, including the sources of soluble and insoluble carbon, nitrogen, phosphate, and metal ions were investigated to improve the biosynthesis and efficiency of the biodemulsifier. The optimal carbon sources were glucose and liquid paraffin. Glucose participated in the biosynthesis of the demulsifier, while liquid paraffin promoted the lipophilicity and secretion of biosurfactants. The absence of yeast extract, ammonium chloride or phosphate (K₂HPO₄/KH₂PO₄) had a negative effect on the production of the biodemulsifier and significantly inhibited its activity. To further enhance the biodemulsifier efficiency, the optimal medium composition was determined using the response surface methodology (RSM) based on the central composite rotation design (CCRD). Using the optimized biodemulsifier production medium: 8.5 g/l glucose; 3% (v/v) liquid paraffin; 1.5 g/l yeast extract; 3.36 g/l NH₄Cl and15 g/l phosphate, the demulsifying ratio increased 35.5% and biodemulsifier yield increased to 2.07 g/l.     

An engineering Escherichia coli mutant with high succinic acid production in the defined medium obtained by the atmospheric and room temperature plasma

Escherichia coli BA002, the ldhA and pflB deletion strain, cannot utilize glucose in nutrient-rich or minimal media anaerobically. Co-expression of heterologous pyruvate carboxylase and nicotinic acid phosphoribosyltransferase in BA002 resulted in a significant increase in cell mass and succinic acid production. Nevertheless, the resultant strain, BA016, still could not grow in a defined medium without tryptone and yeast extract. To solve the problem, a novel atmospheric and room temperature plasma mutation method was employed to generate mutants which can grow in the defined medium. A mutant designated as LL016 was observed to be the best strain that regained the capacity of cell growth and glucose utilization in a defined medium anaerobically. After 120 h of fermentation in the defined medium, 35.0 g/L of glucose was consumed to generate 25.2 g/L of succinic acid. Furthermore, with the highest glucose consumption rate in the dual-phase fermentation, the yield of succinic acid in LL016 reached 0.87 g/g, which was higher than that observed in other strains. From an industrial standpoint, the defined medium is much cheaper than LB medium, which shows a great potential usage for the economical production of succinic acid by LL016.     

Optimization of the medium for glutathione production in Saccharomyces cerevisiae

As a powerful statistical experimental design, uniform design (UD) method has been successfully applied in various fields such as fermentation industry, pharmaceuticals, and others. In this paper, UD was applied to optimize the medium composition for glutathione production in shake-flask culture of Saccharomyces cerevisiae T65. The experiments of nine factors (glucose, yeast extract, peptone, malt extract, molasses, MgSO₄, ZnSO₄, (NH₄)₂HPO₄ and thiamine) and nine levels were carried out according to the uniform design table U₂₇(9⁹). The experimental data was analyzed to obtain the regression model and the optimal medium composition was achieved by optimization with UD 3.0 software. The optimal medium consisted of 70 g/L glucose, 3 g/L yeast extract, 5 g/L peptone, 70 g/L malt extract, 20 g/L molasses, 5.6 g/L MgSO₄, 16 mg/L ZnSO₄, 7 g/L (NH₄)₂HPO₄ and 0.2 mg/L thiamine. The GSH yield at the optimal point achieved 74.6 mg/L, which was 1.81 times higher than that of the control. The application of UD method resulted in enhancement in GSH production.     

Efficient production of cordycepin by the Cordyceps militaris mutant G81-3 for practical use

Cordycepin is one of the most versatile metabolites of Cordyceps militaris. When C. militaris G81-3, the mutant obtained by a proton beam irradiation, was cultivated by liquid surface culture, cordycepin was found to crystallize in the medium due to high cordycepin concentration. Because the cordycepin crystals strongly attached to the mycelial mat, complete recovery of cordycepin was difficult. To prevent cordycepin crystallization, increase of the initial medium volume was examined to decrease the condensation rate by vaporization, in combination with decrease of the initial substrate level. Besides, addition of the water to the culture bottle was examined to cancel out the medium condensation. A 7.4 g/L of cordycepin was obtained without crystallization by the former method. The upper limit of cordycepin production, 14.3 g/L, was obtained by the latter method and this value was the highest level of all the previous reports. A 2.5-fold scale-up of the culture slightly declined the production rate, however, the amount of cordycepin production was properly 2.5 times higher than that of the small scale. In addition, differences in sensitivities to the substrates between the mutant and the wild-type strain are discussed.     

