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Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

Aims: Cell survival and antagonistic activity against Listeria innocua, Listeria monocytogenes and Staphylococcus aureus were investigated after spray-drying three bacteriocin-producing strains of lactic acid bacteria: Carnobacterium divergens, Lactobacillus salivarius and Lactobacillus sakei.

Methods and Results: Bacterial cell concentrates were spray-dried and stored at 4°C and 18°C and 0·3% ERH (equilibrium relative humidity). Enumeration and antagonistic activity were evaluated before and after spray-drying and at regular intervals during storage.

Conclusions: A higher survival rate was obtained when survival was performed at 4°C. With the exception of Carnobacterium divergens which lost the inhibitory activity against Staph. aureus after drying, antagonistic production was not affected by the process nor by the storage. Of the three species studied, Lact. salivarius showed the highest resistance to the spray-drying and storage processes.

Significance and Impact of Study: Spray-drying is a potentially useful process for large scale production of dried powders containing viable organisms with antagonistic activity against pathogens.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

Despite the recent progress in food biotechnology, with the introduction of modern technologies and safety concepts (e.g. HACCP), the problem of food safety and security remains to be solved. Protective cultures and associated antagonistic substances (‘biopreservation’) should be considered an additional factor with the potential for improving the microbiological safety of food. Their implementation should support good manufacturing practices, thereby reducing risks of growth and survival of pathogens and spoilage micro-organisms (Kim and Bhowmik 1990; Mauriello et al. 1999).

Lactic acid bacteria (LAB) have traditionally been used in food processing because of their ability to improve the organoleptic characteristics and healthiness of foodstuffs. Different antimicrobials, such as lactic acid and acetic acid, hydrogen peroxide, carbon dioxide and bacteriocins, produced by these bacteria, can inhibit pathogenic and spoilage micro-organisms, extending the shelf-life and enhancing the safety of food products (Aymerich et al. 2000).

Increasing consumer demand for natural food additives has focused interest on bacteriocins. Bacteriocins of LAB are considered natural biopreservatives, as it is assumed that bacteriocins are degraded by the proteases of the gastrointestinal tract (Cintas 1995), and most of the LAB are considered as GRAS (Generally Recognized as Safe) micro-organisms (Holzapfel et al. 1995).

The application of bacteriocins as food preservatives could be achieved either by using the bacteriocinogenic strain as a starter culture and/or a protective culture, or by using the bacteriocin as a food additive. Therefore, from a commercial point of view, an inexpensive method for large-scale production of cultures containing high levels of viable bacteriocin producers, in a form suitable for product applications, is highly desirable (Gardiner et al. 2000). In previous studies researchers have investigated the production of spray-dried powders of Lactobacillus spp. (Kim and Bhowmik 1990; Teixeira et al. 1995). In addition to maintaining the viability of cultures, it is important that their bacteriocinogenic properties are maintained following the spray-drying process.

Spray-drying can be used to produce large amounts of dairy ingredients relatively inexpensively, the spray-dried powders can be transported at a low cost and can be stored in a stable form for prolonged periods (Gardiner et al. 2000). Because of the advantages of spray-drying, in the present study we investigated the use of this method as a way to preserve bacteriocinogenic strains, e.g. Lact. sakei, Lact. salivarius and Carn. divergens. The purpose of this study was to evaluate the antimicrobial activity of these LAB strains, which are active against L. monocytogenes, L. innocua and Staph. aureus (Hugas et al. 1995; Pilet et al. 1995; Duffes et al. 1999; Dunne et al. 1999; Pascual et al. 1999), before and after spray-drying.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

Bacterial cultures and media

Lact. sakei CTC 494 and Lact. salivarius CTC 2197 were generously supplied by Dr Marta Hugas from Spain. Carn. divergens isolated from trout intestine by Pilet et al. 1995) was generously supplied by ENITIAA (France).

