Process for producing and purifying fructooligosaccharides and products obtained by such processes

Abstract

The present invention relates to a process for the production and purification of fructooligosaccharides (FOS), and to the product obtained by such a process, which contains low or no glucose, fructose or sucrose residues, with different ratios of oligomers (GF2, GF3, GF4, GF5 and GF6) and their isomers, which are used in animal and human nutrition due to their excellent properties of modulating the intestinal microflora and direct and indirect immunomodulation. The process for the production and purification of fructooligosaccharides according to the present invention uses at least two microorganisms or yeasts, simultaneously or separately, in a medium containing sucrose, one of which is unable to produce extracellular invertase, to convert sucrose into fructooligosaccharides (FOS), with the complete consumption of glucose from the medium and the evaporation of alcohol or ethanol during drying of the final product.

Description

[Technical field]

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

[0002] FOS or oligofructose is one of the most popular prebiotic oligosaccharides studied and used in human and animal nutrition because of its recognized bifidobacterial effect.After being fermented by bifidobacteria and lactic acid bacteria, which are preferably 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 in intestinal microflora through the acidification of the environment, inhibiting putrefactive bacteria that cause intestinal inflammation, such as Salmonella, Escherichia coli, Clostridium, Campylobacter and other pathogens, and promoting the growth of beneficial bacteria.

[0003] These organic acids not only lower the pH of the intestine, but are absorbed by the intestinal vesicles and used as an energy source, increase the production of mucin (the protective mucous membrane of the intestinal epithelium), promote the increase of intestinal villi, improve nutrient absorption, strengthen "tight junctions" that enhance the integrity of the intestinal epithelium, promote metabolic normalization, and control blood sugar, triglyceride, and cholesterol levels.

[0004] Modulation of the microbiota not only contributes to healthy intestinal function and balances the diverse hormones produced in the intestinal tract, which today is considered the second brain, but also promotes a secondary immunomodulatory effect, since beneficial microbes produce cytokines (interleukins IL-1, IL-2, IL-4, IL-6, IL-10, etc.) that activate T cells and mobilize cells to fight inflammatory diseases and pathogens, and 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, being recognized by the “tool-like receptor 4” (TLR-4) and promoting the production of interleukin-10, demonstrating that their direct action, i.e., even without being fermented or consumed by probiotic bacteria, can have a profound effect on the innate immune system, even in the earliest parts of the gut.

[0006] FOS are found in many plants and foods, such as chicory (Chichorium intybus), artichoke, Jerusalem artichoke, yacon, onion, banana, tomato, garlic, asparagus, Agave americana, honey, and sugarcane. They are storage carbohydrates and act as osmoregulators in plants. Their formation depends on environmental conditions and developmental stage: low temperatures cause the accumulation of fructans in leaves and roots.

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

[0008] In both plants and microorganisms, its formation is due to hydrolases (EC.3.2.1.26) and transferases (EC.2.4.1.9), such as fructofuranosidases and fructosyltransferases.

[0009] FOS differ by chain size and linkages between fructose molecules, depending on their forming enzyme. According to IUPAC-IUB (International Union of Pure and Applied Chemistry), oligosaccharides are defined as sugars or carbohydrates containing 3 to 10 monosaccharide units in their chain. Thus, fructose oligomers can contain up to 10 fructose molecules in their composition. However, many authors prefer the definition that they are molecules that contain up to 10 degrees of polymerization. Beyond this value, they are called fructan polysaccharides or "fructans", which differ from levan and inulin, whose molecules can contain up to 60 fructose units in their chain.

[0010] The fructose chains can contain bonds of 1F and 6G and 6F type. The first type has fructose units linked by β-2,1 type oxidic bonds. 6G, called neo-FOS, has β-2,6 type oxidic bonds between the fructose and glucose units. As for type 6F, it has β-2,6 type oxidic bonds between the fructose units.

[0011] The best known FOS synthesis process follows the following mathematical model of the action of fructosyltransferase from Aureobasidium pullulans: GFn+GFn → GFn-1+GFn+1 n=1-3 (G: glucose, F: fructose, GF: sucrose)

[0012] This mechanism can be divided into three reactions: 2 GF → GF2+G (1) 2 GF2 → GF3+GF (2) 2 GF3 → GF4+GF3 (3)

[0013] In this model, excess glucose acts as an inhibitor of the fructosyltransferase enzyme and thus exerts a negative feedback effect, i.e., when present in a certain amount in the reaction medium, the reaction stops. As a result, the FOS synthesis process in the prior art reaches a very high level of residual glucose, which accounts for 30% of the total components, with a maximum yield of 60%, leaving sucrose remaining, making spray drying impossible.

[0014] A typical example of a standard FOS reaction using a fructosyltransferase enzyme: [GF] → [G]+[F]+[GF]+[GF2+GF3+GF4] 40% → 12%+1%+3%+24% Yield = [GF2+GF3+GF4] / [GF] = 24% / 40% = 60%

[0015] As a result, excessive glucose concentrations in the medium can be noted, preventing the reaction from continuing and even leading to the formation of larger oligomers, as in the following mathematical example: 2GF → GF2 + G (1) 2 GF2 → GF3 + GF (2) 2 GF3 → GF4 + GF3 (3) 2 GF4 → GF5 + GF4 (4) 2 GF5 → GF6 + GF5 (5) Summary of the Invention [Problem to be solved by the invention]

[0016] The present invention proposes and demonstrates the formation of GF5 and GF6 by the simultaneous removal of glucose with the synthesis of FOS. [Means for solving the problem]

[0017] The most commonly used method in the prior art to remove glucose in the final FOS syrup is the use of chromatography columns. Size exclusion (ion sieving) removes low molecular weight sugars such as glucose, fructose, sucrose, etc., leading to 100% purity of FOS in the refined syrup and 95% purity of FOS in the final product. Figures 1 and 2 compare the purification steps of the conventional FOS production process with the process of the present invention.

