Identification of lactic acid bacteria and yeast from boza

Boza is a low-alcohol beverage produced from the fermentation of barley, oats, millet, maize, wheat or rice. The number of lactic acid bacteria isolated from three boza samples ranged from 9 × 10⁶ to 5 × 10⁷ CFU/mL. Carbohydrate fermentation reactions and PCR with species-specific primers classified the isolates as Lactobacillus paracasei subsp. paracasei, Lactobacillus pentosus, Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus rhamnosus and Lactobacillus fermentum. No filamentous fungi were isolated. Yeasts were isolated from two of the three boza samples, with cell numbers ranging from 1.3 × 10² to 1.8 × 10³ CFU/mL. Results obtained from sequencing of the D1/D2 rDNA region identified the yeasts as Candida diversa, Candida inconspicua, Candida pararugosa, Issatchenkia orientalis, Pichia fermentans, Pichia guillliermondii, Pichia norvegensis, Rhodotorula mucilaginosa and Torulaspora delbrueckii. C. inconspicua has been isolated from human sputum and tongue and is an opportunistic pathogen. R. mucilaginosa is also an opportunistic pathogen implicated in fungaemia, endocarditis and meningitis. P. norvegensis has been associated with septicaemia in humans. Saccharomyces cerevisiae, commonly associated with fermented beverages, has not been detected in any of the boza samples, despite enrichment.     

The P170 expression system enhances hyaluronan molecular weight and production in metabolically-engineered Lactococcus lactis

Recombinant Lactococcus lactis strains based on the P170 expression system were developed for hyaluronan (HA) production, by incorporating genes from the has operon of Streptococcus zooepidemicus and compared with nisin-inducible recombinant L. lactis strains containing the hasABC and hasABD constructs. It was found across all batch and fed-batch experimental studies that HA concentration and molecular weight (MW) were higher for the P170 expression systems than the corresponding NICE-based strains. The highest hyaluronan MW was obtained for all constructs in batch studies at 60 g/L initial glucose concentration, the highest being 2.94 MDa for the P170 strains with hasABC construct (L. lactis APJ3). In fed-batch studies with constant feed rate, the L. lactis APJ3 gave better HA yield (0.03 g/g) than the NICE-based strain. A higher hyaluronan MW was obtained for all strains in pulse fed-batch compared to constant feed experiments, the highest being 2.52 MDa for L. lactis APJ3.     

Antimicrobial activity against Shigella sonnei and probiotic properties of wild lactobacilli from fermented food

Four lactobacilli strains (Lactobacillus paracasei subp. paracasei M5-L, Lactobacillus rhamnosus J10-L, Lactobacillus casei Q8-L and L. rhamnosus GG (LGG), were systematically assessed for the production of antimicrobial substances active towards Shigella sonnei, Escherichia coli and Salmonella typhimurium. Agar-well assay showed that the four lactobacilli strains displayed strong antibacterial activity towards S. sonnei. The nature of antimicrobial substances was also investigated and shown to be dependent on the production of organic acids, in particular the lactic acid. Time-kill assay showed that the viability of the S. sonnei was decreased by 2.7–3.6 log CFU/ml after contact with CFCS (cell-free culture supernatants) of four lactobacilli for 2 h, which confirmed the result of the agar-well assay. Further analysis of the organic acid composition in the CFCS revealed that the content of lactic acid range from 227 to 293 mM. In addition, the aggregations properties, adherence properties and tolerance to simulated gastrointestinal conditions were also investigated in vitro tests. The result suggested that the M5-L, J10-L and Q8-L strains possess desirable antimicrobial activity towards S. sonnei and probiotic properties as LGG and could be potentially used as novel probiotic strains in the food industry.     

