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Home , Lactase, Lactic acid, Lactic acid bacteria

European Journal of Clinical Nutrition (1999) 53, 339±350 ß 1999 Stockton Press. All rights reserved 0954±3007/99 $12.00 http://www.stockton-press.co.uk/ejcn

Review Lactic and the gastrointestinal tract

H Hove1*, H Nùrgaard1 and P Brùbech Mortensen1

1Medical Department CA, Division of Gastroenterology, Rigshospitalet and Paediatric Department L, Gentofte University Hospital, Denmark.

Objective: This review summarises the effects of bacteria on malabsorption, bacterial=viral or associated diarrhoea, and describes the impact of on cancer and the fermentative products in the colon. Results: Eight studies (including 78 patients) demonstrated that lactase de®cient subjects absorbed lactose in better than lactose in , while two studies (25 patients) did not support this. Two studies (22 patients) showed that unfermented acidophilus milk was absorbed better than milk, while six studies (68 patients) found no signi®cant differences. Addition of lactose hydrolysing , lactase, to milk improved lactose malabsorption in seven studies (131 lactose malabsorbers), while one study (10 malabsorbers) demonstrated no improvement. Lactic acid bacteria alleviated travellers' diarrhoea in one study (94 individuals) while a study including 756 individuals was borderline statistically signi®cant. One study (50 individuals) did not ®nd an effect of lactic acid bacteria on travellers' diarrhoea. Six studies (404 infants) demonstrated a signi®cant effect of lactic acid bacteria on infant diarrhoea, while one study (40 infants) did not. Lactic acid bacteria moderated antibiotic associated diarrhoea in three studies (66 individuals), while two studies (117 individuals) were insigni®cant. Conclusions: Lactase de®cient subjects bene®t from a better lactose absorption after ingestion of yoghurt compared with milk and from milk added lactase, whereas ingestion of unfermented acidophilus milk does not seem to improve lactose absorption. The majority of studies support that lactic acid bacteria alleviate bacterial=viral induced diarrhoea, especially in infants, while the effect on antibiotic associated diarrhoea is less clear. Experimental studies indicate an effect of lactic bacteria on human cancer lines, but clinical evidence is lacking. A `stabilising' effect of lactic acid bacteria on the colonic ¯ora has not been documented. Descriptors: lactic acid bacteria; lactobacilli; bi®dobacteria; lactate; lactic acid; lactose malabsorption; antibiotic associated diarrhoea; diarrhoea; ; colonic cancer

Introduction (homofermentative) or at least 50% (heterofer-mentative) lactate producers (Kandler, 1983, Table 1). Interest in the bene®cial effects of lactic acid bacteria dates to Studies on the ¯ora of the gastrointestinal tract report the Russian scientist, E. Metchnikoff (1845 ± 1919), who that numbers of bi®dobacteria may exceed 1011 per gram of proposed that the extended longevity of the Balkan people faeces (Finegold et al, 1983) accounting for 6 ± 25% of all could be attributable to their practice of ingesting fermented cultivable bacteria in faeces (Kitsuoka, 1984; Scardovi, milk products (Metchnikoff, 1908). He believed that gastro- 1986) whereas lactobacilli only constitute 0 ± 1% of the intestinal disturbances occur by intestinal growth of putre- bacteria in the faeces of healthy (Hill & Drasar, factive microbes, and that lactic acid bacteria could minimise 1975; Gorbach, 1971; 1986, Brown, 1977). The exception or prevent the harmful effects of these microbes. The role of of this condition is the dominant presence of bi®dobacteria lactic acid bacteria within the gastrointestinal tract has been in breast-fed infants (Stark & Lee, 1982). Lactic acid one of the most controversial subjects of the area of intestinal bacteria are described to be of nutritional and therapeutic microbial ecology. No other group of bacteria has been bene®t to the host in several clinical conditions. The proposed to be responsible for so many different bene®cial proposed effects of lactic acid bacteria on the intestinal actions, but non-conclusive or insigni®cant results are often tract are mentioned in Table 2. Discussion of these speci®c reported when attempts are made to con®rm that lactic acid health targets follow. bacteria improve the health of the host. Lactic acid bacteria supposedly exert an impact on the Lactic acid bacteria consists of heterogeneous group of small as well as the large intestine. First, bacterial derived gram-positive bacteria, whose main lactase from ingested lactic acid bacteria might enhance the from is lactate. The group comprises cocci of lactose to and in the small (, , ) and rods (lacto- intestine, which are rapidly absorbed or fermented. Sec- and bi®dobacterium), which are either exclusively ondly, lactic acid bacteria may have an impact on the colonic ¯ora in situations in which some sort of imbalance *Correspondence: Hanne Hove, Ph.D., Hovmarksvej 77, 2920 exists. The exact nature of this microbial imbalance and Charlottenlund. Received 14 August 1998; revised 7 December 1998; accepted how it is corrected by the ingestion of lactic acid bacteria is 20 December 1998 not known. Lactic acid bacteria H Hove et al 340 Table 1 The genera of lactic acid bacteria, their fermentation type and main products (Kandler, 1983)

Genus Fermentation type Main product Con®guration of lactate

Streptococcus homofermentative lactate L-lactate Pediococcus homofermentative lactate DL-lactate homofermentative lactate DL-lactate Leuconostoc heterofermentative lactate : acetate D-lactate Bi®dobacterium heterofermentative lactate : acetate L-lactate

