Preparation method of oat plant-based fermented milk enriched with polyphenols based on combined enzymatic hydrolysis-fermentation technology

Abstract

The application discloses a preparation method of oat plant-based fermented milk rich in polyphenols based on a combined enzymolysis-fermentation technology. The preparation method comprises the following steps: after oat is soaked and cleaned, the oat is dried to obtain pretreated oat; the pretreated oat is baked and crushed, and then ball milled to obtain oat ultrafine powder; then, water is added to the oat ultrafine powder to obtain oat milk, the oat milk is stirred and subjected to high-speed shearing, the oat milk is completely dissolved and then gelatinized, and then enzyme is added to perform enzymolysis to obtain an enzymolysis solution; after the enzyme is inactivated, the enzymolysis solution is mixed and homogenized to obtain a homogenized solution, the homogenized solution is sterilized and cooled to obtain a uniform and stable system; finally, a strain is inoculated into the uniform and stable system to perform fermentation and post-ripening to obtain oat plant-based fermented milk. According to the preparation method, a nitrogen source does not need to be additionally added, the polyphenol content of the system and the acidity of the product can be effectively improved, and the fermentation time is shortened.

Description

Technical Field

[0001] This invention relates to the field of fermented milk preparation technology, specifically to a method for preparing oat-based fermented milk enriched with polyphenols using a combined enzymatic hydrolysis-fermentation technology. Background Technology

[0002] In recent years, consumer interest in and research into plant-based dairy products has been growing due to the increasing negative impacts of animal-derived products on human health, animal survival, and the environment. Dairy consumption is declining in many Western countries, while sales of plant-based milk are increasing. Plant-based milk currently accounts for 15% of the total dairy industry, and other plant-based dairy alternatives are gaining popularity. Compared to animal-based milk, plant-based milk consumes less energy in its production and emits fewer greenhouse gases per gram of protein, making it an attractive option for those looking to reduce their carbon footprint.

[0003] Soybeans are the most common ingredient in plant-based fermented milk. Due to their high protein content and beneficial amino acid composition, soybeans are very suitable as a base for plant-based yogurt alternatives. In recent years, other plant ingredients such as coconut and flaxseed have also gained popularity.

[0004] Oats, also known as naked oats, are one of the most nutritious grains, rich in carbohydrates, balanced protein, unsaturated fatty acids, vitamins, and minerals. They are characterized by being "high in three things and low in two," and possess both medicinal and edible properties, earning them FDA recognition as a functional food. However, their high starch content leads to excessively high viscosity during gelatinization, reducing consumer acceptability. Furthermore, because whole oats are used, retaining the bran, oat milk may have poor solubility.

[0005] Current research on oat-based fermented milk mainly focuses on the enzymatic hydrolysis process of oat starch and the influence of fermentation strains on oat fermented milk. Furthermore, the processing of oat products often leads to the loss of key nutrients due to over-processing, resulting in oat milk with low polyphenol content and low acidity, as well as long fermentation time and high cost. Summary of the Invention

[0006] To address the aforementioned shortcomings, this invention provides a method for preparing oat-based fermented milk enriched with polyphenols using a combined enzymatic hydrolysis-fermentation technology. Through research on enzyme and strain formulation and fermentation processes, this invention proposes a preparation method that effectively increases the polyphenol content and acidity of the product without requiring the addition of an extra nitrogen source, while simultaneously shortening the fermentation time.

[0007] The technical solution of the present invention is as follows:

[0008] The preparation method of oat-based fermented milk enriched with polyphenols based on combined enzymatic hydrolysis-fermentation technology includes the following steps:

[0009] (1) Soak and wash the oats, then dry them to obtain pretreated oats;

[0010] (2) The pretreated oats are baked, crushed, and ball-milled to obtain whole oat ultrafine powder;

[0011] (3) Add water to whole wheat ultrafine powder to obtain oat milk, stir, and shear at high speed. After the oat milk is completely dissolved, add enzyme to hydrolyze it to obtain enzymatic hydrolysate.

[0012] (4) After enzyme inactivation, the mixture is prepared and homogenized to obtain a homogenized solution, which is then sterilized and cooled to obtain a uniform and stable system.

[0013] (5) Add the bacterial strain for inoculation, fermentation and post-ripening to obtain oat plant-based fermented milk.

[0014] Further, in step (1), the oats are whole oat grains (oat grains containing bran); the drying temperature is 105℃ and the time is 20-40 min.

[0015] Furthermore, in step (1), drying refers to drying until the surface is dry and no burnt smell is produced.

[0016] Furthermore, in step (2), the ball milling frequency is 4000Hz and the time is 15min.

