We enumerated the predominant gut genera from fecal samples of nine healthy and eight milk-hypersensitive adults both before and after 4 weeks Lactobacillus rhamnosus GG (LGG) supplementation. The anaerobic intestinal microflora of milk-hypersensitive adults was found to resemble that of healthy adults. LGG-consumption resulted in a significant increase in the number of bifidobacteria in healthy but not in milk-hypersensitive subjects, as well as a general increase in bacterial numbers in all other bacterial genera tested in both groups. In conclusion, the composition of the gut microbiota in milk-hypersensitive adults appears to be normal. LGG may have potential in reinforcing the endogenous flora.
Milk hypersensitivity affects approximately 2.5% of the general infant population . In adults, milk-related adverse reactions have mainly been considered to be due to lactose intolerance. However, a recent study on the Finnish population indicated that milk hypersensitivity may be an important source of these adverse reactions and may also be more common than previously reported . It has been estimated that about 3% of 27 years old Finns have milk hypersensitivity .
Milk hypersensitivity is an adverse reaction to milk proteins which is mediated by immune mechanisms and may cause unspecified gastrointestinal disorders in adults. Lactose intolerance due to lactase deficiency results in an inability to break down ingested lactose in the small intestine. The resulting gastrointestinal symptoms are similar to those of milk hypersensitivity . Intestinal inflammation altering the composition of the intestinal flora has recently been demonstrated in allergic infants (Kirjavainen et al., unpublished data; ). This alteration in the microflora may involve an increase in putrefactive and pathogenic bacteria, resulting in maintained or reinforced gut inflammation, and/or a decrease in the number of beneficial bacteria such as bifidobacteria and lactobacilli. Probiotic lactic acid bacteria are used to improve the microbial balance in the gut [6–8]. In hypersensitive subjects, probiotics have been proposed to normalize the intestinal flora and thereby to alleviate inflammation, normalize permeability and reduce food antigen permeation .
The complexity of the digestive tract ecosystem, consisting of over 500 culturable bacterial species amounting to a total of 1014 bacterial cells, makes traditional plate culture and identification of fecal anaerobic bacteria difficult, unreliable and time-consuming. As a result, very little is known regarding the full microbiota composition and how this influences host-microbe interactions and disease. Recent advances in the analysis of such complex microbial ecosystems have involved genetic-based analyses such as the culture-independent method of fluorescence in situ hybridization (FISH). This molecular probing approach has the advantage over traditional plate culture of being rapid, specific and applicable to mixed cultures, also allowing determination of bacteria which are difficult to culture or even unculturable . FISH has been shown to be at least as accurate as conventional cultivation methods in the quantification of predominant groups of anaerobic bacteria in human fecal samples [11,12]. Moreover, the approach allows the processing of stored (frozen) specimens.
We hypothesize that people with milk hypersensitivity may have aberrant microflora, on the basis of evidence that the intestinal microflora in an inflamed gut may be different from that in a healthy gut . Furthermore, oral introduction of an exogenous bacterial strain of intestinal origin, i.e. probiotic Lactobacillus rhamnosus GG (LGG), has been shown to alleviate the clinical symptoms of milk hypersensitivity . Our aims here were thus to determine whether milk hypersensitivity is associated with deviated gut microbiota and to assess LGG as a compositional modulator of the digestive flora.
Materials and methods
Study design and collection of fecal samples
The study was comprised of 12 female and five male volunteers with a mean age of 28 years (from 22 to 41 years). The inclusion criteria were normal lactose tolerance as indicated by lactose tolerance test with ethanol  and that the subjects were free from signs and symptoms of acute infections. All subjects were challenged with 200 ml milk twice a day for 1 week in a double-blind placebo-controlled manner. Based on this challenge and clinical history the participants were divided into milk-tolerant controls (n=9) and milk-hypersensitive subjects (n=8). The control subjects had no reactions following the milk challenge whilst the milk-hypersensitive subjects had immune responses  and unequivocal gastrointestinal reactions such as abdominal bloating/pain, flatulence and diarrhea.
In this double-blind, placebo-controlled cross-over study, subjects were challenged with 1010 CFU LGG bacteria (ATCC 53103, provided by Dr. Maija Saxelin, Valio Ltd., Helsinki, Finland) or placebo in hard gelatin capsules twice a day (morning and evening). Cellulose microcrystalline was the placebo and also the filling in the LGG capsules. The sequence of the oral challenge was randomized, with each challenge period lasting 4 weeks. This enabled the subjects to act as their own controls. Capsules were stored in the refrigerator until consumption. Subjects followed their normal diet but were requested not to consume any products containing probiotics and to keep the diet as constant as possible. None of the milk-hypersensitive subjects followed a strictly milk-free diet. Fecal samples were taken weekly and whole stools stored at −20°C until processing. The samples used in this study were from the end of the control and treatment periods.
The study protocol was approved by the Turku University Ethical Committee and the Foundation for Nutrition Research.
