High-functionality flavored fermented milk and preparation method thereof

Abstract

The invention relates to the technical field of food processing, in particular to high-functionality flavored fermented milk and a preparation method thereof. The preparation method of the high-functionality flavored fermented milk comprises the following steps: (1) sterilizing raw milk to obtain sterilized milk; (2) inoculating a yoghurt starter and lactobacillus reuteri J1 into the sterilized milk, and performing constant-temperature fermentation to obtain coagulated milk; the yoghourt leavening agent consists of streptococcus thermophilus and lactobacillus bulgaricus; and (3) carrying out after-ripening treatment on the coagulated milk to obtain the high-functionality flavored fermented milk. The flavored fermented milk prepared by the preparation method disclosed by the invention can achieve multiple effects of delaying post-acidification, remarkably improving antioxidant activity, optimizing water-holding capacity and viable count and the like on the basis of keeping basic physicochemical properties and sensory quality of the fermented milk, and has a relatively good application prospect in the field of high-quality fermented milk production.

Description

Technical Field

[0001] This invention relates to the field of food processing technology, specifically to a high-functionality flavored fermented milk and its preparation method. Background Technology

[0002] In recent years, with the rise of functional foods, fermented milk has become increasingly popular due to its rich content of probiotics and bioactive substances. Traditional fermented milk production processes are mature, typically using cow's milk as raw material and employing Streptococcus thermophilus and Lactobacillus bulgaricus as starter cultures. The process involves sterilization, inoculation, constant-temperature fermentation, and cooling maturation. Current methods aim to produce fermented milk products with stable acidity, appropriate viscosity, good flavor, and basic nutritional value.

[0003] However, existing fermented milk is mainly used as a general dairy product or a basic probiotic carrier. Its preparation process mainly focuses on controlling the fermentation process and achieving the basic quality of fermented milk (such as acidity, water-holding capacity, and flavor). The antioxidant activity of the product, the number of live functional probiotics (especially highly adhesive strains) and their potential health benefits are not the core focus or technical indicators. As a result, fermented milk products have relatively simple functions and insufficient functional activity.

[0004] Specifically, existing fermented milk preparation processes often suffer from the following problems: 1) Limited product functionality: Traditional fermented milk primarily provides basic nutrition and lactic acid bacteria, lacking scientifically verifiable enhanced antioxidant activity fortified with specific highly adhesive probiotic strains. This makes it difficult to meet the growing market demand for functional fermented milk with clearly defined additional health benefits (such as antioxidants). 2) Insufficient post-acidification control and shelf-life quality stability: During refrigerated storage, traditional starter cultures can lead to a significant and sustained increase in product acidity (post-acidification), affecting the flavor stability and taste of the product during its shelf life, potentially leading to decreased consumer acceptance. 3) Lack of attention to probiotic colonization potential: Traditional processes do not involve screening for the gut adhesion and colonization capabilities of the strains used. Although the product contains live bacteria, the strains may lack good gut colonization ability, thus limiting their potential to continuously exert probiotic effects in vivo. 4) There is room for improvement in the overall quality of the product: Under the premise of ensuring flavor and taste, traditional methods have bottlenecks in improving the water-holding capacity of the product (reducing whey separation) and maintaining a high number of viable bacteria, and have failed to achieve synergistic optimization of these quality parameters by introducing specific functional strains.

[0005] Based on this, the technical problem to be solved by this invention is how to develop a functional fermented milk that not only has excellent basic physicochemical properties and sensory quality, but also has significantly enhanced in vitro antioxidant activity, high viable bacterial count, delayed post-acidification, and is rich in probiotic strains with high intestinal colonization potential, and its matching process, so as to meet the higher consumption demand for fermented milk. Summary of the Invention

[0006] To overcome the shortcomings of existing technologies, the present invention aims to provide a highly functional flavored fermented milk and its preparation method. The flavored fermented milk preparation process provided by the present invention, through mixed fermentation of a yogurt starter and Lactobacillus reuteri J1, can produce a functional fermented milk with characteristics such as high antioxidant activity, high viable cell count, high water-holding capacity, and delayed post-acidification.

