Effects of lotus seed resistant starch and sodium lactate on small intestinal flora and metabolites in rats

The small intestine is an important place for human digestion and absorption, mainly through enzymes to act on sugar, cholesterol, protein and other nutrients, the number of small intestinal microorganisms is much lower than that of the large intestine, however, studies have shown that the imbalance of small intestinal flora has a unique mechanism of action in the occurrence and development of various diseases: 1) small intestinal flora imbalance may lead to irregular arrangement of small intestinal villi and different degrees of breakage, so that mucosal permeability increases and barrier function decreases; 2) A large amount of sodium-potassium-ATPase is expressed in small intestinal epithelial cells, and the content of this enzyme decreases when the flora is dysbiotic, resulting in changes in cell osmotic pressure and damage to cell structure, which may synergistically disrupt the absorption of sugars, proteins, fats and other substances in the small intestine, causing the occurrence
of diabetes and other diseases.
Therefore, increasing the number of probiotics in the small intestine and controlling the reproduction of harmful bacteria by improving diet can be used as important measures
to maintain small intestinal homeostasis, regulate metabolism in the body, and prevent diseases.

Resistant starch (RS) can be fermented and utilized by colonic microorganisms as a substrate, so it has a prebiotic effect to promote the growth and activity of probiotics, and can interact with other prebiotics such as dietary fiber to exert its prebiotic effect
.
Lotus seeds nourish the spleen and kidneys, have a significant impact on the control of blood sugar and insulin levels in people with type 2 diabetes, and the content of amylose in lotus seeds is as high as 42%, which is a good source
of RS.
The surface of lotus seed resistant starch (LRS) after pressure heat and microwave treatment has a special gully-like structure, which can improve the adaptability of probiotics in poor intestinal environment, make better use of carbon sources, promote self-proliferation, and play a probiotic role
in preventing inflammation by participating in insulin secretion and regulating the expression level of various key factors such as antioxidant activity 。 At present, the physiological functions of LRS and sodium lactate (SL) have been studied, but the synergistic effect of LRS and SL has been rarely reported, in order to study whether there is a synergistic probiotic effect between the two, Shang Weixuan, Liu Lu, Zeng Hongliang* and others from the College of Food Science of Fujian Agriculture and Forestry University used LRS and SL as dietary interventions to study the effects of small intestinal flora and metabolic profile in rats, aiming to explore the potential mechanism of LRS and SL affecting small intestinal flora and metabolism.
Provide scientific basis
for the prevention of diabetes and other diseases.

1.
Effects of LRS and SL intervention on the diversity of intestinal flora in rats

The results showed that compared with the NC group, the species richness of LRS group (P<0.
05), SL group (P<0.
01), LRS+SL group (P<0.
05) was significantly improved, and the diversity of LRS group decreased (P<0.
05), which was consistent with Lin Shan's research results, there was no significant change in SL group (P>0.
05), species diversity in LRS+SL group increased significantly (P<0.
05) (Fig.
2A, B), and LRS+ Compared with LRS group (P<0.
001) and SL group (P<0.
05), the species diversity of SL group increased significantly, indicating that the synergy between LRS and SL was conducive to increasing the diversity<b10> of small intestinal flora in rats.
The PCoA analysis of each group of samples is shown in Figure 2C, and the difference test result is 0.
021, which is a significant
difference between samples.
There was little overlap between the NC group and the LRS group and SL group, and more overlap with the LRS+SL group, and the difference was small
.
As shown in Figure 2D, the number of species in the four groups was 118, the NC group had 20 endemic species, the LRS group had 33 endemic species, the SL group had 64 endemic species, and the LRS+SL group had 145 endemic species
.
The results showed that the number of endemic species increased when LRS and SL were added, and the increase effect was most significant
when LRS and SL were synergistic.

2.
Effects of LRS and SL interventions on intestinal colony composition and intergroup differences in rats

As shown in Figure 3A, from the phylum level, the firmicutes, actinobacteriota and proteobacteria were the dominant flora in each group, and the relative abundance of firmicutes and actinobacteriota in the LRS group increased compared with the NC group, and the relative abundance of Actinobacteriota decreased in the LRS group.
The relative abundance of Firmicutes in SL group decreased; The relative abundance of the LRS+SL group Bacteroidota increased
.
Among them, the Firmicutes metabolite butyrate has anti-inflammatory and improved insulin resistance, and its relative abundance increase is conducive to preventing the occurrence and development of obesity and type 2 diabetes, and Firmicutes has a weak ability to utilize or decompose lactic acid, and the relative abundance decreases in the SL group, which is consistent
with the results of Policastro et al 。 The family level was dominated by Lactobacillaceae, followed by Corynebacteriaceae and Peptostreptococcaceae, compared with the NC group, the relative abundance of Lactobacillus family and digestive streptococcaceae in LRS group was up-regulated, and the relative abundance of Corynebacterium family was down-regulated, SL group and LRS+ The SL group showed a decrease in the relative abundance of the family Streptococcaceae (Figure 3B).

