The effect of carbon dioxide on bacterial growth and on uptake processes in bacterial membrane vesicles

The effect of different concentrations of carbon dioxide on growth rates of Escherichia coli, Bacillus sublitis, Bacillus cereus and Pseudomonas aeruginosa was studied by means of a microculture system. For B. subtilis and P. aeruginosa, growth rate was linearly inhibited up to a concentration of 40% carbon dioxide. For E. coli and B. cereus, the inhibition of growth rate followed non-linear curves (concave downwards) in the whole concentration range (5–80%). Uptake of amino acids in E. coli and B. subtilis membrane vesicles was inhibited only half as much as growth, and uptake of glucose in E. coli membrane vesicles was unaffected even by 100% carbon dioxide. These experiments seem to indicate that the main mechanism of action for carbon dioxide is not associated with physical disturbance of the bacterial membrane.     

Pyrococcus yayanosii L-天冬酰胺酶在枯草芽孢杆菌中分泌途径的鉴定及其分泌能力的提高

食品安全（GRAS）菌株枯草芽孢杆菌由于其具有良好的分泌表达能力及易于基因工程操作等特性被广泛用作外源蛋白质的表达菌株。枯草芽孢杆菌中蛋白质的分泌大多依赖于信号肽介导的Sec分泌途径（General secretion pathway）和Tat分泌途径（Twin-arginine translocation pathway）。在本研究中,通过信号肽预测、蛋白质N端测序等手段发现来自于Pyrococcus yayanosii L-天冬酰胺酶在枯草芽孢杆菌中的分泌并不依赖信号肽,该酶通过非经典蛋白质分泌途径（non-classical protein secretion pathway）进行分泌。通过信号肽筛选,发现最适合该酶在枯草芽孢杆菌中表达的信号肽为Tat分泌途径信号肽SP_phoD,并通过共表达分子伴侣PrsA的方式将该L-天冬酰胺酶的分泌量提高了72.11%。     

Inhibitory effects of 4-chlorosalicylic acid on mushroom tyrosinase and its antimicrobial activities

The inhibitory effects of 4-chlorosalicylic acid on the activity of mushroom tyrosinase have been investigated. The results showed that 4-chlorosalicylic acid could strongly inhibit both monophenolase activity and diphenolase activity. The IC₅₀ values were estimated as 1.89 mM and 1.10 mM for monophenolase and diphenolase activities, respectively. For the monophenolase activity, 4-chlorosalicylic acid could not only lengthen the lag time, but also decrease the steady-state rate. For the diphenolase activity, kinetic analyses showed that the inhibition by 4-chlorosalicylic acid was reversible and its mechanism was mixed-II type, which is different from salicylic acid. The inhibition constants (K_I and K_IS) were determined to be 1.51 mM and 0.82 mM, respectively. Furthermore, the antibacterial activity against Escherichia coli, Bacillus subtilis and Staphyloccocus aureus and antifungal activity against Aspergillus niger and Candida boidinii were investigated. The results showed that 4-chlorosalicylic acid was the most effective against E. coli with the MIC of 250 μg/ml and with the MBC of 500 μg/ml.     

Novel transporters from Kluyveromyces marxianus and Pichia guilliermondii expressed in Saccharomyces cerevisiae enable growth on l‐arabinose and d‐xylose

Genes encoding l ‐arabinose transporters in Kluyveromyces marxianus and Pichia guilliermondii were identified by functional complementation of Saccharomyces cerevisiae whose growth on l ‐arabinose was dependent on a functioning l ‐arabinose transporter, or by screening a differential display library, respectively. These transporters also transport d ‐xylose and were designated KmAXT1 (arabinose–xylose transporter) and PgAXT1, respectively. Transport assays using l ‐arabinose showed that KmAxt1p has K_m 263 mm and V_max 57 nm /mg/min, and PgAxt1p has K_m 0.13 mm and V_max 18 nm /mg/min. Glucose, galactose and xylose significantly inhibit l ‐arabinose transport by both transporters. Transport assays using d ‐xylose showed that KmAxt1p has K_m 27 mm and V_max 3.8 nm /mg/min, and PgAxt1p has K_m 65 mm and V_max 8.7 nm /mg/min. Neither transporter is capable of recovering growth on glucose or galactose in a S. cerevisiae strain deleted for hexose and galactose transporters. Transport kinetics of S. cerevisiae Gal2p showed K_m 371 mm and V_max 341 nm /mg/min for l ‐arabinose, and K_m 25 mm and V_max 76 nm /mg/min for galactose. Due to the ability of Gal2p and these two newly characterized transporters to transport both l ‐arabinose and d ‐xylose, one scenario for the complete usage of biomass‐derived pentose sugars would require only the low‐affinity, high‐throughput transporter Gal2p and one additional high‐affinity general pentose transporter, rather than dedicated d ‐xylose or l ‐arabinose transporters. Additionally, alignment of these transporters with other characterized pentose transporters provides potential targets for substrate recognition engineering. Accession Nos: KmAXT1: GZ791039; PgAXT1: GZ791040 Copyright © 2015 John Wiley & Sons, Ltd.     

