Method for producing and purifying fructo-oligosaccharide and product obtained by method

Abstract

The present invention relates to a method for producing and purifying fructooligosaccharides (FOS) having low or no residual content of glucose, fructose or sucrose and products obtained by said method, the FOS having different proportions of oligomers (GF2, GF3, GF4, GF5 and GF6) and their isomers, due to the excellent properties of the FOS in modulating the gut microbiota and direct and indirect immunomodulation, and to the products obtained by said method. Therefore, the feed can be used for nutrition of animals and human beings. The method for producing and purifying fructo-oligosaccharide according to the present invention uses at least two microorganisms or yeasts simultaneously or separately in order to convert sucrose into fructo-oligosaccharide (FOS) and completely consume glucose in the medium and evaporate alcohol or ethanol during the drying of the final product, one of the two microorganisms or yeasts is not able to produce an extracellular invertase in a sucrose-containing medium.

Description

Technical Field

[0001] The present invention relates to a process for the production and purification of fructooligosaccharides (FOS) and the products obtained by this process, which have low or no glucose, fructose or sucrose residues, and which FOS have different ratios of oligomers (GF2, GF3, GF4, GF5 and GF6) and their isomers, and can be used in animal and human nutrition due to their excellent properties for regulating the intestinal microbiota and direct and indirect immunomodulation. Background Art

[0002] FOS or oligofructose is one of the most popular prebiotic oligosaccharides in the research and use of human and animal nutrition, because they have a recognized bifidogenic effect. Because after fermentation, preferably after fermentation by bifidobacteria and lactobacilli present in the intestinal tract, short-chain fatty acids, such as acetic acid, propionic acid, butyric acid and lactic acid, are produced. These compounds indirectly promote the regulation of intestinal microbiota by environmental acidification, which is conducive to the growth of beneficial bacteria, but is not conducive to the growth of spoilage bacteria causing enteritis, such as Salmonella, Escherichia coli, Clostridium, Campylobacter and other pathogens.

[0003] In addition to lowering intestinal pH, these organic acids are absorbed by the intestinal epithelial cells and used as a source of energy, increase the production of mucin (the protective mucous membrane of the intestinal epithelium), promote the increase of intestinal villi, improve the absorption of nutrients, they strengthen the "tight junctions" and promote greater integrity of the intestinal epithelium. In addition, they promote regularization of metabolism and control blood levels of glucose, triglycerides and cholesterol.

[0004] In addition to contributing to the ecological balance and balancing the various hormones produced in the intestine, the regulation of the microbiota is now considered to be the second brain, promoting secondary immunomodulatory effects, since beneficial microorganisms activate T cells, which produce cytokines that recruit cells to fight inflammatory diseases and pathogens, such as interleukins IL-1, IL-2, IL-4, IL-6, IL-10, etc. They also act on the adaptive immune system by increasing the production of IgA, IgG and IgM antibodies by B cells.

[0005] Recent studies have shown that prebiotic oligosaccharides, such as long-chain FOS (lcFOS) and short-chain GOS (scGOS), act directly on the immune system, where they are recognized by the “tool-like receptor” (TLR-4) and produce interleukin 10, thus demonstrating their direct action, i.e., they have a significant impact on the innate immune system even in the very initial part of the intestine, even if they are not fermented or consumed by probiotics.

[0006] FOS can be found in many plants and foods, such as chicory (Chichorium intybus), artichokes, Jerusalem artichokes, yacon, onions, bananas, tomatoes, garlic, asparagus, agave, honey and sugar cane. They are reserve carbohydrates and have an osmoregulatory role in plants. The formation of FOS is attributed to environmental conditions and developmental stage, and is caused by low temperatures leading to the accumulation of fructans in leaves and roots.

[0007] FOS is also produced by many microorganisms, such as Aspergillus, Aureobasidium sp., Fusarium, Penicillium, Gliocladium virens, Sporutrichum thermophile, Scopulariopsis brevicaulis, Rhodothorula, Kluyveromyces marxianus, Klyveromyces marxianus ), Xanthoplylomyces dendrohous, Schwanniomyces occidentalis, Saccharomyces cerevisiae, Bacillus and Zymomonas mobilis.

[0008] Whether in plants or through microbial pathways, the formation of FOS is due to hydrolases (EC.3.2.1.26) and transferases (EC.2.4.1.9) such as fructofuranosidases and fructosyltransferases.

[0009] FOS differ according to the size of the chain and the bonds between the fructose molecules, depending on the enzyme that forms it. According to the definition of IUPAC-IUB (International Union of Pure and Applied Chemistry), oligosaccharides are sugars or carbohydrates that contain from 3 to 10 monosaccharide units in their chain. Thus, fructo-oligomers can contain up to 10 fructose molecules in their composition. However, many authors prefer a definition that they are molecules that contain a degree of polymerization of up to 10. Above this value, they are called fructan polysaccharides or "fructans", varying between levan and inulin, which can contain up to 60 fructose units in their molecular chain.

[0010] Fructose chains can contain bonds of the 1F, 6G and 6F type. The fructose units in the first type are linked by β-2,1 type glycosidic bonds. 6G are called neo-oligofructose (NeoFOS), and they have β-2,6 type glycosidic bonds between fructose and glucose units. As for the 6F type, they have β-2,6 type glycosidic bonds between fructose units.

[0011] The best-known FOS biosynthesis process follows a mathematical model based on the action of fructosyltransferase from Aureobasidium pullulans:

[0012] GFn+GFn→GFn-1+GFn+1n=1–3

[0013] (G: glucose, F: fructose, GF: sucrose)

[0014] The mechanism is divided into three reactions:

[0015] 2 GF → GF2 + G (1)

[0016] 2 GF2 → GF3+ GF (2)

[0017] 2 GF3→ GF4 + GF3 (3)

[0018] In this model, excess glucose acts as an inhibitor of fructosyltransferase, thereby exerting a negative feedback effect, i.e., once a certain amount of glucose is present in the reaction medium, the reaction stops. Therefore, in the prior art, the maximum yield of the FOS synthesis process is 60%, leaving sucrose residual, and the residual glucose reaches a very high level, accounting for up to 30% of the total composition, which makes spray drying unfeasible.

