Characterization and application of a native lactic acid bacterium isolated from tannery fleshings for fermentative bioconversion of tannery fleshings

Lactic acid bacteria (LAB) species isolated from limed and delimed tannery fleshings (TF) were evaluated for their fermentation efficiency and antibacterial property. The native LAB isolates efficiently fermented TF and resulted in a fermented mass with antioxidant properties, indicating their potential for effective eco-friendly bioconversion of TF. From among the LAB isolated, a proteolytic isolate showing better antimicrobial spectrum and reasonably good fermentation efficiency was identified as Enterococcus faecium HAB01 based on various biochemical and molecular tests. This isolate afforded a better degree of hydrolysis (81.36%) of TF than Pediococcus acidilactici (54.64%) that was previously reported by us. The bacteriocin produced by E. faecium was found to be antagonistic to several human pathogens including Listeria, Aeromonas, Staphylococcus and Salmonella. Further, E. faecium HAB01 bacteriocin was thermostable and had a molecular weight of around 5 kDa, apart from being stable at both acidic and alkaline conditions. The bacteriocin was unstable against proteases.     

Pediocin production in milk by Pediococcus acidilactici in co-culture with Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus

The production of pediocin in milk by Pediococcus acidilactici was evaluated in co-culture with the dairy fermentation cultures Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus. The cultures were tested singly and in different combinations in milk (0 or 2% fat content) during incubation at 40°C for up to 10 h. Cell-free milk samples taken every 60 min were tested for bacteriocin activity against Listeria monocytogenes. Pediocin activity was not detectable when P. acidilactici was inoculated into milk as a monoculture. When P. acidilactici was grown in combination with the yogurt starter cultures S. thermophilus and Lb. delbrueckii ssp. bulgaricus, pediocin concentration reached 3,200–6,400 units ml⁻¹ after 8 h of incubation. The results showed that pediocin producing pediococci may be useful adjunct components in mixed cultures of S. thermophilus and Lb. delbrueckii ssp. bulgaricus to amplify the bioprotective properties of fermented dairy foods against Listeria contamination.     

Study of the physicochemical and biological stability of pediocin PA‐1 in the upper gastrointestinal tract conditions using a dynamic in vitro model

Aims: To evaluate the survival of Pediococcus acidilactici UL5 and its ability to produce pediocin PA‐1 during transit in an artificial gastrointestinal tract (GIT). To investigate the physicochemical and biological stability of purified pediocin PA‐1 under GIT conditions. Methods and Results: Skim milk culture of Ped. acidilactici UL5 was fed to a dynamic gastrointestinal (GI) model known as TIM‐1, comprising four compartments connected by computer‐controlled peristaltic valves and simulating the human stomach, duodenum, jejunum and ileum. This strain tolerated a pH of 2·7 in the gastric compartment, while lower pH reduced its viability. Bile salts in the duodenal compartment brought a further 4‐log reduction after 180 min of digestion, while high viable counts (up to 5 × 10⁷ CFU ml^?1 fermented milk) of Ped. acidilactici were found in both the jejunal and ileal compartments. Pediococcus acidilactici recovered from all four compartments was able to produce pediocin at the same level as unstressed cells. The activity of the purified pediocin in the gastric compartment was slightly reduced after 90 min of gastric digestion, while no detectable activity was found in the duodenal, jejunal and ileal compartments during 5 h of digestion. HPLC analysis showed partial degradation of the pediocin peptide in the duodenal compartment and massive breakdown in the jejunal and ileal compartments. Conclusions: Pediococcus acidilactici UL5 showed high resistance to GIT conditions, and its ability to produce pediocin was not affected, suggesting its potential as a probiotic candidate. The physicochemical and biological stability of pediocin was significantly poor under GIT conditions. Significance and Impact of the Study: Pediococcus acidilactici UL5 appears to be a potential probiotic candidate because its capacity to produce pediocin PA‐1 is not affected by the GI conditions as well as the strain shows an acceptable survival rate. Meanwhile, purified pediocin PA‐1 losses activity during GIT transit; microcapsules could be used to deliver it to the target site.     

