A method for preparing levan-type fructooligosaccharides, levan-type fructooligosaccharides and applications thereof

Abstract

The application provides a method for preparing Levan-type fructooligosaccharide, the Levan-type fructooligosaccharide and application thereof, and belongs to the field of animal nutrition and biotechnology. The method comprises the following steps: fermenting Bacillus subtilis BS21 in a fermentation medium containing sucrose; and separating Levan-type fructooligosaccharide from a fermentation product. The Levan-type fructooligosaccharide has a weight average molecular weight Mw of 6-8 kDa, and at least 95% of fructose units contained in the Levan-type fructooligosaccharide are connected through beta-(2,6)-glycosidic bonds. The Levan-type fructooligosaccharide is applied to the preparation of a feed additive for improving animal intestinal health or improving animal growth performance. The application can efficiently prepare the Levan-type fructooligosaccharide with clear structure, the Levan-type fructooligosaccharide has biological activity, can reduce the diarrhea rate of weaned piglets and the feed conversion ratio, and the mechanism is to enrich beneficial bacteria such as lactobacillus johnsonii and clostridium prasaldi in the intestines.

Description

Technical Field

[0001] This invention relates to the fields of animal nutrition and biotechnology, specifically to a method for preparing Levan-type fructooligosaccharides, Levan-type fructooligosaccharides, and their applications. Background Technology

[0002] Fructooligosaccharides (FOS), as a novel type of functional oligosaccharide, have become a research hotspot in the prebiotic field due to their unique probiotic properties, demonstrating broad application potential and market value in multiple fields such as feed, food, and chemicals. As an important member of the FOS family, Levan-type FOS is a fructan formed by fructose residues linked by β-(2,6)-glycosidic bonds. Compared to traditional inulin-type FOS and plant-derived Levan, it possesses superior physiological functions and application advantages, making it a key focus of prebiotic research in recent years. The core value of FOS lies in its selective probiotic effect, which can directionally promote the proliferation of beneficial bacteria in the gut (such as Bifidobacteria and Lactobacillus) while inhibiting the growth and reproduction of harmful pathogens such as Escherichia coli and Salmonella, thereby regulating the intestinal microecological balance and improving the host's intestinal health. Building upon this foundation, Levan-type fructooligosaccharides also possess a variety of additional physiological activities, such as regulating the body's immune function, promoting the absorption of minerals like calcium and magnesium, lowering serum cholesterol, and improving lipid metabolism. In the food industry, they can be added as functional ingredients to yogurt, beverages, baked goods, and infant formula to enhance the nutritional value of these products. In the feed industry, they can be used as green feed additives to replace antibiotics, improve the intestinal health of livestock and poultry, and enhance production performance. In the chemical industry, they can be used to produce biodegradable materials, food preservatives, and cosmetic additives, demonstrating significant industrial application value.

[0003] From the perspective of origin, Levan is mainly produced through two pathways: plant synthesis and microbial synthesis. Plant-derived Levans are mostly low-molecular-weight products, primarily found in the sap and tissues of plants in the Asteraceae and Poaceae families. They have low yields, are difficult to extract, and have a narrow molecular weight distribution, limiting their applications. Microbial-derived Levans, on the other hand, are mostly high-molecular-weight products. Microorganisms offer advantages such as rapid growth, mild culture conditions, and ease of large-scale fermentation, making them the primary route for the production of Levan-type fructooligosaccharides. Currently, the industrial production of Levan-type fructooligosaccharides mainly relies on microbial fermentation technology. Its core principle is to utilize microbial-synthesized levo-glycosylase (β-2,6-fructan: D-glucose-fructose transferase), using sucrose as the sole substrate. Through transfructylation, fructose residues in sucrose molecules are transferred and linked to form β-(2,6)-glycosidic bonds in Levan-type fructooligosaccharides, while glucose is generated as a byproduct. In this reaction process, the key gene responsible for Levan production is the SacB gene. The expression of this gene is sucrose-dependent; only in the presence of sucrose can the SacB gene be activated and express levocyclic glycosylase, thereby catalyzing Levan synthesis. Therefore, sucrose concentration, reaction temperature, pH, and other conditions directly affect the yield and quality of Levan. To date, researchers have discovered that various microorganisms can synthesize and secrete levocyclic glycosylase, thereby producing Levan-type fructooligosaccharides. These mainly include *Fermentomonas motilityis*, *Bacillus natto*, *Bacillus licheniformis*, *Clostridium acetobutyrate*, *Brennia bryophylla*, *Acinetobacter nectaris*, *Lactobacillus reuteri*, *Halomonas*, and *Acetobacter xylinum*. Among them, Bacillus subtilis (especially Bacillus natto strains) has become the most widely studied and most promising strain in the fermentation production of Levan-type fructooligosaccharides due to its advantages such as a powerful enzyme secretion system, rapid growth and reproduction, low culture cost, high safety, and non-pathogenicity. Its synthesized levocyclic glycosylase has high catalytic efficiency, strong substrate specificity, and good stability, and can efficiently catalyze the conversion of sucrose into Levan-type fructooligosaccharides, making it an ideal strain for industrial production. In addition, Lactobacillus reuteri, as a probiotic, not only synthesizes Levan-type fructooligosaccharides in high yields but also synergistically enhances its probiotic effects, giving it unique application advantages in the functional food field.

[0004] Although some progress has been made in the research of microbial fermentation for the production of Levan-type fructooligosaccharides, and some processes have achieved initial small-scale pilot production, existing production processes still face many technical bottlenecks compared to the demands of large-scale industrial production, which seriously restricts the industrialization and application of Levan-type fructooligosaccharides. First, the source of strains is unstable and the yield is low. Currently, most of the strains used for fermentation production are wild-type strains or early-stage genetically modified strains. Wild-type strains have low expression levels of levo-cyclic glycosylase and low Levan synthesis efficiency, and their growth is easily affected by environmental factors (such as temperature, pH, and substrate concentration), resulting in large fluctuations in Levan yield and sucrose conversion rate. On the other hand, the gene stability of early-stage genetically modified strains is poor. After multiple generations of subculturing, the expression level of the SacB gene will decrease significantly, leading to a reduction in Levan yield, which is difficult to meet the cost-effectiveness requirements of industrial production. How to obtain high-yield and stable engineered strains has become a key problem that urgently needs to be solved. Secondly, controlling the molecular weight and distribution of the product is difficult. Levan-type fructooligosaccharides produced by microbial fermentation have a wide molecular weight distribution range, usually containing multiple components from low molecular weight (a few kDa) to high molecular weight (hundreds of kDa). The bioavailability of Levan-type fructooligosaccharides with different molecular weights varies greatly. Among them, the low molecular weight component (degree of polymerization 2-10) has the strongest bioavailability and is the most valuable target product for application. However, there is currently a lack of effective control methods, making it impossible to produce Levan-type fructooligosaccharides with a specific molecular weight range. Furthermore, the high molecular weight component in the product is difficult to separate and purify, which not only affects the uniformity of product quality but also reduces the product's bioavailability and application value. Finally, the functional specificity is unclear. Current research on the probiotic effects of Levan-type fructooligosaccharides mainly focuses on their proliferative effects on common beneficial bacteria such as Bifidobacterium and Lactobacillus. However, there are few studies on their targeted proliferative effects on beneficial bacteria with specific physiological functions, such as Lactobacillus johnsonii and Clostridium plasmidoides. Furthermore, the specific molecular mechanisms by which they regulate the balance of the intestinal microecology and enhance the body's immune function are not yet fully understood. This results in weak product targeting and makes it impossible to develop products with specific functions according to the needs of different populations (such as infants, the elderly, and people with low immunity) and different livestock and poultry.

[0005] Therefore, there is an urgent need in this field for a new method that can efficiently and controllably produce Levan-type fructooligosaccharides with clearly defined functional targets, and to verify its application effects through reliable animal models or clinical trials. Summary of the Invention

[0006] In view of this, the present invention provides a method for preparing Levan-type fructooligosaccharides, Levan-type fructooligosaccharides and their applications, aiming to solve the problems of low yield, uncontrollable molecular weight of products and unclear functional targeting in the production process of Levan-type fructooligosaccharides in the prior art.

[0007] To achieve the above objectives, the present invention provides a method for preparing Levan-type fructooligosaccharides, comprising the following steps: fermenting Bacillus subtilis BS21 in a fermentation medium containing sucrose; and separating Levan-type fructooligosaccharides from the fermentation product.

[0008] Optionally, the Bacillus subtilis BS21 is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 25977.

