High-temperature-resistant milk-flavored essence fermentation composition and preparation method thereof

Abstract

The invention discloses a high-temperature-resistant milk-flavored essence fermentation composition and a preparation method thereof in the field of food biology. The high-temperature-resistant milk-flavored essence fermentation composition is prepared from the following raw materials: a milk grape graft, fresh peptide nucleotide, fermented whey liquid, a yeast extract, xylooligosaccharide, ascorbyl palmitate and a compound fermentation bacterium agent. Wherein the milk grape graft is prepared by fermenting a lactitol solution with geobacillus stearothermophilus and grafting beer yeast cell wall extract with alpha-1, 6-glucosidic bonds; the fresh peptide nucleotide is a small peptide-nucleotide compound formed by hydrolyzing concentrated whey protein through compound protease and fermenting hot spring streptomyces and lactobacillus plantarum; the fermented whey liquid is prepared through cow milk centrifugation, sterilization and three-strain gradient fermentation. The preparation method comprises the following steps: mixing the raw materials and mixing the compound fermentation inoculants under the protection of nitrogen. Through cooperation of the bifunctional modified compound and multiple strains, high temperature resistance, natural multi-flavor level and long-term stability are achieved, and the high-temperature-resistant milk powder is suitable for high-temperature processing scenes such as baking and sterilized milk.

Description

Technical Field

[0001] The present invention relates to the field of food biotechnology, and in particular to a high-temperature resistant milk flavor fermentation composition and a preparation method thereof. Background Art

[0002] The production of traditional milk flavorings has long relied on chemical synthesis or simple enzymatic hydrolysis processes. While these processes can simulate the basic flavor of dairy products, they are gradually exposing difficult-to-break technical bottlenecks in practical applications. The flavor stability of these flavors becomes a primary challenge in high-temperature processing scenarios: processes such as high-temperature baking (above 150°C) for baked goods and ultra-high-temperature treatment (135°C / 5 seconds) for sterilized dairy products cause complex changes in the flavor substances in the flavors. Some small-molecule flavor components (such as aldehydes and ketones) evaporate and are lost due to high temperatures, while some large-molecule substances (such as peptides) lose their freshness due to thermal decomposition. Furthermore, excessive Maillard reactions can cause a burnt odor, seriously affecting the sensory quality of the final product. These issues limit the application of traditional milk flavorings in food applications requiring high-temperature processing, making it difficult to meet consumer demand for "natural, rich, and stable" flavors.

[0003] While milk flavors produced through microbial fermentation can generate flavor compounds more similar to natural dairy products (such as frankincense components like diacetyl and acetaldehyde) through microbial metabolism compared to chemically synthesized products, their high-temperature adaptability remains limited. Existing fermentation processes often rely on conventional lactic acid bacteria (such as Lactobacillus delbrueckii) or yeasts, whose metabolites primarily consist of basic flavor compounds and lack the targeted enhancement of key flavor components (such as lactitol, small peptides, and nucleotides). For example, the small peptides found in conventional fermented whey have a small molecular weight and are easily degraded by molecular chain breakage at high temperatures. While lactitol provides a sweet and smooth sensation, it lacks a heat-protective structure and is susceptible to decomposition or volatilization at prolonged high temperatures. Furthermore, traditional fermentation processes lack precise control over fermentation conditions (such as temperature, pH, and bacterial strain combination), resulting in low flavor production efficiency and a limited variety of flavor compounds. This makes it difficult to achieve the complex flavor profiles associated with the synergistic "creamy richness" and "fermented freshness" characteristic of natural dairy products.

[0004] To improve the high-temperature resistance of fermented flavors, existing technologies attempt to modify flavoring compounds through physical encapsulation (such as cyclodextrin encapsulation) or simple enzymatic hydrolysis (such as protease hydrolysis), but these methods remain unsatisfactory. While physical encapsulation can temporarily isolate some flavoring compounds and reduce volatilization losses at high temperatures, the encapsulation carrier (such as cyclodextrin) is susceptible to structural damage and loss of encapsulation capacity in high-temperature environments. Simple enzymatic hydrolysis only alters the molecular structure of flavoring compounds (such as hydrolyzing large proteins into small peptides) without introducing stable thermal protective groups or synergistic ingredients. The modified small peptides are still susceptible to degradation at high temperatures due to their inherent structural instability. More importantly, these technologies fail to systematically optimize the core flavor components of milk flavors (such as the sweetness of lactitol, the freshness of small peptide-nucleotides, and the naturalness of frankincense). As a result, the modified flavors still suffer from severe flavor loss and a monotonous texture after high-temperature processing, making them difficult to meet the demand for high-quality milk flavors in industrial production. Summary of the Invention

[0005] The purpose of the present invention is to provide a high-temperature resistant milk flavor fermentation composition and a preparation method thereof, which solves the problems of severe flavor loss during high-temperature processing of traditional milk flavors, single flavor levels and poor storage stability at room temperature.

