2015 Volume 21 Issue 5 Pages 751-755
Soy-seasoned salmon roe products are commonly eaten in Japan; long-term preservation of these items requires refrigeration. However, salmon roe products are often contaminated with Listeria monocytogenes, which can potentially cause outbreaks of listeriosis. This study focused on developing a method to inhibit the growth of L. monocytogenes in salmon roe products by treating these foodstuffs with a combination of nisin and commercial pectin-hydrolysate, Neupectin L. We determined that treatment with 0.5 mg g−1 nisin completely inhibited the growth of L. monocytogenes in raw salmon roe. However, treatment with nisin alone did not inhibit L. monocytogenes in soy-seasoned salmon roes. Further work showed that combined treatment with 0.5 mg g−1 nisin and 0.5% Neupectin L completely inhibited the growth of L. monocytogenes during incubation at 12°C. Therefore, this combination is an alternative way to control L. monocytogenes growth in salmon roe products.
Listeriosis, caused by Listeria monocytogenes, is a worldwide health concern. L. monocytogenes is a ubiquitous bacterium that can live in diverse environments such as soil, water, plants, animals, and humans (Weis and Seeliger, 1975; Watkins and Sleath, 1981; Okutani et al., 2004). This bacterium can also contaminate processed foods, such as cheese, butter, meat, and seafood, leading to foodborne listeriosis (Lianou and Sofos, 2007). Yamazaki et al. (2000) reported that 4.5% of raw salmon and 16.7% of smoked fishery products were contaminated by L. monocytogenes, and Handa et al. (2005) also reported that L. monocytogenes was detected in 10% of commercial raw salmon roes purchased in Japan. Salmon roes seasoned with soy sauce and salted salmon roes are often eaten in Japan and other countries as a unique accompaniment to sushi and sashimi. Therefore, an effective way to control the growth of L. monocytogenes is needed, especially since L. monocytogenes can proliferate even in refrigerated food products.
Bacteriocins, which are antimicrobial peptides or proteins synthesized by ribosomes in bacteria, have recently garnered interest as new natural antimicrobial agents. Bacteriocins produced by lactic acid bacteria are well studied and have been proven safe for use in food items and medical preparations (Cotter et al., 2005). The most common bacteriocin used in processed foods is nisin, a product of Lactococcus lactis subsp. lactis that shows antimicrobial activity against various gram-positive pathogenic bacteria (Cintas et al., 1998). Nisin has been most frequently used to prevent the growth of L. monocytogenes in meats, dairy products, and vegetables (Davies et al., 1997; Bari et al., 2005; Theivendran et al., 2006; Arqués et al., 2008). Despite high contamination rates of L. monocytogenes in fishery products, nisin treatment of seafood products is limited compared with other food items. On the other hand, commercial pectin-hydrolysate preparation, Neupectin L, has a broad antimicrobial spectrum including bacteria, yeast, and fungi, and also shows antimicrobial activity in some processed foods such as soba dipping sauce, salted cod roes, and herring roes, without changing food quality. Therefore, Neupectin L is widely used to inhibit the microbial growth in various foods as a natural food preservative (Nozaki and Uezawa, 1985).
In this study, we investigated the effect of a combination of nisin and commercial pectin hydrolysate on the growth of L. monocytogenes in raw salmon roes and soy-seasoned salmon roes.
Bacterial strains and growth conditions L. monocytogenes ATCC7644, IID579 (clinical isolate), and IID580 (clinical isolate) strains were used in this study. These strains were incubated at 30°C for 18 h in tryptic soy broth (BD; Sparks, MD, USA) supplemented with 0.6% yeast extract (TSBYE; BD).
Effects of pH, temperature, and sodium chloride on anti-listerial activity of nisin and potassium sorbate Minimum inhibitory concentrations (MICs) of nisin and potassium sorbate (PS) required for controlling L. monocytogenes ATCC7644 were determined by the broth microdilution method in TSBYE supplemented with 0.5 – 7.0% NaCl, adjusted to pH 5.0 – 7.0. L. monocytogenes ATCC7644 was suspended in TSBYE (6.0 log CFU mL−1) supplemented with either nisin A (Sigma-Aldrich; St. Louis, USA) or PS in a series of 2-fold serial dilutions in 96-well polypropylene microtiter plates (AGC Techno Glass; Shizuoka, Japan), and incubated at 12°C and 30°C. After the OD595 nm of the control samples reached 0.3, the plates were further incubated at 30°C for 2 days or 12°C for 7 days. MICs were determined by observing the turbidity of each well; the lowest concentration of nisin or PS that suppressed growth of L. monocytogenes was recorded as the MIC.
Preparation of liquid soy seasoning and determination of MICs Soy sauce, sweet sake, and water were mixed in a 3:1:1 ratio and heated to remove residual ethanol. This liquid seasoning was diluted with distilled water to 3.0% salinity. MICs of nisin, Neupectin L, and PS against a cocktail of L. monocytogenes ATCC7644, IID579, and IID580 (5.6 log CFU mL−1) in TSBYE or liquid soy seasoning at pH 5.7 or 6.1 were determined by the microdilution method described above. In this study, the “Neupectin L” preparation used was a commercial pectin hydrolysate composed of 28% pectin hydrolysate, 14% lactic acid, and 58% food materials (Asama Chemical; Tokyo, Japan).