[0007] To achieve the above objectives, the first aspect of the present invention adopts the following technical solution:

[0008] A method for preparing a highly functional flavored fermented milk includes the following steps:

[0009] (1) The raw milk is sterilized to obtain sterilized milk;

[0010] (2) Inoculate sterilized milk with yogurt starter and Lactobacillus reuteri J1, and carry out constant temperature fermentation to obtain coagulated milk; the yogurt starter is composed of Streptococcus thermophilus and Lactobacillus bulgaricus;

[0011] (3) The coagulated milk is subjected to post-ripening treatment to obtain the high-functionality flavored fermented milk.

[0012] This invention obtained Lactobacillus reuteri J1 with high adhesion through experimental screening, and then used it in the preparation of fermented milk, which can effectively improve the antioxidant activity, viable bacteria count, water holding capacity and post-acidification function of fermented milk.

[0013] As a preferred option, in step (1), the raw milk is pure milk.

[0014] As a preferred embodiment, in step (1), the sterilization treatment temperature is 80~90℃ and the time is 10~20min. As a more preferred embodiment, the sterilization treatment temperature is 85℃ and the time is 15min.

[0015] As a preferred option, in step (2), the inoculation amount of the yogurt starter is 2.5%~3.5%v / v of the sterilized milk, more preferably 3%v / v.

[0016] As a preferred option, in step (2), the inoculation amount of Lactobacillus reuteri J1 is 3%~5% v / v of the sterilized milk, more preferably 4% v / v.

[0017] As a preferred embodiment, in step (2), the effective lactic acid bacteria concentration in the yogurt starter is (0.8~1.2)×10⁻⁶. 10 CFU / mL, more preferably 1×10⁻⁶ 10 CFU / mL.

[0018] As a preferred embodiment, in step (2), the effective bacterial concentration of *Lactobacillus reuteri* J1 is (0.8~1.2)×10⁻⁶. 8 CFU / mL, more preferably 1×10⁻⁶ 8 CFU / mL.

[0019] As a preferred embodiment, in step (2), the temperature of the constant-temperature fermentation is 34~40℃, and the time is 4~8h. As a more preferred embodiment, the temperature of the constant-temperature fermentation is 37℃, and the time is 6h.

[0020] As a preferred embodiment, in step (3), the post-curing treatment involves storing the coagulated milk at a temperature of 0-4°C for 20-30 hours. As a more preferred embodiment, the post-curing treatment involves storing the coagulated milk at a temperature of 4°C for 24 hours.

[0021] The second aspect of this invention is the following technical solution:

[0022] A high-functionality flavored fermented milk prepared using the method described above.

[0023] The technical solution of the present invention achieves the following significant advantages over the prior art:

[0024] 1) Significantly enhanced antioxidant activity: The fermented milk prepared by the method of this invention has a hydroxyl radical (•OH) scavenging rate of up to 91.37% and a significantly improved iron ion reducing power (OD value), indicating that the product has excellent in vitro antioxidant capacity, which is a functional characteristic that traditional fermented milk does not have or does not highlight.

[0025] 2) Optimize the fermentation and storage process: The addition of specific Lactobacillus reuteri J1 in this invention can promote acid production, shorten fermentation time, and improve production efficiency; at the same time, it can effectively delay the post-acidification process during cold storage, which helps to maintain the flavor and texture stability of the product during its shelf life.

[0026] 3) Improved product texture and increased viable count: At the preferred addition amount of Lactobacillus reuteri J1 in this invention (e.g., 4%), the water-holding capacity of the product is significantly increased to 73.65%, effectively reducing whey separation and improving texture; simultaneously, the viable count of lactic acid bacteria in the product is significantly increased (up to 1.012 × 10⁻⁶). 9 (CFU / g) provides a foundation for the probiotic function.

