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Nutraceutical production with food-grade microorganisms Jeroen Hugenholtz* and Eddy J Smid
Over the past few years a number of new food ingredients labelled as being nutraceuticals have been launched on the food and pharmaceutical market. These include components that have a proven beneficial effect on human health, such as low-calorie sugars and B vitamins. Lactic acid bacteria, in particular Lactococcus lactis, have been demonstrated to be ideal cell factories for the production of these important nutraceuticals. Developments in the genetic engineering of food-grade microoganisms means that the production of certain nutraceuticals can be enhanced or newly induced through overexpression and/or disruption of relevant metabolic genes.
Addresses *Wageningen Centre for Food Sciences and NIZO Food Research PO Box 20, 6710 BA Ede, The Netherlands e-mail:hugenhol@nizo.nl e-mail: ejsmid@nizo.nl Current Opinion in Biotechnology 2002, 13:497-507 0958-1669/02/$ see front matter 2002 Elsevier Science Ltd. All rights reserved. Abbreviations EPS exopolysaccharide FOS fructo-oligosaccharides LAB lactic acid bacteria LDH lactate dehydrogenase MPDH mannitol-phosphate dehydrogenase NICE nisin controlled expression PFK phosphofructokinase

In this review, we describe several nutraceuticals that can be produced through fermentation with food-grade microorganisms. In the description of components that have a proven beneficial effect on human health (i.e. vitamins and lowcalorie sugars) we focus on the strategy to increase production levels in fermented foods. We also discuss a range of components that indirectly improve the health status of humans, through stimulation of certain intestinal microorganisms or through specific growth stimulation of probiotics (see the article by Rastall and Maitin in this issue). The health-promoting activity of these so-called prebiotics is indirect and fully dependent on the action of the intestinal microorganisms, as is discussed. Finally, we mention briefly a range of components with possible, but not proven, stimulatory effect on human health.

Lactic acid bacteria as cell factories

LAB are industrially important microbes that are used all over the world in a large variety of industrial food fermen- tations. Their contribution in these fermentation processes primarily consists of the formation of lactic acid from the available carbon source resulting in a rapid acidification of the unprocessed food material, which is a critical parameter in the preservation of these products. However, besides their lactic acid forming capacity, LAB also have the ability to contribute to other product characteristics such as flavour, texture and nutrition. Next to their most important application, which is undoubtedly in the dairy industry, LAB are also applied at an industrial scale in the fermentation of other raw food materials such as meat and vegetables. L. lactis is by far the most extensively studied LAB, and over the past decades elegant and efficient genetic tools have been developed for use in this bacterium. These tools are of critical importance in metabolic engineering strategies that aim at inactivation of undesired genes and/or (controlled) overexpression of existing or novel genes. Above all, the nisin-controlled expression (NICE) system (Figure 1) for controlled heterologous and homologous gene expression in Gram-positive bacteria [2,3] has proven to be very valuable and has been employed in most of the metabolic engineering strategies discussed here [4,5,6,7]. Initial metabolic engineering of L. lactis has focused, successfully, on the rerouting of pyruvate metabolism. Sugar metabolism was diverted away from lactate production towards the production of-acetolactate, the precursor of diacetyl, by either disruption of lactate dehydrogenase (LDH) or overproduction of NADH oxidase. By combining this strategy with disruption of the ilvB gene, encoding for-acetolactate decarboxylase, very effective diacetyl production from glucose and lactose was achieved [4]. This example shows how a minor pathway in L. lactis can be boosted to become the major metabolic pathway, and

Introduction
The term 'nutraceuticals', launched by Stephen DeFelici in the 1980s, defines a wide range of foods and food components with a claimed medical or health benefit [1]. Over the past five to ten years a large number of new food ingredients labelled as being nutraceuticals have been launched on the food and pharmaceutical market. The beneficial action of these components ranges from the supply of essential minerals or vitamins to protection against several infectious diseases. In the majority of cases, especially in the control of or protection against disease, the health claims are not supported by sound scientific data and we are merely dealing with, rather shaky, circumstantial evidence. Lactic acid bacteria (LAB), in particular Lactococcus lactis, are demonstrated to be ideal cell factories for the production of several important nutraceuticals. Not only do LAB have great potential for in situ production in fermented foods, but they also have huge potential in metabolic engineering strategies. Several examples are presented here where the production of a certain nutraceutical has been enhanced or newly induced through overexpression and/or disruption of relevant metabolic genes.

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Figure 1 Nisin-controlled gene expression (NICE) in L. lactis. The genes nisR and nisK correspond to the two genes originating from the nisine transposon [3] involved in this two-component regulatory system. Addition of small amounts (ng/mL) of nisin results in complete switch-on of the nisin promotor (P*) and subsequent expression of the cloned gene of interest.

Nisin

Nisin induction

Nis K Signal transduction

i

nisR

nisK

EnzymeX

Nis R Regulated gene expression P

gene X

Current Opinion in Biotechnology

demonstrates the power of metabolic engineering in these, relatively simple, LAB. In another example, pyruvate was redirected in the direction of the amino acid alanine. Alanine is an industrially relevant food and pharmaceutical ingredient. It is, in the form of Lalanine, not only a major component of amino- acid-rich medical supplements, but also a substrate for many (bio)chemical synthesis reactions in industry. In addition, alanine has a sweetening power comparable to the carbohydrates glucose and fructose and can serve as a lowcalorie sweetener in foods because of its much lower caloric value. Using metabolic engineering on the level of pyruvate metabolism, high production of alanine was achieved in both L. lactis and in Lactobacillus plantarum. The (heterologous) Bacillus sphaericus alaD gene, encoding for alanine dehydrogenase, was cloned in L. lactis, using the NICE system [3]. The alanine dehydrogenase converts pyruvate to Lalanine in the presence of ammonium. Upon introduction of this system in LDH-deficient lactococcal cells, a complete conversion of the pyruvate pool to alanine was obtained when cells were incubated under the appropriate, high ammonium, conditions [5]. The alanine produced by these cells consisted of a mixture of both stereo-isomers (D- and L-alanine) as a consequence of the endogenous alanine racemase activity of L. lactis (encoded by the alr gene). To obtain a stereospecific L-alanine producing bioreactor, the alr gene was functionally disrupted in the LDH-deficient lactococcal strain (Figure 2). High level expression of alanine dehydrogenase in the resulting cells indeed led to stereospecific L-alanine production as the only end-product of fermentation [5].

These two examples illustrate how the, basically simple, homofermentative metabolism of L. lactis, centred on lactate as sole fermentation product, can be redirected into efficient production of other industrially important (food) ingredients.

