Guidelines for the Evaluation of Probiotics Used in Food (Who) According to Fao/who To Evaluate Foods for Probiotic Effects, the Guidelines Must Be Followed

prebiotics were significantly higher than the control group [12], [13]. The results of several other studies also showed similar results [52], [162]. In a double-blind randomized study on the safety and tolerability of supplementation with a mixture of B. lactis BB 12 and S. thermophilus at different concentrations (1 x 10 6 and 1 x 10 7 CFU/day) in 118 children aged 3 to 24 months with a mean intervention period of 210 days. The results of the study showed that there was no difference in child growth between the study groups [133].

Another study conducted in 14-day-old non-breastfed infants, with feeding of formula containing 2 x 10 7 CFU Bifidobacterium longum BL999 and 4 g/L of a mixture containing 90% galacto-oligosaccharides and 10% fructo-oligosaccharides for 112 days, showed no difference in weight gain between the intervention and control groups with a mean gain of 100 g/L.

about 1.01 kg [122] .

Another study on children aged 18-36 months with probiotic and prebiotic supplementation also showed that after 3 months of intervention, the height gain in the intervention group was significantly higher than that in the control group (4.93 cm vs. 3.89 cm) [15] . However, the results of some studies by other authors showed no difference in the increase in lying length/height in the group supplemented with prebiotic and probiotic compared to the control group [122].

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Studies conducted in several other countries around the world have shown different results. A study conducted in the Netherlands, on 126 infants with prebiotic and probiotic supplementation, 0.24g prebiotic galacto-oligosaccharides/100 ml milk and 1 x 10 7 CFU B.animalis ssp. lactis /g (also known as Bifidobacterium BB12 ) and 1 x 10 7 CFU L. paracasei ssp. paracasei /g (also known as L. casei CRL-431 ), within 6 months showed that the WAZ and HAZ indexes did not differ between the study group and the control group (0.1 vs. 0.17), (0.51 vs. 0.50). After 6 months of intervention, the weight and height gain in the intervention group was 4152 g and

17.7 cm compared with 4282 g and 17.3 cm in control group children and the difference between groups was not statistically significant [158].

Another study in Italy, conducted on 138 non-breastfed infants, fed milk containing 2 x 10 7 CFU Bifidobacterium longum BL999 and 4g/L of a mixture containing 90% GOS and 10% FOS for 112 days, also showed no difference in height and weight gain between the intervention group and the control group, and the average weight gain was approximately 1.01kg.

Height gain/month in boys of the control and intervention groups was 3.51 cm and 3.51 cm, and in girls it was 3.22 cm and 3.22 cm [122].

Another study on 105 infants under 2 months of age showed mixed results. In this study, children in the intervention group (51 children) were supplemented with formula containing Lactobacillus rhamnosus GG and children in the control group were supplemented with formula until they were 6 months old. The results of the study showed that the intervention group had better growth than the control group, the weight and height changes of the intervention group were significantly higher than those of the control group (0.44 ± 0.37 vs. 0.07 ± 0.06, P<0.01 and 0.44 ± 0.19 vs. 0.07 ± 0.06, P<0.01, respectively).

P<0.005) [157]. The results of a meta-analysis of three studies [67], [115], [170], using formula milk for full-term infants, also showed that infants supplemented with prebiotic-containing milk had significantly higher weight gain [119].

Through the research results of the authors, it can be seen that the effects of prebiotics and probiotics on children's weight and height growth are not consistent.

1.2.5.5. Probiotics in the treatment of allergies

Some studies have shown that the number of Bifidobacteria is lower in children with specific allergies than in children without specific allergies. It has been hypothesized that Bifidobacteria are more effective in increasing tolerance to non-bacterial antigens by inhibiting the development of Th2 (proallergic) cells.

In infants with atopic dermatitis given supplemented hydrolyzed formula

L.Rhamnosus (GG) had better clinical improvement than normal hydrolyzed milk, which is thought to be due to the probiotic reducing intestinal permeability [95]. Children with atopic dermatitis who were actively treated with hydrolyzed milk supplemented with L.Rhamnosus (GG) or B.Lactis had better improvement in the severity of skin manifestations than children who drank normal milk. In the supplemented group, serum CD4 decreased and TGF-â1 increased [95].

Several studies have suggested that regular supplementation may stabilize intestinal barrier function and play a role in modulating immune responses that reduce the severity of atopic symptoms, particularly atopic dermatitis associated with cow's milk proteins [95]. A recent study also showed changes in the fecal bacterial composition of children with atopic dermatitis ( reduced fecal Bacteroides and E.Coli levels). Interestingly, serum IgE was correlated with E.Coli levels and in sensitized children, IgE was correlated with Bacteroides levels . Thus, each probiotic appears to influence allergen-induced inflammatory responses and exert a barrier effect against antigens that cause systemic allergic symptoms such as eczema [98].