[0018] On the other hand, the present invention provides, as an alternative, the use of a biotransformation process by using intact microorganisms, both for the purpose of synthesizing FOS and for the purpose of purifying it by removing glucose and other residual sugars from the reaction medium. For that purpose, in addition to the Aureobasidium involved in the synthesis of FOS, the yeast Saccharomyces cerevisiae was used, which selectively consumes only glucose and fructose, but not sucrose and FOS, i.e. oligomers with α-1-2 and β-2-1 type bonds. As a result, the sucrose conversion yield increased to a minimum of 70%, no presence of residual sucrose was observed, and in the syrup and final product, the FOS concentration reached a minimum value of 80-90%, since the ethanol was completely evaporated in the spray dryer drying process.

[0019] When the glucose concentration in the FOS syrup exceeds 3%, the drying process using spray drying technology is not feasible. The reason is the low glass transition temperature (tg) of low molecular weight sugars such as glucose and fructose, which does not occur for sugars with a high degree of polymerization such as kestose (GF2), nystose (GF3) and fructofuranosylnystose (GF4). The removal of glucose by microbiological processes is low cost when compared to the separation of glucose by chromatography as performed by current conventional technology.

[0020] Prior art document (JP Patent Publication 4-293494) teaches the efficient production of fructose-containing oligosaccharides or glycosides useful in the medical and food fields at low cost by treating sucrose with group fructosyl transferase and invertase-deficient yeast in the presence of an acceptor. Sucrose is dissolved in water in the presence of an acceptor (e.g., lactose), and reacted with beta-fructofuranosidase, a fructosyl group transferase produced by Arthrobacter sp. K-1, and simultaneously with invertase-deficient yeast at pH 6.5-7.0 and 35°C for 20 hours. The invertase-deficient yeast is removed by filtration through diatomaceous earth, and the filtrate is treated with activated carbon at 95°C for 1 hour, purified with ion exchange resin, concentrated, and spray-dried to obtain fructose-containing oligosaccharides containing fructose-containing glycosides such as lactosylfructoside or fructosylstevioside.

[0021] However, the present invention is different in that it solves a problem and achieves extraordinary results. Effect of the Invention

[0022] (Resolved Issues) The present invention solves the problem of the person skilled in the art by achieving a better yield in the process of obtaining fructooligosaccharides (FOS) and improving the purity mainly by removing the residues of glucose, fructose or sucrose, which are by-products that make the purification conditions of the final product obtained very difficult and mainly more expensive. Furthermore, the present invention optimizes obtaining the final product in a sustainable way by removing alcohols, especially ethanol, from the final product during the spray dryer drying stage.

[0023] (Unique Effects of the Invention) In addition to obtaining a higher purity end product, using a more sustainable process and using more economical equipment and conditions than those used by those skilled in the art, the present invention also makes it possible to obtain a higher amount of end product of long chain oligomers such as GF5 and GF6, which are important for reaching the colonies of bifidobacteria and lactic acid bacteria in the most distal part of the large intestine, which is not achieved by the short chain oligomers GF2, GF3 and GF4. This is possible because two microorganisms (Aureobasidium and Saccharomyces) are able to live together and act simultaneously together in the process.

[0024] (the purpose) We developed a process for obtaining and purifying fructooligosaccharides (FOS) using yeast, with the objective of: Direct biotransformation of sucrose to FOS with high conversion and yield; Microbial clearance of glucose and Microbial glucose removal using the Saccharomyces cerevisiae yeast, which does not produce extracellular invertase, obtained using CRISPR Cas9 methodology; -Reduction of fructooligosaccharide production costs and the possibility of direct drying of the resulting fructooligosaccharide-containing syrup; generation of FOS with different ratios of short chain oligomers (scFOS such as GF2, GF3, GF4) and long chain oligomers (lcFOS such as GF5 and / or GF6) and significant presence of their isomers; The possibility of producing FOS in oligomeric ratios suited to each intended use in terms of a more effective functional physiological action, a greater regulation of the microflora and further promotion of healthy functioning, in addition to being a direct immunomodulator; - a more sustainable process than those used by those skilled in the art; The purpose of this project is to provide [Brief description of the drawings]

[0025] The diagram is briefly explained below.

[0026] [Figure 1] This is a conventional process for obtaining fructooligosaccharides. [Diagram 2] 1 is a process for obtaining fructooligosaccharides according to the present invention. [Diagram 3] 1 is a diagram illustrating the reaction of FOS formation from a sucrose molecule (GF: sucrose, G: glucose, F: fructose, and GF2, GF3, GF4: FOS). [Figure 4] A: Indicates standard process or conventional technology. [Diagram 5] B: Shows the process referred to in the present invention. [Figure 6] Representative scheme of SUC2 gene and genome editing strategy. A) Location of SUC2 gene on chromosome IX and primer design to edit the gene promoter region using CRISPR / Cas9 technology and consequently delete the region. B) Structure of SUC2 gene after editing the gene promoter. [Figure 7] Canonical correlations of fermentation and fecal quality parameters between negative control (NC) and prebiotic-containing treatments. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0027] Summary of the Invention The present invention relates to a process for the production and purification of fructooligosaccharides (FOS) using at least two microorganisms or yeasts, either simultaneously or separately, in a medium containing sucrose, one of which is unable to produce extracellular invertase, to convert sucrose into FOS, with the process involving complete consumption of glucose from the medium and evaporation of alcohol or ethanol during drying of the final product, and the final product obtained.

[0028] (Detailed Description) The present invention describes novel products containing short-chain fructooligosaccharides GF2, GF3, GF4 (scFOS) and long-chain fructooligosaccharides such as GF5 and GF6 (lcFOS) and their isomers, derived from an innovative purification process, whereby sugar residues such as glucose and fructose are removed by Saccharomyces cerevisiae yeast instead of the traditional removal by chromatographic purification. Moreover, the yield of the sucrose to FOS conversion process was increased to 70% from the order of 60% in the prior art, since the synthesis and purification of FOS occurred simultaneously and the negative feedback effect of excess glucose on the performance of fructosyltransferase enzyme was prevented.

