Lactobacillus rhamnosus GR-1 attenuates foodborne Bacillus cereus-induced NLRP3 inflammasome activity in bovine mammary epithelial cells by protecting intercellular tight junctions

Background:Bacillus cereus is an important pathogen that causes human food poisoning,specifically diarrhea and vomiting.B.cereus can also induce mastitis in dairy cows and has a strong survival ability in milk,as it cannot be inactivated by high-temperature short-time pasteurization.Therefore,B.cereus can enter the market through pasteurized milk and other dairy products,imposing enormous hidden dangers on food safety and human health.Results:In this study,B.cereus 2101(BC)was isolated from milk samples of cows with mastitis.BC grew rapidly with strong hemolysis,making it difficult to prevent mastitis and ensure food security.MAC-T cells were treated with BC and/or Lactobacillus rhamnosus GR-1(LGR-1).Pretreatment with LGR-1 protected the integrity of tight junctions and the expression of zonula occludens-1(ZO-1)and occludin destroyed by BC.Furthermore,LGR-1 pretreatment reduced the expression of NOD-like receptor family member pyrin domain-containing protein 3(NLRP3),caspase recruitment and activation domain(ASC),Caspase-1 p20,gasdermin D(GSDMD)p30,inflammatory factors(interleukin(IL)-1βand IL-18),and cell death induced by BC.Moreover,LGR-1 pretreatment reduced NLRP3 inflammasome activity and increased expressions of ZO-1 and occludin induced by lipopolysaccharides(LPS)+ATP stimulation.MAC-T cells were transfected with NLRP3 si RNA or MCC950 and/or treated with BC and/or LGR-1.NLRP3-si RNA transfection and MCC950 attenuated BC-induced NLRP3 inflammasome activity.Expression of inflammatory cytokines and cell death suggested that the inflammatory pathway might play an important role in the induction of the NLRP3 inflammasome by BC and the protection of LGR-1.Conclusions:These results suggest that LGR-1 might be a probiotic alternative to antibiotics and could be administered to prevent mastitis in dairy cows,thus ensuring food security.

Biofilm formation under high temperature causes the commensal bacteria Bacillus cereus WPySW2 to shift from friend to foe in Neoporphyra haitanensis in vitro model

Although biofilm formation may promote growth,biofilms are not always beneficial to their hosts.The biofilm formation characteristics of Bacillus cereus WPySW2 and its changes at different temperatures were studied.Results show that B.cereus WPySW2 promoted the growth of Neoporphyra haitanensis(an economically cultivated seaweed)at 20℃ but accelerated algal rot at 28℃.Thicker B.cereus WPySW2 biofilms covered the surface of N.haitanensis thalli at 28℃,which hindered material exchange between the algae and surrounding environment,inhibited algal photosynthesis and respiration,and accelerated algal decay.Compared with planktonic bacteria,mature biofilm cells had lower energy consumption and metabolic levels.The biofilm metabolic characteristics of B.cereus WPySW2 changed significantly with temperature.High temperature accelerated biofilm maturation,which made it thicker and more stable,allowing the bacteria to easily adapt to environmental changes and obtain greater benefits from their host.High temperature did not affect the production or increased the abundance of toxic metabolites,indicating that the negative effects of B.cereus WPySW2 on algae were not caused by toxins.This study shows that increased temperature can transform a harmless bacterium into a detrimental one,demonstrating that temperature may change the ecological function of phycospheric bacteria by affecting their morphology and metabolism.

高效苯酚降解菌Bacillus sp. L5-1的分离及其降解特性

从污水处理厂活性污泥中分离筛选出一株高效苯酚降解菌L5-1,经菌落形态观察和16S rDNA基因测序,结果表明菌株L5-1为蜡样芽胞杆菌(Bacillus cereus),美国国家生物信息中心(NCBI)的注册号为MN784421.将苯酚设置为唯一碳源,对其生长和苯酚降解特性展开研究.结果表明:菌株L5-1在10%接种量、温度30~35℃、pH值7~8的条件下,均能高效降解培养基中苯酚(培养基体积为100mL,初始苯酚浓度为500mg/L,14h时降解率>93%).而在最优降解条件下(10%接种量,培养温度为35℃,pH值7.0,NaCl浓度为1%),初始苯酚浓度为500mg/L,菌株在14h内的苯酚降解率可达97.1%;而当初始苯酚浓度为1000mg/L,菌株也可在46h内达到97.71%的降解率.运用Haldance方程动力学模拟菌株在不同浓度苯酚下的生长过程,其最大比生长速率为0.355h^(-1),半饱合常数104.27mg/L,抑制常数为322.83mg/L,R^(2)=0.997.菌株L5-1为目前已报道的Bacillus菌属中降解苯酚能力较强的菌株,为实际处理含酚废水中提供理论参考.