Preparation of soy-seasoned salmon roe products and their chemical analyses Raw salmon roes were purchased from a local supermarket and washed with a 3% saline solution. Liquid seasoning and salmon roes were mixed in a ratio of 2:1 (v/w). After incubation at 4°C for 1 h, the excess liquid seasoning was removed and the soy-seasoned salmon roes were used for further investigation. Salinity and pH of the salmon roe samples were analyzed using the Mohr method and a pH meter (B212; Horiba, Kyoto, Japan), respectively.
Antimicrobial activity of nisin and Neupectin L combination in raw salmon roes and soy-seasoned salmon roe products Raw and soy-seasoned salmon roes were mixed with nisin, Neupectin L, and PS at final concentrations of 0.5 mg g−1, 0.5% (v/v), and 3.0 mg g−1, respectively. After incubation at 4°C for 1 h, the mixture was inoculated with a cocktail of L. monocytogenes ATCC7644, IID579, and IID580 at an initial cell density of 2.2 log CFU g−1. Inoculated raw and soy-seasoned salmon roes were incubated at 12°C and the viable cell counts of L. monocytogenes during the storage period were measured. The viable cell counts of L. monocytogenes were determined with Chromocult Listeria selective agar (Merck KGaA; Darmstadt, Germany) after incubation at 37°C for 24 h.
Organoleptic evaluation of soy-seasoned salmon roe products The color, shape, and flavor of soy-seasoned salmon roe products treated with nisin and Neupectin L were compared with those of untreated soy-seasoned salmon roe products.
Effects of pH, temperature, and sodium chloride on MICs of nisin and potassium sorbate MICs of nisin decreased with lowering of broth pH, such that MICs at pH 5.0 were 0.03 – 0.12 times the MICs at pH 7.0 under most experimental conditions (Table 1). Thomas and Wimpenny (1996) reported that antimicrobial activity of nisin increased with a decline in pH, and our results indicated a similar trend. Moreover, the MICs of nisin at 12°C were significantly lower than those at 30°C. Anti-listerial activities of PS at pH 6.0 and 7.0 were low. Antimicrobial activity of organic acids is due to their incorporation into the bacterial cell, which results in a lowering of pH and ultimately damages essential enzymes. The incorporation of organic acids into the bacterial cell depends on the environmental pH, because only undissociated molecules can enter the cell (Brul and Coote, 1999).
MICs, Minimum inhibitory concentrations.
In the presence of a high level of NaCl, the MICs of nisin increased; MICs in the presence of 3.0 – 7.0% NaCl were 4 – 8 times higher than those in the presence of 0.5% NaCl. Earlier studies have reported that NaCl enhanced nisin activity (Harris et al., 1991; Nilsson et al., 1997; Pawar et al., 2000), but some studies have contradicted these findings (Bell and Lacy, 1985; Bouttefroy et al., 2000). Our results also suggested that NaCl was one of the factors that inhibit the anti-listerial activity of nisin. Monovalent cations such as sodium ions can bind to the bacterial membrane surface and interact with the negatively charged head groups of the phospholipids; this effect has been previously reported for divalent cations (Abee, 1995). This interaction may prevent the first step of nisin adsorption and block the bactericidal effect (Jack et al., 1995). In contrast to nisin, PS caused decreases of MICs with increasing NaCl concentration. However, MICs of nisin were much lower than those of PS even at conditions unsuitable for nisin activity.
MICs of nisin, potassium sorbate, and Neupectin L in liquid soy seasoning Nisin, PS, and Neupectin L were effective against a cocktail of L. monocytogenes ATCC7644, IID579, and IID580 in liquid soy seasoning, as shown in Table 2. MICs of both PS and Neupectin L did not change in TSBYE and liquid soy seasoning. Furthermore, both showed higher anti-listerial activity at a lower temperature. Yokotsuka et al. (1984) reported that pectin hydrolysate showed antibacterial effects against gram-positive and gram-negative bacteria. Those authors concluded that the antibacterial properties of pectin hydrolysate were due to free undissociated carboxyl groups and methyl esters bonded to the carboxyl groups of polygalacturonic acid chains. However, MICs of nisin increased two-fold, except at pH 5.7, at the lower temperature. Antibacterial activity of nisin was inhibited by the presence of NaCl in liquid soy seasoning. Nisin showed high antimicrobial activity at nanomolar concentrations; this peptide's mechanism of action involves binding to lipid II, a membrane-anchored cell wall precursor. This step is followed by blocking of cell wall synthesis and pore formation in the plasma membrane (Breukink et al., 1999; Hasper et al., 2006). Crandall and Montville (1998) reported that a nisin-resistant strain of L. monocytogenes contains more zwitterionic phosphatidylethanol amine and less anionic phosphatidylglycerol and cardiolipin than the wild-type strain, and that requires divalent cations to resist the inhibitory effect of nisin. Crandall and Montville (1998) also suggested models for the antimicrobial activity of nisin against nisin-resistant L. monocytogenes. One model predicted that divalent cations were required to stabilize negative charges, thereby inhibiting the binding of nisin to these anionic sites on the bacterial cell surface. Therefore, we hypothesized that the positive charge of sodium ions in liquid soy seasoning stabilized the negative charge present on the cell surface of L. monocytogenes and inhibited the binding of nisin to lipid II.
