Electronic Journal of Polish Agricultural Universities (EJPAU) founded by all Polish Agriculture Universities presents original papers and review articles relevant to all aspects of agricultural sciences. It is target for persons working both in science and industry,regulatory agencies or teaching in agricultural sector. Covered by IFIS Publishing (Food Science and Technology Abstracts), ELSEVIER Science - Food Science and Technology Program, CAS USA (Chemical Abstracts), CABI Publishing UK and ALPSP (Association of Learned and Professional Society Publisher - full membership). Presented in the Master List of Thomson ISI.
2005
Volume 8
Issue 4
Topic:
Food Science and Technology
ELECTRONIC
JOURNAL OF
POLISH
AGRICULTURAL
UNIVERSITIES
Modzelewska-Kapituła M. , Marin-Iniesta F. 2005. THE POSSIBILITY OF USING Lactobacillus fermentum STRAINS OF HUMAN ORIGIN AS PROTECTIVE CULTURES IN SOFT CHEESE, EJPAU 8(4), #14.
Available Online: http://www.ejpau.media.pl/volume8/issue4/art-14.html

THE POSSIBILITY OF USING LACTOBACILLUS FERMENTUM STRAINS OF HUMAN ORIGIN AS PROTECTIVE CULTURES IN SOFT CHEESE

Monika Modzelewska-Kapituła1, Fulgencio Marin-Iniesta2
1 Chair of Industrial and Food Microbiology, Faculty of Food Science, University of Warmia and Mazury, Poland
2 Faculty of Veterinary Medicine, University of Murcia, Spain

 

ABSTRACT

A possibility to use lactose-negative Lactobacillus fermentum strains, isolated from GI-tract of infants, as protective cultures in soft cheese was studied. The aim of the study was to evaluate if these lactic bacteria strains are capable to inhibit growth of Escherichia coli NCTC 12900 and Listeria monocytogenes LM82 in the environment of soft Spanish cheese. The study demonstrated that all strains of LAB used in the study were able to survive and grow in the environment of the cheese, achieving a population of 105-107 cfu/g. Strains of Lbc. fermentum exhibited some inhibition of Listeria monocytogenes and Escherichia coli growth in a model system of the cheese. After 15 days of incubation at 7°C number of E. coli and L. monocytogenes in cheese samples containing lactic acid bacteria strains were statistically lower (p<0.05) than in control samples with pathogen strains alone. The highest number of cfu for L. monocytogenes (7.94 log10 cfu/g) was observed in a cheese sample without any addition of LAB culture, whereas in cheese samples with Lbc. fermentum 11b, Lbc. fermentum 18b, Lbc. fermentum 22c the population of L. monocytogenes was lower (7.57 log10 cfu/g, 7.37 log10 cfu/g and 7.44 log10 cfu/g respectively). Also the highest number of E. coli was detected in a control sample (7.99 log10 cfu/g). In experimental samples number of E. coli was 7.33 log10 cfu/g in cheese with Lbc. fermentum 11b, 7.36 log10 cfu/g in cheese with Lbc. fermentum 18b and 7.17 log10 cfu/g in cheese with Lbc. fermentum 22c. The low degree of inhibition might be caused by a few factors such as: high inocula of pathogens, lack of acid production by Lbc. fermentum strains, low different from optimal for the LAB temperature of incubation or slower growth rate of lactobacilli in comparison to growth rates of pathogen strains.

Key words: protective cultures, Lactobacillus fermentum, Escherichia coli, Listeria monocytogenes, soft cheese.

INTRODUCTION

Lactic acid bacteria are an important group of industrial starter cultures employed in the production of fermented foods like cheeses, yoghurt, dry sausage and sourdough. They influence organoleptic properties and microbial safety of products [2, 11]. The latter is due to organic acids (lactic and acetic), carbon dioxide, ethanol, hydrogen peroxide, diacetyl and bacteriocins production [3, 7, 17].