[0009] Optionally, the fermentation medium contains 100-120 g / L of sucrose and 2-5 g / L of yeast extract.

[0010] Optionally, the fermentation conditions are: temperature 36-38℃, fermentation time 20-24 hours.

[0011] Optionally, the fermentation medium contains 120 g / L sucrose and 2 g / L yeast extract; and the fermentation conditions are a temperature of 37°C and a fermentation time of 20 hours.

[0012] The present invention also provides a Levan-type fructooligosaccharide, wherein the Levan-type fructooligosaccharide has a weight-average molecular weight (Mw) of 6-8 kDa and contains at least 95% fructose, wherein the fructose units constituting the fructose are linked by β-(2,6)-glycosidic bonds.

[0013] Optionally, the weight-average molecular weight Mw is 7.2 kDa, and the Levan-type fructooligosaccharide is composed of at least 97% fructose.

[0014] The present invention also provides the use of Levan-type fructooligosaccharides as described in any one of claims 6 or 7 in the preparation of feed additives or feed compositions for improving animal gut health or enhancing animal growth performance.

[0015] Optionally, the feed additive or feed composition is formulated for weaned piglets, and its formulation is suitable for reducing diarrhea rate or feed conversion ratio when applied as a 0.5% weight percentage in the diet.

[0016] Optionally, the improvement of animal gut health includes enriching the animal's gut with Lactobacillus johnsonii or Clostridium plasminoides.

[0017] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention, through the use of a specific Bacillus subtilis BS21 strain and an optimized fermentation process, enables the efficient and stable preparation of Levan-type oligofructose with well-defined structures. Specifically, the Levan-type oligofructose prepared by this invention has a specific weight-average molecular weight range and a chemical structure dominated by β-(2,6)-glycosidic bonds, solving the problems of uncontrollable product structure and inconsistent quality in existing technologies, and ensuring the controllability and traceability of product quality.

[0018] 2. The Levan-type fructooligosaccharides with a specific structure provided by this invention possess specific biological activities. By applying them as a feed additive to weaned piglets, diarrhea rates and feed conversion ratios can be significantly reduced at low addition levels (e.g., 0.5%), solving the technical problem of high diarrhea rates and decreased production performance caused by weaning stress.

[0019] 3. This invention elucidates the mechanism of action of its product, namely, the ability to precisely enrich specific core beneficial bacteria (such as Lactobacillus johnsonii and Clostridium plasmidonii) in the animal gut. This clear structure-efficacy relationship solves the problems of unclear functional targeting and unpredictable effects of Levan products in the prior art. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 This is a schematic diagram of the overall process of the technical solution of this invention; Figure 2 These are the ion purification and gel purification results from Example 1 of this invention (A is the main elution peak obtained by ion exchange chromatography; B is the main elution peak obtained by gel filtration chromatography). Figure 3 This is the HPAEC-PAD chromatogram of the mixture of BS21EPS and standard sugar in Example 1 of this invention; Figure 4 This is the molecular weight distribution diagram of BS21EPS based on gel permeation chromatography (GPC) in Example 1 of the present invention; Figure 5 This is a conformational diagram of BS21EPS in Embodiment 1 of the present invention; Figure 6 This is the total ion chromatogram of BS21EPS partially methylated sugar alcohol acetates (PMAAs) in Example 1 of the present invention; Figure 7It is BS21EPS in Embodiment 1 of the present invention. 1 H NMR spectrum; Figure 8 It is BS21EPS in Embodiment 1 of the present invention. 13 C NMR spectrum; Figure 9 This is the heteronuclear single quantum coherence spectrum (HSQC) of BS21EPS in Embodiment 1 of the present invention. Figure 10 This is the homonuclear coherence spectrum (COSY) of BS21EPS in Embodiment 1 of the present invention. Figure 11 This is the heteronuclear multibond correlation spectrum (HMBC) of BS21EPS in Embodiment 1 of the present invention. Figure 12 This is a detailed structural diagram of Levan-type oligofructose in Embodiment 1 of the present invention.

[0022] Figure 13 This is a comparison of the Shannon and Simpson indices of the colonic microbiota in the CON and BS21CEPS groups in Example 2 of this invention; Figure 14 This is the colonic microbial β-diversity analysis based on the dissimilarity between PCoA and Bray-Curtis in Example 2 of the present invention; Figure 15 This is a comparison chart of the relative abundance of major fungal genera in Example 2 of the present invention; Figure 16 This is the species indicator bubble chart (first 20 digits) in Embodiment 2 of the present invention; Figure 17 This is a chord diagram of the microbial composition in Embodiment 2 of the present invention; Figure 18 This is a bar chart of LefSe differential species in Embodiment 2 of the present invention. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other implementation methods obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0024] Before providing a further detailed description of the embodiments of this application, the nouns and terms involved in the embodiments of this application will be explained, and the nouns and terms involved in the embodiments of this application shall be interpreted as follows.

[0025] (1) Levan-type fructooligosaccharides: These are oligosaccharides composed of fructose units with β-(2,6)-glycosidic bonds as the main linkage. In this invention, they preferably have a weight-average molecular weight of 6-8 kDa. Figure 12 As shown, its chemical structure is mainly composed of fructose units linked by β-(2,6)-glycosidic bonds forming the main chain, and may contain some branched structures.

[0026] (2) Bacillus subtilis BS21: refers to a specific strain capable of efficiently utilizing sucrose to produce Levan-type fructooligosaccharides. This strain, after screening and optimization, exhibits high yield, stability, and strong specificity, enabling it to directionally synthesize Levan-type fructooligosaccharides with specific molecular weights and structures. In a preferred embodiment of the present invention, this strain is deposited at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China), with accession number CGMCC No. 25977.

[0027] (3) Weight-average molecular weight (Mw): This refers to a weighted average of the molecular weight of a polymer. Its calculation method better reflects the contribution of macromolecules to the overall average molecular weight, and therefore better reflects the macroscopic physical properties of the polymer, such as viscosity and strength. In this invention, the weight-average molecular weight Mw is determined by gel permeation chromatography (GPC) and is one of the key parameters for defining the core structural characteristics of the Levan-type fructooligosaccharide product of this invention.

[0028] (4) Feed additives: These are substances added in small or trace amounts during feed processing, preparation, and use. They are not the main source of energy or protein, but are used to enhance the nutritional value of basic feed, improve animal production performance, prevent diseases, and ensure animal health. The Levan-type fructooligosaccharide in this invention is used as a functional feed additive.

[0029] (5) Feed conversion ratio (F / G): also known as feed conversion ratio, is a core indicator for evaluating feed utilization efficiency. It refers to the ratio of the total amount of feed consumed by an animal to the weight gain during a specific feeding period. The lower this value, the less feed is consumed per unit of weight gain, and the higher the feed utilization efficiency.

[0030] like Figure 1 As shown, this invention provides a method for preparing Levan-type fructooligosaccharides. This method aims to solve the problems of low production efficiency, uncontrollable product structure, and unclear functional targeting in existing technologies for preparing Levan-type fructooligosaccharides. The core of this method lies in utilizing specific microbial strains for fermentation and efficiently separating the target product from the fermentation products.

[0031] In a basic implementation, the method comprises two main steps. The first step involves fermenting a strain of Bacillus subtilis capable of producing Levan-type oligofructose in a fermentation medium containing a carbon source (e.g., sucrose). Bacillus subtilis, a widely used and safe industrial strain, secretes various extracellular enzymes during its growth and metabolism, including levofloxacin sucrase, which catalyzes the synthesis of Levan from sucrose. By providing suitable nutritional and environmental conditions, this strain can be encouraged to grow rapidly and efficiently synthesize the target product.

[0032] During fermentation, Bacillus subtilis utilizes sucrose in the culture medium as both a substrate and an energy source. A portion of the sucrose is metabolized by the bacteria for its own growth and reproduction, while the other portion undergoes a transglycosylation reaction under the action of levaninase, linking the fructose groups in the sucrose molecules one by one to form a polymer with β-(2,6)-glycosidic bonds as the main chain, namely Levan-type oligofructose, which is then secreted into the culture medium.

[0033] The second step of the method is to separate the Levan-type fructooligosaccharide from the fermentation products. After fermentation, the fermentation broth is a complex mixture containing cell cells, remaining culture medium components, other byproducts of cell metabolism, and the target product, Levan-type fructooligosaccharide. The purpose of the separation step is to separate the target product from these impurities. A typical separation process may include first removing cell cells by solid-liquid separation methods such as centrifugation or filtration to obtain a clear supernatant containing the target product.

