Characterization of Nisin Produced by Lactococcus lactis RP359 Isolated from Kem-Buk-Nud, a Traditional Thai Fermented Food
P Rattanachaikunsopon, P Phumkhachorn
fermented food, nisin
P Rattanachaikunsopon, P Phumkhachorn. Characterization of Nisin Produced by Lactococcus lactis RP359 Isolated from Kem-Buk-Nud, a Traditional Thai Fermented Food. The Internet Journal of Microbiology. 2007 Volume 5 Number 1.
Five hundred colonies of lactic acid bacteria (LAB) isolated from Kem-Buk-Nud, a traditional Thai fermented food, were screened for the presence of nisin gene by using the polymerase chain reaction (PCR) with primers specific to nisin A structural gene. A LAB isolate, RP359, identified as
Nisin is a bacteriocin produced by some strains of
Kem-Buk-Nud is a traditional Thai fermented food mainly produced in the northeastern part of Thailand, especially in Ubon Ratchathani province. Its main ingredients are fresh water fish, salt and pineapple. It is consumed raw with vegetables. Since fermentation of Kem-Buk-Nud is still mediated by indigenous bacteria, it is difficult to obtain consistency of product quality and safety. In order to alleviate the difficulties the use of nisin producing lactic acid bacteria as a starter culture in fermentation of Kem-Buk-Nud is now being considered.
The use of nisin producing lactic acid bacteria as starter cultures in fermentation of foods from which they were isolated has been considered to be a practical way in development of starter cultures because no further strain improvement is required. This study is a very first step towards the development of nisin producing lactic acid bacteria to be used as a starter culture in production of Kem-Buk-Nud. This study focused on screening lactic acid bacteria isolated from Kem-Buk-Nud for the production of nisin using PCR technique and on examining some characteristics of the nisin. Nisin producing lactic acid bacteria obtained from this study will be ultimately developed as starter cultures in fermentation of Kem-Buk-Nud.
Materials and Methods
Bacterial strains and culture conditions. Bacteria used in this study are listed in Table 1.
Isolation and screening of nisin producing LAB from Kem-Buk-Nud samples. Kem-Buk-Nud obtained from different shops in Ubon Ratchathani province, Thailand were used as sources of LAB. Liquid parts of the samples were 10-fold serially diluted in 0.85% (w/v) sterile physiological saline. One hundred microliters of appropriate dilutions were spread on MRS agar plates and incubated at 30 ° C for 48 h. Five hundred colonies of LAB were randomly selected from the samples. To assess which LAB isolated from Kem-Buk-Nud carried a nisin gene, DNA coding for nisin was identified by the polymerase chain reaction (PCR) using
Identification of nisin producing lactic acid bacteria. The nisin producing LAB was identified by using the API 50CHL system as specified by the manufacturer (Bio-Merieux, France).
Detection of antimicrobial activity. Antimicrobial activity of nisins produced by
Determination of minimal inhibitory concentrations (MICs). To determine the MICs of nisins produced by
Effect of enzymes and heat on bacteriocin activity. To evaluate the effect of enzymes on bacteriocin activity, each bacteriocin preparation was treated with the following enzymes at final concentration of 1 mg ml -1 : α-chymotrypsin, trypsin, proteinase K, lipase and α-amylase. Following incubation at 37 ° C for 2 h, enzyme activity was terminated by heating at 100 ° C for 5 min. The residual activity was assayed against
Purification and gel electroporesis of nisins. Purification of the nisins produced by
Nucleotide sequence accession number: The nucleotide sequence described in this study has been deposited in the EMBL/GenBank/DDBJ databases under the accession number AM410671.