Selenium nanoparticles from Lactobacillus paracasei HM1 capable of antagonizing animal pathogenic fungi as a new source from human breast milk

The current study was performed to develop a simple, safe, and cost-effective technique for the biosynthesis of selenium nanoparticles (SeNPs) from lactic acid bacteria (LAB) isolated from human breast milk with antifungal activity against animal pathogenic fungi. The LAB was selected based on their speed of transforming sodium selenite (Na₂SeO₃) to SeNPs. Out of the four identified LAB isolates, only one strain produced dark red color within 32 h of incubation, indicating that this isolate was the fastest in transforming Na₂SeO₃ to SeNPs; and was chosen for the biosynthesis of LAB-SeNPs. The superior isolate was further identified as Lactobacillus paracasei HM1 (MW390875) based on matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and phylogenetic tree analysis of 16S rRNA sequence alignments. The optimum experimental conditions for the biosynthesis of SeNPs by L. paracasei HM1 were found to be pH (6.0), temperature (35˚C), Na₂SeO₃ (4.0 mM), reaction time (32 h), and agitation speed (160 rpm). The ultraviolet absorbance of L. paracasei-SeNPs was detected at 300 nm, and the transmission electron microscopy (TEM) captured a diameter range between 3.0 and 50.0 nm. The energy-dispersive X-ray spectroscopy (EDX) and the Fourier-transform infrared spectroscopy (FTIR) provided a clear image of the active groups associated with the stability of L. paracasei-SeNPs. The size of L. paracasei-SeNPs using dynamic light scattering technique was 56.91 ± 1.8 nm, and zeta potential value was −20.1 ± 0.6 mV in one peak. The data also revealed that L. paracasei-SeNPs effectively inhibited the growth of Candida and Fusarium species, and this was further confirmed by scanning electron microscopy (SEM). The current study concluded that the SeNPs obtained from L. paracasei HM1 could be used to prepare biological antifungal formulations effective against major animal pathogenic fungi. The antifungal activity of the biologically synthesized SeNPs using L. paracasei HM1 outperforms the chemically produced SeNPs. In vivo studies showing the antagonistic effect of SeNPs on pathogenic fungi are underway to demonstrate the potential of a therapeutic agent to treat animals against major infectious fungal diseases.     

Separation of lactobacilli bacteriocins from fermented broths using membranes

Current separation, isolation and purification techniques to obtain highly potent purified lactobacilli and lactococci bacteriocins include chemical precipitation, separation employing solvents and chromatographic techniques. These methods are arduous, costly, with limited scalability, offering low bacteriocin yields (<20%). To address these challenges, the alternatives of ultrafiltration and nanofiltration, as separation methods were tested. Three promising bacteriocin producing strains, Lactobacillus casei NCIMB 11970, Lactobacillus plantarum NCIMB 8014 and Lactococcus lactis NCIMB 8586 were selected to investigate the applicability and feasibility of the method.To facilitate separation, the microorganisms were grown on specially developed low molecular weight medium (LMWM) mainly containing nutritive sources up to 4 kDa molecular weight. Bacterial cells were removed by centrifugation. The clarified broths were filtered using 4 and 1 kDa MWCO. Bacteriocin activity was determined by an antimicrobial activity test using nisin, which has an inhibitory effect on the growth of susceptible microorganisms. Recovery yields using filtration were found to range between 53 and 68%, a high recovery performance.The bacteriocin activity of crude extracts of all the three lactobacilli were between 95 and 105 IU ml^?1. When the substances were separated using ultrafiltration membrane (4 kDa MWCO) their activity was enhanced to 145–150 IU ml^?1, achieving a total potency yield of 44–53%. Further enhancement of yields up to 36% was attained employing nanofiltration (1 kDa MWCO) membranes with an activity increased up to 200 IU ml^?1.Bacteriocin isolation from crude extracts using filtration was found to be effective, offering high recovery yields, optimising their activity as well as presenting a realistic option towards the formulation of these as commercially available antibacterial agents.     

Relationship between the resistance to bile salts and low pH with exopolysaccharide (EPS) production of Bifidobacterium spp. isolated from infants feces and breast milk

The purpose of this study was to investigate a possible relation between resistance to bile salts and low pH with exopolysaccharide (EPS) producing of Bifidobacterium spp. In this study, a total of 31 Bifidobacterium spp. were isolated from breast fed infants feces and breast milk samples. As a result of the identification tests, isolates were identified as Bifidobacterium breve (15 strains), B. bifidum (11 strains), B. pseudocatenulatum (3 strains) and B. longum (2 strains). Bifidobacterium spp. were determined exopolysaccharide (EPS) production. EPS productions observed at chance rations (38.00–97.64 mg/l) among of Bifidobacterium spp. Furthermore, Bifidobacterium spp. were determined resistance to bile salts and low pH. Positive correlations between production of exopolysaccharide and resistance to bile salts (p < 0.01) or low pH (p < 0.01) were found Bifidobacterium spp. This investigation showed that high EPS production of Bifidobacteria may be important in the selection of probiotic strains for resistance to bile salts and low pH.     

