Stability of free and immobilized Lactobacillus acidophilus and Bifidobacterium lactis in acidified milk and of immobilized B. lactis in yoghurt

Estabilidade de Lactobacillus acidophilus e Bifidobacterium lactis nas formas livre e imobilizada em leite acidificado e de B. lactis imobilizado em iogurte

FOOD MICROBIOLOGY

Stability of free and immobilized Lactobacillus acidophilus and Bifidobacterium lactis in acidified milk and of immobilized B. lactis in yoghurt

Estabilidade de Lactobacillus acidophilus e Bifidobacterium lactis nas formas livre e imobilizada em leite acidificado e de B. lactis imobilizado em iogurte

Carlos Raimundo Ferreira Grosso^I; Carmen Sílvia Fávaro-Trindade^II

^IDepartamento de Planejamento Alimentar e Nutrição, Faculdade de Engenharia de Alimentos, Universidade Estadual de Campinas, Campinas, SP, Brasil

^IIDepartamento de Engenharia de Alimentos, Faculdade de Zootecnia e Engenharia de Alimentos, Universidade de São Paulo, São Paulo, SP, Brasil

^{Correspondence}

ABSTRACT

This study evaluated the stability of Bifidobacterium lactis (Bb-12) and of Lactobacillus acidophilus (La-05) both free and immobilized in calcium alginate, in milk and in acidified milk (pH 5.0, 4.4 and 3.8). The stability of immobilized B. lactis in yoghurt (fermented to pH 4.2), during 28 days of refrigerated storage was also evaluated. The efficiency of two culture media (modified MRS agar and Reinforced Clostridial Agar plus Prussian Blue) for counting of B. lactis in yoghurt was determined. Lee's agar was used to count Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus when B. lactis were counted in the MRS medium. B. lactis and L. acidophilus in both free and immobilized forms presented satisfactory rates of survival in milk and acidified milk because the average reduction of the population was only one log cycle after 21 days of storage. The number of viable cells of immobilized B. lactis in yoghurt presented a gradual decline throughout the storage period, passing from 10⁸ cfu/ml to no count after 28 days of storage. When the cultures were not in equilibrium just the selective medium was efficient in counting B. lactis in yoghurt. The results showed that both microorganisms can be added to milk and acidified milk, because their population was only slightly affected during storage. The presence of traditional culture of yoghurt seems to be harmful for survival of immobilized B. lactis and the immobilization in calcium alginate failed as an effective barrier to protect the cells in all analysed treatments.

Key words: probiotics, immobilization, alginate, Lactobacillus acidophilus, Bifidobacterium lactis, yoghurt, milk, Lactobacillus bulgaricus.

ABSTRACT

Este trabalho avaliou a estabilidade de Bifidobacterium lactis (Bb-12) e de Lactobacillus acidophilus (La-05) nas formas livre e imobilizada em alginato de cálcio, em leite e leite acidificado (pHs 5.0, 4.4 e 3.8), e a estabilidade de B. lactis imobilizado em iogurte (fermentado até pH 4.2), durante 28 dias de estocagem refrigerada. Também foi estudada a eficiência de dois meios de cultura (ágar MRS modificado e Reinforced Clostridial Agar, acrescido de Prussian Blue) para enumerar B. lactis em iogurte. Ágar Lee foi usado para enumeração de Streptococcus thermophilus e Lactobacillus delbrueckii ssp. bulgaricus quando B. lactis era enumerado no meio MRS. Ambos os microrganismos, nas formas livre e imobilizada, apresentaram uma taxa de sobrevivência adequada nos leites acidificados, uma vez que houve redução de apenas um ciclo log, após 21 dias de estocagem refrigerada. O número de células viáveis de B. lactis imobilizado mostrou um declínio gradual durante o período de armazenamento do iogurte, passando de 10⁸ ufc/ml até não ter mais contagem na diluição 10^-1. Quando as culturas não estavam em equilíbrio, o meio MRS modificado foi mais eficiente para a contagem de B. lactis em iogurte. Em vista destes resultados pode-se concluir que ambos os microrganismos podem ser incorporados em leite e leite acidificados, haja visto que a redução na população foi pequena durante o período de armazenagem estudado. A presença da cultura tradicional de iogurte parece ter afetado negativamente a sobrevivência de B. lactis e a imobilização não proveu proteção às células, em nenhum dos tratamentos estudados.

