Fermented Milk Products in Infant Nutrition

Fermentation: the anaerobic destiny of pyruvate
The fundamental characteristic of fermented milks results from an intense fermentation process by several lactic acid bacteria, sometimes in association with yeast, acetic bacteria or moulds. The transformation of lactose into lactic acid is the most significant phenomenon during the fermentative process and is responsible for almost all of the fermentation products (3). In the fermentation process, lactose is transported via permease into the cell, where beta-galactosidase hydrolyses the disaccharide into glucose and galactose. Galactose is not fermented and is discharged out of the cell. Glucose, on the other hand, is rapidly phosphorylated and, by the intervention of aldolase, is converted to pyruvic acid in accordance with the glycolytic pathway. Pyruvic acid is then converted to lactic acid by the lactate dehydrogenase enzyme (4).

Yogurt production is the result of the arranged development of Streptococcus thermophilus and Lactobacillus delbrueckii subsp.bulgaricus, microorganisms that remain vital throughout the maintenance period (5). Lactic acid production of the two genera combined is clearly superior to that of a single component. The growth of Streptococcus thermophilus is, in fact, stimulated by amino acids and peptides produced by Lactobacillus bulgaricus from milk proteins (5). On the other hand, the growth of Lactobacillus bulgaricus is stimulated by compounds produced by Streptococcus thermophilus, such as carbon dioxide and formic acid (6).

The interaction between these two microorganisms, with manifestation of microbial synergisms, is favourable to acidification, production of polysaccharides, and formation of the typical yogurt aroma, which is primarily due to the presence of lactic acid and, above all, to acetaldehyde. Small quantities of acetaldehyde (2-4 mg/100g) are enough to confer the typical aroma to yogurt. Also, other carbonylic compounds of fermentation such as acetone and acetoine contribute to create aroma and taste. In the process of yogurt production, around 20-40 percent of lactose present in milk is transformed into lactic acid. With the process of acidification realized in yogurt, significant modifications of the sugars take place; and at the end of the fermentation process, the distribution of sugars is as follows: total sugars 4.8-5.2 g/dL, lactose 3.8-4.0 g/dL, galactose 1.0-1.2 g/dL and traces of glucose. The amount of lactic acid in yogurt ranges between 0.7 and 1.2 g/dL, and the pH value between 3.9 and 4.2. Thus, at the end of the fermentation process, unmodified lactose ranges between 2.5 and 5.5 g/dL, but it does not modify the role of yogurt in the diet of subjects with lactose intolerance. Indeed, the presence of bacterial lactase improves the intestinal hydrolysis of the disaccharide and makes it more digestible. Moreover, fermented milk products show a slower transit through the intestine compared to milk. This allows for a longer action of beta galactosidase of both bacterial and human origin. On the whole, these phenomena could explain the well-known improvement in lactose absorption and diarrhoeal symptoms observed in lactose-malabsorbing children (7, 8).

The duration of the fermentative process depends on the characteristics of the bacteria, the graft type (liquid or freeze-dried crop) and the level of desired acidity in yogurt. This process takes between 3 and 9 hours. Longer fermentation (12-18 hours) combined with lower temperatures (31-35°C) are employed in the production of low acidity yogurt, a product destined for younger children.

During the milk transformation in yogurt, pantothenic acid and vitamin B-12 decrease, while other vitamins increase (folic acid and niacine) (9). Lactic microflora carry out a weak proteolytic activity, which causes the lysis of 1-2 percent of the casein and liberation of amino acid and peptide. Some of these compounds are metabolized by the microorganisms, while others are accumulated in yogurt. In particular, in comparison to milk, smaller amounts of methionine, lysine, threonine, valine and thyroxine and larger amounts of free amino acids are present. In contrast, triglyceride lipolysis is negligible because of the absence of lipase in microorganisms contaminating the milk (10).

Effects of metabolites in fermented milk products
Not only do fermented milks contain such nutrients as carbohydrates, fats, proteins, minerals and vitamins that allow for growth, development and tissue differentiation, but they also contain growth factors, hormones (gastrin, insulin, IGF-I and IGF-II) and molecules with immune-stimulating effects that may have a variety of outcomes:

• The stimulation of the lymphoid tissue associated with the mucosa while the bacteria are transiently colonizing the intestinal lumen (11),
• Cell wall components such as peptidoglycans, polysaccharide and teichoic acid, all of which have been shown to have immunostimulatory properties (12),
• Peptides and free amino acids generated from hydrolysis of milk proteins by proteases from microorganisms. Indeed, a more proteolytically hydrolysed milk results in increased phagocytosis as well as casein peptides and denatured whey proteins that have been shown to stimulate the activities of immune system cells (13). Different peptides deriving from milk protein fermentation may modulate the immune system through direct antimicrobial effects, preventing growth of pathogenic organisms, and by means of anti-inflammatory actions (anti- TNFa effect) (11). Bioactive peptides, produced by proteolytic enzymes of LAB, may act as prebiotics too, stimulating the growth of Bifidobacteria themselves, with synergistic interactions (2).
  

