Functional Fermented Milk Products

Brunser O. Gotteland M, Cruchet S.

Institute of Nutrition and Food Technology (INTA), Univ. of Chile, Santiago, Chile. Avda. El Libano 5524, Macul, Santiago Chile. Phone: 56-2-9781468; E-mail: obrunser@inta.cl

BACK TO THE PROGRAM

The origin of fermented food is lost in the earliest stages of human history. Milk and milk-derived products have constituted a significant part of the diet of all ethnic groups at all ages, including a proportion of the food provided to infants. Because it was difficult to preserve milk unspoiled, all cultures which have had access to the milk and to a variety of other products such as cereals, fish, legumes, fruits etc., have resorted to fermentation with dual aims of improving their taste, texture and digestibility and preserving them for longer periods of time. As many populations consumed routinely these kinds of foods, it is highly probable that children may have received them as part of their diet, even during weaning. (1). In Europe and Western Hemisphere, in parts of Africa and Asia, in Australia and parts of the South Pacific, fermented products derived from cows milk became predominant, even though yogurt originated from Asia and from species other than cows. Products like of kefir, koumiss and kuranga, prepared from the milk of other species and which contained a wide range of microorganisms including bacteria and yeasts, have been widely used in other parts of the world and are also being investigated for their associated microbiota and the resulting compounds (2). The fermented products obtained from American camelids (llamas, alpacas, vicunas, guanacos) and from corn have not been studied in depth in this respect (3). While preparation of fermented foodstuffs was initially an empirical process, interest was progressively aroused about the microorganisms participating in this process, the chemical reactions and the compounds resulting during fermentation, and the health benefits derived from their consumption for variable periods of time, including for the health of the gastrointestinal tract. The improvement of the techniques of food preservation and the massive industrialization of food elaboration during the second part of the last century resulted in a lesser utilization of fermentative procedures and in the subsequent decrease of the intake of fermented foodstuffs. It is tempting to speculate that this phenomenon may have resulted in changes of the resident microbiota of individuals and that this may be related to the increased prevalence of allergies and chronic inflammatory and autoimmune diseases which was simultaneously observed (4, 5). Milk fermentation and metabolite production Fermented products derived from milk result from fermentation of lactose by different bacterial strains. According to the Codex Alimentarius of 1992 yogurt is defined as a coagulated milk product resulting from fermentation of lactose by

Lactobacillus bulgaricus and Streptococcus thermophilus. Other lactic acid bacteria (LAB), such as Lactobacillus, Streptococcus and Bifidobacterium, can be added to yogurt starters to produce fermented milks with specific textural, organoleptic or functional (in the case of probiotic strains) characteristics. The elaboration of fermented milks results from an intense fermentation process by the lactic acid bacteria, sometimes in association with yeast, acetic bacteria or moulds. A permease transports lactose into the bacterial cell where the disaccharide is hydrolysed by beta-galactosidase into glucose and galactose; the latter is exported out of the cell while glucose is phosphorylated and converted first to pyruvic acid by an aldolase and then to lactic acid by the lactate dehydrogenase (6). There is a synergistic relation between S. thermophilus and L. delbrueckii subsp. bulgaricus throughout the process of fermentation: the former use for its growth the amino acids and peptides produced by L. bulgaricus from milk proteins while the growth of L. bulgaricus is stimulated by compounds produced by S. thermophilus such as carbon dioxide and short chain fatty acids (7). Twenty to forty percent of the lactose present in milk is transformed into lactic acid during the process of yogurt elaboration such that in the final product total sugars represent 4.9-5.3 g/dL, with 3.8-4.0 g/dL of lactose, 1.0-1.2 g/dL of galactose and traces of glucose. The lactic acid concentration in yogurt ranges between 0.7 and 1.2 g/dL and the pH between 3.9 and 4.2. The presence of bacterial lactase improves the intestinal hydrolysis of the remnant lactose and makes it more digestible for hypolactasic individuals. In addition, the orocecal transit time of fermented milk products is slower compared with unfermented milk, allowing a more efficient action of both the bacterial beta galactosidase and the residual human intestinal lactase. All these processes may explain why yogurt intake improves lactose digestion and the digestive symptomatology in hypolactasic children (8, 9). During milk fermentation, some vitamins such as pantothenic acid and vitamin B12 decrease while folic acid and niacin increase (10). The proteins in cows milk represent a complex mixture of which about 80% are caseins and consist of four main fractions (S1-, S2-, - and -caseins) which exist in an approximate proportion of 38:11:38:13 (11). One to two percent of the casein is hydrolysed by proteases of LAB, releasing amino acid and peptides which are metabolized by the bacteria or accumulate in the product. Milk triglycerides are not modified during the fermentation process due to the absence of lipase in LAB. In addition, fermented milks also contain growth

