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The antimicrobial activity of acidocin CH5 in MRS broth and milk with added NaCl, NaNO3 and lysozyme
International Journal of Food Microbiology 43 (1998) 33–38
The antimicrobial activity of acidocin CH5 in MRS broth and milk with added NaCl, NaNO 3 and lysozyme Jana Chumchalova´ a , *, Jytte Josephsen b , Milada Plockova´ a b
a Department of Dairy and Fat Technology, Institute of Chemical Technology, Technicka´ 5, 166 28 Prague 6, Czech Republic Department of Dairy and Food Science, The Royal Veterinary and Agricultural University, Food Microbiology, Rolighedsvej 30, DK-1958 Frederiksberg C, Denmark
Received 8 September 1997; received in revised form 18 May 1998; accepted 8 June 1998
Abstract The ability of acidocin CH5, a bacteriocin from Lactobacillus acidophilus CH5 in the form of neutralized and heated supernatant, to prevent the growth of the indicator Lactobacillus delbrueckii subsp. lactis LTI 30 alone or together with other antimicrobials was investigated. The inhibitory activity of acidocin CH5 was higher in MRS broth than in reconstituted skim milk (RSM). In MRS broth and RSM, 1.92 and 32 AU acidocin CH5 / ml, respectively, caused 97 and 89% inhibition of the indicator Lactobacillus delbrueckii subsp. lactis LTI 30. The presence of 5 and 10% milk fat in RSM decreased the inhibitory activity of acidocin CH5 to 20 and 11%, respectively. The inhibitory activity of acidocin CH5 was also reduced in the presence of NaCl, NaNO 3 and lysozyme. In RSM the inhibition was weaker with both acidocin CH5 and NaCl added compared with NaCl alone. In MRS broth the inhibition was stronger with both acidocin CH5 and NaCl added compared with NaCl alone. The inhibition of the indicator Lactobacillus delbrueckii subsp. lactis LTI 30 was stronger with both NaNO 3 and acidocin CH5 in MRS broth (but not in RSM) than with only NaNO 3 present, but the strongest level was obtained with acidocin CH5 alone. Addition of acidocin CH5 and more than 30 mg / ml lysozyme to MRS broth increased the level of inhibition above the level obtained by acidocin CH5 alone. The indicator Lactobacillus delbrueckii subsp. lactis LTI 30 was also sensitive to NaCl, NaNO 3 and lysozyme in both MRS broth and RSM. 1998 Elsevier Science B.V. All rights reserved. Keywords: Bacteriocin; Lactobacillus acidophilus; Lactobacillus delbrueckii subsp. lactis; Lysozyme; Milk; Sodium chloride; Sodium nitrate
1. Introduction The use of bacteriocin-producing lactic acid bacteria or their more or less purified bacteriocins as *Corresponding author. Tel.: 142 2 24353271; fax: 142 2 3119990; e-mail: [email protected]
biopreservatives in food has received increased interest as consumers desire more ‘natural’ food with less chemical preservatives. The activity of bacteriocins in food is highly dependent upon a wide range of physical, chemical and biological factors such as pH, ionic strength, temperature and the type of target microorganism. Bacteriocins are less effec-
0168-1605 / 98 / $19.00 1998 Elsevier Science B.V. All rights reserved. PII: S0168-1605( 98 )00094-4
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J. Chumchalova´ et al. / International Journal of Food Microbiology 43 (1998) 33 – 38
tive inhibitors in the food matrix than in synthetic media, as they are susceptible to enzymatic degradation and non-specific binding to proteins and lipids (Schillinger et al., 1996). Their effectiveness can also be influenced by the preservation method applied and evaluation of the antimicrobial effect of a bacteriocin requires testing in complex food systems (Shelef and Seiter, 1993). In this study the antibacterial activities of acidocin CH5, a bacteriocin produced by Lactobacillus acidophilus CH5 (Chumchalova´ et al., 1995), was investigated in reconstituted skim milk and in MRS broth, alone or in combination with sodium chloride, sodium nitrate and lysozyme. Lactobacillus delbrueckii susp. lactis LTI 30 was selected as the target strain.
2.3. Agar diffusion assay Bacteriocin activity was assayed by the agar diffusion method (Plockova´ et al., 1995b) using approximately 10 5 –10 6 CFU of Lactobacillus delbrueckii subsp. lactis LTI 30 per ml as indicator strain. Zones of growth inhibition were measured after 16 h of incubation. The bacteriocin titer was calculated using the formula BT (AU / ml) 5 2 x 3 1000 /V where x is the number of the last dilution showing inhibition and V is the volume of the supernatant (ml) (Chumchalova´ et al., 1995).
