A non-sugar breast milk oligosaccharide complex prebiotic and methods of making and using same

Abstract

This invention, entitled "A Sugar-Free Human Milk Oligosaccharide Complex Prebiotic, Its Preparation Method, and Its Application," belongs to the field of human milk oligosaccharide production technology. The technical problems to be solved are: resource utilization of by-products, improvement of product resistance to digestion, and regulation of intestinal flora. The key technical points are: preliminary fermentation of recombinant *E. coli*; and based on OD... 600 The fermentation process involves first, second, third, and fourth feedings. When the concentration of human milk oligosaccharides in the fermentation broth no longer increases, fermentation is terminated to obtain the fermentation broth. The fermentation broth is then sterilized, separated from its solid state, purified, concentrated, and dried to obtain the final product. The first to fourth feedings involve adding feeding medium one and feeding medium two, which respectively contain prebiotic product extract one and prebiotic product extract two.

Description

Technical Field

[0001] This invention provides a sugar-free human milk oligosaccharide complex prebiotic, its preparation method, and its application, belonging to the field of human milk oligosaccharide production technology. Background Technology

[0002] Human milk oligosaccharides (HMOs) play an important role in the gut health of infants and young children, among which 2'-fucosylated lactose (2'-FL) is the main active ingredient. Currently, the post-processing of 2'-FL production by microbial fermentation mainly uses chromatographic separation and crystallization to obtain high-purity single components. However, this process suffers from problems such as expensive equipment, low yield, and ineffective utilization of byproducts (such as DFL), resulting in resource waste and high costs.

[0003] On the other hand, a large amount of residue (mainly composed of glucose, galactose, lactose and galactooligosaccharides) is generated during the production of prebiotics. Currently, it is mostly treated as a low-priced by-product or waste liquid and has not been utilized for high-value purposes.

[0004] In addition, most existing prebiotic compositions are made by direct mixing, which can easily lead to uneven mixing and affect the effectiveness of the product.

[0005] Relevant patent documents retrieved: The document, published in China (CN114480240A) on May 13, 2022, discloses a genetically engineered bacterium for producing fucoidosyl lactose and a production method thereof. The method uses glycerol as a carbon source and 0.1-1.0 mM IPTG as an inducer to produce fucoidosyl lactose through fermentation in the presence of lactose. The concentration of glycerol in the fermentation system is 10-30 g / L.

[0006] Relevant non-patent literature retrieved: The journal or book title is *Dairy Science and Technology*, and the document title is "Research Progress on Patent Technology for the Biosynthesis of 2'-fucosylated lactose," Volume 44, No. 5, 2021, published in 2021. This document discloses that the primary condition for producing 2'-fucosylated lactose via biofermentation is the construction of a safe and efficient production strain. Existing technologies related to the synthesis of 2'-FL largely revolve around the construction of recombinant bacteria, optimizing metabolic pathways, overexpressing genes of enzymes involved in 2'-FL synthesis, and searching for safer and more efficient host bacteria. However, there is no improvement on industrial-scale preparation methods.

[0007] The prior art represented by the aforementioned documents has at least the following unresolved technical problems or defects: The utilization rate of fermentation raw materials is low, and the residual liquid generated from prebiotic production is not recycled. The by-product waste liquid of enterprises is only treated at low price or directly discharged, resulting in waste of raw materials and increased production costs. The relevant evidence is that patent CN114480240A only uses conventional glycerol and lactose as fermentation carbon sources and does not involve industrial residual liquid reuse technology.

[0008] The fermentation control method is simplistic, employing only a single IPTG induction and conventional feeding strategy, without considering the OD of the fermentation broth. 600 The changes were precisely controlled in stages, but the overall product yield was low. At the same time, the fermentation byproduct DFL could not be effectively recovered and utilized. The relevant evidence is that the journal article "Research Progress on Patent Technology for Biosynthesis of 2'-fucosylated lactose" shows that the existing 2'-FL fermentation technology focuses on strain modification, and there are obvious shortcomings in the industrial fermentation control process, making it difficult to take into account the comprehensive utilization of the main product and byproduct.

[0009] The product has a high sugar content. The relevant evidence is that conventionally fermented and compounded human milk oligosaccharide prebiotics generally have a sugar content higher than 0.5%, which does not meet the standards for sugar-free products. Summary of the Invention

[0010] The purpose of this invention is to provide: A sugar-free human milk oligosaccharide complex prebiotic, its preparation method and application, and related technologies, to solve technical problems such as resource utilization of by-products, improving product resistance to digestion, and regulating intestinal flora, or combinations thereof.

[0011] Terminology Explanation: Unless otherwise defined, all technical terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter pertains. Unless otherwise stated, all patents, patent inventions, and publications cited throughout this document are incorporated herein by reference in their entirety. Where multiple definitions exist for terms, the definitions provided in this chapter shall prevail.

[0012] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0013] The definition of the standard chemical term can be found in the reference "Modern Industrial Fermentation Regulation", Chu Ju.

[0014] Unless otherwise stated, conventional methods within the scope of the art, such as sugar content detection, shall be used.

[0015] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0016] The terms “optional / arbitrary” or “optionally / arbitrarily” mean that the event or situation described below may or may not occur, including both the occurrence and non-occurrence of the event or situation.

[0017] The “2'-FL” mentioned in this article refers to 2'-fucosylated lactose.

[0018] The “DFL” mentioned in this article refers to lacto-N-difucotetrasaccharide.

[0019] In this article, "GOS" refers to galactooligosaccharides.

[0020] The "sugar content" mentioned in this article refers to the sum of fructose, glucose, sucrose, maltose, and lactose as specified in the "GB28050-2025 National Food Safety Standard for General Rules for Nutrition Labelling of Prepackaged Foods".

[0021] The term "sugar-free" as used in this article refers to the following: in accordance with the provisions of GB28050-2025 National Food Safety Standard for General Rules for Nutrition Labelling of Prepackaged Foods, the "0" threshold is defined as ≤0.5g of sugar content per 100g of product.

[0022] The phrase "concentrated to sugar concentration...% (m / m)" in this article refers to: concentrating to a specified value for the total content of 2'-FL, DFL, and GOS in the liquid.

[0023] The terms "% (m / m)" and "%m / m" used in this article refer to: percentage content by mass.

[0024] The terms "% (m / v)" and "%m / v" used in this article refer to the volume percentage of the mass. For example, x%m / v means that each 100mL of solution contains xg of the substance.

[0025] The terms "‰(m / v)" and "‰m / v" used in this article refer to the volume fraction of mass, for example, x‰m / v means that each 1L of solution contains xg of the substance.

[0026] The terms "% (v / v)" and "%v / v" used in this article refer to volume percentage content.

[0027] The term "ion exchange desalination" as used in this article refers to ion exchange desalination.

