Concentration of acetic (C2:0), propionic (C3:0) and butyric (C4:0) acids obtained after 12 and 24 h
Most plants store starch or sucrose as reserve carbohydrates, but approximately 12-15% of higher plants (representing more than 40,000 species) synthesizes fructans as their main source of carbohydrates . Fructans are found naturally in plants as a heterogeneous mixture of different polymerization degrees, they are a polydisperse mixture. Among plants that store fructans, many are economically important, due to its content of fructans, as it is the case of chicory (
Currently, inulins are extracted from chicory roots, containing fructose chains having a degree of polymerization (DP) from 3 to 60  (Figure 1a). The chemical or enzymatic (endoinulinases) hydrolysis of inulins produces inulins of shorter DP (DP<10), these are called fructooligosaccharides (FOS) [5, 6].
Mexico is considered the origin center of evolution and diversification of the
Agave fructans posses a molecular structure compose of a complex mixture containing highly branched molecules with β(2-1) and β(2-6) linkages, as well as internal and external glucose units, due to the existence of both types of glucose, agave fructans have been classified as graminans (external glucose) and agavins (internal glucose)  (Figure 1b).
All fructans are considered prebiotics molecules that serve as a substrate for the gut microbiota [6, 12-15]. A prebiotic is an ingredient selectively fermented by probiotics (
1.2. Short chain fatty acids (SCFAs)
The gastrointestinal tract is an extremely complex ecosystem containing about 1011 CFU (colony forming units) of bacteria per gram of intestinal content. This large population of bacteria plays a key role in the nutrition and health of the host . The colonic microbiota ferments organic material that cannot be digested otherwise by the host in the upper gut. These include resistant starch, non-digestible carbohydrates (fructans) as well as some proteins and amino acids . The main products of fructans metabolism in the colon are linear SCFAs, mostly acetate (C2:0), propionate (C3:0) and butyrate (C4:0) [19-21] (Figure 2). However, other fermentation products may be lactate, succinate as well as ethanol , which are sometimes only intermediates in the global process of carbohydrates fermentation by the microbiota, and are metabolized in varying degrees to SCFAs by interactions and/or collaboration of present bacteria in the ecosystem, so that generally do not accumulate to any significant extent in the colon . Fructans fermentation also produces a few gases as CO2, CH4, H2 and additionally heat [19, 23]. The presence of both, non-digestible carbohydrates and SCFAs in the colon can positively alter the colonic physiology drastically . Various studies on microbial population have shown that SCFAs production is in the order of C2:0 > C3:0 > C4:0 in a molar ratio of approximately 60:20:20 mainly in the proximal and distal colon [19, 25]. An increased in SCFAs synthesis also creates a more acidic environment in the gut, which is important
1.2.1. Acetic (C2:0), propionic (C3:0) and butyric (C4:0) acids
C2:0 is the principal SCFA produced in the colon, this is readily absorbed and transported to the liver, and therefore is less metabolized in the colon . The presence of acetyl-CoA synthetase in the cytosol of adipose and mammary glands allows the use of C2:0 for lipogenesis once it enters the systemic circulation . C2:0 is the primary substrate for cholesterol synthesis. In the host, it may be absorbed and utilized by peripheral tissues also .
On the other hand, C3:0 is produced via two main pathways: 1) by fixation of CO2 to form succinate, which is subsequently decarboxylated (the “dicarboxylic acid pathway”) and 2) forms lactate and acrylate (the “acrilate pathway”) . C3:0 is also a substrate for hepatic gluconeogenesis and it has been reported that this acid inhibits cholesterol synthesis in hepatic tissue [31, 32]. The ratio of C3:0 to C2:0 in the colon is relevant since it lowers cholesterol synthesis coming from the C2:0 pathway .
Finally, C4:0 is the preferred fuel by the colonic epithelial cells but also plays a major role in the regulation of cell proliferation and differentiation . It is the most important SCFA in colonocytes metabolism, where 70% to 90% of C4:0 is metabolized by the colonocytes. C4:0 is used preferentially over C3:0 and C2:0 in a ratio of 90:30:50 . Approximately 95% of the C4:0 produced by colonic bacteria is transported across the epithelium, but concentrations in portal blood are usually undetectable as a result of a rapid utilization . C4:0 production might also occur through the use of other fermentation products such as C2:0 or lactate that can act as precursors of C4:0.