[0029] A higher proportion of long chain oligomers gives a higher bifidobacterial effect to the FOS product of the invention, because the long chain oligomers reach the more distal parts of the large intestine and are fermented in a wider area of ​​the intestinal wall.When the concentration of short chain oligomers is high, as in the prior art products, their fermentation tends to occur at the beginning of the large intestine and little FOS reaches the distal parts.

[0030] The invention also significantly reduces the cost of producing FOS, since it eliminates the glucose removal step by purification through chromatographic columns, allows direct drying of the syrup by spray dryer, and provides a product in powder form. The aim of the invention is to allow a faster and more efficient process, without investment costs in equipment such as chromatographic columns, by synthesizing and purifying FOS through the microbial bioconversion of sucrose to FOS, the enzymes produced by Aureobasidium pullulans, and the selective consumption of glucose and fructose by using a strain of Saccharomyces cerevisiae that does not produce extracellular invertase. This cost reduction allows the use of this ingredient in the animal nutrition market and in the food industry.

[0031] The process for the production and purification of fructooligosaccharides was developed by using a co-culture of microbial inoculants including Aureobasidium sp. and a Saccharomyces cerevisiae strain that does not produce extracellular invertase, which was inoculated into a medium containing high concentrations of sucrose ranging from 20-60% sucrose for a period of time sufficient to convert sucrose into FOS, a minimum of 12 hours and a maximum of 72 hours, and was instantly consumed as glucose was released into the medium according to the reaction shown in the attached Figure 3, where GF: sucrose, G: glucose, F: fructose, and GF2, GF3, GF4: FOS.

[0032] Increasing the glucose concentration in the medium has a negative "feedback" effect on the action of the fructosyltransferase enzyme, meaning that the FOS synthesis reaction is negatively affected by the presence of glucose until the glucose concentration completely stops the formation of FOS.

[0033] By inhibiting this negative effect of glucose on the action of the enzyme, the present invention makes it possible to increase the conversion of sucrose to FOS. Moreover, it also makes it possible to increase the degree of polymerization of the oligomers formed, which also favors the formation of isomers of the fructooligosaccharides formed. The increase in reaction yield reduces the residual sucrose content, mainly eliminating glucose residues in the product, leading to a product with a purity of at least 80%. Most of the glucose is converted into ethanol and removed by evaporation during drying in a spray dryer.

[0034] One of the outstanding features of the product of the present invention is that long-chain FOS are obtained, therefore the product is not as long as fibre and not as general as the products of the prior art, so the physiological effect caused in the body is different, with a greater bifidobacterial effect, which has a greater impact on the immune system both directly and indirectly.

[0035] In the product of the present invention, a greater bifidobacterial effect was observed in the FOS when compared to the prior art FOS, because the long chain oligomers (GF5 and GF6) are longer and the average size of the other short chain oligomers (GF2, GF3 and GF4) is larger. The longer chain oligomers reach the more distal parts of the colon, so a better distribution of the oligomer ratio allows a more uniform fermentation by good bacteria throughout the colon. In the prior art FOS with a high concentration of short chain oligomers (GF2 and GF3), the fermentation is non-uniform and concentrated in the early part of the colon. As a result, the prior art FOS is more likely to cause SIBO (small intestinal bacterial overgrowth) events than the product according to the present invention.

[0036] The accompanying Figures 4 and 5 show illustrations of chromatograms obtained by analyzing the products on an ion chromatograph, model ICS-5000, manufactured by Thermofisher, using a Carbopac PA100 column.

[0037] In the attached FIG. 4, 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.

[0038] In the attached FIG. 5, 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.

[0039] FIG. 4 (A: standard process; B: process referred to in this invention) shows the standard FOS production process (current prior art) and the process of the present invention, and FIG. 5 shows the clear reduction in glucose as well as the presence of GF5 and GF6 and other intermediate isomers.

[0040] The present invention deals with a method for producing a product containing fructooligosaccharides and short chain fructooligosaccharides GF2, GF3, GF4, long chain fructooligosaccharides GF5 and / or GF6, their isomers and low concentrations of sucrose, as detailed in Figures 1 and 2, comparing the prior art methods and the method of the present invention.

[0041] Raw materials: The present invention uses various sources of sucrose, which can be sugars from sugar cane, beetroot, cane syrup and molasses, preferably VHP (very high polarized) sugars, which are the most concentrated source of sucrose and the lowest cost known. There are few references to the direct use of VHP sugars in the transformation of sucrose to FOS, whether biological or enzymatic. Several studies have shown that the higher the concentration of sucrose in the medium in which the enzymatic synthesis of FOS occurs, the higher the rate of conversion of sucrose to FOS. The process according to the present invention starts with a solution of VHP sugars diluted in water, in which the sugar ratio can vary from 20% to 60%. This solution is inoculated with a culture of Aureobasidium sp. and a genetically modified strain of Saccharomyces cerevisiae PE-2, which does not produce extracellular invertase, at an inoculum amount of 10-20% (v / v) in the final medium, allowing the biotransformation of sucrose to FOS with the simultaneous consumption of glucose.

[0042] In the present invention, a Saccharomyces cerevisiae yeast called PE-2, a wild-type strain isolated from an industrial sugar and ethanol environment in Brazil, was used and the genes involved in invertase production were simply deleted as described below.

[0043] 1. Deletion of the invertase gene in Pe-2 yeast The Saccharomyces cerevisiae yeast used in the purification steps of the present invention has a low or no ability to metabolize the desired products sucrose and fructooligosaccharides, and this lack of ability results in the selective consumption of glucose and fructose in the FOS syrup.