[0017] Furthermore, in step (2), the baking is performed by infrared baking; the pulverization is performed by grinding three times with a universal pulverizer and passing through a 40-mesh sieve.

[0018] Furthermore, in step (2), the baking temperature is 170-190℃ and the time is 10-20 minutes, with the cake being turned over every 2-4 minutes during baking.

[0019] Further, in step (3), the volume ratio of the whole wheat ultrafine powder to water is 1:5-8; the temperature of the water is 50-55℃, preferably, the volume ratio of the whole wheat ultrafine powder to water is 1:6; and the temperature of the water is 55℃.

[0020] Further, in step (3), the stirring speed is 300-500 r / min and the time is 10-20 min; the high-speed shearing speed is 4000-7000 r / min and the shearing time is 10-20 min; preferably, the high-speed shearing speed is 6000 rpm and the shearing time is 10-15 min; the gelatinization temperature is 80-90℃ and the time is 10-20 min.

[0021] Further, in step (3), the enzyme includes one or more of α-amylase, β-amylase, cellulase, and alkaline protease; the addition amount of each enzyme, E / S (percentage of enzyme powder to substrate, wherein the substrate is the gelatinized liquid before enzyme addition), is 0.1%-2%; all enzymes are purchased from Jiangsu Boli Biological Products Co., Ltd.; the enzymatic hydrolysis temperature is 55-65℃.

[0022] Further, in step (3), the specific process of adding enzymes for enzymatic hydrolysis is as follows: first, add α-amylase, β-amylase and cellulase and react at 55-65℃ for 90-120 min, then adjust the temperature to 55-65℃ and pH to 8, and then add alkaline protease to react; the reaction time is 30-60 min.

[0023] Further, in step (4), the preparation involves adding 1%-7% sucrose; the homogenization conditions are 20-30℃ and 30-40MPa, with homogenization performed 3 times; the sterilization is performed at 95℃ for 10 minutes; and the cooling refers to cooling to 43-48℃.

[0024] Further, in step (5), the bacterial strains are Lactobacillus acidophilus and Lactobacillus casei, with a volume ratio of 1:1; the concentration of each strain is 1×10⁻⁶. 8 CFU / mL; the total inoculum amount is 1% (v / v), that is, 1% activated bacterial solution is added to the culture medium base; the fermentation temperature is 37℃, and fermentation ends when the pH value of the fermentation broth is ≤4.5 and the acidity is ≥30°T, and then after-ripening is carried out; the after-ripening temperature is 4℃ and the time is 12-24h.

[0025] An oat-based fermented milk prepared by the aforementioned method.

[0026] The beneficial technical effects of this invention are as follows:

[0027] This invention utilizes a combined enzymatic hydrolysis-fermentation technology. Through the synergistic effect of fermentation and specific enzymes, bound polyphenols are released, thereby increasing the polyphenol content and enriching the product with polyphenols, thus enhancing its antioxidant activity. The enzymatic hydrolysis process effectively increases the acid production rate of lactic acid bacteria, thereby increasing acidity and shortening the fermentation time.

[0028] The nitrogen source required in the fermentation process described in this invention is entirely provided by oat protein from whole oats, without the need for additional nitrogen sources, thus saving production costs.

[0029] This invention uses ball milling to process whole oats, resulting in ultrafine oat flour that is not filtered. This improves grain utilization without producing a rough texture. The finished product has a good texture, a smooth and delicate taste, and a unique oat flavor.

[0030] This invention enables the use of whole oat milk as a fermentation base and fermentation with whole oat milk, without the presence of raw cow's milk, thus meeting the needs of many consumers, such as those allergic to dairy products.

[0031] This invention utilizes α-amylase to hydrolyze the α-1,4 glycosidic bonds in starch to generate small-molecule sugars. During hydrolysis, the ester bonds binding sugars and polyphenols are broken, releasing the polyphenols bound to starch. Pullulanase hydrolyzes starch, resulting in a product containing more free amylose molecules, thus maximizing the utilization of starch raw materials. Simultaneously, it promotes β-amylase activity, facilitating the formation of oligosaccharides and maltose as the main monosaccharides. Cellulase is used to enzymatically hydrolyze whole oat milk, effectively breaking down cellulose and hemicellulose in the bran, releasing bound phenolic acids, especially ferulic acid. Alkaline protease breaks down proteins in fermented oat milk into small-molecule peptides and amino acids, promoting the release of polyphenols bound to proteins. Protease treatment also helps release more free amino acids and small peptides from whole oat milk. These substances are efficient nitrogen sources, further promoting the growth and reproduction of lactic acid bacteria and fermentation acid production.