factors, hormones and immune-stimulating molecules such as peptidoglycans, polysaccharides, teichoic acid (12). Some bioactive peptides deriving from milk protein hydrolysis may also modulate the immune system, as well as inhibit pathogen growth, or they exert anti-inflammatory activities (13, 14). Some peptides such as the caseinmacropeptide may also stimulate the growth of Bifidobacterium populations in the colon (15).

Fermentation and production of hypotensive peptides Fermentation of milk proteins by lactic-acid bacteria may result in the release of tripeptides with blood pressure lowering activities. Two active peptides isoleucylprolyl-proline (Ile-Pro-Pro) and Valyl-Prolyl-Proline (Val-Pro-Pro) have been isolated consistently from casein digests by L. helveticus and have been shown to lower blood pressure in spontaneously hypertensive rats and in humans with mild hypertension (16, 17). Although hypertension is considered a disease of mature and old age, the precursor conditions leading to hypertension are often present at a very young age; furthermore, hypertension secondary to a number of conditions (kidney, endocrine, neurological diseases, etc) are frequent in childhood and for this reason it is important to consider all preventive and therapeutic possibilities that may be useful at a young age, including those resulting from the effect of bacterial fermentation upon food constituents (18-20). In another study, middle-aged individuals with moderate hypertension (systolic readings between 140 and 180 mm Hg and diastolic readings between 90 and 110 mm Hg) received twice daily 150 ml of a milk fermented with L. helveticus LBK-16H for 10 weeks with 7,5 mg/100 g of Ile-Pro-Pro and 10 mg/100 g of Val-Pro-Pro. Contemporarily, the control group received the same product as the experimental group but without the two active peptides. During a four-week run in and during a follow up period of equal duration patients and controls received either a product fermented by a different probiotic or the control product. Blood pressure was monitored at the beginning and at the end of the experimental period with an automatic, 24-hour pressure recorder and, in addition every individual was subjected to nine blood pressure controls in a medical office at regular predetermined intervals. There was a difference of 4,1 0,9 mm Hg in the systolic pressure and a 1,8 0,7 mm Hg between the L. helveticus and the control groups, respectively (21). Reductions in blood pressure of this

magnitude are considered of epidemiologic significance from the point of view of public health (22). Although both Ile-Pro-Pro and Val-Pro-Pro have been reported to be powerful inhibitors of the angiotensin-converting enzyme (ACE), no changes in its activity was observed in any of the participants, suggesting that another mechanism, independent of ACE may be operating. In vivo and in vitro ACE-inhibitory activity originating from the fractionation of caseins has been detected in milk products using different bacterial strains in the fermentation process (13, 23). These peptides are mainly of low molecular weight and some of them only become apparent after the products of the bacterial activity upon the casein fractions are further subjected to proteolysis by pepsin and trypsin in the digestive tract (24). The functional importance of these peptides is that ACE is one of the main molecules that regulate blood pressure through its effects on the synthesis of angiotensin II, a potent vasoconstrictor; at the same time, the process of angiotensin II synthesis induces the degradation of bradykinin, a powerful vasodilator. The net result is that the inhibition of ACE causes lowering of blood pressure (25). In a study carried out in kefir prepared from goats milk, ACE-inhibitory peptides were detected mainly in 16 sequences of amino acids that showed anti-hypertensive activity, two of them being especially potent. Digestion with gastric and pancreatic enzymes further hydrolyzed the original peptides and some of the resulting products also exhibited vasomotor effects (26).