[0044] According to an embodiment of the present invention, a genome editing strategy was used by using modified CRISPR / Cas9 technology for disruptive mutation of SUC-2 gene, where 1109bp of the gene was deleted, preventing functional expression of the encoded protein. Editing was verified by PCR genotyping, growth on sucrose (which is null for edited yeast) and Sanger sequencing.

[0045] The SUC2 gene is located on chromosome IX of the Saccharomyces cerevisiae genome and contains 1599 base pairs. The SUC2 gene is located on the Saccharomyces cerevisiae Pe-2 genome. Through the above genome editing, the SUC2 gene becomes 490 base pairs (see attached Figure 6: Representative diagram of SUC2 gene and genome editing strategy. A) Location of SUC2 gene on chromosome IX and design of primers for genome editing region of gene promoter and deletion of that region using CRISPR / Cas9 technology. B) Structure of SUC2 gene after gene promoter editing), making protein expression completely impossible.

[0046] Genetic engineering was applied to chromosome 9, and tools were used to further reduce this risk of off-target, i.e. cutting at undesired points in the genome by Cas9.

[0047] According to the literature (Ryan OW, Skerker JM, Maurer MJ et al. Selection of chromosomal DNA library 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 highly unlikely to occur in a small genome like Saccharomyces cerevisiae. gRNAs (guide RNAs) are designed for unique sequences in the genome, which makes it impossible for the Cas9 enzyme to recognize other PAM (protospacer adjacent motif) sequences in the experiment.

[0048] Expression products of the genomic region: This deletion abolished the expression of the invertase encoded by SUC2, which was verified by a phenotypic change in which the yeast did not grow in the medium YP + 2% sucrose but grew normally in YP + 2% glucose.

[0049] 2. Co-growth of inoculum for FOS synthesis According to the invention, the inoculum growth can be optimized in a single step, where the Aureobasidium strain responsible for the synthesis of FOS and the Saccharomyces cerevisiae PE-2 strain, which does not produce invertase, are grown together in one single reactor as described above.

[0050] The processes can be performed separately, but by doing so the manufacturing process is optimized.

[0051] For this purpose, a volume of 9x liters of aqueous medium containing 10-20% total reducing sugars (TRS), a volume of 1x liter of Aureobasidium are prepared and grown for 24 hours, after which the genetically modified Saccharomyces cerevisiae are inoculated and incubated for a further 24 hours at a temperature set between 32-35 °C, agitated at 120 rpm, and with an aeration of at least 1 vvm.

[0052] After both cultures were fully grown, the 10x liter inoculum culture was transferred to 90x liter culture medium containing 10-20% TRS (total reducing sugars) and incubated at a controlled temperature between 32-35 °C, agitated at 120 rpm, and with an aeration rate of at least 1 vvm for 24-48 hours to allow for full growth and a concentration of 5 x 10 of each yeast (Aureobasidium sp. and genetically modified Saccharomyces cerevisiae). 7 Grow to a minimum population of CFU / mL.

[0053] 3. FOS synthesis and simultaneous consumption of glucose After growth, as described above, the inoculum is transferred to an aqueous medium of sucrose and / or molasses and / or sugar cane syrup containing 20-60% sucrose in the total medium, at a ratio of 10-20% of the volume of the aqueous medium.

[0054] FOS synthesis occurs in as little as 12 hours. However, the removal of glucose by consumption by Saccharomyces yeasts occurs in 12 to 60 hours, the duration of which varies depending on the initial sucrose concentration of the medium. Thus, this process occurs at 30 to 35 °C for at least 12 hours, but can continue for up to 60 hours.

[0055] Through the present invention, it was possible to improve product purity and yield by converting all or nearly all of the sucrose content to FOS, because the simultaneous consumption of glucose avoids the "negative feedback" effect of glucose on the activity of the fructosyltransferase enzyme and increases the conversion rate of sucrose to FOS.

[0056] The ethyl alcohol produced is 100% evaporated during spray drying, resulting in a high purity final product.

[0057] The following Table 1 (kinetics of formation of fructooligosaccharides in an initial medium containing 44% sucrose on a wet and dry basis, respectively) and Table 2 (prior art and inventive compositions) show the selective consumption of glucose and fructose in the syrup and the high concentrations of FOS obtained in the simultaneous production and purification process (meaning of the abbreviations: scFOS: short chain FOS; LcFOS: long chain FOS). [Table 1]

[0058] In Table 1, it can be clearly observed that from 24 hours onwards, the formation of FOS is accompanied by the consumption of glucose, which allows a higher yield than the prior art process, which has a 60% yield in the conversion of sucrose to FOS. The consumption of glucose leads to the production of ethanol and at the same time to a decrease in dry matter, which is reduced from an initial 45% to 30% at the end of the 72 hour process.

[0059] The formation of ethanol allowed the production of a syrup with high purity in FOS. Since 3-10% of drying aid additives such as silicon oxide or cornstarch are added to the final product composition, the purity of the final product decreases by an average of 10%, taking into account the moisture content of the powder in the composition.

[0060] Table 2 compares the composition of the pure FOS according to the invention with the prior art FOS as produced by Ingredion, as shown in Table 3. [Table 2] [Table 3]

[0061] 4. Microbial Elimination of Glucose The high conversion of sucrose to FOS, as seen in the present invention, has the drawback of releasing a large amount of glucose into the solution in which the synthesis takes place. The presence of high concentrations of glucose, which can exceed 22%, makes it impossible to dry the FOS syrup using spray drying techniques. The solution found according to the present invention was to convert glucose to ethyl alcohol using yeasts that lack invertase activity.

[0062] The present invention significantly reduces the production costs of fructooligosaccharides, since it eliminates the step of removing glucose by chromatographic purification and allows direct drying by spray drying of the FOS syrup (as the product is called before drying) for mass use in the cost-sensitive animal nutrition and food industries.To this end, biochemical transformation of glucose is carried out using yeast or Saccharomyces cerevisiae, a microorganism that does not produce extracellular invertase.