[0027] 4) Introducing functional strains with high colonization potential: The Lactobacillus reuteri J1 strain of this invention is a strain with a high adhesion rate (75.12%) after screening in an in vitro adhesion model. It has excellent intestinal colonization potential and provides the product with potential health benefits beyond traditional fermented milk (such as intestinal barrier support, immune regulation, etc.).

[0028] 5) Balancing Functionality and Sensory Quality: Within the preferred addition range (3%-4%), the product achieves the above-mentioned functional improvements while its sensory scores (color, texture, flavor, and mouthfeel) are superior to those of traditional products, thus achieving a good balance between functionality and acceptability.

[0029] Therefore, the flavored fermented milk prepared by this invention can achieve multiple effects such as delaying post-acidification, significantly improving antioxidant activity, and optimizing water-holding capacity and viable bacteria count while maintaining the basic physicochemical properties and sensory quality of fermented milk. It has good application prospects in the field of high-quality fermented milk production. Attached Figure Description

[0030] Figure 1 The adhesion rate test results are for five different strains of bacteria used in this invention.

[0031] Figure 2 The endpoint pH results of fermented milk obtained under different amounts of Lactobacillus reuteri added in this invention;

[0032] Figure 3 The titration acidity of fermented milk obtained under different amounts of Lactobacillus reuteri added in this invention is shown in the figure.

[0033] Figure 4 The pH value changes of fermented milk obtained under different amounts of Lactobacillus reuteri in this invention were observed during storage at 4°C for 7 days.

[0034] Figure 5 The results of titration acidity changes of fermented milk obtained with different amounts of Lactobacillus reuteri added in this invention were observed after storage at 4°C for 7 days.

[0035] Figure 6 The results show the water-holding capacity of fermented milk obtained under different amounts of Lactobacillus reuteri added in this invention.

[0036] Figure 7 This invention illustrates the effect of different amounts of Lactobacillus reuteri added on the ·OH free radical scavenging rate of fermented milk.

[0037] Figure 8 The effect of different amounts of Lactobacillus reuteri added on Fe in fermented milk obtained in this invention 3+ The influence of free radical OD value. Detailed Implementation

[0038] The technical solutions and effects of the present invention will be clearly and completely described below with reference to specific embodiments. However, those skilled in the art should understand that the embodiments are only used to illustrate the technical solutions of the present invention and should not be regarded as limiting the scope of protection of the present invention.

[0039] In the following examples, the *Lactobacillus reuteri* J1 used was screened from traditional fermented dairy products from Tibet, which has been disclosed in patent application number 2022100075426. *Lactobacillus plantarum* J26 has been disclosed in the existing literature “LI H, CHENGS, HUO J, et al. Lactobacillus plantarum J26 Alleviating Alcohol-Induced Liver Inflammation by Maintaining the Intestinal Barrier and Regulating MAPKSignaling Pathways [J / OL] 2023, 15(1):190[10.3390 / nu15010190”. Lactobacillus gasseri JM-1 has been disclosed in the existing literature “CHENG S, LI H, HUANG Y, et al. Lactobacillus gasseri JM1Isolated from Infant Feces Alleviates Colitis in Mice via Protecting the Intestinal Barrier [J / OL] 2023, 15(1):139[10.3390 / nu15010139”. Lactobacillus paracasei JY-039 has been disclosed in the existing literature “ZHAO J, WANG L, CHENG S, et al. A Potential Synbiotic Strategy for the Prevention of Type 2 Diabetes: Lactobacillus paracasei JY062 and Exopolysaccharide Isolated from Lactobacillus plantarum JY039 [J / OL]2022, 14(2):377[10.3390 / nu14020377”.Lactobacillus rhamnosus JL-1 has been disclosed in the existing literature “LI X, HU D, TIAN Y, et al. Protective effects of a novel Lactobacillus rhamnosus strain with probiotic characteristics against lipopolysaccharide-induced intestinal inflammation in vitro and in vivo [J]. Food Funct, 2020, 11(7):5799-5814”. The aforementioned Lactobacillus reuteri J1 and other comparative strains (Lactobacillus plantarum J26, Lactobacillus gasseri JM-1, Lactobacillus paracasei JY-039, and Lactobacillus rhamnosus JL-1) are all preserved by the Key Laboratory of Dairy Science, College of Food Science, Northeast Agricultural University, and proof of the availability of the biological materials to the public is provided.