Table 2 Studies evaluating the clinical effect of lactic acid bacteria

Positive studies Negative studies

 Yogurt improves lactose absorption compared with milk? (Table 3 a) 8 2  Unfermented acidophilus milk improves lactose absorption compared with milk? (Table 3 b) 2 6  Lactase relieves lactose malabsorption among lactose malabsorbers? (Table 5) 7 1  Lactic acid bacteria reduce incidence of travellers diarrhoea? (Table 6 a) 1 2  Lactic acid bacteria reduce incidence of diarrhoea among infants? (Table 6 b) 6 1  Lactic acid bacteria alleviate antibiotic associated diarrhoea? (Table 7 a) 3 2

Over the past decade there has been increased interest in where intake of marked bi®dobacteria resulted in a rise in bacterial food supplements, called . The de®ni- faecal levels to approximately 108 bi®dobacteria=g faeces tion of a being: `A live microbial feed supplement followed by a gradual decrease after ingestion ceased. which bene®cially affects the host animal by improving its Thus, attempts to increase the number of lactic acid intestinal microbial balance' (Fuller, 1989). There are bacteria in the gastrointestinal tract by ingestion generally several characteristics that are of importance for organisms results in a temporary colonisation of the gut, which used as probiotics (Kim, 1988). These include: the organ- persists as long as the lactic acid bacteria are ingested ism should maintain viability and activity in the carrier (Goldin et al, 1992; Deneke et al, 1988; Saxelin et al, food before consumption, should survive the upper gastro- 1991). After consumption of a bacterium, recovery from intestinal tract, be capable of surviving and growing in the the faeces is not evidence of implantation, even if recovery intestine, be a normal inhabitants of the intestinal tract, and persists for a period after consumption has stopped. Con- eventually produce bene®cial effects when in the intestinal tinued faecal recovery of ingested lactic acid bacteria may tract. Further, the organism must be non-pathogenic and be because of residence time in the large intestine that non-toxic. exceeds microbial generation time.

Survival through the gastrointestinal tract Impact on the The effect of lactic acid bacteria in the intestine requires that the bacteria or at least their survive the acid Relieving in lactose malabsorbers gastric content and are active after the passage of the Lactose is the predominate carbohydrate in milk, and it stomach. Studies of orally administered lactic acid bacteria requires enzymatic hydrolysis to the monosaccharides glu- have demonstrated that the lactic acid bacterial counts in cose and galactose before intestinal absorption. Small the small intestine increase signi®cantly after ingestion intestinal epithelial cells () produce b-galacto- (Robins-Browne et al, 1981). The ability of Bi®dobacter- sidase in childhood, and some people continue to produce ium bi®dum to survive the passage through the upper b-galactosidase throughout life, but globally most adults gastrointestinal tract when ingested in fermented milk are lactose malabsorbers and are as non-milk consumers was investigated by Pochart et al (1992) using in vivo deprived of an important source of protein and calcium. ileal perfusion, and he found that the average number of Although lactose malabsorption is common worldwide, the bi®dobacteria recovered from the terminal ileum consti- symptomatic expression of lactose intolerance is less so. tuted approximately 25% of the number ingested (ingested Lactose intolerance has a substantial psychological compo- dose 1010 bacteria). This is in consistency with a study of nent: among individuals who believed they were lactose ileotomic patients ingesting lactic acid bacteria, where malabsorbers 64% were shown to be lactose digesters bi®dobacteria were cultured from ileostomic contents in (Rosado et al, 1987); most lactase-de®cient people can eight of nine ileostomists within six hours oral administra- consume one glass of milk per day asymptomatically tion of 1010 bacteria, while not present in the ileostomy (Savaiano & Kotz, 1988), and 85% of individuals with ef¯uents of patients during control (Hove et al, discomfort have only mild symptoms (Scrimshaw & 1994). Similarly, faecal levels of speci®c strains of lactic Murray, 1988). acid bacteria increase after ingestion. Goldin et al (1992) Carbohydrate malabsorption increases the delivery of followed the excretion of Lactobacillus gg in faeces 3 and unabsorbed to the colon bacteria and results 7 d after subjects consumed 1011 of Lactobacillus gg as in increased intestinal production and respiratory excretion either concentrate or as a drink. Strain gg increased of hydrogen. Breath hydrogen excretion is therefore used as 4 ± 6 log cycles in almost all subjects, and remained present an indicator of malabsorption of simple carbohydrates as in faeces 7 d after feeding stopped. These results have also (Welsh et al, 1981). A dose-response rela- been obtained with bi®dobacteria (Bouhnik et al, 1992), tionship between breath hydrogen excretion and the amount Lactic acid bacteria H Hove et al 341

Figure 1 Prevalence of adult lactose malabsorption in Europe in percent (Gudmand-Hùyer & Skovbjerg, 1996). (Reprinted by permission of the author and Scandinavian University Press).