[0006] The present invention achieves the above-mentioned purpose through the following technical solutions: A high-temperature resistant milk flavor fermentation composition, wherein the raw materials thereof comprise, by mass percentage: Lactose graft: 20-30%; Fresh peptide nucleotides: 15-25%; Fermented whey liquid: 25-35%; Yeast extract: 8-12%; Xylooligosaccharides: 3-5%; Ascorbyl palmitate: 0.1-0.3%; Composite fermentation agent: 0.2-0.5%; The preparation method of the lactose graft comprises the following steps: A1, preparing lactitol into an aqueous solution, adjusting the pH to 6.7-6.9, inoculating Geobacillus stearothermophilus, and carrying out anaerobic fermentation at 44-46°C; after the fermentation is completed, centrifuging to remove the bacteria, adding brewer's yeast cell wall extract to the supernatant, heating to 54-56°C, and reacting with pullulanase for 4 hours; A2, subsequently adding cyclodextrin glucosyltransferase, reacting at 59-61°C; finally heating to 88-92°C to inactivate the enzyme, and spray drying.

[0007] The core of the lactose-glucan grafted product of this invention lies in the construction of a stable "β-glucan heat-protective shell" through microbial enzymatic reactions. During anaerobic fermentation, Geobacillus stearothermophilus first secretes β-glucanase, which specifically recognizes β-glucans (molecular structures containing β-1,3 and β-1,6 glycosidic bonds) in brewer's yeast cell wall extracts and cleaves them into short-chain oligosaccharides (such as cellotriose and cellotetraose). Subsequently, pullulanase (α-1,6 glycosidic bond hydrolase) and cyclodextrin glucosyltransferase (α-1,6 glycosidic bond transferase) work synergistically. Pullulanase cleaves the α-1,6 glycosidic bond around the C6 hydroxyl group of the lactitol molecule, exposing the reactive site. The cyclodextrin glucosyltransferase then catalyzes the grafting of the C3 hydroxyl group of the β-glucan oligosaccharide with the C6 hydroxyl group of lactitol via an α-1,6 glycosidic bond, forming a "β-glucan-lactitol" covalent complex. This grafted structure significantly hinders the thermal motion of lactitol molecules at high temperatures through the triple-helix steric hindrance effect of β-glucan, reducing its volatilization or decomposition due to molecular chain breakage; at the same time, the hydrophobic outer layer of β-glucan can isolate lactitol from oxygen and inhibit oxidative degradation, thereby retaining more than 90% of the sweet groups of lactitol at a high temperature of 135°C.

[0008] According to a preferred embodiment of the present invention, the lactitol is purchased from Shandong Longli Biotechnology Co., Ltd., and the model is L-AR-01 (food grade, purity ≥98%, crystalline granule type).

[0009] According to a preferred embodiment of the present invention, the Geobacillus stearothermophilus was purchased from the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC) with the collection number CGMCC 1.557 (viable ≥ 1×10 9 CFU / mL, optimal growth temperature 45°C).

[0010] According to a preferred embodiment of the present invention, the brewer's yeast cell wall extract is purchased from Angel Yeast Co., Ltd., model number is AngelYeast BM-01 (β-glucan content ≥70%, food grade, powdered).

[0011] According to a preferred embodiment of the present invention, the pullulanase was purchased from Danisco (Zhangjiagang) Biotechnology Co., Ltd., model Danisco PUL-01 (food grade, optimum temperature 55°C, pH=5.0-7.0, enzyme activity ≥500 U / g).

[0012] According to a preferred embodiment of the present invention, the cyclodextrin glucosyltransferase was purchased from Novozymes (China) Biotechnology Co., Ltd., model Novozyme 435 (food grade, optimum temperature 60°C, pH=6.0-7.0, enzyme activity ≥1000U / g).

[0013] According to a preferred embodiment of the present invention, the yeast extract is purchased from Angel Yeast Co., Ltd., and the model is AngelYeast YE-01 (nucleotide content ≥5%, food grade, liquid).

[0014] According to a preferred embodiment of the present invention, the xylo-oligosaccharide is purchased from Shandong Longli Biotechnology Co., Ltd., and the model is L-XYLO-01 (polymerization degree 2-7, purity ≥95%, powdered).

[0015] According to a preferred embodiment of the present invention, the ascorbyl palmitate is purchased from BASF (China) Co., Ltd., model BASF CYS-01 (active ingredient content ≥98%, food grade, oil-soluble liquid).

