Method and Process of Enrichment of an Agave Fructan in a Prebiotic Drink

Abstract

This invention relates to the development of a method and process of enrichment of a fructan of Agave Tequilana Weber and Oaxacan Agave Angustifolia Haw in a prebiotic drink with unique characteristics that offers the prebiotic effect in probiotic and animal strains, using the in vitro and in vivo method. The developed process of enrichment protects the unique fructan against the polimerization grade change, thus allowing the preservation of its innate prebiotic characteristics.

Claims

1. A prebiotic beverage comprising a mixture of fructans of Agave Tequilana Weber and Agave Angustifolia Haw mixed with agave syrup, wherein the mixture of fructans comprises: a content of soluble solids between 73.48 to 73.80 Brix and pH level of 5.2; a content of soluble carbohydrates within the range of 328 mg/g and 347 mg/g; a complex profile of low polymerization grade fructans (DP3-DP10 fructans) and high polymerization grade fructans (DP>10 fructans) within the presence of neokestose; and wherein the prebiotic beverage maintains between 92.5% and 100% of the fructans mixture.)

2. The prebiotic beverage of claim 1 wherein the mixture of fructans has a prebiotic effect on strains of Bifidobacterium adolescentis (ATCC 15703), Bifidobacterium animalis (27536), Bifidobacterium bifidum (ATCC 29521), Bifidobacterium infantis (ATCC 25962), Lactobacillus acidophillus (ATCC 4356), Lactobacillus casei (ATCC 393), Lactobacillus paracasei (ATCC 25302) and Lactobacillus rhamnosus (ATCC 53103).

3. The prebiotic beverage of claim 1 wherein the mixture of fructans has a prebiotic effect in vivo.

Description

BREIF DESCRIPTION OF THE DRAWINGS

[0013] FIG. 1: Samples sent for analysis B1-B5.

[0014] FIG. 2: Chromatographic profile of carbohydrates and fructanes in the samples B1-B5 (The arrows indicate the different grades of polimerization)

[0015] FIG. 3: Thin-layer chromatography for identification of fructans in prebiotic drinks enriched with Blend

[0016] FIG. 4: Levels of fructose, glucose, sucrose, kestose, DP=5, and total carbohydrates in the prebiotic drinks

[0017] FIG. 5: Growth plates of the prebiotic drink probiotics

[0018] FIG. 6: Cage with mouse of the prebiotic drink in the vivarium

[0019] FIG. 7: Differences shown between ingestion of the feed of the three groups of mice (STD Control, RNE Chicory Inulin, BEB drink)

DETAILED DESCRIPTION OF THE PREFERED EMBODIMENTS

[0020] With the goal of fostering a healthy lifestyle through the consumption of functional products with natural ingredients as a base, throughout the years 2012-2014 an enrichment process has been developed for a prebiotic drink made from a mix of fructans that originate from Agave Tequilana Weber from Jalisco and/or Agave Angustifolia Haw from Oaxaca and agave syrup. This enrichment achieves a prebiotic effect, which has been evaluated in vivo and in vitro.

[0021] First, the ideal mixture of fructan+agave syrup was sought; 5 different blends were analyzed. These were stored at room temperature and their appearance and consistency were analyzed throughout the storage period. The samples did not exhibit changes in appearance and consistency during a period of 70 days of storage. The analyzed blends (FIG. 1) exhibited a content of soluble solids that was similar among the samples (73.48 to 73.80 Brix). The sample blends exhibited homogeneity, presenting pH values of 5.2; that is to say, a slightly acidic pH level. The color of the samples was determined through absorbance at 560 nm in a Biorad Benchmark/Plus Microplate spectophotometer (9) for the agave syrup, using glycerol as reference.

[0022] To determine the ideal blend, the following were analyzed: [0023] 1) Content of soluble carbohydrates, by an enzymatic method in single samples of inulin (5 samples), honey (5 samples), and blend; this in order to assure that the blend was the best option to be used as an ingredient to enrich the drink. The blend samples exhibited a content of fructans in a range of 328 to 347 mg/g. [0024] 2) Carbohydrates profile by TLC: solutions were prepared at a concentration of 50 mg/mL. One microliter (L) of each solution was taken and analyzed by thin-layer chromatography, along with oligosaccharide standards (MOS and FOS). Depending on the staining used, it is possible to differentiate the glucose and fructose derivatives according to the color of the spot (blue and reddish, respectively). The samples exhibited low DP fructans (fructans DP3-DP10) and high DP fructans (fructans DP>10). Additionally, the presence of a small blue spot was observed below the maltose corresponding to neokestose (trisaccharide base of neoseries-type fructans). The abundant spot on the base line of the TLC plate corresponds to fructans (polymers with DP>10 units); therefore it can be assumed that the analyzed samples contain fructans with extensive DP (FIG. 2).

[0025] Once the ideal blend was obtained, the enrichment method was developed for the prebiotic drink, which was evaluated in vivo and in vitro to determine the prebiotic effect. The enrichment was performed through a method of adding the blend in exact quantities and under controlled temperatures. This method is based on the control of the grades of polymerization of the blend and its fructans. This is so the structure is not damaged and to achieve the prebiotic benefit.