of malabsorbed (non-absorbable ) has during the fermented process (McDonough et al, 1987; been found by Rumessen et al (1990), while breath hydro- Gorbach, 1990). Reduced malabsorption may, however, not gen excretion is of limited value in the evaluation of only be associated with the decreased lactose concentra- malabsorption of complex dietary carbohydrates (Nord- tion, but also to high lactase activities in yogurt (Kolars et gaard et al, 1995). al, 1984, Savaiano et al, 1984). In considering how much of The geographic incidence of lactose malabsorption is the improved was due to reduction in lactose and shown in Figure 1 (Gudmand-Hùyer & Skovbjerg, 1996). how much was due to ingested lactase supplied by the Yogurt is made from milk enriched with milk proteins cultures, the following investigations were performed (to improve consistency), which is incubated with two or (Table 4). three species of lactic acid bacteria (that is L. bulgaricus When yogurt was heated in order to inactivate lactase, and S. thermophilus)at42C until the pH drops to approxi- malabsorption measured by breath hydrogen test was still mately 4.5 (Martini et al, 1987). The resulting yogurt is signi®cantly lower than after milk ingestion. Since both cooled and stored until use. products had little or no lactase activity, it was assumed In contrast to yogurt, sweet acidophilus milk is unfer- that the difference was due to reduced lactose in the heated mented, made by adding high concentrations of viable L. yogurt (McDonough et al, 1987). Similarly, when lactose acidophilus cells to cold milk. In storage below 5C L. was added to yogurt so that lactose concentration was equal acidophilus do not multiply and thus, sweet acidophilus to that in milk, the mean breath hydrogen value was milk has the bene®ts of lactase activity, without the acid signi®cantly lower than the value for milk, thus indicating taste of the corresponding fermented product (Martini et al, a response to lactase activity (McDonough et al, 1987). 1991). This was further substantiated by adding commercial lac- It seems evident that lactose intolerant individuals are tase to heated yogurt, in an amount that produced an able to substitute fermented milk (yogurt) for fresh milk, activity level comparable to that found in yogurt, resulting Table 3 (a). in signi®cantly lower breath hydrogen test than for heated The rational for using fermented milk relates to the fact yogurt. With similar lactose content and lactase activity in that lactose content is reduced between 25 and 50 percent heated yogurt added lactase and in yogurt, the two products Lactic acid bacteria H Hove et al 342 Table 3 The ability of fermented and non- in relieving lactose malabsorption. Comparison of yoghurt with regular milk is indicated by (a); while comparison of unfermented acidophilus milk with regular milk is indicated by (b). Comparison of lactose or lactulose with yogurt or heated yogurt with yogurt is indicated by (c)

Number of lactose Results (absorption measured by Reference malabsorbers Test Genera and dose breath hydrogen test)

1981 Payne (b) 11 SAM vs milk L. acidophilus NS between milk and SAM lactose conc.? cfu=d? 1983 Kim (b) 12 SAM vs milk L. acidophilus SAM signi®cantly better absorbed lactose conc.? 1011 cfu=d than milk (P < 0.01) 1983 Newcomer (b) 18 SAM vs milk L. acidophilus NS between milk and SAM lactose conc.? 109 cfu=d 1984 Gilliland (c) 6 Y vs HY L. bulgaricus Y signi®cantly better absorbed than equal lactose conc. S. thermophilus HY (P < 0.05) cfu=d? 1984 Kolars (a) 10 Y vs milk L. bulgaricus Y signi®cantly better 18 g lactose S.thermophilus absorbed than milk (P < 0.01) 1984 Savaiano (a,b) 9 Y, HY and SAM genera? 1) Y sign®cantly better vs milk 109 ±1011 cfu=d absorbed than milk, HY, and SAM 20 g lactose (P < 0.05) 2) NS between milk and SAM 1987 McDonough (a,b) 14 Y, HY, L. bulgaricus 1) Y signi®cantly better Y ‡ lactose, S. thermophilus absorbed than milk, HY, and Y ‡ HY ‡ lactase, SAM, L. acidophilus lactose (P < 0.05) SAM-S vs milk 3Á1011 cfu=d2)Y‡ lactose and SAM-S signi®cantly better 16 g lactose absorbed than milk (P < 0.05) 3) NS between SAM and milk and between Y and HY ‡ lactase. 1988 Wytock (c) 8 Lactose vs 3 L. bulgaricus Two kinds of Y sign®cantly better kinds of Y S. thermophilus absorbed than lactose (P < 0.05) 20 g lactose cfu=d? 1989 Onwulata (a,b) 10 Y, SAM vs milk L. bulgaricus 1) Y signi®cantly better absorbed 18 g lactose S. thermophilus than milk and SAM L. acidophilus 2) NS between SAM and milk cfu=d? 1990 Marteau (a) 8 Y and HY vs genera? 1) Y and HY signi®cantly better milk cfu=d? absorbed than milk 18 g lactose (P < 0.001) 2) NS between Y and HY 1991 Martini (a) 7 Y vs milk L. bulgaricus Y signi®cantly better absorved than 18 g lactose S. thermophilus milk (P < 0.025) 1011 cfu=d 1991 Lin (a,b) 10 Y and SAM vs L. bulgaricus 1) Y signi®cantly better absorbed milk S. thermophilus than milk (P < 0.01) 20 g lactose L. acidophilus 2) SAM signi®cantly better absorbed 4Á1010cfu=d than milk (P < 0.05) 1994 Arrigoni (a) 11* Y vs milk genera? NS between milk and Y 20 g lactose cfu=d? 1994 Kotz (a) 10 High galactosidase L. bulgaricus High galactosidase Y signi®cantly Y vs milk S. thermophilus better absorbed than milk 20 g lactose 2Á108 cfu=d(P < 0.05) 1995 Dehkordi (b) 6 SAM vs milk L. acidophilus NS between milk and SAM 18 g lactose cfu=d? 1995 ShermaÈk (a) 14 Y and HY vs L. bulgaricus NS between milk, Y and HY milk S. thermophilus 12 g lactose cfu=d? 1996 Vesa (c) 14 lactulose vs L. bulgaricus 1) Fermented milk signi®cantly 3 kinds of fermented S. thermophilus better absorved than lactulose milk products Bi®dobacteria 2) NS between the 3 kinds of fermented 18 g lactose vs 10 g L. acidophilus milk lactulose cfu=d?

