Almond-flavored fermented milk and preparation and application thereof

Abstract

This invention discloses an almond-flavored fermented milk, utilizing a strain of lactobacillus with blood pressure-lowering properties. Lactobacillus sp. Almond-flavored fermented milk was prepared by combining Z6 with commercial yogurt cultures. The optimal ratio was determined using sucrose addition, mixed culture ratio, fermentation time, inoculum size, and temperature as single-factor factors, and ACE inhibition rate and sensory score as indicators. Results showed that *Lactobacillus*... Lactobacillus sp. The mixed fermentation of almond-flavored milk with Z6 and commercial strains showed good ACE inhibition rate (52.16±4.46%) and sensory score (88.50±1.80). The optimal processing conditions were: sugar content 7%, mixed culture ratio 8:1, fermentation time 12 h, inoculum size 3%, and temperature 37℃. The physicochemical properties of the fermented milk were measured, with an acidity of 107.43±2.92. ° The physicochemical properties, including temperature (T), viscosity (2713.67±108.21 mPa.s), and water holding capacity (62.38±4.23%), all meet national safety standards. The almond-flavored fermented milk provided in this application exhibits good blood pressure-lowering effects, retains the activity of lactic acid bacteria, broadens the variety of functional yogurts, and provides valuable reference for the development, utilization, and deep processing of almonds.

Description

Technical Field

[0001] This invention belongs to the technical field of fermented beverages, specifically relating to the technical field of nut-flavored fermented milk preparation, and more specifically to the technical field of almond-flavored fermented milk and its preparation and application. Background Technology

[0002] Almonds (Amygdalus communis L.), also known as almonds, are a representative of Xinjiang's specialty dried fruits. They are rich in protein, unsaturated fatty acids, vitamins, and minerals, possessing extremely high nutritional and medicinal value. In recent years, with the rapid development of the food industry, various nut-based beverages have emerged, including almond-based yogurt, health milk, and dairy drinks. Their enormous application potential in lowering blood pressure, lowering blood sugar, anti-oxidation, and antibacterial properties is gradually being revealed.

[0003] Almond protein hydrolysate, obtained through microbial fermentation, is rich in functional polypeptides that are easily absorbed by the human body. Compared to the original fruit, it exhibits more significant antioxidant, blood sugar-lowering, and blood lipid-lowering effects, playing a crucial role in metabolic regulation. In the food industry, almond protein peptides can be used in infant formula, nutritional foods, functional health foods, and sports nutrition products. Currently, there are relatively few dairy products containing almond protein peptides on the domestic market, making it difficult to meet the widespread demand for easy-to-consume, uniquely flavored, and nutritionally rich almond-flavored fermented milk.

[0004] Lactobacillus, a commonly used fermentation starter for yogurt, can improve the flavor characteristics of yogurt and give it a unique aroma when fermented alone. By combining it with other lactic acid bacteria such as Streptococcus thermophilus and Lactobacillus acidophilus for fermentation, the nutritional function and sensory quality of the fermented milk can be enhanced.

[0005] Our team screened and obtained Lactobacillus. Lactobacillussp Z6, after preliminary development, obtained Lactobacillus fermentation broth and mixed it evenly with honey, light calcium carbonate, and aspartame. Then, xanthan gum, CMC-Na, and agar were added and heated until completely dissolved. The mixture was then mixed evenly and bottled for testing to prepare a brown active lactic acid bacteria beverage.

[0006] Developing Lactobacillus based on existing technology Lactobacillussp Z6 is applied to nut fermented milk and has certain health benefits, which can encourage people to utilize Xinjiang almonds, explore Xinjiang's high-quality lactic acid bacteria resources, broaden the application of almond protein peptides in the dairy beverage field, and increase the added value of deep-processed almond products. Summary of the Invention

[0007] Based on the above, this application aims to provide an almond-flavored fermented milk product and its use in the preparation of antihypertensive agents. The optimal ratio was determined using sucrose addition, mixed bacteria ratio, fermentation time, inoculum size, and temperature as single factors, and angiotensin-converting enzyme (ACE) inhibition rate and sensory evaluation as indicators. The almond-flavored fermented milk provided by this application contains richer protein peptides, exhibits good antihypertensive effects, retains the activity of lactic acid bacteria, broadens the variety of functional yogurts, and provides valuable reference for the development, utilization, and deep processing of almonds.

[0008] To achieve this technical objective, the present invention adopts the following technical solution: On the one hand, this application provides an almond-flavored fermented milk, which is composed of fresh milk, almond pulp, sucrose, pectin, and lactobacillus. Lactobacillus sp. Z6 suspension and commercially fermented milk containing commercial strains of Streptococcus thermophilus and Lactobacillus acidophilus.