Low-calorie sugars
Body-weight control is a major concern in Western countries and obesity has been estimated to cost between 2% and 5% of the total health-care expenses of various developed countries. New food products containing low-calorie sugars and/or fatreplacers are in constant progression on the market in response to the consumer's request. We discuss here the production of mannitol, sorbitol, tagatose and trehalose by LAB and propionibacteria.
Production of mannitol and sorbitol

Polyols, such as mannitol and sorbitol, are low-calorie sugars that can replace sucrose, lactose, glucose or fructose in food products as they display equivalent sweetness and taste [8,9]. Mannitol can also serve as antioxidant in biological cells [10] as shown by increased survival of cells during freezing and/or drying in the presence of mannitol [11]. Heterofermentative LAB such as Leuconostoc mesenteroides are known for their ability to produce mannitol in the fermentation of fructose [12]. These bacteria convert part of the fructose for energy generation via the usual heterofermentative pathway, while the rest of the fructose is reduced directly to mannitol. In this way, high yields of mannitol are found, especially when fructose is supplied with, for example, glucose. Mannitol production, at low levels, has also been reported in homofermentative LAB

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Figure 2 Metabolic engineering of L-alanine production in L. lactis. The gene alaD is of Bacillus origin and encodes for alanine dehydrogenase (AlaDH). The AlaDH converts pyruvate to L-alanine in the presence of ammonium and in LDH-deficient lactococcal cells a complete conversion of the pyruvate pool to alanine is

Glucos e
NA Km D+ NAD H

NH
4+ D-Alanine

Racemase

obtained. To obtain a stereospecific L-alanine producing bioreactor, the alr gene encoding

Pyruvat e
=1.15 mM LLDH
K m

AlaDH mM =1.7
+

L-

Alanine

100 %

L-

Alanine

racemase is functionally disrupted resulting in 100% production of L-alanine.

NADHNADH NAD NAD

+

L-

body. It has recently been launched on the food market as a low-calorie sugar, as a prebiotic [16], and as an antiplaque agent by ARLA foods ([17]; http://www.tagatose.dk/). There is no biological source available for extraction of tagatose so the only feasible process for production is an enzymatic isomerisation with arabinose as the substrate [18]. There is only one biochemical pathway that features tagatose and that is in the degradation of lactose by LAB

Lactate
under unusual conditions. In both L. plantarum [13] and L. lactis [14] disruption of LDH resulted in production of a whole new range of metabolites, including mannitol. The mannitol is, most probably, produced by reduction of fructose-6phosphate to mannitol-1-phosphate, which is subsequently dephosphorylated to yield mannitol. With this in mind, metabolic engineering strategies can be designed to induce significant mannitol production in these LAB: for example, overproduction of the mannitol- phosphate dehydrogenase (MPDH) in LDH-deficient L. lactis strains or a decrease in phosphofructokinase (PFK) activity [15]. Higher production of mannitol by L. lactis would also be expected if excretion of this polyol is some- how facilitated, for instance by introducing the mannitol transporter as found in Leuconostoc mesenteroides. Production of sorbitol can be induced in these bacteria in a similar way. By overexpression of the gene coding for sorbitol dehydrogenase, in combination with disruption of the MPDH and the LDH, considerable sorbitol production has been observed in L. plantarum strains. Also in this case, even higher production of sorbitol is expected when the dephosphorylation and the transport (export) of sorbitol is enhanced.
Tagatose production

Tagatose is another carbohydrate that is considered as a potential sucrose replacement. It has almost equal sweetening power as sucrose higher than similar components such as mannitol, sorbitol and erythritol but much lower caloric value because it is poorly degraded by the human

tools, including an efficient gene integration system, and its very efficient system (NICE) for overproduction of enzymes. CurrentOpinioninBiotechnology To induce accu- mulation of tagatose derivatives in lactococcal cells, the initial strategy chosen was to disrupt the lacC and/or lacD genes resulting in production of either tagatose 6-phos- phate or tagatose 1,6-diphosphate. Disruption and a few other microorganisms. The so-called tagatose 6of lacD was accomplished via a two-step recombination phosphate pathway [19] is responsible for breakdown of the process, involving integration of an erythromycin-resistance galactose moiety during lactose metabolism by bacteria such as L. plasmid containing only the lacC and lacF genes via a single lactis. After hydrolysis of lactose-phosphate, crossover event, followed by removal of lacD (or reversion to through the action of phospho--galactosidase, the glucose the wild type) in a second, spontaneous, recombination event is degraded through glycolysis and the galactose-phosphate ('double crossover'). The double crossover event seemed to moiety is converted into tagatose 6-phosphate catalysed by occur, as expected, in almost 50% of cases, so the desired galactose-phosphate isomerase. The tagatose 6-phosphate is then mutant was obtained relatively easily. Growth rates of the phosphorylated forming tagatose-diphosphate, catalysed by lacD- mutant on glucose were identical to the wild type, but tagatose-phosphate kinase, and finally hydrolysed, by tagatose growth on lactose was severely compro- mised in the diphosphate aldolase, to form the glycolytic intermediates engineered strain. This was already a good indication that an dihydroxyaceton-phosphate and glyceraldehyde phosphate. unusual physiological response was occurring in the construct. HPLC analysis focusing on phosphorylated The genes encoding this tagatose pathway ( lacABCD) in L. lactis metabolic intermediates revealed the accumulation of a can be found on the lactose operon [19]. L. lactis was chosen as unique metabolite in the constructed the potential cell factory for the production of tagatose because of its relatively simple carbon metabolism, its broad arsenal of genetic

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Figure 3

Lactate

Glucose

Tre-6P synthase
Glucose-6P

Trehalose-6P

storage. More recently, it has been shown that trehalose has important stabilizing effects on human proteins, preventing protein aggregation as well as formation of pathological conformational forms, and its application against illnesses, such as the Creutzfeld-Jakob disease, has been disclosed [25]. Given the beneficial properties of trehalose, the development of food products in which trehalose is produced in situ at the expense of other sugars, such as lactose, fructose or glucose, is highly desirable. As with many other microorganisms [22,26], trehalose accumulation in Propionibacterium has been shown to occur as a result of different stress conditions, such as low temperature, high osmolarity and other adverse growth conditions [27]. Trehalose is formed from the glycolytic intermediate glucose 6-phosphate via glucose 1-phosphate, UDP-glucose and trehalose 6-phosphate to trehalose [28]. The enzymes that are specific for trehalose production are trehalose 6phosphate synthase and trehalose 6-phosphate phosphatase (Figure 3). The intracellular concentrations of trehalose that are reached vary enormously between different strains and are also strongly dependent on the growth conditions applied. In some fermentations using propionibacteria, high amounts of trehalose were found [27,29]. This demonstrates the actual potential of the use of propioni- bacteria for production of the low-calorie sugar trehalose in (fermented) foods.