2.4. Food additives 2. Materials and methods
2.1. Bacterial strains and media The bacteriocin-producing strain Lactobacillus acidophilus CH5 (Chumchalova´ et al., 1995) was grown under aerobic conditions for 16 h in MRS broth (Merck, Darmstadt, Germany) at 378C. The indicator strain Lactobacillus delbrueckii subsp. lactis LTI 30 was grown in MRS broth, in reconstituted ´ ˇ na Morave, ˇ Czech skim milk (RSM) (KDV, Zabreh Republic) and in RSM with 5 and 10% (w / v) milk fat (Milkos, Strakonice, Czech Republic) and 8.6% non-liquid milk solids under the same conditions as above. All types of milk were supplemented with ˇ ˇ ´ Michalany, 0.5% yeast extract (Imuna, Sarisske Slovak Republic) and treated at 998C for 1 h. Before use Lactobacillus delbrueckii subsp. lactis LTI 30 was subcultured at least three times in the particular medium.
2.2. Preparation of supernatant containing acidocin CH5 The supernatant of a 16 h, 378C culture of Lactobacillus acidophilus CH5 in MRS was adjusted to pH 6.5, boiled for 5 min, cooled, and stored at 2 208C. Before use the supernatant containing acidocin CH5 (SCA) was filter sterilized (0.2 mm, Millipore).
Lysozyme (L 6876) was obtained from Sigma Chem. Co. Sodium chloride and sodium nitrate were obtained from Lachema, Czech Republic. The final concentrations of additives were adjusted to the following concentrations: (i) sodium chloride (NaCl) at 1, 2, 3 and 4% (w / v); (ii) sodium nitrate (NaNO 3 ) at 0.001, 0.01, 0.015 and 0.03% (w / v); and (iii) lysozyme at 0.5, 5, 10 and 50 mg / l.
2.5. Experimental procedure Bottles with (i) MRS broth with 0, 0.32, 1.6, 1.92 and 3.2 AU acidocin CH5 / ml and (ii) RSM and RSM with 5 and 10% milk fat with 0, 0.32, 3.2, 8, 16 and 32 AU acidocin CH5 / ml were inoculated with 1% of a fresh culture of Lactobacillus delbrueckii subsp. lactis LTI 30 and incubated at 378C. The optical density (OD) of the MRS broth at 615 nm and the fat titratable acidity (TA) of the RSM and RSM with 5 and 10% milk was measured over a 24-h period. In the second part of the study the growth of Lactobacillus delbrueckii subsp. lactis LTI 30 in MRS broth with 0.32 AU acidocin CH5 / ml and in RSM with 16 AU acidocin CH5 / ml with or without (i) sodium chloride at 1, 2, 3 and 4% (w / v), (ii) sodium nitrate at 0.001, 0.01, 0.015 and 0.03% (w / v) and (iii) lysozyme at 0.5, 5, 10 and 50 mg / l was examined. The growth in MRS broth without any additives was used as the control. Inoculation was as above.
J. Chumchalova´ et al. / International Journal of Food Microbiology 43 (1998) 33 – 38
35
2.6. Determination of growth
3. Results and discussion
Growth in MRS was measured as the OD at 615 nm (Specol EK, Carl Zeiss, Jena, Germany). In milk, TA determination was carried out by titrating a 10 ml milk sample with 0.25 M NaOH. TA was expressed in 2.5 mmol H 1 / l. The growth rates were calculated as DOD and DTA after 17 h growth as DOD 5 OD ( 17 ) 2 OD ( 0 ) and DTA 5 TA (17 ) 2 TA ( 0 ) (2.5 mmol H 1 / l). The percentage inhibition was calculated as 100 2 DOD (x) / OD ( y) 3 100, where (x) is the sample with antimicrobial (one or two) and ( y) is the sample without antimicrobial.
Fig. 1 shows the growth curve of Lactobacillus delbrueckii subsp. lactis LTI 30 in MRS broth containing 0–1.92 AU acidocin CH5 / ml. At a concentration of 0.32 and 1.6 AU acidocin CH5 / ml, 61 and 75% inhibition, respectively, was observed and nearly complete inhibition (97%) was seen when 1.92 AU acidocin CH5 / ml was used. According to these results the target bacterial strain exhibited a high sensitivity to the crude form of acidocin CH5 in the MRS broth. As a decrease in the OD was not observed it is assumed that cell lysis did not occur. Table 1 shows the inhibitory effect of acidocin CH5 in RSM and RSM with 5 and 10% milk fat added. The largest inhibitory effect was found in RSM without added fat. In RSM alone the inhibitory effect increased almost linearly with the concentration of the bacteriocin. The inhibitory effect from 0.32, 3.20 and 8.0 AU acidocin CH5 / ml was 9, 13 and 17%, respectively, which is very low compared to the inhibitory effect seen in MRS broth where 1.92 AU acidocin CH5 / ml gave 97% inhibition (Fig. 1). The decrease in the inhibitory effect of acidocin CH5 may be due to the action of proteases or other enzymes that can degrade the bacteriocin or due the interactions of acidocin CH5 with milk constituents. The same effect was observed in the study performed by Jung et al. (1992) for nisin. Sakacin A activity against Listeria monocytogenes in a model system of meat particles was also less than in meat juice or a synthetic medium (Schillinger, 1990). In RSM with 5% milk fat the increase of titratable acidity was much lower than for RSM alone (Table
Fig. 1. Influence of acidocin CH5 on the growth (OD at 615 nm) of Lactobacillus delbrueckii subsp. lactis LTI 30 at 378C in MRS broth [(j) 0 AU / ml, (h) 0.32 AU / ml, (d) 1.6 AU / ml, (s) 1.92 AU / ml].
