1. Introduction
According to UNICEF, WHO and the World Bank, the prevalence of stunting was reduced from 23.7% in the year 2000 down to 17.2% in the year 2018 among the children population under 5 years from North Africa [1]. Respective prevalences of moderate and severe wasting were reduced from 6.0% down to 1.6% for the same period of time. In the year 2015, leaders from 193 countries of the world came together and created the sustainable development goal 2030 [2] with a set of 17 goals. Goal number (2) aims to end hunger and malnutrition because low micronutrient intake is a particular problem in some children groups and goal (6) aims to assure the access of hygienic safe foods and drinking water to all. Because young children’s immune systems are not completely mature, they need to be protected from both acute and chronic infections. Infection by dietary pathogens, possibly from unhygienic food preparation and adverse environmental factors are determinant factors causing significant overgrowth of intestinal microorganisms and their metabolites and are key players in the development of malnutrition and ill-health [3,4], which are major constraints in achieving the SDG 2030 in food-insecure settings.
Adding different animal-sources to the diets of young children showed improvement in children’s growth expressed as weight-for-age and height for-age Z-scores [5-7]. However, there is now concern that using milk to improve the nutritional status of Egyptian children is problematic because of the high prevalence of lactose intolerance [8]. The use of dietary supplements (DS) containing probiotics has been increasing in most nations, with the major reason for the improvement of the overall gut health of children. Selected strains of Bifidobacterium spp and Lactobacilli (Lactic acid bacteria) are the main strains with proven probiotic effects [9]. The ability to survive in the gastrointestinal environment is recognized as a fundamental requisite for probiotics [10]. To produce health benefit to the host, the recommended minimum orally ingested dose for a potential effect of live probiotic bacteria per g of product ranged between 108 to 1012 colony-forming units (cfu) per day equivalent to 0.1 up to 100 billion cfu per day [11-14]. The functional aspects of probiotic action in terms of functionality and efficacy are mostly related to the concept of the targeted microbial generation of functional molecules, such as bioactive peptides, during food fermentation [15]. Our previous investigation showed that dietary intervention with the probiotic Lactobacillus acidophilus La1 in yoghurt matrix restored the intestinal permeability of Egyptian children (10.5 years old) due to up regulating the intestinal permeability and lowering the fecal pH values [3]. The global market for probiotics used in food and beverages grow at a rate of 7.9% from $28.6 billion in 2017 to $41.9 billion in 2022 [16]. FAO [17] encourages more extensive studies on probiotic traditional fermented foods because the acid-producing organisms in fermented dairy products could prevent “fouling” in the large intestine.
Sour sobya [SS] a traditional fermented rice milk product containing probiotic Lactobacilli [LAB] is in symbiosis with mixed yeasts spp [18]. This fermented food product had been spotlighted for its antimicrobial and antioxidative activities [19] and its functionalities on promoting different metabolic activities of the colon of adolescent boys [20]. The objective of the present study is to generate adequate dietary intervention data and establish differences in relative efficacy of probiotic yoghurt, with proven probiotic strains originating from the West and of the fermented SS rich in indigenous probiotic bacteria strains. Criteria of efficacy included gut microbiota composition, secretory immunoglobulin A, biomarkers of growth and oxidative stress.
2. Children and Methods
2.1 Design of the Study
The research protocol and all informed consent documents were approved by the NRC Medical Research Ethics Committee (MREC 13-125). Informed written consent was obtained from the mother of each child after a thorough explanation of the study details.
All children were living in urban Giza, whose parents were university graduates and the children were attending day care centers or primary schools. Criteria for inclusion was the age of the children in the range between 3 - 9 years; with no problems of gastrointestinal absorption, no use of antibiotics during the last three months prior to the study; the taste of the supplement was acceptable to the child; the mother was willing to collect stool and urine samples at predetermined dates.