[0019] A classic example of a standard FOS reaction using a fructosyltransferase is:

[0020] [GF]→[G]+[F]+[GF]+[GF2+GF3+GF4]

[0021] 40% → 12% + 1% + 3% + 24%

[0022] Yield=[GF2+GF3+GF4] / [GF]=

[0023] =24% / 40%=60%

[0024] As a result, it can be noted that the concentration of glucose in the medium is too high, preventing the reaction from continuing and possibly even forming larger oligomers, as shown in the following mathematical example:

[0025] 2 GF → GF2 + G (1)

[0026] 2 GF2 → GF3+ GF (2)

[0027] 2 GF3→ GF4 + GF3 (3)

[0028] 2 GF4→ GF5 + GF4 (4)

[0029] 2 GF5→ GF6 + GF5 (5)

[0030] The present invention proposes and demonstrates the formation of GF5 and GF6 by simultaneous removal of glucose and synthesis of FOS.

[0031] In order to remove glucose from the final FOS syrup, the most commonly used method in the prior art is to use a chromatographic column to remove lower molecular weight sugars such as glucose, fructose and sucrose by size exclusion (ion sieving) until the purity of FOS in the purified syrup reaches 100% and the purity of FOS in the final product reaches 95%. Figure 1 and Figure 2 A comparison of the purification steps of a conventional FOS production process and the process of the present invention is shown.

[0032] On the other hand, the present invention provides an alternative, which achieves the dual purpose of synthesizing FOS and purifying it by removing glucose and other residual sugars from the reaction medium by using intact microorganisms for the bioconversion process. Therefore, in addition to the genus Pullulan, which is responsible for the synthesis of FOS, Saccharomyces cerevisiae is used, which selectively consumes only glucose and fructose, but not sucrose and FOS (i.e. oligomers with α,1-2 and β,2-1 bonds). As a result, the sucrose conversion rate is increased to a minimum of 70%, the presence of residual sucrose is not observed, and since the ethanol is completely evaporated during the spray dryer drying process, the FOS concentration in the syrup and the final product reaches a minimum value of 80% to 90%.

[0033] Glucose concentrations greater than 3% in FOS syrup do not allow for the drying process using spray drying technology. This is due to the low glass transition (tg) values ​​of low molecular weight sugars such as glucose and fructose, which are not the case with sugars with a higher degree of polymerization such as GF2, GF3 and GF4. Removing glucose by microbial processes is less expensive than separating it by chromatography, as is currently done in the art.

[0034] Prior art document JPH 04293494 teaches that fructose-containing oligosaccharides or glycosides that can be used in medicine and food fields can be produced efficiently at low cost by treating sucrose with fructosyltransferase and invertase-deficient yeast in the presence of an acceptor. Sucrose is dissolved in water in the presence of an acceptor (e.g., lactose), reacted with β-fructofuranosidase (a fructosyltransferase produced by Arthrobacter sp. K-1), and simultaneously reacted with invertase-deficient yeast, the reaction conditions being pH 6.5-7.0, 35° C., the reaction time being 20 hours, the invertase-deficient yeast being removed by diatomaceous earth filtration, the filtrate being treated with activated carbon at 95° C. for 1 hour, purified with ion exchange resin, concentrated and spray-dried to obtain fructose-containing oligosaccharides (including lactosyl fructoside) or fructose-containing glycosides (e.g., fructosyl stevioside).

[0035] However, the present invention is different in that it solves the following problems and achieves unusual results.

[0036] Problem solved

[0037] The present invention solves the problem of achieving better yields, mainly better purity, in a process for obtaining fructooligosaccharides (FOS) by the skilled person, due to the elimination of glucose, fructose or sucrose residues, and some by-products which make it difficult to obtain better purity and mainly make the purification conditions of the obtained final product more expensive. In addition, the present invention optimizes the acquisition of the final product in a sustainable manner by removing alcohols, in particular ethanol, from the final product during the spray dryer drying stage.

[0038] Extraordinary effects of the present invention

[0039] The present invention, in addition to obtaining a higher purity final product, and using a more sustainable process and more economical equipment and conditions than those used by those skilled in the art, also allows obtaining a final product containing more long-chain oligomers (such as GF5 and GF6), which are very important for reaching the bifidobacterium and lactobacillus colonies in the most distal parts of the large intestine, while the short-chain oligomers GF2, GF3 and GF4 cannot reach. This is possible because the two microorganisms (Usherella pullulans and Saccharomyces cerevisiae) can coexist and work together simultaneously in the process.

[0040] Purpose of the Invention

[0041] A method for obtaining and purifying fructooligosaccharides (FOS) using yeast was developed to present:

[0042] - Direct bioconversion of sucrose to FOS with high conversion and yield;

[0043] - Microbial removal of glucose;

[0044] - Microbial removal of glucose, thanks to the use of Saccharomyces cerevisiae that does not produce extracellular invertase, obtained by using the CRISPR Cas9 approach;

[0045] - Oligofructose has lower production cost;

[0046] - The obtained syrup containing oligofructose can be directly dried;

[0047] - Producing FOS containing different proportions of short-chain oligomers (scFOS such as GF2, GF3, GF4) and a large amount of long-chain oligomers (lcFOS such as GF5 and / or GF6) and their isomers;

[0048] - In addition to acting as a direct immunomodulator, it is possible to produce FOS with a ratio of oligomers more suitable for each purpose of use, including more effective functional physiological effects, greater regulatory effects on the microbiota and greater promotion of ecological balance;

[0049] - More sustainable than methods used by those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] The accompanying drawings are briefly described as follows:

[0051] Figure 1 : Traditional methods for obtaining fructooligosaccharides.

[0052] Figure 2 : A method for obtaining oligofructose according to the present invention.

[0053] Figure 3: Description of the reaction of forming FOS from sucrose molecules. GF: sucrose, G: glucose, F: fructose, GF2, GF3, GF4: FOS.

[0054] Figure 4 : Mark A: Standard method or prior art.

[0055] Figure 5 : Label B: The method described in the present invention.

[0056] Figure 6 : Representative schemes of the SUC2 gene and genome editing strategies. A) Localization of the SUC2 gene on chromosome IX and the design of primers used to edit the gene promoter region and subsequently delete it using CRISPR / Cas 9 technology. B) The structure of the SUC2 gene after editing the gene promoter.