[0032] Furthermore, when using oats as a substrate, the strains used in this invention, specifically *Lactobacillus casei*, have a greater advantage in increasing the total polyphenol content than other strains. This may be because, during fermentation, enzymes secreted by *Lactobacillus casei* hydrolyze the chemical bonds between oat polysaccharides, proteins, or cell walls and polyphenols, leading to the release of a large amount of free polyphenols. At the same time, metabolites may weaken the ether bonds between bound polyphenolic compounds and the cell wall, making them easier to release. Then, cellulase can effectively release bound polyphenols into free polyphenols, greatly increasing the free polyphenol content in oat-based fermented milk. Alternatively, it may be due to the production of new polyphenolic compounds or the enzymatic degradation of large-molecule polyphenolic compounds, resulting in an increase in the free polyphenol content after fermentation.

[0033] The present invention also found that Lactobacillus acidophilus is more suitable for growth than other strains when oats are used as a substrate. Therefore, the two strains were combined for fermentation, which increased the total polyphenol content and acidity in the oat plant-based fermented milk and greatly shortened the fermentation time. Detailed Implementation

[0034] The present invention will now be described in detail with reference to the embodiments.

[0035] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0036] Secondly, the term "an embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment of the invention" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0037] Polyphenols in oats bind to polysaccharides, proteins, and cell wall components in a bound state. These bound polyphenols can be released through fermentation and hydrolysis by corresponding enzymes. Existing research indicates that polyphenols in starch granules can react with amylose to form cage-like inclusions and can also bind to the side chains of amylopectin and the amorphous regions of starch granules.

[0038] This invention protects a method for preparing oat-based fermented milk enriched with polyphenols using a combined enzymatic hydrolysis-fermentation technology, the method comprising the following steps:

[0039] (1) Soak and wash the oats, then dry them to obtain pretreated oats;

[0040] (2) The pretreated oats are baked, crushed, and ball-milled to obtain whole oat ultrafine powder;

[0041] (3) Add water to whole wheat ultrafine powder to obtain oat milk, stir, and shear at high speed. After the oat milk is completely dissolved, add enzyme to hydrolyze it to obtain enzymatic hydrolysate.

[0042] (4) After enzyme inactivation, the mixture is prepared and homogenized to obtain a homogenized solution, which is then sterilized and cooled to obtain a uniform and stable system.

[0043] (5) Add the bacterial strain for inoculation, fermentation and post-ripening to obtain oat plant-based fermented milk.

[0044] In one embodiment of the present invention, in step (1), the oats are whole oat grains (oat grains containing bran); the drying temperature is 105°C and the time is 20 min, 30 min or 40 min.

[0045] In one embodiment of the present invention, in step (1), drying refers to drying until the surface is dry and no burnt smell is produced.

[0046] In one embodiment of the present invention, in step (2), the ball milling frequency is 4000 Hz and the time is 15 min.

[0047] In one embodiment of the present invention, in step (2), the baking is performed by infrared baking; the pulverization is performed by grinding three times with a universal pulverizer and passing through a 40-mesh sieve, and the number of grinding times is not limited to three times.

[0048] In one embodiment of the present invention, in step (2), the baking temperature is 170°C, 180°C, 185°C or 190°C, the time is 10 min, 15 min or 20 min, and the cake is turned over every 2 min, 3 min or 4 min during baking.

[0049] In one embodiment of the present invention, in step (3), the volume ratio of the whole wheat ultrafine powder to water is 1:5; 1:6 or 1:8; the temperature of the water is 50-55℃, preferably, the volume ratio of the whole wheat ultrafine powder to water is 1:6; the temperature of the water is 55℃.

[0050] In one embodiment of the present invention, in step (3), the stirring speed is 300-500 r / min and the time is 10 min, 15 min or 20 min; the high-speed shearing speed is 4000 r / min, 6000 r / min or 7000 r / min and the shearing time is 10 min, 15 min or 20 min; preferably, the high-speed shearing speed is 6000 rpm; the gelatinization temperature is 80℃, 85℃ or 90℃ and the time is 10 min, 15 min or 20 min.

[0051] In one embodiment of the present invention, in step (3), the enzyme includes one or more of α-amylase, β-amylase, cellulase, and alkaline protease; the enzyme addition amount E / S (percentage of enzyme powder to substrate, wherein the substrate is the gelatinized liquid before enzyme addition) is 0.15%, 0.5%, 0.6%, 1%, 1.5%, or 2%; all enzymes are purchased from Jiangsu Boli Biological Products Co., Ltd.; the specific process of enzyme addition for enzymatic hydrolysis is as follows: first, α-amylase, β-amylase, and cellulase are added and reacted at 55℃, 58℃, 60℃, or 65℃ for 90 min, 100 min, 110 min, or 120 min, respectively; then, the temperature is adjusted to 55℃, 58℃, 60℃, or 65℃, and the pH is 8; then, alkaline protease is added and reacted for 30 min, 45 min, or 60 min to obtain the final product.