2.2 Randomized controlled trial (RCT)
Twenty four children were assigned randomly into a 4-arm diet intervention trial with 6 children per group. Control group hadn't receive any supplement; Placebo group had been consuming daily 140 g rice milk porridge; 3- Probiotic yoghurt (PY) had been consuming daily 135 g (1.5 cup) commercial probiotic yoghurt (PY) and Sour sobya (SS) had been consuming daily 140 g commercial fermented sour sobya. The children were home visited daily by a qualified nutritionist for distributing the assigned supplement and for observing and recording compliance.
2.3 Supplements
The probiotic yoghurt was purchased in sealed cups weighing 90 g each, containing Bifidobacterium animalis lactis DN-173 010/CNCM I-2494 strain [Activia] Sour sobya (SS). (Elrahmany, Sayeda Zeinab, Cairo) was purchased twice a week in 500 g container and saved in the refrigerator. Sour sobya (SS) is traditional fermented rice based porridge containing diverse Lactobacilli and Saccharomyces cerevisae with added milk powder, sugar, grated coconut and seasonings. The product is a favorite dessert with sweet sour flavor and is consumed by a considerable segment of the population. Commercial rice milk porridge was purchased from the retail market in 140 g sealed containers.
3. Baseline Characteristics of the Children
Data collected at base line included anthropometric measurements; dietary assessments, collection of stool and urine samples.
Anthropometric measurements were recorded by trained staff according to standard procedures. The age of the child was obtained from the birth certificate; Body weight and standing height measurements were taken, while the child was with the light clothes and bare footed. Based on the data on age, sex, body weight and height for each child, the respective 3 nutrition indices of physical growth; weight for age Z score (WAZ), height for age Z score (HAZ) and weight for height Z score (WHZ) were computed by the EPI INFO software program (CDC). The child whose WAZ measures is below minus two (WAZ < -2) is underweight for his age. The HAZ index identifies linear growth and the child whose HAZ is below (- 2.0) from the WHO child growth standards reference population is considered short for his age, or stunted. The WHZ measures body mass in relation to body length and the child whose WHZ is below (- 2.0) is thin for his height [21].
The basal metabolic rate (BMR) was computed in mega joules (MJ) using the following equations [22]. Boys 3- 10 years = 0.095x body weight + 2.11, Girls 3- 10 years = 0.085 x body weight +2.03. The BMR was expressed in kcal from the relation 1 Mj = 4180 kcal.
3.1 Dietary intake data
Usual dietary intake of food groups and nutrients was estimated by collecting three 24-hour dietary recalls on nonconsecutive days from the mother of each child. The mothers recalled all foods and beverages consumed by their child during the previous 24 hours (beginning from morning meal on the day before up to the morning meal on the day of the interview). All mothers were then asked to estimate the quantities of the mentioned foods and to state the ingredients used for preparing the dishes and their respective quantities. To facilitate the quantity estimates, standardized household measures (cups, plates and bowls etc.) were provided by the interviewer and marked with several levels to indicate different contents. Raw ingredients were converted from household measures to weight estimates by taking reference weights, calculated on the basis of the average of weights on a digital weighing-scale. The average portion sizes consumed in grams of each food composite were converted into the respective estimated daily energy and nutrient intakes by using the Egyptian precise food composition table. The total healthy eating index (HEI-2015) is an overall diet quality assessment tool and it is scored from 0 to 100 [23]. It consists of 13 components including 9 adequacy components (total fruit; whole fruit; total vegetables; greens and beans; whole grains; dairy; total protein foods; seafood and plant proteins; and fatty acids and each ranged from 0 to 5 or 0 to 10 points. Lower scores indicate suboptimal intake for which higher scores reflect higher intakes. Moderation components included refined grains, sodium, and added sugar for which higher scores reflected lower intakes. Children who did not consume any servings of the food groups received zero score and partial consumption resulted in proportional deduction of points and higher scores indicated higher intakes. HEI 2015 total sub-score and component scores were calculated for each day of the 3 collection days.