[0057] Figure 7 : Typical correlations of fermentation and stool quality parameters between negative control (NC) and prebiotic-containing treatments. Summary of the invention

[0058] The present invention relates to a method for producing and purifying oligofructose, wherein at least two microorganisms or yeasts are used simultaneously or separately to convert sucrose into oligofructose (FOS) and completely consume glucose in a medium, and evaporate alcohol or ethanol during the drying process of the final product, thereby obtaining a final product, wherein one of the two microorganisms or yeasts cannot produce an extracellular invertase in a medium containing sucrose. DETAILED DESCRIPTION

[0059] The present invention describes a new product containing short-chain oligofructose (GF2, GF3, GF4 (scFOS)) and long-chain oligofructose (such as GF5 and GF6 (lcFOS)) and their isomers, which is derived from an innovative purification process, in which sugar residues (such as glucose and fructose) are removed by Saccharomyces cerevisiae, rather than by the traditional way of chromatographic purification. In addition, since the synthesis and purification of FOS occur simultaneously, the negative feedback effect of excess glucose production on the performance of fructosyltransferase is prevented, thereby increasing the yield of the sucrose to FOS conversion process from about 60% to 70% in the prior art.

[0060] A higher proportion of longer chain oligomers provides a greater bifidus effect for the FOS product according to the invention, since the longer chain oligomers reach more distal parts of the large intestine and are thereby fermented in a larger area of ​​the intestinal wall, because when short chain oligomers are present in high concentrations, as in products of the prior art, their fermentation tends to occur at the beginning of the large intestine, with little FOS reaching the distal parts.

[0061] The present invention also significantly reduces the cost of producing FOS, since the glucose removal step of purification by chromatography columns becomes unnecessary, allowing the syrup to be directly dried by spray dryer and providing the product in powder form. The present invention (object of the present invention) allows for a faster and more efficient process by microbially bioconverting sucrose to FOS by enzymes produced by Aureobasidium pullulans, and synthesizing and purifying by selectively consuming glucose and fructose using a strain of Saccharomyces cerevisiae that does not produce extracellular invertases, and does not require the investment costs of equipment such as chromatography columns. This reduction in cost makes it possible to use this ingredient in the animal nutrition market and the food industry.

[0062] A method for producing and purifying oligofructose by using a co-proliferation of a microbial inoculum containing the genus Pullulan and a strain of Saccharomyces cerevisiae that does not produce an extracellular invertase is developed, wherein the co-proliferation is inoculated in a medium containing a high concentration of sucrose (20% to 60% sucrose) for a period of at least 12 hours but at most 72 hours, sufficient to convert the sucrose into FOS, and immediately consumes glucose while releasing it into the medium, such as attached Figure 3 Reactions shown, where GF: sucrose; G: glucose; F: fructose; and GF2, GF3, GF4: FOS.

[0063] An increase in the glucose concentration in the medium has a negative "feedback" effect on the action of fructosyltransferase, meaning that the FOS synthesis reaction is negatively affected by the presence of glucose until a concentration is reached that completely stops the formation of FOS.

[0064] By inhibiting this negative effect of glucose on the enzyme action, the present invention can increase the conversion of sucrose to FOS. In addition, it allows the degree of polymerization of the oligomers formed to increase and favors the formation of isomers of oligofructose. The increase in reaction yield leads to a reduction in the residual sucrose content and mainly removes the glucose residues in the product, making it a product with a purity of at least 80%. Most of the glucose is converted into ethanol, which is removed by evaporation during drying in a spray dryer.

[0065] A notable feature of the product of the present invention is the acquisition of long-chain FOS, so that the product is neither as long as the fiber nor as common as the products of the prior art, resulting in different physiological effects in the body: a greater bifidobacterial effect and a greater direct and indirect impact on immunity.

[0066] A greater bifidobacterial effect was observed in the product FOS of the present invention compared to the FOS of the prior art, which is attributed to the larger average size of the longer chain oligomers (GF5 and GF6), as well as the other short chain oligomers (GF2, GF3 and GF4). The longer chain oligomers reach more distal parts of the large intestine, so the better distribution of the oligomer ratio allows more uniform fermentation of beneficial bacteria throughout the large intestine. In the FOS of the prior art, with a high concentration of short chain oligomers (GF2 and GF3), the fermentation is uneven and concentrated in the initial part of the large intestine. Therefore, the FOS of the prior art is more likely to cause SIBO (small intestinal bacterial overgrowth) events than the product according to the present invention.

[0067] Attached Figure 4 and attached Figure 5 Shown is a chromatogram obtained by analyzing the product on an ICS-5000 model ion chromatograph from Thermofisher using a Carbopac PA100 chromatographic column.

[0068] In the attached Figure 4 In the figure, the orange peak (1) represents glucose -2,992, peak (2) represents fructose -3,400, peak (3) represents sucrose -4,050, peak (4) represents GF2 -5,850, peak (5) represents GF3 -10,175, and peak (6) represents GF4 -11,192.

[0069] In the attached Figure 5 In the figure, the orange peak (1) represents glucose-3,000, peak (2) represents fructose-3,408, peak (3) represents sucrose-4,067, peak (4) represents GF2-5,900, peak (5) represents GF3-10,183, peak (6) represents GF4-11,192, peak (7) represents isomers, peak (8) represents GF5, and peak (9) represents GF6.

[0070] Figure 4 The situation of the standard FOS production method (current state of the art) is shown. Figure 5 The conditions of the FOS production method of the present invention are shown (where A: standard method, B: method described in the present invention), Figure 5 In addition to the obvious decrease in glucose, GF5 and GF6 as well as other intermediate isomers are also present.

[0071] The present invention relates to a method for producing oligofructose and a product containing short-chain oligofructose (GF2, GF3, GF4), long-chain oligofructose (GF5 and / or GF6), their isomers and low-concentration sucrose, such as Figure 1 and Figure 2 A comparison is shown between the prior art method and the method of the present invention.

[0072] raw materials :

[0073] The present invention uses sucrose from various sources, which can be sugar from sugar cane, beetroot, cane syrup and molasses, preferably VHP (very high polarization) sugar, which is the most concentrated source of sucrose and the lowest known cost sugar. Few people have mentioned the direct use of VHP sugar to convert sucrose into FOS, whether it is biological conversion or enzymatic conversion. Some studies have shown that the higher the sucrose concentration in the medium of enzymatic synthesis of FOS, the higher the rate of sucrose conversion to FOS. The method according to the present invention starts with a solution of VHP sugar diluted in water, and its proportion can be 20% to 60% sugar. The solution is inoculated with a culture of the genus Pullulan and a genetically modified Saccharomyces cerevisiae (PE-2 Saccharomyces cerevisiae) strain that does not produce extracellular invertase, and the inoculation amount is 10% to 20% (v / v) in the final medium, so that the bioconversion of sucrose to FOS occurs and glucose is consumed at the same time.