[0016] According to a preferred embodiment of the present invention, the composite fermentation bacteria agent was purchased from Shanghai Kaisai Biotechnology Co., Ltd., model number Kaisai Bio-01 (total bacterial activity ≥ 2×10 9 CFU / g, containing Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus plantarum, etc., in powder form).

[0017] According to a preferred embodiment of the present invention, in step A1, the weight volume percentage of the lactitol aqueous solution is 24-26%; the anaerobic fermentation time is 22-26 hours; the centrifugal speed is 12000-13000 rpm, and the centrifugation time is 10-14 minutes; the weight volume percentage of the brewer's yeast cell wall extract is 4-6%; the weight volume percentage of the pullulanase is 0.09-0.11%; and the reaction time is 3-5 hours.

[0018] According to a preferred embodiment of the present invention, in step A2, the weight volume percentage of cyclodextrin glucosyltransferase is 0.04-0.06%; the reaction time is 2-4 hours; the enzyme inactivation time is 15-20 minutes; the inlet air temperature of the spray drying is 200-210°C, and the outlet air temperature is 85-90°C.

[0019] According to a preferred embodiment of the present invention, the preparation method of the fresh peptide nucleotide includes: B1, preparing concentrated whey protein into an aqueous solution, adjusting the pH to 7.1-7.3, adding a composite protease, and hydrolyzing at 54-56°C; inactivating the enzyme at 94-96°C, cooling to 39-41°C, inoculating a mixed bacterial agent of thermophilic Streptomyces and Lactobacillus plantarum, and performing anaerobic fermentation at 39-41°C; B2, centrifuging the fermentation liquid to remove bacteria, filtering the supernatant through an organic filter membrane, adding activated carbon to the filtrate for decolorization, and spray drying to obtain the fresh peptide nucleotide.

[0020] According to a preferred embodiment of the present invention, the concentrated whey protein is purchased from Angel Yeast Co., Ltd., and the model is AngelWhey 80 (protein content ≥80%, food grade, powdered, lactose content ≤0.5%).

[0021] According to a preferred embodiment of the present invention, the composite protease is purchased from Weilan Biotechnology Co., Ltd., model WL-PP-01 (food grade, containing Alcalase 2.4L and Flavourzyme 500L, optimal temperature 55°C, pH 5.0-7.0, enzyme activity ≥1000 U / g).

[0022] According to a preferred embodiment of the present invention, the thermospring Streptomyces was purchased from the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC) with the collection number CGMCC 1.243 (bacterial activity ≥ 1×10 9 CFU / mL, the optimal growth temperature is 70℃, and it produces heat-resistant protease (optimum temperature 70℃, enzyme activity ≥800 U / mL).

[0023] According to a preferred embodiment of the present invention, the plant lactobacillus was purchased from the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC) with the collection number CGMCC 1.557 (bacterial activity ≥ 1×10 9 CFU / mL, the optimal growth temperature is 37°C, and the production of 5'-inosinic acid disodium (IMP) ≥ 500 mg / L).

[0024] According to a preferred embodiment of the present invention, the organic filter membrane is purchased from Hangzhou Beidouxing Membrane Products Co., Ltd., model BDS-PVDF-045 (polyvinylidene fluoride material, pore size 0.45 μm, food grade, high filtration accuracy, molecular weight cut-off ≤ 10 kDa).

[0025] According to a preferred embodiment of the present invention, the activated carbon was purchased from Shanxi Huaqing Environmental Protection Co., Ltd., model HX-AC-01 (granular coconut shell activated carbon, iodine value ≥800 mg / g, food grade, specific surface area ≥1000 m² / g, decolorization efficiency ≥90%).

[0026] The flavor stability of the fresh peptide nucleotides in this invention stems from the dually stable structure of the "small peptide-nucleotide complex." Whey protein concentrate is hydrolyzed by a combined protease (Alcalase 2.4L and Flavorzyme 500L) to produce small peptides with a molecular weight of ≤800 Da (primarily composed of hydrophobic peptide segments such as leucine-proline-glycine and valine-isoleucine-alanine). These peptides' amino termini (-NH2), due to their short and highly hydrophobic nature, are more susceptible to electrostatic bonding with the phosphate groups of nucleotides. During the co-fermentation process with Streptomyces thermophilus and Lactobacillus plantarum, thermophilic proteases secreted by Streptomyces thermophilus further modify the peptide termini, converting the carboxyl groups (-COOH) of some peptides into amide groups (-CONH2), reducing their polarity. Lactobacillus plantarum metabolizes these peptides to produce disodium 5'-inosinic acid (IMP) and disodium 5'-guanylate (GMP), whose phosphate groups form dual "ionic and hydrogen bonds" with the amino termini of the small peptides. This complex is doubly stabilized by hydrophobic interactions (the hydrophobic side chain of the small peptide and the ribose ring of the nucleotide) and electrostatic interactions (amino groups and phosphate groups). It is not easily degraded by peptide chain breakage or nucleotide dephosphorylation at high temperatures. The umami threshold is 40% lower than that of a single small peptide, and the retention rate is ≥88% after treatment at 135℃ / 5min.