[0009] The lactobacillus mentioned Lactobacillussp Z6 suspension uses third-generation activated lactobacillus. Lactobacillussp Z6 was inoculated into MRS liquid medium at a volume ratio of 3.0%, cultured at 37 ℃ until the stationary phase, and obtained by centrifugation at 2500×g for 10 min.

[0010] Lactobacillus Lactobacillussp Z6 was obtained by the Institute of Applied Microbiology, Xinjiang Academy of Agricultural Sciences. This strain is *Lactobacillus*. Lactobacillussp Z6 has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 19870 and deposit date of May 25, 2020. It will be referred to as "Z6" below.

[0011] The commercially available fermented milk used was produced by Inner Mongolia Yili Industrial Group Co., Ltd., product type: flavored fermented milk, product standard code: GB 19302.

[0012] On the other hand, the almond-flavored fermented milk is prepared using the following steps: (1) After filtering and purifying the fresh milk, it is subjected to standardized treatment and preheated to 60 °C; (2) Mix almond pulp and milk in a 1:4 ratio, add 5-9% sucrose and 0.1-0.25% pectin; (3) The mixed raw materials are processed by a colloid mill at a pressure of 90-100 bar to ensure that the liquid is uniform and fine; (4) Heat the homogenized liquid to 90-100 °C and sterilize for 10 min. Then, quickly cool the sterilized liquid to 40-50 °C and add lactobacillus. LactobacillusspZ6 suspension and commercial fermented milk were mixed at a volume ratio of (2~10):1 as a starter culture, stirred evenly, and then bottled. (5) After bottling, inoculate the liquid with 1% to 5% of mixed starter culture and place it in a constant temperature fermentation chamber at 37 to 42 ℃ for 10 to 18 h. After fermentation, quickly cool it down to 0 to 4 ℃ and perform post-ripening treatment to make the flavor and texture of the fermented milk more stable.

[0013] Preferably, the almond pulp in step (2) is prepared by the following steps: after shelling, select almonds free from mold, pests, and obvious damage, and soak them at room temperature for 2-3 hours; add the soaked almonds to a 0.2% NaOH solution and blanch for 30 seconds, then cool, peel, and rinse clean; add water to the rinsed almonds and pulp them at a 1:10 ratio, and add 100 mg / kg V C (Or 0.2% Na2SO3 solution) is used for color protection treatment to prevent oxidation and discoloration. The treated almond pulp is then refrigerated and stored for later use.

[0014] Preferably, the lactobacillus Lactobacillus sp The number of viable bacteria in the Z6 suspension is not less than 1.0 × 10⁻⁶. 8 CFU / mL, commercial fermented milk contains 1.0 × 10⁻⁶ colonies of Streptococcus thermophilus and Lactobacillus acidophilus. 8 CFU / mL.

[0015] Preferably, the amount of sucrose added in step (2) is 7%.

[0016] Preferably, lactobacillus is added in step (4). Lactobacillussp Z6 suspension was mixed with commercial fermented milk at a volume ratio of 8:1 as a starter culture. Preferably, in step (5), the inoculum amount of the mixed fermentation agent is 3%, the fermentation temperature is 37 ℃, and the fermentation time is 12 h.

[0017] Furthermore, this application provides the use of the almond-flavored fermented milk in the preparation of a blood pressure-lowering preparation for health food, wherein the use is for improving the blood pressure-lowering function of the subject, or for use in the preparation of a preparation for improving the blood pressure-lowering function of the subject.

[0018] The blood pressure-lowering function of the target being improved includes enhancing the inhibitory activity of angiotensin-converting enzyme (ACE).

[0019] Furthermore, this application provides a product that helps enhance the inhibitory activity of angiotensin-converting enzyme (ACE), including the almond-flavored fermented milk described above or the almond-flavored fermented milk prepared by the above-described method.

[0020] The use of the almond-flavored fermented milk in the preparation of antihypertensive agents, wherein the use is almond-flavored fermented milk, or the almond-flavored fermented milk is prepared into food together with edible excipients.

[0021] The intended use is for oral administration of the product.