[0040] In the following examples, the raw milk was pure milk, sourced from Inner Mongolia Yili Industrial Group Co., Ltd. The yogurt starter consisted of Streptococcus thermophilus and Lactobacillus bulgaricus, and was a commercial direct-inoculation starter purchased from Angel Yeast Co., Ltd.

[0041] Example 1

[0042] This embodiment provides a highly functional flavored fermented milk, the preparation method of which includes the following steps:

[0043] (1) Pasteurize the raw milk (pure milk) at a temperature of 85°C for 15 minutes. After pasteurization, cool the raw milk to the fermentation temperature to obtain pasteurized milk.

[0044] (2) Inoculate the cooled sterilized milk from step (1) with both yogurt starter and activated Lactobacillus reuteri J1, and ferment at 37°C for 6 hours to obtain coagulated milk. The yogurt starter consists of Streptococcus thermophilus and Lactobacillus bulgaricus. The inoculation amount of the yogurt starter is 3% v / v of the total raw milk volume; the inoculation amount of Lactobacillus reuteri J1 is 4% v / v of the total raw milk volume. The effective lactic acid bacteria concentration of the yogurt starter is 1×10⁻⁶. 10 CFU / mL. The effective bacterial concentration of activated Lactobacillus reuteri J1 is 1×10⁻⁶. 8 CFU / mL.

[0045] (3) The coagulated milk obtained in step (2) is stored at 4°C for 24 hours to complete the ripening process, and the high-functionality flavored fermented milk of this embodiment is obtained.

[0046] Experimental Example 1: Strain Adhesion Rate Test

[0047] The colonization ability of probiotics in the gut depends on their adhesion rate. Good adhesion is not only crucial for successful colonization of probiotics in the host gut, but also an important guarantee for enhancing their survival ability and sustaining their beneficial effects. Therefore, this experiment used a Caco-2 cell model to assess the in vitro adhesion of five strains of lactic acid bacteria, in order to examine the differences in adhesion effects among different lactic acid bacteria.

[0048] The experimental procedure was as follows: Activated *Lactobacillus reuteri* J1, *Lactobacillus gasseri* JM-1, *Lactobacillus plantarum* J26, *Lactobacillus paracasei* TD-039, and *Lactobacillus rhamnosus* JL-1 were centrifuged at 10000×g for 5 min at 4℃. The supernatant was removed, and the resulting bacterial sludge was washed twice with sterile PBS buffer and resuspended in cell culture medium without antibiotics. The bacterial concentration was adjusted to 1×10⁻⁶. 8 CFU / mL was used to obtain a bacterial suspension for later use. Human colon adenocarcinoma cells (Caco-2 cells) cultured to a polarized state were then removed from the cell culture flask at a concentration of 10... 6 Inoculate cells / well into 6-well plates. 24 hours before adding bacteria, replace the culture medium with high-glucose medium without antibiotics. Cultivate until a monolayer of cells covers the 6-well plate. Discard the culture medium in the 6-well plates and add an equal volume of the prepared bacterial suspension. Co-incubate at 37°C in a 5% CO2 incubator for 2 hours, with three replicates per group. At the beginning of co-incubation, dilute and spread the bacterial suspension, and incubate in an anaerobic incubator for 48 hours before colony counting. After 2 hours of co-incubation, wash three times with PBS buffer to remove unadhered cells, trypsin digest to collect adherent cells, then perform serial dilutions and spread the suspension. Incubate in an anaerobic incubator for 48 hours before colony counting and calculating the adhesion rate. The adhesion rate results for different strains are shown below. Figure 1 As shown.