The effect of glucose and maltose concentrations on pyruvate and ascorbate production,antioxidant enzyme activities and LPO levels in Fusarium equiseti

Changes in pyruvate and ascorbate production and antioxidant enzyme activities together with lipid peroxidation levels in Fusarium equiseti were investigated in relation to changes in the concentrations of glucose and maltose as carbon sources in the range of 5–25 g/l in Armstrong Fusarium Medium (AFM). The highest pyruvate concentration obtained at 20 g/l maltose was 67.5 ± 0.69 μg/ml while ascorbic acid reached a maximum value at 25 g/l glucose of 1866±26.1 μg/ml The maximum superoxide dismutas (SOD) activities related to increased pyruvate production were determined in AFM medium containing 20 g/l glucose as 41.49±0.65 and maltose as 61.12±0.8 IU/mg. Catalase (CAT) activity variations showed coherence with SOD activity in a medium containing maltose and reached 219.11±2.8 IU/mg while they were decreased with increasing glucose concentration. glutathione peroxidase (GSH-Px) activities in F. equiseti did not change significantly with glucose and maltose concentration and were determined to be 1.21±0.22 and 1.67±0.15 IU/mg, respectively. Minimum lipid peroxidation levels for each carbon source were determined in both 20 g/l maltose and glucose concentrations as 0.9 and 1.62 nmol MDA/g wet weight.     

Medium optimization for the high yield production of single (+)-terrein by Aspergillus terreus strain PF26 derived from marine sponge Phakellia fusca

Terrein has potential application in the fields of medicine, cosmetology and agriculture, however, the chemical synthesis of terrein with single configuration is a difficult task, and the biosynthesis of terrein always results in low production (ca. 0.33–400 mg/L). In this study, we reported an Aspergillus terreus strain PF26 which could produce (+)-terrein on a high level. After the selection of a suitable basic medium, the component concentrations were optimized using Plackett–Burman design and response surface methodology. Consequently, an optimal medium containing 28.41 g glucose, 23.18 g maltose, 20.00 g mannitol, 8.52 g malt extract, 10.00 g monosodium glutamate 10.00 g NH₄Cl in 1 L ASW was obtained, and a high (+)-terrein production of 3.71 g/L fermentation broth was achieved, which represents the highest fermentation production of (+)-terrein to date. The result highlighted the industry's potential of A. terreus strain PF26 in the production of bioactive (+)-terrein on a large-scale.     

High-level production of erythritol by mutants of osmophilic Moniliella sp.

An erythritol-producing osmophilic yeast-like fungus, Moniliella sp. 440, was isolated from honey and then successively mutated with iterative rounds of N-methyl-N′-nitro-N-nitrosoguanidine (NTG) treatment and selection. Six generations of mutants, named N12115-6, N21105-6, N31074-3, N42208-2, N53199-9, and N61188-12, were selected for and produced erythritol at 151.0, 157.2, 177.8, 191.4, 196.6, and 237.8 g/L, respectively, while the wild type strain produced 113.0 g/L erythritol in media containing 40% glucose and 1% yeast extract. The mutant cells were found to have a short rod-like shape, while the wild type cells have a long rod-like shape. The most efficient erythritol producer, N61188-12, assimilated myo-inositol and weakly assimilated erythritol. However, the wild type strain did not assimilate myo-inositol and assimilated erythritol well. In 250-L and 2000-L pilot-scale fermentors, the erythritol production by N61188-12 was 151.4 g/L and 152.4 g/L, respectively. A simple fed-batch culture of strain N61188-12 in a 2000-L fermentor increased erythritol production to 189.4 g/L after 10 days fermentation.     

Variations of alcohol dehydrogenase activity and fermentative pyruvate,ethanol production of F. equiseti and F. acuminatum depend on the yeast extract and urea concentrations

In this study, pyruvate production of Fusarium equiseti was significantly increased when the yeast extract concentration was raised from 5 to 25 g/L while it was increased to only up to 10 g/L yeast extract in F. acuminatum. Upon supplementation with urea as an alternative nitrogen source, production of pyruvate for both of the Fusarium species were decreased with respect to increase in urea concentration in medium. On the other hand, ethanol production and alcohol dehydrogenase activity of F. equiseti were decreased approximately 1.9- and 1.6-fold with an increase in yeast concentration from 5 to 25 whereas the levels of F. acuminatum were increased 2.3- and 1.8-fold, respectively. In addition, ethanol productions and ADH activities in F. equiseti and F. acuminatum significantly increased on the 12th day up to 15 and 25 g/L urea concentrations, respectively. However, they were significantly decreased under these conditions at higher nitrogen sources. In addition, ethanol production and alcohol dehydrogenase activity in urea supplemented medium were higher than yeast extract supplemented. The results may suggest that the pyruvate, ethanol production and ADH enzyme activity variations and balance between aerobic and anaerobic respiration in F. equiseti and F. acuminatum were effected from yeast extract and urea concentrations in the nutrient medium.     