Abbreviations: Lactose malabsorption was determined by breath hydrogen test; Y: yogurt; HY: heated yogurt; SAM: sweet acidophilus milk; SAM-S: sonicated sweet acidophilus milk (cell membranes disrupted); cfu=d: colony forming units lactic acid bacteria ingested per day; NS: not signi®cantly different * : patients with jejunocolic anastomsis. Lactic acid bacteria H Hove et al 343 would be expected to have comparable utilisation and breath hydrogen excretion and the severity of gastrointest- indeed the breath hydrogen values were not signi®cantly inal symptoms (Dehkordi et al, 1995; ShermaÈk et al, 1995). different (McDonough et al, 1987). In contrast to the above mentioned results, lactose The majority of studies (Table 3a) support that yogurt intolerant individuals who ingest an unfermented milk enhances absorption of lactose when compared with product containing lactic acid bacteria (sweet acidophilus equivalent amounts of lactose in milk. The improved milk) with low concentration of bacterial lactase have no tolerance of lactose when consumed as yogurt containing apparent bene®t from this as compared with milk, Table 3 active live cultures is at least in part related to the inherent (b), and Table 4. If, however, the bacterial cell-membranes galactosidase activity of the yogurt bacteria, which hydro- are damaged by sonication, and intracellular lactase is lyse a part of the ingested lactose (Kotz et al, 1994; released, breath hydrogen values were found in the same SchermaÈk et al, 1995; Sanders, 1993). low level as for heated yogurt (McDonough et al, 1987). In spite of similar average values for the area under the Thus, it seems that the bacterial lactase is not available or is breath hydrogen curve, yogurt and heated yogurt was insuf®cient to exert a measurable effect in sweet acidophi- associated with a delay in the time to breath hydrogen lus milk during digestion, but can be made accessible after rise and the time to peak breath hydrogen when compared disruption of the (Kolars et al, 1984; with milk (ShermaÈk et al, 1995), and studies have McDonough et al, 1987). In accordance, an increase in demonstrated a relationship between the rate of rise in microbial galactosidase of yogurt, enhance lactose absorp- tion compared with conventional yogurt (Kotz et al, 1994). These results are not supported by Kim & Gilliland 1983) Table 4 Lactose and lactase activity of milk and yogurt test products and Lin et al (1991), but are in accordance with Hove et al (McDonough et al, 1987) (1994), who did not ®nd an improved lactose utilisation in lactase de®cient subjects after intake of large amounts of B. Lactose Lactase 10 Product (g=250 ml) (mg glucose=dl) bi®dum (4.2Á10 cells). The ingestion of commerical lactase (often of Control milk 15.7 26 origin) either together with, immediately before or within Yogurt 12.0 3724 5 minutes after milk consumption (meal-time treated milk) Yogurt ‡ lactose 15.7 3724 Heated yogurt 12.0 43 is reported to improve lactose utilisation in most studies, Heated yogurt ‡ lactase 12.0 4138 while refuted in a single study (Onwulata et al, 1989), Sweet acidophilus milk 15.7 1427 (Table 5). Milk treated with a commercial lactase prepara- Sweet acidophilus milk with 15.7 4263 tion 20 ± 24 h prior to ingestion is approximately 90% sonicated cells hydrolysed (Payne et al, 1981). This milk has therefore a Lactase activity was determined by measuring the amount of glucose sweeter taste than regular milk. Aspects of lactic acid released on hydrolysis during incubation at 37C for 2 hours. Values are cultures effect on lactose digestion in lactose malabsorbers means of triplicate analysis. are discussed in detail in review articles (Gorbach, 1990;

Table 5 Ability of commercial lactase in relieving lactose malabsorption in lactose malabsorbers. The b-galactosidase preparations were ingested immediately before or after milk consumption ( Æ 5 min)

References Lactose malabsorbers Treatment Doses Results

1984, Rosado 13 LactAid, Lactase N 18 g lactose LactAcid and Lactase N vs milk 1.5 g LactAid signi®cantly improved 0.4 g Lactase N lactose absorption 1985 Solomons 10 LactAid, Lactase N 18 g lactose LactAid signi®cantly improved vs milk 2 g LactAid lactose absorption 0.3 g Lactase N (P < 0.01). Lactrase N did not improve absorption. 1986 Rosado 21 Lactase N vs 18 g lactose Lactase N signi®cantly milk 0.4 g Lactase N improved lactose absorption 1987 Barillas 9 infants LactAid, Takamine 12 g lactose LactAid and Takamine vs milk 1.0 g LactAid signi®cantly improved 0.3 g Takamine lactose absorption 1988 Lami 52 LactAid vs milk 25 g lactose LactAid signi®cantly improved LactAid? lactose absorption (P < 0.0005) 1989 DiPalma 10 Lactrase vs 50 g lactose Lactrase signi®cantly improved milk 0.5 g Lactrase lactose absorption (P < 0.05) 1989 Onwulata 10 LactAid vs milk 18 g lactose LactAid did not improve LactAid? lactose absorption 1992 Corazza 16 A. niger Lactose? A. niger b-galactosidase b-galactosidase Galactosidase? signi®cantly improved lactose absorption (P < 0.01)

Abbreviations: Lactose malabsorption was determined by the use of breath hydrogen test after ingestion of lactose; A niger: niger derived b- galactosidase. LactAid is a b-galactosidase derived from the yeast Kluyveromyces lactis (pH and temperature optimum 6.8 and 37C). Lactase N, Takamine and Lactrase are b-galactosidase preparations derived from the Asperigillus (Lactase: : pH and temperature optimum 4.4 and 60C; Takamine and Lactrase: Aspergillus oryzae). Lactic acid bacteria H Hove et al 344 Sanders, 1993; Gurr, 1987, Fuller, 1989, Shahani & Ayebo, 1975). It is well established that travellers' diarrhoea to a 1980; Bengmark, 1998). great extent can be prevented by prophylactic intake of , which have been reported to provide protection Prophylaxis against E. coli and antibiotic associated rates in the range of 60 ± 95% (DuPont et al, 1986; Sack, diarrhoea 1986). The use of these agents may cause adverse reactions Although the intestinal ¯ora is a very steady , the and lead to emergence of resistant bacterial strains. There- balance can be disturbed by a number of factors such as fore, the effect of lactic acid bacteria and other probiotics in bacteria, viruses and antibiotics. promoting gastrointestinal health by inhibiting or eliminat- Travellers from United States and Northern Europe ing enteric have been investigated. often suffer diarrhoea when visiting less developed regions Numerous publications based on uncontrolled clinical of the world. The clinical picture varies from a short and observations have suggested a role for lactic acid bacteria mild attack with watery diarrhoea to a severe incapacitating in the prevention of diarrhoea (Beck & Necheles, 1961; disease with a duration of more than a week. In 40 ± 70% of Winkelstein, 1955), and several preparations of lactic acid the cases the etiological agent is enterotoxigenic Escher- bacteria (Paraghurt1, Trevis1, Biotura1) are available ichia coli (E. coli) (Merson et al, 1976; Gorbach et al, without prescription as prophylaxis against diarrhoea.