Tre-6P phosphatase
Pyruvate Glucose-1P UDP-glucose

Trehalose

Propionate
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Trehalose formation in Propionibacteria. Trehalose is formed from the glycolytic intermediate glucose 6-phosphate via glucose 1-phosphate, UDP-glucose and trehalose 6-phosphate.

strain compared with the metabolites of the wild type (i.e. the glucose and fructose phosphates). Spiking the samples with synthesized tagatose-phosphate and tagatose biphos- phate, specially produced for this purpose, gave a strong indication that the unique metabolite was indeed tagatose 1,6diphosphate. Thus, L. lactis was successfully transformed into a tagatose (phosphate) producing cell factory. For use in practical (food) situations, however, strategies will have to developed to dephosphorylate the tagatose diphosphate and excrete the final product, tagatose.
Other low-calorie sugars

Vitamin production
Some vitamins such as folate (B11) are mainly found in products from plant origin. As de novo synthesis of folate does not occur in animals, the natural folate found in meat, eggs or dairy products originates from plant sources. Other B vitamins such as cobalamin (vitamin B12) are naturally found in animal foods products including fish, poultry, milk and milk products, eggs and meat, and are not present in plants. Riboflavin (B2) originates from organ meat, nuts, cheese, eggs, milk and lean meat, but good quantities are also found in green leafy vegetables, fish, legumes, whole grains and yoghurt. These three B vitamins are essential in the human diet. Specific population groups in Western society, such as the elderly, adolescents and other socially isolated groups, run a high risk of insufficient daily intake of these vitamins. Many food-grade bacteria produce excess of B vitamins such as riboflavin (vitamin B2), folate (vitamin B11) and cyanocobalamine (vitamin B 12). This unique property of bacteria offers the possibility to fortify raw food materials such as soya, milk, meat and vegetables with B vitamins by natural means (i.e. without adding food supplements) [30].
Folate

Trehalose is a well-known non-reducing disaccharide synthesized by a wide variety of organisms. It is only partially digested in humans, and therefore is considered a dietetic sugar. It is also poorly metabolised by many other organ- isms, including LAB. The oral bacteria Streptococcus mutans and Streptococcus salivarius for instance, are not able to perform acidification when trehalose is the only carbohy- drate [20]. In comparative studies with human volunteers, mouth rinses with trehalose solutions led to significantly reduced acidification in plaques compared with rinsing with sucrose solutions. By feeding rats with trehalose- containing diets, instead of sucrose, almost complete suppression of dental decay (caries) was observed [20]. Other (biological) activities than can be attributed to trehalose are protection of proteins and whole cells against denaturation under different stress conditions such as heating, drying and freezing. The preserving properties of trehalose on different biological systems have been widely demonstrated with different enzymes [21,22], membranes [22], human cells [23], and tissues [24]. Especially relevant in the context of this overview are the applications of trehalose for preserving different kinds of foodstuffs, keeping the freshness and taste of the product even after prolonged

Folic acid is an essential component in the human diet. It is involved, as a cofactor, in many metabolic reactions, including the biosynthesis of the building blocks of DNA and RNA, the nucleotides. The daily recommended intake for an adult is 200g. For pregnant women a double dose

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is recommended, because folic acid is known to prevent neural-tube defect in newborns [31]. Low folic acid in the diet is associated with high homocysteine levels in the blood and, subsequently, with coronary diseases [32,33]. It is even reported to protect against some forms of cancer [34]. Folate is conspicuously absent in many food products and is considered an essential additive to the general diet. Folic acid is produced by different (green) plants (folium [Latin] means leaf) and by some microorganisms. Vegetables and dairy products are the main source of folic acid for humans. 'Folate' is a non-specific term referring to any folate compound with vitamin activity. The term folic acid is used for the chemically synthesized vitamin. Folate is isolated from natural sources in many different forms. The basic structure of the molecule consists of a pteridine moiety, a p-aminobenzoic acid residue and one or more-linked L-glutamic acid residues. In the general metabolism, folates act as an acceptor and donor of C1 residues. This main biochemical function of folate is mediated by the N-5 and N-10 positions which carry methyl-, methynyl-, methylene-, formimino- or formyl- residues. The pyrazine rings can be reduced to the so-called dihydro- and tetrahydro-form of the folate molecule. Finally, the poly--L-glutamate tail can vary in length between 1 and 10 residues. Folate is an essential cofactor in bacterial metabolism and, as such, will be present in most, if not all, bacteria. Folate is produced in many forms in nature: methylated, hydrated, and with various sizes of polyglutamate tails. Uptake of folate by auxotrophic bacteria and possibly also by human cells, however, is claimed to be restricted to specific folate molecules with polyglutamate tails of three moieties or less. Many bacteria are able to synthesize this cofactor by themselves from simple precursors, such as GTP, paminobenzoic acid and glutamate but some auxotrophic bacteria, including many LAB, have a strict growth requirement for folate. Milk is a well-known source of folate. It contains between 20 and 50g folate/L and thus contributes significantly to the daily requirement of the average consumer. Fermented milk products, especially yoghurt, are reported to sometimes contain even higher amounts of folate [35]. Up to 150g folate/L has been found in yoghurt. This high level is a direct result of the production of additional folate by the LAB in the yoghurt. Of the two LAB species in yoghurt Lactobacillus bulgaricus and Streptococcus thermophilus, only the latter is reported to produce folate [36]. Recently, some other food-grade bacteria were observed to produce folate during milk fermentation [37]. Also Propionibacterium sp., which are well known for their ability to produce vitamin B 12, are capable of producing folate in relatively high amounts [29]. Large differences with respect to production levels were found between different strains and different species. Interestingly, the

Figure 4

Glycolysis

Pentose phosphate pathway

GTP

E4P Shikimate pathway Glutamate Chorismate Folate

Current Opinion in Biotechnology

Tyr

Phe

Trp

PABA

Schematic pathway for folate production in LAB showing the connection with other metabolic pathways (highlighted in yellow). E4P, erythrose-4-phosphate; Tyr, tyrosine; Trp, tryptophan; Phe, phenylalanine; PABA, para-aminobenzoate.