1.2.5.6. Probiotics in the treatment of necrotizing enterocolitis (NEC)

Gut flora helps maintain immune system integrity, prevent infections, produce vitamins, and aid in the development of mucous membranes [108]. A study of 12,000 children supplemented with L. Acidophilus and B. Infantis on the incidence of necrotizing enterocolitis showed a significant reduction in the incidence and mortality of necrotizing enterocolitis in the probiotic group compared with the control group [159].

Thus, there is clear clinical evidence for the effects of probiotics on the intestinal flora, especially in infants. The specific clinical benefits depend on the individual probiotic.

PROBIOTIC

MECHANISM OF CLINICAL BENEFITS

- Increase the ratio of Bifidobacteria and Lactobacilli to harmful bacteria

- Increase mycin production

- Increased intestinal permeability

- Modulate intestinal immune response

- Increase humoral immunity

- (IgA and other antibodies)

- Th1/Th2 response adjustment

- Balance intestinal bacteria

- Reduce the duration of acute diarrhea

- Reduce new cases of acute diarrhea

- Reduces antibiotic-associated diarrhea

- Reduces severity and incidence of allergies and adverse reactions

- Reduced severity and incidence of necrotizing colitis

Figure 1. Mechanism and clinical benefits of probiotics

1.2.6. Safety and dosage of probiotics used

Most Lactobacilli used in food are non-pathogenic, harmless and non-toxic [159]. Many strains of Lactobacilli and Bifidobacteria are widely used in traditional foods and are recognized as safe. To date, there have been more than 70 clinical studies involving more than 4000 children, and no reports of adverse effects on children related to probiotics have been reported. In the FAO/WHO report on probiotics in food, it was stated that “the association between human disease and probiotic use has been limited and all cases have occurred only in critically ill patients” [71]. Lactobacilli- associated bloodstream infections from the external environment, food or feces are very rare. Probiotic-associated L. Rhamnosus infections in immunocompromised patients are uncommon [103]. The mechanism and route of transmission are unknown. Infants can be infected by many bacteria present in the body. However, the fact that Bifidobacteria are abundant in children (especially in breastfed infants) but the pathogenesis is not well documented. Bifidobacteria are present in many foods such as yogurt, including yogurt for babies who have started eating solid foods. In contrast, sometimes

There have also been reports of lactobacilli- associated bloodstream infections , but bacteremia has been associated with Bifidobacteria in commercial products, whether or not Bifidobacteria are present. Bifidobacteria have been used in infant formula for more than 15 years without any reported illnesses or adverse events.

In particular, studies have also shown the safety and good development when using B.Lactis for children from birth [163], for high-risk groups such as premature babies [114], malnourished children, and children of mothers with HIV [56].

As for safety, based on the information currently available, Bifidobacteria , especially B. Lactis , have a good safety profile and are good probiotics for use in infants and young children. Lactobacilli , especially L. Rhamnosus , are generally safe and are suitable probiotics for older children. While there is no data on specific probiotics, the use of probiotics in general should not be recommended for immunocompromised populations. Although the dosage has not been studied and they vary in many studies. There are no published studies on the efficacy

with the use of < 10 7 – 10 10 CFU per single dose or per dose. The daily dose

daily ranged from 10 8 - 10 10 CFU/day. Most of the products containing live bacteria studied contained 10 7 - 10 10 CFU per serving, which provided good control of bacterial viability over the shelf life of the product. Bacterial counts in the distal intestine ranged up to 10 12 CFU/ml of intestinal fluid.

The final issue is how probiotics are delivered. If probiotics are used therapeutically, they must be delivered in 'doses' in the form of capsules or tablets. However, when probiotics are used in pediatrics for the prevention of allergies, antibiotic-associated diarrhea, acute viral diarrhea, and long-term supplementation, probiotics should be included in foods such as yogurt, soft drinks, complementary foods, and formula milk, which will reduce costs compared to daily 'supplementation'.

In North America, some probiotics are used as 'supplements' to soft drinks or formulas and are clearly stated as L.Rhamnosus (GG), L.Casei, L.Reuteri .

Probiotic beverages and infant formulas are widely used around the world, but only B. Lactis is tested and evaluated by the FDA for use in commercial infant formulas.

1.2.7. Guidelines for the evaluation of probiotics used in food (WHO) According to FAO/WHO, to evaluate foods with probiotic effects, the following guidelines must be followed [68]:

1. Identification of probiotic strain/species/variety: Identification of strain, species, and variety is done by phenotypic and genotypic tests.

