Detection and Characterization of a Broad-Range Bacteriocin Produced by Lactobacillus brevis SD-22, Isolated from Traditional Chinese Pickles

ZHANG Guo-qiang，FAN Ming-tao，SHI Jun-ling，FANG Jiang-ping

(1. College of Agriculture and Husbandry, Tibet University, Linzhi 860000, China；2. College of Food Science and Engineering, Northwest A & F University, Yangling 712100, China)

Detection and Characterization of a Broad-Range Bacteriocin Produced by Lactobacillus brevis SD-22, Isolated from Traditional Chinese Pickles

ZHANG Guo-qiang1,2，FAN Ming-tao2，SHI Jun-ling2，FANG Jiang-ping1,*

(1. College of Agriculture and Husbandry, Tibet University, Linzhi 860000, China；2. College of Food Science and Engineering, Northwest A & F University, Yangling 712100, China)

Lactobacillus brevis SD-22 was selected from 329 lactic acid bacteria isolated from traditional Chinese pickles, The strain was found able to produce a bacteriocin strongly inhibited Staphylococcus aureus ATCC 63586 and Escherichia coli ATCC 24936. This bacteriocin, which was designated brevicin SD-22, was purified by ammonium sulphate precipitation for further studies. Brevicin SD-22 exhibited a wide range of antimicrobial activity, strong heat stability ( 121 ℃, 15 min) and pH stability (pH 3.0-8.0). Brevicin SD-22 was sensitive to protease but insensitive to α-amylase. Thus, Brevicin SD-22 has the potential to be applied in food preservation.

Lactobacillus brevis；bacteriocin；characterization

Bacteriocins are ribosomally synthesized, extracellular peptides or proteins with an antibacterial activity usually against closely related species[1]. Some bacteriocins can inhibit broader range of microorganisms, including some food spoilage bacteria and food-borne pathogens. In terms of safety, bacteriocins from lactic acid bacteria (LAB) have attracted more interest than those from other resources, because most of LAB are related to fermented foods[2]. From various foods such as fermented sausage, yoghourt, fermented vegetables, etc., many novel bacteriocin-producing LAB have been isolated and the characteristics of these bacteriocins have been investigated extensively[3].

However, presently industrialized bacteriocins, e.g. Nisin, exhibit certain limitations. On one hand, some bacteriocins are ineffective against Gram-negative spoilage and pathogenic bacteria, yeasts and molds; on the other hand, they do not inhibit all food-borne Gram-positive spoilage and pathogenic bacteria. Therefore, those disadvantages limit the application range of bacteriocins in the food industry.

Until now, screening of LAB producing novel bacteriocins with a broad antimicrobial spectrum and identifying those novel bacteriocins have attracted the most attention. Fermented vegetables contain many species of LAB. Some bacteriocin-producing LAB such as Lactobacillus pentosus B96[4],Lactobacillus plantarum C19[5], Enterococcus faecium BFE 900[6], Lactobacillus plantarum LPCO10[7]and Pediococcus pentosaceus 05-10[8]were successively isolated from fermented vegetables. Traditional Chinese pickles is a widely used traditional China food made from fermented vegetables and its production is a spontaneous fermentation process by a mixed microbial population mainly composed of LAB.

In the present paper, we report the detection, identification and primary characteristics of a novel bacteriocin with a broad inhibitory spectrum produced by Lactobacillus brevis SD-22 from traditional Chinese pickles. Further, this paper is the first report on the selection of bacteriocin-producing Lactobacillus brevis strain from Chinese pickles.

1 Materials and Methods

1.1Materials, reagents and instruments

86 liquor samples of traditional Chinese pickles were collected from the pilot plant of Hubei Biopesticide Engineering Research Center (Wuhan, China).

BCP agar (with bromo-cresol purple and cycloheximide) Eiken Chemical Co. Ltd., (Japan); MRS agar plates Difco (USA); beef liver catalase (50 U/mL), pepsin(1:10000), trypsin (1:250), protease K (40 U/mg) Sigma (USA); other chemicals were analytical reagent.