Chemical Composition and Antioxidative Activity of Echinophora platyloba DC. Essential Oil,and Its Interaction with Natural Antimicrobials against Food‐Borne Pathogens and Spoilage Organisms

Abstract: This study was undertaken to determine the chemical composition and antioxidative capacity of Echinophora platyloba DC. essential oil, and its antimicrobial potency against Listeria monocytogenes, Bacillus cereus, Bacillus subtilis, Staphylococcus aureus, Salmonella typhimurium, Escherichia coli O157:H7, Pseudomonas aeruginosa, Candida albicans, Candida tropicalis, Rhodotorula rubra, and Rhodotorula mucilaginosa. The essential oil was analyzed by GC and GC‐MS; and evaluated for its antioxidative and antimicrobial (singly or in combination with chitosan, nisin, monolaurin, or amphotericin B) activity. Thirty‐three components were characterized representing 95.69% of the total oil composition in which thymol, trans‐ocimene, carvacrol, and (E)‐sesqui‐lavandulol were the major constituents. The oil exhibited high scavenging (IC₅₀: 49.7 ± 2.3 μg/mL) and relative antioxidative activity (RAA%: 85.21 ± 0.4) in 1,1‐diphenyl‐2‐picrylhydrazyl radicals and β‐carotene/linoleic acid bleaching assays, respectively. The oil showed antimicrobial activity against L. monocytogenes, B. cereus, B. subtilis, S. aureus, S. typhimurium, E. coli O157:H7, P. aeruginosa, C. albicans, C. tropicalis, R. Rubra, and R. mucilaginosa. Moreover, R. mucilaginosa and P. aeruginosa were the most susceptible and most resistant organisms, respectively. Regarding the checkerboard data, 47 fractional inhibitory concentration index (FICIs) (≤0.5) indicated synergistic, whereas 7 FICIs (>0.5 to 1) indicated additive effect. Consequently, E. platyloba DC. essential oil could be used as a recommended natural antioxidant and antimicrobial substance for food preservation.     

Functional expression of recombinant sweet-tasting protein brazzein by Escherichia coli and Bacillus licheniformis

Brazzein is an attractive sweetener candidate because of its sugar-like taste, high sweetness, and good stability at high temperature and wide pH range. This study was aimed to express and purify bioactive recombinant brazzein (rBrazzein). The rBrazzein gene was synthesized according to the preferred codons of Bacillus subtilis and successfully expressed in Escherichia coli and Bacillus licheniformis. In E. coli host, lower induction temperature of 30°C increased soluble rBrazzein (Ebrazzein) at high level. In B. licheniformis host, two signal peptides (Sec type and Tat type) were evaluated for the expression of rBarzzein in B. subtilis and B. licheniformis. However, only the Sec-type signal peptide guided the secretion expression of rBrazzein in B. licheniformis. The rBrazzein was expressed steadily and the highest yield reached about 57 mg/L at 36 h by small-scale fermentation. The purification procedure of rBrazzein by B. licheniformis (Bbrazzein) was thus established. Approximately 5 mg/L purified rBrazzein was obtained and the purity was 85%. The conformational state of rBrazzeins was confirmed by circular dichroism. The bioactivities of rBrazzeins were evaluated by sweet taste testing. The Bbrazzein and Ebrazzein were 266 times and 400 times sweeter than sucrose on a weight basis, respectively. The formation of disulfide bonds were both confirmed by LC/MS/MS and MALDI-TOF. The CD analysis indicated that Ebrazzein has a similar secondary structure with natural brazzein, which explained why Ebrazzein had a higher intensity of sweetness. This study demonstrated that B. licheniformis system is useful to produce active recombinant brazzein, and has potential food industry applications.     

