Inhibitory effect of bacteriocin-producing lactic acid bacteria against histamine-forming bacteria isolated from Myeolchi-jeot

Five samples of salted-fermented anchovy were obtained from retail stores in Busan and aseptically collected for these experiments. Each sample (50 g) was diluted with sterile peptone water (450 mL) and homogenized at high speed for 2 min in stomacher. The decimal serial dilutions of the homogenates were then subsequently prepared using a sterile peptone water, and 1.0-mL aliquots of the dilutes were inoculated into brain heart infusion (BHI) agar (BD Difco Co., Sparks, MD, USA) and incubated for 48 h at 37 °C. To isolate histamine-forming bacteria, each strain collected from the plates was sub-cultured for 48 h at 35 °C in decarboxylating broth contained l-histidine monohydrochloride monohydrate (Sigma-Aldrich, St Louis, MO, USA, 1 g/L) and pyridoxal-5′-phosphate (Sigma-Aldrich, 1 mg/L) according to the method of Bover-Cid and Holzapfel (1999) with minor modifications. The cell culture (0.1 mL) was spread on trypticase soy agar (TSA, BD Difco Co.) supplemented with 2.0% (w/v) l-histidine hydrochloride monohydrate. After incubation under anaerobic conditions for 4 days at 35 °C (Anoxomat system, MART Co., Netherland), the colonies with blue or purple color on the plates were considered as histamine-forming bacteria. Meanwhile, to distinguish lactic acid-producing bacteria from salted-fermented anchovy, the homogenized sample was spread directly onto the surface of MRS (BD Difco Co.) agar plates containing 1% CaCO₃. After incubation under aerobic conditions for 48 h at 37 °C, the colonies surrounded by a clear zone were randomly selected from the plates, purified on MRS agar, and examined histamine production as the abovementioned. The identity of histamine-forming isolates was confirmed by using 16S rRNA gene sequence analysis (Chen et al., 2008). Amplification of the isolates was performed with the universal primers UNI-L (5′-AGAGTTTGATCATGGCTCAG-3′) and UNI-R (5′-GTGTGACGGGCGGTGTGTAC-3′). Bacterial cells were cultivated in BHI broth at 37 °C with shaking overnight and centrifuged at 7000×g for 10 min. After washing, the cell pellets were resuspended in PBS (pH 7.0) and lysed by 20% sodium dodecyl sulfate (SDS). After the boiling process for 20 min at 85 °C, the cell debris was removed by centrifugation (13,000×g, 5 min, 4 °C). DNA in the supernatant was precipitated by addition of 70% ethanol and used as template DNA for polymerase chain reaction (PCR). PCR amplification was carried out with a reaction mixture consisting of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl₂, 20 pmol of each primer, 0.2 mM deoxynucleotide triphosphates, 0.5 U of Taq DNA polymerase (Applied Biosystem, Foster City, CA, USA), and template DNA (10 ng). Cycle conditions were an initial denaturation for 4 min at 94 °C, followed by 35 cycles of 30 s denaturation at 94 °C, 30 s annealing at 55 °C, 1 min primer extension at 72 °C, followed by a final extension for 7 min at 72 °C in a PCR Thermal Cycler (Bio-Rad Laboratories Ltd., Canada). To identify the LAB isolates which do not produce histamine, the universal primer used for identification of LAB was 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492RF (5′-GGTTACCTTGTTACGACTT-3′). Thermocycling was carried out using denaturation step at 94 °C for 1 min, annealing at 55 °C for 1 min, and extension at 72 °C for 2 min, for a total of 30 cycles. Amplicons were visualized on a 1.5% agarose gel staining with ethidium bromide to confirm successful amplification and then PCR product was purified using a QIAquick PCR Purification Kit (Qiagen, Valencia, CA, USA). The amplified DNA was directly sequenced with a DNA sequencer (ABI Prism® 3730 Avant Genetic Analyzer, Applied Biosystem) and sequence similarity searching was performed using the National Center for Biotechnology Information (NCBI) Basic Local Align Search Tool (BLAST).