[0034] Subsequently, taking advantage of the low solubility of Levan-type fructooligosaccharides in organic solvents (such as ethanol), a certain amount of organic solvent is added to the supernatant, causing the Levan-type fructooligosaccharides to precipitate from the solution. By collecting, washing, and drying these precipitates, crude Levan-type fructooligosaccharides can be obtained. This method is simple, easily scaled up industrially, and can effectively obtain the target product from complex fermentation systems.

[0035] This invention also provides a Levan-type fructooligosaccharide product prepared by the above method. This product possesses specific physicochemical structural characteristics, which are the basis for its excellent biological functions. In one embodiment, the weight-average molecular weight (Mw) of this Levan-type fructooligosaccharide is in the range of 6-8 kDa. Molecular weight is one of the key factors affecting the bioactivity of polysaccharides; excessively high or low molecular weights may affect their interaction with gut microbiota or immune cells. This invention controls the molecular weight within this specific range to achieve optimal prebiotic effects.

[0036] Besides the molecular weight range, another key structural feature of this product lies in the type and ratio of its glycosidic bonds. Specifically, at least 95% of the fructose units constituting this Levan-type fructooligosaccharide are interconnected via β-(2,6)-glycosidic bonds. Figure 12 As shown, this unique linkage forms the main skeletal structure of the product. Compared to the β-(2,6)-glycosidic bonds commonly found in plant-derived inulin, the β-(2,1)-glycosidic bonds have different spatial conformations and chemical stability. This allows them to be recognized and utilized by specific beneficial gut bacteria, while being difficult for them to be degraded by the host's own digestive enzymes or many harmful bacteria, thus exhibiting highly selective prebiotic properties.

[0037] This chemical structure, defined by its weight-average molecular weight (6-8 kDa) and a high proportion of β-(2, 6)-glycosidic bonds (>95%), distinguishes the Levan-type fructooligosaccharides of this invention from other fructooligosaccharide products in the prior art that have ambiguous structures or are of different types. It is this clear and unique structure that endows it with specific technical effects.

[0038] This invention further provides an application of the aforementioned Levan-type fructooligosaccharides. Specifically, they are used in the preparation of feed additives or feed compositions designed to improve animal gut health or enhance animal growth performance. Animals, especially young animals, are highly susceptible to gut health damage when facing stresses such as weaning, regrouping, and transportation. This damage manifests as excessive proliferation of harmful bacteria, impaired intestinal barrier function, and increased diarrhea rates, thereby affecting their growth and development.

[0039] Adding Levan-type fructooligosaccharides with a specific structure provided by this invention as a functional ingredient to animal basal diets can exert their prebiotic effect. It can be selectively fermented and utilized by specific beneficial microorganisms (such as Lactobacillus and Bifidobacterium) in the complex intestinal environment, thereby promoting the proliferation of these beneficial bacteria, inhibiting the growth of harmful bacteria, and optimizing the intestinal microecological balance. The proliferation of beneficial bacteria and their metabolites (such as short-chain fatty acids) can further improve the intestinal environment, enhance intestinal barrier function, and regulate local and systemic immunity, ultimately resulting in a reduction in diarrhea rates and an increase in feed utilization efficiency, i.e., improved growth performance.

[0040] In a preferred embodiment, based on the above preparation method, the Bacillus subtilis BS21 is Bacillus subtilis with accession number CGMCC No. 25977. Standardizing the preservation of the production strain and legally confirming its accession information is crucial to ensuring that the technical solution can be reliably and consistently implemented by those skilled in the art. Using strains with clearly defined accession numbers avoids problems such as unstable fermentation performance and product quality fluctuations caused by unclear strain origins and passage variations.

[0041] The technical effects of the above-mentioned technical features are as follows: First, they ensure the reproducibility of the method of the present invention. This helps those skilled in the art to obtain the strain from a depository through legal means and operate according to the method described in this invention to reproduce the technical solution of the present invention. Second, they ensure the consistency of product quality. Because a single, well-defined, and stable strain is used, it ensures a high degree of consistency in yield, molecular weight distribution, and chemical structure of Levan-type fructooligosaccharides produced by different batches of fermentation, providing a fundamental guarantee for subsequent standardized application and quality control.

[0042] Furthermore, in another preferred embodiment, the fermentation medium comprises 100-120 g / L sucrose and 2-5 g / L yeast extract. Sucrose is a direct substrate for the synthesis of Levan-type fructooligosaccharides, and its concentration directly affects the final product yield. Yeast extract serves as a high-quality complex nutrient source, providing essential amino acids, vitamins, and trace elements for cell growth.

[0043] Maintaining a sucrose concentration within the range of 100-120 g / L ensures sufficient substrate supply for achieving high product concentrations while avoiding osmotic stress or substrate inhibition effects that may result from excessively high substrate concentrations, thus maintaining good cell physiological condition and enzyme activity. For example, a sucrose concentration of 100 g / L effectively initiates the fermentation process; increasing the concentration to 120 g / L achieves a high product yield. Similarly, controlling the yeast extract concentration within the range of 2-5 g / L satisfies the needs for rapid cell growth and efficient enzyme production while avoiding increased costs or metabolic byproducts due to nutrient overload. For instance, 2 g / L of yeast extract already supports good cell density, while increasing it to 5 g / L further shortens the fermentation lag phase. Therefore, this range of culture medium components represents an optimized result after comprehensively considering product yield and production efficiency.

[0044] In another preferred embodiment, the fermentation conditions are: temperature 36-38℃, fermentation time 20-24 hours. Temperature is one of the most critical environmental factors affecting microbial growth and enzymatic reaction rates. For Bacillus subtilis BS21, the optimal temperature range for its growth and secretion of levofloxacin sucrase is 36-38℃. Below 36℃, cell growth and metabolic rates slow down, prolonging the fermentation cycle; above 38℃, some enzyme proteins may be inactivated, or the cells may enter a stress state, affecting product synthesis. Precisely controlling the temperature within this narrow optimized range is a prerequisite for achieving efficient fermentation.

[0045] Fermentation time needs to be matched with the cell growth curve and product accumulation curve. Within a 20-24 hour culture period, the cells complete the logarithmic growth phase and enter the stationary phase, at which point cell density and extracellular enzyme activity reach high levels, and the accumulation of Levan-type fructooligosaccharides is close to its peak. If the fermentation time is less than 20 hours, product accumulation is insufficient, resulting in a low yield; if it exceeds 24 hours, on the one hand, the substrate may be exhausted, and product growth will cease; on the other hand, the cells may begin autolysis or produce enzymes that degrade Levan, leading to product decomposition and thus reducing the yield. Therefore, this specific fermentation condition is a key combination of process parameters that achieves a synergistic balance between high yield and high efficiency, contributing to improved production efficiency and product yield.

[0046] In a particularly preferred embodiment, the fermentation medium comprises 120 g / L sucrose and 2 g / L yeast extract; and the fermentation conditions are: temperature 37°C, fermentation time 20 hours. This is the optimal combination of process parameters determined through extensive experimental optimization. Under these conditions, an optimal balance is achieved between the growth status of strain BS21, the activity and stability of L-glucan sucrase, the conversion efficiency of the substrate sucrose, and the accumulation rate of the target product.

[0047] The technical advantage of this specific combination lies not only in achieving high product yield and production efficiency, but more importantly, in its ability to directionally and controllably produce Levan-type oligofructose with the specific molecular weight and structure required by this invention. Experiments have demonstrated that, under these specific process conditions, the prepared product exhibits the specific physicochemical properties and biological activities verified in subsequent examples. This method of synergistically optimizing the culture medium components and fermentation conditions reflects the invention's ability to precisely control the production process, providing a technical guarantee for obtaining products with specific biological functions.

[0048] Accordingly, in a particularly preferred embodiment of the product, the Levan-type fructooligosaccharide has a weight-average molecular weight (Mw) of 7.2 kDa, and the Levan-type fructooligosaccharide is composed of at least 97% fructose. For example... Figure 4 As shown, the product prepared under optimal process conditions was analyzed by gel permeation chromatography (GPC). Both the light scattering (LS) and differential refractive index (RI) signals exhibited a relatively concentrated single peak. The calculated weight-average molecular weight (Mw) was precisely 7.237 kDa, the number-average molecular weight (Mn) was 4.524 kDa, and the polydispersity index (PDI) was 1.600. This indicates that the product has a relatively narrow molecular weight distribution, with the main components concentrated around 7.2 kDa.