Table 1 Inhibitory effect of acidocin CH5 in milk with various fat content on Lactobacillus delbrueckii subsp. lactis LTI 30 measured by the increase of titratable acid during 24 h at 378C AU acidocin
Skim milk
CH5 / ml
DTAa (2.5 mmol H 1 / l)
Percent inhibition
DTA (2.5 mmol H 1 / l)
Percent inhibition
DTA (2.5 mmol H 1 / l)
Percent inhibition
0 0.32 3.2 8 16 32
41.0 37.3 35.8 34.2 26.9 4.3
0 9.0 12.7 16.6 34.4 89.5
27.7 26.1 26.5 19.8 29.2 22.1
0 5.8 4.3 28.5 ‘‘0’’ 20.2
31.6 29.6 30.4 30.4 29.2 28.1
0 6.3 3.8 3.8 7.6 11.1
a
Milk with 5% fat
Increase of titratable acidity (TA) after incubation at 378C for 24 h.
Milk with 10% fat
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J. Chumchalova´ et al. / International Journal of Food Microbiology 43 (1998) 33 – 38
1). The same was observed with 10% milk fat added. This indicates that fat by itself inhibited the growth of Lactobacillus delbrueckii subsp. lactis LTI 30. The presence of fat also influenced the activity of acidocin CH5. As seen from Table 1 the effect was pronounced and other studies have also shown that the activity of bacteriocins is diminished in foods that contain fat (Jung et al., 1992). According to published data (Parish and Davidson, 1993) the presence of phospholipids in the milk fat can interfere with the bacteriocin molecules and reduce their inhibitory activity.
3.1. The effect of additives The effect of NaCl alone and together with acidocin CH5 on Lactobacillus delbrueckii subsp. lactis LTI 30 in MRS broth and RSM was determined (Table 2a). Increasing the concentration of NaCl (1 to 4% w / v) in MRS broth and RSM inhibited the growth of Lactobacillus delbrueckii subsp. lactis LTI 30 and at 4% w / v NaCl practically no growth was observed after 24 h at 378C. The inhibition in RSM with NaCl alone was similar. When NaCl at low levels (1 and 2% w / v) was added
Table 2 Growth inhibition of L. delbrueckii subsp. lactis LTI 30 in MRS broth and in RSM in the presence of NaCl, NaNO 3 and lysozyme alone and in combination with 0.32 AU acidocin CH5 / ml in MRS broth and 16 AU acidocin CH5 / ml in RSM (reconstituted skim milk). Growth was determined by measurement of the optical density at 615 nm. Inhibition is expressed as a percentage relative to growth in MRS broth and RSM with no additions Concentration of NaCl (% w / v)
Percent growth inhibition In MRS broth
In RSM
NaCl
NaCl10.32 AU / ml
NaCl
NaCl116 AU / ml
(a) NaCl 0 1 2 3 4
0 15.9 28.6 62.4 100
60.9 23.0 48.0 80.1 100
0 51.9 68.3 85.1 92.8
46.7 33.6 52.9 78.1 92.8
Concentration of NaNO 3 (% w / v)
Percent growth inhibition In MRS broth NaNO 3
( b) NaNO3 0 0.001 0.01 0.015 0.02 0.03 Concentration of lysozyme (mg / l)
0 0 0 0 0 6.3
In RSM NaNO 3 10.32 AU / ml 60.9 56.7 37.0 36.6 36.3 38.0
NaNO 3 116 AU / ml
0 19.5 19.5 21.2 26.4 23.7
35.5 20.9 20.7 18.6 18.6 20.9
Percent growth inhibition In MRS broth Lysozyme
(c) Lysozyme 0 0.5 5 10 50
NaNO 3
0 16.4 6.5 25.4 58.5
In RSM Lysozyme10.32 AU / ml 60.9 24.5 33.5 33.5 82.4
Lysozyme
Lysozyme116 AU / ml
0 14.7 14.2 17.1 17.6
17.6 31.5 39.1 39.1 39.1
J. Chumchalova´ et al. / International Journal of Food Microbiology 43 (1998) 33 – 38