[0027] According to a preferred embodiment of the present invention, in step B1, the weight volume percentage of the concentrated whey protein aqueous solution is 11-13%; the weight volume percentage of the composite protease is 0.07-0.09%; the enzyme inactivation time is 10-15 minutes; the volume ratio of Streptomyces thermophilus to Lactobacillus plantarum in the mixed bacterial agent is (2-4):1; and the anaerobic fermentation time is 18-20 hours.

[0028] According to a preferred embodiment of the present invention, in step B2, the centrifugal speed is 14000-16000 rpm, the time is 15-20 min; the pore size of the organic filter membrane is 0.44-0.46 μm; the weight volume percentage of the activated carbon is 0.1-0.12%; the decolorization time is 30-40 min; the spray drying inlet temperature is 194-196°C, and the outlet temperature is 74-76°C.

[0029] According to a preferred embodiment of the present invention, the method for preparing the fermented whey liquid comprises: removing fat from fresh milk by centrifugation, pasteurizing at 85-86°C for 15-20 minutes, cooling to 36-38°C, and fermenting at 37-38°C for 12-14 hours until the pH drops to 4.5-4.7; and collecting the whey liquid by centrifugation.

[0030] According to a preferred embodiment of the present invention, the fresh milk was purchased from Inner Mongolia Yili Industrial Group Co., Ltd., model number YD-GY-01 (protein content ≥3.2g / 100mL, fat content ≥3.3g / 100mL, total bacterial count ≤3×104 CFU / mL).

[0031] The accumulation of frankincense compounds (such as diacetyl and acetaldehyde) in the fermented whey of this invention relies on the metabolic synergy of strains such as Lactobacillus delbrueckii subsp. After fresh milk is centrifuged, defatted, and pasteurized, Lactobacillus delbrueckii subsp. bulgaricus dominates the fermentation process: β-galactosidase secreted by Lactobacillus delbrueckii converts lactose into galactose (providing a substrate for the lactoglucose graft). Lactate dehydrogenase simultaneously catalyzes the reduction of pyruvate to lactic acid (lowering the pH to 4.5-4.7), inhibiting the growth of contaminants and activating its own encoded thermostable protease (such as PrtB protease). This protease further hydrolyzes β-casein in the whey protein to produce flavor precursors such as β-casomorphin. Furthermore, the galactose produced by lactose conversion is metabolized through the phosphogluconate pathway to produce intermediates such as α-ketoglutarate, which is then decarboxylated to acetaldehyde (the characteristic fresh aroma). Some amino acids, such as leucine, are degraded by Strecker to form isovaleraldehyde (a precursor to creamy aroma), ultimately forming a natural frankincense system dominated by diacetyl (creamy sweetness) and acetaldehyde (fresh grassy aroma). These flavor compounds are adsorbed by macromolecules such as proteins and polysaccharides in the fermentation broth, forming a "flavor-matrix" complex, which reduces volatilization losses at high temperatures.

[0032] The present invention also provides a method for preparing the high-temperature resistant milk flavor fermentation composition, comprising the steps of: S1, adding lactoglucose grafted material, fresh peptide nucleotide, fermented whey liquid, yeast extract, and xylo-oligosaccharide into a three-dimensional mixer and mixing; S2. Then, ascorbyl palmitate was added and the mixture was mixed continuously; finally, the composite fermentation agent was added and the mixture was mixed under nitrogen protection.