Solution for promoting egl3 gene of Trichoderma reesei high-efficiency secretory expression in Escherichia coli and Lactococcus lactis

The objective of this study was to develop a solution for promoting egl3 gene of Trichoderma reesei (coding β-1,4-endoglucanase, EGIII) high-efficiency secretory expression in Escherichia coli and Lactococcus lactis and to investigate the effect of the best recombinant on degrading paper and wheat straw. The coding sequence of the egl3 gene fused with a gene fragment of Usp45 (usp45) of L. lactis was cloned to pMG36e and was expressed in E. coli DH 5α (DH 5α) and L. lactis subsp. lactis MG1363 (MG1363). The maximal productivity in recombinant DH 5α was 226 mU mL⁻¹ for extracellular EGIII and 535 mU mL⁻¹ for intracellular EGIII. The maximal productivity in recombinant MG1363 was 1118 mU mL⁻¹ for extracellular EGIII and 761 mU mL⁻¹ for intracellular EGIII. The plasmid stability in recombinant MG1363 was higher than 85% at 60 generations. Recombinant MG1363 vigorously degraded paper and wheat straw and produced sufficient acids. This study provided EGIII transgenic lactic acid bacteria for processing agricultural byproducts.     

Enhanced exopolysaccharide production and biological activity of Lactobacillus rhamnosus ZY with calcium and hydrogen peroxide

Exopolysaccharides (EPS) are important food and drug additives with beneficial antioxidant, anticancer, and immune-related effects on human health. However, the EPS is limited by low yields and the need for complex culture conditions in fermentation. Here, we report that hydrogen peroxide and calcium stimulated probiotic activity and production of crude exopolysaccharide (c-EPS) by Lactobacillus rhamnosus ZY. Accordingly, supplementation with 3 mM H₂O₂ allowed c-EPS biosynthesis to reach 567 mg/L after 24 h. Addition of both CaCl₂ and H₂O₂ resulted in a c-EPS yield of 2498 mg/L after 12 h, over 9-fold higher than that of an anaerobic culture. We observed that exposure to calcium and hydrogen peroxide made the cells more hydrophobic and led to the over-expression of GroEL, NADH peroxidase, and glyceraldehyde 3-phosphate dehydrogenase, thus increasing energy storage and EPS production. Chromatographic analysis revealed c-EPS was composed mainly of mannose (5.1%), galactose (15.3%), glucose (20–30%), and rhamnose (50–60%). Preliminary in vitro tests revealed that H₂O₂ and CaCl₂ enhanced the 2,2-diphenyl-1-picrylhydrazyl and hydroxyl radical scavenging capacities, resulting in a notable protective effect against oxidative damage in NIH/3T3 cells. Our study provides a simple and cost-effective approach for achieving high yields of good quality EPS using Lactobacillus rhamnosus.     

Lactic acid bacteria isolated from fish gut produce conjugated linoleic acid without the addition of exogenous substrate

The production of conjugated linoleic acid (CLA) by four strains of lactic acid bacteria isolated from fish, i.e., Leuconostoc mesenteroides H20, Leuconostoc mesenteroides H22, Leuconostoc lactis H24 and Lactobacillus pentosus H16, was evaluated in MRS broth and on MRS agar. The bioconversion and production of CLA by resting cells were also assessed. Linoleic acid was detected in cultures grown on agar at percentages of up to 18.3% (w/w) of total fatty acid, and conjugated isomers were found in the fatty acid profiles of Lactobacillus pentosus H16. The percentage of CLA relative to total fatty acid increased from 5.68 ± 1.65% to 23.69 ± 0.79% when resting cells were removed from agar plates and incubated without the addition of exogenous linoleic acid as a substrate. When Lactobacillus pentosus H16 cells were incubated with linoleic acid, cyclization and changes in monounsaturated fatty acid percentages were observed instead of conjugation. These results show that growth on a solid support is required for CLA production. More significantly, an increase in the CLA content could be achieved by incubating resting cells without exogenous substrate.     

Polyphasic characterization of a novel species in the Lactobacillus casei group from cow manure of Taiwan: Description of L. chiayiensis sp. nov.