Antimicrobial activity in fermented foodstuffs A number of bioactive polypeptides have been identified as present in encrypted form in milk proteins; these are released during the fermentation process and/or during the digestion of these proteins by the gastric and pancreatic enzymes in the gastrointestinal tract (13). The active peptides are stored in the proteins as propeptides or as mature C- or N-terminal peptides that are released during proteolysis. Hill and coworkers isolated in 1977 a number of antimicrobial peptides from casein (27). These peptides are very potent and include families or single peptides called caseicidins, isracidins, casocidin-I, kappacin and lactoferricin (28-30). In general, these peptides exert lytic activity on bacteria by becoming inserted in the membrane and assembling to form channels which disrupt it, allowing the income of water and the outward diffusion of electrolytes and small molecules. These antibacterial peptides have specificity for
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prokaryotic membranes. It has been shown recently that fermentation of sodium caseinate by L. acidophilus DPC6026 produces three peptides that represent fragments of isracidin, two of them with potent activity mostly against gram positive but also against gram negative bacteria such as Streptococcus mutans, E. coli O157:H7 and Enterobacter zakazakii. E. zakazakii has been recognized as the etiological agent of a form of meningitis in neonates and milk-based infant formula may have served as its reservoir. The possibility of incorporating the peptides caseicins A or B derived from cows milk protein may represent a protective mechanism against this agent and should increase the interest in producing casein-based ingredients for protective purposes (31, 32).

Fermented foodstuffs in the prevention and treatment of acute diarrhea The use of fermented milk products for their contribution to health has been recorded since Biblical times; these were used in Ancient Egypt and by the Greeks and Romans for their medicinal properties. Cheese has also been used for the same purposes since remote times. The origin of yogurt is not known and was prepared from the milk of buffaloes, cows, donkeys, sheep and goats. Acidified milks were considered as particularly useful in the management of gastrointestinal diseases (33). Marriott and Davidson postulated that acidification of milk would make it easier to digest by the gastrointestinal tract of children and would prevent episodes of diarrhea (34). In vitro studies that showed that acidified milk inhibits the growth of enteropathogens seemed to confirm this observation (35). A number of early studies also supported the idea that the oral administration of some bacteria had a positive effect on the evolution of diarrhea associated with bacteria (36, 37) and it was suggested that this was the result of the positive modulation of immunity by these agents (38). Effects on bacterial diarrhea. The number of studies relating the administration of fermented milk products to the evolution of diarrhea associated with enteropathogenic bacteria is relatively limited. In a study carried out in the Karelian Republic it was shown that administration of L. casei rhamnosus GG (LGG) was not associated in infants and children with shortening of episodes of diarrhea associated with bacterial enteropathogens (39). Clements and coworkers conducted studies to evaluate in adult volunteers the capability of lactobacilli to prevent diarrhea associated with enterotoxigenic E. coli with negative results, even when the LAB were
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administered in high doses and at frequent intervals (40, 41). This has been confirmed by other studies which showed however that although the duration of the diarrhea is not shortened, stool volumes may be significantly decreased (42, 43). The use of fermented products not derived from milk for the management of diarrhea has been evaluated mostly in Africa and in Asia, but again the number of studies is also small. Yartey and coworkers compared the effectiveness of a fermented maize gruel with the same unfermented preparation and with the WHO/UNICEF oral rehydration solution in children with diarrhea. No differences in stool frequency and duration of diarrhea were observed between treatments but the fermented product was better accepted by the children than the unfermented control (44). This is in contrast with the findings of another group in Tanzania who observed that children who regularly consumed lactic-fermented cereal gruels had a 40 % lower frequency of diarrhea during a nine month follow up compared to a control group who were not fed these fermented products (45). Some fermented cereals have been shown to exert antibacterial activity in vitro and to be effective in preventing diarrhea (46, 47). A recent study in Northern Ghana evaluated in 190 children whose median age was 13 months the effect of a spontaneously fermented millet product on diarrhea; the episodes of diarrhea had lasted a median of 48 hours before the administration of the product had been started; 24.4 % of the children were considered to be dehydrated and 90.2 % had malaria parasites in their blood smears; the mean number of episodes of diarrhea in the preceding 12 months was 2.7. The enteropathogens associated with the episodes of diarrhea and the probiotic microorganisms in the product were not been characterized; furthermore, the possible variation in the species of bacteria growing in the fermented product or their counts were not assessed either (48). The product tested did not improve the cure rate or the duration of the episodes of diarrhea compared to the heatinactivated control; furthermore, as a high proportion of the patients were receiving concomitantly antibiotics and anti-malarial drugs it is not known to what extent this affected the results. In a study carried out in Santiago, Chile, 82 weaned infants less than 12 months of age received a milk formula acidified by the addition of L. helveticus and S. thermophilus for a period of six months. A group of 104 infants who were comparable from the anthropometric and socio-economic points of view and who received their medical care in a nearby area served as controls and received a non fermented milk of

