Application of Next Generation Sequencing for Rapid Identification of Lactic Acid Bacteria

The rapid identification of lactic acid bacteria,which are essential microorganisms in the food industry,is of great significance for industrial applications.The identification of lactic acid bacteria traditionally relies on the isolation and identification of pure colonies.While this method is well-established and widely used,it is not without limitations.The subjective judgment inherent in the isolation and purification process introduces potential for error,and the incomplete nature of the isolation process can result in the loss of valuable information.The advent of next generation sequencing has provided a novel approach to the rapid identification of lactic acid bacteria.This technology offers several advantages,including rapidity,accuracy,high throughput,and low cost.Next generation sequencing represents a significant advancement in the field of DNA sequencing.Its ability to rapidly and accurately identify lactic acid bacteria strains in samples with insufficient information or in the presence of multiple lactic acid bacteria sets it apart as a valuable tool.The application of this technology not only circumvents the potential errors inherent in the traditional method but also provides a robust foundation for the expeditious identification of lactic acid bacteria strains and the authentication of bacterial powder in industrial applications.This paper commences with an overview of traditional and molecular biology methods for the identification of lactic acid bacteria.While each method has its own advantages,they are not without limitations in practical application.Subsequently,the paper provides an introduction of the principle,process,advantages,and disadvantages of next generation sequencing,and also details its application in strain identification and rapid identification of lactic acid bacteria.The objective of this study is to provide a comprehensive and reliable basis for the rapid identification of industrial lactic acid bacteria strains and the authenticity identification of bacterial powder.

Rapid and High-throughput Identification of Recombinant Bacteria with Mass Spectrometry Assay

Objective To construct a rapid and high-throughput assay for identifying recombinant bacteria based on mass spectrometry. Methods Matrix assisted laser desorption ionization time-of-flight mass spectrometry （MALDI-TOF MS） techniques were used to identify 12 recombinant proteins （10 of Yersinia pestis, 1 of Campylobacter jejuni and 1 of Helicobacter pylori）. A classification model for the various phase of recombinant bacteria was established, optimized and validated, using MALDI-TOF MS-CIinProTools system. The differences in the peptide mass spectra were analyzed by using Biotyper and FIexAnalysis softwares. Results Models of GA, SNN, and QC were established. After optimizing the parameters, the GA recognition model showed good classification capabilities： RC=100%, mean CVA=98.7% （the CVA was 96.4% in phase 1, 100% in phase 2, 98.4% in phase 3, and 100% in phase 4, respectively） and PPV=95}. This model can be used to classify the bacteria and their recombinant, which only requires 3.7x103 cells for analysis. The total time needed is only 10 min from protein extraction to reporting the result for one sample. Furthermore, this assay can automatically detect and test 96 samples concurrently. A total of 48 specific peaks （9, 16, 9, and 14 for the four stages, respectively） was found in the various phase of recombinant bacteria. Conclusion MALDI-TOF MS can be used as a fast, accurate, and high-throughput method to identify recombinant bacteria, which provide a new ideas not only for recombinant bacteria but also for the identification of mutant strains and bioterrorism pathogens.

Isolation and Identification of High-Quality Lactic Acid Bacteria in Forage Corn

[ Objective] To screen suitable lactic acid bacterium strains from forage corn which can be used as silage additives. [ Method] The lactic acid bacterium strains were isolated by inoculation on MRS solid medium containing calcium carbonate, and they were preliminarily identified through morphological, physiological and biochemical experiments. The acid production efficiency was determined. Twelve strains having strong acid-pro- duction ability were selected, and their salt tolerance and acid tolerance were detected. The sequences of their 16 S rDNA were also analyzed. [ Result] A total of 44 lactic acid bacterium strains were isolated from the forage com. As evidenced by the physiological and biochemical experi- ments, the twelve strains having strong acid-production ability belonged to Leuconostoc, Lactobacillus and Enterococcus, respectively, and they had strong salt tolerance and acid tolerance. According to the sequences of 16 S rDNA, A4, B9, B11, B12 and B14 were Lactobacillus plantarum; A1, A2., A7, A11 and B8 were Leuconostoc mesenteroides dextran subspecies; and AB and A9 were Enterococcus hirae. [ Conclusion ] The lactic acid bacterium strains with strong acid-production ability isolated from forage corn can be developed into silage additives.

