**3. Results**

172 Lipid Metabolism

With all the above, we decided to run an *in vivo* experiment feeding mice with *Agave angustifolia* fructans and evaluating the formation of SCFAs in caecum, colonic sections and

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 *A. angustifolia* (AAO). All diets were made by Laboratory Rodent Diet and were available *ad* 

Agavins were extracted in our laboratory as described by López et al. [10]. Briefly, one hundred grams of milled *Agave angustifolia* stems were extracted twice with 100 ml of 80% v/v ethanol with continuous shaking for 1 h at 55 °C. The sample was filtered and the plant material re-extracted with 100 ml of water for 30 min at 55 °C. The supernatants were mixed; chloroform was used to eliminate the organic fraction. The aqueous phase was concentrated by rotary evaporation under reduced pressure. The sample was dried using a spray dryer and stored in a humidity-free container. RNE were from Beneo Orafti. The average degree

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

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

reaction and spectrophotometric detection of reaction end-products (BioVision).

faeces, as well as the pH drop in all these areas of the gut.

polymerization for agavins is 32[11] and for RNE >23.

**2.3. Faeces and blood samples** 

**2.4. pH and SCFAs** 

**2. Materials and methods** 

**2.1. Animals and diets** 

*libitum* to mice.

**2.2. Chemicals** 

#### **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

#### 174 Lipid Metabolism

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).

Metabolism of Short Chain Fatty Acids in the Colon and Faeces of Mice After a Supplementation of Diets with Agave Fructans 175

**Figure 7.** Concentrations of SCFAs in the caecum and the three sections of the colon of mice fed with a standard diet (STD; ) and supplemented diets containing Raftiline (RNE; ) and *A. angustifolia* Oax. (AAO; ). A) Acetic acid (C2:0); B) Propionic acid (C3:0); and C) Butyric acid (C4:0). Mean values n=8 with their standard errors of the mean for each parameter measured. Mean values with different letters were

significantly different (P≤0.05).

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).


\*[mmol/Kg of fresh weight]. Mean values n=8 with their standard errors of the mean. a,b Mean values with unlike superscript letters were significantly different (P≤0.05).

**Table 3.** Concentration of total short chain fatty acids production in the caecum and the three sections of the colon in mice fed a standard (STD) diet or diet supplemented with Raftiline (RNE-inulin) or *Agave angustifolia* Oax. (AAO-agavins).

Metabolism of Short Chain Fatty Acids in the Colon and Faeces of Mice After a Supplementation of Diets with Agave Fructans 175

174 Lipid Metabolism

(Figure 7c).

SCFAs (Table 3).

**Section** 

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

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

Caecum 73.93a 1.98 70.90a 2.08 83.48b 1.54

Proximal colon 71.98a 2.11 76.93a 2.62 106.92b 4.61

Medial colon 51.44a 1.25 64.26b 2.59 107.25b 2.41

Distal colon 28.58a 0.86 49.65b 2.91 66.84b 2.97

**Table 3.** Concentration of total short chain fatty acids production in the caecum and the three sections of the colon in mice fed a standard (STD) diet or diet supplemented with Raftiline (RNE-inulin) or

\*[mmol/Kg of fresh weight]. Mean values n=8 with their standard errors of the mean. a,b Mean values with unlike

superscript letters were significantly different (P≤0.05).

*Agave angustifolia* Oax. (AAO-agavins).

**STD\* RNE\* AAO\***

**Mean SEM Mean SEM Mean SEM** 

**Figure 7.** Concentrations of SCFAs in the caecum and the three sections of the colon of mice fed with a standard diet (STD; ) and supplemented diets containing Raftiline (RNE; ) and *A. angustifolia* Oax. (AAO; ). A) Acetic acid (C2:0); B) Propionic acid (C3:0); and C) Butyric acid (C4:0). Mean values n=8 with their standard errors of the mean for each parameter measured. Mean values with different letters were significantly different (P≤0.05).

Metabolism of Short Chain Fatty Acids in the Colon and Faeces of Mice After a Supplementation of Diets with Agave Fructans 177

**3.4. Plasma glucose, triglyceride and cholesterol** 

superscript letters were significantly different (P≤0.05).

**4. Discussion** 

or diet supplemented with Raftiline (RNE) and *A. angustifolia* Oax. (AAO).

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 *vs* STD diet (Table 4).

**Diet Glucose (mM) Triglyceride (mM) Cholesterol (mM)** 

**STD** 6.690a 0.25 3.070a 0.09 3.087a 0.10 **RNE** 5.932b 0.27 2.233b 0.27 1.876b 0.14 **AAO** 5.857b 0.31 2.378b 0.18 1.756b 0.09 (Mean values n=8 with their standard errors of the mean for each parameter measured) a,b Mean values with unlike

**Table 4.** Glucose, triglycerides and cholesterol levels in plasma of mice fed with a standard (STD) diet

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 *A. Angustifolia* (AAO) supplemented in the diets. These objectives basically arise from the knowledge on the structural differences between the two fructans types. Raftiline is an inulin type fructan with an average DP of 25 and it is know that its structure is completely linear, therefore containing a terminal glucose molecule. On the other hand, *A. angustifolia* fructans have an average DP of 32, with molecular structures very complex, they are highly branched and present a terminal glucose molecule (graminans) or internal glucose (agavins) [11]. Many reports have established that the structure of undigested carbohydrates and the microbiota present in an ecosystem are determining factors that control fermentation in the gut [50]. It is also know that the profiles of the production and distribution of SCFAs in the gut are influenced not only by the type of consumed carbohydrates, but also by the place where fermentation of those carbohydrates takes place, essentially in the caecum in mice and in the upper colon in humans, but the type of substrate may also affect the site of fermentation [51]. Previous reports mentioned that high DP fructans are fermented more slowly in the caecum and proximal colon, thus reaching the distal colon almost unchanged [6, 34], but bacteria present in this section produce mainly C4:0 acid as the end product of such fermentation [51]. In the present work, inulins were fermented poorly in the caecum and proximal colon but an increment was

**Mean SEM Mean SEM Mean SEM** 

**Figure 8.** pH values in the caecum and the three sections of the colon of mice fed a standard diet (STD; ) or diet supplemented with Raftiline (RNE; ) or *A. angustifolia* Oax. (AAO; ). Mean values n=8 with their standard errors of the mean. Mean values with different letters were significantly different (P≤0.05).