To prepare the bacteriocin solution of the LAB strains, the culture extracts of the isolated LAB were obtained after 24-h incubation at 37 °C in MRS broth. The cultures were centrifuged at 7000×g for 10 min at 4 °C. The cell-free supernatant (CFS) was then adjusted with 1 N of NaOH to pH 6.5 to avoid effect of acid, treated with catalase (200 U/mL) for 30 min at 25 °C to remove hydrogen peroxide, and filtered through a 0.22-μm membrane filter (Millipore Corp., USA). Crude bacteriocin was precipitated from the CFS by 50% (w/v) ammonium sulfate and the precipitates were kept overnight at 4 °C with gentle stirring. After centrifugation (at 12,000×g for 30 min at 4 °C), the resulting pellets were dissolved in 20 mM sodium phosphate buffer (pH 6.5) and dialyzed overnight at 4 °C against distilled water using dialysis membrane (molecular weight cutoff, 1,000; Spectrum Labs., Gardena, CA, USA). The antimicrobial activity of the bacteriocin was assayed by microtiter plate assay (Holo et al., 1991), using histamine-forming bacteria as an indicator strain. In brief, each well of the microtiter plate (BD Falcon, Franklin Lakes, NJ, USA) was filled with 800 μL of BHI broth, 100 μL of a twofold serial dilution of the crude bacteriocin solution or PBS (pH 7.0), and 100 μL of cell cultures (1.0 × 10⁵ CFU/mL) of indicator organism grown in BHI broth. The plates were then placed in an aerobic incubator for 12 h at 37 °C. The extent of growth inhibition was measured spectrophotometrically at 600 nm using microplate reader (Bioteck, Inc., Korea). One bacteriocin unit (BU/mL) was arbitrarily defined as the reciprocal of the highest dilution inhibiting the 50% growth of the indicator strain compared to the control.

Histamine-forming bacteria isolated from the samples were seeded in TSB enriches with l-histidine hydrochloride monohydrate (0.5%) and pyridoxal-HCl (0.0005%) and incubated at 35 °C for 24 h. The cultures (1 mL) were transferred to test tubes containing the same broth (9 mL) and bacteriocin solution (100 and 200 BU/mL) from the LAB isolates and incubated for 24 h at 35 °C. These broth cultures were centrifuged (7000×g, 10 min, 4 °C) and filtered through a 0.22-μm membrane filter. The histamine content in the cultures was performed according to the procedure modified by Eerola et al. (1993) and Mah and Hwang (2003) using high-pressure liquid chromatography (HPLC, Hitachi, Tokyo, Japan). Briefly, 1 mL of the cell cultures or standard histamine solutions was added to 9 mL of 0.4 M perchloric acid (Merck, Darmstadt, Germany) and vigorously mixed. The mixture was then centrifuged at 3000×g for 10 min and the supernatant was filtered through Whatman paper No. 1. The samples (1 mL) were mixed with 2 M sodium hydroxide (200 μL) and saturated sodium bicarbonate solution (300 μL). Subsequently, the samples were added with 2 mL of 10 mg/mL dansyl chloride (Sigma-Aldrich) solution prepared in acetone and the mixture solution was incubated at 40 °C for 45 min. Residual dansyl chloride was removed by addition of 25% ammonium hydroxide (100 μL). After incubation for 30 min at room temperature, the volume of mixture was adjusted to 5 mL with acetonitrile. Finally, the dansyl derivatives were centrifuged at 2500×g for 5 min, and the supernatant was filtered through a 0.22-μm filter. A Nova-Pak C₁₈ column (150 mm × 3.9 mm, Waters, Milford, MA, USA) was used for the separation of histamine, and acetonitrile (solvent B) as the mobile phases at the flow rate of 1 mL/min. The sample volume injected was 20 μL, and the eluted sample was monitored at 254 nm. All experiments were performed in triplicate. Data are expressed as means ± standard deviation (SD) and analyzed with SPSS program (ver. 12.0, SPSS Inc., Chicago, IL, USA). Then data comparisons were performed with paired t test and differences were considered statistically significant when P values were <0.05.