Table 6 The effect of lactic acid bacteria on travellers' diarroea (a), diarrhoea in infants (b) and E. coli induced diarrhoea (c)

References No. individuals Treatment Doses Results

1978 P.-Olano (a) 50* DB, R Lactinex 6Á109=d Results (no. indiv. with Travel to Mexico diarrhoea=no. indiv.) 7=17 lactic acid bact. 2=14 placebo (NS) 1981 Clements (c) 48 DB, R Lactinex 2Á109=d Results (no. indiv. with Challenge with 108- diarrhoea=no. indiv.) 1010E. coli 16=23 lactic acid bact. 17=25 placebo (NS) 1985 Black (a) 94* DB, R L. acidophilus,B. 9Á109=d Results (no. indiv. with Travel to Egypt bi®dum, L. bulgari- diarrhoea=no. indiv.) cus, S. thermophilus 17=40 lactic acid bact. 29=41 placebo (P ˆ 0.019) 1990 Boudraa (b) 45 infants (3 ± 36 m) Yogurt vs milk ? > 5 days: R. Persistent diarrhoea 3=21 yogurt 8=24 milk (P < 0.05) Weight loss > 5%: 0=21 yoghurt 2=24 milk ((P < 0.05) 1990 Oksanen (a) 756 DB, R. Lactobacillus GG 2Á109=d Frequency of diarr. (%): Travel to Turkey 41.0% lactic acid bact. 46.5% placebo (P ˆ 0.065) 1991 Isolauri (b) 71 infants (4 ± 45 m), R Lactobacillus gg 1010 ± 11)=d Duration of diarr. (days): 1) yogurt 1.4 Æ 0.8 yogurt Rotavirus diarrhoea 2) freeze-dried 1.4 Æ 0.8 lactbact. powder powder 2.4 Æ 1.1 placebo (P < 0.001) 1994 Saavedra (b) 55 infants (5 ± 24 m) B. bi®dum, S. termophilus 1010 ± 11=d Results (no. indiv. with DB, R philus diarrhoea=no.indiv.) Rotavirus diarrhoea 2=29 lactic acid bact. 8=26 placebo (P ˆ 0.035) Rotavirus shedding: 3=29 lactic acid bact. 10=26 placebo (P ˆ 0.025) 1995 Majama (b) 49 infants Three groups Duration of diarr. (days): DB, R. 1) Lactobacillus gg 1.8 Æ 0.8 Lactobacillus gg Rotavirus diarrhoea 2) Lactophilus 2.8 Æ 1.2 Lactophilus 3) Yalacta 2.6 Æ 1.4 Yalacta (P ˆ 0.04) 1997 Guarino (b) 61 infants (3 ± 36 m) oral rehydration Æ 6Á109=d Duration of diarr. (days): R. liophylised Lacto- 2.9 Æ 1.2 lactic acid bact. Rotavirus diarrhoea bacillus gg 6.1 Æ 1.7 no lactic acid bact. (P < 0.01) 1997 Shornikova (b) 40 infants (6 ± 36 m) freeze-dried Lacto 1010 ± 11=d Duration of diarr. (days): R. bacillus reuteri 1.7 Æ 1.6 lactic acid bact. Acute diarrhoea 2.9 Æ 2.3 placebo (P ˆ 0.07) 1997 Shornikova (b) 123 infants (1 ± 36 m) oral rehydration Æ 5Á109=d Duration of diarr. (days): DB, R. freeze-dried Lacto- 2.7 Æ 2.2 lactic acid bact. Acute diarrhoea bacillus gg 3.7 Æ 2.8 placebo (P ˆ 0.03)

* Number of patients in the study; results only given for a part of the group. Lactinex: a commercial preparation of lactobacilli, containing dried viable L. acidophilus and L. bulgaricus in equal proportions (5Á108 bacteria per g). Lactobacillus gg: aq lactobacillus strain isolated from healthy humans on the basis of its ability to resist acid and bile, and to adhere to the intestinal mucosa. Lactophilus: subsp. Rhamnosus. Yalacta: combination of S. thermophilus, L. delbruÈckii and L. casei. Abbreviations: No. indiv. with diarr.=no. indiv.: Number of individuals with diarrhoea=total number of individuals; DB.: double blind; R.: randomised; bact.: bacteria, m: months; y: years. Lactic acid bacteria H Hove et al 345 Table 7 The effect of lactic acid bacteria on antibiotic associated diarrhoea (a) and dif®cile induced diarrhoea (b).