Enhancement of Recombinant Subtilisin YaB Production by Bacillus subtilis

Bacillus subtilis is an ideal host for the production of extracellular heterologous proteins. The use of a strong, constitutive promoter is a good means of optimizing the expression of heterologous proteins. In this study, high level extracellular production of subtilisin YaB, a potent meat tenderizer, was achieved by engineering the wild-type subtilisin YaB promoter into an artificial promoter and expressed in Bacillus subtilis host. This artificial promoter provides an efficient tool to produce mutant subtilisin YaB or other recombinant heterologous proteins by B. subtilis at high level.     

Safe preparation of beefy meaty peptide with Bacillus subtilis

Preparation of flavour peptides with microorganisms is an attractive choice for its large-scale production. However, beefy meaty peptide (BMP), as a research hotspot in the field of umami peptide, has been studied in few microorganisms, and furthermore, the safe preparation of BMP has not yet been reported. In this study, multi-copy BMP (8BMP) was successfully expressed and produced in the ‘generally recognized as safe’ Bacillus subtilis (B. subtilis). Firstly, 8BMP with the potentially intense umami was screened out based on molecular docking analysis. Then, it was successfully expressed in the B. subtilis 168 and verified through sensory evaluation. Finally, the scaling capacity of 8BMP was evaluated in a 5-L fermenter, with the highest yield (0.84 g L⁻¹) 6.4 times than that of shake flask, which is conductive for industrial production of BMP and other food peptides.     

Purification of two bacteriocins produced by Enterococcus faecalis DBFIQ E24 strain isolated from raw bovine milk

Enterococcus faecalis DBFIQ E24 was formerly characterised as a producer of antibacterial and antifungal substances. This strain is vancomycin sensitive, nonhaemolytic and gelatinase negative. SDS‐PAGE revealed the presence of an active band located between 1060 and 3500 Da. After the application of a three‐step purification procedure, two antimicrobial peptides were isolated. One of them is mostly active against Bacillus cereus with a molecular mass of 1364 Da, and the other one with a molecular mass of 1686 Da is mainly active against Escherichia coli. These results confirm the technological potential of E. faecalis DBFIQ E24 strain as a GRAS food biopreservative.     

Characterization of traditionally fermented Korean soybean paste, eoyukjang, and isolation of its microorganisms

In this study, traditionally fermented Korean soybean paste, eoyukjang, was characterized and its microorganisms were isolated. The contents of amino-type and ammonia-type nitrogens in the pastes we examined were 89.60 to 98.93 mg/% and 0.32 to 0.30 mM, respectively. Antioxidant activity increased during ripening, with antioxidant activities in 1- and 4-year-old pastes measured at 9.80 and 13.84 μmol of Trolox equivalents/g, respectively. Twenty-two and 19 microorganisms were isolated from soybean pastes under aerobic and anaerobic conditions, respectively. After identification, Bacillus amyloliquefaciens, Bacillus subtilis, and Bacillus vallismortis were dominant. In the enzyme activities, protease and lipoxygenase activities were observed from 0.065 to 0.733 unit/mg protein and 0.016 to 0.19 unit/mg protein, respectively. Amylase activity was, however, broad between 43.1 to 571.8 unit/mg protein.     

CONDITIONS AFFECTING AUTOINDUCER‐2‐BASED QUORUM‐SENSING OF ESCHERICHIA COLI O157:H7 ON FRESH BEEF

ABSTRACT

This study evaluated whether inoculated (none, 1, 5 log colony‐forming units [cfu]/cm²) Escherichia coli O157:H7 would result in detection of autoinducer (AI)‐2‐like activity on beef. Inoculated fresh beef, containing low (LNB) or high (HNB) initial levels of natural flora, was analyzed for bacterial populations and AI‐2‐like activity during aerobic or vacuum‐packaged storage (4, 10, 25C). As expected, no growth of E. coli O157:H7 was detected at 4C, while at 10C, growth was detected only on LNB samples stored aerobically; AI‐2‐like activity was minimal (P ≥ 0.05) at both temperatures. E. coli O157:H7 showed more growth in LNB than HNB, and in aerobically than vacuum‐packaged samples inoculated with 1 log cfu/cm² of the pathogen during storage at 25C. AI‐2‐like activity was generally higher in LNB than HNB samples stored aerobically at 25C, while no significant AI‐2‐like activity was detected in samples stored in vacuum packages. The results indicated that E. coli O157:H7 may exhibit AI‐2‐like activity on aerobically stored beef in the presence of lower initial levels of natural flora, and at temperatures allowing prolific growth of the pathogen. Thus, AI‐2‐based quorum‐sensing of E. coli O157:H7 may not be of importance in beef stored at low temperatures.