The effect of pH on antimicrobial activity of the crude bacteriocin was determined by incubating the crude bacteriocin in various buffers [0.1 M acetate buffer (pH 2.0–5.0), 0.1 M phosphate buffer (pH 6.0–7.0), and Tris HCl (pH 8.0–10.0)] with pH ranging from 2 to 10 for 24 h at 37 °C. To evaluate the heat stability, the crude bacteriocin was heated for 10 min at 80, 100, and 120 °C. Sensitivity of the crude bacteriocin to proteolytic enzymes was examined by incubation of the sample for 1 h at 37 °C after treatment with the following enzymes (1 mg/mL): proteinase K in 50 mM Tris-HCl (pH 7.5), trypsin in 50 mM Tris-HCl (pH 8.0), pepsin in 100 mM Trish-HCl (pH 3.0), papain in 50 sodium phosphate acetate (pH 5.0), and α-chymotrypsin in 20 mM Tris-HCl (pH 8.0). Following incubation, the enzymes were heat inactivated for 3 min at 100 °C. The residual bacteriocin activity was determined by the microtiter plate assay as described earlier. In addition, the bacteriocin treated under each physical or chemical condition were added to TSB containing l-histidine hydrochloride monohydrate (0.5%) and pyridoxal-HCl (0.0005%) and inoculated with initial population of approximately 1.0 × 10⁵ CFU/mL of histamine-forming bacteria. After incubation for 24 h at 35 °C, histamine levels in the cultures were analyzed using HPLC described above. All the experiments were done in triplicate.

The results of the identification of histamine-forming bacteria and bacteriocin-producing LAB found in Myeolchi-jeot are presented in Table 1. Based on 16S rRNA gene sequences, histamine-forming bacteria were identified as B. licheniformis MCH01, Serratia marcescens MCH02, Staphylococcus xylosus MCH03, Aeromonas hydrophila MCH04, and Morganella morganii MCH05, with 98.0–99.9% similarity to the GenBank database. Meanwhile, 53 LAB strains isolated from Myeolchi-jeot were screened for the inhibition activity against histamine-forming bacteria, using a microtiter plate assay. Out of these strains, five strains (9.4%) presented 98.3–100.0% similarity with the 16S rRNA sequences reported for Pediococcus acidilactici MCL11, Leuconostoc mesenteroides MCL12, Enterococcus faecium MCL13, Lactobacillus sakei MCL14, and Lactobacillus acidophilus MCL15 in the GenBank database.

Several fish species including tuna, mackerel, sardines, and anchovy contain high levels of free histidine in their tissues. These fishes subjected to temperature abuse after the catch and before consumption can cause formation of histamine from histidine by bacterial histidine decarboylases (Visciano et al., 2012). Histamine-rich foods may cause food intolerance in sensitive individuals and histamine poisoning that is a common seafood-borne disease causing various symptoms such as rash, nausea, vomiting, fever, diarrhea, headache, itching, flushing, and abdominal cramps (Taylor, 1986). Proteus vulgaris, Proteus mirabilis, Clostridium perfringens, Enterobacter aerogenes, Klebsiella pneumonia, Hafnia alvei, and Vibrio alginolyticus and enterobacteriaceae have been isolated from fish and described as the predominant histamine-forming bacteria (Shalaby, 1996; Kung et al., 2009). Moreover, there have been several reports describing amino acid decarboxylation activity of different genera, such as Acinetobacter, Aeromonas, Bacillus, Cedecea, Citrobacter, Clostridium, Escherichia, Klebsiella, Plesiomonas, Proteus, Pseudomonas, Salmonella, Serratia, Shigella, and Vibrio, and of some lactic acid bacteria (Kim et al., 2009). Our data are partially consistent with results reported by Guan et al. (2011) and Kung et al. (2009). The commonly isolated genera from Myeolchi-jeot were Microbacterium sp., Kocuria sp., Vibrio sp., Psychrobacter sp., Halomonas sp., Brevibacillus sp., Bacillus sp., Enterococcus sp., Tetragenococcus sp., Weissella sp., Lactobacillus sp., and Staphylococcus sp. (Guan et al., 2011). Among bacterial communities in Jeotgal, M. morganii, K. pneumoniae, and H. alvei have been known as the strains causing scombroid poisoning (often called “histamine poisoning”) (Kung et al., 2009). In addition, Staphylococcus epidermidis obtained during the ripening of anchovies showed a powerful histamine-forming activity (Hernández-Herrero et al. 1999).

