Lactic Acid Bacteria and Bifidobacteria with Potential to Design Natural Biofunctional Health-Promoting Dairy Foods

1Today foods are not intended to only satisfy hunger and to provide necessary nutrients for humans, but also to prevent nutrition-related diseases and improve consumers’ health. Increasing consumer demand and interest in obtaining additional benefits from food has stimulated functional foods to emerge on the market, with USA, Europe, and Japan being the dominant markets.

2Although there is no unitary accepted definition, functional foods can be described as an ordinary food that has components or ingredients added to provide a specific health benefit, other than a purely nutritional effect. Ideally, functional food refers to an existing traditional food product that is intended to be consumed as part of a normal diet and has a demonstrated added physiological benefit. Therefore, it could not be in the form of pill or capsule. The concept of biofunctional foods is generally used when this desirable biological, medical, or physiological effect is exerted by microorganisms. The health benefits of these microorganisms can be exerted either directly through the interactions of ingested live microorganisms with the host (probiotic effect), or indirectly by ingestion of the microbial metabolites synthesized during fermentation (bioactive effect).

3Lactic acid bacteria (LAB) have been used to ferment foods for at least 4000 years. Although the probiotic concept has expanded more recently, we have been unconsciously ingesting beneficial microbes with traditional fermented foods since ancient times. Fermented foods are the main carriers to deliver probiotics (Figure 1). Among them, dairy products (in particular fermented milks and yogurt) are by far the most efficient and widely used. Cheese is a dairy product which has a good potential for the incorporation of probiotic cultures due to its specific chemical and physical characteristics compared to fermented milks (higher pH value and lower titrable acidity, higher buffering capacity, greater fat content, higher nutrient availability, lower oxygen content, and denser texture). These conditions facilitate survivability of probiotic strains and tolerance to the low pH conditions encountered during gastric transit. Utilization of probiotics has been optimized in several cheese varieties such as Cheddar, Gouda, Camembert, Cottage type, white-brined, and traditional cheeses, among others. Kefir is another milk-fermented product that has health promoting bacteria. Other non-fermented dairy foods such as low-fat ice cream, chocolate mousse, coconut flan, or infant milk formula have also been supplemented with probiotic strains.

4Probiotic microorganisms are generally LAB belonging to the species Lactobacillus acidophilus, L. gasseri, L. helveticus, L. johnsonii, L. (para)casei, L. reuteri, L. plantarum, L. rhamnosus, and L. fermentum, while members of the genus Bifidobacterium are also used, e.g., Bifidobacterium bifidum, B. longum, B. animalis, and B. breve. On the basis of the currently available literature, probiotics can balance intestinal microbiota, and thereby regulate proper intestinal function and be effective in the prevention or treatment of several gastrointestinal disorders such as infectious diarrhea, antibiotic-related diarrhea, irritable bowel syndrome or Crohn’s disease Other examples of health benefits promoted by probiotics supplied via dairy products are inmmunomodulatory effects (L. casei CRL431), reduction of serum cholesterol level (L. reuteri NCIMB 30242) and antihypertensive effects (L. plantarum TENSIATM).

5Probiotics are defined as ‘live micro-organisms, which when consumed in adequate amounts confer a health benefit on the host’. However, regarding probiotic foods, some considerations are of paramount importance. Firstly, effective levels of the live probiotic in the corresponding food matrix at the time of ingestion are required. In this regard, the minimum effective dose which affects the intestinal environment and provides beneficial effects on human health is considered to be 106–109 live microbial cells per day, although this depends on the particular strain and foodstuff. Since probiotics show beneficial health effects on the host once consumed, another precondition for a bacterial strain to be called probiotic is the ability to survive and colonize (at least transiently) the gastrointestinal tract (GIT), which is in part helped by the buffering capacity of the food matrix. In some particular cases, bacterial viability may not be strictly required. As an example, inactivated and dead L. rhamnosus GG cells can maintain immunological and health-promoting effects.