[0033] The components of the present invention form a "carrier-flavor-protection" trinity system through metabolic networks and physical and chemical reactions: the lactoglucose graft serves as a hydrophobic carrier to encapsulate fresh peptide nucleotides and polar flavor substances (such as diacetyl) in fermented whey, reducing their contact with water and inhibiting hydrolysis; the 5'-inosinic acid disodium supplemented by the yeast extract forms a "nucleotide synergistic network" with the IMP / GMP in the fresh peptide nucleotides, which enhances the perception of umami through cross-activation of taste receptors (such as T1R1 / T1R3); oligoxylose (XOS) serves as a prebiotic to selectively proliferate Lactobacillus plantarum, and the lactic acid produced by its metabolism further lowers the pH of the system, inhibiting the production of amines (such as cadaverine and putrescine) by spoilage bacteria (such as Pseudomonas), reducing flavor deterioration; the palmitic acid chain of ascorbyl palmitate is anchored in the lactic acid of the milk The surface of lipid substances blocks the auto-oxidation chain reaction of unsaturated fatty acids at high temperatures (ROO·free radical scavenging rate ≥80%) and reduces the peroxide value (≤5meq / kg). The functional strains in the composite fermentation agent (Geobacilus stearothermophilus, Streptomyces thermophilus, Lactobacillus plantarum, and Lactobacillus delbrueckii subsp. bulgaricus) achieve dynamic regulation through metabolic complementarity: Geobacilus stearothermophilus maintains high temperature adaptability (optimum growth temperature 45°C) to ensure enzyme activity in the later stages of fermentation (such as before spray drying); Streptomyces thermophilus secretes heat-resistant proteases (optimum temperature 70°C) to adapt to the high temperature environment of the fermentation and drying processes; Lactobacillus plantarum produces acid to regulate pH, providing a suitable microenvironment for other strains; Lactobacillus delbrueckii subsp. bulgaricus continuously converts lactose to ensure the substrate supply for the lacto-glucose graft.

[0034] According to a preferred embodiment of the present invention, in step S1, the mixing speed is 15-20 rpm, and the mixing time is 20-30 min.

[0035] According to a preferred embodiment of the present invention, in step S2, the mixing time is continued for 10-15 minutes; and the mixing time under nitrogen protection is 5-10 minutes.

[0036] The beneficial effects of the present invention are: The heat-resistant milk-flavored fermented composition described in this invention, through multi-dimensional ingredient design and process optimization, exhibits significant flavor stability advantages even in high-temperature processing scenarios. The composition utilizes a lacto-glucan graft as a core carrier, and its internal β-glucan heat-protective shell effectively blocks heat damage to the lactitol molecular structure, reducing flavor loss caused by excessive Maillard reactions. The complex formed by the small peptides in the umami nucleotides and disodium 5'-inosinate and disodium 5'-guanylate maintains a more stable molecular structure at high temperatures, making umami substances less susceptible to decomposition. Furthermore, after the fermented whey liquid undergoes a three-strain gradient fermentation, the volatility of natural frankincense substances (such as diacetyl and acetaldehyde) is reduced, retaining a rich, creamy, and mellow flavor even at high temperatures.

[0037] Compared to commercially available products, the composition of this invention offers a richer and more multifaceted flavor profile. The slightly sweet base provided by the lactoglucan graft complements the fermented, refreshing flavor of the fresh peptide nucleotides. The former mitigates the sharpness of a single umami flavor, while the latter enhances the freshness and richness of the flavor. Naturally occurring frankincense compounds in the fermented whey (such as the refreshing aroma of acetaldehyde and the creamy aroma of diacetyl) serve as a foundation, organically blending sweetness and freshness to create a multi-layered flavor structure with a refreshing sweetness upfront, a mellow freshness mid-tone, and a frankincense finish. This complex flavor profile transcends the limitations of traditional milk flavors, which rely solely on chemically synthesized sweeteners or single enzymatic hydrolysis products. It more closely reflects the flavor characteristics of natural dairy products and significantly enhances the product's sensory quality.

[0038] The composition of the present invention also exhibits outstanding long-term stability. Ascorbyl palmitate, an oil-soluble antioxidant, effectively inhibits the oxidation of unsaturated fatty acids during high-temperature storage, reducing the peroxide value. Antibacterial substances metabolized by the functional strains (such as Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus plantarum) in the composite fermentation agent inhibit the growth of spoilage bacteria and reduce the degradation of flavor substances. DETAILED DESCRIPTION

[0039] The following specific implementation methods are only used to further illustrate the present application and should not be understood as limiting the scope of protection of the present application. Technicians in this field may make some non-essential improvements and adjustments to the present application based on the above application content.

[0040] 1. Implementation Example 1 25 g of lactitol was prepared into an aqueous solution (25% by weight and volume), the pH was adjusted to 6.8, and the solution was inoculated with Geobacillus stearothermophilus and anaerobically fermented at 45°C for 24 h. After the fermentation, the bacteria were removed by centrifugation at 12500 rpm for 12 min. 5 g of brewer's yeast cell wall extract (5% by weight and volume) was added to the supernatant. The solution was heated to 55°C and then 0.1 g of pullulanase (0.1% by weight and volume) was added and reacted for 4 h. Subsequently, 0.05 g of cyclodextrin glucosyltransferase (0.05% by weight and volume) was added and reacted at 59.5°C for 3 h. Finally, the solution was heated to 89°C for 18 min to inactivate the enzyme and spray dried (inlet air temperature 205°C, outlet air temperature 88°C) to obtain a lacto-glucose grafted product.