[0074] In the present invention, a strain of Saccharomyces cerevisiae called PE-2 was used, which is a wild strain isolated from the industrial sugar and ethanol environment of Brazil, which has undergone a simple deletion of the genes related to invertase production, as described below.

[0075] 1. Deletion of the invertase gene in Pe-2 yeast

[0076] The cerevisiae yeast used in the purification step of the present invention has low and / or no metabolic capacity for sucrose and oligofructose as the target product. This lack of capacity leads to the selective consumption of glucose and fructose in the FOS syrup.

[0077] According to one embodiment of the present invention, for the disruptive mutation of the SUC-2 gene, a genome editing strategy was used by using a modified CRISPR / Cas 9 technology, in which 1109 bp of the gene was deleted, thereby preventing the functional expression of the encoded protein. The edit was verified by PCR genotyping, growth on sucrose (zero in the case of edited yeast) and Sanger sequencing.

[0078] The SUC2 gene is located on chromosome IX of the Saccharomyces cerevisiae genome and contains 1599 base pairs. The SUC2 gene is located in the Saccharomyces cerevisiae Pe-2 genome. Through the above genome editing, it now has 490 base pairs (see Figure 6 : Representative scheme of the SUC2 gene and genome editing strategy; A) The location of the SUC2 gene on chromosome IX and the design of primers for the genome editing region of the gene promoter and subsequent deletion using CRISPR / Cas 9 technology; B) The structure of the SUC2 gene after editing the gene promoter), completely rendering it incapable of protein expression.

[0079] Genetic manipulation was applied to chromosome 9, where tools were used to further reduce the risk of off-target effects by enabling Cas 9 to cut at undesired sites in the genome.

[0080] According to the literature (Ryan OW, Skerker JM, Maurer MJ et al. Selection of chromosomal DNA libraries using a multiplex CRISPR system. eLife 2014; 3: e03703; Jakociunas T, Bonde I, Herrgard M et al. Multiplex metabolic pathway engineering using CRISPR / Cas9 in Saccharomyces cerevisiae. Metab Eng 2015; 28: 213-22), off-target effects are unlikely to occur in small genomes (such as Saccharomyces cerevisiae). gRNA (guide RNA) is designed for a unique sequence in the genome, which makes it impossible for the Cas9 enzyme to recognize any other PAM (protospacer adjacent motif) sequence in the experiment.

[0081] Expression products of the genomic region: Following the deletion, the invertase encoded by SUC2 was not expressed, which was confirmed by the change in phenotype, resulting in the yeast not growing in medium YP + 2% sucrose and growing normally in medium YP + 2% glucose.

[0082] 2. Co-growth of inoculum for FOS synthesis

[0083] According to the present invention, the propagation of the inoculum can be optimized as a single step, wherein the A. pullulans strain responsible for synthesizing FOS and the S. cerevisiae PE-2 strain not producing invertase are propagated together in a single reactor, as described above.

[0084] The process can be carried out separately, but by doing it this way the production process is optimized.

[0085] To this end, an aqueous medium with a volume of "9X" liters containing 10% to 20% total reducing sugars (TRS) was first prepared, inoculated with a volume of "1X" liter of Aureobasidium pullulans, grown for 24 hours, inoculated with genetically modified Saccharomyces cerevisiae, and incubated for another 24 hours, the temperature was set between 32°C and 35°C, stirred at a speed of 120 rpm, and the ventilation volume was at least 1 vvm.

[0086] After both cultures were fully grown, "10X" liters of the inoculum culture were transferred to "90X" liters of medium containing 10% to 20% TRS (total reducing sugars) and incubated at a controlled temperature between 32°C and 35°C, with agitation at 120 rpm and aeration of at least 1 vvm, for 24 to 48 hours until fully grown and the minimum population of each yeast (A. pullulans and genetically modified S. cerevisiae) was 5 × 10 7 CFU / ml.

[0087] 3. Synthesize FOS and consume glucose at the same time.

[0088] As described above, after growth, the inoculum is transferred to an aqueous medium of sucrose and / or molasses and / or cane syrup containing 20% ​​to 60% sucrose in the total medium at a rate of 10% to 20% of the volume of the aqueous medium.

[0089] FOS synthesis occurs over a minimum period of 12 hours. However, glucose removal by S. cerevisiae consumption occurs between 12 and 60 hours, depending on the initial sucrose concentration of the medium. Thus, this process occurs between 30°C and 35°C for a period of at least 12 hours, but can last up to 60 hours.

[0090] By means of the present invention, the purity and yield of the product can be improved by converting all or nearly all of the sucrose content into FOS, because the "negative feedback" effect of glucose on fructosyltransferase activity is avoided by consuming glucose at the same time, thereby improving the conversion rate of sucrose into FOS.

[0091] During the spray drying process, the produced ethanol is 100% evaporated, so the final product is of high purity.

[0092] The following Table 1 (kinetics of formation of oligofructose in an initial medium containing 44% sucrose, calculated on a wet and dry basis, respectively) and Table 2 (composition of the product of the prior art and the product of the invention) demonstrate the selective consumption of glucose and fructose in the syrup and show the high concentrations of FOS (meaning of the abbreviations: scFOS, short chain FOS; LcFOS: long chain FOS) obtained in a process in which production and purification are carried out simultaneously:

[0093] Table 1

[0094]

[0095] In Table 1, starting from 24 hours, it can be clearly observed that the consumption of glucose is carried out simultaneously with the formation of FOS. This allows a higher yield than in the prior art process, which has a yield of 60% when converting sucrose to FOS. The consumption of glucose produces ethanol, while the dry matter decreases, which was initially 45%, and drops to 30% at the end of the 72-hour process.

[0096] By forming ethanol, a high-purity FOS syrup can be obtained. In the composition of the final product, 3% to 10% of auxiliary drying additives such as silicon dioxide or corn starch are added, so if the humidity of the powder in the composition is also considered, the purity of the final product is reduced by an average of 10%.

[0097] Table 2 compares the composition of pure FOS according to the present invention with the composition of prior art FOS produced by Ingredion shown in Table 3.

[0098] Composition of FOS commercial products

[0099] Table 2

[0100]

[0101] Composition of pure FOS

[0102] Table 3

[0103]

[0104] 4. Microbial removal of glucose

[0105] The conversion of sucrose to FOS at high conversion rates (as occurs in the present invention) has the disadvantage of releasing large amounts of glucose into the solution where the synthesis occurs. The presence of high concentrations of glucose (which may exceed 22%) would prevent the use of spray drying techniques to dry FOS syrups. The solution found according to the present invention is to convert glucose to ethanol by using yeast that does not have invertase activity.