6Microorganisms involved in dairy fermentations can produce biologically active molecules and enzymes, giving the final food product an additional health value. Unlike the probiotic concept (the bacteria must be ingested alive and produce the beneficial metabolite in the body), the biofunctional concept is generally used when the healthy metabolite emerge in the food product itself during the fermentation process as a consequence of the bacterial metabolic activity. Consequently, the bacteria can act as a microbial factory to enrich foodstuff, for which bacterial viability through the GIT or during the product storage is not absolutely required. The main bioactive compounds produced by LAB during dairy fermentation are vitamins, gamma-aminobutyric acid, bioactive peptides, bacteriocins, enzymes, conjugated linoleic acid, and exopolysaccharides (Table 1).

7There are 13 vitamins that must be obtained exogenously due to the inability of humans to synthesize them; thereby they are essential nutrients in the human diet, and although in small amounts, a daily requirement is necessary to prevent deficiencies. Although most vitamins are present in a variety of foods, human vitamin deficiencies still occur in many countries, mainly because of malnutrition, not only as a result of insufficient food intake but also because of unbalanced diets.

8Although milk contains many vitamins fermentation by LAB and bifidobacteria constitute an effective way to increase vitamin levels in milk. Some bacterial strains included in the genera Lactobacillus and Bifidobacterium can provide an additional source of B vitamins (thiamine, riboflavin, cobalamin, folate, and biotin) during dairy fermentation. Deficiencies in riboflavin (vitamin B2) or thiamine (vitamin B1) can lead to both liver and skin disorders and alterations in brain glucose metabolism, respectively. In this regard, L. casei KNE-1 has been shown to produce thiamine and riboflavin in fermented milk drinks. B. infantis CCRC14633 and B. longum B6 strains have been reported to produce riboflavin and thiamine during soymilk fermentation. It was recently indicated that soymilk fermented by the riboflavin-producing strain L. plantarum CRL2130 was able to prevent ariboflavinosis and experimental colitis in a murine model. Some propionibacteria can produce cobalamin, folic acid, and biotin.

9Folate (vitamin B9) is involved in several vital processes and its deficiency is generally linked to neural tube defects, certain forms of cancer, poor cognitive performance and coronary heart diseases. Even though vitamins are widely present in foods, the prevalence of folate deficiency -especially among women of child bearing age- is a growing concern and thereby folate fortification programs have been implemented in various countries. Rather than incorporating synthetic folate, foods can be naturally fortified with folate synthesized by LAB and bifidobacteria during manufacture of fermented foods. The strains Streptococcus thermophilus CRL803/CRL415 and L. bulgaricus CRL871 were reported to be suitable for the elaboration of yogurt naturally bio-enriched in this vitamin. High folate concentration (up to 150 μg/l) can be reached in yogurt as a result of the ability of S. thermophilus to produce this vitamin. Among bifidobacteria, B. catenulatum ATCC 27539 was shown to produce high levels of folate in vitro, and B. lactis CSCC5127, B. infantis CSCC5187, and B. breve CSCC5181 strains increased folate concentration during fermentation of reconstituted skim milk. Similarly, L. amylovorus CRL887 can be used for natural folate bio-enrichment of fermented milk.

10The deficiency of cobalamin (vitamin B12) can be common, particularly in vegetarians who avoid ingestion of animal protein and use soymilk as an alternative to dairy milk. Animals, plants and fungi are incapable of producing this vitamin, and hence, it is exclusively produced by microorganisms. It has been demonstrated that cobalamin can be synthesized by some bacteria such as L. reuteri ZJ03, Propionibacterium freudenreichii, B. animalis Bb12 in soy-yogurt, kefir and fermented milk, respectively. Microorganisms can biosynthesize two different isoforms, the vitamin and the pseudovitamin. For example, in a recent work, the production of vitamin and pseudovitamin B12 by P. freudenreichii was quantified specifically and shows that at the initial phase of the fermentation both isoforms are biosynthesized at similar levels; however, by the end of the fermentation the pseudovitamin is not detected, most likely because it is converted to the vitamin form. It seems crucial to differentiate between the two isoforms of this vitamin, as the transporter protein in the human GIT has very low affinity for the pseudovitamin, making it un-available to humans.