Anaerobic benzene biodegradation by a pure bacterial culture of Bacillus cereus under nitrate reducing conditions

A pure culture using benzene as sole carbon and energy sources was isolated by screening procedure from gasoline contaminated soil.The analysis of the 16S rDNA gene sequence,morphological and physiological characteristics showed that the isolated strain was a member of genus Bacillus cereus.The biodegradation performance of benzene by B.cereus was evaluated,and the results showed that benzene could be efficiently biodegraded when the initial benzene concentration was below 150 mg/L.The metabolites of anaerobic nitrate-dependent benzene oxidation by strain B.cereus were identified as phenol and benzoate.The results of substrate interaction between binary combinations for benzene,phenol and benzoate showed that the simultaneous presence of benzene stimulated the degradation of benzoate,whereas the addition of benzene inhibited the degradation of phenol.Benzene degradation by B.cereus was enhanced by the addition of phenol and benzoate,the enhanced effects were more pronounced at higher concentration.To our knowledge,this is the first report that the isolated bacterial culture of B.cereus can efficiently degraded benzene under nitrate reducing conditions.

Surface reaction of Bacillus cereus biomass and its biosorption for lead and copper ions

In this study, the surface chemical functional groups of Bacillus cereus biomass were identified by Fourier transform infrared （FTIR） analytical technique. It had been shown that the B. cereus cells mainly contained carboxyl, hydroxyl, phosphate, amino and amide functional groups. The potentiometric titration was conducted to explain the surface acid-base properties of aqueous B. cereus biomass. The computer program FITEQL 4.0 was used to perform the model calculations. The optimization results indicated that three sitesthree pKas model, which assumed the cell surface to have three distinct types of surface organic functional groups based on the IR analysis results, simulated the experimental results very well. Moreover, batch adsorption experiments were performed to investigate biosorption behavior of Cu（Ⅱ） and Pb（Ⅱ） ions onto the biomass. Obviously, the adsorption equilibrium data for the two ions were reasonably described by typical Langmuir isotherm.

Control Effect of Bacillus cereus CGMCC4348 against Botrytis cinerea and Its Mechanism

[ Objectivel The paper was to study antifungal activity and control mechanism of Bacillus cereus CGMCCA348 against Botrytis cinerea, and further find new methods against the disease. [Method] By quantitative bioassay, in vitro experiment and pot test, the antagonistic protein was extracted with ammonium sul- fate precipitation fractionation to determine the control efficiency. ECso of B. cereus CGMCC4348 was 6. 19 mg/L. Average controlling effect of B. cereus CGM- CC4348 on B. cinerea was 75.8%, equal to the control group which was treated by 50 mg/L pyrimethanil. Bacillus cereus CGMCCA348 had good inhibitory effect against B. cinerea. [ Conclusion] The paper provides scientific basis for green control technology against solanaceous vegetable diseases.

Characteristics of Bacillus cereus Spore Counts in Soils w ith Underlying Gold Mineralization in Northwestern Sichuan, China: Results from a Pilot Study

A pilot survey on a microbial mineral exploration method based on so il Bacillus cereus spore counts was carried out across three different gold mi ning regions, which vary in soil type, climate condition and geological setting in northwestern Sichuan, China. B horizon soils from these sites were analyzed for B. cereus spores, Au, Ag, Cu, Pb and Zn. The results show that the numbe rs of B. cereus spores generally increased in soils overlying gold mineraliz ation. Specifically, elevated spore counts were found in samples slightly offset from the outcrops of orebodies, whereas soils directly above the outcrops of or ebodies usually contained low spore counts. However, the background counts of B. cereus spores varied from place to place and were complicated by environmen tal and pedological factors, but the relative ratios of spore counts still were indicative of the underlying gold mineralization.

