Review
Technologies with free and immobilised cells for probiotic bifidobacteria production and protection

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Abstract

Interest in the incorporation of bifidobacteria into fermented products has developed considerably over recent years, with many studies reporting human health benefits associated with the consumption of these bacteria. Due to their slow growth in milk, bifidobacteria are generally propagated in pure cultures and eventually mixed thereafter with other lactic acid bacteria. Data on production of bifidobacteria with conventional free-cell cultures are reviewed, as well as new fermentation processes allowing cell productivity enhancement. Bifidobacteria are sensitive microorganisms with low survival to stresses occurring during their production, storage and consumption. Until now, little work has been published on improving the survival of bifidobacteria to these conditions, but new techniques, such as cell incubation under sublethal conditions and cell propagation in an immobilised biofilm state, are very promising. Combined with encapsulation, these techniques will allow better incorporation of bifidobacteria into established probiotic products, as well as the emergence of new applications.

Introduction

Bifidobacteria, first called Bacillus bifidus, were initially observed and isolated from the faeces of breast-fed infants by Tissier (1900) at the Institut Pasteur in Paris. Controversy concerning the classification of bifidobacteria existed for a long time as they were assigned to several genera such as Bacillus, Bacteroides, Tissieria, Nocardia, Lactobacillus, Actinomyces, Bacterium or Corynebacterium (Mitsuoka, 1984). As the classification and identification of microorganisms at the beginning of the century were based only on their morphology, Orla-Jensen (1924) used new criteria such as metabolic and enzymatic characteristics to separate bifidobacteria from Lactobacillus. This distinction was later confirmed by De Vries and Stouthamer (1967), who demonstrated the presence in bifidobacteria of a specific enzyme, the fructose-6-phosphate phosphoketolase (F6PPK) (Scardovi & Trovatelli, 1965).

Following Tissier's observations, and due to the fact that ageing adults generally have higher levels of putrefactive bacteria such as clostridia and less bifidobacteria than young adults, Metchnikoff (1907) proposed the concept that high numbers of bifidobacteria and other lactic acid bacteria (LAB) in the adult large intestine might be associated with good health and longevity. His theory was that the consumption of fermented milk products, and consequently the ingestion of LAB, can inhibit the production by putrefactive bacteria of toxins that potentially damage intestinal cells and the immune system, which he believed contributed to the ageing process. This statement has led to numerous investigations over the last few decades whose purpose was to feed humans and animals with live bacterial adjuncts, also called probiotics, to improve intestinal health.

The term ‘probiotic’ refers to a preparation of, or a product containing, viable, defined microorganisms in sufficient numbers to alter the microflora in a compartment of the host and, as such, exert beneficial health effects in this host (Schrezenmeir & de Vrese, 2001). Although strains of Bifidobacterium and Lactobacillus are currently the most widely used probiotics for human consumption, other microorganisms including Enterococcus, Streptococcus, Propionibacterium, as well as some yeasts, might also promote human intestinal health (Ouwehand et al., 2003).

Today, dairy food products incorporating bifidobacteria, like fermented milks (Baron, Roy, & Vuillemard, 2000), probiotic cheeses (Boylston, Vinderola, Ghoddusi, & Reinheimer, 2004), drinking and frozen yoghurts (Gilliland, Reilly, Kim, & Kim, 2002), ice creams (Godward & Kailasapathy, 2003) and fermented soya products (Wang, Yu, & Chou, 2002) are well established in the market (Stanton et al., 2001). However, since international standards require that fermented products claiming health benefits contain a minimum of 107 viable probiotic bacteria per gram of product when sold (Ouwehand & Salminen, 1998), many products fail to meet these standards when they are consumed (Shah, Lankaputhra, Britz, & Kyle 1995; Kailasapathy & Rybka, 1997). Indeed, the sensitivity of bifidobacteria to low pH and hydrogen peroxide combined with low viability in dairy products during storage at refrigeration temperature remains a problem in most fermented products (Medina & Jordano, 1994; Lankaputhra & Shah, 1995; Lankaputhra, Shah, & Britz, 1996). Consequently, industrial demand for technologies ensuring bifidobacteria stability in foods remains strong, since high cell survival is important for both economical (a lower cell addition in the product is necessary when stability is high) and health effects.

This review will summarise the latest developments for the production of bifidobacteria to enhance the performance of these fastidious organisms during fermentation, downstream processing and utilisation in commercial products. In addition to cell protection by encapsulation which has drawn the attention of researchers in recent years (Lee & Heo, 2000; Sultana et al., 2000; Sun & Griffiths, 2000; O’Riordan, Andrews, Buckle, & Conway, 2001; Truelstrup Hansen, Allan-Wojtas, Jin, & Paulson, 2002; Lacroix, Grattepanche, Doleyres, & Bergmaier, 2005), particular attention should be paid to cell physiology. For example, it is well known that bacteria in stationary growth phase are much more resistant to environmental stresses compared with exponentially growing cells (Kolter, Siegele, & Tormo, 1993; Hartke, Bouche, Gansel, Boutibonnes, & Auffray, 1994; Rallu, Gruss, & Maguin, 1996). Very few studies have been carried out until now to induce stress responses in bifidobacteria at the cell production stage that can increase technological and probiotic cell characteristics. Consequently, this review also presents recent developments on immobilised cell technology to produce bifidobacteria with increased cell resistance to environmental stress factors encountered by cells from production to consumption.

Section snippets

Taxonomy and growth characteristics

The genus Bifidobacterium belongs to the Actinomyces class, and members are Gram-positive bacteria with high G+C content (>55 mol%) in DNA, whereas all the other LAB belong to the Clostridium branch of the Gram-positive phylum with low G+C content (<54 mol%) in DNA (Schleifer & Ludwig, 1996). Nevertheless, bifidobacteria are generally considered as LAB because of similar physiological and biochemical properties and the sharing of some common ecological niches, such as the gastrointestinal tract (

Technologies for bifidobacteria cell production

Table 1 presents a summary of production data reported in the literature for bifidobacteria.

Free-cell cultures

The ability of microorganisms to grow and survive depends largely on their capacity to adapt to changing environments. Adaptation to adverse environments is usually associated with the induction of a large number of genes, the synthesis of stress-response proteins and the development of cross-resistance to various stresses (Girgis, Smith, Luchansky, & Klaenhammer, 2003). Based on this knowledge, new methods for enhancing the long-term survival of LAB by decreasing the lethal effects of

Microencapsulation for protection of bifidobacteria

Other complementary strategies to fermentation, based on cell protection, have been tested to increase bifidobacteria survival in products and eventually in human intestine. These methods are based on adaptation of the carrier food by adding protective agents or increasing the buffering capacity of the product (Dinakar & Mistry, 1994; Gomes, Malcata, Klaver, & Grande, 1995; Wang, Brown, Evans, & Conway, 1999), as well as on cell encapsulation to provide a physical barrier against the external

Conclusions and perspectives

The increasing interest of consumers in their personal health has led to the development of functional foods, with a major emphasis on foods containing probiotics, which enhance health-promoting microbial flora in the intestine. However, the viability and stability of bifidobacteria has been both a marketing and technological challenge for industrial producers, since bifidobacteria should maintain a suitable level of viable cells during the product's-shelf life without increasing production

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