References No. individuals Treatment Doses Results

1979 Gotz (a) 79 patients (19 ± 88y) Lactinex 2Á109=d No. indiv. with DB, R. diarrhoea=no. indiv. *) Ampicillin therapy 3=36 lactic acid bact. 9=43 placebo (NS) 1987 Colombel (a) 10 volunteers (22 ± 50y) Bi®dobact. longum ? Stools per day: DB, R. 1.2 Æ 0.1 lactic acid bact. Erythromycin 1.9 Æ 0.4 placebo (2 g=d for 3 days) (P < 0.025) Stool weight (g): 145 Æ 16 lactic acid bact. 208 Æ 29 placebo (P < 0.025) 1987 Gorbach (b) 5 patients (24 ± 93 y) Lactobacillus gg 1010=d 4 patients responded C. dif®cile induced with decrease in diarrhoea **) stool frequency and became cytotoxin negative. 1990 Siitonen (a) 16 volunteers R. Lactobacillus gg ? Stools per day are reduced Erythromycin when receiving lactic acid (1.2 g=d for 7 days) bacteria (P < 0.05) 1990 Tankanow (a) 38 infants (5 ± 72 m) Lactinex 2Á109=d No. indiv. with DB, R. diarrhoea=no. indiv. *) Amoxicillin 10=15 lactic acid bact. 16=23 placebo (NS) 1991 Contardi (a) 40 Infants (8 ± 36 m); R. B. bi®dum ? Stools per day: Oral amoxicillin (50 L. acidophilus 2.0 Æ 0.3 lactic acid bact. mg=kg=d) 2.7 Æ 0.5-lactic acid bact. P < 0.001 1995 Biller (b) 4 Infants (5 ± 70 m) Lactobacillus gg 1Á109=d All patients responded within C. dif®cile induced 5 ± 7 d: decrease in stool diarrhoea**) frequency and became cytotoxin negative

Lactinex is a commercial preparation of dried viable L. acidophilus and L. bulgaricus in equal proportions (5Á108 bacteria=1 g (Tankanow et al, 1990)). Abbreviations: No. indiv. with diarr.=no. indiv.: Number of individuals with diarrhoea=total number of individuals; DB.: double blind; R.: randomised; bact.:bacteria; *): both oral and=or injectable ampicillin against infectious disease other than diarrhoea; **) positive test for C dif®cile cytotoxin in the stool; m: months.