[0022] By implementing the technical solution of this invention, the following beneficial effects can be achieved: This application utilizes a strain of Lactobacillus Z6 and a commercially available strain to prepare almond-flavored fermented milk. The ACE inhibition rate was 52.16±4.46%, and the sensory score was 88.50±1.80. Furthermore, the lactic acid bacteria activity was excellent, with a Lactobacillus count of 5.0×10⁻⁶. 8 CFU / mL, thermophilic streptococcus count was 4.5 × 10⁻⁶. 8 CFU / mL. Optimal process conditions were: sugar content 7%, mixed culture ratio 8:1, fermentation time 12 h, inoculum size 3%, and temperature 37 ℃. Acidity 107.43±2.92. ° The physicochemical properties, including temperature (T), viscosity (2713.67±108.21 mPa.s), and water holding capacity (62.38±4.23%), all meet national safety standards. This research provides an application pathway for the development of Lactobacillus Z6, broadens the market prospects for the utilization of Xinjiang almond resources, and offers a new approach to the research and development of functional fermented milk. Attached Figure Description

[0023] Figure 1 The image shows the results of the strain's acid and bile salt tolerance characteristics. Figure 2 The results of ACE inhibition rate determination for different fermented milks are shown. In the diagram, group a represents commercially available fermented milk, group b represents Z6 fermented milk, group c represents a mixture of Z6 and commercially available fermented milk, group d represents Z6 fermented milk (containing almonds), group e represents commercially available fermented milk (containing almonds), and group f represents a mixture of Z6 and commercially available fermented milk (containing almonds). In groups d, e, and f, almond pulp accounts for 20% of the total mass.

[0024] Figure 3 The results show the effect of sugar content on almond-flavored fermented milk; Figure 4 The results show the effect of the mixed culture ratio on almond-flavored fermented milk. Figure 5 The results show the effect of time on almond-flavored fermented milk; Figure 6 The results show the effect of inoculum size on almond-flavored fermented milk. Figure 7 The results show the effect of temperature on almond-flavored fermented milk; Detailed Implementation The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0025] The materials used in this application are: Almonds: Almond Germplasm Resource Nursery, State-owned Forest Farm No. 2, Shache County, Kashgar Region, Xinjiang; Pure Milk: Xinjiang Xiyuchun Dairy Co., Ltd.; Fermented Milk: Inner Mongolia Yili Industrial Group Co., Ltd. (containing Streptococcus thermophilus and Lactobacillus acidophilus); Sucrose: Commercially available; Pectin (65%, derived from citrus peel): Beijing Dingguo Changsheng Biotechnology Co., Ltd.; ACE Inhibitor Activity Detection Kit: Beijing Solarbio Technology Co., Ltd.

[0026] The instruments and equipment used in this application are as follows: L12-P125 high-speed blender: Joyoung Co., Ltd.; JTM-50 vertical colloid mill: Wenzhou Longwan Huwei Machinery Factory; AR224CN analytical balance: Ohaus Instruments (Shanghai) Co., Ltd.; LE438-2MIP67 pH meter: Mettler Toledo Instruments (Shanghai) Co., Ltd.; SPX-250BF biochemical incubator: Shanghai Fuma Experimental Equipment Co., Ltd.; HVE-50 automatic autoclave: HIRAYAMA Corporation, Japan; TS-2101C constant temperature shaking incubator: Jena Analytical Instruments AG, Germany; SW-CJ-1F clean bench: Suzhou Antai Air Technology Co., Ltd.; UV spectrophotometer: Shimadzu Corporation, Japan; NDJ-5S digital rotational viscometer: Shanghai Jiehu Instrument Co., Ltd.

[0027] In this application, SPSS Statistics 20 software was used to analyze the data, and t-tests were used to compare significant differences. Origin 2024 software was used to analyze the data. All experimental results are the average of three trials and are expressed as mean ± standard deviation.

[0028] Lactobacillus in this application Lactobacillus sp The Z6 activation method is described in our team's invention patent application number 202110357517.6, "A Brown Active Lactic Acid Bacteria Beverage and Its Preparation Method": Lactobacillus preserved on slant Lactobacillussp Z6 was inoculated onto a dedicated solid culture medium and cultured at 30°C for 1–2 days to obtain activated lactobacilli. Lactobacillussp. Z6 strain.

[0029] The specific solid culture medium is as follows: 10 g peptone, 5 g beef extract, 4 g yeast powder, 20 g glucose, 1 mL Tween 80, 2 g K2HPO4·7H2O, 3.02 g anhydrous sodium acetate, 2 g triammonium citrate, 0.05 g MnSO4·4H2O, 0.2 g MgSO4·7H2O, 1 L H2O, pH around 5.7, with 2% agar added, sterilized at 121 ℃ for 15 min, and cooled for later use.

[0030] Unless otherwise specified, the technical means used in the following implementation examples are conventional means well known to those skilled in the art.

