Feasibility Studies to Control Acute Diarrhoea in Children by Feeding Fermented Milk Preparations Actimel and Indian Dahi

Feasibility studies to control acute diarrhoea in children by feeding fermented milk preparations Actimel and Indian Dahi

KN Agarwal1* and SK Bhasin2

1Department of Paediatrics, University College of Medical Sciences and GTB Hospital, Delhi, India; and 2Department of Community Medicine, University College of Medical Sciences and GTB Hospital, Delhi, India

The aim of this work was to study feasibility of diarrhoea control in children (6 months to 5 y of age) by feeding fermented milk preparations. The design used was a randomized controlled clinical trial and the study was carried out at the Delhi University College Hospital providing tertiary care, and a nearby community centre Nand Nagri, a resettlement colony in East Delhi. Children suffering from acute diarrhoea (75 patients from the hospital and 75 from the community) were allocated to three groups by double-blind technique. Group 1 was given a fermented milk, Actimel, containing 108 of each Lactobacillus casei DN- 114001, Lactobacillus bulgaricus and Streptococcus thermophilus per gram. Group 2 was given Indian Dahi (Lf 40) containing 108 of each Lactococcus lactis, Lactococcus lactis cremoris and Leuconostac mesenteroides cremoris per gram. Group 3 was given ultra-heat-treated yoghurt preparation (no live bacteria). Actimel was also used as a starter to prepare the curd in order to study the preventive effect of diarrhoea in children in a community. In the hospital study Indian Dahi and Actimel administration reduced mean duration of diarrhoea by 0.3 and 0.6 day (P < 0.001), respectively. The corresponding ﬁgures in the community study were 0.2 and 0.5 day (P < 0.05), respectively. The families using Actimel as a starter showed a reduction in diarrhoeal morbidity episodes by 40% of the children tested in a 3 month follow-up. In conclusion, Actimel, fermented milk containing Lactobacillus casei DN-114001, and Indian Dahi can signiﬁcantly reduce the duration of diarrhoea in children; the former preparation being superior. European Journal of Clinical Nutrition (2002) 56, Suppl 4, S56 – S59. doi:10.1038/sj.ejcn.1601664

Descriptors: acute diarrhoea; Indian Dahi; Lactobacillus casei; children

Introduction the number of episodes as well as duration of diarrhoea In India and neighbouring countries, Dahi (fermented milk (Ullmann & Korzenik, 1998). Dahi has been shown to have curd — Indian yoghurt) has been used as a regular item of the total viable bacterial counts of 5 Â 107 to 5 Â 108=ml; of these diet. It was mentioned in the Ayurveda literature from around 107 to 3.5 Â 108=ml are Lactobacilli, yeast and mould (700 – 600 AD for treatment of diarrhoea. Indian physicians used fat- 5000), coliform (0 – 100) and spores (0 – 30=ml) (Mohanan et al, free curd (butter milk) orally as well as in enemas to treat 1984). Dahi bacterial content and composition vary from place chronic gastrointestinal disorders, eg colitis, chronic diar- to place; some comparative data are summarized in Table 1. rhoea, etc. Dahi along with a usual diet is known to reduce In Rajasthan, Dahi prepared from goat milk had Strepto- coccus paracitrovorus, Lactobacillus acidophilus and Lactobacil- lus bulgaricus in 81.8, 73.5 and 71.2% of batches, respectively (Singh et al, 1991). As many as 250 species of Lactobacillus have been isolated from available Dahi preparations in India *Correspondence: KN Agarwal, D-115=Sector 36, NOIDA, Guatam Budha (Prasad & Ghodekar, 1990). Of these, 72 had no antibiotic Nagar-201301, India. properties. Variable antibiotic properties were noted in E-mail: [email protected] Lactobacillus plantarum S-41, Lactobacillus casei D-76, Guarantor: KN Agarwal Contributors: KNA and SKB planned and monitored the study. Both Lactobacillus bulgaricus D-92, Lactobacillus fermentum C-l O authors were involved in drafting of the manuscript. and Lactobacillus acidophilus D-67. In vitro antimicrobial Fermented milk in diarrhoea treatment KN Agarwal and SK Bhasin S57 Table 1 Proportion of samples having bacterial presence Table 2 Mortality rates in India caused by diarrhoea and cholera