Palavras-chave: probióticos, imobilização, alginato, sobrevivência, Lactobacillus acidophilus, Bifidobacterium lactis, yoghurt, milk, Lactobacillus bulgaricus.

INTRODUCTION

Probiotic supplements contain viable bacteria that beneficially influence health and nutrition when consumed (30). Most commonly they contain Lactobacillus acidophilus and Bifidobacterium, both of which are part of the normal intestinal microbiota (2). Lactobacillus acidophilus, L. casei, Bifidobacterium bifidum, B. longum and Saccharomyces boulardii are frequently used as probiotics in products for humans consumption (27), although other species are also recognised as probiotics. Due to their health benefits probiotic bacteria have been increasingly included in yoghurts and fermented milks during the past two decades (22).

Foods containing these microorganisms are sold in many countries, although their survival in foods is doubtful, since some of the strains are extremely sensitive to a series of factors. Also, methods for counting these organisms have not yet been well established, which is an essential requirement to determine their survival in commercial products (12).

The survival of L. acidophilus and Bifidobacterium spp. in yoghurts has been shown to be a problem, due to their intolerance of acid conditions and the presence of other cultures, such as L. delbrueckii ssp. bulgaricus (17,24). However, microencapsulation or immobilization techniques could provide protection to acid sensitive bifidobacteria and thus increase their survival rate during the shelf life of the yoghurt and during their passage through the gastrointestinal tract (1,5,6,10,23,28).

The method of immobilization by extrusion is the most common approach to make capsules with hydrocolloids. It simply involves preparing a hydrocolloid solution, adding microorganisms to it, and extruding the cell suspension through a syringe needle in the form of droplets to free-fall into a hardening solution or setting bath (15).

The polysaccharide sodium alginate has been most widely used as an immobilizing vehicle (26). It forms a gel when in contact with calcium and multivalent cations (7). Alginate beads (or microparticles) are stable in low pH conditions but swell in weak basic solutions followed by disintegration and erosion (18).

The immobilization of Lactobacillus bulgaricus in calcium alginate offered good protection to the organisms during frozen storage and in ice cream (31). Bifidobacteria immobilized in alginate were more resistant to acid pH values in mayonnaise than the free cells (13).

The objectives of this study were to evaluate the stability of B. lactis and L. acidophilus, both free and immobilized in calcium alginate, in acidified milk, determine the stability of B. lactis in yoghurt, and to verify the efficiency of two culture media, one selective and the other differential, in counting B. lactis in yoghurt.

MATERIALS AND METHODS

Cultures

Lactobacillus acidophilus (La-05) and Bifidobacterium lactis (Bb-12) (Chr. Hansen, Valinhos, Brazil), in the DVS (direct vat set) form, pure and freeze dried, were maintained at -18°C in the proportion of 1g per 150 ml in a sterile solution of 12% reconstituted skim milk. These milks were thawed for inoculation of acidified milks and yoghurts and for preparation of the calcium alginate beads.

Before use a mixed commercial culture of Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus, from Danisco Cultor Niebüll GmbH (Niebüll, Germany), was replicated twice in 12% reconstituted sterile skim milk, at 45ºC for 3 hours.

Culture counts

In the absence of other cultures, L. acidophilus and B. lactis were counted in DeMan Rogosa and Sharp (MRS) agar (Oxoid) by the pour plate technique. A 2% solution of sodium citrate was used to prepare serial dilutions. One ml of each dilution was pour plated in MRS agar. After solidification, the plates were inverted and incubated at 37ºC for 72 h in jars with the Anaerogen (Oxoid) system for generating anaerobic atmosphere. Plating was carried out in duplicate.