PRACTICAL APPLICATIONS

This study presents evidence that Escherichia coli O157:H7 showed autoinducer (AI)‐2 activity and involved in quorum‐sensing on fresh beefcontaining low initial levels of natural flora during aerobic storage at abusive storage temperatures. Thus, AI‐2‐based quorum‐sensing of E. coli O157:H7 may not be important in beef stored at recommended low temperatures.     

Assessment of the microbiological safety of salad vegetables and sauces from kebab take-away restaurants in the United Kingdom

The purpose of this study was to establish the microbiological safety of salad vegetables and sauces served in kebab take-away restaurants. Comparison with published microbiological guidelines revealed that 4.7% of 1213 salad vegetable samples were of unsatisfactory microbiological quality due to Escherichia coli and/or Staphylococcus aureus levels at ≥10² cfu g⁻¹. Another 0.3% of salad samples were of unacceptable quality due to S. aureus at ≥10⁴ cfu g⁻¹ (2 samples) or the presence of Salmonella Kentucky (1 sample). Cucumber was the most contaminated salad vegetable with regards to unsatisfactory levels of E. coli (6.0%) or S. aureus (4.5%). Five percent of 1208 sauce samples were of unsatisfactory microbiological quality due to E. coli, S. aureus at ≥10² cfu g⁻¹ and/or Bacillus cereus and other Bacillus spp. at ≥10⁴ cfu g⁻¹. A further 0.6% of sauce samples were of unacceptable quality due to Bacillus spp. (Bacillus subtilis, Bacillus pumilus, Bacillus licheniformis) at ≥10⁵ cfu g⁻¹ or the presence of Salmonella Agbeni (1 sample). More samples of chilli sauce (8.7%) were of unsatisfactory or unacceptable microbiological quality than any other sauce types. The results emphasize the need for good hygiene practices in kebab take-away restaurants handling these types of ready-to-eat products.     

Structural features of a xylogalactan isolated from Hymenaea courbaril gum

The gum from Hymenaea courbaril (Caesalpiniaceae) produced gum at seed level. The gum is soluble in water, dextrorotatory and less viscous than the gum from Cyamopsis tetragonolobus (guar gum). The polysaccharide, isolated from this gum, contains galactose, glucose, xylose and arabinose. The preparation of degraded products by acid hydrolysis and Smith-degradation process led to obtain degraded gums A and B, and the polysaccharides I and II. Chemical methods in combination with 1D NMR (¹H, ¹³C, DEPT-135) and 2D NMR (COSY, HMQC and HMBC) were applied. The backbone is a xylogalactan; β-d-galactose and β-d-xylose residues are 4-O- and 2-O-linked, respectively. The branches are constituted by xylose, arabinose and galactose. It was also observed 4,6-di-O-substituted galactose residues. This work shows interesting structural features of the polysaccharide isolated from H. courbaril gum.     

Bacterial inactivation and quality changes in fresh-cut avocado treated with intense light pulses

This study investigated the impact of intense light pulses (ILP) on inactivation of Listeria innocua and Escherichia coli as well as quality changes in fresh-cut avocado. Cylinders of avocado inoculated with L. innocua or E. coli were placed in plastic trays, which were sealed with a 64-μm-thick polypropylene film (oxygen permeability of 110 cm³ O₂ m⁻² bar⁻¹ day⁻¹ at 23 °C and 0% RH) and subjected to 15 or 30 pulses at fluencies of 0.4 J/cm² per pulse and then stored for 15 days at 5 °C. In addition to L. innocua and E. coli counts, the headspace atmosphere, pH, colour and firmness were measured. The growth of E. coli and L. innocua was more effectively inhibited when increasing treatment intensity. Hence, significant inactivation was obtained in samples treated with 15 and 30 pulses for L. innocua (2.61 and 2.97 log CFU/g, respectively) and E. coli (2.90 and 3.33 log CFU/g, respectively) just after processing. Oxygen concentrations were significantly reduced, whereas CO₂ and ethanol concentrations increased due to product respiration; however, ethylene production was decreased by the effect of ILP treatments. The use of 30 pulses affected the colour and firmness of fresh-cut avocado, causing browning and softening.     