Lact. sakei and Lact. salivarius were grown in MRS broth (LAB M, Bury, UK) (DeMan et al. 1960) at 30°C and 37°C, respectively. Carn. divergens was grown in Elliker medium (Elliker et al. 1956) at 30°C for 24 h. Working cultures were stored as stab agar medium at 4°C. Fresh stab cultures were prepared from working cultures every month. L. monocytogenes T100 and L. innocua 2030 c (tetracycline resistant), previously isolated from cold smoked fish by Escola Superior de Biotecnologia, were grown in tryptic soy broth (TSB, LAB M) supplemented with 0·6% yeast extract (LAB M) at 37°C. Staph. aureus NCTC 08532 (supplied by Serviços de Microbiologia, ESB, Porto, Portugal) was grown in TSB at 37°C for 24 h.

Preparation of feed solution for spray-drying

Overnight cultures of each strain of lactic acid bacteria were inoculated at 1% v/v into MRS broth and Elliker broth and incubated at 30°C or 37°C for 24 h. After centrifugation at 7000 g for 15 min at 4°C, the cells were resuspended in an equal volume of sterile 11% wv−1 skim-milk powder (Oxoid). Each sample was then directly spray-dried.

Spray-drying and storage

Samples were spray-dried in a laboratory scale apparatus (Niro Atomizer, Gladsaxevej, Denmark). Moisture in spray droplets produced by the atomization of the feed liquid by a vaned wheel (rotary atomizer) rotating at high speed, was evaporated in a vertical co-current drying chamber, 0·8 m diameter and 0·6 m height. Spray-drier conditions were: outlet air temperature 70°C, inlet air temperature 200°C and atomizing air pressure 5 Bar. Powder was collected in a single cyclone separator. Samples of the spray-dried inoculum were stored at 4°C and 18°C in hermetically sealed glass bottles in which the ERH (equilibrium relative humidity) was controlled by equilibrium with silica.

Enumeration

Before drying, the organisms were enumerated on Elliker agar in the case of Carn. divergens and on MRS agar for the other two cultures in the study, by the drop count technique (Miles and Misra 1938). Each sample of spray-dried bacteria was rehydrated to the original volume with deionized water. The cells were allowed to rehydrate for 2 min under vigorous shaking and the suitable dilutions were plated as described above. Plates were incubated at 30°C (Carn. divergens and Lact. sakei) and 37°C (Lact. salivarius) for 24 h before enumeration.

Bacteriocin spectrum of activity

The inhibitory activity of the three bacteriocin producers against the L. innocua, L. monocytogenes and Staph. aureus strains was tested at various intervals during storage by the deferred antagonism test (Barefoot and Klaenhammer 1984). From each lactic acid bacteria, 10 μl were placed on the surface of a solid medium TSYE overlay seeded with 100 μl of an overnight target culture. The plates were examined after overnight incubation at 37°C.

Statistical analysis

Statistical analysis was done with the ANOVA methodology using StatviewTM Package (Abacus Concepts, Berkeley, CA, USA) using as independent variable the storage time.

The experiments were repeated at least three times. Viable counts on MRS agar were converted to log cfu ml−1. Differences were considered significant at P < 0·05. The error bars on the figures indicate the mean standard deviations for the data points.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

In the present study, the use of spray-drying as a way to prepare dairy-based powders containing viable cultures of Lact. sakei, Lact. salivarius and Carn. divergens with antagonistic activity against selected pathogens was investigated. For each strain, three independent experiments were performed.

Survival kinetics of spray-dried cell concentrates during storage

The experimental results for the survival of Lact. salivarius, Lact. sakei and Carn. divergens during drying and subsequent storage at different temperatures (4°C and 18°C) are shown in Table 1 and Fig. 1, respectively. For all strains, no significant differences were observed in the number of viable cells before and after drying. Survival of Carn. divergens and Lact. sakei decreased during the storage time at 4°C and 18°C being the rate of decrease higher at the higher temperature of storage (< 0·05). The survivor curve of Lact. sakei showed the greatest decrease during storage at both temperatures, and no survivors were observed at the end of the 2nd month of storage at 18°C.