Recently a growing interest in incorporating lactic acid bacteria strains as protective cultures into food products has been observed. Protective cultures are bacteria, which may or may not be strains naturally present in foods [18]. They are selected on the basis of their ability to grow in a product and inhibit food poisoning or spoilage organisms. The cultures do not have to provide a desired structure or favour properties of products as in fermentation process or health benefits like probiotics. However under normal storage conditions they should not affect organoleptic properties of a product [8]. Knowledge of the nature of undesired bacteria inhibition by LAB and factors affecting it is essential to receive an efficient preservation system, because the simple addition of protective culture does not guarantee inhibition of food poisoning or spoilage organisms. The interaction will depend on the growth rate of a protective and undesired bacteria and the rate of production of antimicrobial substances, which are determined by a food product features, processing parameters, and the protective culture used. In general, protective cultures should survive in products under refrigeration, grow and exhibit an antagonistic effect at desired temperatures [18]. Protective cultures may be incorporated into different food product for example cheese.

Queso Fresco is a Soft Spanish unripened cheese of high moisture level, low salt content and made without any addition of starter cultures (pH of 6.5). As a result, it is an excellent growth medium for spoilage bacteria and food-borne pathogens like Listeria monocytogenes, Escherichia coli, Salmonella [10].

The aim of the study was to evaluate if lactose-negative strains of Lactobacillus fermentum, previously isolated from GI-tract of infants, with the ability to inhibit the growth of Gram positive and Gram negative bacteria in agar well diffusion method, could be used as protective cultures and inhibit the growth of Escherichia coli and Listeria monocytogenes in soft Spanish cheese. Lactose-negative strains were chosen to prevent changes of organoleptic properties of the cheese.

MATERIAL AND METHODS

Cultures and Media

Cultures of lactic acid bacteria Lbc. fermentum 11b, Lbc. fermentum 18b, Lbc. fermentum 22c were cultivated for 24 h in MRS Broth (Merck) at 37°C.

Cultures of Escherichia coli NCTC 12900 and Listeria monocytogenes LM82, prior to their use in the study, grew in Tryptone Soy Broth (Pancreac) 24 h at 37°C.

For estimation the bacterial number MRS Agar (Merck) for lactobacilli, McConkey Agar (Merck) for E. coli and Palcam Listeria Selective Agar (Merck) for L. monocytogenes, were used. For preparing dilutions quarter-strenght Ringer´s solution was used.

Antimicrobial activity determination

The agar well diffusion method was used for assay of antimicrobial activity of Lbc. fermentum cultures against E. coli and L. monocytogenes strains.

Sterile nutrient agar without glucose (20 cm3 with 18g/l agar) was mixed at approximately 45°C with 0,1 cm3 of a 24 h diluted (1:10) culture of the pathogen strains grown in broth medium at 37°C and poured into Petri’s dishes. After solidification wells were cut with a sterile tubes of 12 mm diameter. Wells were filled with 24 h Lbc. fermentum cultures on MRS broth (Merck) in quantity of 0.3 cm3 containing 108cfu/cm3. Plates were incubated at 37°C and examined after 24 h for clear zones of the pathogen strains inhibition. The diameters of the inhibition zones were measured and the diameter of the well, 12 mm, was subtracted from the total zone diameter.

Cheese Study

Cheese used in the study was white soft pasteurised cheese (Burgo de Arias, Natural, Mantequerias ARIAS, Madrid, Spain) purchased in a food store, produced from cow’s milk, rennet and salt without starter cultures with pH 6.7 and water activity 0.98 (Termoconstanter Novasina). 5g-samples of the cheese were placed into sterile plastic bags (Fibran S.A., Gerona, Spain) and inoculated with 0.1 cm3 of diluted cultures of Lbc. fermentum strains (106 cfu/cm3) and E. coli and L. monocytogenes (105 cfu/cm3). Three types of samples were prepared: control with a pathogen strain, control with Lbc. fermentum strain and experimental with lactic acid bacteria and pathogen strains together. After sealing bags cheese samples were massaged manually from outside to distribute inoculum and then stored at 7°C up to 15 days. At the time of inoculation and selected time intervals during 15 days, samples were homogenized in 45 cm3 of quarter-strenght Ringer’s solution in a Stomacher (IUL Instruments, Barcelona, Spain) and after serial dilutions 0,05 cm3 was surface plated using a spiral plater (Autoplate Model 3000, Spiral Biotech). Plates were incubated for 48 h at 37°C with exception for plates with E. coli, which were incubated at 42°C for 24 h. Prior to the assay the cheese samples were plated to check if any colonies could be detected on MRS Agar, McConkey and Palcam Listeria Selective Agar. No colonies were found which indicated that there was no interference from other microbial flora on the plates. All the microbial assays were performed in duplicate and the reported results are average values.