1.2 Methods

1.2.1 Screening of isolates for antimicrobial activity

The liquor samples (0.1 mL, 10-4-10-6dilution) were spread directly on the surface of BCP agar and then were incubated at 30 ℃ for 1-2 days. Colonies with clear zones around on BCP agar plates were regarded as acid-producing bacteria and they were randomly selected and purified by plating on MRS agar plates. The purified colonies were primarily identified by Gram staining and catalase tests. Only both Gram-positive and catalase-negative strains were selected and stored in MRS broth with 20% glycerol at -20 ℃.

The selected strains were inoculated into the tubes containing 10 mL MRS broth each and statically incubated at 30 ℃ for 18 h. Then 1% of those cultures were inoculated into 250 mL MRS broth individually and incubated at 30 ℃for 24 h without agitation. Bacterial cells were removed by centrifugation (5000×g, 15 min, 4 ℃) and supernatants were examined by the diameters of inhibition zones using agar diffusion assay method with Staphylococcus aureus ATCC 63586 and Escherichia coli ATCC 24936 as indicator strains[9]. Briefly, 100μL of cell-free supernatants were placed into wells (7.80 mm in diameter) on MRS agar plates seeded with the above indicator strains. After incubation at 30 ℃ for 18 h, the diameters of inhibitory zones were determined.

The pH value of the fermented supernatants was adjusted to 6.0 with 2 mol/L NaOH to eliminate the effect of low pH value and the pH of MRS broth was also adjusted to the same value using either lactic acid or acetic acid as control, respectively. After adding beef liver catalase (50 U/mL), the cell-free supernatants (pH 6.0) were incubated at 37 ℃ for 12 h to eliminate the effect of hydrogen peroxide and the same supernatants without catalase were used as control[10]. Diameters of inhibition zones were measured again for final selection.

To determine the possible protein nature of the detected antimicrobial substances, after eliminating hydrogen peroxide and low pH effects, the bacterial cell-free supernatants (pH 6.0) were, respectively, incubated at 37 ℃ overnight with pepsin, trypsinor protease K at a final concentration of 3 mg/mL individually and those without enzyme treatment were used as control. Diameters of inhibition zones were recorded using S. aureus ATCC 63586 as indicator[9].

1.2.2 Strain identification

one perspective strain SD-22 from traditional Chinese pickles was first determined by phenotypical and physiological tests including Gram staining, cell morphology, growth ability, respectively, at 10, 15 ℃ and 45 ℃, also in 3%, 6% and 9% NaCl. Sugar fermentation reactions were obtained by using the API 50 CHL system as the manufacture, recommendations[2,11].

1.2.3 Partial purification of brevicin SD-22

Isolated strain having maximum antimicrobial zone was grown in MRS broth at 37 ℃ for 24 h. After incubation, the broth was centrifuged (5000×g, 10 min, 4 ℃) and the cells were separated out. Supernatant was used as a crude bacteriocin. Different concentrations of ammonium sulphate were added to the supernatant. After stirring on a magnetic stirrer, it was kept undisturbed at 4 ℃ overnight. Precipitates formed were collected by centrifugation (10000×g, 10 min, 4 ℃) and redissolved in 20 mmol sodium phosphate buffer with pH 6.0. Inhibition zone of different fractions was recorded in comparison with the crude bacteriocin.

1.2.4 Antimicrobial spectrum of brevicin SD-22

Partially purified brevicin SD-22 preparations were usedto determine the antimicrobial spectrum against indicator organisms (Table 2). The indicatory LAB were statically incubated in MRS broth at 30 ℃ for 24 h. All the other indicatory bacteria were cultivated overnight in nutrient broth at 37 ℃ with gentle agitation for 12 h. Saccharomyces cerevisiae ACCC 20036 was grown in yeast extract peptone dextrose (YEPD) broth (120 r/min, 30 ℃, 18 h) and Aspergillus niger ACCC 30005 in wort broth (120 r/min, 30 ℃, 24 h). Diameters of inhibition zones were also measured by agar diffusion assay method[9].