[0028] In a first aspect, the present invention provides: a method for preparing a sugar-free human milk oligosaccharide complex prebiotic, comprising the following steps: (1) Preliminary fermentation was carried out using Escherichia coli; (2) When the OD of the fermentation broth 600 When the level reaches 10-15, perform the first feeding. (3) When the OD of the fermentation broth 600 When the level reaches 50-60, perform a second feeding. (4) When the OD of the fermentation broth 600 When the concentration reaches 80-100, perform the third feeding. (5) When the OD of the fermentation broth 600 When the concentration reaches 130-150, perform the fourth feeding. (6) When the concentration of human milk oligosaccharides in the fermentation broth no longer increases, terminate the fermentation to obtain the fermentation broth; (7) The fermentation broth is sterilized, separated from solids and liquids, purified, concentrated and dried to obtain the final product; The first feeding consisted of adding feeding medium one and feeding medium two to maintain a glucose concentration of 10-15 g / L and a lactose concentration of 3-6 g / L. The second feeding involves adding feeding medium one and feeding medium two to maintain a glucose concentration of 10-15 g / L and a lactose concentration of 5-7 g / L. The third feeding involves adding feeding medium one and feeding medium two to maintain a glucose concentration of 5-10 g / L and a lactose concentration of 6-8 g / L. The fourth feeding involves adding feeding medium one and feeding medium two to maintain a glucose concentration of 3-6 g / L and a lactose concentration of 5-7 g / L. The supplemental culture medium consists of: 20-24% magnesium sulfate (m / v), 5-7% yeast extract (m / v), 40-55% prebiotic product extract (m / v), and 2.5-3.5% porcine bone peptone (m / v), with a pH of 6.8-7.0. The prebiotic product residue, on a dry basis, comprises: 40-50% glucose, 7-12% galactose, 20-30% lactose, and 15-20% galacto-oligosaccharides. The supplemental culture medium II comprises: 3-5% (m / v) of compound amino acid powder, 95-99% (m / v) of prebiotic product extract II, and 0.5-0.7% (m / v) of trace metal liquid. The prebiotic product residue solution II, by mass fraction on a dry basis, comprises: 37-46% glucose, 2-6% galactose, 37-46% lactose, and 5-12% galactooligosaccharides.

[0029] In some implementations, the Escherichia coli is a recombinant Escherichia coli.

[0030] Preferably, the recombinant Escherichia coli is: Escherichia coli BLCY-019, classified name: Escherichia coli BLCY-019, accession number: CCTCC NO: M2026303, accession date: January 30, 2026, depositary institution: China Center for Type Culture Collection, accession address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0031] In some implementations, the initial fermentation is carried out at a fermentation temperature of 37°C, a pH of 6.8-7.2, and an aeration ratio of 0.8-1.0 vvm.

[0032] Preferably, the culture medium for the initial fermentation is: potassium dihydrogen phosphate 5-8‰ (m / v), magnesium sulfate 1-2‰ (m / v), diammonium hydrogen phosphate 2-3‰ (m / v), citric acid 1-2‰ (m / v), yeast powder 5-6‰ (m / v), porcine bone peptone 2-3‰ (m / v), glycerol 3-4% (m / v), trace metal solution 0.8-1.0% (m / v), with the balance being water, and the pH is adjusted to 6.8-7.2.

[0033] Preferably, the trace metal solution comprises: Na2EDTA 1.2-1.5‰m / v, zinc sulfate 0.3-0.5‰m / v, manganese chloride 0.2-0.3‰m / v, cobalt chloride 0.04-0.06‰m / v, copper sulfate 0.01-0.03‰m / v, sodium molybdate 0.03-0.05‰m / v, calcium chloride 0.2-0.3‰m / v, ferrous sulfate 0.1-0.3‰m / v, boric acid 0.05-0.1‰m / v, and potassium iodide 0.01-0.05‰m / v.

[0034] More preferably, the zinc sulfate is zinc sulfate heptahydrate (ZnSO4). The manganese chloride is manganese chloride dihydrate (MnCl2·2H2O), and the cobalt chloride is cobalt chloride hexahydrate (CoCl2). The copper sulfate in question is copper sulfate pentahydrate (CuSO4) (6H2O). 5H2O), wherein the sodium molybdate is sodium molybdate dihydrate (Na2MoO4). 2H2O), the calcium chloride is calcium chloride dihydrate (CaCl2) 2H2O), the ferrous sulfate is ferrous sulfate heptahydrate (FeSO4). 7H2O).

[0035] In some implementations, the first feeding also includes: adjusting the temperature to 22-25°C, adding IPTG to a final concentration of 0.1-0.2 mM, and adjusting the ventilation ratio to 1-1.2 vvm.

[0036] In some implementations, the second feeding further includes adding IPTG to a final concentration of 0.08-0.1 mM and adjusting the ventilation ratio to 1.2-1.5 vvm.

[0037] In some implementations, the third feeding also includes adding IPTG to a final concentration of 0.08-0.1 mM and adjusting the ventilation ratio to 1.3-1.5 vvm.

[0038] In some implementations, the fourth feeding also includes adding IPTG to a final concentration of 0.08-0.1 mM and adjusting the ventilation ratio to 1.3-1.5 vvm.

[0039] In some implementations, the total prebiotic content in the fermentation broth is 185-220 g / L.

[0040] Preferably, the total prebiotics include: 140-160 g / L of 2'-FL, 10-15 g / L of DFL, and 30-50 g / L of galactooligosaccharides.

[0041] In some implementations, the sterilization is performed at 85°C for 25-30 minutes.

[0042] In some implementations, the solid-liquid separation is at least one of centrifugation and filtration.

[0043] Preferably, the centrifugation is performed using a disc centrifuge with a centrifugation speed set to 18,000-25,000 rpm.

[0044] Preferably, the filtration is a ceramic membrane filtration.

[0045] More preferably, the ceramic membrane filtration is performed by filtering through a 50nm or 100nm ceramic membrane, controlling the temperature at 30-40℃ and the pressure at 0.2-0.4MPa, and the light transmittance of the filtered liquid is above 70%.

[0046] In some embodiments, the purification is at least one of: immobilized carbon column decolorization, electrodialysis desalination, and ion exchange desalination.

[0047] Preferably, the immobilized carbon column decolorization is performed using a combination of an immobilized carbon column and an Amar filter, with the temperature controlled at 70-80℃, pH at 4.5-5.5, and flow rate at 8-12 m³ / s. 3 / h, the amount of diatomaceous earth used in the Ama filter is 0.05-0.1% (dry basis percentage).

[0048] More preferably, the light transmittance of the immobilized carbon column after decolorization is above 95%.

[0049] Preferably, the electrodialysis desalination is performed using a 20-30 homogeneous membrane, controlling the current at 2.0-3.0A, and using a 5-10% sodium chloride solution as the aqueous phase.