Acid production

pH of each sample after homogenisation was measured with pH meter (Crison).

Statistical analysis

Calculations were performed using Statistica 6.0 (StatSoft). Statistically significant differences between groups were determined with ANOVA test. In all analyses, a value of p<0.05 was considered statistically significant.

RESULTS

The lactic acid bacteria strains used in the study exhibited strong antimicrobial activity against Listeria monocytogenes and Escherichia coli strains in agar well diffusion method (Table 1).

Table 1. Antimicrobial activity of Lactobacillus fermentum strains against Escherichia coli nctn 12900 and Listeria monocytogenes lm82 at 37°C (inhibition zone in mm)

Lbc. fermentum strain

Test bacteria strain

E. coli nctn 12900

L. monocytogenes lm82

Lbc. fermentum 11b

14.0 ± 2.0

12.0 ± 5.3

Lbc. fermentum 18b

14.7 ± 0.6

18.7 ± 5.0

Lbc. fermentum 22c

15.3 ± 3.1

15.3 ± 6.4

Present study demonstrated that all strains were able to survive and grow in the environment of soft cheese. In samples containing LAB strains small but statistically significant inhibition of pathogen growth was observed after 15 days of incubation.

Listeria monocytogenes LM82 grew well in all cheese samples. In control sample, without any LAB strain, achieved a population of 107cfu/g on 5th day of incubation at 7°C. In cheese samples with addition of Lactobacillus fermentum cultures, L. monocytogenes grew slower. Population of 107cfu/g L. monocytogenes achieved on 6th, 7th and 8th day of incubation in cheeses with Lbc. fermentum 11b, Lbc. fermentum 22c and Lbc. fermentum 18b respectively. The highest number of cfu for L. monocytogenes (7.94 log10 cfu/g) was observed in the cheese sample without any addition of LAB culture, whereas in cheese samples with Lbc. fermentum 11b, Lbc. fermentum 18b, Lbc. fermentum 22c the population of L. monocytogenes was lower (7.57 log10 cfu/g, 7.37 log10 cfu/g and 7.44 log10 cfu/g respectively) (Fig. 1). These results indicate inhibitory effect of Lbc. fermentum strains on L. monocytogenes growth in the cheese. LAB also delayed growth of the pathogen. Statistical analyses proved that differences in number of L. monocytogenes between control and experimental chesses were significant at the level of p<0.05.

Figure 1. Growth of Listeria monocytogenes lm82 in spanish soft cheese with and without Lactobacillus fermentum strains during storage at 7°C

Also growth of Escherichia coli was affected by lactic acid bacteria in the cheese. Population of 107cfu/g E. coli achieved on 5th day of incubation at 7°C in control sample containing only E. coli and in cheese samples with E. coli and Lbc. fermentum 11b and E. coli and Lbc. fermentum 18b, and on 6th day in cheese samples containing E. coli with Lbc. fermentum 22c. After 15 days of incubation the highest number of E. coli was detected in control sample (7.99 log10 cfu/g), whereas in experimental ones was 7.33 log10 cfu/g in cheese with Lbc. fermentum 11b, 7.36 log10 cfu/g in cheese with Lbc. fermentum 18b and 7.17 log10 cfu/g in cheese with Lbc. fermentum 22c (figure 2). Although differences in E. coli population in different cheese samples were small, they were statistically significant (p<0.05).

Figure 2. Growth of Escherichia coli NCTC 12900 in spanish soft cheese with and without Lactobacillus fermentum strains during storage at 7°C

All the Lactobacillus strains were able to grow in the cheese environment and achieved a population of about 107cfu/g after 1 (Lbc. fermentum 18b), 2 (Lbc. fermentum 22c) or 5 days of incubation (Lbc. fermentum 11b) in control samples. Population of Lbc. fermentum 11b decreased from 107cfu/g to 105cfu/g after 15 days of incubation whereas population of Lbc. fermentum 18b and Lbc. fermentum 22c remained at the level of 107cfu/g throughout all time of the study (T ables 2, 3, 4).