[0049] Depend on Figure 1 It was found that *Lactobacillus reuteri* J1 exhibited the strongest adhesion ability, with an adhesion rate of 75.12%, significantly better than the other four control strains (*Lactobacillus gasseri* JM-1, *Lactobacillus plantarum* J26, *Lactobacillus paracasei* TD-039, and *Lactobacillus rhamnosus* JL-1, p<0.05). This result indicates that *Lactobacillus reuteri* J1 possesses excellent in vitro intestinal epithelial adhesion potential. Therefore, *Lactobacillus reuteri* J1 was selected as the core probiotic strain for subsequent research on the quality improvement of fermented milk.

[0050] Experimental Example 2: Fermented Milk Index Testing

[0051] Referring to the fermented milk preparation process described in Example 1, the inoculation amount of Lactobacillus reuteri J1 was adjusted to 0%, 3% v / v, 4% v / v, 5% v / v, and 6% v / v of the total raw milk, respectively, while the other processes remained unchanged, to prepare fermented milk products with different amounts of Lactobacillus reuteri J1.

[0052] The fermented milk product prepared above was used as the test sample, and its various indicators (endpoint and titration acidity, post-acidification, water holding capacity, viable count, color, antioxidant activity, and sensory evaluation) were tested to evaluate the impact of Lactobacillus reuteri J1 on the overall quality of fermented milk.

[0053] 2.1 Determination of endpoint pH and titratable acidity

[0054] The endpoint pH and titratable acidity of various fermented milk products were tested. For pH determination, three parallel measurements were performed on each sample using a precision pH meter, and the average value was taken. The titratable acidity was determined as follows: 5g of fermented milk sample was added to an Erlenmeyer flask, followed by 40mL of distilled water and 2 drops of phenolphthalein. The mixture was shaken well and titrated with 0.1mol / L NaOH standard solution until the solution turned pale pink and remained unchanged after shaking for 30 seconds. The volume V (mL) of NaOH standard solution consumed was recorded, and the titratable acidity of the fermented milk was calculated. The endpoint pH results of fermented milk obtained with different amounts of *Lactobacillus reuteri* are shown below. Figure 2 As shown, the titratable acidity determination results of fermented milk obtained with different amounts of Lactobacillus reuteri are as follows: Figure 3 As shown.

[0055] like Figure 2 As shown, with the increase of Lactobacillus reuteri J1 addition, the final pH of the fermented milk showed a significant decreasing trend (p<0.05). Furthermore, the final pH of all experimental groups with added Lactobacillus reuteri J1 was significantly lower than that of the control group using only traditional yogurt starter (Streptococcus thermophilus and Lactobacillus bulgaricus) (i.e., 0% Lactobacillus reuteri J1 addition). This indicates that Lactobacillus reuteri J1 can effectively promote acid production, accelerate the fermentation process, and shorten the time required to reach the gel endpoint.

[0056] like Figure 3 As shown, the titratable acidity of the fermented milk showed a consistent trend with the pH value. The titratable acidity of all experimental groups was higher than 70°T, meeting the acidity requirements of GB 19302-2010 "National Food Safety Standard for Fermented Milk". With increasing amounts of *Lactobacillus reuteri* J1, the titratable acidity showed a significant upward trend (p<0.05). Compared with the control group without *Lactobacillus reuteri* J1, the acidity of all experimental groups with this strain was significantly higher, indicating that *Lactobacillus reuteri* J1 has a strong acid-producing metabolic capacity and can effectively participate in and promote the acidification process during fermentation.

[0057] 2.2 Determination of post-acidification

[0058] Moderate acidification is beneficial for the formation of flavor compounds and improves the flavor of fermented milk, but excessive acidification can affect the product's taste and reduce consumer acceptance. In the post-acidification test, the fermented milk product was stored at 4℃, and the pH and acidity were measured on days 1, 3, 5, and 7 of refrigeration to investigate the post-acidification effect of the strains under refrigeration conditions. The changes in pH and titratable acidity of the fermented milk stored at 4℃ for 7 days are shown below. Figure 4 and Figure 5 As shown.