In controlled clinical trials lactic acid bacteria have containing 1010 ± 11 Lactobacillus casei, a Lactobacillus had varying degrees of success in preventing diarrhoea: casei freeze-dried powder (1010 ± 11), or placebo (pas- in a double-blinded randomised study, 48 volunteers teurised yogurt with only trace amounts of live lactic received either Lactinex (L. acidophilus and L. bulgaricus) acid bacteria). The mean duration of diarrhoea after in total of 2Á108 bacteria=d or placebo and were challenged commencing the therapy was signi®cantly shorter in the with enterotoxin producing E. coli (108 Ð1010). No sig- two groups receiving live lactic acid bacteria in compar- ni®cant differences were noted with respect to attack rate, ison with the placebo group, P < 0.001. Analysis of incubation period, duration of diarrhoea, volume or number speci®c antibody-secreting cells among circulating lym- of liquid stools (Clements et al, 1981), (Table 6 (c)). On the phocytes revealed that lactic acid bacterial therapy contrary, a prospective double blind investigation of tra- resulted in an augmentation of the local immune defence vellers' diarrhoea in 94 tourists travelling to Egypt found re¯ected in an IgA speci®c antibody-secreting cell that daily intake of 1010 lactic acid bacteria (L. acidophilus, response to rotavirus (Kaila et al, 1992). B. bi®dum, L. bulgaricus, S. thermophilus) signi®cantly The use of lactic acid bacteria as prophylaxis against E. reduced the incidence of diarrhoea compared to the placebo coli diarrhoea is supported by both in vitro and in vivo treated group (17 cases of diarrhoea in the lactic acid animal studies. Investigations using rabbit ileal loops have bacteria treated group in comparison with 29 in the placebo shown that lactic acid bacteria signi®cantly reduce the ¯uid treated group, P ˆ 0.02) (Black et al, 1989), Table 6 (a). retention caused by enterotoxigenic E. coli (Foster et al, Rotavirus is a common cause of non-bloody diarrhoea in 1980; Johnson & Calia, 1979). The reduced ¯uid accumu- children accounting for 50 ± 75 percent of episodes of acute lation depended on the administration of a large dose of diarrhoea in children below 3 y referred to the hospital lactic acid bacteria (108=bacteria), whereas the individual (Guarino et al, 1997; Shornikova et al, 1997). No speci®c ingredients in the lactic acid bacterial preparation did not therapy is available for rotavirus, and treatment is limited to demonstrate any anti¯uid response. rehydration. Probiotics has been suggested as a mode of In vivo studies of 28 piglets placed in pens, contami- preventing or moderating the . The group of nated with enterotoxigenic E. coli demonstrated that pro- Isolauri et al (1991) has reported a positive effect of a phylactic feeding with lactic acid bacteria (109=d) human lactic acid bacterial species, Lactobacilli casei signi®cantly reduced the mortaility of E. coli enteropathy strain gg, on the recovery from acute rotavirus induced (1=14 in treatment group in comparison to 7=14 in the diarrhoea in children. The children were randomised to control group) (Nielsen et al, 1988). Further, the average either a lactic acid bacteria fermented milk product daily weight gain (0 ± 14 d post weaning) was signi®cantly Lactic acid bacteria H Hove et al 346 higher in the treatment group (231 g) compared to the teurised yoghurt, Table 7. Further, usual side effects as surviving control group (101 g). abdominal distress, pain and ¯atulence tended to be Similar convincing results has been reported by Under- reduced in the lactic acid bacterial treated group, while dahl et al (1982). When Streptococcus faecium were fed to the difference in faecal volumes was not signi®cantly gnotobiotics (animals which are obtained by hysterotomy different. In ®ve studies of antibiotic associated diarrhoea and kept under sterile conditions and therefore have no including 183 subjects, an alleviating effect of lactic acid intestinal ¯ora) to prevent E. coli induced diarrhoea. Pigs bacteria ingestion was found in 3 studies including 66 fed S. faecium and challenge exposed with E. coli devel- individuals. Therefore, no convincing effect of lactic acid oped mild diarrhoea, but none of the pigs died, and they bacteria on antibiotic associated diarrhoea was demon- continued to eat well and gained weight. Pigs given E. coli strated. The studies of C. dif®cile induced diarrhoea only, developed severe diarrhoea and lost weight, and 5 of included only ®ve and four patients, allowing no conclu- 8 infected pigs died. sions to be drawn. Recent controlled studies have demon- How lactic acid bacteria diminish diarrhoeal disease is strated that another probiotic, the yeast Saccharomyces not known. One proposed mechanism is competitive colo- bouradii, reduce the risk of recurrent Clostridium dif®cile nisation. Competitive colonisation occurs when one intest- associated disease (including patients with Clostridium inal microbe interferes with the colonisation of another. But dif®cile diarrhoea, colitis and pseudomembranous colitis documentation of this phenomenon by observing lactic (McFarland et al, 1994)) and antibiotic associated diarrhoea cultures displacing pathogens or preventing (McFarland et al, 1995; Surawicz et al, 1989). adherence has been dif®cult experimentally. Other studies have demonstrated that lactic acid bacteria stimulate macrophage phagocytosis of viable Salmonella Impact on colon (Hatcher & Lambrecht, 1993), enhance IgA production in intestinal secretions (Perdigon et al, 1990), produce an Prevention of colonic cancer substance (Shahani & Ayebo, 1980; Silvia There is considerable interest in the metabolic activities of et al, 1987), inhibit cell attachment and cell invasion by the intestinal micro¯ora, especially in relation to the enterovirulent bacteria (Bernet et al, 1994), and decrease aetiology of colon cancer. Epidemiological studies have intestinal permeability for macromolecules during rotavirus suggested a correlation between intake of a `Western diet' induced diarrhoea (Isolauri et al, 1993). abundant in beef, fat, and protein but low in ®bre, fruit, and In conclusion, Table 6 shows that seven of nine , and the occurrence of colon cancer. Indeed, a studies since 1985, show an effect of lactic acid bacteria positive correlation has been found in several countries on a 5% signi®cance level while two studies show an between dietary factors such as and animal fat effect on a 7% sign®cance level. Two early studies are consumption and the incidence of large bowel cancer insigni®cant. The overall impression left by the men- (Howel, 1975). Finland is an exception, being a nation tioned human studies is that lactic acid bacterial therapy with a high per capita fat consumption and a relatively low can limit the course of diarrhoeal diseases, especially incidence of colon cancer (Armstrong & Doll, 1975). Dairy rotavirus diarrhoea (Table 6). A possible explanation for products, especially yogurt, are a common compound of the the often found inconsistency of results may be the use of Finnish diet, and possibly as a result, the intestinal micro- different species and subspecies of lactic acid bacteria ¯ora of the Finns harbours high numbers of lactic acid with different af®nity towards the human intestine. Host bacteria (International Agency for Research on Cancer, speci®city in colonisation by individual species has been 1997). In an attempt to explain these epidemiological demonstrated: L. acidophilus, L. fermentum,andL. plan- ®ndings, alterations in the metabolic activity of the intest- tarum are commonly found in the faeces of humans, inal ¯ora have been studied. The studies have involved whereas L. bulgaricus, the organism used in combination measurements of key enzymes: b-glucuronidase, azoreduc- with S. thermophilus to make yogurt, is unable to tase, and nitroreductase, which catalyse the conversion of colonise the bowel. The speci®city is possibly related indirect acting carcinogens to proximal carcinogens in the to the individual lactic acid bacteria ability to colonise large bowel (Goldin & Gorbach, 1984). Oral supplementa- mucosal surface by binding to epithelial cells. Variations tion of the diet with viable bile-resistant L. acidophilus of in lactic acid bacterial preparations and storage of fer- human origin caused a signi®cant decline in these three mented products may also in¯uence results (Gorbach, key-enzymes (Goldin & Gorbach, 1984; Gordin et al, 1990). 