Propionibacteria characteristics relevant to beneficial effects

8.1.1 General features of dairy propionibacteria

Propionibacteria were first described by E. von Freudenreich and S. Orla-Jensen at the end of the 19th century as microorganisms involved in the fermentation of lactate into acetate and propionate with the concomitant production of carbon dioxide during the ripening of Emmental cheese. The first pure culture was isolated from such cheese in 1906 (von Freudenreich and Orla-Jensen, 1906) and the genus Propionibacterium was proposed by Orla-Jensen because of this fermentation in 1909 (Orla-Jensen, 1909).

Propionibacteria are described as pleomorphic rods, 0.5 to 0.8 mm in diameter and 1 to 5 mm in length, often club-shaped, or coccoid, bifid or even branched. Cells occur singly or in characteristic arrangements in V, Y, or Chinese character configuration. The morphology also varies with the physiological stage and the environment, as illustrated in Fig. 8.1. They are non-motile and non-sporing bacteria, anaerobic to aerotolerant and generally catalase positive, which grow in the temperature range 15 to 40°C and in the pH range 5.1 to 8.5 with an optimum at 30°C and neutral pH. They are heterofermentative and metabolise different carbohydrates (including glucose, galactose, fructose and lactose), various alcohols (including glycerol) and organic acids (including pyruvate and lactate, the preferred substrate) to a mixture of propionate, acetate, succinate and carbon dioxide. This particular central carbon metabolic pathway, the propionic fermentation, involves the Wood-Werkman cycle (Wood, 1981) and requires a multimeric transcarboxylase (methyl malonyl CoA carboxyl transferase, EC 2.1.3.1). This enzyme catalyses

Propionibacterium Denitrificans

Fig. 8.1 Variable size and morphology of P. freudenreichii. Morphology is analysed using scanning electron microscopy for bacteria in exponential phase of growth (a), in stationary phase of growth (b), during acid adaptation at pH 5.0 (c) or bile salts adaptation

Fig. 8.1 Variable size and morphology of P. freudenreichii. Morphology is analysed using scanning electron microscopy for bacteria in exponential phase of growth (a), in stationary phase of growth (b), during acid adaptation at pH 5.0 (c) or bile salts adaptation

the reversible transfer of a carboxyl group from methylmalonyl-CoA to pyruvate to form propionyl-CoA and oxaloacetate (second last step before propionate formation) (Deborde, 2002).

Propionibacteria are firmicutes with a high G+C content and are included in the Actinomycetale order. They are thus phylogenetically far from the low G+C firmicutes such as lactic acid bacteria but more related to corynebacteria and mycobacteria. The genus Propionibacterium comprises two distinct groups from different habitats (Cummins and Johnson, 1986). One group includes propionibacteria typically found on the skin and referred to as 'cutaneous propionibacteria'. These bacteria, previously described as anaerobic coryneforms, are involved in the pathology of acnes and may cause opportunistic infections. They are thus not considered for probiotic applications, although the immunomodulatory potential of the P. granulosum species may be of interest (Isenberg et al., 1995). The other group contains strains isolated from cheese and dairy products, and is described as 'dairy propionibacteria' or 'classical propionibacteria'. This includes the species P. freudenreichii, P. acidipropionici, P. jensenii, P. thoenii, P. microaerophilum and P. cyclohexanicum. The species P. acidipropionici and P. freudenreichii, which is divided into two supspecies, freudenreichii and shermanii, are considered for probiotic applications.

Although dairy propionibacteria have been traditionally isolated from dairy products, their natural habitat is the digestive tract of ruminants (Jarvis et al., 1998; Cheong and Brooker, 1999; Rinta-Koski et al., 2001) and they are found in various environments such as soil, fodder, silage, various dairy or vegetable fermented products, dairy plants and also waste waters. Their main application is the ripening of Swiss type cheeses, characterised by round 'eyes' (Noël et al., 1999), in which they are involved in the formation of the characteristic flavour and opening, via the fermentation of lactate to acetate, propionate and CO2 (Langsrud and Reinbold, 1973b), but also the production of branched-chain fatty acids (Thierry et al., 2002, 2004a, 2004b) and in lipolysis (Thierry et al., 2005). However, their metabolic characteristics and the fact that their use in cheese has achieved a 'generally recognised as safe' (GRAS) status allows other applications mainly in the context of food preservation and health promotion, as developed below.

8.1.2 Production of antimicrobial compounds

The production of antimicrobial compounds by safe and food-grade bacteria traditionally used in food processing constitutes a promising alternative to the use of chemical food preservatives.