Differentiation Between Bacillus thuringiensis and Bacillus cereus by 16S rDNA-PCR and ERIC-PCR

16S rDNA and ERIC （Enterobacteia Repetitive Intergenic Consensus Sequences） based on PCR method were tested for the effectiveness of the differentiation of B. thuringiensis and B. cereus. 16S rDNA-PCR primers were designed based on the sequence difference in variable regions of B. cereus 16S rDNA and B. thuringiensis 16S rDNA, 16S rDNA-PCR showed no obvious difference between B. cereus and B. thuringiensis. The only difference was that one 1600-bp amplificon could be obtained from all the three B. Cereus strains, and none amplificon from any B. thuringiensis strains. ERIC was optimized based on previous reports. The genonlic DNA was used for the template of ER1C-PCR, and the following DNA fingerprints were analyzed by the agarose gel electrophoresis. The results showed that DNA fingerprint of three B. thuringiensis strains had a unique amplicon less than 100-bp, while DNA fingerprint of three B. cereus＂ strains had none. Moreover, DNA fingerprint of B. cereus showed a 700-bp amplicon, but didn＇t have any DNA fingerprints ofB. thuringiensis genome. Therefore, ERIC-PCR technique should be able to be used for the differentiation of B. thuringiensis and B. cereus.

Assessment of a short phylogenetic marker based on comparisons of 3’end 16S rDNA and 5’end 16S-23S ITS nucleotide sequences of the Bacillus cereus group

A short phylogenetic marker previously used in the reconstruction of the Order Bacillales and the genus Bacillus was assessed here at a lower taxa level: species in the Bacillus cereus group: B. anthracis, B. cereus, B. thuringiensis and B. weihenstephanensis. This maker is 220 bp in length. It is a combination of 150 bp at the 3’ end of the 16S rDNA and 70 bp at the 5’ end of the 16S-23S ITS sequence. Three additional Bacillus species, B. halodurans, B. licheniformis and B. subtilis, and Clostridium tetani were included for comparison purposes. A total of eight bacterial species and 12 strains were analyzed. A boot- strapped neighbor-joining tree was inferred from comparative analyses of all allelic sequences of the bacterial species and strains under study. Based on its topology, four major Groups were revealed at the 90% nucleotide sequence identities, Group I to IV. Group I contains all al-leles of the Bacillus cereus group. Group II con-tains all alleles of B. halodurans. Group III con-tains all alleles of B. licheniformis and B. subtilis. Group IV contains all alleles of Clostridium tetani. The 220 bp phylogenetic marker used here could resolve different species from different genera. At the genus level, distant species could be dis-tinguished. Very closely-related species, however, were undistinguishable. Species in the B. cereus group, most notably B. cereus, B. anth- racis and B. thuringiensis, could not be distin- guished. After successfully inferring the phylo- genies of the Order Bacillales and the genus Bacillus, we have met the resolving limit of this short phy-logenetic marker: B. cereus, B. anthracis and B. thuringiensis.

BIOSYNTHESIS AND THERMAL PROPERTIES OF POLY(3-HYDROXYBUTYRATE-co-3-HYDROXYVALERATE)WITH LARGE VARIETY OF HYDROXYVALERATE CONTENTS BY BACILLUS CEREUS

Biosynthesis and thermal properties of poly（3-hydroxybutyrate-co-3-hydroxyvalerate） （PHBV） with different HV （hydrovalerate） content produced by a Bacillus cereus strain were investigated. A large variety of HV contents （up to about 90 mol%） of PHBV could be produced by this strain. Combined nitrogen sources containing both yeast extract and ammonium sulphate were better for cell growth and polyhydroxyalkanoates （PHA） production than either yeast extract or ammonium sulphate alone. Propionic acid is more favorable for the production of HV content than that of valeric acid. Finally, thermal properties of PHBV produced by this strain are found close to the results of other groups.

Identification of genes involved in regulating MnSOD2 production and root colonization in Bacillus cereus 905

sodA2-encoding manganese-containing superoxide dismutase(MnSOD2)in Bacillus cereus 905 plays an essential role in antioxidative stress,nutrition utilization,rhizosphere and phyllosphere colonization.However,the genes involved in regulating the sod A2 expression have not been clearly elucidated in B.cereus.In this study,a genome-wide random insertion mutagenesis was constructed by using transposon TnYLB-1 to identify novel genes regulating the sodA2 expression.Seven mutants that changed the sodA2 expression at both m RNA and protein levels were finally obtained.Sequence analysis and BLAST data showed that the genes disrupted by TnYLB-1 in B.cereus 905 shared high conservations with those in the B.cereus type strain,ATCC 14579.These genes encode heat-inducible transcription repressor,chloride channel protein,recombinase A,ferrous iron transport protein,nucleoside diphosphate kinase,and histidine ammonia-lyase.Besides,we also provided the evidence that the genes regulating the sodA2 expression could influence colonization ability of B.cereus 905 on wheat roots.Specifically,those genes downregulating the sodA2 expression significantly reduced bacterial colonization on wheat roots,and mutants with increased MnSOD2 activities could enhance bacterial population densities on wheat roots to a certain degree.Our work provided information that multiple genes are involved in MnSOD2 production and wheat root colonization.The molecular regulatory pathways through which these genes modulate the sod A2 expression and root colonization need to be investigated extensively in the future.