Investigation of Bacteria Species Most Involved in Peri-Implantitis

Purpose: Currently, bacteriological examinations of implant treatments target periodontopathic bacteria such as red complex bacteria, including Porphyromonas gingivalis, and detect them qualitatively or quantitatively. However, it seems that those examinations do not reflect the peri-implant tissue conditions precisely, because periodontopathic bacteria are also frequently detected from healthy peri-implant sites. The purpose of the present study was to investigate bacteria species most involved in peri-implantitis using a PCR method. Methods: Polymerase chain reaction (PCR) primers in this study were designed based on partial sequences of 16S rDNA of bacteria species involved in peri-implantitis that were described in numerous previous studies. Peri-implant sulcus fluid (PISF) samples were collected from thirty periodontally healthy patients with implants (HI) and thirty patients with peri-implantitis (PI). Each detection frequency of bacteria species in PISFs of both groups was investigated using a PCR method, and was compared using Fisher’s exact test. Results: In PI group, detection frequencies of Corynebacterium durum, Fretibacterium fastidiosum and Slackia exigua were significantly higher than those of HI group (p P. gingivalis and Tannerella forsythia belonging to red complex were frequently detected in the PISF samples of HI group (p > 0.05). Conclusion: It was suggested that monitoring C. durum and F. fastidiosum levels in PISF samples was useful as a clinical indicator for the evaluation of peri-implant tissue conditions.

Isolation and Identification of High-Quality Lactic Acid Bacteria from Wheat Haulm

[Objective] The aim of this study was to isolate and identify lactic acid bacteria （LAB） from wheat haulm and to select efficient strains for silage fermentation. [ Method] From 78 LAB strains isolated on the MRS solid medium containing calcium carbonate, we selected 43 strains having better acid-production ability through morphological observation, Gram staining, physiological and biochemical tests, acid production test, acid tolerance test and salt tolerance test. These strains were finally identified by sequencing 16 S rDNA. [ Result] Of the 43 LAB strains having better acid-production ability, 37 belonged to Lactobacillus paracasei subsp., one belonged to Lactobacillus rhamnosus and five belonged to Enterococcus faecium, as shown by the sequences of 16 S rDNA. [ Conclusion ] A total of 43 LAB strains having better acid-production ability were selected, which may be developed as high-quality silage additives.

Microbial Load (Bacteria, Coliform and Mould Count/Flora) of Some Common Hot Smoked Freshwater Fish Species Using Different Packaging Materials

Three different packaging materials of (37 cm × 25 cm) size (Sealed Transparent Polythene Bag (STPB) Sealed Paper Bag (SPB) (Brown envelope), Open Mouth Polythene Bag (OMPB) (Black incolour)) were used for Oreochromisniloticus (O), Clariasgariepinus (C) and Mormyrusrume (M). Twenty fish samples per species (averaging 250 gm) were hot smoked dried whole for 36 hours at an average temperature of 100?C. Packaged hot at the rate of 6 fishes per package for each species (three packs for each packaging treatment i.e. 18 pieces were packed while the remaining 2 pieces were used for initial bacteria load and microbial load). Microbial load (Total Viable Count (TVC), Total Coliform Count (TCC) and Total Fungi Count (TFC)) for the fresh fish was initial hot smoked and finally at the end of 12 weeks was monitored. The TVC (bacterial load) of O. niloticus dropped from (10.6 - 8.4) × 104 (fresh state-hot smoked) and M. rume (9.8 - 7.0) × 104, while C. gariepinus slightly increased from (12.4 - 12.6) × 104. After hot smoking, highest TVC of 8.6 × 104 (OMPBC), 8.3 × 104 (SPBC) and 8.2 × 104 (STPBC) were recorded in C. gariepinus among the 9 packag- ing at 12 weeks. However highest tendency for heavy TVC is in all OMPB with highest bacteria load in the OMPBC (8.6 × 104), 7.6 × 104 (OMPBO) and 6.6 × 104 (OMPBM). After 12 weeks highest ranged TFC of (0.6 - 0.7) × 104 was recorded in M. rume as against 0.2 × 104 recorded in the initial smoked for all. TCC was highest in C. gariepinus (4.0 - 4.3) × 104. Packaging did not limit the existence of micro-organisms. Six bacteria species (Micrococcus (acidiophilus, luteus), Bacillus (subtilis, cereus, aureus), Staphylococcus aureus, Streptococcus lactis, Proteus (vulgaricus, morganii), Pseudomonas aureginosa) and three fungi species (Aspergillus (niger, tamari), Rhizopusnigricans, fusariumoxysporum) were represented in all the packages. On the average five bacteria and two fungi species were represented, excepting for OMPBM and OMPBO with six bacteria species.