1.2.5 Effects of enzymes, temperature and pH on activity of brevicin SD-22

The partially purified brevicin SD-22 preparations were, respectively, treated with trypsin (3 mg/mL), protease K (3 mg/mL) and pepsin (3 mg/mL) and incubated at 37 ℃ for 12 h. In another test, partially purified brevicin SD-22 preparations were incubated, respectively, in either thermostatic water bath at 60, 80, 90 ℃ and 100 ℃ for 20 min or in an autoclave at 121 ℃ for 15 min. By adjusting the pH value in a range from 2.0 to 9.0 and keeping for 12 h, the effect of pH was tested. For all the experiments here, S. aureus ATCC 63586 used as indicator and controls was maintained without any treatment[12]. Bacteriocin activity of all samples was assayed as described[13].

2.1 Screening of isolates for antimicrobial activity

Table 1 Antimicrobial activity of the primarily selected strains after eliminating organic acid effect

A total of 329 acid-producing bacteria isolated from traditional Chinese pickle were cultivated in MRS broth and the cell-free supernatants of 10 strains with distinct antimicrobial activity against both S. aureus ATCC 63586 and E. coli ATCC 24936 indicators were obtained. After eliminating low pH effect, only cell-free supernatant of strain SD-22 still exhibited distinct inhibitory activity against both indicators (Table 1). Further, after catalase treatment, its antimicrobial activity was still the same as the control (data not shown).

After treatment by all the three kinds of protease, the antimicrobial activity of cell-free supernatant of strain SD-22 disappeared. It indicated that the substance with strong antimicrobial activity was sensitive to protease. Therefore, the protein nature of this antimicrobial substance produced by strains SD-22 was verified.

2.2 Strain identification

Cells of strain SD-22 were Gram-positive, like coccusrod shaped and without spore formation. It could grow at 10 ℃and 15 ℃ but not at 45 ℃, as well as not in 3%, 6% and 9% NaCl. Strain SD-2 could produce CO2from glucose and ammonia from arginine. Moreover, it could metabolize L-arabinose, glucose, lactose, raffinose, melibiose, saccharose, gluconate, maltose, D-mannose, ribose, and D-fructose, but not D-xylose, cellobiose, rhamnose, salicin and sorbitol. Carbohydrate fermentation reactions recorded with the API 50 CHL system classified strain SD-22 as a member of L. brevis.

Fermented vegetables are good sources of LAB. Many bacteriocin-producing LAB such as L. pentosus B96, L. plantarum C19, E. faecium BFE 900, L. plantarum LPCO10 and Pediococcus pentosaceus 05-10 were successively isolated from fermented vegetables[4-8].

2.3 Partial purification of brevicin SD-22

An increase in antimicrobial activity after partial purification of crude bacteriocin by ammonium sulphate precipitation took place (Fig. 1). The fraction with the highest bacteriocin activity was precipitated with 30 g/100 mL (by mass per volume) ammonium sulphate. The antimicrobial activity (in terms of inhibition zone diameter) increased from 12 to 23 mm. There was 1.91-fold increase in the partially purified bacteriocin activity than that of crude bacteriocin. Earlier, the inhibitory activity of bacteriocin isolated from malted barley was precipitated from cell free supernatant using 20-30 g/100 mL ammonium sulphate saturation, and resuspended in 20 mmol sodium phosphate buffer, pH 6.0[14].

Fig. 1 Increase in antimicrobial activity of brevicin SD-22 using ammonium sulphate fractionation

2.4 Antimicrobial spectrum of brevicin SD-22Brevicin SD-22 exhibited a broad antimicrobial activity (Table 2). It could significantly inhibit S. aureus,

Table 2 Antimicrobial spectrum of brevicin SD-22

L. monocytogenes and Salmonella typhimurium, moderately against L. plantarum, Lactobacillus delbrueckii subsp.

delbrueckii, Streptococcus thermophilus, E. coli and Bacillus subtilis, less against Lactobacillus acidophilus, Bacillus cereus, Rhizoctonia solani and Septoria nodorum Berk but not against S. cerevisiae and A. niger. From this result, we can see that brevicin SD-22 has inhibitory activity against not only a large range of LAB but also many Gram-positive and Gram-negative bacteria (Table 2). This activity against many Gram-negative bacteria was not frequently seen in bacteriocins from LAB.