Continuous 2-keto-gluconic acid (2KGA) production from corn starch hydrolysate by Pseudomonas fluorescens AR4

A continuous fermentation process for 2-keto-gluconic acid (2KGA) production from cheap raw material corn starch hydrolysate was developed using the strain Pseudomonas fluorescens AR4. The dilution rate and feeding glucose concentration had a significant effect on the cell concentrations, glucose utilization and 2KGA production performance. The optimal operating factors were obtained as: 0.065 h⁻¹ of dilution rate, 180 g/L of feeding glucose concentration, and 16 h of batch fermentation time as the starting point. Under these conditions, the steady state had the 135.92 g/L of produced 2KGA concentration, 8.83 g/L.h of average volumetric productivity, and 0.9510 g/g of yield. In conclusion, the proposed efficient and stable continuous fermentation process for 2KGA production by the strain P. fluorescens AR4 is potentially competitive for industrial production from corn starch hydrolysate in terms of 2KGA productivity and yield.     

Dihydroxyacetone production in an engineered Escherichia coli through expression of Corynebacterium glutamicum dihydroxyacetone phosphate dephosphorylase

Dihydroxyacetone (DHA) has several industrial applications such as a tanning agent in tanning lotions in the cosmetic industry; its production via microbial fermentation would present a more sustainable option for the future. Here we genetically engineered Escherichia coli (E. coli) for DHA production from glucose. Deletion of E. coli triose phosphate isomerase (tpiA) gene was carried out to accumulate dihydroxyacetone phosphate (DHAP), for use as the main intermediate or precursor for DHA production. The accumulated DHAP was then converted to DHA through the heterologous expression of Corynebacterium glutamicum DHAP dephosphorylase (cghdpA) gene. To conserve DHAP exclusively for DHA production we removed methylglyoxal synthase (mgsA) gene in the ΔtpiA strain. This drastically improved DHA production from 0.83 g/l (0.06 g DHA/g glucose) in the ΔtpiA strain bearing cghdpA to 5.84 g/l (0.41 g DHA/g glucose) in the ΔtpiAΔmgsA double mutant containing the same gene. To limit the conversion of intracellular DHA to glycerol, glycerol dehydrogenase (gldA) gene was further knocked out resulting in a ΔtpiAΔmgsAΔgldA triple mutant. This triple mutant expressing the cghdpA gene produced 6.60 g/l of DHA at 87% of the maximum theoretical yield. In summary, we demonstrated an efficient system for DHA production in genetically engineered E. coli strain.     

Microbial levan from Brachybacterium phenoliresistens: Characterization and enhancement of production

Levan producing bacteria was isolated from rhizosphere soil. The molecular identification of this isolate was conducted using 16S rRNA, which resulted in a sequenced region of 1298 base pairs. The sequence alignment in the gene bank indicated that this isolate has a high percentage of similarity (99%) to the retrieved consensus sequence of Brachybacterium phenoliresistens strain phenol-A. The produced levan was characterized using TLC, FTIR, 1H NMR and 13C NMR spectroscopy techniques. The effects of nutritional and physical factors on this isolate’s levan production were investigated. The results demonstrated that the optimal sources for carbon and casein during levan production were sucrose and casein, yielding 7.88 g/land 8.12 g/l of levan, respectively. The highest levan yield (7.97 g/l) was obtained at a sucrose concentration of 300 g/l. At an initial pH of 7.8, this bacterium yielded their highest levan production of 7.88 g/l. The optimal incubation period was 72 h with a yield of 8.58 g/l, the optimal temperature was 30 °C and resulted in 7.87 g/l, and the highest levan production yield was obtained at 150 rpm and yielded 8.12 g/l.     

Alkaline lipase from a novel strain Burkholderia multivorans: Statistical medium optimization and production in a bioreactor

An alkaline lipase from Burkholderia multivorans was produced within 15 h of growth in a 14 L bioreactor. An overall 12-fold enhanced production (58 U mL⁻¹ and 36 U mg⁻¹ protein) was achieved after medium optimization following the “one-variable-at-a-time” and the statistical approaches. The optimal composition of the lipase production medium was determined to be (% w/v or v/v): KH₂PO₄ 0.1; K₂HPO₄ 0.3; NH₄Cl 0.5; MgSO₄·7H₂O 0.01; yeast extract 0.36; glucose 0.1; olive oil 3.0; CaCl₂ 0.4 mM; pH 7.0; inoculum density 3% (v/v) and incubation time 36 h in shake flasks. Lipase production was maximally influenced by olive oil/oleic acid as the inducer and yeast extract as the additive nitrogen. Plackett–Burman screening suggested catabolite repression by glucose. Amongst the divalent cations, Ca²⁺ was a positive signal while Mg²⁺ was a negative signal for lipase production. RSM predicted that incubation time, inoculum density and oil were required at their higher levels (36 h, 3% (v/v) and 3% (v/v), respectively) while glucose and yeast extract were required at their minimal levels for maximum lipase production in shake flasks. The production conditions were validated in a 14 L bioreactor where the incubation time was reduced to 15 h.     