Comparing effects of bovine Streptococcus and Escherichia coli mastitis on impaired reproductive performance

In 2 epidemiological studies, we evaluated the effect of mastitis induced by gram-positive Streptococcus and gram-negative Escherichia coli on impaired reproductive performance in lactating Holstein cows. In the first study, 52,202 cows from 178 dairy farms throughout Israel were divided into groups based on infection before first artificial insemination (AI) with Streptococcus or E. coli, 3 groups with elevated somatic cell count (SCC) without infection by those pathogens [low SCC (200–400) × 10³ cell/mL; medium SCC (401–1,000) × 10³ cell/mL; high SCC, >1,000 × 10³ cell/mL], and uninfected controls. Pregnancy per first AI (P/1stAI) and pregnancy rate at 300 d in milk (PREG 300) were analyzed by the GLIMMIX procedure (SAS); number of AI per pregnancy (AI/P), days open, and rest days (calving to first AI) were analyzed by the MIXED procedure (SAS Institute Inc., Cary, NC). Values of P/1stAI were similarly low for Streptococcus and E. coli (27–28%) versus 42% in controls; PREG 300 was lower for Streptococcus (76%) than for E. coli (79%) versus 88% for uninfected controls and a mean 83% for the elevated SCC groups. Days open and number of AI/P were higher than in controls and similar in Streptococcus and E. coli groups. The second study included 778 cows on 6 dairy farms; the cows were infected before first AI by Streptococcus or E. coli or uninfected. Resumption of cyclicity was determined by an automated activity-monitoring system, and data were sorted by time of infection before or after cyclicity resumed. The Streptococcus group had lower P/1stAI before and after cyclicity (26 and 27%, respectively) than the E. coli group (31 and 34%, respectively) and uninfected controls (42%). Notably, PREG 300 in the Streptococcus group before (73%) and after (67%) cyclicity was much lower than for the E. coli group (85 and 93%, respectively) and the controls (95%). A marked rise in day of cyclicity resumption (∼80 d) was observed in cows that were infected early on. Number of AI/P was higher in the mastitic groups than in uninfected controls. Uterine disease postpartum, although more prevalent among Streptococcus cows, did not substantially alter the larger reduction in P/1stAI and PREG 300 in Streptococcus versus E. coli cows. Thus, long-term Streptococcus-induced mastitis disrupted fertility more than short-term acute E. coli–induced mastitis, resulting in a much higher percentage of Streptococcus cows in late lactation that did not conceive due to reproduction failure.     

Volatile compounds produced by Bacillus species alkaline fermentation of bambara groundnut (Vigna subterranean (L.) Verdc) into a dawadawa-type African food condiment using headspace solid-phase microextraction and GC × GC–TOFMS

The reports on the volatile compounds of a dawadawa-type African food condiment produced from the alkaline fermentation of bambara groundnut (Vigna subterranea (L.) Verdc.) using Bacillus starter cultures are limited. Volatile compounds were isolated from dawadawa-type condiments using headspace solid phase microextraction and analysed by comprehensive gas chromatography coupled to time of flight mass spectrometry. Acids, aldehydes and alcohols accounted for over 70% of the volatile compounds produced in the Bacillus fermented samples. B. subtilis subsp. subtilis SFBA3 produced the highest content of acids (4969.60 µg kg^?1), while the highest content of aldehydes (2811.90 µg kg^?1) and alcohols (1247.60 µg kg^?1) was detected with Bacillus cereus PALB7 and Bacillus licheniformis OALB2, respectively. Sulphur-containing compounds concentration (85.80 µg kg^?1) was highest for Bacillus amyloliquefaciens SFBA2. Maximum 2-methyl butanoic acid and 3-methyl butanoic acid concentrations, indicative of typical dawadawa aroma, were produced by B. subtilis subsp. subtilis SFBA3.     