[0049] Meanwhile, high-performance anion exchange chromatography-pulse amperometric detection (HPAEC-PAD) analysis revealed that the product consists of 97.04% fructose and 2.96% glucose, confirming it as a high-purity fructan. This precise molecular weight and extremely high purity are essential characteristics that distinguish this product from similar products with broad molecular weight distributions or containing more mixed sugars. It is this highly uniform and pure structure that lays the material foundation for its precise and efficient targeted biological functions in subsequent applications, solving the problem of unclear product structures and inconsistent functions in existing technologies.

[0050] In a preferred embodiment, the feed additive or feed composition is formulated for weaned piglets, and its formulation is suitable for application at 0.5% by weight in the diet, which can reduce diarrhea rate or feed conversion ratio. Weaning is the most stressful period in a piglet's life, easily leading to diarrhea and growth retardation, posing a significant challenge to the pig farming industry. This invention precisely focuses on weaned piglets, making it highly relevant to current applications.

[0051] like Figure 13 As shown, animal feeding trials verified the significant effect of this application scheme. In the trial, weaned piglets were fed a diet supplemented with 0.5% of the Levan-type fructooligosaccharides of this invention. The results showed that compared with the control group (CON) fed the basal diet, the diarrhea rate of the experimental group (BS21EPS) decreased significantly from 18.0% to 6.5%, a reduction of 63.8%. At the same time, the feed conversion ratio (F / G) also decreased significantly from 1.70 to 1.59, indicating an improvement in feed utilization efficiency of 6.4%. This significant improvement in both diarrhea rate and feed conversion ratio, achievable at a low dose (0.5%), provides clear and quantifiable efficacy support for the product.

[0052] Furthermore, to elucidate the underlying mechanism of the aforementioned application effects, in another preferred embodiment, the improvement of animal gut health includes enriching the animal's intestines with either *Lactobacillus johnsonii* or *Clostridium plasminogen lysate*. These two bacteria are generally considered beneficial to host health. *Lactobacillus johnsonii* can regulate immunity and inhibit pathogens, while *Clostridium plasminogen lysate* is a major producer of butyrate, a primary energy source for colonic epithelial cells and crucial for maintaining intestinal barrier function.

[0053] like Figure 18As shown, LEfSe differential species analysis of the colon contents of piglets using metagenomics clearly revealed the target of the Levan-type fructooligosaccharide of this invention. The figure shows that *Lactobacillus johnsonii* had the highest linear discriminant analysis (LDA) score in the experimental group (BS21EPS), indicating that it was the species with the most significant difference between the two groups, and its relative abundance was greatly enriched in the experimental group. Simultaneously, the abundance of *Faecalibacterium prausnitzii* also showed a significantly increased trend. This result strongly demonstrates that the Levan-type fructooligosaccharide with a specific structure provided by this invention does not indiscriminately promote the growth of all bacteria, but rather has high targeting, precisely promoting their proliferation and enriching core functional bacteria beneficial to host health. This in-depth revelation of the mechanism of action explains its macroscopic effects of reducing diarrhea and improving growth.

[0054] The technical solution of the present invention will be further illustrated below through two complete embodiments integrating the above-mentioned optimal technical features. These two embodiments aim to fully demonstrate the entire process of preparing Levan-type oligofructose with a specific structure from a specific strain through an optimized process, and successfully applying it to improve the health of weaned piglets, reflecting the synergistic effect between the various technical features.

[0055] Example 1: A process and structural characterization for producing Levan-type fructooligosaccharides using Bacillus subtilis. I. Experimental Objective This embodiment aims to provide a method for utilizing Bacillus subtilis. Bacillus subtilis The specific process for producing Levan oligosaccharides by fermentation with BS21 was described. By purifying and structurally identifying the fermentation products, a high-purity Levan-type oligosaccharide with a well-defined chemical structure was obtained, providing a material basis for subsequent research on its functional application as a feed additive.

[0056] II. Materials and Methods 1. Microbial strains and culture media Bacterial strain: The strain used in the experiment was Bacillus subtilis BS21 (the whole genome sequence of this strain has been deposited in the GenBank database of the National Center for Biotechnology Information in the United States, accession number PRJNA962810). This strain was isolated from the feces of healthy Bama miniature pigs and preserved in glycerol tubes at -20℃. Fermentation medium: sucrose 100 g / L, yeast extract 2 g / L, ammonium sulfate 3 g / L, potassium dihydrogen phosphate 1 g / L, magnesium sulfate heptahydrate 0.6 g / L, manganese sulfate 0.2 g / L. Adjust the initial pH of the medium to 7.0 with hydrochloric acid or sodium hydroxide solution before use.

[0057] 2. Fermentation process (1) Activation of bacterial strain: Take one loop of Bacillus subtilis BS21 from the glycerol tube and inoculate it into a 250 mL Erlenmeyer flask containing 50 mL LB liquid medium. Shake and culture at 37℃ and 150 rpm for 12 hours to obtain the seed culture.

[0058] (2) Fermentation culture: The activated seed culture was transferred to fresh fermentation medium at an inoculation rate of 10% (v / v). The inoculated conical flask was placed in a constant temperature shaker at 37℃ and cultured at a speed of 150 rpm for 20 hours.

[0059] 3. Product Harvesting and Primary Purification (1) Centrifugation: After fermentation, the fermentation broth was centrifuged at 4 ℃ and 6000 rpm for 20 minutes and the supernatant was collected.

[0060] (2) Solvent precipitation: Add 3 times the volume (v / v) of anhydrous ethanol to the supernatant, mix well and let stand at 10 °C for 24 hours.

[0061] (3) Collection and drying: The above mixture was centrifuged again at 4 ℃ and 6000 rpm for 20 minutes, and the precipitate was collected. The precipitate was washed twice with anhydrous ethanol and freeze-dried at room temperature to obtain crude Levan-type oligofructose.

[0062] 4. Fine purification and structural identification 4.1 Removal of impurities from crude polysaccharides (1) Sample pretreatment: In this experiment, the crude polysaccharide extract was purified, including removing protein, fat, and color, and then the purity of the crude polysaccharide was determined by the sulfuric acid-phenol method. The detailed steps are as follows: 600 mL of pure water was added to the crude polysaccharide extract solid to fully dissolve the crude polysaccharide. 0.4-0.6 g of papain and complex protease were added and enzymatically hydrolyzed overnight. 1 / 4 volume of chloroform and n-butanol (4:1, v / v) were added to the aqueous phase and mixed thoroughly. The upper aqueous phase was collected. 1 / 4 volume of petroleum ether was added to the liquid and mixed thoroughly. The lower aqueous phase was collected. 1 / 2 volume of macroporous resin AB-8 was added to the aqueous phase and mixed thoroughly. Adsorption was performed overnight. The liquid was collected and dialyzed in a 3,000 Da dialysis bag for 24-48 h to remove small molecule components. The polysaccharide solution was precipitated with alcohol and dried.

[0063] The purity of crude polysaccharide was determined by the sulfuric acid-phenol method. The specific procedure was as follows: Weigh about 25 mg of polysaccharide solid, dissolve and dilute it with water, take 100 μl of polysaccharide supernatant, add 600 μl of sulfuric acid-phenol reagent (5% phenol solution: concentrated sulfuric acid = 1:5 (v / v)), mix well, let it stand for 10 min in the dark, and measure the absorbance at 490 nm.

[0064] (2) Instrument parameters: Thermo Fisher Scientific (USA) multi-functional microplate reader, model Multiskan GO, was used for quantitative analysis of the target analytes. Corning (USA) microplates were used, and the absorbance difference between wells in parallel experiments at the target wavelength was required to be less than 0.006.

[0065] 4.2 Polysaccharide ion exchange purification (1) Sample pretreatment: Dissolve about 5 g of crude polysaccharide sample in 200 ml of pure water. Centrifuge at 10,000×g for 10 min, and purify the supernatant by passing it through an ion exchange column at a flow rate of 4 ml / min. Elute sequentially with pure water, 0.1 M, 0.2 M and 0.3 M NaCl solutions, collecting one tube for every 15 ml of eluent.

[0066] The total sugar content of the eluent in each collection tube was determined using the sulfuric acid-phenol method. The specific procedure was as follows: 100 μl of diluted polysaccharide supernatant was taken, and 600 μl of sulfuric acid-phenol reagent (5% phenol solution: concentrated sulfuric acid = 1:5 (v / v), the same below) was added. The mixture was stirred, allowed to stand in the dark for 10 min, and the absorbance was measured at 490 nm. An ion purification elution curve was plotted. The elution peak was selected and confirmed (the main peak was selected by default). The eluents from each collection tube corresponding to the same elution peak were combined and concentrated to 1 / 5 of the original volume by rotary evaporation. Dialysis was performed using a 3000 Da dialysis bag for 24-48 h to remove salts.

