Apinun Kanpiengjai

High efficacy bioconversion of starch to lactic acid using an amylolytic lactic acid bacterium isolated from Thai indigenous fermented rice noodles

Amylolytic lactic acid bacterium (ALAB) strain S21 was isolated from Thai indigenous fermented rice noodles and identified as Lactobacillus plantarum, based on 16S rDNA sequence and recA gene analysis. L. plantarum S21 exhibited a specific growth rate (μ) of 0.24 1/h in modified MRS broth containing 10 g/L of starch as the sole carbon source, and a high efficacy in producing lactic acid (9.41, 24.48, 41.84, 74.33, and 94.04 g/L from 10, 25, 50, 75, and 100 g/L of cassava starch, respectively), which are higher values than previously reported for ALAB. Crude amylase from L. plantarum S21 had broad pH stability (3.5–8.0), and hydrolyzed starch to maltose and glucose as the major and minor products. L. plantarum S21 should be considered useful for industrial bioconversion of starch to lactic acid.

Distribution of tannin-'tolerant yeasts isolated from Miang, a traditional fermented tea leaf (Camellia sinensis var. assamica) in northern Thailand.

Miang is a fermented food product prepared from the tea leaves of Camellia sinensis var. assamica, and is traditionally produced in mountainous areas of northern Thailand. Although Miang has a long history and reveals deep-rooted cultural involvement with local people in northern Thailand, little is known regarding its microbial diversity. Yeasts were isolated from 47 Miang samples collected from 28 sampling sites, including eight provinces in upper northern Thailand. A hundred and seven yeast isolates were recovered and identified within 14 species based on the comparison of the D1/D2 sequence of the large subunit (LSU) rRNA gene. Candida ethanolica was determined to be the dominant species that was frequently found in Miang together with minor resident yeast species. All yeast isolates demonstrated their tannin-tolerant capability when cultivated on yeast malt agar (YMA) containing 50g/l tannin, but nine isolates displayed clear zones forming around their colonies, e.g., Debaryomyces hansenii, Cyberlindnera rhodanensis, and Sporidiobolus ruineniae. The results obtained from a visual reading method of tannase revealed that all yeast isolates were positive for methyl gallate, indicating that they possess tannase activity. It is assumed that a tannin-tolerant ability is one of the most important factors for developing a yeast community in Miang. This research study is the first report to describe tannin-tolerant yeasts and yeast communities in traditionally fermented tea leaves.

Molecular structure of cyclomaltodextrinase derived from amylolytic lactic acid bacterium Enterococcus faecium K-1 and properties of recombinant enzymes expressed in Escherichia coli and Lactobacillus plantarum

Gene encoding cyclomaltodextrinase (Cdx) from amylolytic lactic acid bacterium Enterococcus faecium K-1 was cloned and nucleotide sequence was analyzed. The open-reading frame consisted of 1767bp encoding 588 deduced amino acids. Consequently, four typically conserved regions of the glycoside hydrolase family 13 were revealed; however, nine exceeding amino acids (DSYQMTDVP) were found at the 282-290 position in comparison to previously reported cyclomaltodextrinases. This difference is believed to have an influence on the substrate specificity of this enzyme. The recombinant CDases expressed in Escherichia coli BL21 (CDX_E) and Lactobacillus plantarum WCFS1 (CDX_L) with high expression levels of 8041 and 5511U/L were purified by Ni-NTA affinity chromatography. The active form CDX is a dimeric protein with two identical subunits of 62kDa, approximately. Both CDX_E and CDX_L revealed nearly similar properties, but the thermostability of CDX_L was slightly higher. Mn2+ and Co2+ at a concentration of 1mM stimulated the enzyme activity, while the Ag+, Cu2+ and SDS solution completely inhibited enzyme activity. CDX exhibited the highest activity with α-cyclodextrin and β-cyclodextrin, but lower toward pullulan and starch. Importantly, this is the first report describing genes, the molecular structure and properties of cyclomaltodextrinase derived from lactic acid bacteria E. faecium.