#### **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).

**Figure 9.** Concentration of acetic (C2:0), propionic (C3:0) and butyric (C4:0) acids excreted in the faeces of mice fed a standard diet (STD; ) or diet supplemented with Raftiline (RNE; ) or *A. angustifolia* Oax. (AAO; ). Mean values n=8 with their standard errors of the mean. Mean values with different letters were significantly different (P≤0.05).

#### **3.4. Plasma glucose, triglyceride and cholesterol**

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 *vs* STD diet (Table 4).


(Mean values n=8 with their standard errors of the mean for each parameter measured) a,b Mean values with unlike superscript letters were significantly different (P≤0.05).

**Table 4.** Glucose, triglycerides and cholesterol levels in plasma of mice fed with a standard (STD) diet or diet supplemented with Raftiline (RNE) and *A. angustifolia* Oax. (AAO).

#### **4. Discussion**

176 Lipid Metabolism

(P≤0.05).

(Figure 9).

**3.3. SCFAs in the faeces** 

were significantly different (P≤0.05).

**Figure 8.** pH values in the caecum and the three sections of the colon of mice fed a standard diet (STD; ) or diet supplemented with Raftiline (RNE; ) or *A. angustifolia* Oax. (AAO; ). Mean values n=8 with their standard errors of the mean. Mean values with different letters were significantly different

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.** Concentration of acetic (C2:0), propionic (C3:0) and butyric (C4:0) acids excreted in the faeces of mice fed a standard diet (STD; ) or diet supplemented with Raftiline (RNE; ) or *A. angustifolia* Oax. (AAO; ). Mean values n=8 with their standard errors of the mean. Mean values with different letters

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 *A. Angustifolia* (AAO) supplemented in the diets. These objectives basically arise from the knowledge on the structural differences between the two fructans types. Raftiline is an inulin type fructan with an average DP of 25 and it is know that its structure is completely linear, therefore containing a terminal glucose molecule. On the other hand, *A. angustifolia* fructans have an average DP of 32, with molecular structures very complex, they are highly branched and present a terminal glucose molecule (graminans) or internal glucose (agavins) [11]. Many reports have established that the structure of undigested carbohydrates and the microbiota present in an ecosystem are determining factors that control fermentation in the gut [50]. It is also know that the profiles of the production and distribution of SCFAs in the gut are influenced not only by the type of consumed carbohydrates, but also by the place where fermentation of those carbohydrates takes place, essentially in the caecum in mice and in the upper colon in humans, but the type of substrate may also affect the site of fermentation [51]. Previous reports mentioned that high DP fructans are fermented more slowly in the caecum and proximal colon, thus reaching the distal colon almost unchanged [6, 34], but bacteria present in this section produce mainly C4:0 acid as the end product of such fermentation [51]. In the present work, inulins were fermented poorly in the caecum and proximal colon but an increment was

#### 178 Lipid Metabolism

observed on the concentration of C4:0 acid in the distal colon, which agrees well with previous reports. However, mice fed with agave fructans (AAO) produced greater amounts of SCFAs in the caecum and proximal and medial colon than those mice fed with inulins (RNE), suggesting that AAO fructans were easily fermentable, independently of the their high DP, therefore, the difference might be due to the presence of branches in the agave fructans, which could make the molecule more accessible to enzymes (fructosyltransferases), in other words, there are more terminal fructose available for the fructosyltransferases of bacteria. According to the production of SCFAs, proximal and medial colon, were the main sites where fermentation of *A. angustifolia* fructans was carried out. In general, an increment was observed in the production of C3:0 and C4:0 acids in the distal colon of mice that received diets supplemented with fructans. On the other hand, total SCFAs and individual SCFAs concentrations in the different sections of the colon are very important since they have been associated with many diseases of the colon, especially with colon cancer and gastrointestinal disorders. Therefore, increased SCFAs production and a greater delivery of them distally, especially C4:0 acid, may have an important role in preventing some of these diseases and other metabolic problems. Moreover, C3:0 acid has been reported to have a positive metabolic effect, through the inhibition of hepatic cholesterol synthesis from C2:0 acid [32]. Interestingly, a significant decrease in plasma triglycerides and cholesterol levels of animals fed with fructans was observed in this work. Finally, the mice that consumed agave fructans showed the more pronounced drop on pH in the caecum and the three sections of the colon, which creates a more acid environment which is highly beneficial for the grow of bacteria such as *bifidobacteria* and/or *lactobacilli* but is detrimental for the growth of potentially pathogenic species [17,18].

Metabolism of Short Chain Fatty Acids in the Colon and Faeces of Mice After a Supplementation of Diets with Agave Fructans 179

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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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.

### **Author details**

Alicia Huazano-García and Mercedes G. López *Departamento de Biotecnología y Bioquímica, Centro de Investigación y de Estudios Avanzados del IPN, México* 