to MRS broth in combination with 0.32 AU acidocin CH5 / ml the inhibition of the indicator strain was lower than for the bacteriocin alone (Table 2a). However, when 3% NaCl was added together with the bacteriocin the inhibition appeared to be stronger than observed for the NaCl or the bacteriocin alone. The addition of 1% NaCl and 16 AU acidocin CH5 / ml to RSM gave similar results as for MRS. Addition of 2, 3 and 4% NaCl to RSM in combination with the bacteriocin resulted in lower inhibition compared with RSM with NaCl alone, but higher than in RSM with the bacteriocin alone. As seen from Table 2b the addition of more than 0.02% w / v NaNO 3 resulted in a weak growth inhibition of Lactobacillus delbrueckii subsp. lactis LTI 30 in MRS broth. In RSM a lower amount of NaNO 3 (0.001%) had an inhibitory effect. The growth inhibition in RSM did not increase significantly with increasing concentration of NaNO 3 . The antimicrobial effect of the combined addition of NaNO 3 and the bacteriocin appeared to be lower compared with the effect of the bacteriocin alone. The ability of lysozyme to prevent the growth of the indicator organism alone and with the bacteriocin in MRS broth and RSM is presented in Table 2c. Fifty mg / l of lysozyme together with 0.32 AU acidocin CH5 / ml exhibited 58.5% growth inhibition of Lactobacillus delbrueckii subsp. lactis LTI 30 in MRS broth. The growth inhibition caused by 50 mg / l of lysozyme alone or lysozyme together with the bacteriocin in RSM was significantly lower compared with MRS broth (Table 2c). In MRS, acidocin CH5 activity was also inhibited by lysozyme as only concentrations above 50 mg / l lysozyme produced inhibition, which was more than that obtained with acidocin CH5 alone. In RSM, the addition of increasing amounts (10–50 mg / l) of lysozyme did not change the level of inhibition (17%). The addition of 16 AU acidocin CH5 / ml increased the inhibition to 39% for the same concentrations. This shows that in both MRS and RSM media a higher level of inhibition of Lactobacillus delbrueckii subsp. lactis LTI 30 can be obtained by a combination of both bacteriocin and lysozyme than with the components alone. The results show that the supernatant containing bacteriocin acidocin CH5 from Lactobacillus acidophilus CH5 can inhibit the growth of Lactobacillus delbrueckii susp. lactis LTI 30 very
37
efficiently in MRS broth. The inhibitory activity is lower in RSM, especially in RSM containing 5 or 10% added fat. The inhibitory activity of acidocin CH5 is influenced by the presence of NaCl, NaNO 3 , and lysozyme. However, it was, in some cases, possible to increase the level of inhibition of Lactobacillus delbrueckii susp. lactis LTI 30 by acidocin CH5 significantly by addition of other substances such as NaCl or lysozyme compared with the use of the particular substances alone. The results of the present study agree with those of previous investigations in that the efficiency of bacteriocins of lactic acid bacteria decreased in multi-component food systems (Jung et al., 1992; Schillinger et al., 1996). Acidocin CH5 exerts a broad spectrum of activity and it is therefore expected that it possesses great biopreservative potential. It inhibits the growth of bacteria (Chumchalova´ et al., 1995) and fungi (Plockova´ et al., 1997a). From this point of view the strain Lactobacillus acidophilus CH5 has potential as a protective culture in several dairy products but the effect of other factors should be considered.
Acknowledgements This work was supported by the Grant Agency of the Czech Republic (grant 510 / 95 / 0990).