levels produced by Propionibacterium sp. are equally high or even higher than the levels produced by the well-known folate producer S. thermophilus [30]. Another striking obser- vation is the difference in ability to excrete folate by the different propionibacteria. Although some strains, clearly, retain all the folate intracellularly, in other strains almost complete excretion or leakage of the folate is observed. This, presumably, is a result of the different forms of folate that are produced by these propionibacteria. Analysis of the molecular structure, such as the nature of the bound C1 moiety and the presence and length of the polyglutamate tail, will definitely shed more light on this matter. Folate biosynthesis is currently being studied in more detail. Total folate production in L. lactis strain MG1363 is approximately 100 ng/ml. Most of the folate (90%) is accumulated intracellularly, probably because of polyglutamylation of the intracellular folates. Only a small fraction (10%) is released into the environment [38]. Folate biosynthesis involves a multienzyme pathway (Figure 4). The genes involved in folate biosynthesis in L. lactis MG1363 have recently been analysed [38]. The information obtained about the organization of the gene cluster involved in folate biosynthesis allows efficient genetic engineering aimed at increasing the folate biosynthesis level in L. lactis. Using the NICE system in L. lactis strain NZ9000, several of the folate biosynthetic genes have been overexpressed individually or in combination. Overproduction of the first enzyme in folate biosynthesis, GTP cyclohydrolase, leads to threefold increased folate production [7]. In addition, an altered ratio of monoglutamyl-folate over polyglutamyl-folate is observed, explaining a higher release of the folate into the growth medium. The latter modulation of glutamylation level and consequently spatial distribution of the folate produced by L. lactis could lead to improved uptake of this microbially produced vitamin in the human small intestine, thereby

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Figure 5 -L-Rhap 1 2 4)--D-Galp-(1 3 O -D-Galp-1-O-P-OH O

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4)--D-Glcp-(1

4)--D-Glcp-(1

this vitamin for an average adult is about 3g per day [40]. Insufficient vitamin B12 intake leads to pernicious anaemia [41]. Vitamin B12 is a cobalt complex that in its coenzyme form is required by enzymes catalysing several metabolically essential rearrangement reactions. Vitamin B12 or cobalamin is among the most complex molecules found in nature. Only two B12-dependent reactions have been identified in humans, methionine synthetase and methyl malonyl CoA mutase, which is involved in lipid turnover [40]. In bacteria, vitamin B12 serves generally as cofactor in carbon rearrangement reactions that occur during breakdown of amino acids or metabolism of small carbon molecules such as glycerol, ethanolamine and propionic acid. Propionibacteria have long been known for their high production capacity of vitamin B12 and this has led to the development of commercially interesting production processes. Propionibacteria are known for their unique, anaerobic metabolism involving several carbon rearrangement reactions. To catalyse these reactions, the propionibacteria contain a wide variety of enzymes with specific cofactors, such as coenzyme B12 (deoxyadenosylcobalamin), folic acid and biotin. There are, currently, only three microorganisms used for commercial production of vitamin B12, namely Pseudomonas denitrificans, Bacillus megaterium and Propionibacterium [40-43]. The dairy Propionibacterium strains have the huge advantage that they are food-grade and vitamin B12 can be produced in the food, or added to the food, without the need for elaborate processing. Microorganisms produce coenzyme B12 or deoxyadenosylcobalamin via a complicated pathway involving at least 25 conversion steps from the starting precursors uroporphyrinogen III (precursor for heme, F430 and cobalamin), dimethylbenzimidazole and a adenosylmoiety. The biosynthesis of the precursor uropor- phyrinogen III, itself, involves a multistep pathway from the amino acid aminolevulenic acid via porphoblinogen to uroporphyrinogen III. The synthesis of dimethylbenzimi- dazole has not been completely elucidated. It is derived from riboflavin and involves five reactions, one of which seems to require oxygen. Many fermentative processes have been described for the production of vitamin B12 from propionibacteria, and these processes usually focus on growing the bacteria to high cell densities [44]. Varying the nutrient composition of the growth medium, in for example amino acid composition or mineral composition including cobalt ions, usually only effected B12 production when the growth yield of the Propionibacterium was effected. Two experimental findings led to major improvements in the production yields of vitamin B12. Both the addition of the precursor dimethylbenzimidazole and aerobic incubation in the latter phase of fermentation resulted in increased B 12 production. Using either or both of these strategies, production levels of 10 to 35 mg/L could be reached during fermentation of

Basic structure of the repeating unit of the exopolysaccharide produced by L. lactis strain B40. Rha, rhamnose; Gal, galactose; Glc, glucose.

generating improved folate bio-availability in fermented products produced with such strains. To further expand these bio-availability studies, L. lactis strains were constructed that express the-glutamyl hydrolase enzyme derived from rat or human origin [39]. cDNA clones encoding these enzymes were cloned in the NICE system and introduced in L. lactis, and both the human and rat enzymes could be expressed functionally in this organism. The -glutamyl hydrolase expression in growing L. lactis cultures resulted in an inversion of folate spatial distribution (i.e. from mainly intracellular to extracellular accumulation) [38]. These results demonstrate that the expression of human-glutamyl hydrolase in the folate-producer L. lactis leads to intracellular deconjugation of polyglutamyl-folate to monoglutamyl-folate resulting in decreased retention of folate in the cell and, consequently, increased excretion of folate to the environment. Sybesma et al. [7] conclude that expression of heterologous-glutamyl hydrolase in L. lactis increases the extracellular monoglutamyl folate level (i.e. the level of bio-avalaible folate) thereby relieving the requirement for the intestinal hydrolase activity. The work on bacterial folate biosynthesis represents an important first step in the development of novel, functional foods with increased levels of bio-available folate. The identification of-glutamyl hydrolases from (food-grade) bacterial origin would enhance the market introduction of a food-grade microorganism that has improved bioavailable-folate production capacities. Moreover, metabolic engineering of the lactococcal folate biosynthesis pathway itself by changing expression levels of the relevant biosynthetic enzymes is expected to result in significant increases in folate production. These approaches could eventually be combined to generate starter bacteria that can be used for the production of novel fermented dairy products providing 100% instead of the current 15-20% of the human daily requirement for folate intake.
Vitamin B12

Humans cannot synthesize vitamin B12 and must obtain it from organisms that produce it. The daily requirement of

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Propionibacterium in, for example, cheese whey used as cheap growth medium. On the basis of these findings, Hunik [43] described a semi-continuous, large-scale, production process for Propionibacterium. This process resulted in a threefold increase in production level compared to the original, two-phase, batch cultivation. Besides the use of Propionibacterium sp. for industrial scale vitamin production, this food-grade bacterium can also be used for improved in situ vitamin production in, for example, cheese. In this way, fermented dairy products can be fortified with B vitamins in a natural way (i.e. without supplementation).
Riboflavin

ssp. shermanii as starter cultures resulted in a significant increase of the riboflavin content of milk [47]. Since, the riboflavin produced by starter cultures is largely in the free form, the bio-availability is expected to be better than the bioavailability of riboflavin in unprocessed milk [48]. The foodgrade fermentative LAB L. lactis also grows in the absence of riboflavin. On the basis of the genome sequence of L. lactis IL1403 [49], it seems that all genes involved in riboflavin biosynthesis (rib genes) are present in this organism. Work is underway to study the biosyn- thesis pathway and to improve the riboflavin production in this bacterium (see http://www.nutracells.com). Next to the biotechnological vitamin production, there are other ways in which microorganisms are involved as a source of vitamins. It is now well established that the microflora in the human intestines play a significant role in the production of vitamins, and in particular those of the vitamin B group, such as riboflavin [50].

