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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: [email protected]
http://00_60thnestle%20nutrition.pdf/


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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

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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 byL.

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

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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 casein-

macropeptide 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 isoleucyl-

prolyl-proline (Ile-Pro-Pro) and Valyl-Prolyl-Proline (Val-Pro-Pro) have been isolated

consistently from casein digests by L. helveticusand 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 withL. helveticusLBK-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 theL. helveticus

and the control groups, respectively (21). Reductions in blood pressure of this

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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 vivoand in vitroACE-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 ofthe 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 diffusionof 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. acidophilusDPC6026 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. zakazakiihas 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 rhamnosusGG (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 whoregularly 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 heat-

inactivated 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.

thermophilusfor 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

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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.

rhamnosusGG (LGG) has been the most extensively tested, especially for its effects ondiarrhea 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 lactisBb12.

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 favoredL. 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.

caseisubsp rhamnosus, or a combination of S. thermophilusandL. delbruekiiin 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

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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 andHelicobacter pyloricolonization.

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 ofH. pyloricolonization 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. caseiDN-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. animalisand

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 productcontaining L. johnsoniiLa1 or L. paracaseiST11 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

13C-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 withH. 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. pyloriin 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. pylorigrowth 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.

alimentarius15M, L. brevis14G, L. sanfrancisensis7A and L. hilgardii51B with the

purpose of fermenting wheat semolina used for the production of noodles, whose

resulting concentrations of gluten peptides were low; these culinary preparations hadsatisfactory 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. delbrueckiispp. bulgaricus, B.

breve, B. longum andB. 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 vivoresults are as promising as those observed in this

in vitrostudy (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 speciesmost frequently used for production of yogurts and fermented milk products in the

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Western countries, L. acidophilus, L. delbrueckiissp 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 synthesizedby 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 Staphylococcusmay also be altered and their concentrations may, in

some case, correlate with the serum concentrations of IgE. The distribution of

Bifidobacteriumspecies 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 BifidobacteriumBb12. 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 givenLactobacillusGG 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 withL. rhamnosusfor 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

15


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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 (103-

110). 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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107. Lim H-J, Kim S_Y, Lee W-K. (2004). Isolation of cholesterol-lowering lactic

acid bacteria from human intestine for probiotic use. J Vet Sci 5: 391-95.

108. Heber D, Yip I, Ashley JM, Elashoff DA, Go VLW. (1999) Cholesterol-

lowering effects of a proprietary Chinese red-yeast-rice dietary supplement. Am J

Clin Nutr 69: 231-66.

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109. Rossi EA, Vendramini RC, Carlos IZ, de Oliveira MG, de Valdez GF. (2003)

Effect of a new fermented soy milk product on serum lipid levels in

normocholesterolemic adult men. Arch Latinoam Nutr 53: 47-51.

110. St-Onge M-P, Farnworth ER, Savard T, Chabot D, Mafu A, Jones PJH. (2002)

Kefir consumption does not alter plasma lipid levels or cholesterol fractional

synthesis rates relative to milk in hyperlipidemic men: a randomized controlled trial.

BMC Compl Altern Med 2:1.

111. Brashears MM, Gilliland SE, Buck LM. (1998) Bile salt deconjugation and

cholesterol removal from media byLactobacillus casei. J Dairy Sci 81: 2103-10.

112. Pereira DIA, Gibson GR. (2002) Cholesterol assimilation by lactic acid bacteria

and bifidobacteria isolated from the human gut. Appl Environment Microbiol 68:

4689-93.

113. Alonso L, Cuesta EP, Gilliland SE. (2003) Production of free conjugated linoleic

acid by Lactobacillus acidophilus and Lactobacillus casei of human intestinal

origin. J Dairy Sci 86: 1941-46.

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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 formulaAcidified formula Control formula

Age(months)

Children/month

Episodes ofdiarrhea

Incidence Children/month

Episodes ofdiarrhea

Incidence

3-5 7 0 0 18 3 16,7

6-8 81 8 9,9 2=7,1235

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Table 2: Serum cholesterol changes in individuals randomly allocated to receive a

fermented milk containingLactobacillus acidophilusL1 (L1 FM) of human origin or

Lactobacillus acidophilusATCC 43211 (ATCC); placebo = fermented milk without

active bacteria. Values are expressed as mmol/L; means SEM.

Group No Baselineaverage

Week 2 Week 2 minusbaseline

% change

1st study

L1 FM 14 6,27 (0,19) 6,06 (0,17) - 0,21 (0,08) 3,2 (1,2)

ATCC FM 15 6,38 (0,23) 6,30 (0,23) - 0,08 (0,09) 1,2 (1,4)

2nd study

L1 FM 21 6,53 (0,17) 6,27 (0,18) - 0,26 (0,10) 3,8 (1,7)

Placebo 19 6,30 (0,14) 6,40 (0,13) - 0,10 (0,10) 1,9 (1,6)

Combined

L1 FM 35 6,42 (0,13) 6,18 (0,13) - 0,24 (0,07) - 3,6 (1,2)

P vs baseline 0,0015

P vs placebo 0,008

ANOVA 0,03

Modified from Anderson JW. (1999) J Am Coll Nutr 18: 43-50.

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Table 3.- Effect ofLactobacillus casei rhamnosusGG (LGG) on the recovery from acute

diarrhea in infants and preschool children (mean SD)

Group Duration of diarrhea, days

(1) Fermented milk with LGG 1,4 0,8

(2) Freeze-dried LGG 1,4 0,8

(3) Pasteurized yogurt 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% ofcases were associated with rotavirus. The amounts of LGG provided were 10

10-11CFU 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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