References ´ J., Josephsen, J., Plockova, ´ M., 1995. CharacterizaChumchalova, tion of acidocin CH5, a saccharolytic sensitive bacteriocin of Lactobacillus acidophilus CH5. Chem. Mikrobiol. Technol. Lebensm. 17, 145–150. Jung, D.S., Bodyfelt, F.W., Daeschel, M.A., 1992. Influence of fat and emulsifiers on the efficacy of nisin in inhibiting Listeria monocytogenes in fluid milk. J. Dairy Sci. 75, 387–393. Parish, M.E., Davidson, P.M., 1993. Methods for evaluation. In: Branen, A.L., Davidson, P.M. (Eds.), Antimicrobials in Food. Marcel Dekker, New York, pp. 597–615. ´ M., Chumchalova, ´ J., Tomanova, ´ J., 1997. Antifungal Plockova, activity of Lactobacillus acidophilus CH5 metabolites. Potrav. ˇ 15, 39–48. Vedy ˇ ´ ´ M., Chumchalova, ´ J., Husakova, ´ ´ J., Svirakova, ´ E., Plockova, 1995. Agar diffusion assay for nisin quantification using Bacillus stearothermophilus as an indicator organism. Potrav. ˇ 13, 279–289. Vedy
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Schillinger, U., 1990. Bacteriocins of lactic acid bacteria. In: Bills, D.D., Kung, S. (Eds.), Biotechnology and Food Safety. Butterworth-Heinemann Reed, pp. 55–74. Schillinger, U., Geisen, R., Holzapfel, W.H., 1996. Potential of antagonistic microorganisms and bacteriocins for the bio-
logical preservation of foods. Trends Food Sci. Technol. 7, 158–164. Shelef, L.A., Seiter, J.A., 1993. Indirect antimicrobials. In: Branen, A.L., Davidson, P.M. (Eds.), Antimicrobials in Food. Marcel Dekker, New York, pp. 539–569.
The antimicrobial activity of acidocin CH5 in MRS broth and milk with added NaCl, NaNO 3 and lysozyme Jana Chumchalova´ a , *, Jytte Josephsen b , Milada Plockova´ a b
a Department of Dairy and Fat Technology, Institute of Chemical Technology, Technicka´ 5, 166 28 Prague 6, Czech Republic Department of Dairy and Food Science, The Royal Veterinary and Agricultural University, Food Microbiology, Rolighedsvej 30, DK-1958 Frederiksberg C, Denmark
Received 8 September 1997; received in revised form 18 May 1998; accepted 8 June 1998
Abstract The ability of acidocin CH5, a bacteriocin from Lactobacillus acidophilus CH5 in the form of neutralized and heated supernatant, to prevent the growth of the indicator Lactobacillus delbrueckii subsp. lactis LTI 30 alone or together with other antimicrobials was investigated. The inhibitory activity of acidocin CH5 was higher in MRS broth than in reconstituted skim milk (RSM). In MRS broth and RSM, 1.92 and 32 AU acidocin CH5 / ml, respectively, caused 97 and 89% inhibition of the indicator Lactobacillus delbrueckii subsp. lactis LTI 30. The presence of 5 and 10% milk fat in RSM decreased the inhibitory activity of acidocin CH5 to 20 and 11%, respectively. The inhibitory activity of acidocin CH5 was also reduced in the presence of NaCl, NaNO 3 and lysozyme. In RSM the inhibition was weaker with both acidocin CH5 and NaCl added compared with NaCl alone. In MRS broth the inhibition was stronger with both acidocin CH5 and NaCl added compared with NaCl alone. The inhibition of the indicator Lactobacillus delbrueckii subsp. lactis LTI 30 was stronger with both NaNO 3 and acidocin CH5 in MRS broth (but not in RSM) than with only NaNO 3 present, but the strongest level was obtained with acidocin CH5 alone. Addition of acidocin CH5 and more than 30 mg / ml lysozyme to MRS broth increased the level of inhibition above the level obtained by acidocin CH5 alone. The indicator Lactobacillus delbrueckii subsp. lactis LTI 30 was also sensitive to NaCl, NaNO 3 and lysozyme in both MRS broth and RSM. 1998 Elsevier Science B.V. All rights reserved. Keywords: Bacteriocin; Lactobacillus acidophilus; Lactobacillus delbrueckii subsp. lactis; Lysozyme; Milk; Sodium chloride; Sodium nitrate
1. Introduction The use of bacteriocin-producing lactic acid bacteria or their more or less purified bacteriocins as *Corresponding author. Tel.: 142 2 24353271; fax: 142 2 3119990; e-mail: [email protected]
biopreservatives in food has received increased interest as consumers desire more ‘natural’ food with less chemical preservatives. The activity of bacteriocins in food is highly dependent upon a wide range of physical, chemical and biological factors such as pH, ionic strength, temperature and the type of target microorganism. Bacteriocins are less effec-
0168-1605 / 98 / $19.00 1998 Elsevier Science B.V. All rights reserved. PII: S0168-1605( 98 )00094-4
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J. Chumchalova´ et al. / International Journal of Food Microbiology 43 (1998) 33 – 38
tive inhibitors in the food matrix than in synthetic media, as they are susceptible to enzymatic degradation and non-specific binding to proteins and lipids (Schillinger et al., 1996). Their effectiveness can also be influenced by the preservation method applied and evaluation of the antimicrobial effect of a bacteriocin requires testing in complex food systems (Shelef and Seiter, 1993). In this study the antibacterial activities of acidocin CH5, a bacteriocin produced by Lactobacillus acidophilus CH5 (Chumchalova´ et al., 1995), was investigated in reconstituted skim milk and in MRS broth, alone or in combination with sodium chloride, sodium nitrate and lysozyme. Lactobacillus delbrueckii susp. lactis LTI 30 was selected as the target strain.