comparable composition. Patients were contacted by nurses and pediatricians twice weekly for detection of diarrhea. The acidified product exerted a clear preventive effect on new episodes of diarrhea: the incidence of diarrhea, the number of days during which children were affected and the duration of the episodes were significantly lower in the infants receiving the acidified formula. No differences were observed in the species of enteropathogens associated with the episodes of diarrhea; asymptomatic fecal shedding of bacterial enteropathogens decreased throughout the observation period (49) (Table 1). Effects on viral diarrhea. Different species and strains of lactic acid bacteria have been evaluated for their capacity to modify the course of viral diarrhea and L. rhamnosus GG (LGG) has been the most extensively tested, especially for its effects on diarrhea associated with rotavirus infection in children. When administered in association with fermented milk a significant shortening of the evolution of the disease was observed, as the duration of the episodes was 1,4 0,8 days in those receiving the fermented product with the probiotic versus 2,4 0,8 days in the controls (50) (Table 2). A similar effect was observed for L. reuteri when given in progressively higher counts to infants and children with diarrhea due to rotavirus, pointing to a possible dosage effect for these bacteria (51). A recent study in children attending day care centers confirmed the positive effect of L. reuteri on acute diarrhea; the study also included a group of children who received a formula with Bifidobacterium lactis Bb12. Both probiotics were associated with a decrease in the number of episodes of diarrhea and these were shorter and with fewer days with fever; however, there was no effect on respiratory morbidities (52). The comparison of the results obtained with both probiotics favored L. reuteri; this may indicate that there is specificity in the (positive) effects of probiotic microorganisms with respect to the responses they elicit in the host. This demonstration of specificity in the quality of the immune responses towards enteropathogens is in agreement with earlier results obtained in Finland by Majamaa and coworkers, who compared three groups of children who received either LGG, or L. casei subsp rhamnosus, or a combination of S. thermophilus and L. delbruekii in a study of their effects on acute diarrhea: LGG significantly shortened the duration of diarrhea. In analyzing the immune responses elicited by these bacteria, the number of immunoglobulin-secreting cells stimulated was comparable for the three groups, while LGG administration was associated during convalescence with enhancement of IgA

specific antibody-secreting cells to rotavirus and serum IgA antibodies (53). This further supports the idea of the presence of specific capabilities in some of these bacteria to stimulate local and systemic defensive mechanisms. In addition to the specific mechanisms activated by lactic acid bacteria as part of the defensive responses of the body vis a vis pathogenic microorganisms, other processes probably involved in the defense against pathogens relate to the activation of innate immunity and include aspects such as the modulation of the resident microbiota of the gastrointestinal tract, the stimulation of mucus secretion, increased activity of macrophages and neutrophils, changes in intestinal permeability, etc. (14, 54-58).

Fermented foods and Helicobacter pylori colonization. H. pylori is a highly prevalent pathogen which colonizes the human gastric mucosa; it is considered as an aetiological factor for gastroduodenal ulcers and a risk factor for gastric cancers. In developing countries, the infection by H. pylori begins early in life and a high proportion of the paediatric population is colonized by this pathogen. Most of them remain asymptomatic and may not be treated with antibiotics. The antibiotic treatment has a high cost and is not 100% effective because of resistance to these drugs and to problems with patient compliance due to gastrointestinal intolerance. In addition children, when treated, are generally rapidly colonized again with the pathogen. The consumption of fermented products with lactic acid bacteria, including probiotics, have been proposed as alternative solutions to help in the management of H. pylori colonization in at-risk populations. A multicenter, prospective, randomized, double-blind, controlled study was carried out in 86 symptomatic H. pylori-positive children to compare the standard eradication treatment (omeprazole, amoxicillin, and clarithromycin) for 7 days with the same treatment supplemented with a fermented milk containing L. casei DN-114 001 for 14 days (59). Supplementation with the fermented product significantly increased the eradication rate from 57.5% to 84.6% (p=0.0045). In another study, 65 children received the seven-day standard triple therapy supplemented with 250 ml of a commercial yogurt containing B. animalis and L. casei, or milk during 3 months. No differences in the rate of eradication were observed between groups in this study (60). Cruchet et al used a commercial product containing L. johnsonii La1 or L. paracasei ST11 or their respective, heat-inactivated
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control provided daily during four weeks in 326 asymptomatic H. pylori-positive children. No eradication was observed but a significant decrease in the values of the
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C-Urea Breath Test (13C-UBT) was detected in the children receiving live La1