Effect of lignocellulose degradation products on microbial biomass and lipid production by the oleaginous yeast Cryptococcus curvatus

This work investigated effects of lignocellulose degradation products on cell biomass and lipid production by Cryptococcus curvatus. Furfural was found to have the strongest inhibitory effect. For the three phenolic compounds tested, vanillin was the most toxic, while PHB and syringaldehyde showed comparable inhibitions in the concentration range of 0–1.0 g/L. Generally little significant differences on the relative cell biomass and lipid contents at the same concentrations of tested compounds were observed between glucose and xylose as a sole carbon source. At 1.0 g/L of furfural, the cell biomass and lipid content decreased by 78.4% and 61.0% for glucose as well as 72.0% and 59.3% for xylose, respectively. C. curvatus ceased to grow at concentrations of PHB over 1.0 g/L or vanillin over 1.5 g/L. The strain could survive in the presence of syringaldehyde up to 2.0 g/L for glucose or 1.5 g/L for xylose. The compounds’ negative impact was reduced by an increase in inoculum size and a 10% (v/v) seed was detected to be optimal for cell biomass and lipid production. The results demonstrated C. curvatus could effectively utilize most of the dominant monosaccharides and cellobiose existing in lignocellulosic biomass hydrolysate in the presence of toxic compounds.     

Rapid ethanol production at elevated temperatures by engineered thermotolerant Kluyveromyces marxianus via the NADP(H)-preferring xylose reductase-xylitol dehydrogenase pathway

Conversion of xylose to ethanol by yeasts is a challenge because of the redox imbalances under oxygen-limited conditions. The thermotolerant yeast Kluyveromyces marxianus grows well with xylose as a carbon source at elevated temperatures, but its xylose fermentation ability is weak. In this study, a combination of the NADPH-preferring xylose reductase (XR) from Neurospora crassa and the NADP⁺-preferring xylitol dehydrogenase (XDH) mutant from Scheffersomyces stipitis (Pichia stipitis) was constructed. The xylose fermentation ability and redox balance of the recombinant strains were improved significantly by over-expression of several downstream genes. The intracellular concentrations of coenzymes and the reduced coenzyme/oxidized coenzyme ratio increased significantly in these metabolic strains. The byproducts, such as glycerol and acetic acid, were significantly reduced by the disruption of glycerol-3-phosphate dehydrogenase (GPD1). The resulting engineered K. marxianus YZJ088 strain produced 44.95 g/L ethanol from 118.39 g/L xylose with a productivity of 2.49 g/L/h at 42 °C. Additionally, YZJ088 realized glucose and xylose co-fermentation and produced 51.43 g/L ethanol from a mixture of 103.97 g/L xylose and 40.96 g/L glucose with a productivity of 2.14 g/L/h at 42 °C. These promising results validate the YZJ088 strain as an excellent producer of ethanol from xylose through the synthetic xylose assimilation pathway.     

Application of response surface methodology for glucan production from Leuconostoc dextranicum and its structural characterization

Sequential optimization strategy based on statistical experimental designs was employed to enhance glucan production by Leuconostoc dextranicum NRRL B-1146 in flask culture. A two-level Plackett–Burman design was employed first where 11 variables were studied for their influence on glucan production. Sucrose, peptone and yeast extract were the most significant variables improving glucan production. A three-level Box–Behnken factorial design was employed for maximizing the glucan production. A mathematical model was developed to show the effects of each medium component and their combinatorial interactions on glucan production. The optimal medium composition for maximum glucan production was sucrose 5.95%, peptone 0.52% and yeast extract 2.9%. This composition predicted 1063 mg/l glucan, the experimentally found glucan was 1015 ± 4.5 mg/l that showed a good agreement with the predicted value. The purified glucan was homogenous and its structural characteristics investigated by FT-IR, ¹H NMR and ¹³C NMR spectroscopic techniques showed that it contained α-(1 → 6) and α-(1 → 4) linkages.