1.2.2. Production of SCFAs in vitro
|A||< 5||12 h||22.3a||0.50||5.3a||0.20||12.8a||0.20|
|B||= 4.8||12 h||20.2ab||1.50||4.1bc||0.02||10.2b||0.50|
|C||< 10||12 h||26.8a||0.60||4.7ab||0.20||12.8a||0.30|
|D||≈ 10||12 h||20.0ab||0.10||2.1d||0.10||9.3b||0.20|
|E||"/> 10||12 h||19.9ab||1.30||3.6c||0.10||9.2b||0.02|
|F||"/> 20||12 h||14.0b||2.10||0.8e||0.20||5.4c||0.10|
Urías-Silvas & López  analyzed the prebiotic potential of fructans extracted from five different species of
Santiago-García & López  studied the
1.2.3. Production of SCFAs in vivo
Nilsson and Nyman  evaluated the formation of SCFAs in the hindgut of rats fed with lactulose, lactitol, FOS and inulins of different DP and solubility. The major acids formed were C2:0, C3:0 and C4:0. The highest levels of C3:0 acid were found in caecum and proximal and distal colon of rats fed with inulins, whereas the highest levels of C4:0 acid were found in caecum and proximal and distal colon of rats fed with FOS. The authors concluded that the DP and solubility of the used prebiotics were of great importance on SCFAs production.
Similar results were obtained by Licht et al.  who fed rats with different dietary carbohydrates. These authors concluded that C3:0 acid concentrations reached statistical significance in animals fed with inulins, whereas the concentration of C4:0 acid was significantly higher in animals receiving FOS. Klessen et al.  also determined the production of SCFAs in the caecum and colon of germ-free rats associated with contents of human faecal, the rats were fed with inulins of different chain lengths (FOS, inulins and a mixture of FOS-inulins). They observed that FOS produced the greatest amount of C2:0 acid in the colon of the rats whereas inulins increased the concentration of C3:0 acid in the caecum of the animals that consumed this diet. Moreover, FOS, inulins and the mixture of FOS-inulins increased the amount of C4:0 acid in the caecum and colon of the rats fed with the mixture regard to animals fed with standard diet. The authors concluded that the type of diet and the fermentation site in the colon affected the concentration of SCFAs (Table 2). In another work, Levrat et al.  fed rats with different percentages of inulins (5, 10 and 20%), finding that C2:0 acid production was significantly lower in rats fed with 20% inulin diet. Moreover, all percentages of inulin increased the levels of C3:0 acid in the caecum of the rats; the highest concentration was found in animals that consumed the 10% inulin diet whereas C4:0 acid concentration was markedly enhanced in all supplemented diets in spite of the inulin percentage used. In another study, the same authors fed rats with 10% of inulin, they found a higher concentration of C3:0 acid in the portal vein as well as a significant decrease in plasma cholesterol levels of the rats fed with this diet with regard to animals that consumed the standard diet . On the other hand, a study carried out using obese rats that received a diet supplement with inulin, a two-fold greater C3:0 concentration in the portal vein and a decrement on triglyceride accumulation in the liver of these animals was observed . A similar result was seen in hamsters fed with different percentages of inulins (8, 12 and 16%). Plasma cholesterol and triglyceride concentrations were significantly lower with all the percentages of inulins studied with respect to hamsters fed with the standard diet .
To date, there are no published reports on the production of SCFAs
In another study with fructans of
With all the above, we decided to run an
2. Materials and methods
2.1. Animals and diets
Twenty-four male C57BL/6J mice of 12 weeks old at the beginning of the experiment were obtained from the animal facilities of CINVESTAV-Zacatenco (Mexico). The mice were housed in a temperature and humidity controlled room with a 12 h light-dark cycle. They were divided into three groups (eight mice per group) according to diet. Mice were acclimated for 7 days, having free access to a pelleted 5053 standard diet (Laboratory Rodent Diet, USA) and water. During the experimental period (6 weeks), STD mice group were fed with 5053 standard diet, whereas inulins-RNE and agavins-AAO mice groups received a diet prepared by mixing 90 g of 5053 standard diet with 10 g of Raftiline or fructans from
Agavins were extracted in our laboratory as described by López et al. . Briefly, one hundred grams of milled
2.3. Faeces and blood samples
Faeces collection was performed once a week during the experimental period to evaluate the SCFAs. On day 45, mice were anaesthetized by intra-peritoneal injection of sodium pentobarbital solution. Portal vein blood samples were collected in EDTA tubes; after centrifugation for 10 minutes at 2500 r.p.m., plasma was stored at -80 °C. The concentration of serum triglycerides, cholesterol and glucose was measured using kits coupling enzymatic reaction and spectrophotometric detection of reaction end-products (BioVision).
2.4. pH and SCFAs
Segments of the caecum and proximal, medial and distal colon were immediately excised. Caecal and colonic contents of each section were collected in tubes and frozen at -80 °C. The pH measurements were made using a microelectrode (PHR-146, Lazar Research Laboratories, Inc.). Analysis of SCFAs was carried out by gas chromatography and flame ionization detection as described by Pietro Femia et al.  with some modifications. Briefly, 0.05 g of caecal and faecal contents were acidified with 0.05 ml of sulfuric acid and SCFAs were extracted by shaking with 0.6 ml of diethyl ether and subsequent centrifugation at 14000 r.p.m. for 30 s. One microliter of the organic phase was injected immediately into the capillary column (Nukol) of the gas chromatograph coupled to a flame ionization detector. The initial temperature was 80 °C and the final temperature was 200 °C. Nitrogen was used as carrier gas and the quantification of the samples was carried out using calibration curves for C2:0, C3:0 and C4:0 acids. A standard curve for each acid was done for their quantitation in the samples.