Islamabad Bangalore Age (months) Number of children Causes of death (Masaud et al, 1999) (Mohanan et al, 1984) < 12 3854 20, Diarrhoea; 1, cholera Lactobacillus bulgaricus 86 83 12 – 72 11 393 27, Diarrhoea; 3, cholera Streptococcus 80 85 thermophilus Streptococcus lactis 74 7 Lactobacillus helveticus 34 5 Streptococcus cremoris 30 7 deaths in children below 6 y of age (n ¼ 7440) were Lactobacillus casei 20 11 (a) diarrhoea with dehydration — 34%; (b) measles — 7.3%; Lactobacillus acidophilus 14 — (c) pneumonia — 15.1%; and (d) malnutrition — 16.3% (Agarwal et al, 1995, 1998). Diarrhoea under 6 y of age remains the most common cause of death at community activity of Lactobacilli 3.2 Â l04 and 5.9 Â l010=ml on modified level in India. Thus the present study was conducted to find MRS agar were 5.8, 8.3, 6.7, 4.2, 6.7, 7.5 and 6.7 against out the effect of fermented milk preparations on control of Shigella dysenteriae, Salmonella typhosa, Escherichia coli, acute diarrhoea in children. To avoid variability of bacterial Enterobacter aerogenes, Staphilococcus aureus, Bacillus cereus strains (as in traditional Indian Dahi), (a) government dairy and Micrococcus flavus, respectively. Maximum activity was Karnal fermented milk, (b) Actimel fermented milk (Danone, observed in L. casei, L. plantarum, L. bulgaricus, L. acidophilus France), for which efficacy has been shown (Pedone et al, and L. fermentum (Prasad & Ghodekar, 1991). 1999; Agarwal et al, 2001), and (c) ultra-heat-treated milk as L. casei wild lactic culture (L. casei D-34) isolated from control were selected to study control of diarrhoea. Dahi was 34 – 36% cytotoxic to three b tumor cell lines: Hela, HEP-2 and HFS-9. The cytotoxic component was in the protein culture supernatants (anionic=cationic and neutral Methods fractions 1=1=1), and showed toxicity of 50, 70, 70%; 70, 88, The present study was a community-cum-hospital-based 94%; and 50, 89, 90% against Hepa-2, HFS-9 and Hela cell clinical study. The study was carried out by the Departments lines, respectively. The pooled fraction showed a synergistic of Paediatrics and Community Medicine, University College effect. This cytotoxic substance inhibited H-thymidine of Medical Sciences and Guru Teg Bahadur Hospital, incorporation (DNA synthesis in tumour cells; Masud et al, Delhi, India.) Seventy-five children (6 months to 5 y) with 1991). Out of 44 cultures from Dahi only L. casei D48a, diarrhoea were admitted for the study to the hospital. L. plantarum D70a and L. casei B 1922 had a cytotoxic activity Simultaneously, the study was also carried out on 75 children range of 30 – 36% in addition to L. casei D-34 (Manjunath & of the same age with diarrhoea in a slum area named Ranganathan, 1989a,b). Nand Nagri (community study) in one of its dispensaries During this period we came across reports indicating that run by the State Government of Delhi. Children with acute feeding of Bifidobacterium longum containing yoghurt reduced diarrhoeal diseases (three loose stools per day without blood= erythromycin-induced gastrointestinal upset and intestinal mucus) were selected in both studies. The majority of the microflora (Colombel et al, 1987). Infant formula containing parents of these children had less than a fifth-grade educa- Bifidobacterium bifidum and Streptococcus thermophilus reduced tion. All the study children families were in the low socio- incidence of acute diarrhoea and rotavirus shedding (Saavedra economic category. Throughout the community study, et al, 1994). Lactobacillus casei strain DN-114001 has been treatment was given from the OPD of the dispensary and reported in a series of publications to have (a) reduced severity the field workers provided the intervention product twice a of diarrhoea, (b) influenced the intestinal microflora and day at their homes in refrigerated conditions. Both breast-fed (c) stimulated growth and brush border enzyme activity and bottled fed children with or without dehydration were (Guerin et al, 1998). The effect was specifically observed to included for the study purpose. The following children were reduce duration of acute diarrhoea, control of relapsing excluded from the present study after taking a detailed Clostridium difficile colitis, antibiotic-induced gastroenteritis history: (a) those with drug-induced diarrhoea; (b) those with and even diarrhoea due to Vibrio cholerae and enterotoxigenic malabsorption syndrome; (c) persistent diarrhoea cases; E. coli (Isolauri et al, 1991; Raza et al, 1995; Gorbach et al, 1987; (d) those with diarrhoea associated with other systemic Mitra & Rabbani, 1990). Lactobacillus strains have also been diseases like pneumonia; (e) those who had taken antibio- found effective in improving immunogenecity of rotavirus tics; (f) and those with allergy to dairy products. Actimel- vaccine (Isolauri et al, 1991) and shedding of Rotavirus in fermented milk was used as a starter in the community to diarrhoea (Saavedra et al, 1994). A specific strain, Lactobacillus prepare curd, in 20 families (21 children 1 – 5 y, who had curd casei DN-114001, was able to protect mice against infection by daily in diet), and 20 families used traditional home-made Rotavirus (Guerin et al, 1998). curd (19 children 1 – 5 y of age); bacterial strains used were In rural India, mortality is high due to diarrhoea and not determined. The diarrhoeal morbidity was recorded fort- cholera (Table 2). The disease-proportionate percentages of nightly (preliminary studies).