Table 1.   Survival of Lact. sakei, Lact. salivarius and Carn. divergens before and after spray-drying Thumbnail image of
image

Figure 1.   Survival of spray dried Lact. sakei, Lact. salivarius and Carn. divergens during storage at 18°C and 4°C under controlled ERH (< 0·05). (□), (Carn. divergens 18°C; (▮), Carn. divergens 4°C; (▵), Lact. sakei 18°C; (▴), Lact. sakei 4°C; (◊), Lact. salivarius 18°C; (◆), Lact. salivarius 4°C)

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Until the 3rd month of storage there was no significant survival decrease of Lact. salivarius at 4°C and 18°C.

Effect of spray-drying on bacteriocin production

The effect of spray-drying and storage in the dried state on the bacteriocinogenic activity of lactobacilli was investigated. The results obtained by deferred antagonism assay to detect the ability of bacteriocin-producing strains to inhibit L. innocua, L. monocytogenes and Staph. aureus are reported in Table 2.

Table 2.   Sensitivity of three pathogenic micro-organisms to bacteriocins produced by Lact. salivarius, Lact. sakei and Carn. divergens, before and after drying and during storage, at 18°C, as determined by deferred antagonism assay Thumbnail image of

In the case of Carn. divergens there was a loss of antagonistic activity against Staph. aureus immediately after spray-drying but not against the two Listeria spp. During spray-drying and during storage, Lact. salivarius maintained the bacteriocin activity against L. innocua, L. monocytogenes and Staph. aureus at least during the 3 months of storage. Lact. sakei maintained its bacteriocin activity while viable.

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

In this study it was found that the strains investigated, varied considerably in their ability to survive storage in dried state.

Results showed that the storage temperature was a critical parameter affecting the survival of the micro-organisms. In all experiments, survival rates were higher at the lower temperature of storage.

The bacteriocin produced by Lact. sakei CTC 494 (sakacin K), by Lact. salivarius CTC 2197 and by Carn. divergens (divercin V 41) was reported as having an antagonistic activity against both Listeria strains and Staph. aureus (Lact. sakei and Lact. salivarius) and against Listeria spp. (Carnobacterium) (Duffes et al. 1995; Hugas et al. 1995; Pascual et al. 1999; and Pilet et al. 1995). In this study, it was demonstrated that the spray-drying process did not affect this antagonistic activity of Lact. sakei and Lact. salivarius. Similar results were obtained by Gardiner et al. (2000) and Mauriello et al. (1999). Carn. divergens, however, lost its antagonistic activity against Staph. aureus, but not against L. innocua and L. monocytogenes. The production of more than one bacteriocin by a single organism has already been reported (Bhugaloo-vial et al. 1996) and Bhugaloo-vial reported the isolation and classification of two class II a bacteriocins produced by Carn. piscicola V1 which exhibit significant differences in their inhibitory activities against target Gram-positive bacteria. To our knowledge, this is the first time that loss of antagonistic activity during spray-drying is reported. This phenomenon might be explained by a possible loss of the plasmid responsible for that inhibitory activity. Bacteriocin production mediated by plasmids has already been reported (Kim and Bhowmik 1990; Hugas et al. 1995; Pilet et al. 1995).

In conclusion, spray-drying is potentially a useful process for large scale production of dried powders containing viable organisms with antagonistic activity against pathogens. However, this can not be generalized and for each individual organism, it must be investigated if these characteristics are maintained. Furthermore, given the numerous applications of skim milk powders, not only in dairy products but also in food such as instant desserts and mayonnaise, it is possible that the resulting culture containing powders could be used in a wide range of functional food applications, i.e. having antimicrobial properties in those foods.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

Financial support for authors Silva and Carvalho was provided, respectively, by a PhD fellowship issued by PRAXIS XXI (JNICT–BD–19713–99 and JNICT–BD-18512–98, respectively).

References

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References
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