[0067] The purity of polysaccharides purified by gel permeation was determined using the sulfuric acid-phenol method. The specific procedure was as follows: approximately 25 mg of lyophilized polysaccharide sample was weighed, dissolved and diluted with water, and 100 μl of the polysaccharide supernatant was taken. The sulfuric acid-phenol reagent was added, and the mixture was reacted in the dark for 10 min. The absorbance was then measured at 490 nm.

[0068] (2) Instrument parameters: The AKTA purification system from the Life Sciences Division of GE Healthcare (China) was used. For ion purification, DEAE seplife FF weak anion exchange chromatography packing material from Xi'an Lanxiao Technology New Materials Co., Ltd. was selected. A Thermo Fisher Scientific (USA) multi-functional microplate reader (Multiskan GO) was used for quantitative analysis of the target analytes. Corning (USA) microplates were used, and the absorbance difference between wells in parallel experiments at the target wavelength was required to be less than 0.006.

[0069] 4.3 Polysaccharide purification by gel chromatography (1) Sample pretreatment: Take about 1 g of ion-purified polysaccharide sample and add it to 20 ml of pure water.

[0070] Centrifuge at 10,000 × g for 10 min, collect the supernatant and purify it using a gel chromatography column at a flow rate of 1 ml / min; elute with pure water at a rate of 1.5 column volumes, collecting one tube per 10 ml column, and collect all eluent. The total sugar content of the eluent in each collection tube is determined using the sulfuric acid-phenol method. Specifically, take 100 μl of the diluted polysaccharide supernatant, add 600 μl of sulfuric acid-phenol reagent (5% phenol solution: concentrated sulfuric acid = 1:5 (v / v), the same below), mix well, and react in the dark for 10 min. Measure the absorbance at 490 nm. Plot the gel purification elution curve and select the main elution peak. Combine the eluents from each collection tube corresponding to the same elution peak and concentrate them to 1 / 5 of the original volume by rotary evaporation. Dialyze using a 3000 Da dialysis bag for 24–48 h to remove small molecule components. Freeze-dry and identify the purity of the purified polysaccharide using the sulfuric acid-phenol method. The specific procedure is as follows: Weigh 2-5 mg of lyophilized polysaccharide sample, dissolve and dilute it with water, take 100 μl of polysaccharide supernatant, add sulfuric acid-phenol reagent, mix well, let it stand for 10 min in the dark, and measure the absorbance at 490 nm.

[0071] (2) Instrument parameters: The AKTA purification system from the Life Sciences Division of GE Healthcare (China) was used, and the Sephacryl S-400HR gel chromatography packing material from GE was selected for gel purification. Thermo Fisher Scientific (USA) multi-functional microplate reader (Multiskan GO) was used for quantitative analysis of the target analytes. Corning (USA) microplates were used, and the absorbance difference between wells in parallel experiments at the target wavelength was required to be less than 0.006.

[0072] 4.4 Determination of Polysaccharide and Monosaccharide Composition (1) Sample pretreatment: Take a clean chromatographic bottle, weigh an appropriate amount of polysaccharide sample, add 1 ml of 2M TFA acid solution, and heat at 60℃ for 1 hour. Purge with nitrogen and dry. Add 99.99% methanol to clean, then dry again, repeating the methanol cleaning 2-3 times. Add an appropriate amount of sterile water to dissolve, and transfer to a chromatographic bottle for testing. Take an appropriate amount of supernatant, concentrate by rotation or dry with nitrogen. Add 1 ml of 2M TFA solution, and heat at 60℃ for 1 hour. Purge with nitrogen and dry. Add 99.99% methanol to clean, then dry again, repeating the methanol cleaning 2-3 times. Add sterile water to dissolve, and transfer to a chromatographic bottle for testing. Perform direct instrumental analysis or pretreatment and instrumental analysis according to the customer-provided method.

[0073] (2) Instrument parameters: The chromatographic system used was a Thermo ICS 5000+ ion chromatography system (ICS 5000+, Thermo Fisher Scientific, USA), which used an electrochemical detector to analyze and detect monosaccharide components. A Dionex™ CarboPac™ PA20 (150*3.0 mm, 10 μm) liquid chromatography column was used; the injection volume was 5 μl. Mobile phase A (H2O), mobile phase B (0.1M NaOH), mobile phase C (0.1 M NaOH, 0.2M NaAc), flow rate 0.5 ml / min; column temperature 30℃; elution gradient: 0 min A / B / C (95:5:0, V / V), 26 min A / B / C (85:5:10, V / V), 42 min A / B / C (85:5:10, V / V), 42.1 min A / B / C (60:0:40, V / V), 52 min A / B / C (60:40:0, V / V), 52.1 min A / B / C (95:5:0, V / V), 60 min A / B / C (95:5:0, V / V).

[0074] 4.5 Determination of Polysaccharide Molecular Weight Distribution (1) Sample pretreatment: Dissolve the sample in 0.1 M NaNO3 aqueous solution (containing 0.02% NaN3, w / w) to a final concentration of 1 mg / mL, and filter it through a 0.45 μm filter before instrumental analysis. (The 0.02% NaN3 is used as an antibacterial agent.) (2) Instrument parameters: The chromatographic system used was a gel chromatography-differential-multi-angle laser light scattering system. The liquid chromatography system was a U3000 (Thermo, USA), the differential detector was an Optilab T-rEX (Wyatt technology, CA, USA), and the laser light scattering detector was a DAWN HELEOS Ⅱ (Wyatt technology, CA, USA). The specific chromatographic column and elution conditions were as follows: Ohpak SB-805 HQ (300 × 8 mm) and Ohpak SB-803 HQ (300 × 8 mm) gel size exclusion columns were used in series. The column temperature was 45 ℃, the injection volume was 100 μL, the mobile phase was A (0.02% NaN3, 0.1M NaNO3), the flow rate was 0.6 mL / min, and the elution gradient was isocratic for 75 min.

[0075] 4.6 Polysaccharide Methylation Determination Sample pretreatment: Dissolve a small amount of sample (2-3 mg) in 500 μL DMSO. Add 1 mg NaOH and incubate for 30 minutes. Add 50 μL iodomethane solution and react for 1 hour. Add 1 mL water and 2 mL dichloromethane, vortex to mix, centrifuge, and discard the aqueous phase. Repeat washing with water 3 times. Pipette the lower dichloromethane phase and dry under nitrogen. Add 100 μL 2 M TFA and react at 60 °C for 30 minutes. Evaporate to dryness at 30 °C. Add 50 μL 2 M ammonia and 50 μL 1 M NaBD4, mix well, and react at room temperature for 2.5 hours. Add 20 μL acetic acid to terminate the reaction, dry under nitrogen, wash twice with 250 μL methanol, and dry under nitrogen. Add 250 μL acetic anhydride, vortex to mix, and react at 100 °C for 2.5 hours. Add 1 mL water and let stand for 10 minutes. Add 500 μL of dichloromethane, vortex to mix, centrifuge, discard the aqueous phase, and wash with water three times. Take the lower dichloromethane phase and analyze it by GC-MS.

[0076] (2) Instrument parameters: The analytical instrument used in this experiment was an Agilent Technologies Inc. (USA) 6890A-5977B gas chromatography-mass spectrometry (GC-MS) system with an autosampler model of G4567A. The chromatographic system used was an Agilent gas chromatography system (6890A; Agilent Technologies, USA), and the chromatographic column was a TG-200MS (30 m × 0.25 mm × 0.25 µm; Thermo Fisher Scientific, USA). The injection volume was 1 μL, the split ratio was 10:1, the carrier gas was high-purity helium, and the flow rate was 1.5 mL / min. The initial temperature of the column oven was 150 ℃ for 1 minute, then increased to 210 ℃ at a program of 2 ℃ / min, held for 2 minutes, and then increased to 240 ℃ at a program of 2 ℃ / min, held for 3 minutes. The mass spectrometry system used was an Agilent quadrupole mass spectrometer (5977B; Agilent Technologies, USA), equipped with an electron impact ionization (EI) source and a MassHunter workstation. Using the EI source, analytes were detected in full scan mode with an ion source temperature of 200 °C, a quadrupole temperature of 110 °C, an ionization energy of 50 eV, a transfer line temperature of 210 °C, and a mass scan range (m / z) of 50–350.