2.5. Statistical analysis
Results are expressed as mean values with their standard errors of the mean. Statistical differences between groups were evaluated using one-way ANOVA followed by a Tukey test using GraphPad Prism version 5 for Windows. P<0.05 was regarded as statistically significant.
3.1. Feed intake and body weight
The intake of all mice independently of the diet fed ranged between 3.3 and 4.2 g/d with an average of 3.7 g/d, it is worth to mention that the intake fluctuated weekly throughout the study. The feed intake was 9% lower for the AAO group compared to the STD and RNE diets. Mice fed with the diet supplemented with RNE ate 10% more food than even the STD group. Initial body weights ranged from 21.4 to 24.4 g with final body weights ranging between 24.3 and 25.9 g. No significant differences among all groups were noted in body weight even though mice fed AAO reduced their intake.
3.2. Production of SCFAs and pH drop
The total production of SCFAs was greater for the group of mice fed with AAO in the caecum and proximal and medial colon. However in the distal colon, the production of SCFAs were not significantly different among supplemented diets but it did with the STD diet (Table 3).
C2:0 was the most abundant acid formed in the caecum and colon of all mice followed by C3:0 and C4:0 acid. The concentrations of C2:0 acid were significantly higher in the caecum and the first two sections of the colon (proximal and medial) in mice fed with AAO diet compared to RNE or STD groups. However, in the distal colon there were no significant differences on the production of C2:0 acid between groups of mice fed fructans (Figure 7a). The higher concentration of C3:0 acid was found in the caecum of mice fed with AAO diet. This increment was significant with regard to RNE but not for the STD diet. In the proximal and medial colon C3:0 acid production was greater for mice fed AAO, but these enhancements were not significant. Interestingly, in the distal colon of mice fed fructans (AAO and RNE) the enhancement was significantly for the production of C3:0 acid (Figure 7b). The concentration of C4:0 acid increased approximately 24% in the caecum of mice fed with RNE diet. This enhanced was significant with regard to AAO but not for the STD diet. Finally, an increment of C4:0 acid was observed in the medial and distal colon, this change was significant in mice fed with AAO and RNE diets compared to mice fed with the STD diet (Figure 7c).
SCFAs changes were confirmed when the pH was measured in all the same samples. The mice fed AAO diet showed a pronounced pH drop in the caecum and all sections of the colon. The group of mice fed RNE showed significant pH drop only in the medial and distal colon and the pH of the mice fed a STD diet did not change significantly in any sections of the gut (Figure 8). The pH drop changes positively correlated with the total production of SCFAs (Table 3).
3.3. SCFAs in the faeces
The analyses of SCFAs in the collected faeces showed that C2:0 acid was again the most abundant acid in the faeces of all mice followed by C3:0 and C4:0 acids. However, the amounts of C2:0 and C3:0 acids excreted in the faeces were not affected significantly by any dietary treatment. Surprisingly, only the mice fed with agavins (AAO) or inulin (RNE) diets, showed a significant increment on the amount of C4:0 acid compared with the STD diet (Figure 9).
Besides SCFAs some other physiological parameters were determined in the plasma of all mice groups, among them glucose, triglycerides and cholesterol. Glucose concentrations of mice fed AAO and RNE diets were significantly lowered by 12% and 17% respectively when compared to the STD diet. On the other hand, triglycerides concentrations of the supplemented groups compared with the STD were reduced by 37% and 38 % in mice fed AAO and RNE diets, respectively. A reduction of cholesterol concentrations by 36 % and 38 % in animals receiving AAO and RNE diets was also observed
The determination of the production of C2:0, C3:0 and C4:0 acids in the caecum, proximal, medial and distal colon of mice fed with different diets, was performed with the aids to evaluate the profiles of these acids throughout the caecum, large intestine and faeces of mice, and also to be able to establish the main sites of fermentation of inulins such Raftiline (RNE) and fructans extracted from
As a general conclusion, we can mention that the supplementation of diets with inulins or agavins altered the large intestine environment by increasing the amounts of SCFAs and lowering the pH in the colon, consequently reducing few health risks. Finally, we would like to close this work saying that these SCFAs had a positive effect on the host lipid metabolism, since they decreased the levels of triglycerides, cholesterol and glucose in blood of mice fed with supplemented diets.
Based on all the previous data, agave fructans may offer a good prebiotic potential, opening new and excited alternatives as food supplements. Even do, further research is definitely needed on specific health problems and should be performed using supplemented diets with agavins of different structures as well as different mixtures and concentrations, because more knowledge is needed on health issues such obesity, diabetes, colon cancer and in general, gut associated risks that might be improved with this type of ingredients.
Alicia Huazano-García thanks the Consejo Nacional de Ciencia y Tecnología (CONACYT) for her scholarship and also thanks M.S. Patricia Santiago for the agave fructans sample.
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