For quantitative measurements of the number of viable cells of immobilized bacteria, it was necessary to solubilize the alginate beads to liberate the microorganisms. This was made in 2% sterile sodium citrate solution, using the Stomacher 400 (Seward, London, UK), at medium velocity and room temperature, for 2 minutes.

Immobilization process

The immobilization of L. acidophilus and B. lactis in calcium alginate was carried out according to Fávaro-Trindade and Grosso (6).

Evaluation of the stability of immobilized L. acidophilus and B. lactis in acidified milk

Model systems were elaborated to study the viability of incorporating L. acidophilus and B. lactis into acid foods. For this, milk (pH 6.4) previously standardised at 15% solids and sterilised in retort at 121ºC for 8 minutes was used as the standard. Other samples of milk in the same conditions were acidified by the addition of 4N lactic acid in order to achieve pH values of 5.0, 4.4 and 3.8. The calcium alginate beads containing L. acidophilus or B. lactis were added to these acidified milks at a rate of 5% in relation of the milk volume. The cultures of L. acidophilus or B. lactis, dissolved in milk as stated early, were added to these acidified milks at a rate of 5% in relation of the milk volume. All samples were stored at 7ºC for 28 days in a B.O.D. incubator (model TE 390, Tecnal, Piracicaba, Brazil). Counts were made after 0, 7, 14, 21 and 28 days. This experiment was repeated three times for each pH.

Evaluation of the stability of immobilized B. lactis in yoghurt

Yoghurt was prepared from whole milk powder, reconstituted at 15% solids, and sterilized in retort at 121ºC for 8 minutes. Fermentation was carried out at 45ºC with a 2% inoculum of a mixed culture of S. thermophilus and L. delbrueckii ssp. bulgaricus) until a pH of 4.2 was reached. Afterwards, the yoghurt was blended for 5 minutes. The blended product was transferred to plastic cups and the calcium alginate beads containing B. lactis added at a rate of 5% in relation of the yoghurt volume. The contents were gently mixed with a spatula and the cups capped and stored at 7ºC in a B.O.D. incubator (model TE 390, Tecnal, Piracicaba, Brazil) for 28 days. With the purpose of determining the stability of the immobilized B. lactis in the yoghurt, counts of this organism and of S. thermophilus and L. delbrueckii ssp. bulgaricus were made after 0, 7, 14, 21 and 28 days of storage. This experiment was repeated twice.

Evaluation of the culture media employed for counting immobilized B. lactis in yoghurt

L. acidophilus and Bifidobacterium spp, the most commonly used probiotic group, are fastidious organisms and in the laboratory they are grown on complex media such as MRS broth or Reinforced Clostridial Medium (11). Thus, two media were evaluated for the counting of B. lactis in the presence of yoghurt cultures, one selective and the other differential.

The selective medium was MRS deMan, Rogosa and Sharpe agar (Oxoid, Hampshire, UK) supplemented with: 0.5% of a 10% solution of L-cysteine hydrochloride (Synth, São Paulo, Brazil), 0.5% of a dicloxacillin solution (10 mg/100 ml water) (Sigma, Louis, USA) and 1% of a 10% solution of lithium chloride (Vetec, São Paulo, Brazil) (4,9), modified by addition of 0.01% aniline blue (Nuclear, São Paulo, Brazil). A 2% solution of sodium citrate was used to prepare serial dilutions from 10¹ to 10⁸. The spread plate technique was used and after the medium solidification, the plates were inverted and incubated at 37ºC for 72 h in anaerobic jars with the Anaerobac (Probac, São Paulo, Brazil) system.

Lee's agar (20) was used to count Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus when B. lactis was counted in the selective medium. In this case the spread plate technique was used and the plates were inverted and incubated at 37ºC for 48 hours in jars containing the Microaerobac (Probac, São Paulo, Brazil) microaerophyllic generating system.

The differential medium was RCPB, composed of RCA Reinforced Clostridial Agar (Oxoid, Hampshire, UK) with added 0.03% Prussian Blue (Aldrich, USA) (25) and spread plate technique. A 2% solution of sodium citrate was used to prepare serial dilutions from 10¹ to 10⁸. The plates were inverted and incubated at 37ºC for 72h under anaerobiosis in jars with the Anaerobac (Probac, São Paulo, Brazil) system.

