1" target="_self">Table 1) were evaluated. The data suggested that different types of ω-3 LCPUFA in Balb/c mice appeared to affect microbial diversity in the colon microbiome. For example, species enrichment was different among the three
Figure 1. Comparison of gut microbiota composition between the mouse groups. (a) Denaturing gradient gel electrophoresis (DGGE) fingerprinting of V3 region of 16S rRNA genes from faecal bacterial communities; (b) The virtual map of DGGE electrophoresis. Every sample included feces of ten mice and feces in 1, 2 and 3 were same ten ones.
Table 1. Analysis of the microbiota diversity of mice intestine.
experiment groups. The flora diversity in group FO decreased but in PL treatment group the diversity was increased. Then SPSS was used to conduct Principal component analysis (PCA) (Figure 2(a)) and the cluster dendrogram (Figure 2(b)) were also estimated. According to the data, we can found that the mice groups were made into three clusters, with first principal component (PC1) accounting for 63.6% of the variation. The results showed a distinct difference in the three groups, which could be confirmed that ω-3 LCPUFA caused the significant change of gut microbiota.
3.2. The Sequencing of the Significantly Different Bands in DGGE Fingerprints
To find the differences between the bands, 17 bands were selected to sequencing, and the data was shown in Table 2. Combined with (Figure 1(b)), we can found that there were 4 significant bands in the normal group compared with other two groups: 5 (Clostridiales bacterium), 8 (Bacteroides acidifaciens), 9 (Alistipes timonensis JC136), 25 (Clostridiales bacterium). But 17 (Lactobacillus animalis), 23 (Akkermansia muciniphila), 24 (Ruminococcus), 27 (Lactococcus lactis) were more clear in PL group and FO group than normal group and the bands of 23, 27 were superior in PL than FO group. While the 21 band (Streptococcus thermophilus) in PL group was inferior than the other two groups. The relative abundance of these bacteria was analyzed by gray intensity in Figure 1(a), which was depicted in (Figure 3(a)). The abundance of Firmicutes was showed in results, which increased after administrated with ω-3 LCPUFA. Firmicutes are often highly represented in the gut microbiota of healthy individuals and can be reduced in illness  . We demonstrated the effect of triglyceride and phospholipids type ω-3 LCPUFA on the bands of 17, 23, 27, 29 with RT-PCR, the results were shown in (Figures 3(b)-(d)). According to the results we can found that the two types of ω-3 LCPUFA helped improving the microbial structure.
3.3. Triglyceride Type ω-3 LCPUFA and Phospholipids Type ω-3 LCPUFA Improves the Histological Morphology of the Small Intestine in Treated Mice
To verify the activities of the two types ω-3 LCPUFA, we analysed the histological morphology of the small intestines of the mice in this study, the data were showed in Figure 4. Results showed that the morphology of the small intestine was intact in the three groups of mice. As observed, the structures of the
Table 2. The analysis results of DGGE gel bands recovery sequence.
Figure 2. PCA and cluster analysis of DGGE fingerprint. (a) The principal components analysis (PCA) scores plot according to the DGGE fingerprint; (b) Clustering of gut microbiota based on distances between different groups.
(a) (b) (c) (d)
Figure 3. The relative abundance of bacteria analyzed by gray intensity and RT-PCR. (a) The relative abundance of bacteria analyzed by gray intensity; (b) The quantity of Akkeermansia muciniphila analyzed by RT-PCR; (c) The quantity of Lactobacillus analyzed by RT-PCR; (d) The quantity of Lactococcus lactis analyzed by RT-PCR.
Figure 4. Changes in the morphology and the histology of the small intestine. The photographs are representative of 10 mice in each group. Scale bars indicate 100 um.
intestinal villi were complete. However, the histopathological features of the small intestine in the ω-3 LCPUFA-treated mice were greatly improved. The length of the small intestine was increased and the V/C ratio were examined to evaluate the effect ω-3 LCPUFA on the digestion?absorption function of the intestine. The V/C ratio was increased significantly in the ω-3 LCPUFA group, but in PL group was higher than FO group. The data showed that ω-3 LCPUFA can improve the integrity of the histological morphology of the intestinal mucosa.
According to the study of the two types of ω-3 LCPUFA and the results of DGGE, the diversity and structure of the gut microbiota significantly changed in FO and PL group. While the potential role of gut microbiota modulation in two types of ω-3 LCPUFA amelioration is not clearly understood. In the (Figure 1(b)) that the bands 17 (Lactobacillus animalis), 23 (Akkermansia muciniphila), 24 (Ruminococcus), 27 (Lactococcus lactis) were superior in PL and FO group, which are bacterial species that include probiotic strains. So we can conclude that the two types of ω-3 LCPUFA have the ability to improve Lactobacillus animalis, in previous study, which is a potent broad spectrum probiotic strain which inhibited bacterial pathogens, such as A. hydrophila, Pseudomonas aeruginosa and other food spoil pathogens. According to previous study, Lactobacillus animalis would range from preventing against human gastrointestinal bacterial pathogens   . Akkermansia muciniphila is a mucin-degrading bacterium that resides in the mucus layer  . Recently some studies have demonstrated that A. muciniphila administration increased the intestinal levels of endocannabinoids that control inflammation, the gut barrier and gut peptide secretion  . Lactococcus lactis is a food-grade, Gram-positive lactic acid bacterium that is a harmless food industry bacterium, which has been used extensively for producing a variety of peptides, proteins, and oral vaccines. Oral administration of Lactococcus lactis H61 to aged SAMP6 mice was associated with reduced bone-density loss, improved gross lesion and enhanced immune responses, compared with those of controls  . Ruminococcus is considered a keystone species in the human gut that degrades microcrystalline cellulose efficiently and contains the genetic elements necessary for cellulosome production  . Bacterial pathogens, inflammation are harmful for intestinal mucosa. Intestinal mucosal is widely known to be responsible for the defence of the large expanse of mucous membranes that form a barrier between the external environment and the body’s interior. As our result showed in (Figure 4), ω-3 LCPUFA can improve the integrity of the histological morphology. Therefore, the increase of these bacteria may be helpful for intestinal mucosa protection. Intestinal barrier is crucial to control intestinal permeability whose increase is associated with chronic inflammatory conditions  . This may be indicated that the function of LCPUFA ω-3 may be related to enhancing intestinal immunity.
We have described that phospholipids type ω-3 LCPUFA can improve the richness of bacteria, while triglyceride type ω-3 LCPUFA decrease it. The genetic diversity of the gut microbiota contributes to the overall development, provides the host with many beneficial functions and development of immune system. The 21 band (Streptococcus thermophilus) in PL group was inferior than the other two groups, in which can improve anti-inflammatory properties. This may be one of the reasons why Phospholipids type ω-3 LCPUFA has superiority for human health than Triglyceride type ω-3 LCPUFA.
The present study suggests that Phospholipids type ω-3 LCPUFA and Triglyceride type ω-3 LCPUFA could improve the structure of gut microbiota, increase the quantity of probiotic strain and regulate histological morphology of the small intestines. The two types ω-3 LCPUFA have different impacts on microbiota regulating function. Phospholipids type ω-3 LCPUFA is more superior than triglyceride type ω-3 LCPUFA. The results might provide a new theoretical basis for explaining the functions of ω-3 LCPUFA and the relationship between diet fat and gut microbiota.
This work was supported by the National Natural Science Foundation of China (Changhu Xue 31330060), the National Natural Science Foundation of China (Yuming Wang 31371757).
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