[0050] More preferably, the conductivity after electrodialysis desalination can be reduced to 1000-1500 μs / cm.

[0051] Preferably, the ion-exchange desalination involves passing the material after electrodialysis desalination sequentially through a cation exchange column, an anion exchange column, another cation exchange column, another anion exchange column, and another cation exchange column for deep desalination, controlling the material temperature at 35-45℃ and the flow rate at 2-4 BV / h.

[0052] More preferably, the conductivity after desalination by the ion exchange is 10-50 μs / cm.

[0053] Preferably, the concentration is carried out using a six-effect evaporator, with a concentration temperature of 65-75℃ and a vacuum degree of -0.06~-0.1Mpa.

[0054] In some implementations, the concentration is: concentrated to a sugar concentration of 65-75% (m / m).

[0055] In some embodiments, the pH of the feed medium is 6.8-7.0.

[0056] In some embodiments, the pH of the supplemental culture medium 2 is 6.8-7.0.

[0057] In some embodiments, the compound amino acid powder comprises: proline 10.3-13.8% (m / m), valine 25.3-34.5% (m / m), and glutamic acid 50-65% (m / m).

[0058] Secondly, the present invention provides a sugar-free human milk oligosaccharide complex prebiotic prepared by the above preparation method.

[0059] In some embodiments, the sugar-free human milk oligosaccharide complex prebiotic has a moisture content of ≤3% and a sugar content of ≤0.5%, and by mass fraction includes 2'-FL 70-75%, DFL 5-10%, and GOS 15-25%.

[0060] Thirdly, the application of the sugar-free human milk oligosaccharide complex prebiotics prepared by the above method in the preparation of products that regulate intestinal flora function.

[0061] In some implementations, the regulation of gut microbiota includes: promoting the proliferation of probiotics, inhibiting the growth of pathogenic bacteria, and increasing the retention rate of prebiotics.

[0062] In some implementations, the product is food, health supplement, or medicine.

[0063] Fourthly, the present invention provides: a product for regulating intestinal flora function, comprising the sugar-free human milk oligosaccharide complex prebiotic prepared by the above preparation method.

[0064] The present invention has at least the following beneficial effects: Compared with existing technologies, this invention has better technical effects in terms of product performance and intestinal regulation efficacy.

[0065] According to experimental tests, this invention increases the total prebiotic content of the fermentation broth from less than 170g / L in the comparative example to 185-220g / L.

[0066] According to experimental tests, this invention increases the proportion of beneficial bacteria in in vitro mixed culture from 60-62% in the comparative example to 66-68%.

[0067] According to experimental tests, this invention reduces the proportion of harmful bacteria in in vitro mixed culture from 38-40% in the comparative example to 32-34%.

[0068] Furthermore, based on the present invention: Based on the comparison of Example 1 and Comparative Examples 1-3, this invention uses a prebiotic extract with specific components as a fermentation feed material and precisely controls the fermentation process, achieving new technical effects such as resource utilization of by-products, stable product composition ratios, strong product resistance to digestion, and efficient regulation of intestinal flora. The combined technical effect is superior to the sum of the effects of each individual technique. Detailed Implementation

[0069] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0070] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all instruments, devices, equipment, reagents, products, etc., used in the embodiments of the present invention are obtained through conventional commercial means.

[0071] The recombinant *Escherichia coli* used in the embodiments and comparative examples of this invention is *Escherichia coli* BLCY-019, classified and named as *Escherichia coli* BLCY-019, with accession number CCTCC NO: M2026303, accession date: January 30, 2026, depositary institution: China Center for Type Culture Collection, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province. It should be noted that this invention does not limit the type of recombinant *Escherichia coli*, and any equivalent substitutions made for the type of recombinant *Escherichia coli* are within the scope of protection of this invention.

[0072] The sources of some materials and reagents are shown in Table 1.

[0073] Table 1

[0074] The prebiotic product residue solution one and prebiotic product residue solution two used in Examples 1-3 of this invention are: the residual liquid after chromatographic separation in the production process of galactooligosaccharides, which is concentrated under vacuum (50°C, vacuum degree -0.08Mpa) until the total concentration of glucose, galactose, lactose and galactooligosaccharides is 50-60% (m / v).

[0075] Among them, prebiotic residue one comes from the production process of type 90 galactooligosaccharide, and prebiotic residue two comes from the production process of type 70 galactooligosaccharide.

[0076] The prebiotic product residue solution one and prebiotic product residue solution two used in Comparative Examples 1-2 of this invention are solutions directly compounded according to their composition, with a total concentration of glucose, galactose, lactose, and galactooligosaccharides of 50-60% (m / v).

[0077] Example 1 A method for preparing a sugar-free human milk oligosaccharide complex prebiotic includes the following steps: (1) Seed culture The preserved recombinant Escherichia coli glycerol tube seed was inoculated into shake flask seed culture medium at an inoculation rate of 0.8‰ (v / v) and cultured at 37℃ with shaking for 10 h to obtain shake flask seed solution.

[0078] The shake flask seed culture medium is prepared as follows: peptone 9‰ m / v, yeast powder 5‰ m / v, sodium chloride 9‰ m / v, with the remainder being water.

[0079] The above shake flask seed solution was inoculated into a 1000L seed tank at an inoculation rate of 0.9‰ (v / v) and cultured at 37℃ for 12h to obtain the seed tank seed solution.

[0080] The seed culture medium in the seed tank is prepared as follows: 9‰ m / v peptone, 6‰ m / v corn steep liquor, 9‰ m / v sodium chloride, with the remainder being water.

[0081] (2) Fermentation production The seed culture from the seed tank described in (1) was inoculated into a 10000L fermenter for fermentation. After inoculation, the fermentation temperature was controlled at 37℃, the pH at 7.0, the aeration ratio at 0.9vvm, and the stirring speed at 125rpm for cultivation.

[0082] The initial fermentation medium was prepared as follows: potassium dihydrogen phosphate 6.5‰ (m / v), magnesium sulfate heptahydrate 1.5‰ (m / v), diammonium hydrogen phosphate 2.5‰ (m / v), citric acid 1.5‰ (m / v), industrial yeast powder 5.5‰ (m / v), porcine bone peptone 2.5‰ (m / v), glycerol 3.5% (m / v), trace metal solution 0.9% (m / v), with the remainder being water, and the pH was adjusted to 7.0.

[0083] The method for preparing the trace metal liquid is as follows: Na2EDTA: 1.5‰ (m / v); Zinc sulfate heptahydrate (ZnSO4) 7H2O): 0.45‰ (m / v); Manganese chloride dihydrate (MnCl2) 2H2O): 0.2‰ (m / v); Cobalt chloride hexahydrate (CoCl2) 6H2O): 0.05‰ (m / v); Copper sulfate pentahydrate (CuSO4) 5H2O): 0.03‰ (m / v); Sodium molybdate dihydrate (Na2MoO4) 2H2O): 0.04‰ (m / v); Calcium chloride dihydrate (CaCl2) 2H2O): 0.25‰ (m / v); Ferrous sulfate heptahydrate (FeSO4) 7H2O): 0.3‰ (m / v); Boric acid (H3BO3): 0.1‰ (m / v); Potassium iodide (KI): 0.01‰ (m / v).