[0106] The invention significantly reduces the cost of producing oligofructose, since the step of removing glucose by chromatographic purification becomes unnecessary, allows the direct drying of FOS syrup (the name of the product before drying) by spray drying, and enables its large-scale use in the animal nutrition and food industries, which are more cost-sensitive. To this end, the biochemical conversion of glucose is carried out by using yeast or microorganisms (Saccharomyces cerevisiae) that do not produce extracellular invertase.

[0107] 5. Direct drying of syrup

[0108] The presence of glucose in the FOS syrup at a concentration higher than 3% does not allow a direct drying process in a spray dryer. This is due to the glass transition temperatures (Tg) of the compounds being different from each other. Glucose and fructose have low tg values ​​and do not allow spray drying, which is not the case with sugars with a higher degree of polymerisation, such as GF2, GF3 and GF4. The production of FOS by a microbial process and the removal of glucose is characterised by the low cost of the process compared to separation of glucose by chromatography as is currently done in the prior art.

[0109] Get the final product:

[0110] In the present case, we chose to use A. pullulans for the bioconversion of sucrose because it produces FOS containing only those shorter chain oligomers (GF2, GF3 and GF4, GF5 and / or GF6) that have the greatest bifidus effect.

[0111] Because the FOS of the present invention may be partially or fully purified, their concentration may be between 70% and 95% pure.

[0112] The FOS obtained according to the invention contains short chain oligomers (scFOS) such as GF2, GF3, GF4 and / or long chain oligomers (lcFOS) such as GF5 and / or GF6, which have the greatest bifidus effect, while the former have greater immunomodulatory capacity, which is the biggest difference between the products in question. The larger the chain size without reaching the polymer size, the greater the bifidus effect of the product. This is due to the fact that the efficacy of intermediate chains such as GF5 and GF6 is greater than that of GF2 and GF3, and is far superior to long chain polymers (such as inulin and levan), which can be used more as soluble fiber, helping to transport faster through the intestine, but with less bifidus effect or regulation on the intestinal microbiota.

[0113] Likewise, smaller oligomers (e.g., GF2, GF3) have been shown to have immunomodulatory activity, acting directly on immune system cells present in the intestinal epithelium, such as macrophages, T cells, B cells, etc.

[0114] The greater the bifidobacteria effect, the more the number of beneficial bacteria such as lactobacilli and bifidobacteria increases, and therefore, the greater the degree of acidification they cause in the intestine, the less suitable it is for Salmonella and E. Coli to grow, thereby improving its efficiency as a growth promoter and population control agent. In the formulation of the present invention, the ratio of GF2 can be 10% to 45%, the ratio of GF3 can be 30% to 55%, the ratio of GF4 can be 10% to 25%, the ratio of GF5 can be 3% to 13% and the ratio of GF6 can be 0% to 8%. Preferably, the ratio of the FOS oligomers of the present invention can vary in the range of GF2 between 15% and 40%, GF3 between 35% and 50%, GF4 between 13% and 23%, GF5 between 3% and 10% and GF6 between 0% and 6%.

[0115] The bioconversion method used in the present invention has the advantage of controlling the proportion of short-chain oligomers formed by the synthesis time. In the present invention, the shorter the synthesis time, the greater the proportion of GF2 relative to GF3, GF4 and GF5. Each species has its own different microbial flora. Therefore, in the present invention, the FOS composition for birds will be different from the FOS composition for monogastric animals, fish, ruminants or humans. The ratio between short-chain oligomers should vary depending on the purpose of using FOS.

[0116] According to one embodiment of the present invention, the method for producing and purifying oligofructose adopts at least two microorganisms or yeasts simultaneously or separately to convert sucrose into oligofructose (FOS) and completely consume glucose in the medium, and evaporate alcohol or ethanol during the drying process of the final product, wherein one of the two microorganisms or yeasts is unable to produce extracellular invertase in the medium containing sucrose.

[0117] According to another embodiment of the present invention, the method for producing and purifying oligofructose employs at least two microorganisms or yeasts simultaneously or separately to convert sucrose into oligofructose (FOS) and completely consume glucose in the medium, and evaporate alcohol or ethanol during the drying process of the final product, wherein one of the two microorganisms or yeasts is unable to produce an extracellular invertase in a medium containing sucrose.

[0118] According to another embodiment of the present invention, the method for producing and purifying oligofructose uses an aqueous medium containing 20% ​​to 60% by mass of sucrose, wherein the inoculum concentration of the genus Pullulan and the genetically modified Saccharomyces cerevisiae that cannot produce extracellular invertase is 5×10 7CFU / ml, maintained at a temperature between 30°C and 35°C under stirring and aeration until the glucose present in the medium is completely removed and the ethanol is removed by drying the final product.

[0119] According to another embodiment of the present invention, the method for producing and purifying oligofructose is prepared by preparing an inoculum containing yeast in the following manner: an aqueous medium containing 10% to 20% total reducing sugar (TRS) is inoculated with Pullulanus at a volume ratio (v / v) of 1:10 for medium: Pullulanus, and after its growth, the genetically modified Saccharomyces cerevisiae is inoculated at the same volume ratio of 1:10 for inoculum: aqueous medium, and maintained at a temperature between 32°C and 35°C under stirring and aeration conditions, wherein, after the two cultures or yeasts are fully grown, the inoculum is transferred to a new medium containing 10% to 20% TRS at a volume ratio (v / v) of 1:10 for inoculum: medium, respectively, and maintained at the same temperature and under stirring and aeration conditions until it is fully grown and the minimum population of each yeast is 5×10 7 CFU / ml, removing ethanol when drying the final product.

[0120] According to a preferred embodiment of the present invention, first, a volume of "9X" liters of an aqueous medium containing 10% to 20% total reducing sugar (TRS) is prepared, in which a volume of "1X" liter of Aureobasidium is inoculated, and after 24 hours of growth, the genetically modified Saccharomyces cerevisiae is inoculated and incubated for at least another 24 hours at a temperature between 32°C and 35°C, under stirring and aeration conditions. After both cultures have grown completely, the "10X" liter inoculum culture is transferred to a "90X" liter medium containing 10% to 20% TRS (total reducing sugar), and incubated at a controlled temperature between 32°C and 35°C, under stirring and aeration conditions, until fully grown and the minimum population of each yeast (Aureobasidium and genetically modified Saccharomyces cerevisiae) is 5×10 7 CFU / ml; the inoculum is then transferred to an aqueous medium of sucrose and / or molasses and / or cane syrup containing 20% ​​to 60% sucrose in the total medium at a ratio of 10% to 20% of the volume of the aqueous medium, wherein the synthesis of FOS occurs over a minimum period of 12 hours, and the removal of glucose by consumption by said genetically modified Saccharomyces cerevisiae occurs between 12 hours and 60 hours, this period depending on the initial sucrose concentration of the medium, and the temperature is between 30°C and 35°C.