Evaluation of mono or mixed cultures of lactic acid bacteria in type II sourdough system

The aim of this study was to evaluate the use of mono and mixed lactic acid bacteria (LAB) cultures to determine suitable LAB combinations for a type II sourdough system. In this context, previously isolated sourdough LAB strains with antimicrobial activity, which included Lactobacillus plantarum PFC22, Lactobacillus brevis PFC31, Pediococcus acidilactici PFC38, and Lactobacillus sanfranciscensis PFC80, were used as mono or mixed culture combinations in a fermentation system to produce type II sourdough, and subsequently in bread dough production. Compared to the monoculture fermentation of dough, the use of mixed cultures shortened the adaptation period by half. In addition, the use of mixed cultures ensured higher microbial viability, and enhanced the fruity flavor during bread dough production. It was determined that the combination of L. plantarum PFC22 + P. acidilactici PFC38 + L. sanfranciscensis PFC80 is a promising culture mixture that can be used in the production of type II sourdough systems, and that may also contribute to an increase in metabolic activity during bread production process.     

Expanded bed adsorption as a unique unit operation for the isolation of bacteriocins from fermentation media

Expanded bed adsorption using a strong cation exchanger allowed the direct isolation of amylovorin L471, a bacteriocin from Lactobacillus amylovorusDCE 471, from the fermentation medium. The pH of the loading and elution buffer were optimised in a packed bed with cell-free culture supernatant. Bound bacteriocin was eluted with 1.0 M NaCl. The highest recovery (30%) was obtained at the lowest pH (3.6). At higher pH values the recovery was lower, namely 12%, 15% and 7% at pH 4.5, 6.5 and 8.0, respectively. In expanded bed mode, direct isolation of the bacteriocin from the fermentation medium at pH 3.6 (loading and elution) initially resulted in a recovery of 12%. After optimisation of the pH (loading and elution at pH 3.6 and 6.5, respectively), the recovery for amylovorin L471 increased up to 30% and higher. Recovery of enterocin A from Enterococcus faeciumCTC 492 fermentation medium averaged 15% (loading and elution at pH 3.6 and 6.0, respectively). With pediocin, produced by Pediococcus acidilactici ATCC 8042, 26% recovery was obtained at a pH of 6.5 during loading and elution. Low recoveries can be ascribed to non-optimal operation conditions (pH of loading and elution buffer), inactivation of the bacteriocin on a cationic resin, and the formation of more insoluble and less active, strongly hydrophobic bacteriocin aggregates upon further purification.     

酒醅中精氨酸利用菌株的分离筛选及其对浓香型白酒中瓜氨酸积累的影响

【目的】分析浓香型白酒酒醅发酵过程中氨基甲酸乙酯前体物质瓜氨酸含量显著增加的原因,确定酒醅中能够利用精氨酸并积累瓜氨酸的微生物,为解析白酒中氨基甲酸乙酯的形成机制提供研究基础和理论依据。【方法】采用高通量筛选技术,从浓香型白酒酒醅中分离具有高精氨酸利用能力和高瓜氨酸积累特性的菌株,并通过基因型和表现型验证以及模拟窖内发酵验证它们对瓜氨酸积累的贡献。【结果】共筛选获得20株具有高精氨酸利用能力的菌株,其中Lactococcus garvieae LD3,Bacillus amyloliquefaciens BG5,Pediococcus acidilactici PH7和Staphylococcus pasteuri SH11具有较高的瓜氨酸生成能力,并可使酒醅中瓜氨酸含量显著增加。【结论】筛选获得的4类微生物均能够通过ADI途径代谢积累瓜氨酸,是导致酒醅瓜氨酸含量增加的原因。     