[0084] When fermentation broth OD 600When the concentration reaches 13, adjust the fermentation temperature to 24℃. Add IPTG (isopropyl-β-D-thiogalactoside) to the fermentation broth to a final concentration of 0.15mM. Simultaneously, begin feeding fed medium I and fed medium II. By controlling the flow rate, maintain the glucose concentration in the fermentation broth at 12.5g / L and the lactose concentration at 4.5g / L. Increase the aeration ratio to 1.1vvm and adjust the stirring speed to 175rpm.

[0085] The supplemental culture medium I comprises: 22% (m / v) magnesium sulfate heptahydrate, 6% (m / v) industrial yeast extract, 48% (m / v) prebiotic product residue, and 3% (m / v) porcine bone peptone. The pH is adjusted to 6.9. The prebiotic product residue I is composed of: 46% glucose, 10% galactose, 26% lactose, and 18% galacto-oligosaccharides, by mass fraction on a dry basis.

[0086] The supplemental culture medium II comprises: 4% (m / v) compound amino acid powder, 97% (m / v) prebiotic product residue II, and 0.6% (m / v) trace metal solution, adjusted to pH 6.9. The compound amino acid powder contains: 12.0% (m / m) proline, 29.5% (m / m) valine, and 58.5% (m / m) glutamic acid. The prebiotic product residue II is composed of: 42.5% glucose, 5% galactose, 42.5% lactose, and 10% galacto-oligosaccharides, on a dry basis by mass fraction.

[0087] The contents of the above-mentioned prebiotic product residue solution one and prebiotic product residue solution two are all added amounts on a dry basis.

[0088] When fermentation broth OD 600 When the concentration reaches 55%, add IPTG to a final concentration of 0.09 mM into the fermentation broth. Simultaneously, feed medium 1 and feed medium 2 are added, and the glucose concentration is controlled at 12.5 g / L and the lactose concentration at 6 g / L by adjusting the feed rate. The aeration ratio is adjusted to 1.3 vvm and the stirring speed is adjusted to 250 rpm.

[0089] When fermentation broth OD 600 When the concentration reaches 90%, add IPTG to a final concentration of 0.09 mM into the fermentation broth. Simultaneously, feed medium 1 and feed medium 2 are added, and the glucose concentration is controlled at 7.5 g / L and the lactose concentration at 7 g / L by adjusting the feed rate. The aeration ratio is adjusted to 1.4 vvm and the stirring speed is adjusted to 350 rpm.

[0090] When fermentation broth OD 600When the concentration reaches 140, add IPTG to the fermentation broth to a final concentration of 0.09 mM. Simultaneously, feed medium 1 and feed medium 2 are added, and the glucose concentration is controlled at 4.5 g / L and the lactose concentration at 6 g / L by adjusting the feed rate. Maintain an aeration ratio of 1.4 vvm and a stirring speed of 350 rpm.

[0091] When the concentration of human milk oligosaccharides in the fermentation broth no longer increases, stop feeding both feed medium 1 and feed medium 2, and maintain an aeration ratio of 1.4 vvm and a stirring speed of 350 rpm until the consumption of glucose and lactose reaches at least 0 g / L, then terminate the fermentation.

[0092] Tests showed that under these fermentation conditions, the yield of 2'-FL in the fermentation broth was 157 g / L, the yield of DFL was 13 g / L, the yield of GOS was 39 g / L, and the total prebiotic content was 209 g / L.

[0093] (3) Purification and drying Sterilization: After fermentation, the fermentation liquid described in (2) is sterilized at 85°C for 25 min.

[0094] Centrifugation: Use a disc centrifuge at 22,000 rpm to remove bacterial cells and collect the supernatant.

[0095] Ceramic membrane filtration: The supernatant was filtered through 50nm and 100nm ceramic membranes, with the operating temperature controlled at 35℃ and the operating pressure at 0.3MPa. The light transmittance of the filtered liquid was measured to be 74%.

[0096] Immobilized carbon column decolorization: The ceramic membrane filtrate was decolorized by passing it through an immobilized carbon column in combination with an Ama filter. The feed temperature was controlled at 75℃, the pH at 5.0, and the system flow rate at 10 m³ / h. The amount of diatomaceous earth added to the Ama filter was 0.08% of the dry basis of the feed. Testing showed that the transmittance of the decolorized feed was 97%.

[0097] Electrodialysis desalination: 25 pairs of homogeneous membranes were used for electrodialysis desalination, with a controlled current of 2.5A and a 7% sodium chloride solution as the aqueous electrode phase. Testing showed that the conductivity of the feed solution decreased to 1200 μS / cm after electrodialysis.

[0098] Cation-exchange desalination: The material after electrodialysis desalination is passed sequentially through a cation-anion-cation-anion-cation exchange column for deep desalination, with the material temperature controlled at 40℃ and the flow rate at 3 BV / h. After ion-exchange desalination, the conductivity of the solution decreases to 30 μS / cm.

[0099] Concentration: The material after ionization and exchange is concentrated using a six-effect evaporator. The concentration temperature is controlled at 70℃ and the vacuum degree is -0.08MPa, and the material is concentrated to a sugar concentration of 70%.

[0100] Drying: The above 70% sugar concentration concentrate was transferred to a belt vacuum dryer for drying to obtain sugar-free human milk oligosaccharide complex prebiotic powder. The drying temperature in zone one was 170℃, the drying temperature in zone two was 135℃, the drying temperature in zone three was 60℃, and the vacuum degree was -0.09MPa.

[0101] (4) Product Analysis The product was found to have a moisture content of 1.2% by mass. On a dry basis, its composition is: 2'-FL 74.8%, DFL 6.5%, GOS 18.5%, and sugar content 0.20%.

[0102] Example 2 A method for preparing a sugar-free human milk oligosaccharide complex prebiotic includes the following steps: (1) Seed culture The seed culture method is the same as in Example 1.

[0103] (2) Fermentation production The seed culture from the seed tank described in (1) was inoculated into a 10000L fermenter at an inoculation rate of 5% (v / v). After inoculation, the fermentation temperature was controlled at 37°C, pH 6.8, aeration ratio of 0.8vvm, and stirring speed of 100 rpm for cultivation.

[0104] The initial fermentation medium was prepared as follows: potassium dihydrogen phosphate 5‰ (m / v), magnesium sulfate heptahydrate 1‰ (m / v), diammonium hydrogen phosphate 2‰ (m / v), citric acid 1‰ (m / v), industrial yeast powder 5‰ (m / v), porcine bone peptone 2‰ (m / v), glycerol 3% (m / v), trace metal solution 0.8% (m / v), with the balance being water, and the pH was adjusted to 6.8. The preparation method of the trace metal solution was the same as in Example 1.