European Journal of Clinical Nutrition Fermented milk in diarrhoea treatment KN Agarwal and SK Bhasin S58 The Hospital Ethical Committee approved the study. Haemoglobin and Serum Naþ and Kþ were also estimated. Written consent was obtained from one of the parents. The data collected were entered in the computer using SPSS A detailed history regarding number, consistency of stools program. In order to find out the statistically significant and presence of mucus and blood was obtained. The infor- difference in the duration of control of diarrhoea amongst mation was recorded on a pre-structured schedule. All the three groups, the ANOVA test was applied. children were treated as per the recommended WHO diarrhoea treatment plan (WHO, 1988). The diarrhoea was considered controlled when the consistency of stools Results turned semi-solid or solid. The study period was 1 April Stool examination revealed that, of the 109 stool samples 1999 to January 2000. sent for culture, Vibrium cholera 01 (Ogawa) was seen in 18, One group of children was provided a fermented milk Salmonella in 3 and E. coli in 2. These children were excluded containing 108 Lactobacillus casei DN-114001 per gram (Acti- from the study. Twenty-four dropped out or were lost to mel by Danone, France). A second group was provided with follow-up. One-hundred and fifty children were followed up. Dahi, a type of yoghurt used in India, and the third group The stool sample in five patients showed rotavirus. No received ultra-heat-treated fermented milk (no bacteria), in ova=cyst was seen. All products were well accepted by the both the hospital and community studies. Fermented milk children. Of these, 75 children were studied in the hospital with Lactobacillus casei, and Indian Dahi and ultra- and 75 in the community. The sex ratio did not differ. The heat-treated-yoghurt (UHY) were provided by M=S Britannia proportion of patients with 1, 2, 3, 4 and 5 days of duration India Ltd, Delhi. All the three preparations were supplied of diarrhoea was 40, 15, 32, 10 and 3%, respectively. to us every week under refrigerated conditions. The Mild dehydration was present in 54% of the cases and consumption regimen of all the three preparations was one moderate dehydration in 31%. These could be managed on serving (100 ml) given three times a day. Children aged oral rehydration fluids. between 6 and 12 months received two servings a day. The remainder had no dehydration. No serum Naþ or Kþ These preparations were given double blind along with the level alterations were observed. The age and sex of the rehydration therapy. The three preparations used were children, literacy of the parents and duration of diarrhoea similar in all aspects such as packaging, taste, colour, in both the community and hospital study were matched. quantity etc. and were stored at 10 – 4C. The composition The haemoglobin level of the children varied between 86 of the three preparations had a similar protein content of and 98 g=l (mean 101 g=l). Ninety two percent of the study 2.8% in the preparation. UHY sugar was marginally higher children were undernourished as compared with the 50th and the fat content was double as compared with the other percentile weight for age (Agarwal & Agarwal, 1994). two preparations. This was also reflected in the higher solid Serum albumin level varied from 45 to 62 g=l. The main content in UHY preparation. The UHY preparation did not outcome measure evaluated was days taken for control of have any bacteria; Indian Dahi contained 108 Lactococcus diarrhoea. lactis, Lactococcus lactis cremoris and Leuconostac mesenteroids It was seen that, for both the hospital and the community cremoris per gram, while Actimel contained 108 of each study, diarrhoea was controlled earlier by 0.6 and 0.5 day, Lactobacillus casei DN-114001, Lactobacillus bulgaricus and respectively, in those receiving Actimel. The time taken to Streptococcus thermophilus per gram. Cultures were enumer- control diarrhoea also decreased by 0.3 and 0.2 day in the ated on a specific medium 8 days after production. group that received Indian Dahi, although it was less effec- Patients were allotted to each of these three groups based tive than with the group receiving Lactobacillus casei pre- on the random table provided by the Biostatistical Unit. paration (Table 3). In this short duration of treatment, the Stool samples available by 11 a.m could only be examined change in weight (loss) was < 2%. Lactobacillus casei were for microbiology. The following microbiological tests were seen in all the stool samples in children given Lactobacillus conducted: (i) direct microscopy for possible ova and cysts; casei preparation. (ii) profile of anaerobic bacterial flora; (iii) isolation and identification of Vibrium cholera 01 (Ogawa), Salmonella serovars, Shigella species, Escherichia coli-EPEC, ETEC and EHEC, and Rotavirus. The patients having Shigella, Salmonella Table 3 Effect of Actimel and Indian Oahi in control of acute diarrhoea and Vibrium cholera were excluded from the study. Using acid (in days) MRS agar identification of Lactobacillus casei in faeces was carried out. Hospital (n ¼ 75) Community (n ¼ 75) Plates (Lab M, Amersham, Aylerbury, England) were incu- n Mean s.d. n Mean s.d. bated anaerobically for 72 h at 37C and checked for isolates. Catalase-negative, Gram-positive bacteria were labelled as UHY 25 2.1 0.72 25 2.4 0.9 Lactobacilli. Lactobacillus isolates were further characterized Indian Dahi 27 1.8 0.61 25 2.2 1.0 Actimel preparation 23 1.5 0.48 25 1.9 0.8 and identified by criteria outlined in the anaerobe laboratory P < 0.01 P < 0.05 manuals (Holdman et al, 1974; Finigold, 1977).