[0063] 5. Direct drying of syrup The presence of glucose in concentrations above 3% in FOS syrups makes them unable to be directly dried in a spray dryer. This occurs due to the different glass transition temperatures (tg) of the compounds. Glucose and fructose cannot be spray dried due to their low tg values, but this does not occur for sugars with a higher degree of polymerization, such as kestose (GF2), nystose (GF3) and fructofuranosyl nystose (GF4). The production of FOS and the removal of glucose by microbiological processes is characterised by a low-cost process compared to the current conventional technique of glucose separation by chromatography.

[0064] Obtaining the final product In the present invention, we chose to use Aureobasidium for the biotransformation of sucrose because the FOS produced by Aureobasidium contain only short chain oligomers (GF2, GF3, GF4, GF5, and / or GF6) that are most effective in producing bifidobacteria.

[0065] The FOS of the present invention can be partially or completely purified, so that its concentration can vary from 70-95% purity.

[0066] The FOS obtained according to the invention contain short chain oligomers (scFOS) such as GF2, GF3, GF4 and / or long chain oligomers (lcFOS) such as GF5 and / or GF6, which are the ones with the greatest bifidobacterial effect, while the former have a greater immunomodulatory potential, which is a major differentiating factor of the products in question. The larger the chain size, the greater the bifidobacterial effect of the product, without reaching the polymer size. This is due to the fact that the effectiveness of medium chains such as GF5 and GF6 is greater than GF2 and GF3, and much better than the long chains of polymers such as inulin and levan, which can be more used as soluble fiber, contribute to a faster passage through the intestinal tract and have a less bifidogenic or modulating effect on the intestinal flora.

[0067] Similarly, smaller oligomers such as GF2 and GF3 have been shown to have immunomodulatory activity, acting directly on cells of the immune system present in the intestinal epithelium, such as macrophages, T cells, and B cells, among others.

[0068] The greater the bifidobacteria effect, the greater the population of beneficial bacteria such as lactobacilli and bifidobacteria, leading to increased acidification in the intestinal tract and an environment that is difficult for Salmonella and E. coli to live in, increasing the efficiency as a growth promoter and population regulator. In the formulation of the present invention, the ratio of GF2 may vary in the range of 10-45%, the ratio of GF3 may vary in the range of 30-55%, the ratio of GF4 may vary in the range of 10-25%, the ratio of GF5 may vary in the range of 3-13%, and the ratio of GF6 may vary in the range of 0-8%. Preferably, the ratio of the FOS oligomer of the present invention may vary between GF2 15-40%, GF3 35-50%, GF4 13-23%, GF5 3-10%, and GF6 0-6%.

[0069] The biotransformation process used in the present invention has the advantage of controlling the ratio of short chain oligomers formed over the synthesis time. In the present invention, the shorter the synthesis time, the greater the ratio of GF2 to GF3 and GF4 and GF5. Each species has its own microflora that is different from the others. Therefore, in the present invention, the FOS composition used in birds is different from the FOS composition used in monogastric, fish, ruminant or human. The ratio between the short chain oligomers should vary depending on the intended use of the FOS.

[0070] According to an embodiment of the present invention, the fructooligosaccharides production and purification process uses at least two microorganisms or yeasts, simultaneously or separately, one of which is of the genus Aureobasidium and the other yeast is unable to produce extracellular invertase, to convert sucrose into fructooligosaccharides (FOS) in a medium containing sucrose, with the complete consumption of glucose from the medium and the evaporation of alcohol or ethanol during drying of the final product.

[0071] According to an embodiment of the present invention, the fructooligosaccharides production and purification process uses at least two microorganisms or yeasts, one of which is of the genus Aureobasidium and the other yeast is a genetically modified Saccharomyces cerevisiae unable to produce extracellular invertase, in a medium containing sucrose, simultaneously or separately, to convert sucrose into fructooligosaccharides (FOS), with the complete consumption of glucose from the medium and the evaporation of alcohol or ethanol during drying of the final product.

[0072] According to yet another embodiment of the present invention, the process for producing and purifying fructooligosaccharides uses an aqueous medium containing 20-60% by mass of sucrose, and comprises culturing Aureobasidium sp. and Saccharomyces cerevisiae genetically modified so as not to produce extracellular invertase, each of the two yeast strains being 5×10 7 At a minimum population inoculum concentration of CFU / ml, the medium is left at 30°C-35°C with stirring and aeration until the glucose present in the medium is completely removed and the ethanol is removed by drying the final product.

[0073] According to yet another embodiment of the present invention, the process for producing and purifying fructooligosaccharides comprises the steps of: preparing an inoculum containing said yeast by inoculating Aureobasidium from an aqueous medium containing 10%-20% total reducing sugars (TRS) in a medium:Aureobasidium volume ratio (v / v) of 1:10, respectively, and after growth, inoculating a genetically modified Saccharomyces cerevisiae in the same inoculum:aqueous medium volume ratio of 1:10 and maintaining at a temperature of 32°C-35°C with stirring and aeration; after complete growth of both cultures or yeasts, transferring the inoculum to a fresh culture medium containing 10%-20% TRS in an inoculum:medium volume ratio (v / v) of 1:10, respectively, and growing to completion at the same temperature with stirring and aeration; and obtaining 5×10 yeasts of each yeast. 7 A minimum population of CFU / ml is maintained and the ethanol is removed when the final product is dried.