[0052] In one embodiment of the present invention, in step (4), the preparation involves adding 1%, 3%, 5%, or 7% sucrose; the homogenization conditions are 20°C, 25°C, or 30°C, 30MPa, 35MPa, or 40MPa, and homogenization is performed 3 times; the sterilization is performed at 95°C for 10 minutes; and the cooling refers to cooling to 43°C, 45°C, or 48°C.

[0053] In one embodiment of the present invention, in step (5), the bacterial strains are Lactobacillus acidophilus (accession number: CICC6089) and Lactobacillus casei (accession number: CICC6117), and the inoculation volume ratio of the two strains is 1:1; the concentration of each strain is 1×10⁻⁶. 8CFU / mL; the total inoculum amount is 1% (v / v), that is, 1% activated bacterial solution is added to the culture medium base; the fermentation temperature is 37℃, and fermentation ends when the pH value of the fermentation broth is ≤4.5 and the acidity is ≥30°T, and then after-ripening is carried out; the after-ripening temperature is 4℃ and the time is 12h, 16h, 20h or 24h.

[0054] An oat-based fermented milk prepared by the aforementioned method.

[0055] The present invention will be further described below through specific embodiments and comparative examples.

[0056] Example 1:

[0057] The preparation method of oat-based fermented milk enriched with polyphenols using a combined enzymatic hydrolysis-fermentation technology includes the following steps:

[0058] (1) Carefully select high-quality whole oat grains, soak them for half an hour, wash them repeatedly 3 times, place them in an oven, and dry them at 105℃ for 30 minutes to obtain pretreated oat grains.

[0059] (2) The obtained pretreated oat grains were baked at 180°C for 10 minutes, and turned over once every 2 minutes to give them a rich roasted aroma. The roasted grains were first coarsely crushed 3 times by a universal grinder, passed through a 40-mesh sieve, and then ground in a ball mill for 15 minutes (the ball mill frequency was 4000Hz) to obtain whole oat ultrafine powder.

[0060] (3) Add 6 times the volume of 55℃ deionized water to whole oat ultrafine powder, stir the oat milk for 20 min at a speed of 500 rpm / min, perform high-speed shearing at 6000 rpm for 15 min, and gelatinize the fully sheared oat milk at 85℃ for 15 min. Then perform enzymatic hydrolysis. Add 1% α-amylase, 0.6% β-amylase and 0.5% cellulase to the gelatinized oat milk. The enzymatic hydrolysis temperature is 55℃ and the time is 120 min. Adjust the pH to 8 with sodium hydroxide, adjust the temperature to 60℃, add 0.15% alkaline protease and heat for 30 min to obtain the enzymatic hydrolysate.

[0061] (4) After enzyme inactivation, add 7% sucrose (mass fraction of sucrose in oat milk) to the oat milk. After stirring evenly, homogenize under the conditions of 25℃ and 35MPa, homogenize 3 times; then sterilize at 95℃ for 10min, and quickly cool to 45℃ to obtain a homogeneous and stable system.

[0062] (5) Inoculate with the starter culture, ferment, and measure the pH and acid value at regular intervals. Fermentation ends when the pH of the fermentation broth is ≤4.5 and the acidity is ≥30°T. After fermentation, proceed with post-ripening to obtain oat-based fermented milk. The fermentation strains are *Lactobacillus casei* and *Lactobacillus acidophilus*, mixed in a 1:1 ratio, with a concentration of 1×10⁻⁶ for each strain. 8 The total inoculum concentration was 1% (v / v); the fermentation temperature was 37℃; the post-ripening temperature was 4℃; and the fermentation time was 18h. The experimental results of the relevant physicochemical indicators of oat plant-based fermented milk are shown in Table 1 below.

[0063] Example 2:

[0064] The preparation method of oat-based fermented milk enriched with polyphenols using a combined enzymatic hydrolysis-fermentation technology includes the following steps:

[0065] (1) Carefully select high-quality whole oat grains, soak them for half an hour, wash them repeatedly 3 times, place them in an oven, and dry them at 105℃ for 20 minutes to obtain pretreated oat grains.

[0066] (2) The obtained pretreated oat grains were baked at 180°C for 10 minutes, and turned over once every 2 minutes to give them a rich roasted aroma. The roasted grains were first coarsely crushed 3 times by a universal grinder, passed through a 40-mesh sieve, and then ground in a ball mill for 15 minutes (the ball mill frequency was 4000Hz) to obtain whole oat ultrafine powder.