At the genus level, the relative abundance of Lactobacillus was the highest, followed by Corynebacterium and Romboutsia, the relative abundance of Lactobacillus in the LRS group was upregulated and Corynebacterium was down-regulated, the relative abundance of Romboutsia in the LRS group increased, and the relative abundance of Romboutsia decreased in the remaining two groups (Figure 3C).

。 The top 15 species with significant differences in the relative abundance in Figure 3C were screened, and combined with Figure 3E and Figure 3F, the relative abundance of Dietzia in the LRS group was lower than that of the other three groups, and the levels of Vagococcus in the LRS group, SL group and LRS+SL group were significantly lower than those in the NC group (P<0.
01).
<b14> 。 Compared with the remaining groups, the LRS+SL group showed significant effects
in promoting unclassified_f__Ruminococcaceae growth (P<0.
01) and inhibiting the reproduction of Allobaculum (P<0.
05).
。 Among them, Lactobacillus is considered to be a probiotic that protects the intestinal barrier, regulates immune response, fights pathogenic bacterial infection, regulates metabolism, and prevents inflammation; Dietzia can act as an antioxidant and prevent cancer by producing natural carotenoids (canthaxanthin); Vagococcus has been linked to human intestinal infections, similar to pathogenic bacteria<b16> such as Enterococcus and Carnobacterium.

At the genus level, compared with the NC group, the relative abundances of Vagococcus (P<0.
01), Dietzia (P<0.
05), Enterorhabdus (P<0.
05) and Coriobacteriaceae_UCG-002 in the LRS group were significantly or significantly reduced, norank_f__Erysipelotrichaceae, Lachnospiraceae_NK4A136_ The relative abundance of group increased significantly (P<0.
05) (Figure 4A).
<b10> Studies have shown that Enterorhabdus is considered to be one of the human intestinal pathogenic bacteria, which participates in the fermentation of undigested protein in the intestine, which may disrupt intestinal homeostasis, and its products ammonia, putrescine, etc.
are closely related to the occurrence of colorectal cancer; Coriobacteriaceae_UCG-002 has been shown to be relatively abundant in the gut microbiota of morbidly obese individuals and may have potentially metabolically deranged effects
.
The relative abundance of Lactococcus in the SL group was significantly reduced (P<0.
05), and the relative abundance of unclassified_f__Micrococcaceae and unclassified_f__Aerococcaceae was significantly increased (P<0.
05) (Figure 4B).
<b12> 。 The relative abundance of Vagococcus and Allobaculum in LRS+SL group was significantly reduced (P<0.
01), and the relative abundance of unclassified_f__Ruminococcaceae (P<0.
01) and Lachnospiraceae_NK4A136_group (P<0.
05) was significantly or significantly increased (Figure 4C).
<b13> 。 In addition, Lachnospiraceae and Ruminococcaceae, both Firmicutes, are common flora in the human gut and can hydrolyze starches and sugars to produce butyrate and short-chain fatty acids
.
At the same time, both the LRS group and the SL group showed significantly lower relative abundance of unclassified_f__Ruminococcaceae (P<0.
05) and a very high relative abundance of Allobaculum (P<0.
01) compared with LRS+SL group (Fig.
4D, E).
<b15> 。 The relative abundance of Allobaculum was positively correlated with the expression of ANGPTL4, a key regulator of lipid metabolism and a circulating medium of gut microbiota and fat deposition, and experiments have shown that high-fat dietary treatment also significantly increased
the relative abundance of Allobaculum in mice.

It can be seen that the dietary intervention of LRS and SL is conducive to the optimization of small intestinal flora structure and the maintenance of intestinal homeostasis, which is consistent
with the results of Zeng Hongliang et al.
In addition, the synergistic effect of LRS and SL promotes the proliferation of unclassified_f__Ruminococcaceae and inhibits the proliferation of Allobaculum, which has the potential effect
of preventing metabolic disorders.