[0074] According to a preferred embodiment of the present invention, a volume of "9x" liters of aqueous medium containing 10-20% total reducing sugars (TRS) and "1x" liter of Aureobasidium are first prepared, and after 24 hours of growth, the genetically modified Saccharomyces cerevisiae are inoculated and incubated and maintained at a temperature of 32-35°C for at least another 24 hours with stirring and aeration. After complete growth of both cultures, the "10x" liters of inoculated culture are transferred to "90x" liters of culture medium containing 10-20% TRS (total reducing sugars), and grown to completion, with each yeast (Aureobasidium sp. and genetically modified Saccharomyces cerevisiae) being at 5x10 7 Incubate at a controlled temperature of 32-35°C with stirring and aeration until a minimum population of CFU / mL is reached, then transfer the inoculum to an aqueous medium of sucrose and / or molasses and / or sugarcane syrup containing 20-60% sucrose in the total medium at a ratio of 10-20% of the volume of the aqueous medium, synthesis of FOS occurs in a minimum of 12 hours, whereas removal of glucose by consumption of the genetically modified Saccharomyces yeast occurs in 12-60 hours, the period of which varies depending on the initial sucrose concentration of the medium and the temperature varies from 30-35°C.

[0075] According to a more preferred embodiment of the present invention, first, a volume of “9×” liters of aqueous medium containing 10-20% total reducing sugars (TRS) is prepared, which is inoculated with a volume of “1×” liters of Aureobasidium, and after 24 hours of growth, the genetically modified Saccharomyces cerevisiae is inoculated and incubated for another 24 hours at a temperature set at a minimum of 32-35° C., agitated at 120 rpm, and aerated at 1 vvm. After complete growth of both cultures, the “10×” liters of inoculated culture is transferred to a “90×” liters of culture medium containing 10-20% TRS (total reducing sugars), and after complete growth, each yeast (Aureobasidium sp. and genetically modified Saccharomyces cerevisiae) is 5×10 7Incubate at a controlled temperature of 32-35°C with agitation at least 120 rpm and aeration at 1 vvm until a minimum population of CFU / mL is reached, then transfer the inoculum to an aqueous medium of sucrose and / or molasses and / or sugar cane syrup containing 20-60% sucrose in the total medium at a ratio of 10-20% of the volume of the aqueous medium, synthesis of FOS occurs in a minimum of 12 hours, whereas removal of glucose by consumption of the genetically modified Saccharomyces yeast occurs in 12-60 hours, the period of which varies depending on the initial sucrose concentration of the medium and the temperature varies from 30-35°C.

[0076] Purified fructooligosaccharides (FOS) are free of glucose and ethanol in the final product.

[0077] Below are examples to better illustrate the scope of the present invention, but these examples should not be used to limit the scope of the present invention. EXAMPLES

[0078] Example 1: Fructooligosaccharide (FOS) production: Aureobasidium strains involved in the synthesis of FOS and Saccharomyces cerevisiae PE-2 strain, which does not produce extracellular invertase, were used in one reactor. To do this, 200 L of aqueous medium containing 15% total reducing sugars (TRS) was first prepared and inoculated with 20 L of Aureobasidium. After 24 h of growth, the genetically modified Saccharomyces cerevisiae PE-2 was inoculated and this was incubated for another 24 h at a temperature set between 33 °C, agitated at 120 rpm, and with an aeration of at least 1 vvm.

[0079] After complete growth of both cultures, the 200 L inoculum was transferred to 2000 L of culture medium containing 15% TRS (total reducing sugars) and grown to completion at a controlled temperature of 33 °C, shaking at 120 rpm, and aeration of at least 1 vvm, resulting in a population of 5 × 10 7Incubation was continued for 36 hours until a minimum population of CFU / mL was achieved.

[0080] After growth, the inoculum was transferred to an aqueous medium of sucrose cane syrup containing 40% sucrose in the total medium at a ratio of 15% of the inoculum volume.

[0081] The synthesis of FOS occurred in as little as 12 h. However, the removal of glucose by consumption by Saccharomyces yeasts occurred after 40 h, depending on the initial sucrose concentration of the medium, the temperature was 33 °C, and the time was 40 h.

[0082] The ethyl alcohol produced was 100% evaporated during spray drying so the final product now contained high purity.

[0083] The FOS produced present only short chain oligomers (GF2, GF3 and GF4, GF5 and / or GF6), which are the oligomers with the greatest bifidobacterial effect at the following mass ratios of oligomers (between 24% for GF2, 35% for GF3, 32% for GF4, 7% for GF5 and 2% for GF6).

[0084] Application of FOS obtained in cats: The study was conducted in a randomized block design with time as a blocking factor in cats receiving different types of prebiotics, including the FOS of the present invention (yFOS) and the prior art FOS produced by Ingredrion (scFOS). The study consisted of 6 treatments replicated over 3 periods, with 12 cats per period (2 cats per treatment), for a total of 6 replicates per treatment (n=36 total).

[0085] The objective of this experiment was to show that the inclusion of prebiotics FOS, GOS, and inulin in cat food promotes the intestinal fermentation of organic matter, changes the concentration of fecal fermentation products, and therefore the apparent digestibility results of the food. Furthermore, the objective was to show that there is a significant difference in performance between the FOS of the present invention (yFOS) and the prior art FOS (scFOS) manufactured by Ingredion. Six experimental diets were formulated, one was a negative control (NC) diet without any added prebiotics, and the other five contained 0.8% FOS (yFOS), 0.8% GOS (GOS), 0.8% inulin (INU) of the invention from Yessinergy, 0.8% FOS (scFOS) from the prior art manufactured by Ingredion, and 0.8% combination of yFOS and inulin (yFOS INU), respectively. The inclusion of prebiotics was made to replace cellulose since it is a fiber but is not fermentable for cats. All feeds were formulated according to the recommendations of FEDIAF (2018), the acronym for the Federation of European Pet Food Industry Associations.

[0086] At the end of the digestibility study, all cats were maintained for an additional period for daily collection of fresh feces within 15 minutes after defecation for determination of pH, volatile fatty acids (VFA), fecal ammonia nitrogen and fecal lactate until the full 21-day period was completed.