[0067] (3) Add 6 times the volume of 55℃ deionized water to whole oat ultrafine powder, stir the oat milk for 20 min at a speed of 500 rpm / min, perform high-speed shearing at 6000 rpm for 15 min, and gelatinize the fully sheared oat milk at 85℃ for 15 min. Then perform enzymatic hydrolysis. Add 0.5% α-amylase, 0.6% β-amylase and 0.5% cellulase to the gelatinized oat milk. The enzymatic hydrolysis temperature is 55℃ and the time is 120 min. Adjust the pH to 8 with sodium hydroxide, adjust the temperature to 60℃, add 0.15% alkaline protease and heat for 30 min to obtain the enzymatic hydrolysate.

[0068] (4) After enzyme inactivation, add 7% sucrose to the oat milk. After stirring evenly, homogenize under the conditions of 25℃ and 35MPa for 3 times; then sterilize at 95℃ for 10 minutes and quickly cool to 45℃ to obtain a homogeneous and stable system.

[0069] (5) Inoculate with the starter culture, ferment, and measure the pH and acid value at regular intervals. Fermentation ends when the pH of the fermentation broth is ≤4.5 and the acidity is ≥30°T. After fermentation, proceed with ripening to obtain oat-based fermented milk. The fermentation strains are *Lactobacillus casei* and *Lactobacillus acidophilus*, mixed in a 1:1 ratio, with a concentration of 1×10⁻⁶ for each strain. 8The total inoculum concentration was 1% (v / v); the fermentation temperature was 37℃; the post-ripening temperature was 4℃; and the fermentation time was 18h. The experimental results of the relevant physicochemical indicators of oat plant-based fermented milk are shown in Table 1 below.

[0070] Example 3:

[0071] The preparation method of oat-based fermented milk enriched with polyphenols using a combined enzymatic hydrolysis-fermentation technology includes the following steps:

[0072] (1) Carefully select high-quality whole oat grains, soak them for half an hour, wash them repeatedly 3 times, place them in an oven, and dry them at 105℃ for 30 minutes to obtain pretreated oat grains.

[0073] (2) The obtained pretreated oat grains were baked at 180°C for 10 minutes, and turned over once every 2 minutes to give them a rich roasted aroma. The roasted grains were first coarsely crushed 3 times by a universal grinder, passed through a 40-mesh sieve, and then ground in a ball mill for 15 minutes (the ball mill frequency was 4000Hz) to obtain whole oat ultrafine powder.

[0074] (3) Add 6 times the volume of 55℃ deionized water to whole oat ultrafine powder, stir the oat milk for 20 min at a speed of 500 rpm / min, perform high-speed shearing at 6000 rpm for 15 min, and gelatinize the fully sheared oat milk at 85℃ for 15 min. Then perform enzymatic hydrolysis. Add 1.5% α-amylase, 0.6% β-amylase and 0.5% cellulase to the gelatinized oat milk. The enzymatic hydrolysis temperature is 55℃ and the time is 120 min. Adjust the pH to 8 with sodium hydroxide, adjust the temperature to 60℃, add 0.15% alkaline protease and heat for 30 min to obtain the enzymatic hydrolysate.

[0075] (4) After enzyme inactivation, add 7% sucrose to the oat milk. After stirring evenly, homogenize under the conditions of 25℃ and 35MPa for 3 times; then sterilize at 95℃ for 10 minutes and quickly cool to 45℃ to obtain a homogeneous and stable system.

[0076] (5) Inoculate with the starter culture, ferment, and measure the pH and acid value at regular intervals. Fermentation ends when the pH of the fermentation broth is ≤4.5 and the acidity is ≥30°T. After fermentation, proceed with post-ripening to obtain oat-based fermented milk. The fermentation strains are *Lactobacillus casei* and *Lactobacillus acidophilus*, mixed in a 1:1 ratio, with a concentration of 1×10⁻⁶ for each strain. 8 The total inoculum concentration was 1% (v / v); the fermentation temperature was 37℃; the post-ripening temperature was 4℃; and the fermentation time was 18h. The experimental results of the relevant physicochemical indicators of oat plant-based fermented milk are shown in Table 1 below.

[0077] Example 4:

[0078] The preparation method of oat-based fermented milk enriched with polyphenols using a combined enzymatic hydrolysis-fermentation technology includes the following steps:

[0079] (1) Carefully select high-quality whole oat grains, soak them for half an hour, wash them repeatedly three times, place them in an oven, and dry them at 105℃ for 40 minutes to obtain pretreated oat grains.