[0121] According to a more preferred form of implementation of the invention, first, a volume of "9X" liters of an aqueous medium containing 10% to 20% of total reducing sugars (TRS) is prepared and inoculated with a volume of "1X" liter of A. pullulans, after 24 hours of growth, inoculated with genetically modified S. cerevisiae, and incubated for a minimum of 24 hours, the temperature is set between 32°C and 35°C, stirring at a speed of 120 rpm, and aeration of 1 vvm. After both cultures have grown completely, the "10X" liter inoculum culture is transferred to "90X" liters of a medium containing 10% to 20% of TRS (total reducing sugars), incubated for 24 to 48 hours at a controlled temperature between 32°C and 35°C, stirring at a speed of 120 rpm, and aeration of at least 1 vvm, until fully grown and the minimum population of each yeast (A. pullulans and genetically modified S. cerevisiae) is 5×10 7 CFU / ml; the inoculum is then transferred to an aqueous medium of sucrose and / or molasses and / or cane syrup containing 20% ​​to 60% sucrose in the total medium at a rate of 10% to 20% of the volume of the aqueous medium, the synthesis of FOS occurs over a minimum period of 12 hours, and the removal of glucose by consumption by the genetically modified Saccharomyces cerevisiae occurs between 12 hours and 60 hours, this period depending on the initial sucrose concentration of the medium, and the temperature is between 30°C and 35°C.

[0122] In the final product, purified fructooligosaccharides (FOS) are free of glucose and ethanol.

[0123] The following are examples to better illustrate the scope of the present invention; however, these examples should not be used for the purpose of limiting the present invention.

[0124] Embodiment 1:

[0125] Production of Fructooligosaccharides (FOS):

[0126] The A. pullulans strain responsible for synthesizing FOS and the S. cerevisiae PE-2 strain that does not produce extracellular invertase were used in a single reactor. To this end, 200 L of aqueous medium containing 15% total reducing sugars (TRS) were first prepared, inoculated with 20 L of A. pullulans, grown for 24 hours, and then inoculated with genetically modified S. cerevisiae PE-2 and incubated for another 24 hours, with the temperature set between 33°C, stirring at a speed of 120 rpm, and aeration of at least 1 vvm.

[0127] After both cultures were fully grown, 200 L of the inoculum was transferred to 2000 L of medium containing 15% TRS (total reducing sugars) and incubated at a controlled temperature of 33°C with shaking at 120 rpm and aeration of at least 1 vvm for 36 hours until fully grown and the minimum population of each yeast (A. pullulans and S. cerevisiae) was 5 × 10 7CFU / ml.

[0128] After growth, the inoculum was transferred to an aqueous medium of sucrose-sugar cane syrup containing 40% sucrose in the total medium at a volume ratio of 15% for the inoculum.

[0129] The synthesis of FOS occurs within a minimum period of 12 hours. However, the removal of glucose by consumption by S. cerevisiae occurs after 40 hours, a period that depends on the initial sucrose concentration of the medium, and lasts for 40 hours at a temperature of 33°C.

[0130] During the spray drying process, the ethanol produced is 100% evaporated, so the final product is now of high purity.

[0131] The produced FOS is present only as those short-chain oligomers with the greatest bifidogenic effect (GF2, GF3 and GF4, GF5 and / or GF6), and the mass proportions of the oligomers are as follows: GF2 is 24%, GF3 is 35%, GF4 is 32%, GF5 is 7% and GF6 is 2%.

[0132] The resulting FOS is applied to cats:

[0133] The experiment was conducted by feeding cats with different types of prebiotics, including FOS of the present invention (yFOS) and FOS of the prior art produced by Ingredrion (scFOS), in a randomized block design with period as the blocking factor. The experiment consisted of six treatments, repeated in three periods, with 12 cats per period (two cats per treatment), and each treatment was repeated six times in total (total n=36).

[0134] The purpose of this experiment was to demonstrate that the addition of the prebiotics FOS, GOS and inulin to the cat's diet alters the concentration of fecal fermentation products as a result of increased intestinal fermentation of organic matter and thus altered apparent digestibility of the diet. In addition, the purpose was to demonstrate that the FOS of the invention (yFOS) differs significantly in performance from the prior art FOS (scFOS, produced by Ingredion).

[0135] Six experimental diets were formulated, one was a negative control (NC) diet without any prebiotics added, and the other five contained: 0.8% FOS of the present invention (yFOS, obtained from Yessinergy); 0.8% GOS (GOS); 0.8% inulin (INU); 0.8% FOS of the prior art (scFOS, produced by Ingredion); 0.8% combination of yFOS and inulin (yFOS+INU). Prebiotics were added to replace cellulose because cellulose, although a fiber, is not fermentable for cats. All diets were formulated according to the recommendations of the European Federation of the Pet Food Industry (FEDIAF) (2018).

[0136] At the end of the digestibility testing, all cats were housed for an additional period until a total housing period of 21 days was completed and fresh feces were collected daily, up to 15 minutes after defecation, and pH, volatile fatty acids (VFA), fecal ammonia nitrogen, and fecal lactic acid were determined.

[0137] To measure fecal pH, two grams of fresh feces (collected within 15 minutes after defecation) were diluted in 6 ml of milliQ water and the pH was assessed using a precision digital pH meter.

[0138] To determine the concentration of VFA (acetic acid, propionic acid, butyric acid, isobutyric acid, isovaleric acid, valeric acid, 4-methylvaleric acid, hexanoic acid and heptanoic acid) and fecal ammonia nitrogen, 5 grams of fresh feces were collected, quickly homogenized and mixed with 15mL of 16% formic acid solution (1:3w / v). The mixture was kept refrigerated and centrifuged three times at 4,500G for 15 minutes each time, using the supernatant and discarding the precipitate. To measure VFA, the samples were labeled and stored in a refrigerator (-15°C) and subsequently submitted to the Chemistry Laboratory of the State University of Maringa for analysis. VFA in feces was determined by gas chromatography.