2.3. Agar diffusion assay Bacteriocin activity was assayed by the agar diffusion method (Plockova´ et al., 1995b) using approximately 10 5 –10 6 CFU of Lactobacillus delbrueckii subsp. lactis LTI 30 per ml as indicator strain. Zones of growth inhibition were measured after 16 h of incubation. The bacteriocin titer was calculated using the formula BT (AU / ml) 5 2 x 3 1000 /V where x is the number of the last dilution showing inhibition and V is the volume of the supernatant (ml) (Chumchalova´ et al., 1995).
2.4. Food additives 2. Materials and methods
2.1. Bacterial strains and media The bacteriocin-producing strain Lactobacillus acidophilus CH5 (Chumchalova´ et al., 1995) was grown under aerobic conditions for 16 h in MRS broth (Merck, Darmstadt, Germany) at 378C. The indicator strain Lactobacillus delbrueckii subsp. lactis LTI 30 was grown in MRS broth, in reconstituted ´ ˇ na Morave, ˇ Czech skim milk (RSM) (KDV, Zabreh Republic) and in RSM with 5 and 10% (w / v) milk fat (Milkos, Strakonice, Czech Republic) and 8.6% non-liquid milk solids under the same conditions as above. All types of milk were supplemented with ˇ ˇ ´ Michalany, 0.5% yeast extract (Imuna, Sarisske Slovak Republic) and treated at 998C for 1 h. Before use Lactobacillus delbrueckii subsp. lactis LTI 30 was subcultured at least three times in the particular medium.
2.2. Preparation of supernatant containing acidocin CH5 The supernatant of a 16 h, 378C culture of Lactobacillus acidophilus CH5 in MRS was adjusted to pH 6.5, boiled for 5 min, cooled, and stored at 2 208C. Before use the supernatant containing acidocin CH5 (SCA) was filter sterilized (0.2 mm, Millipore).
Lysozyme (L 6876) was obtained from Sigma Chem. Co. Sodium chloride and sodium nitrate were obtained from Lachema, Czech Republic. The final concentrations of additives were adjusted to the following concentrations: (i) sodium chloride (NaCl) at 1, 2, 3 and 4% (w / v); (ii) sodium nitrate (NaNO 3 ) at 0.001, 0.01, 0.015 and 0.03% (w / v); and (iii) lysozyme at 0.5, 5, 10 and 50 mg / l.
2.5. Experimental procedure Bottles with (i) MRS broth with 0, 0.32, 1.6, 1.92 and 3.2 AU acidocin CH5 / ml and (ii) RSM and RSM with 5 and 10% milk fat with 0, 0.32, 3.2, 8, 16 and 32 AU acidocin CH5 / ml were inoculated with 1% of a fresh culture of Lactobacillus delbrueckii subsp. lactis LTI 30 and incubated at 378C. The optical density (OD) of the MRS broth at 615 nm and the fat titratable acidity (TA) of the RSM and RSM with 5 and 10% milk was measured over a 24-h period. In the second part of the study the growth of Lactobacillus delbrueckii subsp. lactis LTI 30 in MRS broth with 0.32 AU acidocin CH5 / ml and in RSM with 16 AU acidocin CH5 / ml with or without (i) sodium chloride at 1, 2, 3 and 4% (w / v), (ii) sodium nitrate at 0.001, 0.01, 0.015 and 0.03% (w / v) and (iii) lysozyme at 0.5, 5, 10 and 50 mg / l was examined. The growth in MRS broth without any additives was used as the control. Inoculation was as above.
J. Chumchalova´ et al. / International Journal of Food Microbiology 43 (1998) 33 – 38
35
2.6. Determination of growth
3. Results and discussion
Growth in MRS was measured as the OD at 615 nm (Specol EK, Carl Zeiss, Jena, Germany). In milk, TA determination was carried out by titrating a 10 ml milk sample with 0.25 M NaOH. TA was expressed in 2.5 mmol H 1 / l. The growth rates were calculated as DOD and DTA after 17 h growth as DOD 5 OD ( 17 ) 2 OD ( 0 ) and DTA 5 TA (17 ) 2 TA ( 0 ) (2.5 mmol H 1 / l). The percentage inhibition was calculated as 100 2 DOD (x) / OD ( y) 3 100, where (x) is the sample with antimicrobial (one or two) and ( y) is the sample without antimicrobial.