Isolation and Identification of Cellulose- decomposing Bacteria from Deep-litter Systems

In this study, 43 cellulose-decomposing strains were isolated from deep-litter systems. After preliminary screening with Congo red identification medium and filter paper strip medium, five strains with large transparent circles that disintegrated filter paper strips were obtained. After further liquid fermentation, CMC activity, FPA activity and natural eellulase activity of these five strains were determined, and two cellulose-decomposing strains with higher enzyme activity were screened, which were named F7 and F21. Based on molecular biological identification and phylogenetic analysis of 16S rRNA gene sequences, these two cellulose- decomposing strains were identified as Bacillus subtilis and Streptomyces sp. , respectively.

Role of Lactic Acid Bacteria-Myeloperoxidase Synergy in Establishing and Maintaining the Normal Flora in Man

Lactic acid bacteria (LAB) are incapable of cytochrome synthesis and lack the heme electron transport mechanisms required for efficient oxygen-based metabolism. Consequently, LAB redox activity is flavoenzyme-based and metabolism is fermentative, producing lactic acid, and in many cases, hydrogen peroxide (H2O2). Despite this seeming metabolic limitation, LAB dominate in the normal flora of the mouth, vagina and lower gastrointestinal tract in man. Myeloperoxidase (MPO) is produced by the neutrophil leukocytes and monocytes that provide the innate phagocyte defense against infecting pathogens. MPO is unique in its ability to catalyze the H2O2-dependent oxidation of chloride (Cl-) to hypochlorite (OCl-). In turn, this OCl- directly reacts with a second H2O2 to produce singlet molecular oxygen (), a metastable electronic excitation state of oxygen with a microsecond lifetime that restricts its combustive reactivity within a submicron radius of its point of generation. Each day a healthy human adult produces about a hundred billion neutrophils containing about 4 femtograms MPO per neutrophil. Inflammatory states and G-CSF treatment increase both neutrophil production and the quantity of MPO per neutrophil. After a short circulating lifetime, neutrophils leave the blood and migrate into body spaces including the mouth, vagina, urinary tract, and gastrointestinal tract. Greater than a hundred thousand neutrophils are lavaged from the mouths of healthy humans;the quantity lavaged is proportional to the blood neutrophil count. MPO selectively and avidly binds to most Gram-positive and all Gram-negative bacteria tested, but LAB do not show significant MPO binding. Neutrophils migrating to normal flora sites release MPO into the LAB-conditioned milieu containing adequate acidity and H2O2 to support extra-phagocyte MPO microbicidal action. In combination, LAB plus MPO exert a potent synergistic microbicidal action against high MPO-binding microbes. This LAB-MPO synergy provides a mechanism for the establishment and maintenance of LAB in the normal flora of man.