[0080] (2) The obtained pretreated oat grains were baked at 180°C for 10 minutes, and turned over once every 2 minutes to give them a rich roasted aroma. The roasted grains were first coarsely crushed 3 times by a universal grinder, passed through a 40-mesh sieve, and then ground in a ball mill for 15 minutes (the ball mill frequency was 4000Hz) to obtain whole oat ultrafine powder.

[0081] (3) Add 6 times the volume of 55℃ deionized water to whole oat ultrafine powder, stir the oat milk for 20 min at a speed of 500 rpm / min, perform high-speed shearing at 6000 rpm for 15 min, and gelatinize the fully sheared oat milk at 85℃ for 15 min. Then perform enzymatic hydrolysis. Add 2% α-amylase, 0.6% β-amylase and 0.5% cellulase to the gelatinized oat milk. The enzymatic hydrolysis temperature is 55℃ and the time is 120 min. Adjust the pH to 8 with sodium hydroxide, adjust the temperature to 60℃, add 0.15% alkaline protease and heat for 30 min to obtain the enzymatic hydrolysate.

[0082] (4) After enzyme inactivation, add 7% sucrose to the oat milk. After stirring evenly, homogenize under the following conditions: 25℃, 35MPa, homogenize 3 times; then sterilize at 95℃ for 10 min, and quickly cool to 45℃ to obtain a homogeneous and stable system.

[0083] (5) Inoculate with the starter culture, ferment, and measure the pH and acid value at regular intervals. Fermentation ends when the pH of the fermentation broth is ≤4.5 and the acidity is ≥30°T. After fermentation, proceed with post-ripening to obtain oat-based fermented milk. The fermentation strains are *Lactobacillus casei* and *Lactobacillus acidophilus*, mixed in a 1:1 ratio, with a concentration of 1×10⁻⁶ for each strain. 8 The total inoculum concentration was 1% (v / v); the fermentation temperature was 37℃ for 16 hours; and the post-ripening temperature was 4℃ for 18 hours. The experimental results of the relevant physicochemical indicators of the oat-based fermented milk are shown in Table 1 below.

[0084] Comparative Example 1:

[0085] Similar to Example 1, except that enzymatic hydrolysis and fermentation time were not performed (see Table 1), while other treatment conditions remained the same. The experimental results of the relevant physicochemical indicators of the obtained oat plant-based fermented milk are shown in Table 1 below.

[0086] Comparative Example 2:

[0087] Similar to Example 1, except that the enzymes used in Example 1 were replaced with 1% α-amylase and 0.6% β-amylase, and the fermentation time is shown in Table 1. Other treatment conditions were the same. The experimental results of the relevant physicochemical indicators of the obtained oat plant-based fermented milk are shown in Table 1 below.

[0088] Comparative Example 3:

[0089] Similar to Example 1, except that the enzymes used in Example 1 were replaced with 1% α-amylase, 0.2% pullulanase, and 0.15% alkaline protease. The fermentation time is shown in Table 1, and other treatment conditions are the same. The experimental results of the relevant physicochemical indicators of the obtained oat plant-based fermented milk are shown in Table 1 below.

[0090] Comparative Example 4:

[0091] The enzymes used in Example 1 were replaced with 1% α-amylase, 0.6% β-amylase, and 0.5% cellulase. The fermentation time is shown in Table 1, and other treatment conditions remained the same. The experimental results of the relevant physicochemical indicators of the obtained oat plant-based fermented milk are shown in Table 1 below.

[0092] Comparative Example 5:

[0093] The bacterial strain used in Example 1 was changed to *Lactobacillus plantarum* (i.e., *Lactobacillus plantarum* subsp. *plantarum*, preservation number: CICC 22158), and the fermentation time is shown in Table 1. Other treatment conditions were the same. The experimental results of the relevant physicochemical indicators of the obtained oat plant-based fermented milk are shown in Table 1 below.

[0094] Comparative Example 6:

[0095] The bacterial strain used in Example 1 was changed to Lactobacillus acidophilus, and the fermentation time is shown in Table 1. Other treatment conditions remained the same. The experimental results of the relevant physicochemical indicators of the obtained oat plant-based fermented milk are shown in Table 1 below.

[0096] Comparative Example 7:

[0097] The bacterial strain used in Example 1 was changed to Lactobacillus casei, and the fermentation time is shown in Table 1. Other treatment conditions remained the same. The experimental results of the relevant physicochemical indicators of the obtained oat plant-based fermented milk are shown in Table 1 below.

[0098] Comparative Example 8:

[0099] The bacterial strain used in Example 1 was changed to *Lactobacillus bulgaricus* (i.e., *Lactobacillus delbrueckii* subsp. bulgaricus, purchased from Beijing Aubowei Biotechnology Co., Ltd., bio-52481). The fermentation time is shown in Table 1, and other treatment conditions remained the same. The experimental results of the relevant physicochemical indicators of the obtained oat plant-based fermented milk are shown in Table 1 below.