All plant and animal cells contain riboflavin. Milk, milk products, meat, eggs and green leafy vegetables are the most common sources of riboflavin in our diet. In addition, fortified cereals and bakery products significantly con- tribute to our daily intake. Riboflavin from animal sources is better absorbed than from vegetable sources. Riboflavin is active in the transfer of electrons in numerous essential oxidation-reduction reactions. It participates in many metabolic reactions of carbohydrates, fats and proteins and in energy production via the respiratory chain. Furthermore, riboflavin coenzymes are essential for the conversion of pyridoxine (vitamin B6) and folic acid into their coenzyme forms and for the transformation of tryptophan to niacin. Clinical symptoms of riboflavin deficiency are rarely seen in developed countries; however, the subclinical stage of deficiency, characterized by a change in biochemical indices, is common. In children, this may result in less than normal growth. Riboflavin deficiency usually occurs in combination with deficiencies of other water-soluble vitamins. Riboflavin deficiency can also occur as a result of trauma, including burns and surgery, and has been observed in patients with chronic debilitating disorders (e.g. rheumatic fever, tuberculosis, subacute bacterial endocarditis), diabetes, hyperthyroidism and liver cirrhosis. Other groups at risk are the elderly, women taking oral contraceptives, children and adolescents from low socio-economic backgrounds, children with chronic heart disease, people who exclude milk products from their diet and infants undergoing prolonged phototherapy for hyperbilirubinaemia. Various bacteria, yeast and fungi are commercially employed to synthesize riboflavin, using cheap natural materials and industrial wastes as growth medium. The industrial production of riboflavin is a good example of a successful biotechnological process performed by microorganisms [45]. The produced vitamins are used for food and feed fortification. The source of origin determines to a large extent the bioavailability of riboflavin. In milk, a sizeable part of the riboflavin exists in the co-enzyme form, bound covalently to protein [46]. It has been reported that fermentation of cow milk with L. lactis and Propionibacterium freudenreichii

Prebiotics
The human intestine harbours an abundant and extremely diverse microflora with an estimated number of more than 400-500 microbial species [51]. Going from the stomach to the colon, the absolute number of bacteria dramatically increases up to 1012 CFU/ml gut content [52]. The intestinal microflora consists of species that exert either beneficial or harmful effects or both on the host. Species belonging to the group of bifidobacteria and lactobacilli are generally associated with health-promoting and protective properties such as activation of the immune system, inhibition of pathogenic bacteria, alleviation of lactose intolerance, and production of vitamins, largely from the B group [53]. Therefore, it seems desirable to increase the number of bifidobacteria sp. and lactobacilli in the colon. The diet can have a profound effect on the composition of the intestinal microflora [54]. A classic example of the effect of dietary factors is that human milk, in contrast to cow milk, contains relatively high levels of Nacetyl-neuramic acid. This compound appears to be an excellent substrate for bifidobacteria. This might explain for the higher proportion of bifidobacteria in the gastrointestinal tract of breast-fed infants compared with formula-fed children [55]. Prebiotics can be defined as non-digestible food ingredients that beneficially affect the host by selectively stimulating the growth and/or activity of one or a limited number of bacterial species already present in the colon, thereby improving the health of the host [53]. In principle, any foodstuff that reaches the colon (e.g. non-digestable carbo- hydrates, some peptides and certain lipids) can be classified as potential prebiotics [56]. Most oligosaccharides are predominantly fermented in the colon part of the gastrointestinal tract. Fructooligosaccharides (FOS) are -D-fructans with various degrees of polymerization. Inulin is a well-known example of a FOS that, upon ingestion, increases the content of bifidobacteria in the human gastrointestinal tract. This effect is known in the literature

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as the bifidogenic effect of prebiotic compounds. Besides the FOS, galacto-oligosaccharides, gluco-oligosaccharides, transgalacto-oligosaccharides, isomalto-oligosaccharides and xylo-oligosaccharides have also been found to exert bifidogenic effects (see [51]). Most potential bifidogenic compounds that have been studied are carbohydrates of plant origin. However, fructans produced by bacteria and fungi have also been identified as bifidogenic compounds, probably serving very different functions. Most bacterial fructans are high molecular weight polymers of the levan type (i.e. they are composed of-2,6-linked fructose molecules). They are, most of the time, part of the exopolysaccharide (EPS) capsule of the microbial cell. EPS protects the cells from desiccation, helps in surface attachment, and, in some plant pathogenic species, is involved in preventing the invading bacteria from being recognized by the host defence system [57,58]. The only bacterial species known so far that produces an inulin-type fructan is Streptococcus mutans, a human pathogen involved in dental caries [59]. Food-grade bacteria are also a potential source of nondigestable carbohydrates and potentially bifidogenic prebiotics. Many LAB are capable of producing EPSs [60]. Homopolysaccharides, such as dextran, mutan and levan are produced by several species of Lactobacillus, Streptococcus and Leuconostoc. Hetero- or homo-polysaccharides, as well as oligosaccharides composed of repeating units, are produced by a large group of LAB. The structures of the EPSs produced by LAB are diverse. Different L. lactis strains, for example, produce EPSs with different repeating units [61-63]. Although biodegradability of EPS is important for its bioactive properties, only limited information is available on this topic. The EPS from L. lactis B40 (Figure 5) was tested for biodegradability and found to be extremely resistant towards degradation by microorgan- isms from fecal and soil samples [64]. The polysaccharides produced by LAB can be considered as 'food-grade' additives and have received considerable attention based on their relevance for dairy-product properties such as texture and mouthfeel. With respect to their properties as nutraceuticals, claims have been made about Lactococcus EPS with regard to cholesterol-lowering activity [65], immunomodulation [66] and antitumour activity [67]. Up to now, no studies on selective stimulation of the growth of Bifidobacterium by lactococcal EPS have been published. However, their non- digestible nature and stool-bulking effect also groups these compounds among the dietary fibres and thereby generates an additional claim as nutraceutical. Therefore, specific oligosaccharides and polysaccharides produced by lactococcal strains could have a wide range of applications in the pharmaceutical and food industry. Significant progress in the understanding of EPS biosynthesis pathways, genetics, kinetics and physics has been made in recent years [68]. This knowledge is now being applied for metabolic engineering of strains with increased

EPS production and modified EPS composition. By overproduction of fructose diphosphatase in L. lactis, increased EPS formation on fructose as growth substrate was reported [69]. Recently, Boels and co-workers [6] also demonstrated that overexpression of UDP-glucose pyrophosphorylase and UDP-galactose epimerase increased the intracellular pools of UDP-glucose and UDP-galactose, both precursors of lactococcal EPS. These types of studies provide important knowledge for targeting bottlenecks in the EPS biosynthesis or for creating EPSs with novel properties.