As shown in Table 2, the five LAB strains isolated from Myeolchi-jeot were found to produce an antibacterial compound with inhibitory activity against histamine-producing bacteria such as B. licheniformis MCH01, S. marcescens MCH02, S. xylosus MCH03, A. hydrophila MCH04, or M. moarganii MCH05. The crude bacteriocin of P. acidilactici MCL11 inhibited the growth of B. licheniformis MCH01 and S. xylosus MCH03. In particular, P. acidilactici MCL11 showed the strongest antimicrobial activity (512 BU/mL) against S. xylosus MCH03. The antimicrobial compound produced by L. mesenteroides MCL12 (128 BU/mL) and L. sakei MCL14 (256 BU/mL) strains showed activity against S. marcescens MCH02. The crude bacteriocin produced by E. faecium MCL13 showed an antimicrobial activity of 64 and 256 BU/mL against S. xylosus MCH03 and A. hydrophila MCH04, respectively. L. acidophilus MCL15 was also found to have a bacteriocin activity against A. hydrophila MCH04, which was weaker than that of E. faecium MCL13.

Joosten and Nuńez (1996) reported that the bacteriocin-producing enterococci and L. lactis strains completely inhibited the outgrowth of histamine producer L. buchneri St2A; therefore, no histamine formation was detected in the cheeses made with bacteriocin-producing starters. Our results are in agreement with the findings of previous studies. Gómez-Sala et al. (2015) demonstrated that analysis of 1245 LAB isolates obtained from fish, seafood, and fish products showed that 197 exerted direct antimicrobial activity against 20 spoilage and food-borne pathogenic microorganisms. Furthermore, LAB isolates selected on the basis of their direct antimicrobial activity were identified as E. faecium, E. faecalis, Pediococcus pentosaceus, Weissella cibaria, L. sakei subsp. carnosus, L. sakei subsp. sakei, Lactobacillus curvatus, and L. mesenteroides subsp. cremoris based on 16S rDNA sequences. L. mesenteroides HK4, HK5, and HK11 and Streptococcus salivarius HK8 strains isolated from Jeotgal were also chosen by preliminary bacteriocin activity test (Cho and Do, 2006). L. lactis subsp. lactis VR84 produced nisin Z induced the death of the histamine producing strain S. thermophilus PRI60. However, L. lactis subsp. lactis EG46-produced lacticin 481 did not show a lethal action against PRI60 strain, but were able to reduce its growth extent and histamine accumulation (Tabanelli et al., 2014). Furthermore, the bacteriocin produced by L. casei was able to inhibit the activity of the histamine-forming bacteria such as Pseudomonas sp., Proteus morganii, and Micrococcus sp. (Nugrahani et al., 2016). S. xylosus no. 0538 obtained from a salted and fermented anchovy (Myeolchi-jeot) possessed not only the greater capability to degrade histamine but a detectable ability to degrade tyramine as well. In addition, this strain was also found to produce the bacteriocin-like inhibitory substance(s) and have the highest antimicrobial activity against B. licheniformis strains defined as amine producers. S. xylosus no. 0538 exhibited significantly greater ability to degrade histamine, degrading histamine to about 62–68% of its initial concentration within 24 h (Mah and Hwang, 2009).

As shown in Table 3, the five histamine-producing bacteria isolated from Myeolchi-jeot had strong ability to produce histamine. Among the tested strains, the highest level of histamine (2869.4 ± 49.0 mg/L) formation was observed for the M. morganii MCH05 strain. In addition, the concentration of histamine produced by S. xylosus MCH03 (2257 ± 30.7 mg/L) strain was higher than that produced by B. licheniformis MCH01 (1699.3 ± 35.6 mg/L), S. marcescens MCH02 (1987.2 ± 27.8 mg/L), and A. hydrophila MCH04 (1655.5 ± 41.2 mg/L). Meanwhile, the treatment with 100 and 200 BU/mL of the bacteriocin obtained from the tested LAB significantly reduced the histamine content of the five histamine-producing bacteria. After 24 h of incubation in the presence of the bacteriocin (200 BU/mL) of P. acidilactici MCL11, histamine contents of B. licheniformis MCL01 and S. xylosus MCH03 were reduced by 49 and 27%, respectively, as compared to the control (without bacteriocin). The histamine accumulation of S. xylosus MCH03 and A. hydrophilia MCH04 at 24 h of incubation in the presence of the bacteriocin of E. faecium MCL13 was significantly inhibited compared with the control group. The bacteriocin obtained from L. sakei MCL14 was effective in degrading histamine produced by S. marcescens MCH02 and M. morganii MCH05 strains. These bacteriocins produced by L. mesenteroides MCL12 and L. acidophilus MCL15 strains also exhibited excellent histamine degradation ability. The histamine degradation ability of these bacteriocins increased in a concentration-dependent manner. The reduction of histamine content by treatment with these bacteriocins may be related to antagonistic activity of these antibacterial substances against histamine-producing bacteria.