[0100] Comparative Example 9:

[0101] The bacterial strain used in Example 1 was changed to Streptococcus thermophilus (purchased from Beijing Aubowei Biotechnology Co., Ltd., bio-72807). The fermentation time is shown in Table 1, and other treatment conditions are the same. The experimental results of the relevant physicochemical indicators of the obtained oat plant-based fermented milk are shown in Table 1 below.

[0102] Comparative Example 10:

[0103] The bacterial strains used in Example 1 were changed to a 1:1 mixture of Lactobacillus acidophilus and Lactobacillus bulgaricus. The fermentation time is shown in Table 1, and other treatment conditions remained the same. The experimental results of the relevant physicochemical indicators of the obtained oat plant-based fermented milk are shown in Table 1 below.

[0104] Comparative Example 11:

[0105] The bacterial strains used in Example 1 were changed to a 1:1 mixture of Lactobacillus acidophilus and Streptococcus thermophilus. The fermentation time is shown in Table 1, and other treatment conditions remained the same. The experimental results of the relevant physicochemical indicators of the obtained oat plant-based fermented milk are shown in Table 1 below.

[0106] Comparative Example 12:

[0107] The bacterial strains used in Example 1 were changed to a 1:1 mixture of Lactobacillus casei and Lactobacillus bulgaricus. The fermentation time is shown in Table 1, and other treatment conditions remained the same. The experimental results of the relevant physicochemical indicators of the obtained oat plant-based fermented milk are shown in Table 2 below.

[0108] Comparative Example 13:

[0109] The bacterial strain used in Example 1 was changed to a 1:1 mixture of Lactobacillus casei and Streptococcus thermophilus. The fermentation time is shown in Table 1, and other treatment conditions remained the same. The experimental results of the relevant physicochemical indicators of the obtained oat plant-based fermented milk are shown in Table 1 below.

[0110] Comparative Example 14:

[0111] The bacterial strain used in Example 1 was changed to a 1:1 mixture of Lactobacillus bulgaricus and Streptococcus thermophilus. The fermentation time is shown in Table 1, and other treatment conditions remained the same. The experimental results of the relevant physicochemical indicators of the obtained oat plant-based fermented milk are shown in Table 1 below.

[0112] Test example:

[0113] The lactic acid bacteria prepared in Examples 1-4 and Comparative Examples 1-14 were tested. The specific test methods are as follows:

[0114] Extraction of phenolic compounds: Collect 4 mL samples from the examples and comparative examples, mix with 20 mL of 80% ethanol, and place in an ultrasonic cleaner at 25°C for 20 minutes. Centrifuge the resulting liquid at 4000 rpm for 10 minutes at 4°C, and repeat the above steps three times. Collect the supernatant and concentrate it using a vacuum rotary evaporator at 40°C. Maintain the liquid containing free phenolic compounds in a constant volume of 10 mL with methanol and store in the dark. Add the remaining precipitate to hexane to remove lipids. Then, hydrolyze the precipitate by adding 20 mL of 4M NaOH, shaking for 1 hour, and adjust the pH to 2.0–3.0 with 6M HCl. Extract the mixture with 20 mL of ethyl acetate, then sonicate for 20 minutes and centrifuge at 4000 rpm for 10 minutes. Repeat the operation three times to collect the supernatant and evaporate it under vacuum at 40°C. Maintain the liquid containing bound phenolic compounds in a constant volume of 10 mL with methanol and store in the dark.

[0115] Phenolic content determination: 0.25 mL of the extract was mixed with 1 mL of distilled water, and 0.25 mL of Folin-Ciocalteu phenol reagent was added for 6 minutes. Then, 2.5 mL of 7% Na₂CO₃ and methanol were added to a total volume of 10 mL. The resulting liquid was incubated in the dark at room temperature for 90 minutes. Subsequently, the absorbance was measured at 760 nm on a spectrophotometer. The phenolic compounds were quantified using a gallic acid standard calibration curve. Results are expressed as milligallic acid equivalents (GAE) / L sample.

[0116] pH value determination: pH value was determined using a PB-10 high-precision acidity meter.

[0117] Acidity determination: The determination of acidity in fermented milk was carried out in accordance with GB 5009.239-2016 "National Food Safety Standard - Determination of Acidity in Food", using the first method, the phenolphthalein indicator method.

[0118] Determination of viable bacteria count: The viable bacteria count of lactic acid bacteria was determined according to the method in GB 4789.35-2016 "National Food Safety Standard for Microbiological Examination of Food - Examination of Lactic Acid Bacteria".