Statistical analysis

A means difference analysis was used to check for significant differences between the values obtained according to Tukey's Test, with the help of software STATISTICA 6.0.

RESULTS AND DISCUSSION

Evaluation of the stability of L. acidophilus and of B. lactis in free and immobilized forms in acidified milk

Free L. acidophilus showed good stability in acidified milk: the viable count did not change at pH 6.4 and 5 and was reduced by only one log cycle after 14 days of storage at pH 4.4 and 3.8 (Table 1). The population of immobilized L. acidophilus was reduced by one log cycle after 14 days of refrigerated storage at all pHs values (Table 2). This result is in agreement with that reported by Laroia and Martin (17), in which L. acidophilus survived in great numbers in a frozen fermented product with pH values varying from 3.9 to 4.6, and with that reported by Gilliland and Speck (8), where L. acidophilus remained viable in milk acidified with lactic acid.

The survival of free B. lactis in acidified milks was considered satisfactory, since a reduction of only one log cycle was registered after 21 days of storage at initial pH of 5.0, and between 7 and 14 days at pH 4.4.

The pH 3.8 was more harmful to the free B. lactis only after 28 days of refrigerated storage, showing a reduction of two log cycles. The immobilized cells of B. lactis suffered a reduction of only one log cycle after 14 days of refrigerated storage, at pH value 3.8 (Table 4).

This result differs from those of Laroia and Martin (17), in which B. lactis failed to survive in products with pH 3.9 to 4.6. However, according to Lankaputhra et al. (16), the resistance during refrigerated storage in acid products varies according to the species of Bifidobacterium. These researchers tested the viability of 9 species of Bifidobacterium in acidified milk at pH 4.3, 4.1, 3.9 and 3.7, stored under refrigeration for 42 days, and showed that only three species, B. infantis 1912, B. longum 1941 and B. pseudolongum 20099, were capable of surviving well. The remaining species (B. bifidum 1900 and 1901, B. adolescentis 1920, B. breve 1930, B. longum 20097 and B. thermophilum 20210) were destroyed by the low pH or presence of H₂O₂.

Evaluation of the stability of immobilized B. lactis in yoghurt

One of the requirements for microorganisms to be used for therapeutic purposes is that they remain viable in the food used as a vehicle for their consumption (14). B. lactis immobilized in calcium alginate was incorporated into high acid yoghurt (pH 4.2) and a gradual decline in the number of viable cells was shown throughout the storage period (Table 5).

According to Laroia and Martin (17) and to Martin and Chou (21), the low pH of fermented products is harmful to some species/strains of Bifidobacterium. However, the present study was shown that free and immobilized B. lactis incorporated into acidified milks (pH 4.4 and 3.8) presented a much higher survival rate than in yoghurt at similar pH values, since the maximum reduction in viable count was two log cycles after 28 days of storage (Tables 3 and 4), whilst in yoghurt the whole population was destroyed in the same period (Table 5). Thus, the survival of the free and immobilized B. lactis in yoghurt may have been affected by other factors, such as inhibitory substances produced by the yoghurt culture or an excess of dissolved oxygen. The alginate matrix failed to function as a barrier to these factors. This result was different from that reported by Khalil and Mansour (13), in which immobilization was effective in the protection of Bifidobacterium bifidum and B. infantis cells immobilized in alginate and incorporated into mayonnaise (pH 4,4). Immobilization in alginate also improved the survival of Lactobacillus bulgaricus in a milk based dessert (31). Lee and Heo (19) showed that B. longum encapsulated in Ca-alginate spheres survived to simulated gastroenteric juice (pH 1.55) significantly better than free cells. This study has indicated that survival of alginate immobilized bacteria decreased with the decrease of sphere size (diameters 1-2,6 mm) and increased with the increase of alginate concentration (1-3%).