Assessment of Pediococcus acidilactici as a Potential Silage Inoculant

Eighteen Pediococcus strains were screened for their potential as silage inoculants. Pediococcus acidilactici G24 was found to be the most suitable, exhibiting a short lag phase on both glucose and fructose, a rapid rate of acid production, a high sugar-to-lactate conversion efficiency, no detectable breakdown of proteins or lactic acid, and the ability to grow within a broad range of pH and temperature. When tested in laboratory silos using grass with a water-soluble carbohydrate content of 24 g/kg of aqueous extract, P. acidilactici G24 stimulated the natural Lactobacillus plantarum population and accelerated the rates of lactic acid production and pH decrease. After 6 days of fermentation, the inoculated silage exhibited a 12% decrease in ammonia nitrogen and an 11% increase in crude protein levels compared with uninoculated controls. The use of an L. plantarum inoculant at a rate of 10⁴ bacteria per g of grass in conjunction with P. acidilactici G24 produced no additional beneficial effect. Inoculation of grass with a water-soluble carbohydrate level of 8 g/kg of aqueous extract with P. acidilactici G24 led to no acceleration in the rate of L. plantarum growth or pH decrease. However, after 7 days of fermentation the inoculated silage had a 14% lower ammonia nitrogen protein content than did uninoculated controls. The results suggest that P. acidilactici G24 may be useful as a silage inoculant for crops with a sufficiently high water-soluble carbohydrate level.     

Agarose/agar assay system for the selection of bacteriocin-producing lactic fermentation bacteria

The direct selection of bacteriocin-producing lactic fermentation bacteria was possible by plating diluted cultures of Pediococcus acidilactici on mixed agarose agar layers with the amount of each component incrementally adjusted to 1.2% (w/v). Between 0.5 and 1% agarose, the increased flexibility of the solidified support layer allowed its removal from Petri dishes without tearing and its smooth layering on the surface of 1.5% (w/v) standard agar medium seeded with Listeria innocua as the test organism. Selection of bacteriocin-producing clones was based on the size of inhibition zones visible in the bottom agar layer under colonies growing on the agarose/agar top layer. The lack of contact with the test organism permitted the transfer of superior clones from the surface of the agarose/agar layer directly into an appropriate nutrient medium.     

Lignocellulose conversion of ensiled Caragana korshinskii Kom. facilitated by Pediococcus acidilactici and cellulases

To explore the biofuel production potential of Caragana korshinskii Kom., Pediococcus acidilactici and an exogenous fibrolytic enzyme were employed to investigate the fermentation profile, structural carbohydrates degradation, enzymatic saccharification and the dynamics of bacterial community of C. korshinskii silage. After 60 d of ensiling, all additives increased the fermentation quality. The highest lactic and acetic acids and lowest non-protein nitrogen (NPN) and ammonia nitrogen (NH₃-N) were observed in P. acidilactici and Acremonium cellulase (PA + AC) treated silage. Additionally, all additives significantly increased the ferulic acid content and fibre degradability with the highest values obtained from PA + AC silage. The bacterial community in all silages was dominated by P. acidilactici throughout the entire fermentation process. The bacterial community was also modified by the silage additives exhibiting a relatively simple network of bacterial interaction characterized by a lower bacterial diversity in P. acidilactici (PA) treated silage. The highest 6-phospho-beta-glucosidase abundance was observed in PA-treated silage at the mid-later stage of ensiling. PA treatment exhibited lower structural carbohydrates degradation but performed better in lignocellulose conversion during enzymatic saccharification. These results indicated that pretreating C. korshinskii improved its silage quality and potential use as a lignocellulosic feedstock for the production of bio-product and biofuel.     