Bioconversions
Food-grade bacteria can also be used for the bioconversion of food components into components with beneficial effects. This opens new ways to improve the food quality. Propionibacterium freudenreichii ssp. shermanii was found to convert linoleic acid into conjugated linoleic acid (CLA) [70]. The latter compound has raised extensive interest because of its beneficial physiological effects and anti- carcinogenic properties [71,72]. Eukaryotic microorganisms, such as the non-pathogenic ciliate Tetrahymena thermophila, also possess potentially interesting enzymatic activities for improving the nutri- tional quality of food products. This organism can convert cholesterol from milk into provitamin D 3 and related compounds [73].

Other nutraceuticals: antioxidants

Almost everyday a new food component is claimed as having specific beneficial effects for human health. The resulting list of actually established 'nutraceuticals' is constantly growing and includes many metabolites that can be produced by the food-grade microorganisms discussed so far. The nutraceutical-status of many antioxidants is still very dubious. Compounds such as the thiols glutathione, thioredoxin and rubredoxin [74-76] serve as electron acceptors in metabolic reactions and, as such, can be considered antioxidants. Glutathione is present in some LAB such as L. lactis [77,78], while conspicuously absent in many others [79] including Lactobacillus plantarum (Y Li and D Molenaar, personal communication). In the latter microorganism other thiols such as thioredoxin presumably serve in the redox-reactions for electron transfer. With the full knowledge of the genome sequence, it is now possible to increase the production and/or accumulation levels of these thiol-compounds in the cell and in the fermented products. This provides extremely useful study material for establishing once and for all whether these antioxidants have any real nutritional effects. The above mentioned low-calorie sugars mannitol and trehalose are also reported to react with oxygen radicals and, as such, have antioxidant activity [10]. However, to date, there is no evidence that increased uptake of these metabolites actually raises the antioxidant level in the human blood or in human cells. Therefore, apart from providing protection of food constituents (protein, flavour)

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against oxidation, the nutraceutical activity of these antioxidants is still very much in doubt.

6.

Boels IC, Ramos A, Kleerebezem M, de Vos WM: Functional analysis of the Lactococcus lactis galU and galE genes and their impact on sugar nucleotide and exopolysaccharide biosynthesis. Appl Environ Microbiol 2001, 67:3033-3040. Sybesma WFH, Starrenburg MJC, Mierau I, Kleerebezem M, de Vos WM, Hugenholtz J: Production of B-vitamins in lactic acid bacteria by using metabolic engineering. In Abstracts of 102nd General Meeting American Society for Microbiology: 2002 May 19-23; Salt Lake City, USA: Washington DC: American Society for Microbiology:K-76. Debord B, Lefebvre C, Guyot-Hermann AM, Hubert J, Bouche R, Guyot JC: Study of different forms of mannitol: Comparative behaviour under compression. Drug Develop Ind Pharm 1987, 13:1533-1546. Dwivedi BK: Low Calorie and Special Dietary Foods. CRC Press, Inc; West Palm Beach; 1978.

Conclusions
We have described the use of food-grade microorganisms, often starter bacteria, for the in situ production, in fermented foods, of nutraceuticals. The nutraceuticals fall basically into three categories: first, alternative, low-calorie sugars; second, polysaccharides and oligosaccharides; and third, B vitamins. A final category of miscellaneous components, such as antioxidants, has no proven benefits for human health. We have presented several examples where significant production of these beneficial nutraceuticals can be induced in fermented foods either by selecting specific highproducing LAB (or propionibacteria) or by inducing highproduction through engineering of the responsible metabolic pathways. Some of these selected and con- structed strains will undoubtedly find their way into novel fermented foods with increased nutritional values.

7.

8.

9.

10. Shen B, Jensen RG, Bohnert HJ: Mannitol protects against oxidation by hydroxyl radicals. Plant Phys. 1997, 115:527-532. 11. Efiuvwevwere BJO, Gorris LG, Smid EJ, Kets EPW: Mannitolenhanced survival of Lactococcus lactis subjected to drying. Appl Microbiol Biotechnol 1999, 51:100-104. 12. Soetaert W, Buchholz K, Vandamme EJ: Production of D-mannitol and D-lactic acid by fermentation with Leuconostoc mesenteroides. In Agro Food Ind High-Tech; 1995:41-44. 13. Ferain T, Schanck AN, Delcour J: 13C nuclear magnetic resonance analysis of glucose and citrate end products in an ldhL-ldhD double-knockout strain of Lactobacillus plantarum. J Bacteriol 1996, 178:7311-7315. 14. Neves AR, Ramos A, Shearman C, Gasson MJ, Almeida JS, Santos H: Metabolic characterization of Lactococcus lactis deficient in lactate dehydrogenase using in vivo 13C-NMR. Eur J Biochem 2000, 267:3859-3868. 15. Wisselink HW, Weusthuis RA, Eggink G, Hugenholtz J, Grobben GJ: Mannitol production by lactic acid bacteria: a review. Int Dairy J 2002, 12:151-162. 16. Bertelsen H, Jensen BB, Buemann B: D-tagatose - a novel low-calorie bulk sweetener with probiotic properties. World Rev Nutr Diet 1999, 85:98-109. 17. Zehner LR: D-tagatose as a low-calorie carbohydrate sweetener and bulking agent. European Patent Application EP0257626, 1988.

Update
Major progress has been made recently ([38]; www.nutracells.com) in the area of riboflavin and vitamin B12 production. Overexpression in L. lactis of the ribA gene using the NICE system (Figure 1) resulted in significant, three- to fourfold higher production of riboflavin. Even higher production was found when riboflavin production was deregulated by selecting roseoflavin-resistant mutants. In the field of vitamin B12 production, a major new development is the discovery of a lactic acid bacterium, Lactobacillus reuteri, that is able to produce a cobalamin. This opens new possibilities for producing fermented products with an increased vitamin B12 content.

Acknowledgements
We acknowledge the European Commission for sponsoring the RTD Project QLK1-CT-2000-01376 'Nutra Cells' (http://www.nutracells.com).

