DETERMINING EFFICACY

15.6 DETERMINING EFFICACY

15.6.1 I N V ITRO S YSTEMS

Various in vitro model systems have been validated and allow a reproduction of the physicochemical events encountered in the different parts of the gastrointestinal tract. Available models are of various degrees of complexity, from single-stage

TABLE 15.5 Synbiotics Tested in Animals and In Vitro

Probiotic

Prebiotic

Experimental Model

Outcomes

Reference

Lactobacillus acidophilus 74-2

Fructo-

In vitro batch culture using

Increased bifidobacteria populations;

Gmeiner et al. 57

oligosaccharides

human fecal flora

increased production of propionate; increased -galactosidase activity; decrease -glucuronidase activity

Bifidobacterium longum

Inulin

Rats with carcinogen

Synergistic action of pro-/prebiotic

Rowland et al. 58

Bifidobacterium Fructo-

Mice induced with colon

Decrease in aberrant crypt foci

Gallaher and Khil 59

oligosaccharides

cancer

Asahara et al. 60 Functional F

Bifidobacterium breve

Trans-

Mice infected with

Decrease in excretion of Salmonella

oligosaccharides

Salmonella typhimerium

ood Carboh

ydrates

Probiotics, Prebiotics, and Synbiotics

TABLE 15.6 Synbiotic Tested in Clinical Trials

Experimental Design

Primary Endpoint

Reference

Bifidobacterium breve +

Kanamori et al. 61 Lactobacillus casei

Galacto-

Capsule, 3 g/day

1 infant with

Increase in short-chain fatty acid

oligosaccharides

(1.10 9 CFU/g)

laryngotracheo-

production; bowel movement

esophageal cleft

resumed

Bifidobacterium sp.

Inulin

Fermented milk

Randomized, placebo

Increase in bifidobacteria with

Bouhnik et al. 50

with 18 g/day

controlled, n = 12

fermented milk; no additional

prebiotic or

effect of inulin

placebo

Bifidobacterium lactis Bb12

Galacto-

Yogurt

Randomized, n = 30

Decrease in B. longum; no

Malinen et al. 62

oligosaccharides

persistence of probiotic after treatment was stopped

Bifidobacterium longum 913 +

Kiessling et al. 7 Lactobacillus acidophilus 145

Fructo-

300 g of yogurt

Crossover placebo-

Increase in HDL concentration;

oligosaccharides

with 1%

controlled study, n = 29;

decrease in LDL/HDL

Lactobacillus paracasei

n = 12

Positive effect on microflora;

Morelli 26

persistence for a few days after treatment was stopped

Lactobacillus acidophilus +

Fisberg 63 Bifidobacterium infantis

Fructo-

Supplement

Parallel, double-blind,

GI tolerance; decreased number

oligosaccharides

randomized study, n = 626

of sick days; catch-up growth; decreased constipation

Lactobacillus plantarum v299

Oat fiber

Parallel, randomized,

Decrease of pancreatic sepsis

Oláh et al. 64

double-blind study, n = 43

after surgical intervention

Functional Food Carbohydrates

fermenters to a cascade of bioreactors simulating the physiological differences between each part of the colon. 65–68 Generally, temperature, pH, redox potential, and transit time are controlled. Reproduction of peristaltic movements may vary from stirring to differential pulse movements. The gastrointestinal secretion and digestive

absorption are also simulated with some models. 67 The latest advances in the area are models simulating the attachment of microbial cells to an artificial intestinal

membrane (biofilm reactor). 69 In vitro models allow the study of the human gut ecosystem in controlled laboratory conditions while preserving the diversity of gut microbiota. Fermentative activities of a large array of substrates can be tested; these models have thus been proven useful in the initial steps of development of new/emerging pre- and probiotics.

15.6.2 A NIMAL M ODELS

Animal models are a more realistic representation of the mammalian intestinal tract. 65 These models allow detailed studies of the systemic effects and of the host–response resulting from the manipulation of the gut microflora. Gastrointestinal disorders can

be induced in some animals models, such as ulcerative colitis, 70 colorectal cancers, 71 and necrotizing enterocolitis. 72 Because immune response and microbial effects of probiotics, prebiotics, and synbiotics are often species specific, human flora-associ- ated animals are of preferential use for assessing the effectiveness of organisms or carbohydrates under study. 73–75

15.6.3 E X V IVO M ODELS

Biopsies of intact and pathologic tissues allow an investigation of the ecological niches present in the gut and a characterization of the microflora attached to the

intestinal epithelium. 76 Most of the in vitro or animal models use fecal microflora as starting inoculum. Although fecal microflora is a good representation of the

luminal microflora, bacteria adhering to the epithelium are likely to differ. 68 The development of molecular tools has greatly improved the possibilities of exploring

microflora from biopsies’ tissues. Characterization of the epithelium-adhering micro- flora may consequently advance greatly in the next few years.