3.2 The estimated average nutrient requirement (EAR)
The food intake data were converted into weights and the combined data were converted into respective energy and the estimated nutrient intakes was calculated using the data values from the precise Egyptian food composition table using a computer aided nutritional analysis program. The probability of nutrient adequacy (PA) was calculated by comparing the actual intake with indices used to evaluate the overall nutritional quality of the children diets. The age and sex-specific FAO/ WHO essential minerals and vitamins [24] were used as reference for expressing the individual daily intake of a nutrient into the respective probability of nutrient adequacy (PA). The requirement for zinc was based on medium bioavailability [24]. The percentage of reference for each nutrient was capped at 100% daily value (DV) to avoid overvaluing by single nutrient. Validation test. Ratios of estimated daily energy intake (kcal) / BMR (kcal) below 0.5 were excluded from the calculation.
3.3 Microbiological examination and enumeration
Culture media for the enumeration of Bifidobacterium in the presence of yogurt bacteria was prepared according to Antunes et al. [25]. The plates were incubated in anaerobic conditions at 37˚C for 48 - 72 h, after which LAB colonies in each plate were counted. The results are expressed as the log10 cfu per gram of probiotic yoghurt. The viable Lactobacilli in the SS was carried out by serial dilutions of SS from 1/10 to 1/107 and plating 100 μl aliquots on the selective MRS agar media (de Man, Rogosa and Sharpe; Difco, Sparks, MD, USA) supplemented with 3% ethanol and 0.5% cycloheximide to inhibit yeast growth [26]. The plates were incubated at 37°C under anaerobic environment using anaerobic gas packs (Oxoid, Uni path Ltd, Basing stoke, UK), capable of producing 1800 cm3 hydrogen and 350 cm3 carbon dioxide. The plates were incubated at 30°C and examined after 5 days for the bacterial growth and the counts were expressed as CFU/gram SS. Eight Lactobaccilli colonies (Gram positive isolates) were isolated from the highly diluted plates and were further identified by matrix-assisted laser desorption/ionization timeof- flight mass spectrometry (MALDI-TOF MS) protein analysis. The method had been described earlier in detail by many investigators [27-29]. The yeasts in SS were enumerated on acidified yeast extract 10 g; peptone 20 g; glucose 20 g; agar 15 g per liter [YEPG] adjusted to pH 3.5. The plates were incubated at 30°C for 72 h and colonies were enumerated and the yeast isolates were identified with API 50 CHL test (BioMerieux, Marcy-l’Etoile, France) following instructions of the manufacturer.
3.4 Fecal microbiological examination and enumeration
Baseline fecal collection was performed within 24 hours before the beginning of the RCT and the 2nd stool collection was completed by last day of the RCT. The children evacuated their feces into containers and the mothers were asked to save them on ice until transported to the laboratory for bacterial count. The microbiological testing was completed within 24 hours of stool collection and the remaining stool sample was kept frozen at -20˚C for further analysis.
Ten grams of stool sample were mixed with 90 ml of sterile physiological saline (8.5 g NaCl /l) to make an initial dilution of one tenth (1/10). The suspension was used for making suitable serial dilutions up to 10-8 by incorporating 1 ml into 9 ml of saline in sterile tubes and vigorous mixing. Aliquots (100μl) of each dilution were subsequently plated on the following selective medias: Total anaerobic bacteria (Columbia blood agar; Bio Mérieux ); Bacteroides (Bacteroides Bile Esculi); Bifidobacteria were grown on modified Beerens Columbia agar base + cysteine [30]; Enterobacteriaceae on MacConkey broth (BGMB); Lactobacilli on anaerobic deMan-Rogosa-Sharpe (MRS) agar with vancomycin [31]. The plates were incubated at 37°C for 3 days under anaerobic conditions using Gas Pak (H2 and CO2) anaerobic systems (BBL Microbiology Systems, USA). The colonies were counted and expressed as log10 colonies forming unit (CFU) per gram fecal material. Yeasts and molds were enumerated on pour plates of potato dextrose agar, PDA (Oxoid, UK). The plates were incubated at 25°C for 5 days .The colonies on the plates were counted (CFU), and the numbers were expressed as log10 cfu per g fecal material.