Table 2. Changes of Lactobacillus fermentum 11b population in cheese samples during incubation at 7°C (cell counts presented as log10 cfu/g))

Time [days]

Cheese with
Lbc. fermentum
11b

Cheese with
Lbc. fermentum 11b
and E. coli

Cheese with
Lbc. fermentum 11b
and L. monocytogenes

0

6.20 ± 0.20

6.20 ± 0.28

6.20 ± 0.08

1

6.72 ± 0.03

6.41 ± 0.08

6.63 ± 0.37

2

7.18 ± 0.04

6.93 ± 0.13

6.81 ± 0.45

5

7.55 ± 0.30

7.90 ± 0.23

7.41 ± 0.50

6

7.18 ± 0.05

7.92 ± 0.20

7.48 ± 0.24

7

6.56 ± 0.20

7.08 ± 0.29

6.65 ± 0.26

8

6.24 ± 0.23

6.76 ± 0.20

6.22 ± 0.30

9

6.08 ± 0.30

6.59 ± 0.52

6.12 ± 0.28

13

5.58 ± 0.28

6.18 ± 0.13

5.68 ± 0.37

14

5.43 ± 0.06

5.88 ± 0.08

5.32 ± 0.03

15

4.93 ± 0.11

5.74 ± 0.08

5.00 ± 0.15

Table 3. Changes of Lactobacillus fermentum 18b population in cheese samples during incubation at 7°C (cell counts presented as log10 cfu/g))

Time [days]

Cheese with
Lbc. fermentum 18b

Cheese with
Lbc. fermentum 18b
and E. coli

Cheese with
Lbc. fermentum 18b
and L. monocytogenes

0

6.88 ± 0.09

6.88 ± 0.23

6.88 ± 0.20

1

7.42 ± 0.11

6.70 ± 0.18

7.11 ± 0.18

2

7.38 ± 0.17

6.87 ± 0.04

6.81 ± 0.09

5

7.57 ± 0.54

7.19 ± 0.09

7.02 ± 0.15

6

6.99 ± 0.18

7.46 ± 0.17

6.91 ± 0.15

7

6.91 ± 0.13

7.46 ± 0.24

6.83 ± 0.16

8

6.97 ± 0.06

6.84 ± 0.07

6.95 ± 0.07

9

6.80 ± 0.09

6.74 ± 0.09

7.15 ± 0.29

13

6.88 ± 0.15

6.76 ± 0.13

6.92 ± 0.30

14

6.72 ± 0.29

6.48 ± 0.06

6.78 ± 0.30

15

6.70 ± 0.18

6.62 ± 0.09

6.96 ± 0.10

Table 4. Changes of Lactobacillus fermentum 22c population in cheese samples during incubation at 7°C (cell counts presented as log10 cfu/g)

Time [days]

Cheese with
Lbc. fermentum 22c

Cheese with
Lbc. fermentum 22c
and E. coli

Cheese with
Lbc. fermentum 22c
and L. monocytogenes

0

6.78 ± 0.08

6.78 ± 0.18

6.78 ± 0.23

1

6.81 ± 0.04

6.99 ± 0.21

7.17 ± 0.16

2

7.01 ± 0.50

7.43 ± 0.24

7.15 ± 0.09

5

7.68 ± 0.17

7.07 ± 0.08

7.13 ± 0.12

6

7.32 ± 0.15

7.04 ± 0.23

7.00 ± 0.24

7

7.25 ± 0.07

6.86 ± 0.43

6.97 ± 0.18

8

7.18 ± 0.21

6.95 ± 0.85

7.15 ± 0.09

9

7.29 ± 0.18

6.92 ± 0.23

7.00 ± 0.11

13

7.25 ± 0.17

7.11 ± 0.11

7.08 ± 0.22

14

7.23 ± 0.17

7.11 ± 0.45

6.94 ± 0.39

15

7.08 ± 0.15

7.08 ± 0.07

7.19 ± 0.09

DISCUSSION

It is well documented that LAB have a potential to produce antimicrobial substances such as organic acids and bacteriocins [3, 5, 12], which can inhibit the growth of undesired bacteria. Also they can be used to control the fermentation process by suppressing the growth of competing microflora [22].