Prolific histamine formers in Indian anchovy were identified as M. morganii, P. vulgaris, and E. aerogenes, and produced high histamine content of 104.1–203.0 mg/100 mL (Rodtong et al., 2005). Kim et al. (2009) noted that the histamine contents in fish, squid, and shellfish samples remarkably increased up to 36.6–2123.9 mg/kg after 24 h of storage at 25 °C, while the contents began to gradually increase after 2–3 days of storage at 4–10 °C. The dominant microbial group in these samples was enterobacteria throughout the storage period. In our results, the histamine-producing isolates from Myeolchi-jeot were identified as B. licheniformis, S. marcescens, S. xylosus, A. hydrophila, and M. morganii and these strains were capable of producing histamine in the range of 1655.5 to 2869.4 mg/L. Consequently, in raw fish, histamine content is linked to the type of histamine-forming bacteria, the type of seafood, and temperature/time storage conditions (Visciano et al., 2012).

Regarding to the inhibition of histamine formation, Zaman et al. (2011) observed that Staphylococcus carnosus FS19 and Bacillus amyloiquefaciens FS05 isolated from fish sauce which possess amine oxidase activity were found to be effective in reducing biogenic amine accumulation, and histamine concentration was reduced by 27.7 and 15.4% by FS19 and FS05, respectively, which is in disagreement with our observations that the LAB isolates tested in our study showed histamine degradation capacity by production of the antimicrobial substance such as bacteriocin. The histamine levels produced by B. licheniformis MCH01, S. marcescens MCH02, S. xylosus MCH03, A. hydrophila MCH04, and M. morganii MCH05 were reduced by 21–77% in the presence of the bacteriocin (200 BU/mL) obtained from the tested LAB.

These bacteriocins obtained from P. acidilactici MCL11 and L. sakei MCL14 remained stable after incubation at pH 4.0–8.0. However, no the bacteriocin activity was recorded under extremely acidic condition (pH 2.0). The bacteriocin of L. mesenteroides MCL12 and E. faecium MCL13 remained stable after incubation for 24 h at pH from 4.0 to 10.0, but not when kept at pH 2.0. The bacteriocin of L. acidophilus MCL15 remained active after 24 h of exposure to pH values ranging from 3.0 to 9.0 at 37 °C. The activity of the bacteriocin produced by P. acidilactici MCL11, L. mesenteroides MCL12, and E. faecium MCL13 remained almost completely after heating for 10 min at 80 °C; however, the bacteriocin activity of these strains was partially destroyed after 10 min at 100 °C. In particular, the bacteriocin produced by E. faecium MCL13 was inactivated after 10 min at 120 °C, whereas the inhibitory activity of L. sakei MCL14 was 100% stable to heat treatment at 100 °C for up to 10 min, and the antimicrobial activity of L. acidophilus MCL15 was not affected by the heat treatment for 10 min at 120 °C. The activity of the bacteriocin produced by P. acidilactici MCL11 was destroyed after treatment with papain, pepsin, and proteinase K, but not when treated with α-chymotrypsin and trypsin. However, the treatment with papain and proteinase K had no effect on the activity of the bacteriocin of L. mesenteroides MCL12. Treatment of the bacteriocin produced by E. faecium MCL13 with papain and trypsin did not result in any activity loss, but the bacteriocin activity partially inactivated by treatment with pepsin, proteinase K, and α-chymotrypsin. Meanwhile, the bacteriocin of L. sakei MCL14 was destroyed by all the proteolytic enzymes tested such as papain, pepsin, proteinase K, α-chymotrypsin, and trypsin. Complete inactivation in antimicrobial activity of the bacteriocin produced by L. acidophilus MC15 was observed after treatment with α-chymotrypsin and trypsin. The histamine-degrading ability of the bacteriocin inactivated by some physico-chemical treatments was significantly reduced compared to the control group (Table 4).