Seed treatment with lactic acid bacteria against seed-borne pathogens of spring wheat

Antifungal potential of lactic acid bacteria (LAB) such as Lactobacillus sakei KTU05-6, Pediococcus acidilactici KTU05-7 and Pediococcus pentosaceus KTU05-8, KTU05-9 and KTU05-10 was tested on naturally contaminated wheat seeds. LAB influence on fungal growth on kernels, seedling diseases and seed germination was examined by laboratory and field experiments. KTU05-10 was selected and later used for seed treatment as solitary strain and in two- or three-component mixtures with KTU05-7 and KTU05-6. The occurrence of Fusarium spp. on wheat kernels in agar plate assays was decreased by seed treatment with all LAB cultures, and the efficacy of each strain depended on incubation temperature. Inoculation of wheat kernels with strains of solitary KTU05-10 and in mixtures with KTU05-7 and KTU05-6 significantly reduced the incidence of Fusarium spp., Bipolaris sorokiniana and Alternaria spp. LAB influence on seed germination and seedling diseases was observed in laboratory and field experiments, but in most cases, this influence was insignificant.     

Production of fuel ethanol from bamboo by concentrated sulfuric acid hydrolysis followed by continuous ethanol fermentation

An efficient process for the production of fuel ethanol from bamboo that consisted of hydrolysis with concentrated sulfuric acid, removal of color compounds, separation of acid and sugar, hydrolysis of oligosaccharides and subsequent continuous ethanol fermentation was developed. The highest sugar recovery efficiency was 81.6% when concentrated sulfuric acid hydrolysis was carried out under the optimum conditions. Continuous separation of acid from the saccharified liquid after removal of color compounds with activated carbon was conducted using an improved simulated moving bed (ISMB) system, and 98.4% of sugar and 90.5% of acid were recovered. After oligosaccharide hydrolysis and pH adjustment, the unsterilized saccharified liquid was subjected to continuous ethanol fermentation using Saccharomycescerevisiae strain KF-7. The ethanol concentration, the fermentation yield based on glucose and the ethanol productivity were approximately 27.2 g/l, 92.0% and 8.2 g/l/h, respectively. These results suggest that the process is effective for production of fuel ethanol from bamboo.     

Next generation sequencing,biochemical characterization,metabolic pathway analysis of novel probiotic Pediococcus acidilactici NCDC 252 and it’s evolutionary relationship with other lactic acid bacteria

Pediococcus acidilactici NCDC 252 is a facultative anaerobe of dairy origin that possessed all studied in vitro probiotic attributes and several useful enzyme activities. Its whole genome was sequenced and analysed for its evolutionary relationship with other lactic acid bacteria (LAB). This is a novel sequence and first report of genome sequence of P. acidilactici of dairy origin. Its genome is relatively larger than other studied genomes of P. acidilactici and is comprised of 40 scaffolds that totals to 3,243,337 bases and 44.5% GC content. A total of 3054 coding sequences (CDS) were identified by RAST and DIAMOND servers. The genome also encoded different enzyme activities required for utilization of various carbohydrates. This was also confirmed by carbohydrate utilization studies. The genome also encoded genes for probiotics properties. The phylogenetic analysis of P. acidilactici NCDC 252 genome was done using Maximum Parsimony and Maximum Likelihood methods to study its evolution and relatedness to other LABs based upon their 16S rDNA sequences. The strain exhibited highest resemblance to Lactobacillus plantarum WCFS1 and is also much close to P. acidilactici based on similarity of ribosomal protein. The strain seems to have acquired some genes for its adaptation in dairy/environmental niche. This genome sequence is novel with genome more similar to L. plantarum and biochemical and phenotypic characteristics of P. acidilactici.

     

Identification and succession of lactic acid bacteria during fermentation of 'urutan', a Balinese indigenous fermented sausage