Sample preparation and FISH analysis
The fecal samples were weighed and phosphate-buffered saline (PBS; 8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4 and 0.24 g KH2PO4 l−1) added to make up a 1/10 solution (w/v). The sample was homogenized and a 5 ml portion removed and vortexed with glass beads for 30 s, then centrifuged at 300×g for 2 min. Bacterial cells were fixed and FISH performed as described by Langendijk and associates . Briefly, the bacterial cells were fixed in a 4% (v/v) paraformaldehyde solution overnight at 4°C. The cells were then washed twice in PBS and resuspended in 1 ml of PBS:ethanol (1:1, v/v). A portion of the cell suspension was then hybridized overnight in hybridization buffer (HB; 20 mM Tris-HCl, 0.9 M NaCl) with a Cy3 indocarbocyanin-labeled probe (Table 1). Cells were washed with the hybridization buffer, filtered through a 0.2 µm polycarbonate filter (Millipore Corporation) and mounted on a glass slide with Slow Fade (Molecular Probes Inc.). Total cell counts were determined using 4′,6-diamidino-2-phenylindole (DAPI) as a nucleic acid dye. Fluorescent cells were counted visually using a Nikon Microphot intestinal microflora epifluorescence microscope mounted with Cy3 and DAPI specific filters. Fifteen random microscopic fields were counted per assay.
|Lab158||GGTATTAGCA(T/C)CTGTTTCCA||Lactobacillus sp., Enterococcus sp.|||
The Mann-Whitney U-test and the Wilcoxon signed rank test were used in statistical comparisons. P-values less than 0.05 were considered statistically significant. Posterior power calculations were performed using computer software SPSS for Windows™ release 9.0.1 (SPSS, Inc., Chicago, IL, USA).
Results and discussion
To our knowledge, this is the first study to compare the intestinal microflora of milk-hypersensitive and healthy adults using the FISH probing technique. There were no differences in the number of bacteria belonging to the major genera between healthy and milk-hypersensitive subjects prior to supplementation. Posterior power calculations indicated that if future expanded studies comparing the presence of these genera between healthy and milk-hypersensitive adults were conducted, approximately 70–200 subjects per group should be included (depending on the genera studied). The observed differences in the log means would then be significant at a 5% significance level with 80% probability.
After supplementation with LGG, there were a significantly greater number of bifidobacteria in fecal samples from the healthy group than in those from milk-hypersensitive subjects. After treatment with LGG there was also a significant increase in numbers of bacteroides, clostridia and total bacteria in both the healthy and milk-hypersensitive subjects (Fig. 1a and b) and in the case of milk-hypersensitive subjects a trend towards increased lactobacilli/enterococci (P=0.07).
It is conceivable that the difference in bifidobacteria numbers between the groups is due to the presence of a factor in milk-hypersensitive subjects which did not allow LGG to induce proliferation of bifidobacteria. One such factor might be the concomitant induction of strain antagonistic to bifidobacteria. Alternatively, it has been shown that the immune response to LGG can vary between milk-hypersensitive and healthy people . In theory, this difference in response may directly or indirectly (via an antagonistic strain) affect bifidobacterial proliferation. A less specific increase in the other genera tested was observed in both healthy and milk-hypersensitive groups. Similar data have been obtained by plate counting, including a study by Benno and colleagues  showing that consumption of LGG results in increased numbers of probiotics such as lactobacilli and bifidobacteria.
Studies on the effect of LGG and other probiotic supplementations on the gastrointestinal microflora are few. It has been shown, however, that LGG supplementation does not increase the total number of lactobacilli; rather the proportion of LGG within this population increases . Any beneficial effects exerted by this strain are therefore likely to be due to specific properties of the organism and their transient persistence in the gut, rather than an increase in total lactobacilli. In this context, we detected no marked increase in lactobacilli after LGG supplementation.
Many species of bacteroides are putrefactive by reason of their proteolytic activities, and clostridia are potential pathogens by toxin formation. In both the control and milk-hypersensitive subjects there was a significant increase in the number of these microorganisms. This is in tentative agreement with a previous study using pre-term infants, in which LGG supplementation failed to reduce the number of potential pathogens . Thus, our results do not support the hypothesis that the mechanism by which LGG exerts a beneficial effect on milk-hypersensitive subjects is via a reduction in the number of pathogens. In fact, our data would indicate that LGG supplementation had a nonspecific proliferation-enhancing effect on the anaerobic gut flora. It thus seems more likely that the anti-inflammatory properties of LGG are due to factors other than modulation of the composition of the intestinal flora, for example the ability to degrade milk proteins to smaller peptides and amino acids  and to directly down-regulate the inflammatory response . However, as very little research has been done in this area, more work is needed for conclusive results to be obtained.
In conclusion, our results indicate that the major bacterial genera inhabiting the intestine are not involved in the etiology and/or are not disturbed by the immunological events associated with milk hypersensitivity. LGG may have potential use in fortifying the endogenous flora, however, current data imply that the previously reported down-regulation of milk-induced inflammatory responses in milk-hypersensitive subjects is not due to modulation of the microbial composition in the gut.
We thank Tuija Poussa, MSc, for able statistical consultations. E.A. was supported by a scholarship from the Center for International Mobility (CIMO), Finland and P.V.K. by a grant from the Academy of Finland.
fluorescent in situ hybridization
Lactobacillus rhamnosus GG