11Biotin (vitamin B7) deficiency can be caused by inadequate dietary intake or some inborn genetic disorders that affect its metabolism. Subclinical deficiency can cause mild symptoms, such as hair thinning or skin rash typically on the face. Biotin can be synthesized by some LAB in dairy products, e.g., L. helveticus MTCC 5463 increased biotin content in fermented milks. Some propionibacteria can also produce biotin.

12Vitamin K is an important promoter of bone and cardiovascular health. It has been associated with the inhibition of arterial calcification and stiffening, and the reduction of vascular risk. This vitamin is nearly non-existent in junk food, with little being consumed even in a healthy Western diet. Its deficiency has been implicated in several clinical ailments such as intracranial hemorrhage in newborn infants and possible bone fracture resulting from osteoporosis. Vitamin K occurs in two forms: firstly, phylloquinone (vitamin K1), which is present in green plants, and secondly, menaquinone (vitamin K2), which is produced by some intestinal bacteria. Menaquinone can be biosynthesized by some LAB species (mainly Lactococcus lactis) commonly used in industrial fermentation of cheese, buttermilk, sour cream, cottage cheese, and kefir. Other LAB have been screened for the ability to produce menaquinone; these included strains from the genera Lactococcus, Bifidobacterium, Leuconostoc, and Streptococcus. Although the MK forms are ubiquitous in bacteria, it should be noted that some genera such as Lactobacillus have lost the functional ability to produce them.

13Gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter of the central nervous system (CNS). Several important physiological functions of GABA have been characterized, such as neurotransmission, induction of hypotension, diuretic effects, antidiabetic, relaxing and tranquilizer effects. In fact, some GABAA-receptor agonist drugs (e.g., benzodiazepines) are important pharmacological agents used for clinical treatment of anxiety.

14Gamma-aminobutyric acid is biosynthesized through α-decarboxylation of glutamate, an enzymatic conversion which is catalyzed by glutamate decarboxylase (GAD). Several food-grade LAB have been reported to exhibit GABA-producing ability. Among them, most of the GABA-producing strains are lactobacilli (L. brevis, L. paracasei, L. delbrueckii, L. buchneri, L. plantarum, L. helveticus), Streptococcus thermophilus, and Lactococcus lactis. Some, Bifidobacterium spp. were also reported to produce GABA, although with lower capacity than LAB.

15Some fermented dairy products enriched in GABA using GABA-producing LAB as starters have been developed. The strains L. casei Shirota, S. salivarius fmb5 and L. plantarum NDC75017 were utilized to ferment and enrich milk in GABA. More recently, yogurt enriched with 2 mg GABA/ml was produced using the strain S. thermophilus APC151. Also, fermented soya milk (using L. brevis OPY-1 as source of GABA), or cheese (Lactococcus lactis as source of GABA) have been produced. Thus, GABA has potential as a health-promoting bioactive component in foods.

16During milk fermentation, LAB, making use of their proteolytic system can transform milk proteins into biologically active peptides. These peptides can exert a wide range of effects, such as antimicrobial, antihypertensive, antithrombotic, immunomodulatory, and antioxidative. The most studied mechanism of bioactive peptides is the antihypertensive action displayed by the inhibition of the angiotensin-I-converting enzyme (ACE; peptidyldipeptide hydrolase, EC 3.4.15.1) which regulates blood pressure. ACE inhibitory peptides have been isolated from a variety of fermented dairy products including cheese, fermented milks and yogurt. The best known ACE-inhibitory biopeptides, Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP), have been identified in milk fermented by L. helveticus. In addition, other dairy starter cultures industrially used in the manufacture of fermented dairy products (e.g., L. helveticus, L. delbrueckii ssp. bulgaricus, L. plantarum, L. rhamnosus, L. acidophilus, Lactococcus Lactis, or S. thermophilus) can generate bioactive peptides. Other ACE-inhibitory peptides such as β-casein f(72-81), Ser-Lys-Val-Tyr-Pro-Phe-Pro-Gly-Pro-Ile (SLVYPFPGPI) have been produced by L. delbrueckii ssp. bulgaricus LB340 in fermented milk.