Fig. 1 shows the growth curve of Lactobacillus delbrueckii subsp. lactis LTI 30 in MRS broth containing 0–1.92 AU acidocin CH5 / ml. At a concentration of 0.32 and 1.6 AU acidocin CH5 / ml, 61 and 75% inhibition, respectively, was observed and nearly complete inhibition (97%) was seen when 1.92 AU acidocin CH5 / ml was used. According to these results the target bacterial strain exhibited a high sensitivity to the crude form of acidocin CH5 in the MRS broth. As a decrease in the OD was not observed it is assumed that cell lysis did not occur. Table 1 shows the inhibitory effect of acidocin CH5 in RSM and RSM with 5 and 10% milk fat added. The largest inhibitory effect was found in RSM without added fat. In RSM alone the inhibitory effect increased almost linearly with the concentration of the bacteriocin. The inhibitory effect from 0.32, 3.20 and 8.0 AU acidocin CH5 / ml was 9, 13 and 17%, respectively, which is very low compared to the inhibitory effect seen in MRS broth where 1.92 AU acidocin CH5 / ml gave 97% inhibition (Fig. 1). The decrease in the inhibitory effect of acidocin CH5 may be due to the action of proteases or other enzymes that can degrade the bacteriocin or due the interactions of acidocin CH5 with milk constituents. The same effect was observed in the study performed by Jung et al. (1992) for nisin. Sakacin A activity against Listeria monocytogenes in a model system of meat particles was also less than in meat juice or a synthetic medium (Schillinger, 1990). In RSM with 5% milk fat the increase of titratable acidity was much lower than for RSM alone (Table
Fig. 1. Influence of acidocin CH5 on the growth (OD at 615 nm) of Lactobacillus delbrueckii subsp. lactis LTI 30 at 378C in MRS broth [(j) 0 AU / ml, (h) 0.32 AU / ml, (d) 1.6 AU / ml, (s) 1.92 AU / ml].
Table 1 Inhibitory effect of acidocin CH5 in milk with various fat content on Lactobacillus delbrueckii subsp. lactis LTI 30 measured by the increase of titratable acid during 24 h at 378C AU acidocin
Skim milk
CH5 / ml
DTAa (2.5 mmol H 1 / l)
Percent inhibition
DTA (2.5 mmol H 1 / l)
Percent inhibition
DTA (2.5 mmol H 1 / l)
Percent inhibition
0 0.32 3.2 8 16 32
41.0 37.3 35.8 34.2 26.9 4.3
0 9.0 12.7 16.6 34.4 89.5
27.7 26.1 26.5 19.8 29.2 22.1
0 5.8 4.3 28.5 ‘‘0’’ 20.2
31.6 29.6 30.4 30.4 29.2 28.1
0 6.3 3.8 3.8 7.6 11.1
a
Milk with 5% fat
Increase of titratable acidity (TA) after incubation at 378C for 24 h.
Milk with 10% fat
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J. Chumchalova´ et al. / International Journal of Food Microbiology 43 (1998) 33 – 38
1). The same was observed with 10% milk fat added. This indicates that fat by itself inhibited the growth of Lactobacillus delbrueckii subsp. lactis LTI 30. The presence of fat also influenced the activity of acidocin CH5. As seen from Table 1 the effect was pronounced and other studies have also shown that the activity of bacteriocins is diminished in foods that contain fat (Jung et al., 1992). According to published data (Parish and Davidson, 1993) the presence of phospholipids in the milk fat can interfere with the bacteriocin molecules and reduce their inhibitory activity.