2. In vitro screening of potential probiotics. Necessary to assess the safety of probiotics and to understand the probiotics and their mechanism of action. Commonly used tests are resistance to gastric acid, bile acids, adhesion to intestinal mucosal cells, activity against potential pathogenic bacteria, reduction of adhesion to the surface of pathogenic bacteria, bile salt hydrolysis activity, resistance to sperm (probiotics used in the vagina).

3. Safety: There must be evidence of the safety and non-contamination of food by probiotics.

4. Studied on animals and humans. Research to evaluate effectiveness and efficiency on humans.

5. Food labels: include strain/species/breed, number of viable bacteria at the end of the shelf life, effects, storage conditions, and contact information when needed.

1.3. DIARRHEA

1.3.1. Epidemiology of diarrhea:

An estimated 2.5 million cases of diarrhoea occur each year in children under five years of age, a rate that has remained relatively stable over the past two decades. The most severe cases are in Africa and South Asia, which account for more than half of the world's cases and also have the highest rates of diarrhoeal death, especially among children. Children under two years of age have the highest incidence, which is also

is the period when children are fed complementary foods in addition to breast milk. This rate gradually decreases as children grow older. Mortality due to diarrhea has decreased over the past two decades, from 5 million deaths in children under 5 years of age at the beginning of the previous decade to 1.87 million in 2003 and 1.5 million in 2004, along with a general downward trend in child mortality from all causes. Children under 3 years of age have an average of 3-4 episodes of diarrhea per year. However, diarrhea remains the second leading cause of death in children under 5 years of age globally, after pneumonia [169].

In Vietnam, children suffer from diarrhea on average 2.2 times/year and it is 22.0% of the causes of death in children under 5 years old [1]. According to Nguyen Yen Binh's research, among the 5 agents causing diarrhea in children, Rotavirus accounts for the highest rate (39.3%), followed by E.coli (21.0%), Shigella (6.7%), Campylobacter (6.0%) and less

The most common is Salmonella (1.0%) [1].

1.3.2. Definition:

Diarrhea is defined as abnormally loose stools (loose stools, watery stools, stools with mucus, blood, etc.) 3 or more times in 24 hours [3]. Loose stools are unformed stools, except for breastfed babies who usually have several loose stools a day. For these babies, determining actual diarrhea must be based on an increase in the number of stools or an increase in the degree of looseness of the stools that mothers consider abnormal [3].

1.3.3. Classification of diarrhea: There are 3 main clinical forms of acute diarrhea that can affect human life and require different treatment methods.

1.3.3.1. Acute watery diarrhea (including cholera):

Acute diarrhea, lasting no more than 14 days, usually about 5-7 days, accounting for about 80% of all cases of diarrhea [3]. The main cause of the disease is V. cholerae or E. coli bacteria , as well as rotavirus . Loose diarrhea causes dehydration and electrolyte loss, patients often die due to exhaustion because of

severe dehydration and electrolyte loss. Children who are malnourished or have weakened immune systems are at greater risk of death from diarrhea than healthy children.

1.3.3.2. Acute bloody diarrhea (Dysentery syndrome):

This is a diarrheal disease with a common syndrome called dysentery syndrome, including: fever, abdominal cramps, tenesmus, stools often contain blood and mucus. In particular, stools have a lot of white blood cells, mainly polymorphonuclear leukocytes when examined under a microscope. Accounting for about 10-20% of all cases of diarrhea.

The main causes are Shigella, E.Coli (EIEC), Campylobacter jejuni [3]. Children with dysentery syndrome are at high risk of blood infection, malnutrition and dehydration. Dysentery is a major contributor to the morbidity and mortality of diarrhea. Dysentery is particularly severe in malnourished children and dysentery itself causes more dangerous consequences for malnutrition than acute watery diarrhea.

1.3.3.3. Prolonged diarrhea:

Persistent diarrhea is defined as an episode of diarrhea lasting 14 days or longer, accounting for 5-10% of all cases of diarrhea. Up to 3-23% of acute diarrhea episodes become persistent diarrhea. This rate is highest in children aged 1-2 years. Persistent diarrhea often leads to rapid deterioration of nutritional status and has a high mortality rate due to severe extraintestinal infections and dehydration.

1.3.3.4. Osmotic diarrhea:

Osmotic diarrhea occurs when too much water is drawn into the intestines. This can result from digestive disorders (e.g., pancreatic disease or celiac disease), in which nutrients are retained in the intestines and taken with the water. Osmotic diarrhea can also be caused by osmotic laxatives (which draw water into the intestines to relieve constipation). In healthy people, too much magnesium, vitamin C, or undigested lactose can also cause osmotic diarrhea and intestinal distention. In people with lactose intolerance, it is difficult to absorb

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