3.
Differences in intestinal metabolic profiles in rats after LRS and SL intervention

The OPLS-DA results for metabolic profiling data are shown
in Figure 5.
In the anion mode, the metabolic modes of LRS group and NC group, LRS+SL group and NC group, LRS group and LRS+SL group, SL group and LRS+SL group can be completely separated, indicating that LRS and LRS and SL synergy can effectively intervene in the change of
metabolic mode.

Under the conditions of VIP>1, P<0.
05, FC<1 or FC>1, the screened differential metabolites were compared and classified with the KEGG database, and the results are shown in Figure 6, and the specific differential metabolites are shown in Table 1
。 In summary, LRS and SL are conducive to the synthesis of steroid hormones, and LRS can promote the increase of the levels of (R)-lipoic acid, ginkgolide A, α-curcumin and other substances in rats when acting alone, which is conducive to regulating amino acid and lipid metabolism.
SL intervention regulates levels of metabolites such as steroid hormones and phospholipids; When the two work together, the regulatory effect on amino acid metabolism and vitamin metabolism is more obvious, and may improve the body's resistance to harmful metabolites
.

Figure 7 shows
the functional pathways that differ significantly between LRS, SL, LRS+SL and NC groups, and between LRS groups, SL groups, and LRS+SL groups.
where the horizontal axis represents the relative importance of metabolites in the pathway and the bubble size represents the pathway importance value
.
There are two functional pathways that differ greatly from the LRS group in the NC group: steroid hormone, cysteine and methionine metabolism pathways (Figure 7A).

The steroid hormone functional pathway is related to steroids such as estrogen, corticosterone, dehydroepiandrosterone and their derivatives, and is positively correlated with
the abundance of Oscillibacter, a bacterium that regulates bile acid metabolism and lipid metabolism 。 There were four functional pathways with obvious differences between NC and SL groups: steroid degradation, steroid hormone biosynthesis, primary bile acid synthesis, and glutathione metabolism pathway (Figure 7B).

Among them, primary bile acid synthesis plays an important role
in combating cholestasis.
Glutathione metabolism is an important antioxidant and metabolic regulatory pathway in the body, which can scavenge nitrogen and oxygen free radicals and prevent liver diseases
.
The control group had three functional pathways with obvious differences from the LRS+SL group: aminoacyl-tRNA biosynthesis, β β-alanine metabolism, and staurosporine biosynthesis pathway (Figure 7C).

。 There were two functional pathways with large differences between the LRS group and the LRS+SL group: glutathione metabolism, ascorbate and aldarate metabolism (Figure 7D).

。 There were three functionally different pathways between the SL group and the LRS+SL group: flavone and flavonol, flavonoid biosynthesis, and glycerophospholipid metabolism (Figure 7E).

Amino acid metabolic pathways such as β-alanine metabolism and lipid metabolism pathways such as glycerophospholipid metabolism are all related to
the prevention of diseases such as metabolic syndrome.
In summary, LRS and SL both show the important role of amino acid metabolism and hormone metabolism regulation when they act alone, and SL also has a promoting effect in optimizing liver function and enhancing liver defense system.
When the two work synergistically is conducive to maintaining lipid metabolism, vitamin metabolism stability and promoting the synthesis and expression
of biologically active substances.

4.
Correlation analysis results of microbiota diversity and metabolic spectrum

Correlation analysis of different genera and differential metabolites of small intestinal flora is shown in Figure 8, Globicatella, Rothia, Rhodococcus, Bacillaceae, Gemella, Acinetobacter, Ralstonia, and LysoPC (24:1(15Z)) (P<0.
05), Dehydroinduced vomiting rosinoxylol (P<0.
001), capsaicin, oleoyl ethanolamide, corticosterone, estrogen, (R)-lipoic acid, estriol, ginkgolide A (P<0.
05) were significantly positively correlated, and L-serine, aschine, LysoPC (20:2(11Z, 14Z)), and LysoPC (18:1(9Z)) (P<0.
05).
<b10> 。 Lachnospiraceae_UCG-001 was significantly positively associated
with LysoPC(24:1(15Z))(P<0.
05), dehydrovostigator roflagolol (P<0.
001), capsaicin (P<0.
05), oleoyl ethanolamide (P<0.
01), and corticosterone (P<0.
01).
Enterorhabdus was negatively correlated with estrogen, (R)-lipoic acid and ginkgolide A (P<0.
01).
<b12> Allobaculum was negatively correlated with citrulline (P<0.
01) and positively correlated with vitamins such as LysoPC (17:0) and histidine (P<0.
05).
<b13>

Conclusion