Study on Relationship Between Differential Proteins of Bacillus cereus LBR-4 and Its Salt Tolerance Mechanism

In order to explore the salt tolerance mechanism of Bacillus cereus LBR-4 with salinity of 14%NaCl,differential proteomic analysis of the whole protein of LBR-4 strain expressed under 14%NaCl high salinity condition and normalculture condition(1%NaCl)was studied by two-dimensional electrophoresis and mass spectrometry.The isoelectric point of most detected proteins was between pH 4-7 and the molecular weight distribution was 10-70 ku.Compared with the normal culture condition,the expression level of 118 protein spots in the whole protein expression map changed significantly(accounting for 25.2%of the total protein spots).The expression level of 78 protein spots increased significantly,including 22 new protein spots that appeared under high salt stress.The expression levels of 40 protein spots decreased significantly,including 18 protein spots that disappeared under high salt stress.By mass spectrometry,six distinct differentially expressed protein spotswere dihydroxy acid dehydratase,cell division protein FtsZ,iron sulfur cluster synthesis protein SufD,unknown carboxylase YngE,hypothetical acetaldehyde dehydrogenase DhaS and phenylalanine acid tRNA ligase alpha subunit.It was speculated that under high salt stress,the cells had protective measures and the secretion of intracellular compatible solutes increased.The iron and sulfur clusters involved in various physiological reactions also activated the stressful suf synthesis pathway,and therate of cell division and reproduction was also slowed down and ensured the normal progress of physiological reactions inthe cells.

Phenotypic plasticity in Bacillus cereus strains isolated from various Antarctic habitats

We studied five strains of psychrotolerant Bacillus cereus （B. cereus） isolated from Antarctic snow （BCsn）, ice （BCic）, lake water （BCwt）, sediment （BCsd）, and soil （BCsl） samples in terms of their growth, biochemical properties, and heat shock re- sponses. Analyses of growth kinetics at 4℃ showed that BCsn had the fastest generation time （16.1 h）, whereas BCWT had the slowest （30.8 h）. Strain BCsd formed the largest zone of lipid hydrolysis （18 mm） whereas BCsn formed the smallest zone （3 mm）.Only BCsd produced gelatinase. These physiological differences illustrate adaptations of B. cereus isolates to different niches. Strains BCsl and BCwr were resistant to all 12 of the antibiotics tested. Strains BCsn, BCio, and BCsd were resistant to cell wall synthesis inhibitors （penicillin and ampicillin） and susceptible to protein synthesis inhibitors （tetracycline and streptomycin）. A carbon-substrate utilization assay revealed that BCsn, BCic and BCwr could specifically utilize D-glucose-6-phosphate, salicin, and 2＇-deoxyadenosine, respectively, indicating a degree of metabolic diversity among these Antarctic B. cereus strains. An analy- sis of heat shock proteins （HSPs） produced in response to a 60℃ heat treatment revealed significant variations in the amounts of HSP33 （p = 0.01, df= 4）, HSP44 （p = 0.003, dr= 4）, and HSP60 （p = 0.04, df= 4） among the strains. This emphasizes the impor- tance of HSPs in bacterial taxonomy. These results show that there are considerable adaptive variations among B. cereus strains from extremophilie environments. This could be significant in evaluating the taxonomy and evolution of this species.

A Potential Unhairing Protease from a Newly Isolated Bacillus cereus

[Objective] This study aimed to illustrate a protease, secreted from a newly isolated Bacillus cereus, and show its potential application in eeofriendly tannery processing. [Method] The skins, such as bovine, ovine, hircine and porcine skins, were treated alone by the protease, which was secreted from a newly isolated Bacillus cereus, for 12 h without sulfide till the hairs were removed entirely. And then the difference of histological appearances treated with and without proteases was analyzed to reveal the dehairing mechanism. [ Result] Histological examination of skins dehaired by the crude enzymes revealed that no obvious chan- ges were observed in the dennis. Therefore it may be speculated that the proteases would firstly help in the selective breakdown of keratin tissue in the follicles viz. hydrolyzing the outer epithelial sheath of hair roots of the epidermis, provoking depilation, thereby intact hairs were pulled out without breaking other tissues of the skin. [ Conclusion] The protease produced by Bacillus cereus, strain SZ-4 could be in a great measure applied in unhairing in beamhouse of leather making exhibiting considerable unhairing capability without affecting the grain of skins as well as the tensile strength of the leather.

