Modulation of mice fecal microbiota by administration of casein glycomacropeptide

Casein glycomacropeptide (GMP) is known to promote the in vitro growth of Bifidobacteria and Lactobacilli. In this paper, we used conventional culture techniques and fluorescent in situ hybridization (FISH) techniques to investigate the effect of casein GMP on mice fecal microbiota. The population structure of the intestinal microbiota, including Lactobacillus, Bifidobacteria, Enterococcus, coliforms and Enterobacteriaceae, was tested and compared. After consecutive administration of casein GMP for 15 days, numbers of Lactobacillus and Bifidobacteria increased significantly (P<0.01), numbers of Enterobacteriaceae and Coliforms decreased significantly (P<0.05) while no significant changes were observed for Enterococcus. The detection limits of FISH technique were significantly lower (P<0.01) than the traditional culture method. These results suggested that consumption of casein GMP had a prebiotic effect on male BALB/c mice. Casein GMP helped establish a healthier intestinal microbiota. Additionally, FISH was proved to be a rapid and relatively low-cost detection method that can be used to further our understanding of human intestinal microbiota.


Introduction
All animal intestinal tracts, as well as those of humans, harbor complex microorganism communities.These bacterial communities are comprised of up to 400 to 500 bacterial species forming a complex ecosystem.][3] The intestinal microbiota play both positive and negative roles.A healthy intestinal microbiota can help digest food, and metabolize endogenous and exogenous compounds, and can potentiate the immune system and prevent pathogen colonization in the gastrointestinal tract (GIT). 2,4However, an unbalanced gut microbiota can cause diseases such as diarrhea and inflammatory bowel disease (IBD).
Therefore, sustaining a well-balanced gut microbiota by promoting the colonization of beneficial bacteria is crucial to human health.
It has been observed that some Lactobacillus and Bifidobacterium strains have a beneficial effect on hosts by adhering to the surface of intestinal cells and colonizing the gastrointestinal tract. 5,6Thus, analyzing intestinal microbiota in the research for functional food components that promote beneficial gut bacteria had been one of the objectives in this field.
Casein glycomacropeptide (GMP) is a casein-derived peptide, cleaved from κ-casein by chymosin at 105Phe-106Met during cheese making.The concentration of casein GMP ranges from 1.2 to 1.5 g/L in sweet whey, comprising 15-20% of the total protein. 7Casein GMP is a peptide with many known biological functions, including binding of cholera and Escherichia coli enterotoxins, 8 inhibition of bacterial and viral adhesion, 9 suppression of gastric secretions, 10 modulation of immune system responses, 11 and promotion of bifidobacterial growth whose prebiotic activity was attributed to its glycosylated N-acetylneuraminic acid component. 12,13ecause of its unique carbohydrate composition and versatile biological activities, casein GMP is considered a potential ingredient for functional foods and nutraceuticals.Idota et al. 14 found that casein GMP had promoted in vitro growth in Bifidobacteria and Lactobacilli, but the effect of casein GMP on the intestinal microflora has not been tested.
Identification and enumeration of gastrointestinal microbiota commonly use traditional culture techniques.Culture-based studies provided us with the first insights into the diversity and complexity of the gastrointestinal tract ecosystem.However, the conventional culture method has a number of disadvantages, for example, it is laborious and time-consuming.It is also limited by the possibility of culturing the studied microorganisms, and a certain expertise and specialized equipment are required to isolate strict anaerobes. 15Such drawbacks have often hindered related research.
Recent years have seen the development of new molecular techniques, especially those based on 16S rRNA sequences.These molecular techniques are essential for the study of microbial diversity, including understanding the distribution, function and adaptation of such microorganisms in different environments.The techniques are also important in understanding the relationship between microbial diversity and specific metabolic activities.
Fluorescence in situ hybridization (FISH) was first used to detect the bacterial ecosystem in 1988. 168][19] In this study, to investigate the modulation of mice fecal microbiota upon administration of casein GMP, we applied both a culture-dependent method and FISH to evaluate changes in gut microflora.
Depending on the probes chosen, FISH can be used to detect bacteria from different phylogenetic term.Specific oligonucleotide probes and primers have been designed for many bacterial species that are known to be present in the GIT.But because the complex microflora of the human gut is difficult to study with probes at a species level due to the vast diversity of this ecosystem, in this study, we chose probes that are designed to recognize major genera or groups of microbes. 17(group A) received the basal diet and an equal volume of normal saline per day.The casein GMP group (group B) was administered the basal diet and 0.5 mg/mL casein GMP (Tatua Dairy Co-operative Co., Ltd, New Zealand) at the dose of 0.2 mL per day; casein GMP purity (of protein) 71%; sialic acid on protein 5.6%.