Purification,structural data and biological properties of polysaccharide from Prunus amygdalus gum

This work demonstrates the efficiency of almond gum polysaccharides (AGPs) as bioactive compounds. AGPs were first extracted using H₂O₂, in the presence of NaOH, at different times and temperatures. The optimal extraction conditions were 4% H₂O₂ and 2 N NaOH, for 7 h at 50 °C, leading to an extraction yield of 58.2% (w/w). After a purification step, the retained AGPs were characterised using high‐performance liquid chromatography showing a molecular weight of 99.3 kDa. The monosaccharide composition of AGPs were assessed using gas chromatography–mass spectrometry. AGPs were found to be a complex heteropolysaccharide with a repeating unit mainly composed of galactose, arabinose, xylose, mannose, rhamnose, and glucuronic acid with the respective ratios: 45:26:7:10:1:11. The acidic nature of the polysaccharide is due to the presence of glucuronic acid. Total antioxidant activity, free radical‐scavenging activity and reducing power assay of AGPs were investigated. The obtained results showed high antioxidant activities of AGPs. Furthermore, beyond 60 mg mL^?1, AGPs exhibited bacterial growth inhibition for five pathogenic strains: Escherichia coli, Staphylococcus aureus, Enterococcus feacalis, Pseudomonas aeruginosa and Salmonella typhimurium.     

Antimicrobial Activity of Monoacyl Hexose Coexistent with Lysozyme against Gram-Positive Bacilli

The antimicrobial activities of myristoyl, palmitoyl, or stearoyl hexoses, which were glucose, mannose, and galactose, coexistent with lysozyme against three Gram-positive bacteria, Bacillus coagulans, Bacillus subtilis, and Bacillus licheniformis, were measured in order to investigate the availability of monoacyl hexose as an antimicrobial co-agent. The lysozyme exhibited an antimicrobial activity against Bacillus subtilis and Bacillus licheniformis, but there was no significant difference between the dependencies of the antimicrobial activity against the two bacteria on the lysozyme concentration. However, the antimicrobial activities of the monoacyl hexoses coexistent with the lysozyme were different between those against the two bacteria. The stearoyl hexose coexistent with the lysozyme exhibited the highest antimicrobial activity against two bacteria. It was indicated that the antimicrobial action of the monoacyl hexose would be exerted parallel with the bacterial lysis of lysozyme. Stearoyl hexose, which has a high hydrophobicity, coexistent with the lysozyme could exhibit a higher antimicrobial activity than only the lysozyme.     

Development of efficient enzymatic production of theanine by γ‐glutamyltranspeptidase from a newly isolated strain of Bacillus subtilis,SK11.004

BACKGROUND: Theanine, a unique amino acid found almost exclusively in tea plants, has various favourable physiological and pharmacological functions in humans. Gamma‐glutamyltranspeptidase (GGT, EC 2.3.2.2) is considered to be the most effective enzyme for the production of theanine. In fact, GGT can catalyse the transfer of γ‐glutamyl moieties from γ‐glutamyl compounds to water (hydrolysis) or to amino acids and peptides (transpeptidation). RESULTS: A novel strain, SK11.004, which produces GGT with high theanine‐forming ability was isolated from fermented shrimp paste and identified as Bacillus subtilis through its physiological and biochemical properties as well as its 16S rDNA sequence analysis. Theanine (18.9 mmol L^?1) was synthesised by GGT (0.06 U mL^?1) through transfer reaction in the presence of glutamine (20 mmol L^?1) as a donor and ethylamine HCl (50 mmol L^?1) as an acceptor at pH 10 and 37 °C for 4 h, the conversion rate being up to 94%. CONCLUSION: The enzymatic synthesis of theanine using GGT from a newly isolated strain Bacillus subtilis SK11.004 was found to be an efficient method. Moreover, compared with others, the GGT from B. subtilis SK11.004 exhibited the highest ratio of transferring activity to hydrolytic activity using glutamine, suggesting a high potential application in the production of theanine and other functional γ‐glutamyl compounds. Copyright © 2010 Society of Chemical Industry     

Flocculation Properties of Poly(��-Glutamic Acid) Produced from Bacillus subtilis Isolate

Bacillus subtilis R 23 produced extracellular biopolymer showing excellent flocculation activity. The biopolymer was confirmed as poly(γ-glutamic acid) (PGA) using high-performance liquid chromatography profile and product characterization. The production, characteristics, and flocculation properties of PGA were studied. PGA produced by B. subtilis R 23 was devoid of any polysaccharides and had a molecular weight of 6.2 × 10⁶ Da. The flocculating activity of PGA in the kaolin suspension was markedly stimulated by the addition of bivalent and trivalent cations in optimum concentration. The pH of reaction mixture also influenced the flocculating activity. Response surface methodology was used to establish the optimum parameters for maximum flocculating activity and to study their interactions. A maximum flocculating activity of 30.32 ± 1.4 1/optical density was obtained with 7.5 mg/L of PGA in combination with 8.0 mM of Ca⁺² at pH 7.5.