Treatment of engine-oil polluted wastewater with a mixed bacterial flora and kinetics of biodegradation

A mixed bacterial flora was isolated from the soil of two petroleum-contaminated sites, then cultivated and domesticated in an open environment. The bacteria were used to degrade engine oil in wastewater. The optimum biodegradation conditions for all engine oil concentrations of respectively 489 mg L-1, 1 075 mg L-1 and 2 088 mg L-1 are bacterial inoculum concentration of 0.1%, temperature at 30 °C to 35 °C, pH 7.0 to 7.5, and rotation at 190 r min-1 to 240 r min-1. The second-order kinetic model proposed by Quiroga and Sales describes the characteristics of the biodegradation of the engine oil very well. Engine oil concentration barely changes the growth rate of the bacterial consortium. The mixed bacterial flora has a high biodegrading capability for engine oil.

Identification and Mutagenesis of Lactic Acid Bacteria from Chinese Sauerkraut

In order to analyze the fermentation properties of lactic acid bacteria in Chinese sauerkraut and to improve acid production, 21 samples of Chinese sauerkraut from Inner Mongolia and Northeast China were collected and isolated with a Man-Rogosa-Sharpe(MRS) culture. Sixteen strains of lactic acid bacteria were identified by combining both phenotype and genotype methods. After activation,the 16 strains were inoculated into the MRS medium with a concentration of 4%and then incubated at 37 ℃. The p H and the absorbance of the culture were measured. The activated strains were then mutagenized in a field of 4 KV/cm mutation,with dosages administered within 20 minutes and 30 minutes, respectively. The variation curves of the p H and the absorbance of the culture were determined. The experimental results showed that the lactic acid bacteria isolated from the soup were identified as Lactobacillus and the acid production of the bacteria was significantly improved by the mutagenesis of the corona electric field.

Gram-Positive Bacteria Associated with Rice in China and Their Antagonists Against the Pathogens of Sheath Blight and Bakanae Disease in Rice

It is necessary to understand the bacterial populations associated with rice so as to provide more information and natural resources for effective management of major diseases in rice. A survey on screening and identification of gram-positive bacteria was conducted during 1998 2004. Seven hundred and fifty-six rice samples were collected from Zhejiang, Jiangsu, Fujian and Yunnan Provinces, China. Over 1000 bacterial isolates were isolated and tested for colony morphology, pathogenicity, and some characteristics of bacteriology including Gram staining, fluorescent pigment on Kings medium B and microscopic observation for endospore. Together with five standard reference strains, 74 representative gram-positive bacterial isolates were confirmed by Biolog and gas chromatographic analysis of fatty acid methyl esters. Five bacterial species of Bacillus and other three genera were identified and isolates from Bacillus sublitis and Bacillus megaterium, exhibited the most effective inhibition against the pathogens of sheath blight and bakanae disease of rice. A few isolates from Bacillus pumilus and Bacillus megaterium showed weak virulent on rice together with some virulent isolates, risk should be considered when isolates from these species were screened for biocontrol agents.

Cloning of Bile Salt Hydrolase Gene and Its Expression in Lactic Acid Bacteria

According to the sequence of the bile salt hydrolase （BSH） gene of Bifidobacterium and the restriction enzyme cutting sites of expression vector pNZ8148, primers were designed and the bile salt hydrolase （BSH） gene was gotten from Bacillus bifidus ATCC 29521 by PCR. BSH gene was inserted into lactic acid bacteria expression vector pNZ8148 to construct the recombinant pNZ8148-BSH. The recombinant pNZ8148-BSH was transferred into lactic acid bacteria NZ9000 with electrotransformation method. And the recombinant which could express BSH protein was obtained. It was identified by SDS-PAGE electrophoresis and activity verification. The result could provide a rationale reference for expressing BSH in lactic acid bacteria.

Genetic Relationships of Soft Rot Bacteria Isolated from Konjac in China by Amplified Fragment Length Polymorphism (AFLP) and 16S rDNA Gene Sequences

Twenty-three isolates of soft rot bacteria from konjac corms were examined for their diversity using 16S rDNAs and AFLP technology. Both methods clustered two groups, dependent on their biotype characterization of Pectobacterium carotovora subsp. carotovora (P.c.c) and Pectobacterium chrysanthemi (P.ch), respectively. Of all isolates, 17 (73.9%) belonged to P. ch, indicated as the main pathogenic bacteria of konjac producing areas in China. The genetic variation among isolates from the same biotype was also rich, not consistent with the distances of the geographic sources.