Two Gram-stain-positive, rod-shaped, non-motile, catalase-negative and facultative anaerobic strains, NCYUAS^T and BCRC 18859 (=NRIC 1947), were isolated from cow manure of Taiwan and coconut juice of Philippines, respectively. Comparative sequence analysis of 16S rRNA gene revealed that the novel strains were members of the genus Lactobacillus. These two strains had 100% of 16S rRNA gene sequence similarity and 98.6% of average nucleotide identity (ANI) value based on whole genome sequences. On the basis of 16S rRNA gene sequence similarity, the type strains of Lactobacillus casei (99.6% similarity), Lactobacillus paracasei subsp. paracasei (99.1%), L. paracasei subsp. tolerans (99.1%), Lactobacillus rhmnosus (99.0%) and ‘Lactobacillus zeae’ (99.7%) were the closest neighbors to these novel strains. The results of phenotypic and chemotaxonomic characterization, multilocus sequence analysis (MLSA) based on the sequences of three housekeeping genes (dnaK, pheS and yycH), whole-genome sequence (WGS)-based comparison by ANI and in silico DNA–DNA hybridization (isDDH), species-specific PCR and whole-cell MALDI-TOF MS spectral pattern analyses demonstrated that the novel two strains represented a single, novel species within the L. casei group, for which the name Lactobacillus chiayiensis sp. nov., is proposed. The type strain is NCYUAS^T (=BCRC 81062^T = NBRC 112906^T).     

Intestinal Lactobacillus sp. is associated with some cellular and metabolic characteristics of blood in elderly people

The higher counts or particular groups (Firmicutes/Bacteroidetes) of intestinal microbiota are related to host metabolic reactions, supporting a balance of human ecosystem. We further explored whether intestinal lactobacilli were associated with some principal cellular and metabolic markers of blood in 38 healthy >65-year-old persons. The questionnaire, routine clinical and laboratory data of blood indices as much as the oxidized low-density lipoprotein (ox-LDL) and baseline diene conjugates in low-density lipoprotein (BDC-LDL) of blood sera were explored. The PCR-based intestinal Lactobacillus sp. composition and counts of cultivable lactobacilli (LAB) were tested. The facultative heterofermentative lactobacilli (Lactobacillus casei and Lactobacillus paracasei) were the most frequent (89 and 97%, respectively) species found, while Lactobacillus acidophilus, Lactobacillus plantarum and Lactobacillus reuteri were present in almost half of the elderly persons. The number of species simultaneously colonizing the individuals ranged from 1 to 7 (median 4). In elderly consuming probiotics the LAB counts were significantly higher than in these not consuming (median 7.8, range 4.2–10.8 vs. median 6.3, range 3.3–9.7 log cfu/g; p = 0.005), adjusted (OR = 1.71, CI95 1.04–2.82; p = 0.035) for age and body mass index (BMI). The colonization by L. acidophilus was negatively related (r = ?0.367, p = 0.0275) to L. reuteri, staying significant after adjusting for age, sex and BMI (OR = 0.16, CI95 0.04–0.73; p = 0.018). However, the blood glucose concentration showed a tendency for a negative correlation for colonization with Lactobacillus fermentum (r = ?0.309, p = 0.062) adjusted for BMI (Adj. R² = 0.181; p = 0.013) but not for age and sex. The higher white blood cells (WBC) count was positively related (r = 0.434, p = 0.007) to presence of Lactobacillus reuteri adjusted for age, sex and BMI (Adj. R² = 0.193, p = 0.027). The lower values of ox-LDL were predicted by higher counts of cultivable lactobacilli adjusted by sex, age and BMI (r = ?0.389, p = 0.016; Adj. R² = 0.184 p = 0.029). In conclusion, the pilot study of elderly persons shows that the intestinal lactobacilli are tightly associated with WBC count, blood glucose and content of ox-LDL which all serve as risk markers in pathogenesis of inflammation, metabolic syndrome and cardiovascular disease (CVD).     