[0139] Ammonia nitrogen in feces was measured by using Kjeldahl method. The extract prepared for VFA measurement was used. The extract was thawed at room temperature, and 2 mL aliquots were transferred to test tubes, diluted in 13 mL distilled water, and distilled in a nitrogen distiller (Tecnal T-036 / 1 Piracicaba, Brazil). 5 mL potassium hydroxide KOH (2 mol / L) was distilled, and nitrogen was received in a conical flask equipped with 10 mL solution (boric acid 0.97 N). Next, each sample was titrated with HCL (0.005 N).

[0140] Normality and homogeneity of variance of the variables measured in each experiment were first analyzed. Apparent digestibility coefficients were compared between treatments using the Tukey test with a probability of 5%. For fermentation products (ammonia, SCFA and BCFA), pH and fecal DM, considering the interdependence between them, multivariate statistics were applied by canonical correlation analysis when multivariate analysis of variance (MANOVA) showed a probability value of less than 5% for the null hypothesis, and this was considered significant.

[0141] In the following Table 4, regarding the fermentation parameters in feces (short-chain fatty acids (mmol / kg feces MS), where NC = negative control; yFOS = fructooligosaccharides according to the invention; GOS = galacto-oligosaccharides; INU = inulin; scFOS = FOS produced by Ingredion; FOS+INU = FOSy (FOS according to the invention) combined with INU:

[0142] Table 4

[0143] NC yFOS GOS INU scFOS INU+FOS Acetic acid 26.8 26.1 28.1 20.4 17.1 22.6 Propionic acid 8.0 7.0 9.1 5.2 6.0 8.2 Butyric acid 4.4 4.5 4.1 2.7 4.1 4.9 Isobutyric acid 1.6 1.8 1.7 1.5 1.5 1.7 Isovalerate 1.8 2.3 1.8 1.6 1.8 1.8 Valeric acid 3.2 3.7 2.5 2.4 2.8 3.1 4-Methylvaleric acid 0.49 0.67 0.31 0.4 0.22 0.24 Hexanoic acid 0.24 0.35 0.24 0.25 0.22 0.24 Heptanoic acid 1.9 2.2 2.1 1.7 1.8 1.9 Total VFA 39.2 37.6 41.3 28.3 27.1 35.7 Total SCFA 7.1 8.6 6.2 5.9 6.3 6.9 Ammonia (mg / kg DM) 1.8 2.5 2.1 1.7 1.9 2.1 Lactate (mmol / kg DM) 11.6 12.4 25.6 14.5 16.9 18.7 Fecal pH 6.2 6.1 6.0 6.1 5.9 6.1 Fecal MS (%) 40.4 35.6 34.9 39.0 39.9 36.4

[0144] As shown in Table 4, the formation of intestinal fermentation products such as lactate, propionate and acetate was more related to the content of GOS in the diet, while others (butyrate, BCFA and ammonia) showed greater correlation with the content of yFOS (invention), proving that these two treatments provided more intestinal fermentation than other treatments. As expected, fecal pH was negatively correlated with fermentation products.

[0145] Attached Figure 7 Representative correlations of fermentation and stool quality parameters between negative control (NC) and prebiotic-containing treatments are shown.

[0146] Although higher ammonia levels and greater fecal softness are undesirable in domestic cat feces, the purpose of the experiment was to show that the FOS of the invention has a greater overall bifidus effect than other prebiotics available on the market. In this respect, the values ​​of the total amount of volatile fatty acids (total VFA) and the total amount of mainly short-chain fatty acids (total SCFA) are high, showing the excellent performance of the product of the invention. A more important indicator of greater fermentation throughout the large intestine in the yFOS and yFOS+INU treatments is that they produce more butyric acid. Butyric acid is currently the most desired by nutritionists.

[0147] In the following Table 5, blood parameters related to monocyte and granulocyte phagocytosis are given (per μL of blood), where NC = negative control; yFOS = fructooligosaccharides according to the invention; GOS = galacto-oligosaccharides; INU = inulin; scFOS = FOS produced by Ingredion; FOS+INU = FOSy (FOS according to the invention) combined with INU:

[0148] Table 5

[0149] NC yFOS GOS INU scFOS FOS+INU Phagocytic monocytes ％ 56.9 65.5 60.6 54.5 57.5 47.4 N 160,384 269,163 214,144 282,573 321,787 268,610 Phagocytic granulocytes ％ 1.4 1.6 1.5 1.1 1.2 0.7 N 2,683 3,837 4,514 6,399 4,682 5,305 White blood cell count N 304,048 398,000 349,480 543,563 504,288 648,356

[0150] Table 5 shows that when treated with yFOS, the concentration of monocytes and phagocytic granulocytes was the highest, and the concentration of total leukocytes was also low, which is also very ideal. This demonstrates the superiority of the product of the present invention, yFOS, in regulating the immune system of cats. It also shows that yFOS increases the stimulatory effect of the cellular immune system and reduces the stimulatory effect of the humoral immune system, i.e., it has an anti-inflammatory effect.

[0151] Likewise, Table 6 contains the results of fecal IgA concentrations, where NC = negative control; yFOS = fructooligosaccharides according to the invention; GOS = galacto-oligosaccharides; INU = inulin; scFOS = FOS produced by Ingredion; FOS+INU = FOSy (FOS according to the invention) combined with INU:

[0152] Table 6

[0153] NC yFOS GOS INU scFOS FOS+INU Fecal IgA (μg / mL) 1.06 3.09 4.76 5.95 7.32 2.06

[0154] The results shown in Table 6 reconfirm the results shown in Tables 4 and 5, demonstrating the superiority of the treatment with the product yFOS of the present invention in terms of bifidobacterial effect and immune system modulation compared to other treatments (including treatment with scFOS, which represents an important commercial brand of FOS in the prior art). It is again confirmed that yFOS increases the stimulatory effect of the cellular immune system and reduces the stimulatory effect of the humoral immune system, i.e., has an anti-inflammatory effect.

Claims

1. A method for producing and purifying oligofructose, It is characterized in that At least two microorganisms or yeasts are used simultaneously or separately, one of which is unable to produce an extracellular invertase in a medium containing sucrose, to convert sucrose into fructooligosaccharides (FOS) and to completely consume the glucose in the medium and evaporate the alcohol or ethanol during the drying process of the final product.

2. The method for producing and purifying oligofructose according to claim 1, It is characterized in that The obtained FOS contained long-chain oligomers GF5 and GF6, and other short-chain oligomers GF2, GF3 and GF4 of larger average size.