Strains and probes
Lactobacillus helveticus TS206 strain was optimized and preserved in our laboratory.The 16S rRNA-targeted oligonucleotide probes (Invitrogen, USA) used in this study are listed in Table 1.The EUB338 probe was linked with 6-carboxy-x-rhodamine (ROX) at the 5′ end.The group-specific probes were linked with fluoresceine isothiocyanate (FITC) at their 5′ ends.

Fluorescent in situ hybridization analysis
Before performing FISH hybridization, the specificity of probes was tested using L. helveticus TS206.Briefly, L. helveticus TS206 was cultured in MRS broth at 37°C for 12 h.The bacterial cells were harvested and pelleted by centrifugation (3500×g for 10 min at 4°C), washed once with phosphate-buffered saline (PBS, pH 7.2), and adjusted to a final concentration of 10 -7 CFU/mL.The suspension was hybridized with Lacb722 probe as described below.As a negative control, the specificity of Bif164 probe was also tested by L. helveticus TS206 in a similar manner.
FISH hybridization was performed as described by Franks et al. with slight modifications. 20After administration of casein GMP for 15 days, fresh stool were collected from the Group A (control group) and Group B (casein GMP group) mice in sterile plastic centrifugal tubes (5 mL; RNase-free), weighed and kept for no longer than 12 h at 4°C before processing.Portions (0.3 g) of each stool were suspended in 2.7 ml of filtered (0.2-um-pore-size filter, Millipore Cor-poration, USA) PBS (130 mM NaCl, 3 mM NaH 2 PO 4 •2H 2 O, 7 mM Na 2 HPO 4 •12H 2 O, pH 7.2) and vortexed to homogenize the samples.The suspension was centrifuged at 700 g for 2 min to remove debris.The supernatant was diluted 1:3 with PFA (4% (w/v) paraformaldehyde in PBS), fixed at 4°C for 16 h, and stored in 50% (v/v) ethanol/PBS at -70°C for use. 21he suspension fixed with PFA as described above was incubated with 10 ul of 10 mg/ml lysozyme dissolved in 100 mM Tris-HCl (pH 7.2) for 20 min at 37°C before hybridization.The fixed cell suspension (10 ml) was applied to a pre-cleaned glass slide (RNase-free) and dried at room temperature.The cells were then dehydrated with a series of solutions containing 50%, 80%, and 99.5% ethanol (3 min for each concentration).The cells fixed on the glass slide were hybridized with 25 uL hybridization buffer overnight at 46°C in a humid chamber.The hybridization buffer (0.9 M NaCl, 20 mM Tris-HCl (pH 7.2), 0.1% SDS, 30% (v/v) formamide, 10% (w/v) dextran sulfate) contained 20 ng/uL of fluorescently labeled probe.The fixed cell suspension can be diluted with PBS (pH 7.2) based on the quantity of the target organism.After hybridization, the cells were subsequently transferred into washing buffer (0.9 M NaCl, 20 mM Tris-HCl (pH 7.2), filtered through a 0.2-um-pore-size filter), incubated at 48°C for 30 min to remove non-specific binding of the probe, rinsed briefly with distilled water, and air-dried.

Enumeration of bacteria in the fecal samples by fluorescent in situ hybridization
Fluorescent cells were counted in triplicate for appropriately diluted samples.Digital images of the slides were taken using a Nikon Eclipse Ti-U fluorescent microscope (Nikon, Japan).These images were analyzed and fluorescent cells were counted using NIS-Element BR image analysis software (Nikon, Japan).Depending on the amount of fluorescent cells, 15 randomly chosen microscopic fields were imaged.