[0041] 12 g of concentrated whey protein was prepared into an aqueous solution (weight volume percentage 12%), the pH was adjusted to 7.2, 0.08 g of composite protease (weight volume percentage 0.08%) was added, and hydrolysis was carried out at 55°C for 3 h; the enzyme was inactivated at 95°C for 12 min, then cooled to 40°C, and a mixed bacterial agent of thermophilic Streptomyces and plantarum Lactobacillus was inoculated (volume ratio 3:1), and anaerobically fermented at 40°C for 19 h; the fermentation broth was centrifuged at 15000 rpm for 18 min to remove the bacteria, the supernatant was filtered through an organic filter membrane (pore size 0.45 μm), 0.11 g of activated carbon (weight volume percentage 0.11%) was added to the filtrate for decolorization for 35 min, and spray dried (inlet air temperature 195°C, outlet air temperature 75°C) to obtain fresh peptide nucleotides.

[0042] Take 100g of fresh milk, centrifuge to remove fat, pasteurize at 85.5℃ for 18min, cool to 37℃, inoculate with fermentation agent, ferment at 37.5℃ for 13h until the pH drops to 4.6, and collect the whey liquid by centrifugation.

[0043] 25 g of the lactoglucose grafted material prepared above, 20 g of fresh peptide nucleotides, 30 g of fermented whey liquid, 10 g of yeast extract, 4 g of xylo-oligosaccharides, and 0.2 g of ascorbyl palmitate were added to a three-dimensional mixer and mixed at a speed of 18 rpm for 25 min; then ascorbyl palmitate was added and mixing was continued for 12 min; finally, 0.3 g of a composite fermentation agent was added under nitrogen protection and mixed for 8 min to obtain a high-temperature resistant milk flavor fermentation composition.

[0044] Example 2 The specific preparation method is the same as that in Example 1, except that the following weight ratios are weighed: 22g of lactose grafted material, 23g of fresh peptide nucleotides, 32g of fermented whey liquid, 9g of yeast extract, 5g of xylo-oligosaccharides, 0.25g of ascorbyl palmitate, and 0.4g of a composite fermentation agent. Preparation of the lactose grafted material: A 24.5% (weight / volume) aqueous solution of lactitol, anaerobic fermentation for 23 hours, centrifugation at 12,800 rpm (for 11 minutes), pullulanase reaction for 3.5 hours, and cyclodextrin glucosyltransferase reaction for 3.5 hours. Preparation of the fresh peptide nucleotides: A 12.5% (weight / volume) aqueous solution of whey protein concentrate, a 3.5-hour composite protease reaction, enzyme inactivation for 13 minutes, and fermentation for 19.5 hours. Preparation of the fermented whey liquid: Pasteurization for 17 minutes, fermentation for 12.5 hours.

[0045] Example 3 The specific preparation method is the same as that in Example 1, except that the following weight ratios are weighed: 28g of lactose grafted material, 18g of fresh peptide nucleotides, 34g of fermented whey liquid, 11g of yeast extract, 3g of xylo-oligosaccharides, 0.15g of ascorbyl palmitate, and 0.35g of a composite fermentation agent. Preparation of the lactose grafted material: 25.5% by weight of a lactitol aqueous solution, anaerobic fermentation for 25 hours, centrifugation at 12,200 rpm (for 13 minutes), pullulanase reaction for 4.5 hours, and cyclodextrin glucosyltransferase reaction for 2.5 hours. Preparation of the fresh peptide nucleotides: 11.5% by weight of a whey protein concentrate aqueous solution, 2.5 hours of composite protease reaction, 14 minutes of enzyme inactivation, and 18.5 hours of fermentation. Preparation of the fermented whey liquid: 19 minutes of pasteurization and 13.5 hours of fermentation.

[0046] Comparative Example 1 The specific preparation method is the same as that of Example 1, except that the following are weighed in mass ratio: 18 g (less than 20%) of lactoglucose graft, 20 g of fresh peptide nucleotide, 30 g of fermented whey liquid, 10 g of yeast extract, 4 g of xylo-oligosaccharide, 0.2 g of ascorbyl palmitate, and 0.3 g of composite fermentation agent.

[0047] Comparative Example 2 The specific preparation method is the same as that of Example 1, except that the following are weighed in mass ratio: 25 g of lactoglucose graft, 26 g of fresh peptide nucleotide (higher than 25%), 30 g of fermented whey liquid, 10 g of yeast extract, 4 g of xylo-oligosaccharide, 0.2 g of ascorbyl palmitate, and 0.3 g of composite fermentation agent.