[0105] When fermentation broth OD 600 When the concentration reaches 10, adjust the fermentation temperature to 22℃. Add IPTG to the fermentation broth to a final concentration of 0.1mM. Simultaneously begin feeding fed medium 1 and fed medium 2. By controlling the flow rate, maintain the glucose concentration in the fermentation broth at 10g / L and the lactose concentration at 3g / L. Increase the aeration ratio to 1.0vvm and adjust the stirring speed to 150rpm.

[0106] The fed culture medium I comprises: 20% (m / v) magnesium sulfate heptahydrate, 5% (m / v) industrial yeast powder, 40% (m / v) prebiotic product residue, and 2.5% (m / v) porcine bone peptone, adjusted to pH 6.8. The prebiotic product residue I is composed of: 50% glucose, 7% galactose, 28% lactose, and 15% galacto-oligosaccharides, by mass fraction on a dry basis.

[0107] The supplemental culture medium II comprises: 3% (m / v) compound amino acid powder, 95% (m / v) prebiotic product residue II, and 0.5% (m / v) trace metal solution, adjusted to pH 6.8. The compound amino acid powder contains 10.3% (m / m) proline, 25.3% (m / m) valine, and 64.4% (m / m) glutamic acid. The prebiotic product residue II is composed of: 39% glucose, 6% galactose, 43% lactose, and 12% galacto-oligosaccharides, on a dry basis by mass fraction.

[0108] The contents of the above-mentioned prebiotic product residue solution one and prebiotic product residue solution two are all added amounts on a dry basis.

[0109] When fermentation broth OD 600 When the concentration reaches 50%, add IPTG to a final concentration of 0.08 mM into the fermentation broth. Simultaneously, feed medium 1 and feed medium 2 are added, and the glucose concentration is controlled at 10 g / L and the lactose concentration at 5 g / L by adjusting the feed rate. The aeration ratio is adjusted to 1.2 vvm and the stirring speed is adjusted to 200 rpm.

[0110] When fermentation broth OD 600 When the concentration reaches 80%, add IPTG to a final concentration of 0.08 mM into the fermentation broth. Simultaneously, feed medium 1 and feed medium 2 are added, and the glucose concentration is controlled at 5 g / L and the lactose concentration at 6 g / L by adjusting the feed rate. The aeration ratio is adjusted to 1.3 vvm and the stirring speed is adjusted to 300 rpm.

[0111] When fermentation broth OD 600 When the concentration reaches 130, add IPTG to the fermentation broth to a final concentration of 0.08 mM. Simultaneously, feed medium 1 and feed medium 2 are added, and the glucose concentration is controlled at 3 g / L and the lactose concentration at 5 g / L by adjusting the feed rate. Maintain an aeration ratio of 1.3 vvm and a stirring speed of 300 rpm.

[0112] When the concentration of human milk oligosaccharides in the fermentation broth no longer increases, stop feeding both feed medium 1 and feed medium 2, and maintain an aeration ratio of 1.3 vvm and a stirring speed of 300 rpm until the consumption of glucose and lactose reaches at least 0 g / L, then terminate the fermentation.

[0113] Tests showed that under these fermentation conditions, the yield of 2'-FL in the fermentation broth was 140 g / L, the yield of DFL was 12 g / L, the yield of GOS was 38 g / L, and the total prebiotic content was 190 g / L.

[0114] (3) Purification and drying Sterilization: After fermentation, the fermentation liquid described in (2) is sterilized at 85°C for 25 min.

[0115] Centrifugation: Use a disc centrifuge at 18,000 rpm to remove bacterial cells and collect the supernatant.

[0116] Ceramic membrane filtration: The supernatant was filtered through 50nm and 100nm ceramic membranes, with the operating temperature controlled at 30℃ and the operating pressure at 0.2MPa. The light transmittance of the filtered liquid was measured to be 70%.

[0117] Immobilized carbon column decolorization: The ceramic membrane filtrate was decolorized by passing it through an immobilized carbon column in combination with an Ama filter. The feed temperature was controlled at 70℃, the pH at 4.5, and the system flow rate at 8 m³ / h. The amount of diatomaceous earth added to the Ama filter was 0.05% of the dry basis of the feed. Testing showed that the transmittance of the decolorized feed was 95%.

[0118] Electrodialysis desalination: 20 pairs of homogeneous membranes were used for electrodialysis desalination, with a controlled current of 2.0 A and a 5% sodium chloride solution as the aqueous electrode phase. Testing showed that the conductivity of the feed solution decreased to 1500 μS / cm after electrodialysis.

[0119] Cation-exchange desalination: The material after electrodialysis desalination is passed sequentially through a cation-anion-cation-anion-cation exchange column for deep desalination, with the material temperature controlled at 35℃ and the flow rate at 2 BV / h. After ion-exchange desalination, the conductivity of the solution decreases to 50 μS / cm.

[0120] Concentration: The material after ionization and exchange is concentrated using a six-effect evaporator. The concentration temperature is controlled at 65℃ and the vacuum degree is -0.06MPa, and the material is concentrated to a sugar concentration of 65%.

[0121] Drying: The above 65% sugar concentration concentrate was transferred to a belt vacuum dryer for drying to obtain sugar-free human milk oligosaccharide complex prebiotic powder. The drying temperature in zone one was 160℃, the drying temperature in zone two was 130℃, the drying temperature in zone three was 50℃, and the vacuum degree was -0.08MPa.

[0122] (4) Product Analysis The product was found to have a moisture content of 1.5% by mass. On a dry basis, its composition is: 2'-FL 74.0%, DFL 6.5%, GOS 19.2%, and sugar content 0.3%.

[0123] Example 3 A method for preparing a sugar-free human milk oligosaccharide complex prebiotic includes the following steps: (1) Seed culture The seed culture method is the same as in Example 1.

[0124] (2) Fermentation production The seed culture from the seed tank described in (1) was inoculated into a 10,000 L fermenter for fermentation at an inoculation rate of 10% (v / v). After inoculation, the fermentation temperature was controlled at 37°C, pH 7.2, aeration ratio of 1.0 vvm, and stirring speed of 150 rpm for cultivation.

[0125] The initial fermentation medium was prepared as follows: 8‰ potassium dihydrogen phosphate (m / v), 2‰ magnesium sulfate heptahydrate (m / v), 3‰ diammonium hydrogen phosphate (m / v), 2‰ citric acid (m / v), 6‰ industrial yeast powder (m / v), 3‰ porcine bone peptone (m / v), 4% glycerol (m / v), 1.0% trace metal solution (m / v), with the remainder being water, and the pH was adjusted to 7.2. The preparation method of the trace metal solution was the same as in Example 1.