MICs, Minimum inhibitory concentrations; TSBYE, Tryptic soy broth supplemented with 0.6% yeast extract.
Growth of L. monocytogenes in raw salmon roes and soy-seasoned salmon roe products The pH of raw salmon roe products prepared in the present study ranged from 5.9–6.1. The pH of soy-seasoned salmon roe treated with 0.5 mg g−1 nisin alone or with 3.0 mg g−1 PS alone were also 6.1. However, the pH of soy-seasoned salmon roe containing 0.5% Neupectin L alone or the combination of 0.5 mg g−1 nisin and 0.5% Neupectin L were 5.6 and 5.7, respectively. The low pH of the samples treated with Neupectin L appears to be due to the presence of lactic acid in the Neupectin L preparation. Salinities of the raw and soy-seasoned salmon roes were 0.49% and 1.50%, respectively (data not shown). These treatments did not affect the color, shape, or flavor of the salmon roes.
The growth of L. monocytogenes in raw salmon roes stored at 12°C is shown in Fig. 1a. In the control sample, L. monocytogenes multiplied rapidly, with the viable cell count reaching approximately 7 log CFU g−1 after a storage period of 4 days. In the samples treated with 3.0 mg g−1 PS or 0.5% Neupectin L alone, the growth rates of L. monocytogenes decreased slightly compared to that of the control sample. However, viable counts of L. monocytogenes in both samples still exceeded 6 log CFU g−1 during the storage periods. In the samples treated with 0.5 mg g−1 nisin alone or a combination of 0.5 mg g−1 nisin + 0.5% Neupectin L, L. monocytogenes was completely inhibited, and cell densities remained below the detectable limit (2 log CFU g−1) during storage periods. These results indicated that 0.5 mg g−1 nisin inhibits the growth of L. monocytogenes in raw salmon roes.
Growth of Listeria monocytogenes in raw salmon roes (a) and soy-seasoned salmon roe products (b) at 12°C. Symbols represent control (●), 0.5 mg g−1 nisin A (▲), 3.0 mg g−1 potassium sorbate (■), 0.5% Neupectin L (○), and the combination of 0.5 mg g−1 nisin A + 0.5% Neupectin L (□). Arrows indicate viable cell counts of L. monocytogenes were below the detectable limit (2 log CFU g−1). The results are shown as the mean ± standard deviation from three independent experiments.
Although Takahashi et al. (2011) reported that Nisaplin (a commercial antimicrobial agent containing nisin) inhibited the growth of L. monocytogenes in raw salmon roes, the anti-listerial activity of this agent in soy-seasoned salmon roe products was not reported. The growth of L. monocytogenes in soy-seasoned salmon roe products incubated at 12°C is shown in Fig. 1b. L. monocytogenes in control samples increased to 7 log CFU g−1 during storage periods, and its growth rate in soy seasoned roe was slightly lower than that in raw salmon roes, probably due to higher salinity of liquid soy seasoning. In the samples treated with 3.0 mg g−1 PS or 0.5% Neupectin L alone, the viable cell counts of L. monocytogenes reached around 5 log CFU g−1 after storage for 7 days. In samples treated with 0.5 mg g−1 nisin, viable cell counts of L. monocytogenes remained below the detectable limit (2 log CFU g−1) during the first 2 days of storage. Subsequently, L. monocytogenes growth increased to 4 log CFU g−1 after 7 days of storage. On the other hand, combined treatment with 0.5 mg g−1 nisin and 0.5% Neupectin L inhibited the growth of L. monocytogenes to levels below the detectable limit throughout the storage period. These results suggested that combined treatment with these two antimicrobial substances is an effective way to inhibit the growth of L. monocytogenes in soy-seasoned salmon roe products because of additive antibacterial effects. Bari et al. (2005) reported that application of nisin in combination with organic acids showed significant decreases of L. monocytogenes, and Ahamad and Marth (1989) also reported that 0.05% lactic acid prolonged generation times of L. monocytogenes incubated at 13°C. Since 0.07% lactic acid was present in the soy-seasoned salmon roe products used in this study, lactic acid present in Neupectin L was also considered to act as an additional mild antibacterial agent.
In conclusion, we have demonstrated an effective way to control L. monocytogenes by the combination of nisin and Neupectin L in soy-seasoned salmon roe products. This treatment may be used to make fishery products safer for consumption without altering food quality and taste.
Acknowledgements Neupectin L used in this study was kindly provided by Dr. Mizuo Yajima (Asama Chemical Co., Ltd., Tokyo, Japan). This work was supported by a research project ensuring food safety from farm to table (FP-6105), funded by the Ministry of Agriculture, Forestry and Fisheries of Japan.