[0087] To measure fecal pH, 2 grams of fresh feces (collected within 15 min after defecation) was diluted with 6 mL of milliQ water and the pH was assessed using a precision digital pH meter.

[0088] To measure the concentrations of VFAs (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 15 mL of 16% formic acid solution (1:3 w / v). The mixture was centrifuged three times, 15 min each, with the supernatant discarded, while still refrigerated, at 4,500 G. To measure VFAs, samples were identified and stored in a freezer (-15 °C) and then submitted for analysis to the Chemistry Laboratory of the State University of Maringá. Measurement of VFAs in feces was performed by gas chromatography.

[0089] Fecal ammonia nitrogen was determined by applying the Kjeldahl method. Extracts prepared for VFA measurements were used. Extracts were thawed at room temperature, 2 mL aliquots were transferred to test tubes, diluted with 13 mL distilled water, and distilled in a nitrogen still (Tecnal T-036 / 1, Piracicaba, Brazil). Distillation was performed with 5 mL of potassium hydroxide KOH 2 mol / L and nitrogen into an Erlenmeyer flask containing 10 mL of solution (boric acid 0.97 N). Each sample was then titrated with HCL (0.005 N).

[0090] The variables measured in each experiment were preliminarily analyzed for normality and equality of variance. The apparent digestibility coefficients between treatments were compared by using the Tukey test with a probability of 5%. Considering that for fermentation products (ammonia, SCFA and BCFA), pH and fecal DM, which show interdependence among themselves, multivariate statistics were applied through an analysis of standard correlations, which considered that if the multivariate analysis of variance (MANOVA) presented a probability value of less than 5% for the null hypothesis, this was also significant.

[0091] Table 4 below shows the fermentation parameters in the feces (short chain fatty acids (mmol / kg fecal MS), where NC = negative control, yFOS = fructooligosaccharides according to the invention, GOS = galactooligosaccharides, INU = inulin, scFOS = FOS produced by Ingredion, FOS+INU = FOSy (FOS according to the invention) related to INU. [Table 4]

[0092] As shown in Table 4, the formation of intestinal fermentation products such as lactate, propionate and acetate was more strongly correlated with the inclusion of GOS in the diet, while the formation of others (butyrate, BCFA and ammonia) was more strongly correlated with the inclusion of yFOS (invention), demonstrating that these two treatments promote intestinal fermentation more than the other treatments. Fecal pH was inversely correlated with the fermentation products, as expected.

[0093] Accompanying FIG. 7 presents the standard correlations of fermentation and fecal quality parameters between the negative control (NC) and prebiotic-containing treatments.

[0094] Although high ammonia content and soft feces are undesirable in the feces of domestic cats, the purpose of the experiment was to show the overall strong bifidobacterial effect of the FOS of the present invention when compared with other prebiotics available on the market.In this aspect, the high values ​​of total volatile fatty acids (total VFA) and mainly total short chain fatty acids (total SCFA) indicate the superior performance of the product of the present invention.Another more important indicator that there was a greater fermentation through the large intestine in yFOS and yFOS+INU treatments was the higher production of butyric acid.Currently, butyric acid is the most desired by nutritionists.

[0095] Table 5 below shows blood parameters (per μL of blood) related to monocyte and granulocyte phagocytosis, where C=negative control, yFOS=fructooligosaccharides according to the invention, GOS=galactooligosaccharides, INU=inulin, scFOS=FOS produced by Ingredion, FOS+INU=FOSy (FOS according to the invention) related to INU. [Table 5]

[0096] Table 5 shows that with yFOS treatment, there is the highest concentration of monocytes and phagocyte granulocytes, and a low concentration of total white blood cells, which is also highly desirable. This demonstrates the superiority of yFOS, the product of the present invention, in regulating the immune system of cats. It also shows that yFOS increases the antagonism of the cellular immune system and decreases the antagonism of the humoral immune system, i.e., has an anti-inflammatory effect.

[0097] Similarly, Table 6 contains the results for fecal IgA concentration, where C=negative control, yFOS=fructooligosaccharides according to the invention, GOS=galactooligosaccharides, INU=inulin, scFOS=FOS produced by Ingredion, FOS+INU=FOSy (FOS according to the invention) related to INU. [Table 6]

[0098] The results shown in Table 6 reaffirm those shown in Tables 4 and 5 and demonstrate the superiority of treatment with yFOS, the product of the present invention, in terms of its bifidobacterial effect and its superiority in terms of regulating the immune system when compared to other treatments, including treatment with scFOS, an important commercial brand of FOS in the prior art. It was reaffirmed that yFOS increases the cellular immune system and decreases the humoral immune system, i.e. has an anti-inflammatory effect.

Claims

1. A process for the production and purification of fructooligosaccharides, comprising the steps of using at least two microorganisms or yeasts, either simultaneously or separately, in a medium containing sucrose, one of which is unable to produce extracellular invertase, to convert sucrose into fructooligosaccharides (FOS), with complete consumption of glucose from the medium and evaporation of alcohol or ethanol during drying of the final product.

2. The process for producing and purifying fructooligosaccharides according to claim 1, characterized in that the obtained FOS has long chain oligomers GF5 and GF6, and the average size of the other short chain oligomers GF2, GF3 and GF4 is large.

3. The process for producing and purifying fructooligosaccharides according to claim 1, characterized in that the ratios of oligomers in the fructooligosaccharides (FOS) are, by mass ratio relative to the total mass, 10-45% GF2, 30-55% GF3, 10-25% GF4, 3-13% GF5, and 0-8% GF6.

4. The process for producing and purifying fructooligosaccharides according to claim 3, characterized in that the proportions of oligomers in the FOS vary between 15-40% GF2, 35-50% GF3, 13-23% GF4, 3-10% GF5, and 0-6% GF6, in mass ratios relative to the total mass.