[0077] 4.7 Polysaccharide NMR Scan Analysis (1) Sample pretreatment: Dissolve an appropriate amount of purified polysaccharide in D2O to prepare a polysaccharide solution with a concentration greater than or equal to 40 mg / mL. Transfer the dissolved solution to an NMR tube, adding 0.5 mL. Place the NMR tube in the NMR spectrometer to scan one-dimensionally. 1 H spectrum, 13 C spectrum, DEPT135, two-dimensional COSY, HSQC, TOCSY, NOESY and HMBC spectra.

[0078] (2) Instrument parameters: This project uses a Bruker (Germany) 500 MHz nuclear magnetic resonance spectrometer for quantitative analysis of the target analytes, with a scanning temperature of 25 ℃. Liquid probe QXI 1H / 31P / 13C / 15N 5 mm quad-resonance reverse detection probe (Z-gradient, ATM Acc); Technical parameters: Signal-to-noise ratio (1H): 888; Resolution (Hz): 0.32 (rotating) BBFO 1H-19F, 31P-15N, 1H decoupling / observe multi-nuclear forward detection probe (Z-gradient, ATM); Technical parameters: Signal-to-noise ratio (1H): 798; Resolution (Hz): 0.26 (rotating); Signal-to-noise ratio (13C): 328; Resolution (Hz): 0.1.

[0079] III. Test Results In this invention, the purity and monosaccharide composition analysis, molecular weight and distribution determination, and nuclear magnetic resonance spectral characterization and signal attribution of the product were all entrusted to Sanshu Biotechnology (Nantong, China) Co., Ltd. The specific measurement results are as follows.

[0080] 3.1 Results of polysaccharide impurity removal and ion / gel purification The extracted crude polysaccharide underwent multi-step purification, including impurity removal, ion exchange, and gel purification, as follows: Figure 2 As shown in Table 1, the entire process aimed to gradually improve the purity of the polysaccharide sample. The final product ZG-D1N1 achieved a purity of 83.7%, thus realizing the final purification goal.

[0081] Table 1. Efficiency and Product Analysis of Multi-Step Polysaccharide Purification Process 3.2 Purity and Monosaccharide Composition Analysis of the Product To verify the monosaccharide composition of the target product, the hydrolyzed sample was first analyzed by HPLC. Figure 3 By comparing the retention time with that of monosaccharide standards, it was qualitatively confirmed that the hydrolysis products of the sample contained only two monosaccharides, glucose (Glc) and fructose (Fru), thus excluding interference from other sugars.

[0082] 3.3 Determination of molecular weight and its distribution The molecular weight and distribution of the products were determined by gel permeation chromatography (GPC). Figure 4 The GPC elution curve showed a single and symmetrical peak, indicating good product homogeneity. Calculations showed that the number-average molecular weight (Mn) of this fructan was 4.524 kDa, the weight-average molecular weight (Mw) was 7.237 kDa, and the polydispersity index (PDI) was 1.600, indicating that it is a low-to-medium molecular weight polysaccharide with a narrow molecular weight distribution. Notably, the slope of the double logarithmic curve of the root mean square (RMS) radius versus molar mass was 0.36. Figure 5 As shown, the morphology of BS21EPS polysaccharide is close to spherical.

[0083] 3.4 BS21EPS Methylation Analysis Results Based on this, the purified fructan components were quantitatively analyzed: Figure 6 The methylation product analysis showed that the signal intensities of the fructose-related peaks (6-Fru (f), t-Fru (f), 1,6-Fru (f)) were significantly higher than those of the glucose peak (t-Glc (p)). Based on peak area integration and external standard curve calculations using the chromatographic workstation, the monosaccharide composition of this component was 97.04% fructose and 2.96% glucose. This result indicates that the sample is a high-purity fructan containing only a small amount of glucose terminal residues.

[0084] 3.5 Nuclear magnetic resonance spectral characterization and signal assignment of fructan To resolve the fine structure of fructan, a one-dimensional (e.g., Figure 7 and Figure 8 (as shown) and two-dimensional (as shown) Figure 9 , Figure 10 and Figure 11 (As shown) NMR analysis. The 1H NMR spectrum of the sample showed no significant absorption peaks in the anomeric hydrogen region (δ 4.4–5.3 ppm, where δ 4.71 ppm is the HOD solvent peak), while the non-anomeric hydrogen signals associated with the sugar ring protons were mainly concentrated in the δ 3.1–4.3 ppm region. The 13C NMR spectrum identified three main signals in the anomeric carbon region, with chemical shifts of δ 104.17, 103.77, and 104.08 ppm, respectively, denoted as sugar residues A, B, and C.

[0085] By analyzing two-dimensional spectra such as COSY and HSQC, the 1H and 13C signals of each sugar residue were systematically assigned. Taking sugar residue A as an example, the chemical shifts of H3-H6 were determined to be 4.10, 4.02, 3.87, and 3.81 (3.48) ppm by continuous correlation peaks in the COSY spectrum; the corresponding C3-C6 chemical shifts were determined to be 76.26, 75.17, 80.26, and 63.36 ppm by the HSQC spectrum. The complete chemical shift assignment results of each sugar residue are shown in Table 2.

[0086] Combining the results of one-dimensional and two-dimensional NMR analyses, the fine structure of this fructan can be determined: its main chain consists of repeated linkages of →6)-β-D-Fruf-(2→ units (sugar residue A), with a branch consisting of →1,6)-β-D-Fruf-(2→ units (sugar residue C) attached at position C6 of the main chain, and the end is a β-D-Fruf-(2→ unit (sugar residue B). The complete structure is shown below. Figure 12 As shown.

[0087] Table 2 Sugar residues 1 H and 13 Chemical shift of C IV. Results Analysis This study successfully obtained high-purity, structurally defined fructans (Levan-type oligofructoses) by establishing a systematic fermentation process and through separation, purification, and structural characterization. The key characteristics of the product were also analyzed in depth, laying a solid material foundation for subsequent applications.

[0088] 4.1 Stability and deterministic analysis of fermentation and purification processes The established process route in this study exhibited good stability and reproducibility. The fermentation stage was carried out under well-defined parameters (37 °C, 150 rpm, 20 h), providing a stable environment for fructan biosynthesis. In downstream processing, the purification strategy of ethanol precipitation combined with ion exchange-gel filtration chromatography proved to be highly efficient and feasible.

[0089] This method not only effectively separates fructans from impurities such as proteins and pigments, but also produces a final product with a narrow polydispersity index (PDI = 1.600), indicating that this purification route can yield products with high molecular weight uniformity. This result demonstrates the strong determinism of this process and its potential for large-scale production.

[0090] 4.2 Chemical Structure Identification Through isolation, purification, and structural characterization of the fermented polysaccharide, the complete structure of the fructan was finally determined as follows: the main chain consists of repeated linkages of →6)-β-D-Fruf-(2→ units, with a branch consisting of →1,6)-β-D-Fruf-(2→ units connected at the C6 position of the main chain, and the end is a β-D-Fruf-(2→ unit. A schematic diagram of its structural fragment is shown below. Figure 10 As shown.

[0091] 4.3 Correlation between product quality and structural characteristics All analytical results point to the same conclusion: the fructan prepared by this process has high purity (97.04% fructose monosaccharide) and a well-defined primary structure.

[0092] A successful purification process is a prerequisite for obtaining high-quality structural data, while clear structural characterization results, in turn, prove the effectiveness of the purification process. This high degree of consistency between "process-structure-quality" indicates that this study not only achieved the preparation of Levan-type fructooligosaccharides, but also established a standardized process for precise quality control and evaluation.

[0093] Example 2: Effects of Levan-type fructooligosaccharides on growth performance, gut health, and microbiota of weaned piglets I. Experimental Objective This embodiment aims to verify the application effect of Levan-type fructooligosaccharides produced by fermentation of Bacillus subtilis BS21 as a feed additive through animal experiments.

[0094] II. Materials and Methods 2.1 Experimental Animals and Experimental Design Ninety-six healthy, 28-day-old Duroc × Landrace × Large White crossbred weaned piglets of similar weight (8.0 ± 0.3 kg) were selected and randomly divided into two treatment groups using a completely randomized block design. The control group (CON) was fed a corn-soybean meal basal diet, while the fructan group (BS21EPS) received 0.5% (w / w) of Levan-type crude polysaccharide prepared by fermentation of Bacillus subtilis BS21 (preparation method see Example 1) in addition to the basal diet. Each group had 6 replicates (pens), with 8 piglets per pen, for a total of 96 piglets, and the experimental period was 28 days.