[0059] Depend on Figure 4 and Figure 5 It was found that all fermented milk samples exhibited a rapid decrease in pH and a significant increase in acidity during the early storage period (1-3 days). After 3 days, the changes tended to level off, and all samples were accompanied by whey separation. The control group (with 0% Lactobacillus reuteri J1 addition) showed a greater decrease in pH and an increase in acidity during storage than the Lactobacillus reuteri J1-added groups. This indicates that the addition of Lactobacillus reuteri J1 can effectively delay the post-acidification process of fermented milk during refrigeration, which is beneficial for maintaining the flavor and texture stability of the product during its shelf life.

[0060] 2.3 Determination of water-holding capacity

[0061] The water-holding capacity of each fermented milk was tested as follows: A 50 mL centrifuge tube was taken and recorded as mass W0. A 10 mL sample of fermented milk was added and recorded as mass W1. The tube was centrifuged at 3000 rpm for 10 min, allowed to stand for 10 min, and the supernatant was poured off and recorded as mass W2. The test was performed in triplicate. The formula for calculating water-holding capacity is: Water-holding capacity (%) = 100% × (w2 - w0) / (w1 - w0). The changes in water-holding capacity of fermented milk obtained under different amounts of *Lactobacillus reuteri* are shown below. Figure 6 As shown.

[0062] Depend on Figure 6 It was found that with the increase of *Lactobacillus reuteri* J1 addition, the water-holding capacity showed a trend of first increasing and then decreasing. When the addition amount was 4%, the water-holding capacity reached the highest value of 73.65%, which was significantly higher than that of the control group and other addition groups (p<0.05). The change in water-holding capacity was closely related to the acidity of the system and the protein gel structure. Under moderate acidification conditions, the interaction between protein molecules was enhanced, which could form a more dense and regular three-dimensional network structure, thereby improving the ability to encapsulate water. However, excessive acidity would lead to excessive cross-linking of proteins in the gel network, causing structural shrinkage and whey precipitation, and the water-holding capacity would decrease accordingly. This invention shows that 4% *Lactobacillus reuteri* J1 addition achieved a better balance between the degree of acidification and the stability of the gel structure, thereby achieving the maximum improvement in the water-holding capacity of fermented milk.

[0063] 2.4 Determination of viable bacterial count

[0064] The method for determining the viable bacteria count in fermented milk is as follows: Take 5g of fermented milk and perform serial dilutions, then take 10g of each diluted sample. -5 10 -6 10 -7 Three gradients were performed. 100 µL of the sample was spread onto a solid MRS plate and incubated at 37°C for 48 h. The colony count was recorded. Each gradient was performed twice. The results of the viable cell count in the fermented milk are shown in Table 1. The results in Table 1 are expressed as mean ± standard deviation. Comparisons are made in each column, with different lowercase letters representing significant differences.

[0065] Table 1. Effects of different amounts of Lactobacillus reuteri added on the viable count of fermented milk Table 1 shows that the viable count of lactic acid bacteria in fermented milk initially increased and then stabilized with increasing amounts of *Lactobacillus reuteri* J1. The peak viable count was reached at 4% (1.012 ± 0.019) × 10⁻⁶. 9 The CFU / g concentration was significantly higher than that of the control group (p<0.05). Further increases in *Lactobacillus reuteri* J1 (5%–6%) resulted in a stable viable count without further significant increase. This phenomenon may be related to the saturation of nutrient utilization in the fermentation system. Excessively high cell density leads to substrate limitation or metabolite accumulation, thereby inhibiting the continued growth of cell numbers. The results indicate that 4% addition of *Lactobacillus reuteri* J1 can effectively increase the viable probiotic count in the final product, providing a technical basis for the development of highly active probiotic fermented dairy products.

[0066] 2.5. Color Measurement

[0067] The L*, a*, and b* values ​​of each fermented milk were measured using a colorimeter, and calibrated with a white plate before use. The measurement results of the color parameters (L, a, b) of the fermented milk are shown in Table 2. The results in Table 2 are expressed as mean ± standard deviation. Each column is compared, and different lowercase letters represent significant differences.