In the present study inhibition of pathogenic strains by lactic acid bacteria was observed in all cases. However it was not as strong as in agar well diffusion method at 37°C in which lactic acid bacteria strains exhibited strong antimicrobial activity against Listeria monocytogenes and Escherichia coli strains. It could be explained by the fact that culture medium (MRS containing 2% of glucose) used in agar well diffusion method was strongly acidic and diffusion of acid into agar medium caused the inhibition of pathogens growth. In cheese samples acid production did not occur and that is why strong growth inhibition was not observed.

Decrease in L. monocytogenes counts observed by Morgan et al. [13] in soft cheese, could be due to the combined inhibitory effect of low pH and the activity of starters. Inhibition of L. monocytogenes by a low pH has been observed for various cheeses [6, 9, 10, 14, 16, 19]. It is also well recognized that lactic acid bacteria can cause inhibition of the pathogen growth by production of antagonistic compounds [15]. Mendoza et al. [10] eliminated the influence of low pH using in their study lactose-negative strain of Lactococcus lactis subsp. diacetylactis and still they observed growth inhibition of L. monocytogenes, E. coli O157:H7 and P. aeruginosa at 7°C. Also in the present study the effect of acidification was eliminated, because all the strains were lactose-negative and acidification of the cheese samples did not occur (data not shown). It is possible that differences in pathogens numbers between control and experimental samples were caused only by nutrient competition between LAB and pathogenic strains.

Also, the temperature of incubation might play a significant role in the process of the growth inhibition [18]. Psychotrofic strains of LAB isolated from commercial fresh salads inhibited different pathogens at different temperatures: Aeromonas hydrophila and Listeria monocytogenes at 8ºC but not at 37ºC and Staphylococcus aureus and coliforms conversely [21]. Mesophilic strains used in this study were of human origin thus the process of growth inhibition of pathogens in cheese might be more efficient at their optimal temperature of 37°C than at 7°C.

Another aspect, which should be taken into consideration when the aim of the addition of the culture is only the inhibition of undesired bacteria in a product, is the size of inoculum, which influence not only the development of preservation factors, but also on the sensory quality of the product as well as the cost effectiveness of the method [18]. Generally high inocula are needed (106-109cfu/g). Successful inhibition of psychrotrofic bacteria in milk at refrigerated temperatures required at last 108cfu/cm3 of lactic acid bacteria [4] but on the other hand extensively high population of the culture may restrict its growth, which is necessary for bacteriocin production [22]. In the study of Mendoza et al. [10] sufficient inoculum of Lactococcus lactis subsp. diacetylactis, which suppressed the growth of pathogens, was on the level of 107cfu/cm3. In the present study inoculum was equal 1.6·106cfu/g (Lbc. fermentum 11b), 7.0·106cfu/g (Lbc. fermentum 18b) and 6.0·106cfu/g (Lbc. fermentum 22c) which could be not sufficient for inhibition of pathogens in a low temperature. As reported by Breid et al. [1] for exerting inhibitory effect in a food product growth rate of the protective culture should be higher to that of undesired bacteria. Enterococcus faecium failed to inhibit L. monocytogenes at 3°C and 5°C because a higher growth rate of the pathogen [20]. Indeed, in the present assay the growth rates of pathogens were higher or at least equal to those of lactic acid bacteria.

CONCLUSIONS

Lactose-negative strains of Lactobacillus fermentum of human origin exhibited some inhibition of Listeria monocytogenes LM82 and Escherichia coli NCTC 12900 growth in a model system of the cheese. This unsatisfactory small, but statistically significant, extend of inhibition might be caused by a few factors such as low temperature of incubation, absence of acid and bacteriocin production by the LAB, slower growth rate of lactobacilli in comparison to growth rates of pathogen strains or not sufficient inoculum of LAB. Also, the extremely high initial number of pathogenic microflora which would not probably occur in a food product played a significant role.

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Monika Modzelewska-Kapituła
Chair of Industrial and Food Microbiology,
Faculty of Food Science,
University of Warmia and Mazury, Poland
Plac Cieszynski 1, 10-957 Olsztyn, Poland
email: monikamodz@tlen.pl

Fulgencio Marin-Iniesta
Faculty of Veterinary Medicine, University of Murcia, Spain
University of Murcia, Spain

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