1980). These results were only partly con®rmed by Marteau The best results seem to have been obtained by using et al (1990) in a study where 9 subjects ingested lactic acid Lactobacillus gg (Isolauri et al, 1991, Siitonen et al, 1990), bacteria (L. acidophilus, B. bi®dum) for 3 weeks. He which is a lactobacillus strain initially isolated from healthy reported of unchanged levels of faecal azo-reductase and humans. The strain was originally selected for its tolerance b-glucuronidase, while only nitroreductase decreased to acid and bile and the ability to adhere to human small during the observation period. intestinal cells. The studies were extended in an animal model of colon The results of lactic acid bacteria treatment against cancer induced by the chemical carcinogen, 1,2-dimethyl- antibiotic associated diarrhoea are shown in Table 7 (a). hydrazine dihydrochloride (DMH). The activation of DMH Siitonen et al (1990) studied 16 subjects, who ingested to a potent carcinogen occurs in the large intestine, and the 400 mg erythromycin three times daily for seven days bacterial enzyme-b-glucuronidase is involved in this pro- together with either 125 ml Lactobacillus gg fermented cess. Suppression of this enzyme might reduce DMH yogurt or 125 ml pasteurised regular yoghurt. The subjects activation and subsequent tumour formation. In experi- receiving Lactobacillus gg yogurt were colonised with ments DMH-treated animals with given L. acidophilus in these bacteria even during antibiotic treatment and had powdered form and compared with controls (Goldin & fewer daily defecations than the group ingesting pas- Gorbach, 1980). At 20 weeks, 40% of the L. acidophilus Lactic acid bacteria H Hove et al 347 treated animals had colon tumours versus 77% of the controls, of large quantities of B. bi®dum (Hove et al, 1994). When P ˆ 0.02. At 36 weeks, however, 73% of the L. acidophilus in isolated culture, B. bi®dum had a speci®c fermentative treated animals and 83% of the controls had colon tumours. pattern, but this pattern could not be reencountered in the These studies show that the addition of lactic acid bacteria to ileostomic outputs of nine ileostomists after oral ingestion the diet may delay colon tumour formation by prolonging the of large quantities of B. bi®dum. induction, indicating that lactobacilli may slow tumor devel- As B. bi®dum, L. acidophilus had a speci®c fermentation opment in experimental animals. pattern when in isolated culture, but after incubation in Lactic acid bacteria have shown antineoplastic properties mixed lactic acid bacteria=faecal incubations the speci®c in a variety of cancer cell lines of both human and animal pattern disappeared although L. acidophilus was added in origin. The literature substantiating this in vitro effect has unphysiological large amounts constituting 50 ± 90% of the increased tremendously over the last decade. In brief, lactic total ¯ora. The fermentation products in mixed L. acid- acid bacteria reduce tumour cell viability (McGroatry et al, ophilus=faecal incubations were related to the type of 1988; Sekine et al, 1985; Reddy et al, 1973; Reddy et al, 1983; added substrate rather than to the addition or not of acid Kato et al, 1981), suppress induced carcinogenesis in the bacteria (Hove et al, 1994). This does not imply that lactic and colon (Reddy & Riverson, 1993), inhibit mutagenic acid bacteria do not contribute to the forma- activity (Hosono et al., 1986; Renner & MuÈntzner, 1991), tion in the mixed homogenates, but rather that the cap- and bind potent mutagenic metabolic compounds (Morotomi ability for saccharide fermentation represented by the & Mutai, 1986) and food mutagen (Zhang et al, 1990). added bacteria already exists in the faecal ¯ora. New However, in spite of a wealth of indirect evidence, no direct perspectives of modulating the colonic ¯ora has recently data have yet proven cancer suppression in humans, as a result been introduced by Gibson et al. (1995), who showed that of consumption of lactic cultures in fermented or unfermented oral ingestion of a diet rich in the indigestible carbohy- dairy products. drates oligofrutose and inulin signi®cantly increases the High concentrations of faecal bile have also been number of bi®dobacteria. Therefore, the prospect of lactic associated with the development of colon cancer, and the acid bacteria therapy may be a change in diet which lithocholic acid=deoxycholic acid ratio has been reported to subsequently alters colonic ¯ora rather than ingestion of be increased in patients with colon cancer compared to cultures of lactic acid bacteria themselves. controls (Owen et al, 1987). Ingestion of L. acidophilus for 6 weeks have been demonstrated to lower concentrations of Lactic acid bacteria as pathogenic organisms total bile acid and deoxycholic acid (Lidbeck et al, 1991), Although with these organisms are rare, it has although the changes were not signi®cant. been reported that lactic acid bacteria may play a role in a variety of serious infections. These include endocarditis Correcting of a colonic `imbalance' (Axelrod et al, 1973), bacteraemia (Bayer et al, 1978), Lactic acid bacteria may have an impact on the colonic gastrointestinal infections (Bourne et al, 1978), and splenic ¯ora in situations where some sort of imbalance exists. The abscess (Sherman et al, 1987). A risk factor is immuno- exact nature of this microbial imbalance and how it is suppressive therapy (Sherman et al, 1987) and poor oral corrected by the ingestion of lactic acid bacteria has not hygiene where dental procedures can cause endocarditis. been substantiated. A prerequisite for an effect on the Treatment of lactic acid bacterial infections can be dif®cult colonic ¯ora is that a substantial number of ingested since eradication is complicated by the often deep-seated lactic acid bacteria reach the large bowel. The work of location, the antimicrobial resistance to antibiotics, and the others (Robins-Browne et al, 1981; Hove et al, 1984; problem in identifying the organisms and thereby to initiate Goldin et al, 1992; Saxelin et al, 1991; Lidbeck et al, apppropriate treatment (Sherman et al, 1987). In a single 1991) indicate that ingested lactic acid bacteria do reach the case L. acidophilus ingestion has been associated with the caecum. It is, however, questionable whether an ingested development of D-lactic (Mason, 1986). dose of lactic acid bacteria in the range of 1010 ± 11 bacteria is able to in¯uence the colonic ¯ora, which number approximately 1013 bacteria (1011 g), a number of 100 ± Conclusion 1000 times higher than the ingested amount of lactic acid Lactic acid bacteria appear to alleviate lactose malabsorp- bacteria. This question is even more pertinent in light of the tion in lactose malabsorbers when administered in fermen- widely held belief that a dietary culture, even one posses- ted dietary milk products (yogurt, Table 3 (a)), but not in sing in vitro adhering capabilities, is highly unlikely to infermented milk products (set acidophilus milk, Table displace any bacterial strain that colonises a healthy human 3(b)). Lactic acid bacteria seem to shorten the course of intestinal tract (Savage, 1977). Human gastrointestinal infectious diarrhoea especially in infants with rotavirus microbiology is notably a dif®cult ®eld to study because diarrhoea (Table 6 (b)) and reduce the risk of travellers' of limits on direct experimentation and the dif®cult phy- diarrhoea (Table 6 (a)). The ability of lactic acid bacteria to siological requirements of the intestinal microbes. 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