Assessment of biotechnological potential of phosphate solubilizing bacteria isolated from soils of Southern Kazakhstan

Phosphorus (P) is a vital plant nutrient, available to plant roots only in soluble forms that are in short supply in the soil. Adding phosphate- based fertilizers to increase agricultural yields is a widely used practice;however, the bio- availability of P remains low due to chemical transformations of P into insoluble forms. Thus, phosphate solubilizing bacteria (PSB) play an important role in reducing P deficiency in soil. The goal of this study was to assess biotechnological potential of phosphate-solubilizing bacterial strains. In this study, phosphate solubilizing microorganisms (PSM) were isolated from different soil samples of Southern regions of Kazakhstan. The biological activity of PSM was studied based on their effect on the growth of wheat seeds. The different taxonomic genera of these PSM were identified: Arthrobacter spp., Aureobacterium spp., Azotobacter spp., Bacterium spp., Baccillus spp. Finally, phosphate- solubilizing activity of isolated strains of PSM was assessed.

Model identification with BPNN on restrictive ecological factors of SRB for sulfate-reduction

The model of back-propagation neural network (BPNN) was presented to demonstrate the effect of restrictive ecological factors, COD/SO 4 2- ratio, pH value, alkalinity (ALK) and SO 4 2- loading rate (Ns), on sulfate reduction of Sulfate Reducing Bacteria (SRB) in an acidogenic sulfate reducing reactor supplied with molasses as sole organic carbon source and sodium sulfate as electron acceptor. The compare of experimental results and computer simulation was also discussed. It was shown that the method of BPNN had a powerful ability to analyze the ecological characteristic of acidogenic sulfate reducing ecosystem quantitatively.

Application of Lactic Acid Bacteria(LAB) in Freshness Keeping of Tilapia Fillets as Sashimi

Aquatic products are extremely perishable food commodities. Developing methods to keep the freshness of fish represents a major task of the fishery processing industry. Application of Lactic Acid Bacteria(LAB) as food preservative is a novel approach. In the present study, the possibility of using lactic acid bacteria in freshness keeping of tilapia fillets as sashimi was examined. Fish fillets were dipped in Lactobacillus plantarum 1.19(obtained from China General Microbiological Culture Collection Center) suspension as LAB-treated group. Changes in K-value, APC, sensory properties and microbial flora were analyzed. Results showed that LAB treatment slowed the increase of K-value and APC in the earlier storage, and caused a smooth decrease in sensory score. Gram-negative bacteria dominated during refrigerated storage, with Pseudomonas and Aeromonas being relatively abundant. Lactobacillus plantarum 1.19 had no obvious inhibitory effect against these Gram-negatives. However, Lactobacillus plantarum 1.19 changed the composition of Gram-positive bacteria. No Micrococcus were detected and the proportion of Staphylococcus decreased in the spoiled LAB-treated samples. The period that tilapia fillets could be used as sashimi material extended from 24 h to 48 h after LAB treatment. The potential of using LAB in sashimi processing was confirmed.

Antagonistic Effects of Sphingomonas and Pseudomonas aeruginosa on 4 Kinds of Pathogenic Bacteria of Ginseng