whereas no differences were observed in the other groups. Interestingly, the magnitude of the decrease in the UBT values induced by La1 intake correlated with the basal values of UBT before treatment (61). In another study with a small number of participants, a yogurt containing L. gasseri OLL 2716 (LG21) was administered daily for 8 weeks to 12 children colonized with H. pylori. A 13C-UBT was performed and the levels of serum pepsinogen I and II were measured after 4 and 10 weeks of yogurt intake. No significant differences in the 13C-UBT values before and at 4 and 10 weeks after ingestion were observed. The PG I/II ratio 4 weeks after the onset of ingestion was significantly higher than before ingestion; as the PG I/II ratio is inversely correlated with the level of inflammation in H. pylori-infected gastric mucosa, an increase in this ratio may indicate a decrease of the H. pylori-associated gastric mucosal inflammation in these children (62). These results, in addition to others obtained in adults, suggest that fermented milk products and probiotics may be used to maintain low gastric densities of H. pylori in asymptomatic, colonized subjects and that when administered together with antibiotics in symptomatic patients, these could be useful in increasing the eradication rates and in decreasing the severity of gastric inflammation and of adverse effects. Such protective effects may be due to the inhibition of H. pylori growth through the release of bacteriocins or of organic acids by some strains of Lactobacillus or Bifidobacterium, and to the decrease of the adhesion of the pathogen to gastric epithelial cells (63). In addition, probiotics and fermented milk products may have a role in the stabilization of the gastric barrier function, in the decrease of mucosal inflammation, and the stimulation of the healing of the gastric mucosa; this is probably related to the antioxidant and anti-inflammatory properties of these bacteria (64).

Fermented foods and intestinal motility There are few studies in children that have focused on the relationship between fermented foods and intestinal motility. Most of these have been carried out in adults and, particularly, in the elderly. Although it is logical to suppose that the presence of fermented foods and their associated microflora probably have beneficial effects on the motor activities of the gut (65, 66), this has not been clearly demonstrated.
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Gluten fermentation in the management of celiac disease Celiac patients should remain on a strict gluten-free diet for life; however, this is a complicated proposition and many of them abandon the diet. Another problem with maintaining a gluten-free diet is that wheat flour and gluten have useful properties that are applicable to the industrial processing of foodstuffs and are widely employed to thicken, increase the consistency, palatability and mouth of a great number of preparations. The possibility of using the fermentative capabilities of bacteria to hydrolyze gluten and gliadin to destroy their pathogenic capacity for celiac patients is being actively explored in many laboratories. The rationale of this is that many bacteria, including lactobacilli, have in their genetic program an extensive repertoire of proteases that allow them to hydrolyze the peptide sequences in gluten, hordein and avenin associated with the small intestinal lesions in celiac disease (67): the resulting products would be harmless for the patients. Di Cagno and coworkers demonstrated that it was possible to decrease considerably the concentration of gliadin using a mixture of L. alimentarius 15M, L. brevis 14G, L. sanfrancisensis 7A and L. hilgardii 51B with the purpose of fermenting wheat semolina used for the production of noodles, whose resulting concentrations of gluten peptides were low; these culinary preparations had satisfactory sensory qualities when tasted by an expert panel (68). The same group showed that the VSL#3 probiotic preparation has the capability of decreasing in celiac patients the toxic capability of wheat flour after prolonged fermentation, as evidenced by the absence of CD3+ lymphocytes in jejunal biopsies incubated in vitro with the resulting gliadin digests. VSL#3 is a mixture of S. thermophilus, L. plantarum, L. acidophilus, L. casei, L. delbrueckii spp. bulgaricus, B. breve, B. longum and B. infantis (69). The gluten-derived T-cell epitopes in gluten responsible for the pathogenesis of the intestinal lesion in celiac disease are resistant to digestion by endoluminal proteases because they are rich in proline. For this reason prolyl oligopeptidases appear as another logical approach to the neutralization of the deletereous capacity of gliadin-derived peptides. A prolyl endoprotease obtained from Aspergillus niger has recently been shown to be stable at a wide range of pH and to resist digestion by pepsin. It degraded all the synthetic T-cell stimulatory peptides tested derived from gliadin as well as the intact protein and with greater speed than prolyl oligopetidase. Tests in patients are
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required to verify whether the in vivo results are as promising as those observed in this in vitro study (70). The products of the fermentation of gliadin have been the object of a few preliminary assays in celiac patients on a gluten-free diet but the results have not been clear cut, due mostly to methodological problems (71).