'Urutan' is a Balinese traditional fermented sausage, which is made of lean pork and fat mixed with spices, sugar, and salt. The mixture is stuffed into cleaned pig intestine and fermented under uncontrolled condition during sun drying for 5 days. The investigation showed that lactic acid bacteria (LAB) were the dominating bacteria during 'urutan' fermentation. Among the 71 isolates obtained, lactobacilli dominated by 77.5% and the other 22.5% were pediococci. Based on physiological characteristics, the isolates were classified into 13 groups: nine belonged to the lactobacilli and the other four were pediococci. One isolate representing each group was chosen randomly, and then was identified by 16S rDNA sequence comparison. Phylogenetic relationship positioned three groups to Lactobacillus plantarum and four groups were closely related to L. farciminis. Two groups were identified as obligate heterofermentative lactobacilli: one was L. fermentum and the other was distantly related to L. hilgardii. Two groups belonging to the pediococci were strains of Pediococcus acidilactici and the other two were closely related to P. pentosaceus. A dramatic succession occurred during fermentation of 'urutan'. Three species mainly dominated the process wherein the initial growth was started by L. plantarum then followed by the growth of P. acidilactici, and finally, L. farciminis was found to be predominant at the last stage of fermentation.     

Characterization and Determination of Origin of Lactic Acid Bacteria from a Sorghum-Based Fermented Weaning Food by Analysis of Soluble Proteins and Amplified Fragment Length Polymorphism Fingerprinting

The group that includes the lactic acid bacteria is one of the most diverse groups of bacteria known, and these organisms have been characterized extensively by using different techniques. In this study, 180 lactic acid bacterial strains isolated from sorghum powder (44 strains) and from corresponding fermented (93 strains) and cooked fermented (43 strains) porridge samples that were prepared in 15 households were characterized by using biochemical and physiological methods, as well as by analyzing the electrophoretic profiles of total soluble proteins. A total of 58 of the 180 strains were Lactobacillus plantarum strains, 47 were Leuconostoc mesenteroides strains, 25 were Lactobacillus sake-Lactobacillus curvatus strains, 17 were Pediococcus pentosaceus strains, 13 were Pediococcus acidilactici strains, and 7 were Lactococcus lactis strains. L. plantarum and L. mesenteroides strains were the dominant strains during the fermentation process and were recovered from 87 and 73% of the households, respectively. The potential origins of these groups of lactic acid bacteria were assessed by amplified fragment length polymorphism fingerprint analysis.     

Bacteriocin plasmids ofPediococcus acidilactici

Summary Pediococcus acidilactici strains E, F and H isolated from fermented sausages produced bacteriocins which were protein in nature and inhibitory to a variety of spoilage and pathogenic microorganisms often encountered in foods. These strains harbored two to three plasmids ranging in size from 7.4 to 40.2 megadaltons. Curing experiments and plasmid profile analysis indicated the involvement of plasmid DNA with bacteriocin activity in all three strains. Carbohydrate fermentation and antibiotic resistance phenotypes did not appear to be associated with bacteriocin plasmids. Both bacteriocin activity and resistance determinants were linked in strain H and mediated by a 7.4-megadalton plasmid, whereas in strains E and F these two traits were not linked.     

Multilocus Hybridization Typing in Pediococcus acidilactici Strains

In previous a study we demonstrated the presence of several genomic subpopulations within a collection of Pediococcus acidilactici strains isolated from different environments, through a multilocus typing analysis taking into consideration housekeeping conserved loci and protein coding genes of the primary metabolism. In this study, representative strains of five genomic subpopulations previously described (I, II, III, V, VII) were analyzed by restriction analysis of chromosomal DNA and subsequent hybridization assays using as probes amplified fragments obtained from five housekeeping genes (16S rDNA, rpoC, ldhD, ldhL, and metS). A computer similarity and clustering analysis of hybridization data showed the subdivision of P. acidilactici strains in five distinct genotypes according to the grouping previously obtained confirming that pediocin AcH/PA-1 producer strains represent one genomic lineage within the species P. acidilactici. Received: 30 January 2001 / Accepted: 16 May 2001     