Amylolytic Enzymes Acquired from L-Lactic Acid Producing Enterococcus faecium K-1 and Improvement of Direct Lactic Acid Production from Cassava Starch

An amylolytic lactic acid bacterium isolate K-1 was isolated from the wastewater of a cassava starch manufacturing factory and identified as Entercoccus faecium based on 16S rRNA gene sequence analysis. An extracellular α-amylase was purified to homogeneity and the molecular weight of the purified enzyme was approximately 112xa0kDa with optimal pH value and temperature measured of 7.0 and 40xa0°C, respectively. It was stable at a pH range of 6.0–7.0, but was markedly sensitive to high temperatures and low pH conditions, even at a pH value of 5. Ba2+, Al3+, and Co2+ activated enzyme activity. This bacterium was capable of producing 99.2% high optically pure L-lactic acid of 4.3 and 8.2xa0g/L under uncontrolled and controlled pH at 6.5 conditions, respectively, in the MRS broth containing 10xa0g/L cassava starch as the sole carbon source when cultivated at 37xa0°C for 48xa0h. A control pH condition of 6.5 improved and stabilized the yield of L-lactic acid production directly from starch even at a high concentration of starch at up to 150xa0g/L. This paper is the first report describing the properties of purified α-amylase from E. faecium. Additionally, pullulanase and cyclodextrinase activities were also firstly recorded from E. faecium K-1.

Starchy effluent from rice noodle manufacturing process as feasible substrate for direct lactic acid production by Lactobacillus plantarum S21

In utilization of both starch containing wastewater and gelatinized starchy waste, Lactobacillus plantarum S21 demonstrated the high capability of lactic acid production directly from starchy effluent and maintained its potency even at high concentration of initial starchy substrate of 40, 60 and 80 g/L by maximum yielding 1.00±0.06, 0.89±0.03, 0.90±0.07 g/g substrate, productivity of 0.79±0.06, 0.98±0.00, 1.23±0.07 g/L·h and production efficiency of 94.6, 78.8, and 74.3%, respectively, at 48 h fermentation. This is the first report on direct conversion of starchy wastes to lactic acid by amylolytic lactic acid bacterium using high concentration of starchy substrate.

Effective enhancement of polylactic acid-degrading enzyme production by Amycolatopsis sp. strain SCM_MK2-4 using statistical and one-factor-at-a-time approaches

ABSTRACT This study aims to find the optimal medium and conditions for polylactic acid (PLA)-degrading enzyme production by Amycolatopsis sp. SCM_MK2-4. Screening of the most effective components in the enzyme production medium by Plackett–Burman design revealed that the silk cocoon and PLA film were the most significant variables enhancing the PLA-degrading enzyme production. After an response surface methodology, a maximum amount of PLA-degrading enzyme activity at 0.74u2009Uu2009mL−1 was predicted and successfully validated at 95% after 0.39% (w/v) silk cocoon and 1.62% (w/v) PLA film were applied to the basal medium. The optimal initial pH value, temperature, and inoculum size were evaluated by a method considering one-factor-at-a-time. The values were recorded at an initial pH in the range of 7.5–9.0, a temperature of 30–32°C, and an inoculum size of 4–10%. The highest activity of approximately 0.95u2009Uu2009mL−1 was achieved after 4 days of cultivation using the optimized medium and under optimized conditions in a shake flask. Upscaling to the use of a 3-L stirred tank fermenter was found to be successful with a PLA-degrading activity of 5.53u2009Uu2009mL−1; which represents a 51-fold increase in the activity compared with that obtained from the nonoptimized medium and conditions in the shake flask.

X-ray-induced mutation of Bacillus sp. MR10 for manno-oligosaccharides production from copra meal

ABSTRACT The present study demonstrates the effectiveness of X-ray radiation in strain improvement for defective lipase production by Bacillus sp. MR10 for further application in the fermentative production of manno-oligosaccharides (MOS) from agricultural by-product, defatted copra meal (DCM). The mutants obtained were screened based on their defective lipase activity together with their β-mannanase production performance. Among 10 selected mutants, the strain M7 was the highest promising mutant regarding the smallest lipase activity (0.05 U/ml) and the retained β-mannanase activity similar to the parental strain (22 U/ml) were detected. The mutant M7 effectively hydrolyzed DCM to MOS with low-degree of polymerization (DP) oligomers including mannotriose (M3), mannotetraose (M4), and mannopentose (M5) as the main products. Although the pattern of DCM hydrolysis products of mutant M7 was distinctly different from wild type, the biochemical and catalytic properties of purified β-mannanase of mutant were similar to those of wild type. Both purified β-mannanases with apparent molecular mass of 38u2009kDa displayed optimal activity at pH 5–7 and 45–55°C. Co2+ and Hg2+ nearly completely inhibited activities of both enzymes, whereas Ba2+, Fe3+, and 2-mercaptoethanol obviously activated enzyme activities. Both enzymes showed high specificity for locust bean gum, konjac mannan, DCM, and guar gum. Thus, the mutant M7 has a potential for commercial production of high-quality MOS from low-cost DCM for further application in the feed industry.