[0119] The test results are shown in Table 1.

[0120] Table 1 Test data for the examples and comparative examples

[0121]

[0122]

[0123] Examples 1-4 optimized the α-amylase dosage during enzymatic hydrolysis. Since oat flour contains a large amount of starch, studying the optimal α-amylase dosage is essential. Table 1 shows that both mild and excessive enzymatic hydrolysis can destabilize the oat milk system, hindering fermentation, leading to decreased acidity and prolonged fermentation time. The amount of α-amylase added has the greatest impact on the stability of the oat milk system, and using 1% α-amylase yields the most significant results.

[0124] As shown in the comparative examples, the polyphenol content was significantly reduced without enzymatic hydrolysis. Changing the type of enzyme resulted in longer fermentation times and lower acidity compared to the examples, leading to lower polyphenol content. Changing the type of bacteria or using a single strain resulted in lower total polyphenol content and slower acid production, particularly in Comparative Example 9, where the fermentation time reached 20 hours with low acidity and polyphenol content when using only *Streptococcus thermophilus*. Furthermore, the examples and comparative examples demonstrate that the presence of *Lactobacillus casei* increases polyphenol content compared to other strains, especially the combination of *Lactobacillus casei* and *Lactobacillus acidophilus*, which is more conducive to increasing polyphenol content while shortening fermentation time.

[0125] This invention provides a method for preparing oat-based fermented milk enriched with polyphenols based on a combined enzymatic hydrolysis-fermentation technology. This method effectively increases the total polyphenol content and the acid production rate of lactic acid bacteria, and can increase the acidity of oat-based fermented milk in a short time while shortening the fermentation time.

[0126] The above description is merely a preferred embodiment of the present invention, and the present invention is not limited to the above embodiments. It is understood that other improvements and variations that are directly derived or conceived by those skilled in the art without departing from the spirit and concept of the present invention should be considered to be included within the protection scope of the present invention.

Claims

1. A method for preparing oat-based fermented milk enriched with polyphenols using a combined enzymatic hydrolysis-fermentation technology, characterized in that, The preparation method is as follows: (1) After soaking and washing the oats, they are dried to obtain pretreated oats; (2) The pretreated oats are baked, crushed, and ball-milled to obtain whole oat ultrafine powder; (3) Add water to whole wheat ultrafine powder to obtain oat milk, stir, and shear at high speed. After the oat milk is completely dissolved, add enzyme to hydrolyze it to obtain enzymatic hydrolysate. (4) After enzyme inactivation, the mixture is prepared and homogenized to obtain a homogenized solution, which is then sterilized and cooled to obtain a uniform and stable system; (5) Add the starter culture for inoculation, fermentation, and post-ripening to obtain oat plant-based fermented milk; In step (3), the enzymes include α-amylase, β-amylase, cellulase, and alkaline protease; the amount of each enzyme added is 0.1%-2%; In step (5), the bacterial strains are Lactobacillus acidophilus and Lactobacillus casei, and the volume ratio of the two strains added is 1:1; In step (3), the specific process of adding enzymes for enzymatic hydrolysis is as follows: first, add α-amylase, β-amylase and cellulase and react at 55-65℃ for 90-120 min, then change the enzymatic hydrolysis conditions to carry out proteolytic hydrolysis, that is, adjust the pH to 8 with sodium hydroxide, adjust the temperature to 60℃, add alkaline protease and react for 30-60 min.

2. The preparation method according to claim 1, characterized in that, In step (1), the oats are whole oat grains.

3. The preparation method according to claim 1, characterized in that, In step (2), the baking temperature is 170-190℃ and the time is 10-20 minutes. The cake is turned over every 2-4 minutes during baking.

4. The preparation method according to claim 1, characterized in that, In step (3), the volume ratio of the whole wheat ultrafine powder to water is 1:5-8; the temperature of the water is 50-55℃.

5. The preparation method according to claim 1, characterized in that, In step (3), the stirring speed is 300-500 r / min and the time is 10-20 min; the high-speed shearing speed is 4000-7000 r / min and the shearing time is 10-20 min; the gelatinization temperature is 80-90℃ and the time is 10-20 min.

6. The preparation method according to claim 1, characterized in that, In step (3), the high-speed shearing speed is 6000 rpm and the shearing time is 10-15 min.

7. The preparation method according to claim 1, characterized in that, In step (4), the preparation involves adding 1%-7% sucrose.

8. The preparation method according to claim 1, characterized in that, In step (4), the pressure of homogenization is 30-40 MPa and the temperature is 20-30 ℃; the cooling refers to cooling to 43-48 ℃.

9. An oat-based fermented milk prepared by the preparation method according to any one of claims 1-8.

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