[0026] Water previously submitted to a resin filtering process, activated carbon, and softening filter in which the water must contain no more than 30 ppm of calcium carbonate were mixed at 45 C. with the blend, at a proportion of 11:1 respectively. This allowed the blend fructans to become soluble and dispersed in a very large aqueous base, which in turn allowed resistance to the pasteurization temperature of 60 C. for 30 minutes so that the structure was not damaged and the prebiotic benefit was retained. Pasteurization was performed in a cauldron with heating by gas and resistances. The mixture was constantly stirred at no more than 60 rpm with a propeller stirrer. Afterwards, the mixture was stirred as it cooled without any sort of cold exchange, as fructans undergo structural changes if exposed to sudden temperature changes. It was necessary to pay close attention to the operating conditions so the enriched mixture would not be contaminated (isolated cauldron in a fresh area at no more than 28 C.).

[0027] To determine whether the developed enrichment process was ideal, TLC analysis was performed on the beverage, which determined the presence of fructans and their profile in the enriched prebiotic drink. The presence of fructans was so high that the drink underwent dilutions. The presence of fructans with a DP higher than 10 was observed. (FIG. 3) The quantity of fructans was determined by the enzymatic technique, which showed that the drinks obtained 3.7-4% of fructans. This proves the success of the enrichment process, as 4% of fructans was added to the drink. To correct this value, carbohydrates were individually measured by liquid chromatography (HPAEC-PAD); a value of 3.9% of fructans was exhibited(FIG. 4).

[0028] To achieve a scientific evaluation and validity for this enrichment process of the blend in the prebiotic drink, a validation was performed with in vitro studies using different prebiotic strains, and in vivo using laboratory mice for the study.

[0029] Within the inulin in vitro evaluations, readings by thin-layer chromatography and high-performance anion-exchange chromatography coupled to an amperometric detector for the detection of fructans were performed. As well, the total of soluble solids was measured in the drink in Brix. Finally, the quantity of present fructans was quantified through an enzymatic method.

[0030] Through in vitro evaluations, the prebiotic potential of the drinks was determined by the probiotic bacteria study, using the Man, Rogosa, and Sharpe culture medium, also known as the MRS medium. (FIG. 5) The probiotic bacterias studied were of the bifidobacteria (six strains) and Lactobacilli (four strains) types. Of the Bifidobacterium genus: B. adolescentis (ATCC15703), B. animalis (27536), B. bifidum (ATCC29521), B. breve (ATCC, 15700), B. longum (15707), B. infantis (ATCC25962); and four strains of the Lactobacillus genus: L. acidophillus (ATCC4356), L. casei (ATCC393), L. paracasei (ATCC25302), and L. rhamnosus (ATCC53103).

[0031] It was concluded that the prebiotic effect of the enrichment process of the drinks exists. There was growth in: [0032] 1) 4 strains of the Bifidobacterium genus: B. adolescentis (ATCC 15703), B. animalis (27536), B. bifidum (ATCC 29521), B. infantis (ATCC 25962) [0033] 2) 4 strains of the Lactobacillus genus: L. acidophillus (ATCC 4356), L. casei (ATCC 393), L. paracasei (ATCC 25302), and L. rhamnosus (ATCC 53103).

[0034] The Bifidobacteria longum and Bifidobacteria breve probiotics did not grow in any of the mediums, regardless of the fact that it was attempted several times. The in vivo study was performed with ten and eight mice of the C57B1/6J line from the vivarium at Zacatenco, Mexico, aged 12 weeks. They were used at the beginning of the experiment, under controlled temperature and humidity and a light/dark cycle of 12 hours. (FIG. 6) The amount of ingested feed was measured weekly, as well as water consumption, mice weights, feces accumulation (residual carbohydrates were measured), and blood samples were taken from the mice's tails to determine glucose. After 5 weeks, the mice were put down and the large intestines and colon contents were collected to measure pH and SCFA. Additionally, blood was taken from the portal veins to determine triglycerides (TGA) and cholesterol.

[0035] The results showed the following: [0036] 1) The mice that consumed the prebiotic drink ate less (due to the fiber in the drink and their feeling of fullness)

[0037] FIG. 7 [0038] 2) The mice that consumed the prebiotic drink showed a smaller content of triglycerides and cholesterol than the other two groups, but the difference is not major. [0039] 3) The feces of the BEB mice show a larger content of residual carbohydrates than the other two groups and a larger production of SCFAs. [0040] 4) The prebiotic drink achieves the prebiotic effect within the digestive system of the mice.

[0041] To conclude, the experimental design tests for the enrichment process of the drink were performed, as well as the ideal blend development, in the pilot production plant for the prebiotic drink. The technological project had an execution period of 24 months in order to favor the technical enrichment process of a drink with a single fructan, thus achieving its innate benefit for those consuming the prebiotic drink.

[0042] It is important to mention that the drink possesses unique characteristics of pH and acidity to maintain the shelf life of the contained fructan during the shelf life of the product.

BIBLIOGRAPHIC REFERENCES

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