Examples of activated peptides liberated in fermented milk products, for instance, are ACE- inhibitory peptides that have antihypertensive effects and hemodynamic properties (immunomodulating function). Other effects of different bioactive peptides are represented by modulation of absorption processes in the intestine by opioid peptides, inhibition of platelet aggregation by antithrombotic peptides, and the transport of minerals, especially calcium, by caseinphosphopeptide-like molecules. To characterize the effects of metabolites produced by LAB, supernatants of different samples of fermented milk prepared through ultracentrifugation have been analyzed and compared with unfermented milk supernatants. Thoreux, et al, have investigated the throphic action and nutrient efficiency of fermented milk on IEC-6 intestinal cells in culture (14). Fermented milk supernatants were more effective than those of unfermented milk in stimulating mitochondrial function, DNA synthesis and cAMP production (a second messenger of trophic activation), suggesting the existence of specific trophic effects depending on the LAB species that have been involved. Milk fermentation seems then to produce potent trophic factors able to promote cell growth, proliferation and differentiation.

Another study (15) has shown a dose-dependent inhibition of LPS-induced TNF-alpha secretion by human peripheral mononuclear blood cells due to non-protein molecules (<3000 daltons) contained in culture supernatants of lactic acid bacteria (Bifidobacterium breve and Streptococcus thermophilus). Commensal bacteria also display a TNF-alpha inhibitory capacity but to a lesser extent. Accordinlgy, these results underline the beneficial effect of commensal bacteria in intestinal homeostasis and may explain the role of some probiotic bacteria in alleviating digestive inflammation.

In studies on adults, problems with study design, lack of appropriate controls, inappropriate route of administration, sole use of in vitro indicators of the immune response, and short duration of most of the studies limit the interpretation of the results and the conclusions drawn from them. Nevertheless, these studies, in toto, provide a strong rationale for the hypothesis that increased yogurt consumption, particularly in immunocompromised populations such as the elderly, may enhance the immune response, which would, in turn, increase resistance to immune-related diseases (16).

Review of clinical trials in children
At present, the development of new infant formulas able to interact and modulate the intestinal flora is believed to be an important new approach to positively influencing the incidence of gastro-intestinal, respiratory, and allergic diseases in early childhood. These kinds of formulas may consist of either fermented formulas or formulas containing probiotics and prebiotics.

A new infant formula fermented with Bifidobacterium breve C50 and Streptococcus thermophilus 065 increases the bifidobacteria population in animals (mice with a human microflora), in healthy human volunteers and also in newborn babies (16). These results could be linked to the metabolites produced during the natural processes of fermentation. Thibault, et al., (17) have examined the clinical effects of this type of formula on the incidence of diarrhoea in healthy infants aged 4 to 6 months. No reduction in the incidence of diarrhoea episodes has been observed between two groups, while the fermented formula fed group showed less severity than the standard formula fed group. The reduction of severity was indicated by a lower number of dehydration cases and a lower number of medical consultations. The results of this study support the hypothesis that the use of a fermented milk formula could be a nutritional solution to reduce severity, and more specifically the medical consultation rate, for infantile forms of diarrhoea. Beyond the health benefit, this development could influence the health costs of diarrhoea in pediatric populations, since diarrhoea episodes in children have a high social cost.

Boudraa, et al., (18) compared the effects of an infant formula with those of the same formula after undergoing microbial fermentation on the duration of diarrhoea in young children with acute watery diarrhoea. This study confirms that young, healthy children, with watery diarrhoea, can be successfully fed with the fermented product of a standard infant formula. The beneficial effects in children with acute diarrhoea, and especially in those with carbohydrate malabsorption, reported in other studies, could be explained by facilitated lactose digestion and absorption promoted by metabolic activities in fermented milk products.
In a multicentre, double blind, randomised trial, Pedone, et al., (19) investigated whether supplementation of healthy children with milk fermented by yogurt cultures and Lactobacillus casei could affect the incidence of acute diarrhoea when compared with traditional yogurt. Data demonstrated that consumption of milk fermented with yogurt cultures and L. casei had a greater influence in decreasing the incidence of diarrhoea than standard yogurt alone while it also shortened the duration of acute diarrhoea. The results of this trial suggest an additional benefit of L. casei in acute diarrrhoea in children, compared with standard yogurt. In a separate investigation (20), the same group has evaluated the effect of L. Casei on a group of healthy, ambulatory children with mild diarrhoea that did not require hospitalisation. The trial failed to demonstrate an effect on the incidence of diarrhoea, but it did show the clinical benefits of fermented milk in decreasing the severity of acute diarrhoea episodes in children in an everyday situation with no other modification of their usual diet.

Very recently, an RCT trial has shown that antipoliovirus response can be triggered by a fermented product that is able to favor intestinal bifidobacteria (21). Whether the effect has been achieved through the bifidogenic effect of the formula or directly linked to compounds (peptides above all) produced by the fermentation process remains to be investigated.

Conclusions
Fermented milk products represent a rich source of nutrients and may improve lactose digestion through the breaking down of lactose into glucose and galactose by bacterial enzymes (22). Fermented milk products also help enhance the immune system with modulation of the cellular immune response through bioactive peptides, whose activity may extend beyond the immune functions by some still unclear mechanisms. There is now proof that fermentation products (23), fermented milks (24) and probiotics used for fermentative purposes (25, 26) may all contribute to health benefits, but clear study designs are needed to clarify roles and specific domains for these activities. In the meantime, the role of fermented milk products in feeding infants and children should be carefully considered, starting at weaning (27).

References

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This material has been prepared by members of the IFM's Advisory Committee on Child Health and Nutrition. It does not necessarily represent the views of the Infant Food Manufacturers Association. October 2004