[0048] Comparative Example 3 Weigh according to the mass ratio: 25g of lactoglucose grafted material, 20g of fresh peptide nucleotide, 30g of fermented whey liquid, 10g of yeast extract, 4g of oligoxylose, and 0.2g of ascorbyl palmitate (omit the composite fermentation agent).

[0049] 2. Performance Testing The compositions prepared in Examples 1-3 and Comparative Examples 1-3 were subjected to performance tests according to the following methods: 1. High-temperature resistance test: Gas chromatography-mass spectrometry (GC-MS, instrument model: Agilent 7890B-5977B, chromatographic column: HP-5MS capillary column (30m×0.25mm×0.25μm), carrier gas: helium (flow rate: 1mL / min), column temperature program: initial 50°C for 2 minutes, then increase to 250°C at 5°C / min and hold for 5 minutes) was used to determine the total retention rate of flavor substances (diacetyl, acetaldehyde, and δ-decalactone) before and after heating (calculation formula: retention rate = (peak area after heating / peak area before heating) × 100%). 2. Flavor Evaluation: Ten professional sensory panelists (trained in ISO 8586-1:2012) conducted a blind evaluation of the heated samples for frankincense (diacetyl / acetaldehyde), fermented flavor (small peptide-nucleotide complex), and subtle sweetness (sweetness of lacto-glucose grafted compounds). The scoring scale was 1-10 (10 being the best). The frankincense was described as "rich creamy aroma with a hint of grassy fragrance," the fermented flavor as "fresh fermented flavor without rancidity," and the subtle sweetness as "natural lactose sweetness without a cloying sweetness." 3. Microbial stability test: The samples were stored in a 25°C incubator for 6 months. Samples were taken at 0, 1, 3, and 6 months, and the number of spoilage bacteria (Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 6538) was detected by plate count method. The total colony count (CFU / g) was calculated. 4. Verification of thermal protection structure: The microstructure of the lactoglucan graft was observed using a scanning electron microscope (SEM, ZEISS Gemini 300) (accelerating voltage 5 kV, working distance 10 mm), and the β-glucan content was determined using an anthrone colorimetric method (detection wavelength 620 nm, standard β-glucan solution). 5. Umami stability test: High performance liquid chromatography (HPLC, instrument model: Waters e2695, chromatographic column: C18 column (4.6 mm × 250 mm × 5 μm), mobile phase: acetonitrile-0.1% formic acid aqueous solution (15:85), flow rate 1 mL / min, detection wavelength 254 nm) was used to determine the retention rates of small peptides (molecular weight ≤ 800 Da, pretreated with ultrafiltration tubes with a molecular weight cutoff of 10 kDa) and nucleotides (disodium 5'-inosinate and disodium 5'-guanylate, separated by ion exchange chromatography) (calculation formula: retention rate = (peak area after heating / peak area before heating) × 100%).

[0050] 6. Performance test results: Table 1: Performance test results of various embodiments and comparative examples

[0051] As can be seen from Table 1, the present invention effectively solves the problems of severe flavor loss during high-temperature processing, single flavor level, and poor storage stability at room temperature of traditional milk flavors through multi-dimensional component combination and process optimization. The test data show that the high-temperature resistance of Examples 1-3 (total flavor retention rate of 88%-92%) is significantly higher than that of the comparative example (75%-82%), indicating that the thermal protection structure of the lactoglucan graft (β-glucan content of 3.6%-4.2%) effectively blocks the decomposition and volatilization of flavor substances caused by high temperature, solving the problem of flavor loss during high-temperature processing. In the flavor level score, the frankincense (9.1), fermented freshness (8.8), and slightly sweetness (8.5) of Example 1 are all better than those of the comparative example (the highest is 7.9). This is due to the multi-component synergy of the lactoglucan graft (sweet base), fresh peptide nucleotides (fermented fresh source), and fermented whey (natural frankincense), which avoids the dominance of a single flavor substance and enriches the flavor level. The flavor degradation rate at room temperature for 6 months is only 2.8%-3.5% (6.3%-8.2% for the comparative example), and the number of microorganisms is undetectable or extremely low (<100 CFU / g for Examples 1-3 and 1.2×10 4 CFU / g), attributed to the synergistic antibacterial effect of the complex fermentation agent (inhibiting spoilage bacteria metabolism) and xylooligosaccharides (prebiotics regulating bacterial flora), significantly improving storage stability. Furthermore, the positive correlation between β-glucan content and small peptide and nucleotide retention rates further validates the key role of thermal protection structures in the stability of umami substances, comprehensively addressing the core pain points of traditional milk flavors.

[0052] The above-described embodiments merely illustrate several implementations of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, and all such variations and improvements fall within the scope of protection of the present invention.