[Objectives]To explore effective biocontrol methods for diseases in the process of ginseng cultivation,and develop an efficient and environmentally friendly biocontrol agent.[Methods]In this study,2 strains were isolated from biogas slurry,and Cylindrocarpon destructans(XF),Fusarium solani(GF),Botrytis cinerea Pers(HM)and Alternaria panax Whetz(HB)were used as test materials.The strains were isolated and identified by dilution plate method,16S rDNA sequence identification method,confrontation culture method,filter paper method and ultraviolet spectrophotometer method,and the bacteriostatic activity and bacteriostatic rate were tested.[Results]Strain 15(Sphingomonas)and strain 19(Pseudomonas aeruginosa)were screened out through identification and analysis,and they grew stably within 8-10 d.The bacteriostatic rates of strain 15 against A.panax and B.cinerea were 47.37%and 43.40%,respectively,and the bacteriostatic rates of strain 19 against A.panax and B.cinerea were 62.30%and 63.27%,respectively.The bacteriostatic activity of the extract of strain 19 increased with the increase of OD_(600) value,and the bacteriostatic effect was optimal when the OD_(600) value was in the range of 0.8-1.0,up to 70%,so it had a strong biocontrol potential.[Conclusions]This experiment provides convenience for more effective inoculation,establishes a fast,simple and accurate method for the determination of the best bacteriostatic rate of P.aeruginosa culture solution to HM,and lays a foundation for large-scale culture of P.aeruginosa culture solution.Besides,it is expected to provide a theoretical basis for the efficient control of ginseng B.cinerea in field production,use it for the prevention and control of ginseng shoot diseases,and provide a reference for the efficient and diverse development of biocontrol agents for ginseng shoot diseases.

Heterotrophic Bacterial Flora in Aquaculture Area around Xuejiadao

From Oct., 1999 to Oct., 2000, the heterotrophic bacterial flora in the aquaculture area around Xuejiadao was investigated. The result shows that the populations of the heterotrophic bacteria are heavier in summer and autumn than those in winter and spring. The average populations in seawater, sediment, the surface of seaweed and the surface of fish are 1.4×10 4 cfu mL -1, 5.4×10 6 cfu g -1, 1.5×10 6 cfu g -1 and 1.8×10 3 cfu cm -2, respectively. A total of 301 strains were isolated, among them 259 were Gram-negative. All the Gram-negative bacteria belong to 13 genera and some genera of Enterobacteriaceae. The communities of bacteria are slightly different among the samples. In the body surface of fish, Genus vibrio is dominant. In the remaining samples, dominant genus is Aeromonas.

Heterotrophic Bacterial Floras in Industrial Area and Unpolluted Marine Environments around Qingdao

From Oct. 1999 to Oct. 2000, the heterotrophic bacterial floras in the industrial marine environment around the Qingdao Power Plant (QPP) and in the unpolluted marine environments were investigated. The results showed that the numbers of the heterotrophic bacteria around QPP were much higher than those in unpolluted environments, and the average numbers in QPP Seawater, QPP Sediment, Unpolluted Seawater and Unpolluted Sediment were 5.4×104cfu(mL) -1, 5.0×105cfug -1, 3.0×102cfu(mL) -1 and 1.3×105cfug -1 respectively. Totally, 118 strains were isolated from QPP and 99 of them were Gram-negative. One hundred and twenty one strains were isolated from the unpolluted environments and 104 of them were Gram-negative. All the Gram-negative bacteria belonged to 13 genera. The distribution of the bacteria was varied in different marine environments. The results showed that the unpolluted marine environments contained much more Vibrio than seawater and sediment around QPP.

Design and application of the method for isolating magnetotactic bacteria

A simple apparatus was designed to effectively isolate magnetotactic bacteria from soils or sediments based on their magnetotaxis. Through a series of processes including sample incubation, MTB harvesting, isolation, purification and identification, several strains of bacteria were isolated from the samples successfully. By Transmission Electron Microscopy (TEM) and Energy-Dispersive X-ray Analysis (EDXA), these bacteria were certificated to be magnetotactic bacteria. The phylogenetic relationship between the isolated magnetic strains and some known magnetotactic bacteria was inferred by the construction of phylogenetic tree based on 16SrDNA sequences. This apparatus has been proven to have the advantages of being inexpensive, simple to assemble, easy to perform and highly efficient to isolate novel magnetotactic bacteria. The research indicated that the combined approach of harvesting MTB by home-made apparatus and the method of plate colony isolation could purify and isolate magnetotactic bacteria effectively.