[0068] Table 2. Effects of different amounts of Lactobacillus reuteri added on the color of fermented milk

[0069] As shown in Table 2, with the increase of Lactobacillus reuteri J1 addition, the brightness value (L) decreased significantly (p<0.05), indicating a decrease in sample brightness. The redness value (a) showed a trend of first increasing and then decreasing, but there was no significant difference among the groups (p>0.05). The yellowness value (b) increased significantly with increasing addition (p<0.05), reflecting a gradual increase in the yellow hue of the product. Therefore, the addition of Lactobacillus reuteri J1 mainly affects the brightness and yellowness of fermented milk.

[0070] 2.6 Antioxidant Activity Assay

[0071] The scavenging ability of hydroxyl radicals (·OH) is a key indicator for evaluating in vitro antioxidant activity; a higher scavenging rate indicates stronger antioxidant capacity. The procedure for determining OH radical scavenging ability is as follows: Take 1 mL of fermented milk, add 9 mL of anhydrous ethanol and mix well to obtain a mixed sample; take 4 mL of the mixed sample, add 0.5 mL each of 9 mmol / L salicylic acid solution, 9 mmol / L FeSO4 solution, and 8.8 mmol / L H2O2 solution, mix, and place in a water bath at 37℃ for 30 min. Then measure the absorbance at 510 nm (A). i Simultaneously, distilled water was used to replace the fermented milk, and the blank absorbance (A0) was measured. Pure water was used instead of the hydrogen peroxide solution, and the background OD value of the fermented milk (A0) was measured. j The formula for calculating the clearance rate is: Clearance rate (%) = 100% × [A0 - (A...] i -A j )] / A0.

[0072] The ferric ion reducing power (FRAP) method reflects the ability of Fe³⁺ to be reduced to Fe²⁺ by measuring the absorbance (OD value) of the system at 700 nm; a higher OD value indicates a stronger reducing power. 3+ The procedure for determining the reducing power was as follows: 1 g of fermented milk was dissolved in 9 mL of 95 v / v% ethanol solution to obtain a mixed sample; 2 mL of the mixed sample was taken, and 2.5 mL of 0.2 mol / L phosphate buffer (pH 6.6) and 2.5 mL of 10 g / L potassium ferricyanide aqueous solution were added. The mixture was then placed in a 50℃ constant temperature water bath for 20 min. 2 mL of 0.1 g / mL trichloroacetic acid aqueous solution was added, and the mixture was stirred and centrifuged at 3000 r / min for 10 min. 2 mL of the supernatant was taken, and 0.4 mL of 1 g / L FeCl3 aqueous solution and 2 mL of distilled water were added. The mixture was reacted at room temperature for 10 min, and the absorbance OD value was measured at 700 nm.

[0073] The results of the scavenging rate of ·OH free radicals in fermented milk obtained with different amounts of Lactobacillus reuteri are as follows: Figure 7 As shown. The effect of different amounts of Lactobacillus reuteri added on Fe... 3+ The effect of free radical OD value as follows Figure 8 As shown.

[0074] like Figure 7As shown, with the increase of *Lactobacillus reuteri* J1 addition, the ·OH scavenging rate of fermented milk showed a trend of first increasing and then decreasing. The scavenging rate of all added groups was significantly higher than that of the control group without *Lactobacillus reuteri* J1 (p<0.05). Among them, the scavenging rates of the 4% and 5% addition groups were greater than 85%, with the 5% addition group having the highest scavenging rate of 91.37%. This result indicates that the addition of *Lactobacillus reuteri* J1 can significantly enhance the in vitro antioxidant capacity of fermented milk.

[0075] like Figure 8 As shown, the changes in antioxidant capacity measured by the FRAP method were consistent with the above trend. With increasing *Lactobacillus reuteri* J1 addition, the OD value also showed a trend of first increasing and then decreasing. All addition groups were significantly higher than the control group (p<0.05), with the OD values ​​of the 4%, 5%, and 6% addition groups being higher than 0.17, indicating superior Fe... 3+ Reducing capacity. Results from both methods consistently indicate that the addition of Lactobacillus reuteri J1 effectively enhances the antioxidant activity of fermented milk.