3.1. The effect of additives The effect of NaCl alone and together with acidocin CH5 on Lactobacillus delbrueckii subsp. lactis LTI 30 in MRS broth and RSM was determined (Table 2a). Increasing the concentration of NaCl (1 to 4% w / v) in MRS broth and RSM inhibited the growth of Lactobacillus delbrueckii subsp. lactis LTI 30 and at 4% w / v NaCl practically no growth was observed after 24 h at 378C. The inhibition in RSM with NaCl alone was similar. When NaCl at low levels (1 and 2% w / v) was added
Table 2 Growth inhibition of L. delbrueckii subsp. lactis LTI 30 in MRS broth and in RSM in the presence of NaCl, NaNO 3 and lysozyme alone and in combination with 0.32 AU acidocin CH5 / ml in MRS broth and 16 AU acidocin CH5 / ml in RSM (reconstituted skim milk). Growth was determined by measurement of the optical density at 615 nm. Inhibition is expressed as a percentage relative to growth in MRS broth and RSM with no additions Concentration of NaCl (% w / v)
Percent growth inhibition In MRS broth
In RSM
NaCl
NaCl10.32 AU / ml
NaCl
NaCl116 AU / ml
(a) NaCl 0 1 2 3 4
0 15.9 28.6 62.4 100
60.9 23.0 48.0 80.1 100
0 51.9 68.3 85.1 92.8
46.7 33.6 52.9 78.1 92.8
Concentration of NaNO 3 (% w / v)
Percent growth inhibition In MRS broth NaNO 3
( b) NaNO3 0 0.001 0.01 0.015 0.02 0.03 Concentration of lysozyme (mg / l)
0 0 0 0 0 6.3
In RSM NaNO 3 10.32 AU / ml 60.9 56.7 37.0 36.6 36.3 38.0
NaNO 3 116 AU / ml
0 19.5 19.5 21.2 26.4 23.7
35.5 20.9 20.7 18.6 18.6 20.9
Percent growth inhibition In MRS broth Lysozyme
(c) Lysozyme 0 0.5 5 10 50
NaNO 3
0 16.4 6.5 25.4 58.5
In RSM Lysozyme10.32 AU / ml 60.9 24.5 33.5 33.5 82.4
Lysozyme
Lysozyme116 AU / ml
0 14.7 14.2 17.1 17.6
17.6 31.5 39.1 39.1 39.1
J. Chumchalova´ et al. / International Journal of Food Microbiology 43 (1998) 33 – 38
to MRS broth in combination with 0.32 AU acidocin CH5 / ml the inhibition of the indicator strain was lower than for the bacteriocin alone (Table 2a). However, when 3% NaCl was added together with the bacteriocin the inhibition appeared to be stronger than observed for the NaCl or the bacteriocin alone. The addition of 1% NaCl and 16 AU acidocin CH5 / ml to RSM gave similar results as for MRS. Addition of 2, 3 and 4% NaCl to RSM in combination with the bacteriocin resulted in lower inhibition compared with RSM with NaCl alone, but higher than in RSM with the bacteriocin alone. As seen from Table 2b the addition of more than 0.02% w / v NaNO 3 resulted in a weak growth inhibition of Lactobacillus delbrueckii subsp. lactis LTI 30 in MRS broth. In RSM a lower amount of NaNO 3 (0.001%) had an inhibitory effect. The growth inhibition in RSM did not increase significantly with increasing concentration of NaNO 3 . The antimicrobial effect of the combined addition of NaNO 3 and the bacteriocin appeared to be lower compared with the effect of the bacteriocin alone. The ability of lysozyme to prevent the growth of the indicator organism alone and with the bacteriocin in MRS broth and RSM is presented in Table 2c. Fifty mg / l of lysozyme together with 0.32 AU acidocin CH5 / ml exhibited 58.5% growth inhibition of Lactobacillus delbrueckii subsp. lactis LTI 30 in MRS broth. The growth inhibition caused by 50 mg / l of lysozyme alone or lysozyme together with the bacteriocin in RSM was significantly lower compared with MRS broth (Table 2c). In MRS, acidocin CH5 activity was also inhibited by lysozyme as only concentrations above 50 mg / l lysozyme produced inhibition, which was more than that obtained with acidocin CH5 alone. In RSM, the addition of increasing amounts (10–50 mg / l) of lysozyme did not change the level of inhibition (17%). The addition of 16 AU acidocin CH5 / ml increased the inhibition to 39% for the same concentrations. This shows that in both MRS and RSM media a higher level of inhibition of Lactobacillus delbrueckii subsp. lactis LTI 30 can be obtained by a combination of both bacteriocin and lysozyme than with the components alone. The results show that the supernatant containing bacteriocin acidocin CH5 from Lactobacillus acidophilus CH5 can inhibit the growth of Lactobacillus delbrueckii susp. lactis LTI 30 very
37
efficiently in MRS broth. The inhibitory activity is lower in RSM, especially in RSM containing 5 or 10% added fat. The inhibitory activity of acidocin CH5 is influenced by the presence of NaCl, NaNO 3 , and lysozyme. However, it was, in some cases, possible to increase the level of inhibition of Lactobacillus delbrueckii susp. lactis LTI 30 by acidocin CH5 significantly by addition of other substances such as NaCl or lysozyme compared with the use of the particular substances alone. The results of the present study agree with those of previous investigations in that the efficiency of bacteriocins of lactic acid bacteria decreased in multi-component food systems (Jung et al., 1992; Schillinger et al., 1996). Acidocin CH5 exerts a broad spectrum of activity and it is therefore expected that it possesses great biopreservative potential. It inhibits the growth of bacteria (Chumchalova´ et al., 1995) and fungi (Plockova´ et al., 1997a). From this point of view the strain Lactobacillus acidophilus CH5 has potential as a protective culture in several dairy products but the effect of other factors should be considered.
Acknowledgements This work was supported by the Grant Agency of the Czech Republic (grant 510 / 95 / 0990).
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