Optimization of Pulsed-field Gel Electrophoresis Procedure for Bacillus cereus

In order to develop a rapid and reliable method for B. cereus genotyping, factors influencing PFGE results, including preparation of bacterial cells embedded in agarose, lysis of embedded cells, enzymatic digestion of intact genomic DNA, and electrophoresis parameters allowing for reproducible and meaningful DNA fragment separation, were controlled. Optimal cellular growth （Luria-Bertani agar plates for 12-18 h） and lysis conditions （4 h incubation with 500 μg/mL lysozyme） produced sharp bands on the gel.

Isolation,Identification of Bacillus Thuringiensis/Cereus and Its Enhancement on Protein Wastewater Treatment by Rhodobacter Sphaeroides

In order to enhance the degrading protein capability of purple non-sulfur bacteria(PNSB),an effective strain,L2,was used to co-culture with Rhodobacter sphaeroides ATCC17023.The effects of added strain on protein removal of R.sphaeroides were investigated.Results showed that strain L2,being identified as Bacillus thuringiensis/cereus,had a high potential for producing protease with a production of 295 U/m L.The optimal B.thuringiensis/cereus(40 μL) could significantly increase protein degradation of R.sphaeroides.Protein removal and biomass production were improved by 483% and 67%,respectively.R.sphaeroides/total biomass production was more than 95%.Theoretical analysis revealed that R.sphaeroides syntrophically interacted with B.thuringiensis/cereus.Protein degradation of B.thuringiensis/cereus provided small molecule substrates(VFAs) for R.sphaeroides growth and cells materials synthesis.

Antibiotics Resistance Profile of Bacillus cereus Strains Isolated from Soil and Pepper in Brazzaville

Introduction: Bacillus cereus and spores produced in various ecological niches are responsible for toxic infections in humans. This study is conducted to determine the antibiotics resistance profile of B. cereus strains isolated from soil and pepper consummated in Brazzaville. Methodology: An antimicrobial susceptibility test of 16 B. cereus strains from soil and peppers was performed using 11 antibiotics by the Kirby-Bauer’s diffusion on disc method. Results: Results revealed 100% (16/16) of resistance in penicillin G, amoxicillin, ceftazidime, rifampicin, and colistin, also 18.75% (3/16), 11.76% (2/16), and 18.75% (3/16) of resistance in doripenem, vancomycin and chloramphenicol respectively. In addition, we have observed 100% (16/16), 81.25% (13/16), 76.47% (13/16), 35.29% (5/16), 35.50% (6/16), and 12.5% (2/16) of sensitivity to line-zolid, tigecycline, ciprofloxacin, vancomycin, doripenem and chloram-phenicol respectively. However, all strains have been multidrug resistant (MDR) to betalactams, polypeptides, and ansamycins. Moreover, 7 strains (43.75%) have been variably multiresistant. One strain, Ri10 has been resistant to beta-lactams, polypeptides, ansamycins, cyclins and glycopeptides. No strain was ultraresistant (XDR) or largely insensitive (PDR) to different antibiotics. Conclusion: This study reveals that 51% of strains have been resistant to antibiotics, 32% are sensitive, and 17% have intermediate resistance. These results partly explain the high rate of gastroenteritis observed in Brazzaville due to food poisoning.

Application of Antimicrobial Bacillus subtilis Strain as a Starter Culture to Improve Qualities and Safety of Fermented Soybean (SIENG) Produced in Cambodia