Fermentation of carbohydrates: solving the problem of lactose maldigestion and intolerance Congenital deficiency of lactase activity is exceptional (72). Lactose malabsorption develops sometimes after the intestinal mucosa has been damaged by pathogens or, more frequently, due to the spontaneous disappearance of its activity in adolescents and adults (73). Lactose maldigestion by individuals lacking the specific hydrolytic enzyme, lactose-florizin hydrolase, a -galactosidase, or having low levels of enzyme activity in the brush border of enterocytes of the upper part of the small intestine may experience symptoms if the disaccharide load is high enough (lactose intolerance). The symptoms associated with lactose intolerance are bloating, flatulence, cramping pain and liquid stools sometimes expelled explosively (74). Affected individuals learn instinctively how much lactose and lactose-containing products they can tolerate without suffering unpleasant symptoms. The best laboratory test for documentation of lactose malabsorption is the measurement of hydrogen in breath after administration of a lactose load (75). The relationship between the amount of lactose ingested and the magnitude of the symptoms is not lineal as there is considerable variation in the responses of individuals to the same dose of this disaccharide (76). Because many of the bacteria associated with the production of fermented milk products have lactase activity, intake of these products improves the symptoms of this deficiency: yogurts and fermented milks improve the efficiency of lactose digestion. The reasons for this are that the bacteria used to produce them survive their passage through the acidic lumen of the stomach and their -galactosidase is released into the lumen of the upper part of the small intestine by the bile salts, although some degree of integrity of the bacterial bodies is required for proper lactase activity (77). The species most frequently used for production of yogurts and fermented milk products in the
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Western countries, L. acidophilus, L. delbrueckii ssp bulgaricus, S. thermophilus, etc., have high levels of lactase activity (7). By comparison some probiotics added to these preparations have rather low lactase activity and they do not add importantly to the lactase activity in the duodenum but may release it in the colon, where there is no undigested lactose (74). Furthermore, the -galactosidase of many of these non probiotic and probiotic species may have different pH optimums which differ from that required for optimal digestion in the environment of the lumen of the gastrointestinal tract. Fermented milk products represent a useful, economic adjuvant for management of lactose intolerance. One question repeatedly asked when feeding yogurt and fermented products to infants and small children is whether the D(-)lactate synthesized by the acidifying microorganisms may cause acidosis as the human body lacks the enzyme required for its metabolism. Studies carried out in Santiago in six-month old infants demonstrated that this is not the case (78), a finding that was corroborated recently by Connolly and coworkers (79).

Probiotics and fermented foods for control of inflammatory bowel disease In recent years it has been postulated that the two most frequent inflammatory diseases of the gastrointestinal tract, ulcerative colitis and Crohndisease, are somehow associated to alterations in the resident microbiota of the gastrointestinal tract and in the responses this elicits in the local and systemic mechanisms of immunity. As a logical result of this hypothesis, considerable interest was awakened on the effects of probiotics and fermented foods on the severity, remission and relapse-free interval in these diseases. In a preliminary study in four male patients with Crohns disease whose main age was 14,5 years (range 10-18 years), Guandalini observed that it became possible to taper corticosteroids in three of them because of clinical improvement in their symptoms (80). A randomized, controlled study published contemporarily contradicted the previous results because it did not reveal any improvements on clinical or endoscopic recurrences or in the severity of the lesions (81). Other studies have tended to confirm in general these latter results, including those of a multicenter evaluation in which a large number of children were carefully followed while they received LGG in addition to their standard maintenance therapy (low dose corticosteroids on alternate

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days, 6-mercaptopurin, aminosalicylates or azathioprine) (82-84). Whether other lactic acid bacteria or probiotics may exert beneficial effects remains largely unanswered.