Assay of stool secretory immunoglobulin A (s-Ig A). The Immu Chrom ELISA kit (Bio Vender research Diagnostics, Cat # RIC 6100, Germany) was used and the instruction of the manufacturer was followed. Weighed portions (80-120 mg) were diluted, vortexed and centrifuged at 13000 rpm for 5 minutes. The supernatant was diluted 1:250 in wash buffer. Standards, controls and stool samples were simultaneously run in the ELISA and the concentration of s-IgA was read at 405 nm in ELISA reader against a standard curve and the results were expressed in mg per gram stool.
3.5 Urine collection
First urine collection was performed within 24 hours before the beginning of the RCT (baseline) and the second urine sample was collected at the end of the 3 week RCT. The children were asked to evacuate morning urine into containers and aliquots were immediately frozen at -20°C until further analysis.
4. Urinary Total Hydroxyl Proline (HPRO)
The total urinary hydroxyl proline was determined by acid hydrolysis by mixing urine aliquots (100μl) with equal volume of 12 normal hydrochloric acid in air tight screw pyrex tubes and the tubes were heated at 125°C for 2 h. Upon cooling the pH of the acid hydrolysate was adjusted to 6.0 by sodium carbonate / citrate /1- isopropanol buffer solution. The decolorization with active charcoal; the formation of pyrole derivative with tosylchloramine and the development of the color with p– aminobenzaldehyde (Ehrlich's reagent followed the standard method [32]. The intensity of the developed pink color was recorded at 560 nm against a blank. The results are expressed as μg total HPRO per ml urine.
Urinary creatinine was determined by the classical alkaline picric acid - sodium hydroxide reagent [33,34].
Urinary hydroxyl proline creatinine ratio (HPRO) The urinary hydroxyproline is expressed as total hydroxyproline / creatinine (mg/g). The hydroxyl proline index [HPRO-I] is defined as hydroxyl proline creatine ratio /per kg body weight.
Determination of urinary thiobarbituric acid [U-TBA]. The colorimetric method was followed using the TBA reaction [35] and the results were expressed as urinary TBA creatinine ratio (mg/g).
5. Statistical Analysis
All data were expressed as the arithmetic means with standard error. The data were examined for the total sample of children as well as stratified by gender and by two distinct age groups: < 6 years and school-age children ≥ 6 years. Numerical data were presented in Whisker box plots showing median and interquartile ranges. Paired t-test was used to compare within groups between baseline and subsequent to treatment. Treatments were examined for significance by one way ANOVA followed by Duncan test. Pearson correlation coefficients were computed and P= 0.05 indicates significant statistical differences. SPSS version 18 software program and Excel sheet were used for the statistical analysis.
6. Results
The characteristics of the children are presented in Table (1). The mean anthropometric indices of all children were within the norms exceeding the cutoff point of -2.0. However, WHZ of 15.8% of the boys were < -2.0 denoting that they were thin for their height (wasted). Wasting represents the failure to receive adequate nutrition during the period immediately before the survey. Children less than 6 years of age tended to be slightly undernourished (WAZ = -1.0 ± 1.42) compared to the respective mean value (0.18 ± 0.25) among those > 6 years of age; but the difference didn’t reach significant value (P > 0.05). The stunting rate (HAZ < -2.0) was statistically higher among the girls (54.4%) compared with the boys (Chi-square test = 8.28; P < 0.01). The basal metabolic rate (BMR) correlated significantly with HAZ (r = 0.4732; P < 0.01) and WHZ (r = 0.5038; P < 0.01).
Mean values ± standard error. Within the same row, mean values are significantly different (P < 0.05), if they don’t share the same alphabet (Student’s t- test).