内蒙古呼伦贝尔地区传统发酵乳中乳酸菌的多样性分析

【目的】对内蒙古呼伦贝尔地区传统发酵乳制品中乳酸菌资源的生物多样性进行研究。【方法】采用纯培养和16S rRNA基因序列分析法对内蒙古呼伦贝尔地区传统发酵乳中的乳酸菌进行多样性分析。【结果】从8份传统发酵乳制品(6份酸牛奶和2份酸马奶)样品中分离到24株乳酸菌,通过16S rRNA基因序列分析和系统进化关系分析将24株乳酸菌鉴定为2株Lactobacillus kefiranofaciens、2株Lactobacillus kefiri、5株Lactobacillus paracasei、3株Lactobacillus plantarum、1株Lactobacillus rhamnosus、6株Lactococcus lactis subsp.lactis、2株Leuconostoc mesenteroides subsp.dextranicum、2株Streptococcus thermophilus和1株Enterococcus faecium。【结论】Lactococcus lactis subsp.lactis为内蒙古呼伦贝尔地区传统发酵乳制品的优势菌种,占总分离株的25%,其次为Lactobacillus paracasei,占总分离株的20.83%。     

Biological control of fruit-rot of jackfruit by rhizobacteria and food grade lactic acid bacteria

Fungal species causing fruit rot of jackfruit have been isolated from seven different locations of Birbhum and Burdwan districts of West Bengal, India. Each isolate showed more or less similar microscopic characteristics. A representative strain VBAM1, isolated from a severely infected jackfruit was identified as Rhizopus stolonifer by 28S rDNA sequence homology. Increased reducing sugar content in pectin broth indicates pectinase production by the pathogen. The pathogen was not inhibited by ⩾500 μg/ml of Mancozeb and Bavistin. Copper oxychloride, Blytone 50% a.i. showed antifungal activity at comparatively lower concentration (200 μg/ml). Two rhizospheric bacterial strains, Burkholderia cenocepacia VBC7 and Pseudomonas poae VBK1, and three different strains of Lactococcus lactis subsp. lactis can produce significant zones of inhibition against the pathogen in dual culture overlay plates. They induced mycelia breakage of pathogen as evidenced from scanning electron micrographs. When applied to whole plants, the strains reduced or prevented disease and when applied postharvest to Rhizopus inoculated fruit delayed and/reduced disease incidence. These agents were also re-isolated from the applied surfaces and survived for long time when mixed with suitable carrier base indicating stability in a formulation over time.     

Enantioselective synthesis of ethyl-(S)-3-hydroxy-3-phenylpropanoate (S-HPPE) from ethyl-3-oxo-3-phenylpropanoate using recombinant fatty acid synthase (FAS2) from Kluyveromyces lactis KCTC 7133 in Pichia pastoris GS115

Various yeast strains were examined for the microbial reduction of ethyl-3-oxo-3-phenylpropanoate (OPPE) to ethyl-(S)-3-hydroxy-3-phenylpropanoate (S-HPPE), which is the chiral intermediate for the synthesis of a serotonin uptake inhibitor, Fluoxetine. Kluyveromyces lactis KCTC 7133 was found as the most efficient strain in terms of high yield (83% at 50 mM) and high optical purity ee > 99% of S-HPPE. Based on the protein purification, activity analysis and the genomic analysis, a fatty acid synthase (FAS) was identified as the responsible β-ketoreductase. To increase the productivity, a recombinant Pichia pastoris GS115 over-expressing FAS2 (α-subunit of FAS) of K. lactis KCTC7133 was constructed. In the optimized media condition, the recombinant P. pastoris functionally over-expressed the FAS2. Recombinant P. pastoris showed 2.3-fold higher reductase activity compared with wild type P. pastoris. With the recombinant P. pastoris, the 91% yield of S-HPPE was achieved at 50 mM OPPE maintaining the high optical purity of the product (ee > 99%).     

Characterization of the LP28 strain-specific exopolysaccharide biosynthetic gene cluster found in the whole circular genome of Pediococcus pentosaceus