Fermented milk in the management of allergy Atopic diseases such as eczema, allergic rhinitis and asthma are chronic allergic disorders whose prevalence has increased five times during the past 20 years in the developed countries (85). There is growing evidence suggesting an inverse association between infections early in life and atopy, according to the hygiene hypothesis (86). The higher frequency of (food) allergies is probably related to the industrial processing of food, to changes in food consumption pattern and to alterations of the intestinal microbiota. It is estimated that food allergies affect 3.5 % of adults and 8 to 10 % of children; the allergens most frequently involved are egg, peanuts, milk, fish, nuts, shellfish, wheat, kiwi and mustard. Various studies have shown a relation between allergic conditions and the composition of the gut microbiota. The levels of bifidobacteria in stools from allergic infants, particularly those with atopic eczema, are significantly lower than those in healthy subjects (87-89). Some other bacterial populations such as Clostridium, Bacteroides and Staphylococcus may also be altered and their concentrations may, in some case, correlate with the serum concentrations of IgE. The distribution of Bifidobacterium species in the colon of allergic infants is characterized by higher levels of B. adolescentis and B. longum and lower levels of B. bifidum, while the opposite situation has been observed in healthy infants (90). This may predispose to the future development of allergies as the former species are considered to be more characteristic of the adult-type microbiota and associated with higher levels of the pro-inflammatory cytokines TNF- and IL-12 by the macrophage-like cell line J774.1 in vitro (91). The gut microbiota participates in the establishment of immune oral tolerance by reorienting the Th2 responder phenotype of newborns towards the Th-1 cell-mediated immune response and through the stimulation of TGF- and IgA secretion. In addition, the microbiota is also involved in the regulation of the gut mucosal barrier, which blocks the transfer of antigens across the mucosa; these antigens originate from the environment, including foods, and are capable of initiating immune responses which are altered in children with atopic eczema (92).

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Based on these findings, it has been proposed that intake of fermented food products containing LAB could be used to modulate the homeostasis of the gut microbiota and thus decrease symptomatology in infants at risk for allergy (93). About 13 clinical trials have been carried out to evaluate whether probiotic intake may alleviate symptoms in infants with atopic eczema. They are randomized, double-blind and placebo-controlled, and carried out in Finland by the same group of investigators using mostly LGG and sometimes Bifidobacterium Bb12. LGG consumption decreases SCORAD as well as the fecal -1 antitrypsin and TNF-, plasmatic sCD4 and the eosinophil protein X in urine (94, 95). Kalliomaki et al were the first to report a decrease of atopic eczema in infants with family history of atopy when their mothers were given Lactobacillus GG four weeks prepartum and during lactation (23% vs 46%, in the probiotic and placebo groups, respectively; RR 0.51 [95% CI 0.32-0.84]) (96). Interestingly, this protective effect of LGG against atopic eczema was subsequently shown to persist until 4 years of age (97). Administration of probiotics to mothers was also associated with a significant increase in TGF-2 levels in milk (98). In another study in 230 children with atopic dermatitis, the daily administration of LGG for 4 weeks induced a greater decrease of SCORAD than in the placebo group but only in the subgroup of IgE-sensitized children (99). In contrast with these results, Brouwer et al. did not observe any improvement in SCORAD neither in its inflammatory parameters (eosinophil protein X in urine, blood eosinophils, fecal -1 antitrypsin) and cytokines production (IL-4, IL-5 and IFN-) by peripheral blood mononuclear cells, in 50 infants less than five months receiving a hydrolyzed whey-based formula alone or supplemented with LGG or with L. rhamnosus for three months (100). Some other studies have used probiotic strains (L. paracasei 33) in children with allergic rhinitis, observing an improvement of the quality of life of these children compared with the placebo group (101).