Claims

1. A high temperature resistant milk flavor fermentation composition, characterized in that: In terms of mass percentage, the raw materials include: Lactose graft: 20-30%; Fresh peptide nucleotides: 15-25%; Fermented whey liquid: 25-35%; Yeast extract: 8-12%; Xylooligosaccharides: 3-5%; Ascorbyl palmitate: 0.1-0.3%; Composite fermentation agent: 0.2-0.5%; The preparation method of the lactose graft comprises the following steps: A1, preparing lactitol into an aqueous solution, adjusting the pH to 6.7-6.9, inoculating Geobacillus stearothermophilus, and carrying out anaerobic fermentation at 44-46°C; after the fermentation is completed, centrifuging to remove the bacteria, adding brewer's yeast cell wall extract to the supernatant, heating to 54-56°C, and reacting with pullulanase for 4 hours; A2, subsequently adding cyclodextrin glucosyltransferase, reacting at 59-61°C; finally heating to 88-92°C to inactivate the enzyme, and spray drying.

2. The high temperature resistant milk flavor fermentation composition according to claim 1, characterized in that In step A1, the weight volume percentage of the lactitol aqueous solution is 24-26%; the anaerobic fermentation time is 22-26 hours; the centrifugation speed is 12000-13000 rpm, and the centrifugation time is 10-14 minutes; the weight volume percentage of the brewer's yeast cell wall extract is 4-6%; the weight volume percentage of the pullulanase is 0.09-0.11%; and the reaction time is 3-5 hours.

3. The high temperature resistant milk flavor fermentation composition according to claim 1, characterized in that In step A2, the weight volume percentage of cyclodextrin glucosyltransferase is 0.04-0.06%; the reaction time is 2-4 hours; the enzyme inactivation time is 15-20 minutes; the inlet air temperature of the spray drying is 200-210°C, and the outlet air temperature is 85-90°C.

4. The high temperature resistant milk flavor fermentation composition according to claim 1, characterized in that The preparation method of the fresh peptide nucleotide comprises the following steps: B1, preparing concentrated whey protein into an aqueous solution, adjusting the pH to 7.1-7.3, adding a composite protease, and hydrolyzing at 54-56° C.; inactivating the enzyme at 94-96° C., cooling to 39-41° C., inoculating a mixed bacterial agent of Streptomyces thermophilus and Lactobacillus plantarum, and performing anaerobically fermenting at 39-41° C.; B2, centrifuging the fermentation liquid to remove bacterial cells, filtering the supernatant through an organic filter membrane, adding activated carbon to the filtrate for decolorization, and spray drying to obtain the fresh peptide nucleotide.

5. The high temperature resistant milk flavor fermentation composition according to claim 4, characterized in that In step B1, the weight volume percentage of the concentrated whey protein aqueous solution is 11-13%; the weight volume percentage of the composite protease is 0.07-0.09%; the enzyme inactivation time is 10-15 minutes; the volume ratio of Streptomyces thermophilus to Lactobacillus plantarum in the mixed bacterial agent is (2-4):1; and the anaerobic fermentation time is 18-20 hours.

6. The high temperature resistant milk flavor fermentation composition according to claim 4, characterized in that In step B2, the centrifugation speed is 14,000-16,000 rpm, and the time is 15-20 minutes; the pore size of the organic filter membrane is 0.44-0.46 μm; the weight volume percentage of the activated carbon is 0.1-0.12%; the decolorization time is 30-40 minutes; the spray drying inlet air temperature is 194-196°C, and the outlet air temperature is 74-76°C.

7. The high temperature resistant milk flavor fermentation composition according to claim 1, characterized in that The preparation method of the fermented whey liquid comprises: removing fat from fresh milk by centrifugation, pasteurizing at 85-86° C. for 15-20 minutes, cooling to 36-38° C., fermenting at 37-38° C. for 12-14 hours until the pH value drops to 4.5-4.7; and collecting the whey liquid by centrifugation.

8. A method for preparing the high temperature resistant milk flavor fermentation composition according to any one of claims 1 to 7, characterized in that the steps include: S1, adding lactoglucose grafted material, fresh peptide nucleotide, fermented whey liquid, yeast extract, and xylo-oligosaccharide into a three-dimensional mixer and mixing; S2. Then, ascorbyl palmitate was added and the mixture was mixed continuously; finally, the composite fermentation agent was added and the mixture was mixed under nitrogen protection.

9. The preparation method according to claim 8, characterized in that In step S1, the mixing speed is 15-20 rpm and the mixing time is 20-30 min.

10. The preparation method according to claim 8, characterized in that In step S2, the mixing time is continued for 10-15 minutes; the mixing time under nitrogen protection is 5-10 minutes.

Images