The overall diet quality expressed as healthy eating index (HEI) averaged 60.7 ± 11.8 (29.0 - 89.54) and correlated significantly with WAZ (r = 0. 4306; P < 0.01). The ideal HEI is 80 points and very low HEI has 34 points; 17% of the recorded diets had HEI between 29 up to 50 points. The HEI components scores calculated from the 3 day dietary recall showed that on daily basis, 50% of the children consumed vegetables, while only 16.7% consumed fruits. 4.3 and 12.5% of the children didn’t consume any vegetables or fruits on any of the 3 d dietary recalls (Figure 1). The estimated daily intakes of vegetable and fruit averaged between 50 g and 70 g, respectively. Half of the children consumed dairy product protein daily and different types of cheese contributed considerably. According to the HEI-2015, diets containing less than 8% saturated fatty acids per 1000 k calories has a component score of 10 points; the respective score in the diet of our children averaged 3.15 ± 0.48 points. The average score for whole grains was lower in the diets of the young children < 6 years compared with the respective value among those ≥ 6 years (Figure 2). The total fat intake expressed as% of energy intake was significantly higher (P < 0.05) among younger children < 6 years (35.9 ± 1.2% EI) compared to the respective mean value intake (31.0 ± 1.15% EI) among children ≥ 6 year (Table 1). All children consumed at least one source of animal protein on all days based on 24 h dietary recall with mean daily intake of 19 ± 1.5 g (range 1.7- 58.4 g).
6.1 Diet supply of vitamins and minerals
The estimated daily intake of 8 vitamins, calcium and zinc were calculated and the results were then expressed as % estimated average requirements [FAO/WHO; 2004]. Large percentage of the children did not meet the requirements for vitamin A, B2 and for calcium intakes (Table 1). The average supply of 7 vitamins was higher in the diets of young children (< 6 years) compared with the respective mean values among those ≥ 6 years.
The gut microbiota composition at baseline didn’t show variations between boys and girls or age groups. Baseline mean levels of the urinary biomarkers hydroxyl proline index (U-HPRO-I) and thiobarbituric acid were also comparable between the groups (Table 1).
6.2 Composition of the supplements
Table (2) presents the chemical composition and the viable probiotic counts of the supplements. The 135 g daily portion of PY provided 1.4 × 109 cfu total Bifidum bacteria. The portion size of SS (140 g daily) provided 4.2 ± 0.62 × 109 cfu total diverse lactic acid bacteria and 1.79 ± 0.14 × 109 cfu of yeast. The proteomic spectra of MALDI-TOF identified 5 out of 7 isolates as Lactobacillus rhamnosus with high score values. The API analysis identified the sobya yeast as Sacharomyces cerevisia.
6.3 The effect of dietary intervention on the gut microbiota counts
The fecal Lactobacilli counts increased 18 and 21 fold at day 21 over the respective mean baseline counts among the children consuming the PY and SS, respectively [Table 3]. No significant changes in the counts could be detected in the fecal samples of the control or the placebo groups. The mean fecal Bifidobacteria increased significantly in the feces of children consuming the Bifido probiotic yoghurt (log10 7.79 up to log10 9.3 cfu per g stool). The mean yeast counts increased significantly only in the stool of children consuming the SS for 3 weeks (Table 3). The mean counts of the pathogenic bacteria Enterobacteriaceae was reduced significantly at day 21 in the feces of the children consuming PY, SS and also in the placebo group compared with the respective baseline counts (Table 3). The total anaerobic counts increased significantly in the feces of children consuming the PY or the SS, denoting richness of the gut microbiome (Table 3).
The baseline fecal secretory IgA level averaged 3.4 ± 0.48 mg / g fecal material and increased significantly following the 3 week intervention with PY or SS (P < 0.02), compared with the respective pre feeding levels; while their levels remained unchanged among the control and placebo groups (Table 4, Figure 3).
Baseline urinary HPRO-I averaged 2.3 (0.92 - 4.6); whereby 2/3 of all children were below the acceptable HPRO-I level for normal growth (≥2.5) [35,36] . The proportion of boys (54%) with acceptable U-HPRO-I values were significantly higher than the respective proportion of girls (19%) ( χ2 = 3.1, df = 1, 0.1 < P> 0.05).