Fermented foods play a very important role in Cambodian health and nutrition,as well as other developing countries where food preservation methods may be limited.SIENG,a Khmer fermented soybean product,naturally contains both beneficial and pathogenic microorganisms.Traditional fermentation that relies on natural microbial flora and environmental conditions results in variable product quality and can lead to spoilage.A starter culture such as Bacillus subtilis can ensure the safety and stability of the products.The objective of this study is to control the growth of Gram positive pathogens contaminated into traditional fermented soybean(SEING)by using antimicrobial Bacillus subtilis isolated from the same kind of food.Out of 120 SIENG samples,49 B.cereus strains were isolated,and 12 of B.cereus were positively synthesized compared with the lyophilized control enterotoxin.Two of these strains(BTM8-7 and BTM8-8)produced high levels of enterotoxin.We identified five Bacillus strains with the ability to fight against indicator pathogenic microorganisms.Among the five strains,B.CeM6-2 had the highest activity level against Lactobacillus plantrum ATCC 8014 and the largest diameter.B.CeM6-2 tolerated up to 20 h at 30℃ and 22 h at 37℃.In testing the strains with PK and PK-PMSF enzymes,bacteriocin produced by the strain B.CeM6PK untreated and B.CeM6-2PK-PMSF had a significantly stronger ability to suppress all the pathogenic indicators from 0 h to 47 h compared to the B.CeM6-2PK.Moreover,CeM6-2 outperformed the Miyagino strains,as it actively produced bacteriocin that fought against all four indicator strains of Gram positive and lactic acid groups,especially against Listeria monocytogenes,Streptococcus pyrogene,Leuconostoc mesenterids and L.plantarum,from 0 h-58 h and 0 h-40 h at 35℃.CeM6-2(1%)strain had the highest ability to fight against B.cereus at 24 h and at 34 h to 44 h incubation as well.CeM6-2(10:0 mL)and CeM6-2(9:1 mL)have the strongest ability to fight against B.cereus at room temperature(48 h and 72 h).The longer incubation and time at room temperature produce the highest level of bacteriocin.Thus,bacteriocins produced by B.CeM6-2 can be used as a preservative in food processing industries to avoid food spoilage even in higher temperatures and time.

Comparative Analysis of Various Strains of Plant Growth Promoting Rhizobacteria on the Physiology of Garlic (Allium sativum)

Garlic is a most important medicinal herb belonging to the family Liliaceae. Both its leaves and bulb are edible. The current study was based on evaluating the growth promoting potential of plant growth promoting rhizobacteria (PGPR) on garlic (Allium sativum L.) growth and biochemical contents. Garlic cloves were inoculated with 3 kinds of PGPRs, Pseudomonas putida (KX574857), Pseudomonas stutzeri (Kx574858) and Bacillus cereus (ATCC14579) at 108 cells/mL prior to sowing. Under natural conditions, plants were grown in the net house. The PGPR significantly enhanced % germination, leaf and root growth and their biomass also increased the diameter of bulb and fresh and dry weight. The flavonoids, phenolics, chlorophyll, protein and sugar content were also significantly increased due to PGPR inoculation. The Pseudomonas stutzeri was found most effective for producing longer leaves with moderate sugar, high flavonoids (129%) and phenolics (263%) in bulb over control (Tap). The Pseudomonas putida exhibited a maximum increase in bulb diameter and bulb biomass with maximum phenolics and flavonoid contents.

Transcriptomic analysis of biofilm formation by Bacillus cereus under different carbon source conditions

Background:Previous studies found differences in the utilization of different carbon sources during biofilm formation by Bacillus cereus.Illumina HiSeq high-throughput sequencing technology was used to investigate the changes in gene transcript levels in Bacillus cereus biofilm bacteria under different carbon source conditions.Results:Compared with the control group,the number of differentially expressed genes in the glucose,maltose,lactose,and skim milksupplemented groups was 351,1136,133,and 487,respectively.The results showed that the pathways involved in the differentially expressed genes were mainly distributed in glycolysis and pentose phosphate pathway,tricarboxylic acid cycle,amino acid metabolism,and fatty acid metabolism.The gene expression of enzymes related to acetoin synthesis from pyruvate was mostly upregulated in the glucose-supplemented group.The gene expression of enzymes related to pyruvate synthesis of branched-chain amino acids in the maltose-supplemented group was mostly upregulated.In the lactose-supplemented group,the gene expression of acetoin biosynthesis from pyruvate was upregulated.Pyruvate production through glycolysis pathway increased in the skim milk-supplemented group,but the metabolic capacity of the tricarboxylic acid cycle did not change significantly.Conclusion:The content of pyruvate stored by Bacillus cereus biofilm bacteria through glycolysis or pentose phosphate pathway increased,but the carbon flux into the tricarboxylic acid cycle did not increase,which suggested that carbon fluxes in the extracellular polysaccharide synthesis pathway of the biofilm may be increased,resulting in increased biofilm biomass formation.