3. The method for producing and purifying oligofructose according to claim 1, It is characterized in that The oligofructose (FOS) contains oligomers in the following ratios relative to the total mass: GF2 is 10% to 45%, GF3 is 30% to 55%, GF4 is 10% to 25%, GF5 is 3% to 13%, and GF6 is 0% to 8%.

4. The method for producing and purifying oligofructose according to claim 3, It is characterized in that The proportion of the mass of FOS oligomers relative to the total mass varies between 15% and 40% for GF2, 35% and 50% for GF3, 13% and 23% for GF4, 3% and 10% for GF5 and 0% and 6% for GF6.

5. The method for producing and purifying oligofructose according to claim 1, It is characterized in that Sugars from sugar cane, beetroot, cane syrup and molasses are used as sources of sucrose.

6. The method for producing and purifying oligofructose according to claim 1 or 5, It is characterized in that The medium contains 20 to 60 mass % of sucrose.

7. The method for producing and purifying oligofructose according to claim 1, It is characterized in that One of the microorganisms or yeasts is Aureobasidium sp.

8. The method for producing and purifying oligofructose according to claim 1, It is characterized in that One of the microorganisms or yeasts is a genetically modified Saccharomyces cerevisiae.

9. The method for producing and purifying oligofructose according to claim 1, It is characterized in that The genetically modified Saccharomyces cerevisiae is a wild strain called PE-2 isolated from the industrial sugar and alcohol environment in Brazil, which has undergone a simple deletion of the gene related to the production of invertase.

10. The method for producing and purifying oligofructose according to claim 1, It is characterized in that The genetically modified Saccharomyces cerevisiae as the yeast selectively consumes only glucose and fructose, and does not consume sucrose and FOS, ie, oligomers having α,1-2 and β,2-1 type bonds.

11. The method for producing and purifying oligofructose according to claim 8, It is characterized in that Genetic manipulation has been applied to chromosome 9.

12. The method for producing and purifying oligofructose according to claim 1, It is characterized in that At least two microorganisms or yeasts are used simultaneously or separately, one of which is unable to produce an extracellular invertase in a medium containing sucrose, to convert sucrose into fructooligosaccharides (FOS) and to completely consume the glucose in the medium and to evaporate the alcohol or ethanol by evaporation during the drying process of the final product.

13. The method for producing and purifying oligofructose according to claim 12, It is characterized in that At least two microorganisms or yeasts are used simultaneously or separately, one of which is unable to produce an extracellular invertase in a medium containing sucrose, to convert sucrose into fructooligosaccharides (FOS) and to completely consume the glucose in the medium and evaporate the alcohol or ethanol during the drying process of the final product.

14. A method for producing and purifying oligofructose according to any one of the preceding claims, It is characterized in that An aqueous medium containing 20% ​​to 60% by mass of sucrose is inoculated with Pullulan and a genetically modified Saccharomyces cerevisiae that is unable to produce an extracellular invertase, and the inoculum concentration is such that the minimum population of each of the two yeasts is 5×10 7 CFU / ml, maintained at a temperature between 30°C and 35°C under stirring and aeration until the glucose present in the medium is completely removed and the ethanol is removed by evaporation during the drying of the final product.

15. A method for producing and purifying oligofructose according to any one of the preceding claims, It is characterized in that The inoculum containing the yeast is prepared by inoculating Pullulan with an aqueous medium containing 10% to 20% total reducing sugar (TRS) at a volume ratio (v / v) of 1:10 for medium: Pullulan, and after its growth, inoculating the genetically modified Saccharomyces cerevisiae at the same volume ratio of 1:10 for inoculum: medium, maintaining at a temperature between 32°C and 35°C under stirring and aeration conditions, and after the two cultures or yeasts are fully grown, transferring the inoculum to a new medium containing 10% to 20% TRS at a volume ratio (v / v) of 1:10 for inoculum: medium, respectively, maintaining at the same temperature and stirring and aeration conditions until fully grown and the minimum population of each yeast is 5×10 7 CFU / ml.

16. A method for producing and purifying oligofructose according to any one of the preceding claims, It is characterized in that First, a volume of "9X" liters of aqueous medium containing 10% to 20% total reducing sugar (TRS) is prepared and inoculated with a volume of "1X" liter of Pullulan, after 24 hours of growth, inoculated with genetically modified Saccharomyces cerevisiae and incubated for at least another 24 hours at a temperature between 32°C and 35°C with stirring and aeration. After both cultures have grown completely, the "10X" liter inoculum culture is transferred to a "90X" liter medium containing 10% to 20% TRS (total reducing sugar) and incubated at a controlled temperature between 32°C and 35°C with stirring and aeration until fully grown and the minimum population of each yeast (the Pullulan and the genetically modified Saccharomyces cerevisiae) is 5×10 7 CFU / ml; the inoculum is then transferred to an aqueous medium of sucrose and / or molasses and / or cane syrup containing 20% ​​to 60% sucrose in the total medium at a ratio of 10% to 20% of the volume of the aqueous medium, wherein the synthesis of FOS occurs over a minimum period of 12 hours, and the removal of glucose by consumption by the genetically modified Saccharomyces cerevisiae occurs between 12 hours and 60 hours, this period depending on the initial sucrose concentration of the medium, and the temperature is between 30°C and 35°C.

17. The product obtained by the method according to any one of claims 1 to 16, Features Contains purified fructooligosaccharides (FOS) and is glucose and ethanol free in the final product.

18. The product according to claim 17, It is characterized in that The obtained fructooligosaccharides (FOS) present long-chain oligomers GF5 and GF6, and other short-chain oligomers GF2, GF3 and GF4 of larger average size.

19. The product according to claim 17 or 18, It is characterized in that The mass ratio of GF2 is 10% to 45%, the mass ratio of GF3 is 30% to 55%, the mass ratio of GF4 is 10% to 25%, the mass ratio of GF5 is 3% to 13% and the mass ratio of GF6 is 0% to 8%.

20. The product according to claim 19, It is characterized in that The mass ratios of the FOS oligomers varied between 15% and 40% for GF2, 35% and 50% for GF3, 13% and 23% for GF4, 3% and 10% for GF5 and 0% to 6% for GF6.

21. The product according to any one of claims 17 to 20, Features Contains fructooligosaccharides (FOS) and 3 to 10% by mass of drying auxiliary additives known to those skilled in the art, relative to the total mass.

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