Propionic acid and its salts are widely used as antifungal agents in the industry. Food-grade propionate can be produced by propionic fermentation instead of chemical synthesis. The short chain fatty acids produced during food fermentation by dairy propionibacteria, mainly propionate, thus gained increased interest because of inhibitory effects towards undesirable microorganisms. Propionibacterial cultures are also considered in the context of food protection as biopreservatives. A commercial product, Microgard™, consisting of a pasteurised skim milk fermented by P. freudenreichii subsp. shermanii, exhibits inhibitory activity associated with organic acids (Daeschel, 1989). It inhibits several Gram-negative bacteria (Pseudomonas, Salmonella and Yersinia) and several fungi, but not Gram-positive bacteria (Al-Zoreky et al., 1991). Propionibacteria were effective at protecting fermented milks and bread sour dough against spoilage by yeast and moulds (Suomalainen and Mayra-Makinen, 1999). The amount of short chain fatty acids and the antifungal activity were shown to vary widely depending on the medium and on the species within propionibacteria (Lind et al., 2005). Finally, co-cultures of lactic acid bacteria and propionibacteria were shown to be more effective in avoiding these food spoilages (Suomalainen and Mayra-Makinen, 1999; Schwenninger and Meile, 2004).

Undesirable flora can also be inhibited by bacteriocins. Bacteriocins are secreted bacterial peptides, which may be covalently modified and have an antimicrobial activity against bacteria closely related to the producer strain (Cotter et al., 2005). However, bacteriocins produced by Gram positive bacteria may have broad spectra including both Gram positive and negative bacteria. Bacteriocins of propionibacteria were reviewed by Holo et al. in 2002 (Holo et al., 2002). The first description of bacteriocin production by dairy propionibacteria deals with P. thoenii P127, which produces propionicin PLG-1 (Lyon and Glatz, 1991). It is active against P. thoenii, P. jensenii and P. acidipropionici, but not P. freudenreichii, and also against several lactic acid bacteria and Gram-negative bacteria, yeasts and moulds. In vitro, PLG-1 failed to inhibit growth of common food-borne pathogens of the geni Bacillus, Staphylococcus, Clostridium, Yersinia and Salmonella. However, it was active in fermented milk against psychrotrophic spoilage or pathogenic organisms including Listeria monocytogenes, Pseudomonas fluorescens, Vibrio parahaemolyticus and Yersinia enterolitica (Lyon and Glatz, 1993; Lyon et al., 1993). Jenseniin G is produced by P. jensenii P126 (later reclassified as P. thoenii), it is active against propionibacteria, lactocci and lactobacilli, while no other bacteria, yeasts or moulds were shown to be inhibited (Grinstead and Barefoot, 1992). However, this bacteriocin was reported to inhibit the outgrowth of clostridial spores (Ekinci and Barefoot, 1999; Holo et al., 2002). Thoeniicin 447, produced by P. thoenii, is active against Propionibacterium acnes (Van der Merwe et al., 2004) and may thus be useful against this opportunistic pathogen.

A growing number of bacteriocins are still discovered in the different species of dairy propionibacteria (Brede et al., 2004; Van der Merwe et al., 2004). Broad screening of strains with appropriate indicators revealed the production of bacteriocin is widespread within dairy propionibacteria (Miescher, 1999). As an example, propionicins SM1 and SM2, firstly described by S. Miescher et al. in P. jensenii are encoded by a plasmid which was also detected in P.acidipropionici and P. freudenreichii (Rehberger and Glatz,

1990; Kiatpapan et al., 2000). Only a limited number of propionibacteria bacteriocins have been fully sequenced and characterised. Moreover, other antimicrobial compounds with either wide or narrow spectra of inhibition still are to identify in propionibacteria (Holo et al., 2002). This clearly opens new perspectives for the improvement of food quality and health.

8.1.3 Production of vitamins

The peculiar central carbon metabolism of propionibacteria is characterised by the transfer and rearrangement of C1-compounds. The Wood-Werkman cycle involves a multimeric transcarboxylase (EC 2.1.3.1) responsible for the formation of propionyl-CoA and oxaloacetate. These reactions are catalysed by enzymes with specific cofactors including vitamin B12 (cobalamin), B9 (folic acid) and biotin.