The populations of L. delbrueckii ssp. bulgaricus and S. thermophilus suffered reductions of two and one log cycles, respectively, after 21 days of storage (Tables 5 and 6). This result confirms the predominance of S. thermophilus during the refrigerated storage of yoghurt prepared with addition of B. lactis, as reported by Rybka and Kailaspathy (29).

Evaluation of culture media for the counting of B. lactis in yoghurt

S. thermophilus, L. delbrueckii ssp. bulgaricus and B. lactis were easily differentiated in RCPB by the distinct morphologies of their colonies. L. delbrueckii ssp. bulgaricus grew forming colonies with diameters of 2 to 3 mm, each with a small white clearly defined centre surrounded by a relatively large blue halo; S. thermophilus grew forming colonies with white centres (less clearly defined than those of L. delbrueckii ssp. bulgaricus) and a blue halo with a diameter of about 1 mm; B. lactis formed very small cylindrical white colonies (approximately 0.5 mm in diameter). This result was similar to that reported by Ongoo and Fleet (25), with the difference that these authors obtained larger colonies for S. thermophilus than for L. delbrueckii ssp. bulgaricus. This difference could have been due to the use of strains from different companies, so from different origins.

After 7 days of storage (Table 6) counting of B. lactis in RCPB became impossible, because the colonies of S. thermophilus and L. delbrueckii ssp. bulgaricus dominated the whole plate, since their numbers were much higher than that of B. lactis.

When the supplemented MRS medium was used, B. lactis grew in adequate numbers, forming small brilliant blue colonies with diameters of approx. 0.5 mm. The area immediately around the colonies were more blue than the rest of the medium. L. delbrueckii ssp. bulgaricus failed to grow in any of the dilutions plated and S. thermophilus showed limited growth, in numbers much lower than expected for the 10^-1 to 10^-3 dilutions, forming small light blue colonies with diameters of approx. 0.2 mm. The blue color of the colonies was much lighter than B. lactis, and there was no surrounding blue area, as observed for B. lactis.

In the plates containing mixtures of the three microorganisms, the supplemented MRS was efficient as a selective medium for the counting of B. lactis, since L. delbrueckii ssp. bulgaricus did not grow, while S. thermophilus showed only limited growth. It was possible to count the viable cells of B. lactis even when this number was several log cycles lower than that of the other cultures present. In Lee's agar, B. lactis failed to grow under the conditions used, and S. thermophilus and L. delbrueckii ssp. bulgaricus could be distinguished from each other by a difference in the colour of the colonies, the former forming bright yellow colonies and the latter cream coloured colonies.

According to Dave and Shah (3), some media containing antibiotics inhibit the growth of Bifidobacterium spp., the count therefore not being representative of the true number of viable cells present in the product analysed. However, this problem was not observed in this work, when the supplemented MRS medium, which contained antibiotic, was used.

CONCLUSIONS

One of the requirements for microorganisms to be used as dietary adjuncts is the need to maintain viability and activity in the carrier food before consumption. In this study, free and immobilized B. lactis and L. acidophilus presented a good survival rate in milk and acidified milk. On the other hand, the survival of immobilized B. lactis in yoghurt was considered to be unsatisfactory, that is, immobilization in calcium alginate failed as an effective barrier to protect the cells.

Both RCPB and supplemented MRS were efficient media for counting B. lactis in yoghurt whilst the cultures were in equilibrium; when L. delbrueckii ssp. bulgaricus and S. thermophilus prevailed, the differential medium (RCPB) did not allow for the counting of B. lactis, the selective medium (MRS) being more efficient.

ACKNOWLEDGEMENTS

The authors are grateful to CNPq for the financial support. The microorganisms were kindly provided by Chr. Hansen, Brazil.

Submitted: December 08, 2002; Returned to authors: March 14, 2003; Approved: March 04, 2004.

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Publication Dates

• Publication in this collection
  16 Nov 2004
• Date of issue
  June 2004

History

• Accepted
  04 Mar 2004
• Received
  08 Dec 2002
• Reviewed
  14 Mar 2003