饲用微生物的分离鉴定及其对杏鲍菇菌糠发酵的效果

【目的】菌糠的营养素含量齐全,但纤维素含量过高是阻碍其饲料化利用的主要因素。故本研究筛选适合于发酵杏鲍菇菌糠的微生物菌株,以改善其饲用品质。【方法】首先,本研究采用纤维素-刚果红、苯胺蓝和MRS-Ca (De Man, Rogosa, Sharpe-Ca)筛选培养基,结合纤维素、木质素酶活力及抑菌活性的测定,从EM (effective microorganisms)原液发酵的杏鲍菇菌糠中分离筛选具有较强纤维素、木质素降解能力及抑菌能力的细菌/真菌。通过细菌16S rRNA和真菌18S rDNA基因序列分析确定菌株所属种属。其次,将筛选出的菌株菌液等体积混合制成复合菌剂用于固态发酵杏鲍菇菌糠。测定不同发酵时长菌糠营养成分含量以确定最佳发酵时间,并与相同工艺条件下EM原液发酵的杏鲍菇菌糠进行饲用品质比较。【结果】筛选并鉴定得到纤维素酶活性较高的特基拉芽孢杆菌(Bacillus tequilensis)菌株P11、发酵毕赤酵母(Pichia fermentans)菌株R8和马克斯克鲁维应变酵母(Kluyveromyces marxianus)菌株MU5;木质素酶活性较高的解淀粉芽孢杆菌(Bacillus amyloliquefaciens subsp.plantarum)菌株MU7;抑菌活性较高的类肠膜魏斯氏菌(Weissella paramesenteroides)菌株R4和乳酸片球菌(Pediococcus acidilactici)菌株R9。使用以上菌株复合发酵杏鲍菇菌糠7 d后,各项指标达到稳定。与EM原液发酵的杏鲍菇菌糠相比,复合菌剂发酵杏鲍菇菌糠的NDF和ADF分别显著降低了19.6%和21.44%(P0.05);CP (crude protein)、CA (crude ash)和EE (ether extract)含量分别显著提高了10.44%、5.26%和123.53%(P0.05)。【结论】本研究筛选得到的芽孢杆菌、酵母菌和乳酸菌优势菌株复合后用于发酵杏鲍菇菌糠可以很好地改善其饲用品质,效果优于生产中常用市售EM原液。     

Cloning and Expression of the Pediocin Operon in Streptococcus thermophilus and Other Lactic Fermentation Bacteria

Production of pediocin in Pediococcus acidilactici is associated with pMBR1.0, which encodes prepediocin, a pediocin immunity protein, and two proteins involved in secretion and precursor processing. These four genes are organized as an operon under control of a single promoter. We have constructed shuttle vectors that contain all four structural genes, the chromosomal promoter ST_P2201 from Streptococcus thermophilus, and repA from the 2-kbp S. thermophilus plasmid pER8. The recombinant plasmid, pPC318, expressed and secreted active pediocin in Escherichia coli. Streptococcus thermophilus, Lactococcus lactis subsp. lactis, and Enterococcus faecalis were electrotransformed with pPC418, a modified vector fitted with an erythromycin resistance tracking gene. Pediocin was produced and secreted in each of the lactic acid bacteria, and production was stable for up to ten passages. The expression of pediocin in dairy fermentation microbes has important implications for bacteriocins as food preservatives in dairy products. Received: 7 June 1999 / Accepted: 6 July 1999     

Ascorbic acid-induced loss of a pediocin-encoding plasmid in Pediococcus acidilactici CFR K7

A strain of Pediococcus acidilactici CFR K7 grown in presence of 1 mm ascorbate for 18 h resulted in 35% colony-forming units (CFU) irreversibly losing their ability to produce pediocin. Agarose gel electrophoresis of plasmid DNA and dot-blot hybridization showed concomitant loss of a 7.8 kb plasmid coding for pediocin from these colonies. Sensitivity of these colonies to nitrofurantoin was also observed after ascorbate treatment. Since ascorbic acid is a readily available and non-hazardous compound in contrast to other conventional plasmid curing agents, the possibility of its use in determining plasmid-encoded traits in food grade lactic acid bacteria is also proposed. This revised version was published online in July 2006 with corrections to the Cover Date.