[0126] When fermentation broth OD 600 When the concentration reaches 15, adjust the fermentation temperature to 25°C. Add IPTG to the fermentation broth to a final concentration of 0.2 mM. Simultaneously, begin feeding fed medium 1 and medium 2, controlling the flow rate to maintain the glucose concentration at 15 g / L and the lactose concentration at 6 g / L in the fermentation broth. Increase the aeration ratio to 1.2 vvm and the stirring speed to 200 rpm.

[0127] The preparation method of the supplemental culture medium one is as follows: 24% (m / v) magnesium sulfate heptahydrate, 7% (m / v) industrial yeast powder, 55% (m / v) prebiotic product residue, and 3.5% (m / v) porcine bone peptone, adjusted to pH 7.0. The prebiotic product residue one consists of: 40% glucose, 12% galactose, 30% lactose, and 18% galacto-oligosaccharides, on a dry basis by mass fraction.

[0128] The preparation method of the supplementary culture medium II is as follows: 3.5% (m / v) of compound amino acid powder, 99% (m / v) of prebiotic product residue II, and 0.7% (m / v) of trace metal solution, adjusted to pH 7.0. The compound amino acid powder contains 13.8% (m / m) proline, 34.5% (m / m) valine, and 51.7% (m / m) glutamic acid. The composition of prebiotic product residue II is: 46% glucose, 4% galactose, 42% lactose, and 8% galacto-oligosaccharides, by mass fraction on a dry basis.

[0129] The contents of the above-mentioned prebiotic product residue solution one and prebiotic product residue solution two are all added amounts on a dry basis.

[0130] When fermentation broth OD 600When the concentration reaches 60%, add IPTG to a final concentration of 0.1 mM to the fermentation broth. Simultaneously, feed medium 1 and feed medium 2 are added, adjusting the feed rate to control the glucose concentration at 15 g / L and the lactose concentration at 7 g / L. The aeration ratio is further increased to 1.4 vvm, and the stirring speed is increased to 300 rpm.

[0131] When fermentation broth OD 600 When the concentration reaches 100%, add IPTG to a final concentration of 0.1 mM into the fermentation broth. Simultaneously, feed medium 1 and feed medium 2 are added, adjusting the feed rate to control the glucose concentration at 10 g / L and the lactose concentration at 8 g / L. Increase the aeration ratio to 1.5 vvm and the stirring speed to 400 rpm.

[0132] When fermentation broth OD 600 When the concentration reaches 150, add IPTG to the fermentation broth to a final concentration of 0.1 mM. Simultaneously, feed medium 1 and feed medium 2 are added, adjusting the feed rate to control the glucose concentration at 6 g / L and the lactose concentration at 7 g / L. Maintain an aeration ratio of 1.5 vvm and a stirring speed of 400 rpm.

[0133] When the concentration of human milk oligosaccharides in the fermentation broth no longer increases, stop feeding both feed medium 1 and feed medium 2, and maintain an aeration ratio of 1.5 vvm and a stirring speed of 400 rpm until the consumption of glucose and lactose reaches at least 0 g / L, then terminate the fermentation.

[0134] Tests showed that under these fermentation conditions, the yield of 2'-FL in the fermentation broth was 151 g / L, the yield of DFL was 11.5 g / L, the yield of GOS was 42 g / L, and the total prebiotic content was 204.5 g / L.

[0135] (3) Purification and drying Sterilization: After fermentation, sterilize the fermentation broth at 85℃ for 30 minutes.

[0136] Centrifugation: Solid-liquid separation was performed using a disc centrifuge at 25,000 rpm, and the supernatant was collected.

[0137] Ceramic membrane filtration: The supernatant was filtered through 50nm and 100nm ceramic membranes, with the operating temperature controlled at 40℃ and the operating pressure at 0.4MPa. Testing showed that the transmittance of the filtered liquid reached 72%.

[0138] Immobilized carbon column decolorization: The ceramic membrane filtrate was decolorized by passing it through an immobilized carbon column in combination with an Ama filter. The feed temperature was controlled at 80℃, the pH at 5.5, and the system flow rate at 12 m³ / h. The amount of diatomaceous earth added to the Ama filter was 0.1% of the dry basis of the feed. Testing showed that the transmittance of the decolorized feed increased to 98%.

[0139] Electrodialysis desalination: 30 pairs of homogeneous membranes were used for electrodialysis desalination, with a controlled current of 3.0 A and a 10% sodium chloride solution as the aqueous electrode phase. After electrodialysis, the conductivity of the feed solution decreased to 1000 μS / cm.

[0140] Deep desalination via ion exchange: The electrodialysis material is sequentially passed through a "cation-anion-cation-anion-cation" ion exchange column, with the material temperature controlled at 45℃ and the flow rate at 4 BV / h. After ion exchange desalination, the conductivity of the solution decreases to 15 μS / cm.

[0141] Concentration: The material after ionization and exchange is concentrated using a six-effect evaporator. The concentration temperature is controlled at 75℃ and the vacuum degree is -0.1MPa, and the material is concentrated to a sugar concentration of 75%.

[0142] Drying: The above 75% sugar concentrate was transferred to a belt vacuum dryer for drying. The drying temperature was 180℃ in zone one, 140℃ in zone two, and 70℃ in zone three, with a vacuum degree of -0.1MPa. After drying, the powder was pulverized through a 40-mesh sieve to obtain sugar-free human milk oligosaccharide complex prebiotic powder.

[0143] (4) Product Analysis Testing revealed that the product obtained in this embodiment is a white to pale yellow powder with a moisture content of 1.6%. On a dry basis, its composition is: 2'-FL 73.6%, DFL 5.7%, GOS 20.4%, and sugar content 0.30%.

[0144] Comparative Example 1 The prebiotic product residue in the supplemented culture medium of Example 1 was replaced with a mixture of glucose 30%, galactose 20%, lactose 23%, and galactooligosaccharides 27% by mass fraction on a dry basis. All other components were the same as in Example 1.

[0145] When the concentration of human milk oligosaccharides in the fermentation broth no longer increases, stop feeding both feed medium 1 and feed medium 2, and maintain an aeration ratio of 1.4 vvm and a stirring speed of 350 rpm until the consumption of glucose and lactose reaches at least 0 g / L, then terminate the fermentation.

[0146] Tests showed that under these fermentation conditions, the yield of 2'-FL in the fermentation broth was 110 g / L, the yield of DFL was 6 g / L, the yield of GOS was 49 g / L, and the total prebiotic content was 165 g / L.

[0147] After drying, the product obtained in Comparative Example 1 was tested and found to be a white to pale yellow powder with a moisture content of 1.6%. On a dry basis, the product composition was: 2'-FL 65.2%, DFL 3.2%, GOS 30.3%, and sugar content 1.30%.