We have previously isolated a lactic acid bacterium (LAB), Pediococcus pentosaceus LP28, from the longan fruit Euphoria longana. Since the plant-derived LAB strain produces an extracellular polysaccharide (EPS), in this study, we analyzed the chemical structure and the biosynthesizing genes for the EPS.The EPS, which was purified from the LP28 culture broth, was classified into acidic and neutral EPSs with a molecular mass of about 50 kDa and 40 kDa, respectively. The acidic EPS consisted of glucose, galactose, mannose, and N-acetylglucosamine moieties. Interestingly, since pyruvate residue was detected in the hydrolyzed acidic EPS, one of the four sugars may be modified with pyruvate. On the other hand, the neutral EPS consisted of glucose, mannose, and N-acetylglucosamine; pyruvate was scarcely detected in the polysaccharide molecule.As a first step to deduce the probiotic function of the EPS together with the biosynthesis, we determined the whole genome sequence of the LP28 strain, demonstrating that the genome is a circular DNA, which is composed of 1,774,865 bp (1683 ORFs) with a GC content of 37.1%. We also found that the LP28 strain harbors a plasmid carrying 6 ORFs composed of 5366 bp with a GC content of 36.5%. By comparing all of the genome sequences among the LP28 strain and four strains of P. pentosaceus reported previously, we found that 53 proteins in the LP28 strain display a similarity of less than 50% with those in the four P. pentosaceus strains. Significantly, 4 of the 53 proteins, which may be enzymes necessary for the EPS production on the LP28 strain, were absent in the other four P. pentosaceus strains and displayed less than 50% similarity with other LAB species. The EPS-biosynthetic gene cluster detected only in the LP28 genome consisted of 12 ORFs containing a priming enzyme, five glycosyltransferases, and a putative polysaccharide pyruvyltransferase.     

Polyphasic characterization of two novel Lactobacillus spp. isolated from blown salami packages: Description of Lactobacillus halodurans sp. nov. and Lactobacillus salsicarnum sp. nov.

Microbiota analysis of blown pack spoiled salami revealed five distinguishable Lactobacillus isolates we could not assign to a known species. Two of the isolates (TMW 1.2172^T and TMW 1.1920) are rod-shaped, whilst three isolates (TMW 1.2098^T, TMW 1.2118 and TMW 1.2188) appear coccus shaped or as short rods. All isolates are Gram-stain positive, facultative anaerobic, catalase and oxidase negative, non-motile and non-sporulating. Phylogenetic analysis of the 16S rRNA, dnaK, pheS and rpoA gene sequences revealed two distinct lineages within the genus Lactobacillus (L.). The isolates are members of the Lactobacillus alimentarius group with Lactobacillus ginsenosidimutans DSM 24154^T (99.4% 16S similarity), Lactobacillus versmoldensis DSM 14857^T (97.9%) and Lactobacillus furfuricola DSM 27174^T (97.7%) as phylogenetic closest related species and L. alimentarius DSM 20249^T (97.7%) and Lactobacillus paralimentarius DSM 13961^T (97.5%) as closest relatives, respectively. Average Nucleotide Identity (ANI) and digital DNA-DNA hybridization (dDDH) values between the isolates and their close related type strains are lower than 80% and 25%, respectively. For both designated type strains, the peptidoglycan type is A4α l-Lys-d-Asp and the major fatty acids are C_16:0, C_18:1ω9c and summed feature 7. Based on phylogenetic, phenotypic and chemotaxonomic analysis we demonstrated that the investigated isolates belong to two novel Lactobacillus species for which we propose the names Lactobacillus salsicarnum with the type strain TMW 1.2098^T = DSM 109451^T = LMG 31401^T and Lactobacillus halodurans with the type strain TMW 1.2172^T = DSM 109452^T = LMG 31402^T.     

Efficient production of 2,3-butanediol in Saccharomyces cerevisiae by eliminating ethanol and glycerol production and redox rebalancing

2,3-Butanediol is a promising valuable chemical that can be used in various areas as a liquid fuel and a platform chemical. Here, 2,3-butanediol production in Saccharomyces cerevisiae was improved stepwise by eliminating byproduct formation and redox rebalancing. By introducing heterologous 2,3-butanediol biosynthetic pathway and deleting competing pathways producing ethanol and glycerol, metabolic flux was successfully redirected to 2,3-butanediol. In addition, the resulting redox cofactor imbalance was restored by overexpressing water-forming NADH oxidase (NoxE) from Lactococcus lactis. In a flask fed-batch fermentation with optimized conditions, the engineered adh1Δadh2Δadh3Δadh4Δadh5Δgpd1Δgpd2Δ strain overexpressing Bacillus subtilis α-acetolactate synthase (AlsS) and α-acetolactate decarboxylase (AlsD), S. cerevisiae 2,3-butanediol dehydrogenase (Bdh1), and L. lactis NoxE from a single multigene-expression vector produced 72.9 g/L 2,3-butanediol with the highest yield (0.41 g/g glucose) and productivity (1.43 g/(L·h)) ever reported in S. cerevisiae.     