Analysis by conventional culture techniques
Fresh stool were collected in sterile plastic centrifugal tubes, weighed (approx.0.1 g) and processed immediately.After thorough mixing, 10-fold dilutions were prepared in sterile saline.Aliquots of adequate dilutions were plated in triplicate onto different selective media: Lactobacillus were enumerated on MRS broth; Enterobacteriaceae on Violet Red Bile Dextrose Agar (VRBDA); 22 Bifidobacteria on Tryptone-Peptone-Yeast Extract (TPY) agar with 2 ug/L dicloxacillin; 23 Enterococcus on Bile Esculin Azide Agar; and coliforms on MacConkey Agar.The plates described above were incubated anaerobically at 37°C for 48 h except for the coliforms that were incubated aerobically at 37°C for 24 h (24).All bacterial counts (colony-forming units [CFU] per g of wet feces) were transformed to logarithms (log 10 CFU) for statistical analysis.Media (Hopebio) used in this study were obtained from Qingdao Hope Bio-Technology Co. Ltd, China.

Data analyses
Data are expressed as means±SD.The differences in bacterial counts between different samples or between different techniques were determined by t-test using SPSS for Windows, version 11.5 (IBM SPSS Statistics, USA).P>0.05 was considered significant.

Analysis of the fecal microbiota by conventional culture techniques
The differences in target microbiota counts of fecal microbiota at different time points are shown in Table 2. Lactobacillus and Bifidobacteria in the fecal sample increased after supplementing with GMP.Their counts reached 109 and 1010 CFU/g (wet weight).After 15 days of treatment, the differences compared to their basal level were significant (P<0.01).Enterobacteriaceae and coliforms in the fecal sample decreased significantly after 15 days of casein GMP treatment (P<0.05).During the whole trial, there was no significant change in Enterococcus in the control group or in the experimental group.

Specificity of 16S rRNA-targeted oligonucleotide probes
Using the above-mentioned methods, the specificity of 16S rRNA-targeted oligonucleotide probes was analyzed.It was confirmed that the probes only hybridize with their target strains.Specifically, more than 90% of the Lacb722 probes were specifically hybridized with their corresponding target strain L. helveticus TS206 (Figure 1A), whereas no fluorescent signal was detected using Bif164 probes (Figure 1B).

Article
Table 1.16S rRNA-targeted oligonucleotide probes for fluorescent in situ hybridization analysis.

Comparison of conventional cultivation method and fluorescent in situ hybridization
The trend was consistently detected by both methods, but the level of bacteria detected by FISH was much higher than that detected by conventional cultivation techniques.The accuracy of the culture-dependent approach is limited by the selectivity of the media.FISH is limited by the specificity of the probe.However, the advantage of FISH technique is obvious in that it can enumerate culturable and unculturable bacteria.More importantly, the accuracy of FISH is much higher than the culturedependent approach, reaching above 90%, and this technology is also fast, practical, and better reflects the main gastrointestinal microbiota profile.

Discussion
Early works have found that casein GMP promotes the in vitro growth of Bifidobacteria and Lactobacilli, but the related research in vivo is still lacking.Our study has demonstrated that use of casein GMP can significantly increase the intestinal concentration of Lactobacillus and Bifidobacteria.These intestinal microbiota are essential to the health of the host and play a role in regulating immune responses, inhibiting exogeneous or harmful bacteria, and facilitating digestion and absorption of food ingredients. 2The accumulation of probiotics (Lactobacillus and Bifidobacteria) with administration of casein GMP to the male BALB/c mice facilitates competitive exclusion of pathogens, thereby promoting non-specific host resistance to microbial pathogens.Related studies have also demonstrated that Bifidobacteria can potentiate the secretion of secretory IgA (SIgA) 25 that prevents the spreading of intestinal bacteria across the gut mucosa by coating intestinal bacteria and blocking their interaction with the epithelium. 26he consumption of casein GMP can signifi-   cantly decrease the intestinal concentration of potential pathogens such as Enterobacteriaceae and coliforms, which might be associated with the sialic acid component (N-acetylneuraminic acid) in casein GMP.The sialic acid unit of casein GMP has been found to inhibit a variety of toxins, viruses and bacteria that caused infection through receptor association 27 since sialic acid receptors found on the surface of most bacteria are required for their initial infection. 27Casein GMP, with an exogenous source of sialic acid might, therefore, inhibit bacterial adhesion by blocking the cell surface receptors preventing the bacteria from targeting the epithelial cell lining.Thus, the consumption of casein GMP has a prebiotic effect on the establishment of healthy intestinal microbiota, as well as prevention of infection.The intestinal tract is a complex ecosystem of microorganisms.Therefore, it is important to establish an accurate, rapid and stable method of analysis to understand the human gut microbial community.In our study, we applied both culture method and FISH techniques in the analysis of mice feces.Both are able to monitor the difference in the numbers of major representatives of the intestinal flora.Cultural-based studies were able to provide a basic understanding of the diversity and complexity of the GIT ecosystem; however, they only allow the detection of culturable bacteria, are time-consuming and results are frequently biased due to the selectivity of the culture medium.So estimations of conventional bacteria by cultivation methods are inaccurate.On the other hand, it is well known that anaerobic bacteria are predominant in the GIT, 40-80% of the total microscopic counts can not be recovered by culture. 3So it is impossible to accurately examine the intestinal microflora using traditional culture techniques.Molecular approaches based on nucleic acid such as FISH and quantitative real time polymerase chain reaction (qPCR) can overcome the shortcomings of the culture method.FISH is limited by the specificity of the probe and the efficacy of the probe binding to the bacterial target.The probes (EUB338, Enter1432, Bif164, Lacb722, ENC221) in our study have been widely used in related research and have been shown to have a reliable specificity.FISH can enumerate culturable and unculturable bacteria and detect 90% of the total intestinal microorganism.Therefore, for accurate and fast identification, FISH is probably more appropriate than conventional culture techniques because FISH can provide information about the presence, number, morphology and spatial distribution of microorganisms.Moreover, FISH gives a detailed picture of the microenvironments without any selective purification or amplification steps.