[0148] Comparative Example 1 shows that the proportion of component one of the feed culture medium was out of range, which led to a decrease in the fermentation yield of the target product and the proportion of components in the dried product did not meet the standard.

[0149] Comparative Example 2 The prebiotic product residue in the supplemented culture medium II of Example 1 was replaced with the following composition by mass fraction on a dry basis: 35% glucose; 6% galactose; 55% lactose; and 4% galactooligosaccharides. All other components remained the same as in Example 1.

[0150] When the concentration of human milk oligosaccharides in the fermentation broth no longer increases, stop feeding both feed medium 1 and feed medium 2, and maintain an aeration ratio of 1.4 vvm and a stirring speed of 350 rpm until the consumption of glucose and lactose reaches at least 0 g / L, then terminate the fermentation.

[0151] Tests showed that under these fermentation conditions, the yield of 2'-FL in the fermentation broth was 98 g / L, the yield of DFL was 5 g / L, the yield of GOS was 25 g / L, and the total prebiotic content was 128 g / L.

[0152] After drying, the product obtained in Comparative Example 2 was tested and found to be a white to pale yellow powder with a moisture content of 1.9%. On a dry basis, its composition was: 2'-FL 66.4%, DFL 3.5%, GOS 29.5%, and sugar content 0.60%.

[0153] Comparative Example 2 shows that the ratio of the two components of the supplemental culture medium was out of range, which led to a decrease in the fermentation yield of the target product and the component ratio of the finished product after drying did not meet the standard.

[0154] Comparative Example 3 The only difference between Comparative Example 3 and Example 1 is that only one feeding was performed, i.e., when OD 600 When the concentration reaches 55, feed medium one and feed medium two are added at once. By controlling the flow rate, the glucose concentration in the fermentation broth is controlled at 12.5 g / L and the lactose concentration is controlled at 4.5 g / L.

[0155] When the concentration of human milk oligosaccharides in the fermentation broth no longer increases, stop feeding both feed medium 1 and feed medium 2, and maintain an aeration ratio of 1.4 vvm and a stirring speed of 350 rpm until the consumption of glucose and lactose reaches at least 0 g / L, then terminate the fermentation.

[0156] Tests showed that under these fermentation conditions, the yield of 2'-FL in the fermentation broth was 75 g / L, the yield of DFL was 7 g / L, the yield of GOS was 20 g / L, and the total prebiotic content was 102 g / L.

[0157] After drying, the product obtained in Comparative Example 3 was tested and found to be a white to pale yellow powder with a moisture content of 1.5%. On a dry basis, its composition was: 2'-FL 71.3%, DFL 5.0%, GOS 20.7%, and sugar content 3.0%.

[0158] Comparative Example 3 shows that with only one feeding, the final glucose and lactose concentrations cannot be guaranteed to be consumed simultaneously to 0 g / L. In the end, one of them will be left unconsumed, resulting in an excessively high sugar content in the product.

[0159] Comparative Example 3 shows that only one feeding resulted in a decrease in the fermentation yield of the target product and an excessively high sugar content in the dried product.

[0160] Detection Example 1 The in vitro digestibility of the products obtained in the examples and comparative examples was tested as follows: a sugar-free human milk oligosaccharide complex prebiotic was prepared into a 10% sugar concentration solution using PBS buffer, and simulated oral digestion, gastric digestion, and small intestinal digestion were performed according to the steps.

[0161] The specific process includes: First, simulating oral digestion: α-amylase is added to the sugar solution at a dosage of 7.5 U / mL, and the mixture is placed in a 37°C water bath shaker for 5 min; then, simulating gastric digestion: the pH of the reaction solution is adjusted to 3.0 using 1M HCl, and pepsin is added at a dosage of 100 U / mL, mixed well, and placed in a 37°C water bath shaker for 2 h; finally, simulating small intestinal digestion: the pH of the reaction solution is adjusted to 6.5 using 1M sodium hydroxide, and α-amylase, trypsin, and lipase are added at dosages of 10 U / mL, 5 U / mL, and 10 U / mL, respectively, mixed well, and placed in a 37°C water bath shaker for 8 h.

[0162] Samples were taken before and after oral digestion, gastric digestion, intestinal digestion, and again before and after intestinal digestion. HPLC analysis was performed to detect changes in component composition. The retention rate of the sugar-free human milk oligosaccharide complex prebiotic was calculated. Three replicates were set up for each group, and the average value was calculated. Retention rate = (Prebiotic content after simulated digestion / Initial prebiotic content) × 100%. The experimental results are shown in Table 2.

[0163] Table 2. Retention rate of sugar-free human milk oligosaccharides and prebiotics after simulated in vitro digestion.

[0164] The prebiotic retention rates in Examples 1-3 are as high as 99.85%-99.90%, indicating that the products of the present invention are hardly degraded in the simulated human digestive tract environment, and can effectively ensure the activity of prebiotics.

[0165] Detection Example 2 The products obtained in the examples and comparative examples were subjected to in vitro beneficial bacteria proliferation experiments, and the methods are as follows: Culture medium preparation: blank culture medium, MRS medium without added carbon source; control culture medium: MRS medium using glucose as carbon source, with a carbon source content of 2%; experimental culture medium: MRS medium using sugar-free human milk oligosaccharide complex prebiotic liquid after in vitro digestion as carbon source, with a final carbon source content of 2%. Activation and culture of bacteria: Five probiotics, namely Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium adolescentis, Bifidobacterium bifidum, and Lactobacillus acidophilus, and two pathogenic bacteria, Escherichia coli and Cocci, were activated separately and then mixed and inoculated into the above culture medium. The cultures were incubated in a constant temperature incubator at 37°C for 12 hours (mean logarithmic growth phase). Index testing: Samples were taken every 3 hours for pH and OD levels. 600 Detection: After centrifugation of the bacterial culture, the precipitate was collected for relative quantitative analysis. The changes in the proportion of harmful and beneficial bacteria were compared. Three parallel groups were set up for each group, and the average value was calculated. The experimental results are shown in Table 3.

[0166] Table 3

[0167] Examples 1-3 show that the product of the present invention can effectively promote the proliferation of probiotics such as Bifidobacterium and Lactobacillus, while inhibiting the growth of pathogenic bacteria such as Escherichia coli and fecal coli.

[0168] Comparative Examples 1 and 2 demonstrate that when the composition of the prebiotic extract changes, the prebiotic component ratio of the resulting product becomes unbalanced, and compared with the example, the ability to regulate the intestinal flora is reduced. The sugar content of Comparative Examples 1-3 is >0.5%, which is also not conducive to the proliferation of beneficial bacteria.