Synthesis of γ-Aminobutyric Acid by Lactic Acid Bacteria Isolated from a Variety of Italian Cheeses

The concentrations of γ-aminobutyric acid (GABA) in 22 Italian cheese varieties that differ in several technological traits markedly varied from 0.26 to 391 mg kg⁻¹. Presumptive lactic acid bacteria were isolated from each cheese variety (total of 440 isolates) and screened for the capacity to synthesize GABA. Only 61 isolates showed this activity and were identified by partial sequencing of the 16S rRNA gene. Twelve species were found. Lactobacillus paracasei PF6, Lactobacillus delbrueckii subsp. bulgaricus PR1, Lactococcus lactis PU1, Lactobacillus plantarum C48, and Lactobacillus brevis PM17 were the best GABA-producing strains during fermentation of reconstituted skimmed milk. Except for L. plantarum C48, all these strains were isolated from cheeses with the highest concentrations of GABA. A core fragment of glutamate decarboxylase (GAD) DNA was isolated from L. paracasei PF6, L. delbrueckii subsp. bulgaricus PR1, L. lactis PU1, and L. plantarum C48 by using primers based on two highly conserved regions of GAD. A PCR product of ca. 540 bp was found for all the strains. The amino acid sequences deduced from nucleotide sequence analysis showed 98, 99, 90, and 85% identity to GadB of L. plantarum WCFS1 for L. paracasei PF6, L. delbrueckii subsp. bulgaricus PR1, L. lactis PU1, and L. plantarum C48, respectively. Except for L. lactis PU1, the three lactobacillus strains survived and synthesized GABA under simulated gastrointestinal conditions. The findings of this study provide a potential basis for exploiting selected cheese-related lactobacilli to develop health-promoting dairy products enriched in GABA.     

d- and l-lactic acid production from fresh sweet potato through simultaneous saccharification and fermentation

The aim of this study was to develop a bioprocess for l- and d-lactic acid production from raw sweet potato through simultaneous saccharification and fermentation by Lactobacillus paracasei and Lactobacillus coryniformis, respectively. The effects of enzyme and nitrogen source concentrations as well as of the ratio of raw material to medium were investigated. At dried material concentrations of 136.36–219.51 g L⁻¹, yields of 90.13–91.17% (w/w) and productivities of 3.41–3.83 g L⁻¹ h⁻¹ were obtained with lactic acid concentrations as high as 198.32 g L⁻¹ for l-lactic acid production. In addition, d-lactic acid was produced with yields of 90.11–84.92% (w/w) and productivities of 2.55–3.11 g L⁻¹ h⁻¹ with a maximum concentration of 186.40 g L⁻¹ at the same concentrations of dried material. The simple and efficient process described in this study will benefit the tuber and root-based lactic acid industries without requiring alterations in plant equipment.     

Optimization of the aeration and agitation speed of Aeribacillus palidus 418 exopolysaccharide production and the emulsifying properties of the product

The specific properties of exopolysaccharides (EPS) from thermophilic microorganisms have attracted interest in their optimized production. In this study, the ability of Aeribacillus pallidus 418 to grow and produce polysaccharide in a 5-l stirred tank bioreactor was investigated. Agitation rates of 100, 200, 600, 900, and 1100 revolutions per minute (rpm), at an air flow rate of 0.5 gas volumes per unit medium volume per minute (vvm), and aeration rates of 0.25, 0.5, 1.0, and 1.5 vvm, at an agitation rate of 900 rpm, were examined. A maximum EPS yield of 170 μg/ml has been registered in a single impeller bioreactor equipped with an original Narcissus impeller at agitation speed of 900 rpm, with an aeration rate of 0.5 vvm. The bioprocess oxygen uptake rate (OUR) and oxygen mass transfer coefficient (K_La) were evaluated. The emulsifying properties of the specific EPS produced by A. pallidus 418 were determined. Stable oil-in-water emulsions, a low level of separated water phase and high dispersion stability were found, which together demonstrate the prospects for the industrial exploration of EPS production. Enhanced synergism between the A. pallidus 418 synthesized EPS and various commercially used hydrocolloids was observed; superior synergy was achieved in combination with xanthan gum.