The bacteriocin activity of P. acidilactici MCL11 was not affected by treatment with α-chymotrypsin and trypsin, but was lost after incubation with proteolytic enzymes such as papain, pepsin, and proteinase K. This bacteriocin was stable at up to 80 °C for 10 min and was in the pH range of 4.0–8.0. This is in disagreement with results recorded for pediocin SA-1. Pediocin SA-1 from P. acidilactici NRRLB5627 was inhibitory to several food spoilage bacteria and food-borne pathogens such as Listeria spp. and found to be very effective against the anaerobic Clostridium sporogenes and Clostridium thiaminolyticum (Papagianni and Anastasiadou, 2009). This bacteriocin was heat stable for up to 60 min at 121 °C, not impaired even following incubation at 30 °C for 1 week at pH values ranging between 3.0 and 12.0, and found to be resistant to treatment with trypsin, α-chymotrypsin, pepsin, and papain, but not to proteinase K (Anastasiadou et al., 2008).

The bacteriocin produced by L. mesenteroides MCL12 showed slight resistance to proteolytic enzymes such as α-chymotrypsin and trypsin. Treatment of this bacteriocin with papain and proteinase K had no effect on activity. The activity of this bacteriocin did not decrease after heat treatment at 80 °C for 10 min and the stability of the antimicrobial activity was observed at pH range of 4.0 to 10.0. Characteristics of the bacteriocin produced by L. mesenteroides MCL12 were widely different from those of the mesentericin Y105. Mesentericin Y105, the bacteriocin from L. mesenteroides, had a narrow inhibitory spectrum limited to Listeria genus. Neither the Gram-negative and other Gram-positive indicator bacteria nor the related LAB species were inhibited when tested by the well-diffusion assay. This bacteriocin exhibited excellent stability under heating and acidic conditions. However, all of the proteolytic enzymes such as pronase, proteinase K, trypsin, chymotrypsin, and pepsin totally inhibited the antimicrobial activity of this bacteriocin (Héchard et al., 1992).

The bacteriocin activity of E. faecium MCL13 was stable the pH range between 3.0 and 10.0. However, a reduction in the activity was observed when the bacteriocin was exposed to 100 °C for 10 min. The bacteriocin activity was not affected by the presence of papain and trypsin. Unlike our results, the bacteriocins produced by the E. faecium strain showed a broader spectrum of activity against indicator strains of Enterococcus spp., Listeria spp., Clostridium spp., and Propionibacterium spp. This bactercioin was inactivated by α-chymotrypsin, proteinase K, trypsin, pronase, pepsin, and papain, but not by lipase, lysozyme, and catalase. The bacteriocin was heat stable and displayed highest activity at neutral pH (Toit et al., 2000).

Jiang et al. (2012) reported that sakacin LSJ618 produced by the strain L. sakei LSJ618 exhibited inhibitory activity against food-spoiling bacteria and food-borne pathogens, including the Gram-positive L. monocytogenes, Staphylococcus aureus, Sarcina spp., Micrococcus luteus, and the Gram-negative Proteus spp. and Escherichia coli, but not against most of the LAB tested. This bacteriocin was completely inactivated by pepsin, papain, trypsin, and lipase, was stable between pH 2.0 and 8.0, and was heat resistant (30 min at 121 °C), which is partially in agreement with our observations. The bacteriocin of L. sakei MCL14 retained their activity at pH 4.0–8.0 and was thermally stable over a wide temperature range up to 100 °C for 10 min. Also, the bacteriocin activity was unstable after treatment with all the proteolytic enzymes like papain, pepsin, proteinase K, α-chymotrypsin, and trypsin.

The activity of the bacteriocin produced by L. acidophilus MCL15 was stable a pH range between 2.0 and 10.0, and remained constant after heating at 80, 100, and 120 °C for 10 min. However, the bacteriocin activity was destroyed or diminished after treatment with proteolytic enzymes such as papain, pepsin, proteinase K, α-chymotrypsin, and trypsin. The same results were recorded for the bacteriocin of L. acidophilus IBB 801. Acidophilin 801 obtained from L. acidophilus IBB 801 strain displayed a narrow inhibitory spectrum, being active particularly towards closely related lactobacilli and two Gram-negative pathogenic bacteria including E. coli Row and Salmonella panama 1467, whereas acidolin and acidophilin produced by L. acidophilus strains showed a wide inhibitory spectrum against Gram-positive and Gram-negative bacteria. The antimicrobial activity of acidophilin 801 was insensitive to catalase but sensitive to proteolytic enzymes such as trypsin, proteinase K, and pronase, heat-stable (30 min at 121 °C), and maintained in a wide pH range (pH 3.0–10.0) (Zamfir et al., 1999).