18. Kim P, Yoon SH, Roh HJ, Choi JH: High production of D-tagatose, a potential sugar substitute, using immobilized L-arabinose isomerase. Biotechnol Prog 2001, 17:208-210. 19. van Rooijen RJ, van Schalkwijk S, de Vos WM: Molecular cloning, characterization and nucleotide sequence of the tagatose-6phosphate pathway gene cluster of the lactose operon of Lactococcus lactis. J Biol Chem 1991, 266:7176-7181. 20. Neta T, Takada K, Hirasawa M: Low-cariogenicity of trehalose as a substrate. J Dent 2000, 28:571-576. 21. De Carlo S, Adrian M, Kalin P, Mayer JM, Dubochet J: Unexpected property of trehalose as observed by cryo-electron microscopy. J Microsc 1999, 196:40-45. 22. Felix CF, Moreira CC, Oliveira MS, Sola-Penna M, Meyer-Fernandes JR, Scofano HM, Ferreira-Pereira A: Protection against thermal denaturation by trehalose on the plasma membrane H+-ATPase from yeast. Synergetic effect between trehalose and phospholipid environment. Eur J Biochem 1999, 266:660-664. 23. Guo N, Puhlev I, Brown DR, Mansbridge J, Levine F: Trehalose expression confers desiccation tolerance on human cells. Nat Biotechnol 2000, 18:168-171. 24. Wu SF, Suzuki Y, Kitahara AK, Wada H, Nishimura Y: Skin flap storage with intracellular and extracellular solutions containing trehalose. Ann Plast Surg 1999, 43:289-294. 25. Singer MA, Lindquist S: Thermotolerance in Saccharomyces cerevisiae: the Yin and Yang of trehalose. Trends Biotechnol 1998, 16:460-468. 26. Welsh DT, Reed RH, Herbert RA: The role of trehalose in the osmoadaptation of Escherichia coli NCIB 9484: interaction of

References and recommended reading

Papers of particular interest, published within the annual period of review, have been highlighted as:

of special interest of outstanding interest

1. 2.

Pszczola DE: The nutraceutical initiative: a proposal for economic and regulatory reform. Food Biotechnol 1992, 46:77-79. Kleerebezem M, Beerthuyzen MM, Vaughan EE, de Vos WM, Kuipers OP: Controlled gene expression systems for lactic acid bacteria: transferable nisin inducible expression cassettes for Lactococcus, Leuconostoc and Lactobacillus spp. Appl Environ Microbiol 1997, 63:4581-4584. Kuipers OP, de Ruyter PGGA, Kleerebezem M, de Vos WM: Quorum sensing-controlled gene expression in lactic acid bacteria. J Biotech 1998, 64:15-21. Hols P, Kleerebezem M, Schank AN, Ferain T, Hugenholtz J, Delcour J, de Vos WM: Conversion of Lactococcus lactis from homolactic to homoalanine fermentation through metabolic engineering. Nat Biotechnol 1999, 17:588-592.

3.

4.

Hugenholtz J, Hols P, Starrenburg MJC, de Vos WM, Kleerebezem M: Metabolic engineering of Lactococcus lactis leading to high diacetyl production. Appl Environ Microbiol 2000, 66:4112-4114. This report demonstrates how powerful metabolic engineering can be in L. lactis. Almost complete rerouting of sugar metabolism towards diacetyl, instead of lactic acid, was observed in the described mutants. 5.

506

Food biotechnology

trehalose, K+ and glutamate during osmoadaptation in continuous culture. J Gen Microbiol 1991, 137:745-750. 27. Cardoso F, Gaspar P, Ramos A, Hugenholtz J, Santos H: Effect of environmental conditions on trehalose production by Propionibacterium. In Abstract Book of the 3rd International Symposium on Propionibacteria. 2002 July 8-11; Zurich, Switzerland: p 41:P29.

50. Hill MJ: Intestinal flora and endogenous vitamin synthesis. Eur J Cancer Prev 1997, 6(Suppl 1):S43-S45. 51. Moore WEC, Holdeman LV: Human fecal flora: the normal flora of 20 Japanese-Hawaiians. Appl Microbiol 1974, 27:961-979. 52. Kleessen B, Bezirtzoglou E, Mtt J: Culture-based knowledge on biodiversity, development and stability of human gastrointestinal microflora. Micro Ecol Health Dis 2000, 12:53-63. 53. Gibson GR, Roberfroid MB: Dietarymodulation of the colonic microbiota: introducing the concept of prebiotics. J Nutr 1995, 125:1401-1412. 54. Gil A, Rueda R: Modulation of the intestinal microflora by specific dietary components. Micro Ecol Health Dis 2000, 12:31-39. 55. Benno Y, Sawaka K, Mitsuoka T: The intestinal microflora of infants: composition of fecal flora in breast-fed and bottle-fed infants. Microbiol Immunol 1984, 28:975-986. 56. Fooks LJ, Fuller R, Gibson GR: Prebiotics, probiotics and human gut microbiology. Int Dairy J 1999, 9:53-61. 57. Kasapis S: Rhizobium trifolii capsular polysaccharide: a novel biopolymer with striking physical properties. Int J Food Sci Technol 1994, 29:29-38. 58. Hettwer U, Gross M, Rudolph K: Purification and characterization of an extracellular levansucrase from Pseudomonas syringae pv. phaseolicola. J Bacteriol 1995, 177:2834-2839. 59. Sato S, Kuramitsu HK: Isolation and characterization of a fructosyltransferase gene from Streptococcus mutans GS-5. Infect Immun 1986, 52:166-170. 60. Kleerebezem M, van Kranenburg R, Tuinier R, Boels IC, Zoon P, Looijesteijn E, Hugenholtz J, de Vos WM: Exopolysaccharides produced by Lactococcus lactis: from genetic engineering to improved rheological properties? Antonie Van Leeuwenhoek 1999, 76:357-365. 61. Nakajima H, Hirota T, Toba T, Itoh T, Adachi S: Structure of the extracellular polysaccharide from the slime forming Lactococcus lactis subsp. cremoris SBT 0495. Carbohydr Res 1992, 224:245-253. 62. van Casteren WH, de Waard P, Dijkema C, Schols HA, Voragen AG: Structural characterisation and enzymic modification of the exopolysaccharide produced by Lactococcus lactis subsp. cremoris B891. Carbohydr Res 2000, 327:411-422. 63. Gruter M, Leeflang BR, Kuiper J, Kamerling JP, Vliegenthart JF: Structure of the exopolysaccharide produced by Lactococcus lactis subspecies cremoris H414 grown in a defined medium or skimmed milk. Carbohydr Res 1992, 231:273-291. 64. Ruijssenaars HJ, Stingele F, Hartmans S: Biodegradability of food-associated extracellular polysaccharides. Curr Microbiol 2000, 40:194-199. 65. Nakajima H, Suzuki Y, Kaizu H, Hirota T: Cholesterol lowering activity of ropy fermented milk. J Food Sci 1992, 57:1327-1329. 66. Kawaguchi M, Fujioka Y, Kishimoto M, Matsuma T, Ichikawa T: Dietary fiber and phophorylated oligosaccharide protect small intestinal mucosa of rat from oxidative damages in vitro. J Jap Soc Food Sci Technol 1999, 46:487-490. 67. Kitazawa H, Toba T, Itoh T, Kumano N, Adachi S, Yamaguchi T: Antitumoral activity of slime-froming, encapsulated Lactococus lactis subsp. cremoris isolated from Scandinavian ropy sour milk, "viili". Animal Sci Technol 1991, 62:277-283. 68. Boels IC, van Kranenburg R, Hugenholtz J, Kleerebezem M, d Vos WM: Sugar catabolism and its impact on the biosynthesis and engineering of exopolysaccharide production in lactic acid bacteria. Int Dairy J 2001, 11:723-732. 69. Looijesteijn PJ, Boels IC, Kleerebezem M, Hugenholtz J: Regulation of exopolysaccharide production by Lactococcus lactis subsp. cremoris by the sugar source. Appl Environ Microbiol 1999, 65:5003-5008. 70. Rainio A, Vahvaselka M, Suomalainen T, Laakso S: Production of conjugated linaloic acid by Propionibacterium freudenreichii ssp. shermanii. Lait 2002, 82:91-101. 71. Kritchevsky D: Antimutagenic and some other effects of conjugated linoleic acid. Br J Nutr 2000, 83:459-465.