[0076] 2.7 Sensory Evaluation

[0077] The sensory evaluation process was as follows: Ten professionals with sensory training were selected to conduct sensory evaluations of fermented milk containing different concentrations of Lactobacillus reuteri. The final score was the average of the ten participants. The scoring criteria are shown in Table 3. The results of the sensory evaluation are shown in Table 4.

[0078] Table 3. Sensory Evaluation Criteria for Fermented Milk

[0079] Table 4. Effects of Lactobacillus reuteri addition on the sensory properties of fermented milk

[0080] According to the sensory evaluation results in Table 4, when the addition amount of *Lactobacillus reuteri* J1 was 3% and 4%, the total score of the fermented milk was higher than that of the control group, and it also performed better in sub-items such as color, texture, flavor, and mouthfeel, indicating that adding this strain helps improve the overall sensory quality of the product. However, when the addition amount was further increased to 5% and 6%, the total score and the scores of each sub-item all decreased, and even fell below the control group, with the decline in flavor and mouthfeel scores being particularly significant. Therefore, adding an appropriate amount of *Lactobacillus reuteri* J1 can improve the texture of fermented milk and enhance its flavor and mouthfeel, but excessive addition will lead to flavor deterioration, decreased mouthfeel, and negative impact on color and texture. Therefore, the addition range of 3%–4% can enhance the functional characteristics of the product while maintaining or improving its overall sensory acceptability.

[0081] In summary, the flavored fermented milk and its preparation process provided by this invention can achieve multiple effects such as delaying post-acidification, significantly improving antioxidant activity, and optimizing water-holding capacity and viable bacteria count while maintaining the basic physicochemical properties and sensory quality of fermented milk. It has good application prospects in the field of high-quality fermented milk production.

[0082] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the protection scope of the present invention.

Claims

1. A method for preparing a high-functionality flavor fermented milk, characterized by, The method comprises the following steps: (1) subjecting raw milk to sterilization treatment to obtain sterilized milk; (2) inoculating yogurt starter and Lactobacillus reuteri J1 into the sterilized milk to perform constant-temperature fermentation to obtain coagulated milk; the yogurt starter is composed of Streptococcus thermophilus and Lactobacillus bulgaricus; (3) subjecting the coagulated milk to post-ripening treatment to obtain the high-functionality flavor fermented milk.

2. The method for preparing a high-functionality flavor fermented milk according to claim 1, characterized by, In step (1), the raw milk is pure milk.

3. The method for preparing a high-functionality flavor fermented milk according to claim 1, characterized by, In step (1), the sterilization treatment is performed at a temperature of 80-90°C for 10-20 min.

4. The method for producing high-functionality flavor fermented milk according to any one of claims 1 to 3, characterized by, In step (2), the inoculation amount of the yogurt starter is 2.5%-3.5% v / v of the sterilized milk.

5. The method for producing high-functionality flavor fermented milk according to any one of claims 1 to 3, characterized by, In step (2), the inoculation amount of the Lactobacillus reuteri J1 is 3%-5% v / v of the sterilized milk.

6. The method for producing high-functionality flavor fermented milk according to any one of claims 1 to 3, characterized by, In step (2), the effective lactic acid bacteria concentration in the yogurt starter is (0.8-1.2)×10 10 CFU / mL.

7. The method for producing high-functionality flavor fermented milk according to any one of claims 1 to 3, characterized by, In step (2), the effective bacteria concentration of the Lactobacillus reuteri J1 is (0.8-1.2)×10 8 CFU / mL.

8. The method for producing high-functionality flavor fermented milk according to any one of claims 1 to 3, characterized by, In step (2), the constant-temperature fermentation is performed at a temperature of 34-40°C for 4-8 h.

9. The method for preparing high-functionality flavor fermented milk according to any one of claims 1 to 3, characterized by, In step (3), the post-ripening treatment is to store the coagulated milk at a temperature of 0-4°C for 20-30 h.

10. High-functionality flavor fermented milk prepared by the preparation method according to any one of claims 1-9.

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