17On an industrial scale, two fermented milk products with antihypertensive claims, CalpisTM and Evolus®, have been tested extensively in rats and in clinical trials, and are commercialized as functional foods. Evolus® is available in the market as a L. helveticus fermented milk -produced in Finland- proven to decrease the systolic blood pressure in hypertensive subjects due to the actions of L. helveticus bioactive peptides. CalpisTM is defined as a milk product marketed in Japan (Calpis Co. Ltd.) with antihypertensive properties. It is prepared by fermenting skimmed milk with L. helveticus and Saccharomyces cerevisiae, which produce VPP and IPP peptides from β-casein and κ-casein.

18Bacteriocins are ribosomally synthesized antimicrobial peptides produced by a particular bacterium that are active against other competitor bacteria; thereby they constitute an important part of the microbial defense system. Such bacteriocin-producing strains may offer potential as an alternative to antibiotics, and may be useful as a means of controlling pathogen carriage, therefore being highly suitable as microbial food additives (Table 2). Bacteriocins from LAB have attracted much interest because they are frequently produced by commercially useful strains that are generally regarded as safe (GRAS) for human consumption. These antimicrobial molecules are among the beneficial peptides intrinsically synthesized by some LAB during milk fermentation and they have been traditionally used as naturally produced food biopreservatives. In addition, they may function in the GIT as potential natural biotherapeutic agents facilitating the competition of probiotic strains and/or inhibition of pathogens; thereby they are potential contributors to the microbiota balance and human health.

19Nisin is the most well-known bacteriocin used as food preservative due to its antibacterial effect against Listeria, clostridia spores and LAB associated to spoilage. Nisin has been approved as a food additive (E234) in the European Union according to Directive 95/2/EC (EC, 1995) in the following products: semolina and tapioca puddings (3 mg/kg); ripened and processed cheese (12.5 mg/kg), clotted cream (10 mg/kg), and Mascarpone cheese (10 mg/kg). It is also permitted in over 40 countries world-wide including USA, Australia, South Africa, Russia, and India for use as an antimicrobial agent in a variety of food products. Nisin-containing Camembert and semihard cheeses with prolonged shelf-life were made using Lactococcus lactis (strains CNRZ150 or TAB50, respectively) as nisin producers. Apart from nisin, plantaricins are very well-known bacteriocins. For example, plantaricin C is a broad spectrum bacteriocin produced by L. plantarum, isolated from ripening cheese. Plantaricins have been reported to produce an immunomodulatory effect on dendritic cells. However, bacteriocins other than nisin have so far only few and limited authorized uses in foods. Consequently, the use of bacteriocin-producing bacteria as starter culture for in situ biosynthesis during milk fermentation becomes an effective alternative strategy to incorporate bacteriocins in dairy foods. Similarly, the lacticin 3147-producing strain Lactococcus lactis DPC3147 used as a protective culture in Cheddar cheese reduced numbers of Listeria monocytogenes to <10 cfu/g within 5 days at 4°C. Other bacterial species such as L. acidophilus can be added as an adjunct in many food fermentation processes to contribute to bacteriocin production and food preservation. Other LAB strains such as L. plantarum WHE92 used as adjunct to the starter culture reduced Listeria monocytogenes, Listeria innocua, and Escherichia coli O157:H7 counts in cheese as a consequence of the production of plantaricin. Using a similar concept, Danisco developed a freeze-dried culture of Pediococcus acidilactici (marketed as CHOOZIT Flav 43) for use as a bacteriocin-producer adjunct in Cheddar and semihard cheeses.

20Studies of the direct impact of dairy foods containing bacteriocins on human health and microbiome are still limited. In vivo antimicrobial activity of nisin and lacticin 3147 has been recently demonstrated in a murine infection model. A nisin-producing Lactococcus lactis CHCC5826 modified the microbiota composition of human microbiota-associated rats increasing bifidobacteria levels and decreasing Enterococcus/Streptococcus populations. Lacticin 3147 has the potential to be employed in the treatment of Clostridium difficile diarrhea and to eliminate the pathogen when added to an anaerobic fecal fermentation.