European Journal of Clinical Nutrition Fermented milk in diarrhoea treatment KN Agarwal and SK Bhasin S59 Discussion Danan CG, Ghabanet C, Pedone C, Popot F, Vaissade P, Bouley C et al (1998): Milk fermented with yoghurt cultures and Lactobacillus Marteau et al (2001) reviewed studies related to control of casei compared with yoghurt and gelled milk. Influence on diarrhoea in infants by using Lactobacillus rhamnosus strain intestinal microflora in healthy infants. Am. J. Clin. Nutr. 67, GG; Lactobacillus casei strain Shirota; Enterococcus faecium 111 – 117. SF68, Saccharomyces boulardii, Lactobacillus reuteri, Bifidobac- Finigold SM (1977): Anaerobic Bacteria in Human Disease. New York: Academic Press. terium bifidum and Streptococcus thermophilus. These had a Gorbach SL, Change TW & Goldin B (1987): Successful treatment of curative as well as a preventive effect on diarrhoea in relapsing Clostridium difficile colitis with Lactobacillus GG. 2, children. In addition, Lactobacillus rhamnosus strain GG, 1519 – 1521. Saccharomyces boulardii and mixed strains of Lactobacillus þ Guerin DC, Meshin JC, Lambre F, Charpilienne A, Sepezah M, Bouley C, Cohen J & Andrieux C (1998): Development of a Bifidobacterium þ Streptococci significantly prevented travel- heterologous model in germ free suckling rats for studies of ler’s diarrhoea. Many of these strains were able to prevent rotavirus diarrhea. J. Virol. 72, 9298 – 9302. antibiotic-induced diarrhoea too. Holdman LV, Cato EP & Moor WEC (1974): Anerobe Laboratory In the present study such curative as well as preventive Manual. Blacksburg, VA: Polytechnic Institute and State University. Lactobacillus casei effects were observed by administering Isolauri E, Juntunen M, Rautanan T, Sillanaukee P & Kaivula T DN-114001 preparation. It was superior to Indian Dahi in (1991): A human Lactobacillus strain (L. casei sp. strain GG) reducing the duration of diarrhoea. Administration of promotes recovery from acute diarrhea in children. Pediatrics 88, yoghurt as well as Lactobacillus casei DN-114001 fermented 90 – 97. Manjunath N & Ranganathan B (1989a): Cytotoxic activity of Lacto- milk reduced severity of diarrhoea in normal children over a bacilli isolated from plant and dairy sources. Indian J. Exp. Biol. period of 6 months (Pedone et al, 1998; Agarwal et al, 2001). 127,72– 75. In infants fed these preparations for one month, the number Manjunath N & Ranganathan B (1989b): A cytotoxic substance of enterococci increased in faecal matter and proteolytic produced by a wild culture of Lactobacillus casei D-34 against tumour cells. Indian J. Exp. Biol. 27,141– 145. yoghurt. Those on Lactobacillus casei DN-114001 preparation Marteau PR, de Verse M, Cellier CJ & Schrezenmeir J (2001): Protec- showed an increase in Lactobacilli per gram faeces and tion from gastrointestinal diseases with the use of probiotics. Am. activity of B-glucuronidase B-glucoside (harmful enzymes) J. Clin. Nutr. 73(Suppl), 430S – 436S. decreased (Danan et al, 1998). In mice, both preparations Masud T, Sultana K & Shah MA (1991): Incidence of lactic acid bacteria isolated from indigenous Dahi. Asian Australasian J. Ani. increased lactase activity in the small intestine (Thoreux Sci. 4,329– 331. et al, 1998). These possible gut alterations may be of help Mitra AK & Rabbani GH (1990): A double-blind, controlled tiral of in the control of diarrhoea. It may be mentioned here that bioflorin (Streptococcus faecium SF68) in adults with acute diarrhea Indian Dahi has no lactase activity. due to Vibro cholerae and enterotoxigenic Escherichia coli. Gastro- enterology 99, 1149 – 1152. The results of the present study with Lactobacillus casei Mohanan KR, Shankar PA & Laxminarayana H (1984): Microflora of DN-114001, Actimel and Indian Dahi, Karnal Dairy, show Dahi prepared under house hold conditions of Bangalore. J. Food that these fermented milks could be used as a tool in the Sci. Technol. 21,45– 46. control of diarrhoea. Further regular use will also reduce the Pedone CA, Bemabeu AO, Postaire ER, Bouley CF & Reinert P (1999): The effect of supplementation with milk fermented by Lactobacillus number episodes of diarrhoea, as suggested by the commu- casei (strain DN-114001) in acute diarrhea in children attending nity study using Actimel as a starter. day care centers. Int. J. Clin. Pract. 53, 179 – 184. Prasad MM & Ghodekar DR (1990): Effect of mutagenic agents on the antibiotic-producing lactobacilli isolated from Indian fermented milk products (Brief Communication). In 13th International Dairy Acknowledgements Congress, Montreal, IL 2, p 368. We are grateful to Dr JM Antoine, MD from Danone, CIRDC, Prasad MM & Ghodekar DR (1991): Anti-microbial activity of lacto- France and Dr KL Gaba from M=S Britannia India Ltd. Delhi bacilli isolated from fermented milk products. Cult. Dairy Prod. 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