By using PCR with primers specific to nisin A structural gene, one LAB isolate, designated RP359 was shown to give a PCR product corresponding to a 231 bp nisin gene amplified from genomic DNA of
To analyze the amplified PCR product of LAB strain RP359, it was subjected to nucleotide sequencing. Results showed that the sequence was 231 bp long with an open reading frame (ORF) (Figure 1). The comparison between the sequence of nisin A gene and that of the ORF revealed a single nucleotide substitution from G to A at the position corresponding to the third base of the fifteenth codon of the ORF. To confirm the nucleotide substitution, PCR amplification and nucleotide sequencing of the PCR product were repeated for LAB strain RP359 and the same results were obtained. This point mutation was considered to be a silent mutation because it did not cause difference between amino acid sequence deduced from the ORF sequence and that of nisin A (Fig. 1).
To examine whether the silent mutation in nisin gene of
Purified nisins of
RP359 nisin and nisin A exhibited the same response to enzymes and heat treatments. They were sensitive to α-chymotrypsin and proteinase K but resistant the other enzymes including trypsin, lipase and α-amylase. No change in their antimicrobial activities was detected from the samples treated with heat at 100 ° C for 10, 20 and 30 min.
To investigate antimicrobial spectrum of RP359 nisin and nisin A, they were tested for antimicrobial activity using agar diffusion assay against a wide range of indicator organisms comprising LAB and food-borne pathogens. It was found that all strains that were sensitive to RP359 nisin were also sensitive to nisin A, and vice versa. The sizes of the inhibition zones varied considerably between the different indicator strains. However, among all of the sensitive strains, the inhibition zones produced by RP359 nisin were larger than those produced by nisin A (Table 1). When
The MICs of RP359 nisin and nisin A were determined in liquid cultures of nisin-sensitive indicator strains. It was found that the MICs of both nisins were identical in all cases (Table 1). These results demonstrate that there is no difference in specific activities of RP359 nisin and nisin A against all tested indicator strains.
a ATCC, American Type Culture Collection; DMST, Department of Medical Science Thailand; DSM, Deutsche Sammlung von Mikroorganismen; TISTR, Thailand Institute of Scientific and Technological Research.
b Values were obtained with identical concentrations (1µg ml -1 ) of bacteriocins in the agar diffusion assay. They were the average of two experiments.
In this study, we intended to screen LAB isolated from Kem-Buk-Nud for the production of nisin, not other bacteriocins because of beneficial properties of nisin and its suitability for our future uses. Nisin is the only bacteriocin accepted as safe to be used as a food preservative. It has been well tested and confirmed as non-toxic when consumed orally and has proved to be a safe food preservative (Delves-Broughton, 1990). The susceptibility of nisin to enzymatic degradation is an advantage for its use in food, as nisin is quickly digested and would not affect the intestinal flora or be absorbed into the blood stream.
The detection frequencies of nisin producing strains from LAB isolated from various foods were substantially different. Among traditional Thai fermented foods, one nisin producing strain was found from 500 LAB isolated from Kem-Buk-Nud whereas the same number of nisin producing strain was found from 14,020 LAB isolated from nham, a Thai style fermented meat (Noonpakdee
Nucleotide sequences of the coding regions of nisin A gene and RP359 nisin gene differed in only one nucleotide. This nucleotide substitution did not cause difference in amino acid sequences and some characteristics (spectrum of antimicrobial activity and sensitivity to enzymes and heat) between both nisins. Only difference between nisin A and RP359 nisin that could be detected was the size of the inhibition zones obtained from agar diffusion assays. From the results, it could be concluded that this difference was not resulted from the difference in production level or specific antimicrobial activity between nisin A and RP359 nisin. It is possible that the nucleotide substitution (silent mutation) in RP359 nisin gene may affect the physicochemical properties (eg., solubility, stability, aggregation behavior, and diffusion) of the nisin which in turn result in ability to produce large size of inhibition zones. Silent mutations in several genes have been reported to be responsible for changing in folding, solubility and aggregation behavior of the proteins encoded by the mutated genes (Cortazzo
Dr. Pongsak Rattanachaikunsopon, Department of Biological Science, Faculty of Science, Ubon Ratchathani University, Warin Chamrap, Ubon Ratchathani 34190, Thailand. Tel. and Fax: +66(45)288380. E-mail: email@example.com