Following the 3 week intervention with PY or SS, the urinary HPRO - I in every child belonging to both groups reached or exceeded the acceptable U-HPRO-I value of 2.5. The mean values were significantly higher compared with the respective pre feeding levels or to the control and the placebo (Table 4 and Figure 4). The baseline U- TBA concentration averaged 138.6 ± 29.6 mg / g creatinine. The 3 week dietary intervention with PY or SS reduced the U-TBA concentration roughly by half the respective initial levels (Table 4, Figure 5); while the respective mean values remained almost unchanged among the control and the placebo groups.
7. Discussion
In recent years there has been increasing focus on improving children’s diet as part of an overall strategy for preventing children under nutrition. Dietary indexes in paediatric populations is a rapidly expanding area of research because they assess nutritional adequacy and are predictive of child growth; whereby weight status is the most commonly measured health related outcome, followed by height/ length [37]. The WAZ of our children correlated significantly with age (r = 0.5726; P < 0.05); with HEI (r = 0.3821; P < 0.01) and with dietary animal protein intake (r = 0.2945; p < 0.05). HAZ correlated only with HEI (r = 0.2000, P < 0.05). The average HEI-2015 for the diet of both children groups; < 6 years (62 ± 1.6) and above ≥ 6 years old (64.16 ± 1.33) were higher than the respective HEI figures of 55.3 and 51.2 reported for US child population < 6 years and 6-11 years old, respectively [38]. In China, the developed HEI averaged 52.4 and was associated with education [39].
The high saturated fatty acids intakes (17.8 ± 0.94% EI) in the diets of our children is quite similar to that reported previously for populations from developed countries [40] and is attributed to high intakes of meat & dairy products and no sea foods. Acceptable mean level of 8.3 ± 0.1% saturated fatty acids/EI was reported among Korean children 3-5 years old [41]. A source of animal proteins was consumed at least once daily by our children and it was reported that ¾ of the Middle Eastern region consume daily animal sourced foods (ASF) [42].
Meta analysis demonstrated that the probiotic supplement had no effect on the gain in body weights among children from developed countries, yet probiotic supplements improved the growth of all children from developing countries [43]. Indeed, the daily supplementation with PY or SS was associated with significant improvement in the linear growth taking the net positive increases in U-HPRO index (Δ day 21 minus baseline values of each child) as the biomarker of growth. No such changes could be detected in the U-HPRO index values among the placebo group, though it provided 230 kcal per day derived from rice milk porridge. Negative response was also reported among Egyptian stunted children 42 months old, who consumed daily supplement providing additional 191 kcal derived from plant formula consisting of sesame: dates: dried apricots: coconut: peanut and cocoa in the proportions of 30:20: 20: 15: 10: 5 weight percent. The criteria of response was HAZ and serum ferritin didn’t differ at the end of the 8 week supplementation that the respective placebo group based on sugar syrup [44].
It is hypothesized that infection by pathogenic Enterobacter bacteria has negative impact on demarcated variations in the gut health which impair growth. This hypothesis is supported in the present study by the existence of high significant negative correlation coefficient (r = - 0.4454; P < 0.01) between the net increases in the U- HPRO-I (Δ day 21 minus baseline value of each child) and the respective net decreases in the fecal count of log10 Enterobacteriaceae (Δ day 21 minus baseline values of each child). Such relation could not be detected by anthropometric measurements, denoting the usefulness of HPRO-I biomarker before growth spurt could be detected by anthropometry in the response in growth rate over short periods of time and in close monitoring of treatment regimens. The availability of potential biomarkers with high prognostic value to assist in making the prospect of catch-up growth in poor settings has high priority [45]. Hydroxy proline [HPRO] is a collagen-related marker, since it is present in all fibrillar collagens and tissue breakdown contribute to urinary HPRO [46]. Mean HPRO-I of 2.5 ± 0.9 and 1.6 ± 0.6 were reported among healthy well-nourished and in under-weight children, respectively [35-36].