Tissue culture is another system often employed to characterize attachment properties of emerging pro- and prebiotics. Tissue cultures, although validated sys- tems, encounter limitations, as models are often derived from cancer cell lines. Limitations are also seen because tissue cultures need to be kept aerobic, whereas most of the probiotics are anaerobic strains. Tissue cultures may give an indication of the immune response after exposure to prebiotics or probiotics, but the models often need optimization to reflect the real conditions encountered at the intestinal site, particularly in a pathological phase (such as the inflammatory stage).

15.6.4 H UMAN T RIALS

Definitive assessments of pro- and prebiotic effects are only achieved by results of well-designed human feeding studies. Ideally, trials should be double blind,

randomized, and placebo controlled (similar to phase 2 trials in a drug development

Probiotics, Prebiotics, and Synbiotics

procedure). Comparative studies at multiple centers are advantageous. In the case of prebiotics, the choice of placebo is not always clarified; some studies use

nondegradable polysaccharides (starch, maltodextrin) or readily digested saccha- rides (glucose/lactose). 2 Records of food intake and bowel habits during the trial period provide generally useful information. If the product is designed to be used as replacement therapy or as a complement therapy in a particular disease state, sample size, exclusion criteria, and primary endpoints must be well defined. Criteria such as history of drug administration, genetic susceptibility, and family history must be taken into account. There may also be a need for comparative studies with standard therapy (phase 3 study). The effectiveness of probiotic studies may involve comparison between well-established strains and new strains or combinations of strains. Similarly, synbiotic trials may require a specific design measuring the potential synergistic effect between the synbiotic components. Follow-up studies (phase 4) are useful to determine the long- term effect of probiotic and prebiotic use. 19

15.6.5 N EW M OLECULAR T OOLS FOR A NALYZING G UT M ICROFLORA (B IOMARKERS )

Modern molecular techniques have led to the possibility of characterizing the com- plete gut microflora in situ. They have enabled both the qualitative and quantitative

monitoring of phylogenetically related bacterial groups without the need for tradi- tional cultivation techniques that only select for those bacteria that are culturable in the laboratory (viable but nonculturable (VBNC)).

Analysis of 16S rDNA gene profiles obtained directly from feces has greatly expanded estimates of species diversity within the microflora. About 70% of clones correspond to novel bacterial lineages, whereby the majority fell into three dominant

groupings: Bacteroides spp., Clostridium coccoides, and Clostridium leptum. 77 In feeding trials, a range of 16S rDNA gene probes designed to target the most important groups of bacteria present in the gut microflora are applied to monitor the changes in bacterial numbers. This technique, known as fluorescent in situ hybridization (FISH), allows a quantification of microbial populations. With the increase in the isolation and identification of novel bacterial species, however, the number of probes available is rapidly expanding, and so is the number of species monitored. With an increasing level of analysis, there is a need for high-throughput techniques allowing an integrated analysis of these changes while conserving the information on micro- bial diversity.

Bacterial community analysis using 16s rDNA gene fingerprinting techniques such as T/DGGE allows a qualitative whole community analysis of samples.

T/DGGE separates polymerase chain reaction (PCR) amplicons according to their sequence variation. The profiles obtained can distinguish a change in a specific

species or, indeed, monitor the overall changes in diversity in response to the application of a functional food or probiotic. Another approach that is being developed is real-time quantitative PCR, which, though expensive, is less time- consuming than FISH and ultimately will be more robust in quantifying bacterial

numbers. 78 A combination of both quantitative and qualitative molecular

Functional Food Carbohydrates

approaches is extremely useful in evaluating the efficacy of pro-, pre-, and synbi- otics in boosting human health. In addition to monitoring bacterial change follow-

ing probiotic or prebiotic ingestion, there is a need to understand how these changes affect the expression of genes both in the microbial population and in the intestinal epithelium. Only this kind of information will lead us to understand the mecha- nisms whereby probiotics and prebiotics are effective. More and more bacterial genomes are being sequenced, and with the rapid development of DNA microarray technology, cross talk (transcriptomics, gene expression) between probiotics and human mucosa cells is very much on the horizon. Such data can be subsequently used to predict both the proteomics and the metabolomics of the effects of pre-, pro-, and synbiotics on gut health.