[0095] 2.2 Experimental Diets and Feeding Management Diet design: Two groups of piglets were fed a control diet and an experimental diet, respectively. The experimental diet was formulated by replacing part of the corn with 0.5% Levan-type fructan to ensure that the two diets were basically consistent in terms of major nutrients. The specific diet composition and nutrient levels are shown in Table 3.

[0096] Feeding and management: During the 28-day trial period, all piglets had free access to feed and water, and were immunized and managed according to standard procedures.

[0097] Table 3. Composition and nutrient levels of the experimental diet (%, feeding basal level) Note: 1 The premixed feed composition per kilogram of feed is as follows: Vitamin A (calculated as vitamin A acetate) 12,000 IU; Vitamin D (calculated as vitamin D3) 2,500 IU; Vitamin E (calculated as DL-α-tocopherol acetate) 30 IU; Vitamin B... 12 12 micrograms (μg); Vitamin K (as sodium menadione bisulfite) 3 milligrams (mg); D-pantothenic acid (as calcium pantothenate) 15 mg; Niacin 40 mg; Choline (as choline chloride) 400 mg; Manganese (as manganese oxide) 30 mg; Iron (as ferric sulfate) 90 mg; Copper (as copper sulfate) 10 mg; Iodine (as ethylenediamine dihydroiodide) 0.35 mg; Selenium (as sodium selenite) 0.3 mg.

[0098] 2.3 Measurement Indicators and Methods To systematically evaluate the comprehensive effects of Levan-type fructooligosaccharides on the growth performance, nutrient utilization, and overall health of weaned piglets, this study established and measured the following core indicators. All measurements were performed in accordance with standard operating procedures to ensure the accuracy and comparability of the data.

[0099] 2.3.1 Growth performance indicators Measurement indicators: average daily gain (ADG, g / d), average daily feed intake (ADFI, g / d), and feed conversion ratio (F / G).

[0100] Measurement periods: Data were collected for 0-14 days, 15-28 days and the entire period of 0-28 days.

[0101] Measurement method: Piglets were weighed on an empty stomach in the early morning at the beginning and end of each stage (days 0, 14, and 28). The daily feed input and output of each repeat pen were accurately recorded and calculated.

[0102] 2.3.2 Diarrhea rate Measurement indicator: Diarrhea rate (%).

[0103] Measurement periods: The incidence of diarrhea was recorded for 0-14 days, 15-28 days and the entire period of 0-28 days.

[0104] Measurement method: Diarrhea rate (%) = [Total number of diarrhea episodes / (Total number of piglets × Number of test days)] × 100%.

[0105] 2.3.3 Apparent Total Intestinal Digestibility (ATTD) Measurement indicators: apparent intestinal digestibility of total energy (GE), dry matter (DM), organic matter (OM), crude protein (CP), neutral detergent fiber (NDF), and acid detergent fiber (ADF).

[0106] Sample source: Fresh fecal samples collected at the end of the experiment were mixed and prepared.

[0107] Determination methods: The endogenous indicator method (chromium trioxide) was used. The content of each nutrient in the diet and feces was determined according to the AOAC (2006) standard method; the total energy was determined using an oxygen bomb calorimeter; and the chromium content was determined using atomic absorption spectrometry.

[0108] The formula for calculating digestibility is: Nutrient ATTD (%) = [1 - (Cr2O3 content in diet / Cr2O3 content in feces) × (Nutrient content in feces / Nutrient content in diet)] × 100%.

[0109] 2.3.4 Serum biochemical, antioxidant, and immune indicators Sample source: Serum separated from blood collected from the vena cava on day 28 of the experiment.

[0110] Measurement indicators and methods: The following indicators were measured using a commercially available ELISA kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) by spectrophotometry or enzyme-linked immunosorbent assay (ELISA): Antioxidant indicators: catalase (CAT) activity, glutathione peroxidase (GSH-Px) activity, malondialdehyde (MDA) content, superoxide dismutase (SOD) activity, and total antioxidant capacity (T-AOC).

[0111] Concentrations of inflammatory factors: C-reactive protein (CRP), interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α).

[0112] 2.3.5 Colonic microbiota analysis All genomic DNA was extracted from fermentation samples and amplified by PCR (ABI GeneAmp®, 9700; fuses: 338F and 806R), and quantified using qubit 4.0 (Thermo Fisher Scientific, USA). Uniform amplicon was polymerized in equimolar quantities and paired for sequencing on an Illumina PE300 platform (Illumina San Diego, USA) according to the standard protocol of Majorbio Biopharmaceuticals Ltd. (Shanghai, China). Microbiome bioinformatics was performed, and microbial ecology was studied with quantitative insights (QIIME2). Species annotation was performed in the Silva database using QIIME2 software. Metagenomic functions were predicted by PICRUSt2 (a phylogenetic survey of unobserved communities) based on representative ASV sequences. Bioinformatics analysis of the gut microbiota was performed using the Majorbio Cloud platform (https: / / cloud.majorbio.com). Microbial community similarity across different samples was determined by principal coordinate analysis (PCoA) based on Bray-curtis dissimilarity using the Vegan v2.5-3 software package.

[0113] 2.3.6 Antioxidant and immune indicators of colon tissue Sample source: Supernatant of colon tissue homogenate collected at the end of the experiment.

[0114] Measurement indicators and methods: Corresponding to serum indicators, the same commercially available kits and measurement methods were used to analyze the levels of CAT, GSH-Px, MDA, SOD, T-AOC, CRP, IL-1β, IL-6, and TNF-α in colonic contents or tissues to assess local oxidative stress and inflammatory status in the intestine.

[0115] 2.3.7 Statistical Analysis This invention employs standardized statistical methods in data analysis: the experimental design follows the principle of randomization and a control group is established to ensure fair comparison; all continuous data undergo normality and homogeneity of variance tests, and those meeting the criteria are expressed as "mean ± standard error (Mean ± SEM)"; independent samples t-tests are used for comparisons between two groups, while one-way ANOVA and Duncan's post-hoc test are used for comparisons between multiple groups; if the data do not meet the parametric test criteria, non-parametric methods such as the Mann-Whitney U test are used; all statistical analyses are performed using SPSS professional software, and P < 0.05 is used as the criterion for judging statistical significance.

[0116] III. Test Results 3.1 Growth performance and diarrhea rate indicators As shown in Table 4, compared with the control group, the BS21EPS group significantly improved feed utilization efficiency (P<0.05) and significantly reduced diarrhea rate (P = 0.01) while maintaining the same growth rate and feed intake (no significant difference in average daily weight gain and average daily feed intake).

[0117] Table 4 Growth performance and diarrhea rate 3.2 Total intestinal digestibility of nutrients Adding BS21EPS to the diet did not improve the apparent digestibility of nutrients. P > 0.05 (as shown in Table 5).

[0118] Table 5 Apparent intestinal digestibility of each nutrient 3.3 Serum biochemical, antioxidant and immune indicators Dietary supplementation with BS21EPS significantly reduced the level of pro-inflammatory responses in the body, specifically manifested in a highly significant decrease in the concentrations of key pro-inflammatory cytokines (IL-1β, TNF-α) in serum. P <0.05). Meanwhile, this treatment had no significant effect on the body's overall antioxidant status and some immune indicators (such as the anti-inflammatory factor IL-10) (as shown in Table 6).

[0119] Table 6 Antioxidant and immune indicators in serum 3.4 Analysis of the structural impact of BS21CEPS on the gut microbiome ecosystem 3.4.1 Analysis of α-diversity and β-diversity 3.4.1.1 Alpha Diversity Analysis Alpha diversity reflects the richness and evenness of the microbial community within a single sample, and the results are as follows: Shannon index, such as Figure 13 As shown: The Shannon index of the CON group was significantly higher than that of the BS21CEPS group ( P A value <0.05 indicates that the control group samples have a richer variety of microbial species and a more even distribution of species. The Simpson index, such as... Figure 13 As shown: The Simpson index of the BS21CEPS group shows an upward trend ( P = 0.092), which is close to the significance level, indicating that there is a significant trend in the evenness of the microbial community between the two groups.

[0120] 3.4.1.2 β-diversity analysis β-diversity reflects the degree of difference in microbial community structure among different samples, and the results are as follows: PCoA analysis, such as Figure 14 As shown: Principal coordinate analysis at the species level revealed a clear separation trend between the CON group (blue) and the BS21CEPS group (pink) (R = 0.341, P = 0.016), indicating a significant difference in the microbial community structure between the two groups. Bray-Curtis analysis of variance, as shown... Figure 14 As shown, the statistical test results showed P=0.016, further verifying that the difference in the composition of the two groups of microbial communities was statistically significant; the difference between the two groups was larger in the BS21CEPS group, suggesting that the treatment had a more significant impact on the community structure.