5. 2. The process for producing and purifying fructooligosaccharides according to claim 1, characterized in that sugars derived from sugar cane, beetroot, sugar cane syrup and molasses are used as sucrose sources.

6. The process for producing and purifying fructooligosaccharides according to claim 1 or 5, characterized in that the medium contains 20 to 60% by mass of sucrose.

7. The process for producing and purifying fructooligosaccharides according to claim 1, characterized in that one of the microorganisms or yeasts is of the genus Aureobasidium.

8. 2. The process for producing and purifying fructooligosaccharides according to claim 1, characterized in that one of the microorganisms or yeasts is a genetically modified Saccharomyces cerevisiae.

9. 9. The process for the production and purification of fructooligosaccharides according to claim 8, characterized in that the genetically modified Saccharomyces cerevisiae is a wild strain, called PE-2, isolated from an industrial sugar and alcohol environment in Brazil, which has a simple deletion of the gene responsible for invertase production.

10. The process for producing and purifying fructooligosaccharides according to claim 9, characterized in that the genetically modified Saccharomyces cerevisiae selectively consumes only glucose and fructose, and does not consume sucrose and FOS, i.e., oligomers having α-1-2 type and β-2-1 type bonds.

11. 9. The process for the production and purification of fructooligosaccharides according to claim 8, characterized in that the genetic manipulation is applied to chromosome 9.

12. 2. The process for the production and purification of fructooligosaccharides (FOS) according to claim 1, characterized in that in a medium containing sucrose, at least two microorganisms or yeasts are used simultaneously or separately, one of which is of the genus Aureobasidium and the other is a yeast incapable of producing extracellular invertase, for the conversion of sucrose into fructooligosaccharides (FOS), accompanied by the complete consumption of glucose from the medium and the evaporation of alcohol or ethanol during drying of the final product.

13. 13. The process for the production and purification of fructooligosaccharides (FOS) according to claim 12, characterized in that at least two microorganisms or yeasts are used simultaneously or separately, one of which is of the genus Aureobasidium and the other is another genetically modified Saccharomyces cerevisiae yeast unable to produce extracellular invertase, in a medium containing sucrose, for the conversion of sucrose into fructooligosaccharides (FOS), with the complete consumption of glucose from the medium and the evaporation of alcohol or ethanol during drying of the final product.

14. Using an aqueous medium containing 20 to 60% by mass of sucrose, Aureobasidium sp. and a genetically modified Saccharomyces cerevisiae strain unable to produce extracellular invertase were cultured at 5 × 10 ７ The process for the production and purification of fructooligosaccharides according to claim 1, characterized by maintaining a minimum population inoculation concentration of CFU / ml at a temperature between 30°C and 35°C, with stirring and aeration, until the glucose present in the medium is completely removed and the ethanol is removed by evaporation during drying of the final product.

15. The inoculum containing the yeast is prepared from an aqueous medium containing 10%-20% total reducing sugars (TRS) inoculated with Aureobasidium in a volume ratio of medium:Aureobasidium (v / v) of 1:10, respectively, and after growth, inoculated with genetically modified Saccharomyces cerevisiae in the same volume ratio of inoculum:medium (v / v) of 1:10, and maintained at a temperature of 32°C-35°C with stirring and aeration. After complete growth of both cultures or yeasts, the inoculum is transferred to a new culture medium containing 10%-20% TRS in a volume ratio of inoculum:medium (v / v) of 1:10, respectively, and grown to completion, with a minimum population of 5×10 for each yeast. ７ The process for producing and purifying fructooligosaccharides according to claim 1, characterized in that the fructooligosaccharides are kept at the same temperature with stirring and aeration until the fructooligosaccharides reach CFU / ml.

16. First, a volume of "9x" liters of aqueous medium containing 10-20% total reducing sugars (TRS) was prepared, inoculated with a volume of "1x" liters of Aureobasidium, and after 24 hours of growth, inoculated with genetically modified Saccharomyces cerevisiae and maintained by incubation for another 24 hours at a temperature of 32-35°C with at least stirring and aeration. After complete growth of both cultures, the "10x" liters of inoculated culture was transferred to a "90x" liters of culture medium containing 10-20% TRS (total reducing sugars), and after complete growth, each yeast (Aureobasidium sp. and genetically modified Saccharomyces cerevisiae) was allowed to grow at a concentration of 5x10 ７ 2. The process for the production and purification of fructooligosaccharides according to claim 1, characterized in that the inoculum is incubated at a controlled temperature of 32-35°C with stirring and aeration until a minimum population of CFU / mL is reached, and then the inoculum is transferred to an aqueous medium of sucrose and / or molasses and / or sugar cane syrup containing 20-60% sucrose in the total medium at a ratio of 10-20% of the volume of the aqueous medium, the synthesis of FOS occurs in a minimum of 12 hours, while the removal of glucose by consumption of the genetically modified Saccharomyces yeast occurs in 12-60 hours, the period of which varies depending on the initial sucrose concentration of the medium, and the temperature varies between 30-35°C.

17. 2. A product obtained by the process of claim 1, characterized in that the final product is free of glucose and ethanol and contains purified fructooligosaccharides (FOS).

18. 18. The product according to claim 17, characterized in that the fructooligosaccharides (FOS) obtained have long oligomers GF5 and GF6, with the other short oligomers GF2, GF3 and GF4 having a larger average size.

19. 19. Product according to claim 18, characterized in that the mass proportion of GF2 varies from 10 to 45%, the mass proportion of GF3 varies from 30 to 55%, the mass proportion of GF4 varies from 10 to 25%, the mass proportion of GF5 varies from 3 to 13% and the mass proportion of GF6 varies from 0 to 8%.

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

21. 18. Product according to claim 17, characterized in that it comprises fructooligosaccharides (FOS) and 3-10% by weight, based on the total weight, of drying aid additives known to those skilled in the art.

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