[0031] Example 1: An almond-flavored fermented milk This application provides an almond-flavored fermented milk, composed of fresh milk, almond pulp, sucrose, pectin, and lactobacillus. Lactobacillus sp. Z6 suspension and commercially fermented milk containing commercial strains of Streptococcus thermophilus and Lactobacillus acidophilus.

[0032] Lactobacillus Lactobacillussp. Z6 was obtained by the Institute of Applied Microbiology, Xinjiang Academy of Agricultural Sciences. This strain is *Lactobacillus*. Lactobacillussp. Z6 has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 19870 and deposit date of May 25, 2020. It will be referred to as "Z6" below.

[0033] The lactobacillus mentioned Lactobacillussp. Z6 suspension uses activated lactobacilli. Lactobacillus sp Z6 was inoculated into MRS liquid medium at a volume ratio of 3.0%, cultured at 37 ℃ until the stationary phase, and obtained by centrifugation at 2500×g for 10 min.

[0034] Example 2: Preparation method of almond-flavored fermented milk This application also provides almond-flavored fermented milk prepared using the following steps: (1) After filtering and purifying the fresh milk, it is subjected to standardized treatment and preheated to 60 °C; (2) Mix almond pulp and milk in a 1:4 ratio, add 5% to 9% sucrose and 0.1% to 0.25% pectin; (3) The mixed raw materials are processed by a colloid mill at a pressure of 90-100 bar to ensure that the liquid is uniform and fine; (4) Heat the homogenized liquid to 90-100 °C and sterilize for 10 min. Then, quickly cool the sterilized liquid to 40-50 °C and add lactobacillus. Lactobacillus sp.Z6 suspension and commercial fermented milk were mixed in a volume ratio of (2~10):1 as a starter culture, stirred evenly, and then bottled. (5) After bottling, inoculate the liquid with 1% to 5% of the mixed starter culture and place it in a constant temperature fermentation chamber at 37 to 42 ℃ for 10 to 18 h. After fermentation, quickly cool it down to 0 to 4 ℃ and perform post-ripening treatment to make the flavor and texture of the fermented milk more stable.

[0035] Preferably, the almond pulp in step (2) is prepared by the following steps: after shelling, select almonds free from mold, pests, and obvious damage, and soak them at room temperature for 2-3 hours; add the soaked almonds to a 0.2% NaOH solution and blanch for 30 seconds, then cool, peel, and rinse clean; add water to the rinsed almonds and pulp them at a 1:10 ratio, and add 100 mg / kg V C (Or 0.2% Na2SO3 solution) is used for color protection treatment to prevent oxidation and discoloration. The treated almond pulp is then refrigerated and stored for later use.

[0036] Preferably, the lactobacillus Lactobacillussp The number of viable bacteria in the Z6 suspension is not less than 1.0 × 10⁻⁶. 8 CFU / mL, commercial fermented milk contains 1.0 × 10⁻⁶ colonies of Streptococcus thermophilus and Lactobacillus acidophilus. 8 CFU / mL.

[0037] Preferably, the amount of sucrose added in step (2) is 7%.

[0038] Preferably, lactobacillus is added in step (4). Lactobacillussp. Z6 suspension was mixed with commercial microbial strains at a volume ratio of 8:1 as a fermentation agent.

[0039] Preferably, in step (5), the inoculum amount of the mixed fermentation agent is 3%, the fermentation temperature is 37 ℃, and the fermentation time is 12 h.

[0040] Example 3: Application of Almond-Flavored Fermented Milk Furthermore, this application provides the use of the almond-flavored fermented milk in the preparation of antihypertensive preparations or health foods, wherein the use is for improving the antihypertensive function of a subject, or for the preparation of preparations for improving the antihypertensive function of a subject.

[0041] The blood pressure-lowering function of the target being improved includes enhancing the inhibitory activity of angiotensin-converting enzyme (ACE).

[0042] Furthermore, this application provides a product that helps enhance the inhibitory activity of angiotensin-converting enzyme (ACE), including the almond-flavored fermented milk described above or the almond-flavored fermented milk prepared by the above-described method.

[0043] The present invention also provides the uses of the almond-flavored fermented milk, specifically: to be used directly as a blood pressure-lowering functional food; or as a raw material to be combined with edible excipients to prepare blood pressure-lowering functional foods.

[0044] The intended use is for oral administration of the product.