B12 and B9 are two distinct hydrosoluble vitamins, their main source are eggs and dairy products for B12, and cereals and vegetables for B9. They are both involved in haematopoiesis and in the biosynthesis of methionine, thymidine and purine. Deficiency in B vitamins causes anaemia due to decreased synthesis of nuclei basis and is also involved in neurodegenerative diseases. The daily recommended intake of B12 is 2.4 mg for adults and 2.6 mg for pregnant women while that of B9 is 300 mg for adults and 400 mg for pregnant women. Indeed, this last is known to prevent neural-tube defects during development.

Dairy propionibacteria have long been used for the industrial production of vitamin B12 by fermentation. Pseudomonas denitrificans can also be used in that aim, but propionibacteria allow the production of food-grade vitamin, either in food during fermentation, or as a food additive (Hugenholtz et al., 2002). The industrial production of B12 by P. freudenreichii fermentation is a two-stage process which can lead to the production of 330g of B12 from 1 kg of propionibacterial biomass (Deborde, 2002). B9 is naturally present in milk, but its concentration can be enhanced in fermented milks. The yogurt starter Streptococcus thermophilus was shown to produce B9 which is consumed by lactobacilli (Crittenden et al., 2003). Different strains of dairy propionibacteria produce varying amounts of B9, which can be higher than that produced by S. thermophilus (Hugenholtz et al., 2002). Interestingly, an almost complete excretion of B9 into the medium was observed for some strains.

8.1.4 Use in fermented food products

The main fermented food product containing propionibacteria is Swiss-type cheese. In addition to ripening, propionibacteria participate in microbiological safety and nutritional quality of cheeses. Propionibacteria being able to ferment a wide variety of substrates, they can acidify different non-dairy food products. Their antimicrobial properties afford increased shelf-life of the fermented products.

Babuchowski et al. reported the use of dairy propionibacteria in fermented vegetables such as sauerkraut, red beet juice and vegetable salads. This resulted in increases in B9, B12, propionic and acetic acid contents, inhibition of harmful and pathogenic microorganisms and the extension of the shelf-life of the products (Babuchowski et al., 1999). This was later confirmed in a pilot study on the fermentation of root vegetables (mainly beetroots and turnips) (Jagerstad et al., 2004).

Dairy propionibacteria were also used in fermented milks where they improve nutritional quality. Suomalainen and Mayra-Maliken reported the use of propionibacteria in conjunction with lactic acid bacteria to avoid spoilage by yeasts, moulds and Bacillus species in fermented milk and in bakery products (Suomalainen and Mayra-Makinen, 1999). In another study, P. freudenreichii NIZO B2336, which produces large amounts of riboflavin, was incorporated into yoghurts and ingested by rats with were fed a riboflavin-deficient diet (LeBlanc et al., 2006). The resulting fermented milk had enhanced concentration of riboflavin and its ingestion suppressed the symptoms of ariboflavinosis in rats, while the conventional yoghurt without propionibacteria failed to have such health effect. Furthermore, P. freudenreichii was shown to convert in vitro free linoleic acid into conjugated linoleic acid, mainly the cis-9, trans-11 and trans-9, cis-11 octadecadienoic isomers (Rainio et al., 2001; Jiang et al., 1998b). In addition, P. freudenreichii was shown to convert free linoleic acid, added to skim milk by the incorporation of hydrolysed soil oil, during fermented milk production and storage (Xu et al., 2005). The main isomer produced was rumenic acid, the cis-9, trans-11-octadecadienoic acid. Considering the potent beneficial effects of ruminic acid (Wahle et al., 2004), in particular on carcinogenesis (Lavillonniere et al., 2003; Lock et al., 2004), this can constitute another health-promoting potential for dairy propionibacteria. However, enhancement of free rumenic acid in dairy products, as a result of probiotic growth, remains to be demonstrated. Indeed, the CLA content of dairy fat is already elevated and subjected to variations linked to cattle feed or breed (Sieber et al., 2004). Further research in this field is thus necessary to evidence the positive role of probiotics, via CLA production, in dairy or non-dairy fermented products.

8.1.5 Recent use as probiotics

Most of the first publications on dairy propionibacteria dealt with their peculiar metabolism and their use in cheese technology. However, there is a growing interest on their use as probiotics. Indeed, considering that they benefit of the GRAS status in dairy products fermentation, that they adapt to different environments and substrates, and that they produce metabolites with potent health-promoting effects, it is tempting to speculate on their efficacy as probiotics. There is presently much more experimental data on the probiotic applications of bacteria belonging to the Lactobacillus and Bifidobacterium species than on propionibacteria. However, several reports suggest that they may have a positive impact on the gut health, function and comfort. These promising characteristics are summarised in Table 8.1 and further described below.

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