Reestablishing microbiota-mediated colonization resistance (CR) by the administration of potential probiotics could markedly develop immune response for healthy gut microbiota, which disfavor colonization of enteric pathogens and reduce infections, particularly those caused by antibiotic-resistant bacteria [47].
The increase in fecal Lactobacilli counts following the 3 week intake of PY (16.3 ± 0.24%) or the SS (16.7 ± 0.24%) confirmed previous reports that Lactobaccilli survive the gastric transit and reach the gut. Higher % recoveries of Lactobaccilli were detected in the feces ranging from 28.4% of Lactobacillus casei DN - 114 after a dosage of log 109cfu [48] up to 1000 fold excess specific L. rhamnosus-related bacteria after dosage of 1.55 x 1010 cfu L rhamnosus GG [49]. The fecal Lactobacilli and its ratio to the total anaerobic bacteria are typically used as an indicator of healthy gut [50].
The increases in fecal Bifidobacteria output during the PY period was associated with reduction in the urinary TBARS concentrations indicating potential anti-oxidative activities of Bifidobacterium. This finding is in good agreement with previous report on the reducing concentrations of fecal TBARS following consumption of Bifidobacterium bifidum or Lactobacillus acidophilus yoghurt med by Iranian adults [51].
The gut microbiota plays major role in regulating mucosal and systemic immunity and it is estimated that roughly 3/4 of the colon anaerobic bacteria is coated with secretory immunoglobulin A (s-Ig A) in absence of colonic mucosal inflammation [52]. The significant increases in fecal S-Ig A concentration following the 3 week dietary intervention with PY or SS are in good agreement with previous reports showing that 3 week probiotic supplementation increased fecal secretory IgA concentrations in preterm infants [53] . This unique probiotic property has implication on their potential role in preventing gastrointestinal infections and lowering respiratory tract infections [54].
The urinary thiobarbituric acid (U-TBA) is formed by the reaction with malondialdehyde (MDA) of lipids and is a biomarker of the oxidative stress [55] and the excessive free radicals so generated would have the potential to cause enormous pathological damage. In the present study the 3 week supplementation with PY or SS successfully reduced the concentration of urinary TBA roughly to half the respective initial concentrations; suggesting that probiotic bacteria inhibited the excessive generated free radicals. This finding supports the results reported previously on adolescents demonstrating the potential anti oxidative activity of probiotic yoghurt and SS [20].
Lactobacillus rhamnosus Yoba 2012 is the world’s first generic probiotic strain formulated by culturing Western probiotic L. rhamnosus GG with Streptococcus thermophilus to optimize synergistic propagation of both strains in wide range of fermented food matrices with shelf life viability of 2 years [56].
8. Conclusion and Recommendations
A monitoring and evaluation framework, operational and technical guidance as well as program tools for nutrition is urgently needed for giving high priority to food safety unit and research area of lactic acid fermentation as a technique to reduce the risk of having pathogenic microorganisms grow in the food.
Developments of novel traditional fermented foods containing endogenous probiotic strains particularly from cereal origin at low cost and retain good viability/ survival in the product during storage to up regulate disordered intestinal function and re-establish gutmicrobial balance.
The expansion in conducting well controlled randomized controlled trials to define and optimize several issues and mechanistic studies of relevance to probiotic functionalities on up regulating the gut-microbial balance.
Competing Interests
The authors declare that they have no competing interests.
Author Contributions
RB recruited the children and was responsible for conducting the randomized controlled trial, collecting the stool and urine samples, sharing in the microbiological examinations and chemical analysis. MS was responsible for the chemical analysis of the biological fluids and the immunological assays of the stool. LH designed the concept of the study, carried out the statistical analysis and wrote the manuscript. MT was responsible for the microbiological of the supplements and the stool samples; MA shared in the microbiological examinations of the samples. AA shared in the conceptualization of the study and the design of the study.
Acknowledgments
The authors are grateful to the participants and their mothers, who without their cooperation this study could not be completed. Assay. Thanks are due to Dr Hesham Abdel Latif – Faculty of Agriculture- Cairo university for the ANOVA analysis.