[0121] 3.4.2 Structural remodeling of the core gut bacterial community and specific enrichment of functional bacteria Analysis of fecal metagenomics at the species level in experimental animals revealed that BS21CEPS treatment specifically remodeled the gut microbiota structure, such as... Figure 15 As shown, under the premise of maintaining overall stability of the gut microbiota, the abundance of core probiotic strains and functional strains in the intestines of the treated animals underwent significant structural adjustments. Compared with the control group, the abundance of *Lactobacillus johnsonii* (L. johnsonii) in the BS21CEPS treatment group was significantly higher. Lactobacillus_johnsonii Clostridium plasminoides ( ) Faecalibacterium_prausnitzii ), Strychnos nucifera ( Lachnospira _sp) and Argas ( Agathobacter_ The relative abundance of *Sp.* increased significantly or extremely significantly; while the relative abundance of *Oscillatoria* decreased significantly. The abundance of most other genera, including *Lactobacillus reuteri*, did not change significantly.

[0122] The above data confirms that BS21CEPS can precisely and significantly increase the abundance of beneficial functional bacteria, represented by Lactobacillus johnsonii and Clostridium plasmidonii, and inhibit specific bacterial genera without disrupting the overall stability of the gut microbiota, thereby optimizing the microbiota structure and exerting biological functions to improve gut health.

[0123] 3.4.3 Analysis of the top 20 differentially expressed species based on a bubble chart of taxonomic indicators The most core and significant effect of BS21CEPS treatment is the highly specific enrichment of Lactobacillus johnsonii (Lactobacillus). Lactobacillus_johnsonii ), and accompanied by a tailed bacteriophage ( Caudoviricetes_ The relative decrease in abundance of sp., while the impact on the abundance of most other species did not reach a statistically significant level, such as Figure 16 As shown in the figure, this further clarifies that its role in regulating the gut microbiota is highly specific, primarily targeting the proliferation of beneficial bacteria.

[0124] 3.4.4 Comparative Analysis of Gut Microbiota Structure Between Two Groups of Samples Based on Circular Chord Diagrams like Figure 17 As shown in the microbial composition chord diagram, BS21CEPS treatment significantly modulates the species composition of the microbial community. Compared with the control group, the treated group showed a significantly higher species composition. Lactobacillus_johnsoni i Ruminococcus The relative abundance of species such as _sp. increased significantly, while Clostridium The decrease in the abundance of species such as _sp. indicates that BS21CEPS has a directional regulatory effect on the target bacterial community and can be used as an effective means to regulate the microbial community.

[0125] 3.4.5 Identification of Microbial Species with Significant Differences Between the Two Sample Groups like Figure 18 Linear discriminant analysis (LDA) was used to compare the microbial communities of the experimental group and the control group treated with BS21CEPS. The results showed that there was a statistically significant difference in species abundance between the two groups. In the BS21CEPS treatment group... Lactobacillus johnsonii , Parabacteroides sp. AM44_16、 Acinetobacter radioresistens Species such as [list of species] showed significant enrichment; while in the control group, [list of species] Phascolarctobacterium succinatutens, Bacteroides sp .、Clostridioides difficile Species such as [list of species] are dominant. These differences confirm that BS21CEPS can directionally regulate the microbial community structure, significantly promoting the proliferation of beneficial bacteria and inhibiting the growth of potentially harmful bacteria, providing crucial experimental evidence for its application in the field of probiotic regulation.

[0126] 3.5 Antioxidant and immune indicators of colon tissue BS21CEPS treatment did not significantly alter the activity of antioxidant enzymes or the levels of oxidative damage markers in colonic tissue. However, at the colonic tissue level, the core effect of Leven treatment was a significant downregulation of pro-inflammatory responses (IL-1β, TNF-α). P < 0.05), and consequently reduce the body's compensatory anti-inflammatory response (IL-10) ( P < 0.05), thereby adjusting the overall immune status of the colon to a new homeostasis with a lower level of inflammation (as shown in Table 7).

[0127] Table 7. Antioxidant and inflammatory factor indices in colon tissue IV. Results Analysis 4.1 Growth performance and nutrient digestion efficiency In terms of growth performance, the BS21CEPS diet significantly improved feed utilization efficiency and significantly reduced diarrhea rate without changing the average daily weight gain and average daily feed intake of the experimental animals, demonstrating a positive impact on animal production performance.

[0128] 4.2 Immune and Inflammatory Regulatory Effects The regulatory effect of dietary supplementation with BS21CEPS on the body's inflammatory response was clear and significant: the concentrations of key pro-inflammatory cytokines IL-1β and TNF-α in serum and colon tissue were significantly reduced, directly demonstrating its potent anti-inflammatory activity. Notably, the anti-inflammatory factor IL-10 in colon tissue also showed a simultaneous downward trend. This result suggests that BS21CEPS does not simply promote the anti-inflammatory response, but rather adjusts the overall colonic immune status to a new homeostasis of low inflammation by strongly inhibiting pro-inflammatory signaling, thus avoiding damage to intestinal tissues from excessive inflammatory responses. Furthermore, this treatment had no significant effect on overall antioxidant status or some immune indicators, further demonstrating its highly targeted effect, primarily focusing on the regulation of inflammatory pathways.

[0129] 4.3 Directed remodeling of the gut microbiota BS21CEPS treatment exhibits high specificity and precision in regulating the gut microbiota: Specific enrichment of core functional bacteria: After treatment, *Lactobacillus johnsonii* (… Lactobacillus johnsonii Clostridium plasminoides ( ) Faecalibacterium prausnitzii The abundance of probiotics and short-chain fatty acid-producing bacteria, represented by [specific strain name], was significantly or extremely significantly increased. These strains play a key role in maintaining the intestinal barrier, inhibiting pathogens, and regulating immunity, and are the core functional flora for improving intestinal health.

[0130] Inhibition of potentially harmful bacteria: *Vibrio spp.*, *Clostridium difficile* ( Clostridioides difficile The abundance of bacterial genera such as ( ) associated with intestinal inflammation or disease was significantly reduced, further optimizing the bacterial community structure.

[0131] Maintenance of overall community stability: While the core functional bacteria underwent significant changes, the overall structure of the gut microbiota remained stable, and the abundance of most genera was not significantly affected, indicating that the regulatory effect of BS21CEPS is directional and mild, and does not disrupt the ecological balance of gut microbiota.

[0132] LDA differential species analysis further confirmed that BS21CEPS can directionally promote the proliferation of beneficial bacteria and inhibit potentially harmful bacteria. This precise regulatory mode provides key experimental support for its application as a probiotic regulator.

[0133] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for preparing Levan-type oligofructose, characterized in that, Includes the following steps: Bacillus subtilis BS21 was fermented in a fermentation medium containing sucrose. Levan-type fructooligosaccharides were isolated from the fermentation products.

2. The method according to claim 1, characterized in that, The Bacillus subtilis BS21 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 25977.

3. The method according to claim 1 or 2, characterized in that, The fermentation medium contains 100-120 g / L of sucrose and 2-5 g / L of yeast extract.

4. The method according to claim 1 or 2, characterized in that, The fermentation conditions are: temperature 36-38℃, fermentation time 18-24 hours.

5. The method according to claim 1 or 2, characterized in that, The fermentation medium contains 120 g / L sucrose and 2 g / L yeast extract; and the fermentation conditions are a temperature of 37°C and a fermentation time of 20 hours.

6. A Levan-type fructooligosaccharide obtained by the method for preparing Levan-type fructooligosaccharides as described in any one of claims 1-5, characterized in that, The Levan-type fructooligosaccharide has a weight-average molecular weight (Mw) of 6-8 kDa and contains at least 95% fructose, wherein the fructose units constituting the fructose are linked by β-(2, 6)-glycosidic bonds.

7. The Levan-type fructooligosaccharide according to claim 6, characterized in that, The weight-average molecular weight Mw is 7.2 kDa, and the Levan-type fructooligosaccharide is composed of at least 97% fructose.

8. The use of the Levan-type fructooligosaccharide according to any one of claims 6 or 7 in the preparation of feed additives or feed compositions for improving animal gut health or enhancing animal growth performance.

9. The application according to claim 8, characterized in that, The feed additive or feed composition is formulated for weaned piglets, and its formulation is suitable for application at 0.5% by weight in the diet to reduce diarrhea rate or feed conversion ratio.

10. The application according to claim 8, characterized in that, The improvement of animal gut health includes enriching the gut of the animal with Lactobacillus johnsonii or Clostridium plasmidii.

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