28. Deborde C, Corre C, Rolin DB, Nadal L, de Certaines JD, Boyaval P: Trehalose biosynthesis in dairy Propionibacterium. J Magn Res Anal 1996, 2:297-304. 29. Hugenholtz J, Hunik J, Santos H, Smid EJ: Nutraceutical production by propionibacteria. Lait 2001, 82:103-112. 30. Smid EJ, Starrenburg, M, Mierau I, Sybesma W, Hugenholtz J: Increase of folic acid levels in fermented foods. Inn Food Technol 2001, Feb-March:13-15. 31. Wald N: Prevention of neural tube defects. Results of the Medical Research Council vitamin study. Lancet 1991, 338:131-137. 32. Boushey CJ, Beresford AA, Omenn GS, Moltulsky AG: A quantitative assessment of plasma homocysteine as a risk factor for vascular disease. J Am Med Assoc 1996, 274:1049-1057. 33. Brattstrom L: Vitamins as homocysteine-lowering agents. J Nutr 1996, 126:1276S-1280S. 34. Ames BN: Micronutrient deficiencies cause DNA damage and cancer. Food Sci Agric Chem 1999, 1:1-15. 35. Alm L: Effect of fermentation on B-vitamin content of milk in Sweden. J Dairy Sci 1980, 65:353-359. 36. Rao DR, Reddy AV, Pulusani SR, Cornwell PE: Biosynthesis and utilization of folic acid and vitamin B12 by lactic cultures in skim milk. J Dairy Sci 1984, 67:1169-1174. 37. Lin MY, Young CM: Folate levels in cultures of lactic acid bacteria. Int Dairy J 2000, 10:409-414. 38. Hugenholtz J, Sybesma W, Nierop Groot M, Wisselink W, Ladero V, Burgess K, van Sinderen D, Piard J-C, Eggink G, Smid EJ et al.: Metabolic engineering of lactic acid bacteria for the production of nutraceuticals. Antonie van Leeuwenhoek 82: 217-235. 39. Yao R, Schneider E, Ryan TJ, Galivan J: Human gamma glutamyl hydrolase: cloning and characterization of the enzyme expressed in vitro. Proc Natl Acad Sci USA 1996, 93:10134-10138. 40. Roth JR, Lawrence JG, Bobik TA: Cobalamin (coenzyme B12): Synthesis and biological significance. Annu Rev Microbiol 1996, 50:137-181. 41. Battersby AR: How nature builds the pigments of life: The conquest of vitamin B12. Science 1994, 264:1551-1557. 42. Hollriegl V, Lamm L, Rowold J, Horig J, Renz P: Biosynthesis of vitamin B12. Different pathways in some aerobic and anaerobic microorganisms. Arch Microbiol 1982, 132:155-158. 43. Hunik J: Improved process for the production of vitamin B12. WO Patent, Patent No 00/37669. 44. Hettinga DH, Reinbold GW: The propionic acid bacteria - a review. J Milk Food Technol 1972, 35:295-301. 45. Stahmann KP, Revuelta JL, Seulberger H: Three biotechnical processes using Ashbya gossypii, Candida famata, or Bacillus subtilis compete with chemical riboflavin production. Appl Microbiol Biotechnol 2000, 53:509-516. The authors describe a couple of examples where production of riboflavin, through microbial fermentation, is at least as effective and economically feasible as the chemical synthesis. This information can serve as a stimulus for the development of fermentation processes for the production of several other vitamins. 46. Rougheed ZK, McCormick DB: Qualitative and quantitative assessment of flavins in cow's milk. J Nutr 1990, 120:382-388. 47. Laxminarayana H, Shankar PA: Fermented milk in human nutrition. Ind Dairyman 1980, 32:121-126.

48. Singh R, Deodhar AD: Relative bioavailability of riboflavin in cow's milk and fermented milk using rat bioassay. Int Dairy J 1994, 4:59-71. 49. Bolotin A, Wincker P, Mauger S, Jaillon O, Malarme K, Weissenbach J, Ehrlich SD, Sorokin A: The complete genome sequence of the lactic acid bacterium Lactococcus lactis ssp. lactis IL1403. Genome Res 2001, 11:731-753.

Nutraceutical production with food-grade microorganisms Hugenholtz and Smid

507

72. Lee KN, Kritchevsky D, Pariza MW: Conjugated linoleic acid and atherosclerosis in rabbits. Atherosclerosis 1994, 108:19-25. 73. Valcarce G, Munoz L, Nusblat A, Nudel C, Florin-Christensen J: The improvement of milk by cultivation with ciliates. J Dairy Sci 2001, 84:2136-2143. 74. Carmel-Harel O, Storz G: Roles of the glutathione- and thioredoxin-dependent reduction systems in Escherichia coli and Saccharomyces cerevisiae reponses to oxidative stress. Annu Rev Microbiol 2000, 54:439-461. 75. Newton GL, Arnold K, Price MS, Sherrill C, delCardayre SB, Aharonowitz Y, Cohen G, Davies J, Fahey RC, Davis C: Distribution

of thiols in microorganisms: mycothiol is a major thiol in most actinomycetes. J Bacteriol 1996, 178:1990-1995. 76. Smirnova GV, Krasnykh TA, Oktyabrsky ON: Role of glutathione in the response of Escherichia coli to osmotic stress. Biochemistry (Mosc) 2001, 66:973-978. 77. Fernandes L, Steele JL: Glutathione content of lactic acid bacteria. J Dairy Sci 1993, 76:1233-1242.

78. Wiederholt KM, Steele JL: Glutathione accumulation in Lactococci. J Dairy Sci 1994, 77:1183-1188. 79. Fahey RC, Brown WC, Adams WB, Worsham MB: Occurrence of glutathione in bacteria. J Bacteriol 1978, 133:1126-1129.