21Lactic acid bacteria associated to dairy fermentations possess enzymes which can be produced in situ during fermentation of dairy foods and have bioactive potential on the consumer. Examples are hydrolytic enzymes that may exert potential synergistic effects on digestion and alleviate symptoms of intestinal malabsorption. A well-known example is the β-galactosidase activity, which can achieve lactose degradation and thereby improve health and reduce symptoms of lactose intolerant consumers. Yogurt and other conventional starter cultures and probiotic bacteria in fermented and unfermented milk products improve lactose digestion and alleviate symptoms of intolerance in lactose malabsorbers. These beneficial effects are due to microbial β-galactosidase.

22Conjugated linoleic acid (CLA) is a polyunsaturated fatty acid (PUFA) that can be biosynthesized by LAB and bifidobacteria through bioconversion of linoleic acid (LA; cis-9,cis-12 C18:2). The two isomers that have been shown to have bioactive potential are cis-9,trans-11 (c9,t11) and trans-10,cis-12 (t10,c12). The health-promoting properties of CLA include anticarcinogenic, antiatherogenic, anti-inflammatory, and antidiabetic activity, as well as the ability to reduce body fat. Although it is a native component of milk, the amount consumed in foods is far from that required in order to obtain desired beneficial effects. Thus, increasing the CLA content in dairy foods through milk fermentation with specific LAB offers a promising alternative. An effective way to increase CLA uptake in humans is to increase CLA levels in dairy products by using strains with high production potential. A number of food-grade LAB and bifidobacteria were reported to produce CLA in milk products, as is the case of Lactococcus lactis LMG, L. rhamnosus C14, L. casei CRL431, L. acidophilus Lac1, L. plantarum-2, B. bifidum CRL1399 and B. animalis Bb12. Some of these strains were also used as adjunct cultures for the manufacture of high CLA-content buffalo cheese. The CLA-producing starter culture of Lactococcus lactis CI4b enhanced levels of total CLA in Cheddar cheese. Similarly, L. bulgaricus LB430 and S. thermophilus TA040 are suitable for production of CLA-enriched yogurt.

23In addition, it has been shown that specific microorganisms such as L. plantarum PL60 or B. breve NCIMB702258, are able to produce CLA following dietary administration in animal models and following the administration as a freeze-dried product in humans. Thus, intestinal CLA production by bacteria may contribute to enhance CLA supply in addition to the CLA provided by the producing strain in fermented milks during the manufacture.

24Exopolysaccharides (EPS) are complex extracellular carbohydrate polymers that can be produced by some LAB in situ during dairy fermentations. Some of them promote selective growth of bifidobacteria, thus playing a role on the host microbiota and immune system. In this regard, EPS derived from yogurt fermented with L. delbrueckii ssp. bulgaricus OLL1073R-1 exerted immune-stimulatory effects in mice. Yogurt, Swiss-type, and Cheddar cheeses represent suitable food matrices for the delivery of the hypocholesterolemic EPS-producer strain L. mucosae DPC 6426. Other microorganisms with potential to produce EPS in cheese are P. freudenreichii KG15/KG6, Lactococcus lactis SMQ-461 or S. thermophilus MR-1C. Significant levels of EPS can also be produced in kefir by L. plantarum YW11. Recently, EPS produced by bifidobacteria have attracted the attention due to their immune modulation capability.

25Exopolysaccharides can also improve the quality, sensory and rheological properties of the final food product, which in many cases results in a reduction of the amount of chemical stabilizers required, thus favoring a more natural product. For example, strains of B. longum subsp. infantis CCUG 52486 and S. thermophilus were suitable to produce yogurt and fermented ice-cream with improved viscosity and texture and reduced syneresis as a consequence of their high EPS production.