2.5 Effects of enzymes, temperature and pH on brevicin SD-22

Table 3 Effects of enzymes, temperature and pH on brevicin SD-22

As shown in Table 3, complete inactivation in antimicrobial activity was observed after treatment of brevicin SD-22 with proteinase K, trypsin and pepsin, which revealed that the antimicrobial substance was of proteinaceous nature. However, treatment with α-amylase had no effect on bacteriocin activity. This suggested that the peptide was not glycosylated or its activity was not dependent on glycosylation[15].

Brevicin SD-22 remained stable after incubation for 12 h at pH values from 3 to 8, but its activity slightly reduced at pH 9. The results demonstrated that the bacteriocin was resistant to acidic condition.

Brevicin SD-22 proved to be relatively heat stable at moderate temperatures or 100 ℃ for 20 min. Even after treated at 121 ℃ for 15 min, inhibitory activity still could be detected. But complete loss of its activity was observed when treated at 121 ℃ for 20 min. This heat stability would be a very useful characteristic as food preservative, because many food-processing procedures involve a heating step.

3 Conclusion

Out of 329 LAB strains isolated from traditional Chinese pikles, strain SD-22 that is able to produce a bacteriocin strongly inhibits S. aureus and E. coli was screened. This strain was identified as L. brevis by phenotypical and physiological tests and API 50 CHL system. This is the first bacte-riocin-producing strain of L. brevis isolated from fermented vegetables.

Precipitation by ammonium sulphate is the most commonly used procedure[16]. After partial purification of crude bacteriocin by ammonium sulphate precipitation, brevicin SD-22 exhibited a wide range of antimicrobial activity against not only a large range of LAB but also many food-borne spoilage and pathogenic Gram-positive and Gram-negative bacteria. And it has strong heat stability and pH stability. Brevicin SD-22 was sensitive to protease but insensitive to α-amylase. The heat stability and antimicrobial spectrum of brevicin SD-22 suggested that it was a novel broad-spectrum bacteriocin produced by L. brevis[17].

These studies might hopefully lay the groundwork for future application of brevicin SD-22 and its producer, and broaden the application range of L. brevis as producing starter and bio-preservative in the food industry.

The further work is to purify this bacteriocin to homogeneity which shall be followed by DNA and amino acid sequences and action modes of brevicin SD-22, and to research the effects of L. brevis SD-22 on food quality and the production of this novel brevicin SD-22 in various fermentation conditions.

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一株从泡菜中分离的产细菌素乳杆菌的鉴定及细菌素特性研究

张国强1,2，樊明涛2，师俊玲2，方江平1,*
(1.西藏农牧学院，西藏 林芝 860000；2.西北农林科技大学食品科学与工程学院，陕西 杨凌 712100)

从泡菜中分离的329株乳酸菌中，筛选出一株产细菌素的乳酸菌，编号为SD-22。经生理生化实验鉴定为短乳杆菌(Lactobacillus brevis)。在排除过氧化氢干扰和有机酸抑菌作用后，brevicin SD-22对革兰氏阳性细菌和革兰氏阴性细菌具有较好的抑制作用，对部分真菌也有抑制作用，但对酵母菌和青霉无抑制作用，具有良好的热稳定性和pH值稳定性，经胰蛋白酶、胃蛋白酶和蛋白酶K处理后抑菌活性完全消失，对α-淀粉酶不敏感。因此初步认为该菌株是一株产广谱细菌素的乳酸菌。

乳杆菌；细菌素；特性

TS201.3

A

1002-6630(2011)03-0171-05

2010-04-03

“十一五”国家科技支撑计划项目(N2006BAC01A04；2007BAC06B08)

张国强(1982—)，男，讲师，博士研究生，研究方向为食品微生物与蛋白质组学。E-mail：guoqiangz2008@yahoo.com.cn

*通信作者：方江平(1967—)，男，副教授，博士研究生，研究方向为农产品加工与储藏。E-mail：xzfjp@sina.com