Conclusions
Casein GMP is one of the biologically active components of milk.In this paper, we investigated the effect of casein GMP on modulating the intestinal microbiota in vivo using both a conventional culture method and FISH.The results showed that casein GMP had obvious biological capability to modulate the intestinal flora; more specifically, numbers of Lactobacillus and Bifidobacteria increased significantly (P <0.01), whereas numbers of Enterobacteriaceae and coliforms decreased significantly (P <0.05).Casein GMP showed a prebiotic effect as it helped to modulate the intestinal microbiota and establish intestinal microbiota that is considered to be healthier.The study provides theoretical support for casein GMP to be developed into an excellent source of functional food.

Male 6 -
week old BALB/c mice (n=20; provided by The Academy of Military Medical Sciences of China, China) were fed normal fodder for one week and divided equally into two groups thereafter.The control group N o n -c o m m e r c i a l u s e o n l y R=G/A, Y=T/C, M=A/C, K= G/T, S= G/C, W= A/T, H=A/C/T, B=G/T/C, V=G/C/A, D=G/A/T, N=G/A/T/C Analysis of the fecal microbiota by fluorescent in situ hybridization techniques

Figure 1 .
Figure 1.A) The positive hybridizing signal of Lactobacillus helveticus TS206 detected by Lacb722 probe.Lactobacillus helveticus TS206 was used to confirm the specificity of Lacb722 probe.L. helveticus TS206 was incubated in MRS broth at 37°C for 12 h.The bacterial cells were pelleted by centrifugation(3500×g, 10 min, 4°C) and washed once with phosphate-buffered saline (PBS, pH 7.2).The result of the FISH experiment with the L. helveticus TS206 showed that the specificity of the Lacb722 probe was high with the labeling efficiency above 90%.B) The positive hybridizing signal of Lactobacillus helveticus TS206 detected by Bif164 probe.Lactobacillus helveticus TS206 was used to verify the specificity of Bif164 probe.L helveticus TS206 was incubated in MRS broth at 37°C for 12 h.The bacterial cells were pelleted by centrifugation (3500×g, 10 min, 4°C), and washed once with phosphate-buffered saline (PBS, pH 7.2).The result of the FISH experiment with the L. helveticus TS206 revealed that the labeling efficiency was 0%.

Table 3 . Enumeration (log CFU/g of feces) and percentage of Lactobacillus, Bifidobacteria, Enterococcus spp, Enterobacteriaceae in faecal samples before and after 5, 10, 15 days of assumption of casein glycomacropeptide by for fluorescent in situ hybridization. Time Fuorescent in situ Percentage of target points hybridization counts microbiota in fecal Target (log CFU/g of feces, n=10) total bacteria microbiota Means±SD
* Significant differences among target microbiota counts of fecal microbiota in different periods ( * P<0.05, °P<0.01).D, administration period (days).