26At present, the status of probiotic-based products is full of ambiguities because various regulatory agencies in different countries are defining and categorizing probiotics differently. Despite the emerging interest of consumers toward probiotics and functional foods, in Europe the regulatory framework is still not harmonized and no health claim for probiotics alone (except yogurt starters) has been approved. Paradoxically, probiotics or bioactive bacteria have been introduced into the market as dietary supplements or natural health products (capsules, tablets, and powders). Japan was the very first global jurisdiction for implementing a regulatory system for functional foods and nutraceuticals in 1991, and is currently acting as global market leader where probiotics are available as both foods and drugs. The government has designated Foods for Specific Health Uses (FOSHU), which classifies health claims into different subcategories (gastrointestinal health, cholesterol moderation, hypertension moderation, lipid metabolism moderation, sugar absorption moderation, mineral absorption, and bone and tooth health). In China, State Food and Drug Administration (SFDA) has regulated all health foods including functional foods and nutraceuticals, and a well-developed market for functional foods is established. Currently USA is regulating probiotics as a variety of products as per their intended usage and regulatory bodies are Dietary Supplement Health and Education Act (DSHEA) and Food and Drug administration (FDA). Dietary supplements are considered as ‘foods’ and are regulated by DSHEA and do not need FDA approval before being marketed. However, probiotics and dietary supplements containing a new dietary ingredient without a marketing history are regulated by FDA. In conclusion, a harmonized categorization of probiotics and functional foods may help to regulate these products whenever solid clinical documentation is available to support any health effects and health messages in human subjects. The appropriate level of evidence for determining a health benefit for probiotics should always be put ahead of commercial and labeling industrial interests.

27A goal of the dairy industry is to develop novel dairy products with increased nutritional and/or health promoting properties. Food-grade bacteria have the potential to fortify fermented dairy food products with bioactive metabolites by a natural process, thereby reducing the need for fortification with costly chemically synthesized supplements. Nowadays, a number of commercial sources have available synthetic formulations of bioactive substances for their use as a dietary supplement. The use of health-supporting bacteria for naturally enriching dairy foods with bioactives could be a suitable alternative to food fortification with chemical formulations.

28The starter cultures must be carefully selected, since the ability of microbial cultures to produce bioactive metabolites is generally a strain-dependent trait and varies considerably among strains within the same species. The yield of bioactive synthesis and the concentration of such compound in dairy products is another critical strain-dependent factor. In this regard, the dose of bioactives ingested with the corresponding food product should remain over the minimum required to meet the human requirements and/or have the claimed therapeutic level on the consumer, according to existing clinical recommendations and studies. An open question when using co-cultures or strain combinations is their interaction in terms of nutrient availability, bacterial growth, as well as the bioactive production yield. In some cases, metabolites (i.e., vitamins etc.) produced by one of the strains could be consumed by the other strains, thus decreasing the final content in food.

29Generally, the biosynthetic pathways are genetically encoded. In this regard, the increasing availability of bacterial genome sequences over the last decade has provided a major contribution to the knowledge about microbial production of bioactive molecules. However, the presence of the genes required for the biosynthesis of a particular biomolecule should not be assumed as synonym of metabolite production. Typical exceptions to the correlation genotype-phenotype occur when the genes are not active or when the metabolite is intracellularly biosynthesized and a release system is lacking. This is indeed one of the major bottlenecks during biosynthesis of some vitamins that needs to be overcome through the use of alternative strategies such as autolytic mutants and metabolic engineering.

30Consideration should also be given by manufacturers to the optimum conditions for bioactive compound biosynthesis by LAB during technological processes. The content and activity of a bioactive compound in the dairy fermented foodstuffs is the result of the type of food matrix, the individual bacterial strain properties as well as the processing conditions and storage time. In this regard, it should be noted that the high bioactive biosynthetic rates observed in culture media might not always be extrapolated to dairy products. Therefore, factors such as optimal temperature for microbial growth and viability, food composition or bioactive stability and shelf-life in the final foodstuff are of paramount importance to reach the maximum concentration and activity in the final product.

31Overall, the current review updates knowledge about LAB, bifidobacteria and propionibacteria with potential to enrich dairy food products with health-promoting bio-metabolites. Promising applications at commercial level emerge; however, adequate selection of strains is vital to increase the concentration and bioavailability of such biomolecules in fermented foods. The use of LAB and bifidobacteria able to synthesize bioactive components in fermented foods could help to provide these compounds in foods, this being in compliance with current regulatory rules.

32DL provided the general concept, and drafted part of the manuscript. CG and ER wrote part of the manuscript. All authors revised and approved the manuscript.

33The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.