[0169] Verification of technical effectiveness and / or analysis of technical problem solving This invention addresses the problems of high equipment costs, low by-product utilization, uneven product blending, and easy digestibility and degradation of prebiotics in existing processes. It utilizes the residual prebiotic extract from existing enterprises as a fermentation raw material, combining segmented and precise fermentation control to synthesize multi-component active substances, eliminating the need for chromatographic separation and crystallization processes throughout the entire process. Testing shows that the total prebiotic content of the fermentation broth can reach 185-220 g / L; the finished product has stable components, with a total sugar content ≤0.5%, and a retention rate of 99.85%-99.90% after simulated digestion. It also effectively proliferates beneficial intestinal bacteria and inhibits harmful bacteria, achieving high-value utilization of industrial by-products, reducing production costs, and ensuring stable and excellent product efficacy.

[0170] In solving the above problems or overcoming the above defects, the present invention encountered the following difficulties and obstacles: The applicant has experimented with various prebiotic residues (such as Comparative Examples 1-2), but not all of them can be directly used for the preparation of human milk oligosaccharide complex prebiotics. Using a single residue as a feedstock is also insufficient; only by using residues one and two, with their specific types and proportions as feedstock culture media as described in this application, can fermentation be precisely controlled to achieve high yields of human milk oligosaccharide complex prebiotics. Excessively high or low sugar content will lead to metabolic imbalance, resulting in reduced purity or yield of the complex prebiotics. The two residue feedstock culture media must be compatible with the four-stage OD of this invention. 600 A precise feeding strategy, in conjunction with other measures, dynamically adjusts the concentration of glucose and lactose fed according to the metabolic needs of the microorganisms at each stage. Only in this way can a dynamic balance be achieved between the carbon source flow rate and the microbial synthesis metabolism. Otherwise, carbon source excess will lead to metabolic inhibition or carbon source deficiency will lead to product synthesis stagnation.

[0171] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for the preparation of a non-digestible human milk oligosaccharide complex prebiotic, characterized in that, Includes the following steps: (1) Preliminary fermentation was carried out using Escherichia coli; (2) When the OD600 of the fermentation broth reaches 10-15, the first feeding is carried out; (3) When the OD600 of the fermentation broth reaches 50-60, a second feeding is carried out; (4) When the OD600 of the fermentation broth reaches 80-100, the third feeding is carried out; (5) When the OD600 of the fermentation broth reaches 130-150, the fourth feeding is carried out; (6) When the concentration of human milk oligosaccharides in the fermentation broth no longer increases, terminate the fermentation to obtain the fermentation broth; (7) The fermentation broth is sterilized, separated from solids and liquids, purified, concentrated and dried to obtain the final product; The first feeding consisted of adding feeding medium one and feeding medium two to maintain a glucose concentration of 10-15 g / L and a lactose concentration of 3-6 g / L. The second feeding involves adding feeding medium one and feeding medium two to maintain a glucose concentration of 10-15 g / L and a lactose concentration of 5-7 g / L. The third feeding involves adding feeding medium one and feeding medium two to maintain a glucose concentration of 5-10 g / L and a lactose concentration of 6-8 g / L. The fourth feeding involves adding feeding medium one and feeding medium two to maintain a glucose concentration of 3-6 g / L and a lactose concentration of 5-7 g / L. The supplemental culture medium consists of: magnesium sulfate 20-24% m / v, yeast powder 5-7% m / v, prebiotic product extract 40-55% m / v, and porcine bone peptone 2.5-3.5% m / v. The prebiotic product residue, on a dry basis, comprises: 40-50% glucose, 7-12% galactose, 20-30% lactose, and 15-20% galacto-oligosaccharides. The supplemental culture medium II comprises: 3-5% m / v of compound amino acid powder, 95-99% m / v of prebiotic product extract II, and 0.5-0.7% m / v of trace metal solution; The prebiotic product residue solution II, on a dry basis by mass fraction, comprises: 37-46% glucose, 2-6% galactose, 37-46% lactose, and 5-12% galacto-oligosaccharides. The Escherichia coli is: Escherichia coli BLCY-019, classified and named: Escherichia coli BLCY-019, accession number: CCTCC NO: M2026303, accession date: January 30, 2026, depositary institution: China Center for Type Culture Collection.

2. The production method according to claim 1, characterized by, The preliminary fermentation in step (1) is as follows: fermentation temperature is 37℃, pH is 6.8-7.2, and aeration ratio is 0.8-1.0vvm; The culture medium for the initial fermentation is: potassium dihydrogen phosphate 5-8‰ m / v, magnesium sulfate 1-2‰ m / v, diammonium hydrogen phosphate 2-3‰ m / v, citric acid 1-2‰ m / v, yeast powder 5-6‰ m / v, porcine bone peptone 2-3‰ m / v, glycerol 3-4% m / v, and trace metal solution 0.8-1.0% m / v, with the pH adjusted to 6.8-7.2; The trace metal solution is as follows: Na2EDTA: 1.2-1.5‰m / v, zinc sulfate: 0.3-0.5‰m / v, manganese chloride: 0.2-0.3‰m / v, cobalt chloride: 0.04-0.06‰m / v, copper sulfate: 0.01-0.03‰m / v, sodium molybdate: 0.03-0.05‰m / v, calcium chloride: 0.2-0.3‰m / v, ferrous sulfate: 0.1-0.3‰m / v, boric acid: 0.05-0.1‰m / v, potassium iodide: 0.01-0.05‰m / v.

3. The production method according to claim 1, characterized by, The first feeding step (2) also includes: adjusting the temperature to 22-25℃, adding IPTG to a final concentration of 0.1-0.2mM, and adjusting the ventilation ratio to 1-1.2vvm; The second feeding step (3) also includes: adding IPTG to a final concentration of 0.08-0.1 mM and adjusting the ventilation ratio to 1.2-1.5 vvm; The third feeding step (4) also includes: adding IPTG to a final concentration of 0.08-0.1 mM and adjusting the ventilation ratio to 1.3-1.5 vvm; The fourth feeding step (5) also includes: adding IPTG to a final concentration of 0.08-0.1 mM and adjusting the ventilation ratio to 1.3-1.5 vvm.

4. The production method according to claim 1, characterized by, The sterilization described in step (7) is: sterilization at 85℃ for 25-30 minutes; The solid-liquid separation in step (7) is at least one of centrifugation and filtration; The purification in step (7) is at least one of immobilized carbon column decolorization, electrodialysis desalination, and ion exchange desalination; The concentration described in step (7) is: concentration to a sugar concentration of 65-75% m / m.

5. The preparation method according to claim 1, characterized in that, The pH of the first feeding medium is 6.8-7.0; the pH of the second feeding medium is 6.8-7.

0. The compound amino acid powder contains: proline 10.3-13.8% m / m, valine 25.3-34.5% m / m, and glutamic acid 50-65% m / m.

6. The application of the sugar-free human milk oligosaccharide complex prebiotic prepared by the preparation method according to any one of claims 1-5 in the preparation of products that regulate intestinal flora function.

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