Expression and comparative characterization of complete and C-terminally truncated forms of saccharifying α-amylase from Lactobacillus plantarum S21

Lactobacillus plantarum S21 α-amylase possesses 475 amino acids at the C-terminal region identified as the starch-binding domain (SBD) and has been previously reported to play a role in raw starch degradation. To understand the specific roles of this SBD, cloning and expression of the complete (AmyL9) and C-terminally truncated (AmyL9ΔSBD) forms of α-amylase were conducted for enzyme purification and comparative characterization. AmyL9 and AmyL9ΔSBD were overproduced in Escherichia coli at approximately 10- and 20-times increased values of volumetric productivity when compared to α-amylase produced by the wild type, respectively. AmyL9ΔSBD was unable to hydrolyze raw starch and exhibited substrate specificity in a similar manner to that of AmyL9, but it was weakly active toward amylopectin and glycogen. The hydrolysis products obtained from the amylaceous substrates of both enzymes were the same. In addition, AmyL9ΔSBD showed comparatively higher Km values than AmyL9 when it reacted with starch and amylopectin, and lower values for other kinetic constants namely vmax, kcat, and kcat/Km. The results indicated that the C-terminal SBDs of L. plantarum S21 α-amylase contribute to not only substrate preference but also substrate affinity and the catalytic efficiency of the α-amylase without any changes in the degradation mechanisms of the enzyme.

A soil bacterium Rhizobium borbori and its potential for citrinin-degrading application

Red mold rice is the fermentation product of ordinary white rice with various Monascus spp. and is popular in Asian countries as a food additive and for its beneficial effects on human health. However, most Monascus spp. not only produce valuable secondary metabolites, such as monacolin K, but also the mycotoxin citrinin, which limits the applicability of red mold rice in medical therapy and food production. The aim of this study was to isolate, screen and identify citrinin-degrading bacteria that can be used for citrinin degradation in industrial and medical applications. A total of 96 bacteria were obtained from 74 samples of soil, water and wastewater collected from Chiang Mai, northern Thailand. Of these, 14 isolates exhibited growth in a mineral medium containing 5xa0ppm crude citrinin, among which only five demonstrated citrinin-degrading ability without any loss of monacolin K after culture at 30xa0°C for 72xa0h. Isolates PS21 and PS45 degraded citrinin the fastest of all five isolates and also retained the lowest residual citrinin concentration. Based on 16S rRNA gene sequence analysis, isolates PS21 and PS45 were identified as Enterococcus cloacae and Rhizobium borbori, respectively. When R. borbori PS45 and E. cloacae PS21 were cultured in a mineral medium containing 5xa0ppm pure citrinin at 30xa0°C for 120xa0h, the residual citrinin contents were 1.83 and 2.82xa0ppm, respectively. The citrinin-degrading activities of both isolates were found predominantly in the cell-free supernatant. Based on its higher citrinin-degrading activity and safety, we propose R. borbori PS45 as a suitable microorganism for citrinin reduction in red mold rice products.

Efficient secretory expression of gene encoding a broad pH-stable maltose-forming amylase from Lactobacillus plantarum S21 in food-grade lactobacilli host

The native and the N-terminal signal peptide sequence deleted gene encoding for α-amylase from Lactobacillus plantarum S21 were cloned into the inducible lactobacilli expression vectors pSIP409 and pSIP609 and expressed in L. plantarum WCFS1 and food-grade L. plantarum TGL02, respectively. Only the native amylase gene was expressed and secreted extracellular amylase at a level of approximately 2000 U/L with 90xa0% secretion efficiency from both hosts. The purified extracellular amylase from the L. plantarum TGL02 retained unique properties of the wild-type enzyme, particularly the broad pH stability (4.0–8.0) and maltose-forming activity. The results indicate high compatibility of L. plantarum S21 signal peptide sequence to both recombinant lactobacilli hosts. The recombinant lactobacilli exhibited high efficiency for direct lactic acid production from starch as found with L. plantarum S21. The efficient compatible signal peptide is also expected to be applied in secretory expression for production of valuable proteins in food-grade lactobacilli host.