Effect of fermented foodstuffs on blood lipids One aspect of the functional capabilities of fermented foodstuffs and their bacteria that has been explored with considerable interest relates to their effects on lipid metabolism, especially the triglyceride and cholesterol blood levels. Mann et al. carried out the first studies in the Massai of Kenya and reported that the intake of large volumes (4-5 liters per day) of fermented milk was associated with a decrease of up to 18% in
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their blood cholesterol (102). However, this study had methodological problems derived from the fact that the subjects were drinking large volumes of fermented milk daily and that they gained weight (the energy intake was 5500 Kcal/day) despite an intensive program of physical exercise. This study awoke considerable interest and was followed by others in which it was shown that fermented milk products have cholesterol lowering effects, especially on total and LDL levels and that some of the products tested even induced modest increases of HDL cholesterol in healthy, and especially in moderately hypercholesterolemic adults; these effects are not exclusive of one species or strain of bacteria and have been also observed with soy-based products but not with kefir (103110). Each publication on these subjects proposes different mechanisms to explain the changes observed: incorporation of cholesterol to the membranes of bacteria, synthesis of conjugated linoleic acid, deconjugation of bile salts, etc. (111-113), a suggestion that a number of factors, rather than a single one, interact to produce the final result. These studies have all been conducted in adults of varying ages and little is known about the possible responses in children and their repercussion on long-term follow-up with products that have demonstrated positive effects at older ages. It is tempting to postulate that the bacteria used in the preparation of fermented products which decrease cholesterol levels may leave some imprint in the metabolism of younger subjects, probably on the enzymes participating in its endogenous synthesis. In summary, fermented foods have a long history as components of the diet of all human groups; the recent application of modern research methodologies is demonstrating that the bacteria and the products of the fermentation processes in which they participate intervene in a variety of activities that are positive for health and wellbeing in all age groups.

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Table 1.- Incidence of episodes of acute diarrhea in relation to age in children who received an acidified formula or a control non acidified formula
Acidified formula Age (months) 3-5 6-8 9-11 12-15 TOTAL Children/ month 7 81 150 165 403 Episodes of diarrhea 0 8 19 12 39 Incidence 0 9,9 12,6 7,3 9,7 2=7,1235 p<0,005 2=7,6726 p<0,005 2=15,085 p<0,005 159 329 586 41 71 137 25,8 21,6 23,3 Children/ month 18 Control formula Episodes of diarrhea 3 Incidence 16,7

Z= 3,517 p<0,001 From Brunser O. et al. (1989) Acta Paediatr Scand 78: 259-64.

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Table 2: Serum cholesterol changes in individuals randomly allocated to receive a fermented milk containing Lactobacillus acidophilus L1 (L1 FM) of human origin or Lactobacillus acidophilus ATCC 43211 (ATCC); placebo = fermented milk without active bacteria. Values are expressed as mmol/L; means SEM.
Group No Baseline average Week 2 Week 2 minus baseline % change

1st study L1 FM ATCC FM 2nd study L1 FM Placebo Combined L1 FM P vs baseline P vs placebo ANOVA Modified from Anderson JW. (1999) J Am Coll Nutr 18: 43-50. 35 6,42 (0,13) 6,18 (0,13) 0,0015 0,008 0,03 - 0,24 (0,07) - 3,6 (1,2) 21 19 6,53 (0,17) 6,30 (0,14) 6,27 (0,18) 6,40 (0,13) - 0,26 (0,10) - 0,10 (0,10) 3,8 (1,7) 1,9 (1,6) 14 15 6,27 (0,19) 6,38 (0,23) 6,06 (0,17) 6,30 (0,23) - 0,21 (0,08) - 0,08 (0,09) 3,2 (1,2) 1,2 (1,4)

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Table 3.- Effect of Lactobacillus casei rhamnosus GG (LGG) on the recovery from acute diarrhea in infants and preschool children (mean SD) Group (1) Fermented milk with LGG (2) Freeze-dried LGG (3) Pasteurized yogurt Duration of diarrhea, days 1,4 0,8 1,4 0,8 2,4 1,1 1 vs 3 and 2 vs 3: F=8,70; p < 0,001

The study was carried out in 71 well-nourished children 4 to 45 months of age; 82% of cases were associated with rotavirus. The amounts of LGG provided were 1010-11 CFU in 125 g twice daily of fermented milk; freeze dried LGG: 1010-11 CFU once daily; pasteurized preparation 125 g twice daily. Modified from Isolauri E et al. (1991). Pediatrics 88: 90-97.

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