[0045] Example 4: Determination of the Probiotic Characteristics of Strains (1) Test of lactic acid bacteria acid tolerance Following He Shanshan's experimental method with slight modifications, the activated third-generation Z6 strain was inoculated into MRS liquid medium at a volume ratio of 3.0% and cultured at 37 ℃ until the stationary phase. After centrifugation at 2500×g for 10 min, the collected bacterial cells were resuspended in media with pH values ​​of 2.0, 3.0, 4.0, and 5.0, respectively, and incubated at 37 ℃ for 3 h. The bacterial suspensions were then collected, and plate counting was performed according to GB 4789.2-2022 "National Food Safety Standard - Microbiological Examination of Food - Determination of Total Colony Count". The survival rate (C, %) was calculated using the following formula.

[0046]

[0047] In the formula: N1 is the number of viable bacteria under tolerance treatment conditions, CFU / mL; N0 is the number of viable bacteria in normal MRS broth medium, CFU / mL.

[0048] (2) Lactic acid bacteria bile salt tolerance test The bacterial cells were collected and resuspended in MRS liquid medium with bile salt concentrations of 0.50, 0.75, 1.00, and 1.25 g / L, respectively. The bacterial suspensions were collected after static incubation at 37 °C for 3 h. The plate counting method and survival rate calculation formula were the same as (1).

[0049] (3) Simulated gastric and intestinal fluid test The artificial gastric and intestinal fluids were prepared according to Chen Qi's method, with slight modifications to the experiment.

[0050] The bacterial suspension was mixed with simulated gastric juice and intestine at a ratio of 1:5 and vortexed for 2 min to ensure thorough mixing. It was then incubated at 37 °C for 3 h to simulate the growth of the strain in a human digestive environment. Culture samples were collected at 0 h and 3 h, diluted and spread on MRS solid medium, and incubated at 37 °C for 24 h. Viable cell counts were then determined to assess the survival ability of the strain in the simulated digestive environment.

[0051] (4) Probiotic properties of Lactobacillus Z6 1. Acid and bile salt resistance Probiotics must overcome a series of physiological barriers to function effectively in the gastrointestinal tract. Among these barriers, the acidic environment of gastric juice is the most critical, with the pH typically varying between 1.5 and 3.0 depending on the diet. This necessitates that probiotics possess acid-resistant properties to survive after passing through gastric juice. Upon entering the intestinal fluid, probiotics encounter bile salts in the small intestine at concentrations of 0.3–3 g / L. Bile secreted by the small intestine can damage cell membranes, altering their permeability and thus affecting the interaction between cells and the surrounding environment, leading to a reduced survival rate of probiotics. Therefore, probiotics must also exhibit good tolerance to bile salts. Figure 1 The survival rate of Z6 was highest at pH 5, reaching 102.58 ± 1.09%, and then decreased with decreasing pH, failing to survive at pH 2. Regarding bile salt tolerance, lower bile salt concentrations did not affect the survival rate of Z6. The highest survival rate (110.77 ± 2.37%) was observed at a bile salt concentration of 0.75 g / L. Significant inhibitory effects began to appear at concentrations greater than 1.0 g / L, with the survival rate dropping to 80.95 ± 4.48% at a bile salt concentration of 1.25 g / L. This indicates that Z6 exhibits good tolerance to bile salts, which can mitigate the effects of bile salt stress to some extent, allowing it to colonize the intestine with high activity after entering the gut. This may be related to the content of its bile salt hydrolase and the production of extracellular polysaccharides.

[0052] 2. Resistance to gastric and intestinal fluids Pepsin and trypsin in gastric and intestinal fluids have antibacterial properties and act as a natural barrier to prevent microorganisms from surviving in the intestines. Therefore, the ability to tolerate gastric and intestinal fluids is a key factor affecting the effectiveness of probiotics. After Z6 was cultured in artificial gastric and intestinal fluids for 3 hours, the survival rate was above 80%, reaching 88.50±0.88% and 88.13±0.85%, respectively.

[0053] Table 1: Results of strains' tolerance to gastric and intestinal fluids

[0054] Example 5: Comparison of ACE inhibitory activity and flavor Commercially available fermented milk, Z6 fermented milk, a mixture of Z6 and commercially available fermented milk, Z6 fermented milk (containing almonds), commercially available fermented milk (containing almonds), and a mixture of Z6 and commercially available fermented milk (containing almonds) were collected separately. The ACE inhibitory activity and flavor of these samples were compared. ACE inhibitory activity was measured using an angiotensin-converting enzyme inhibitor activity assay kit. Ten professional sensory evaluators (male:female = 1:1) then conducted a comprehensive evaluation of the almond fermented milk based on four aspects: flavor, color, texture, and mouthfeel. Detailed scoring criteria are shown in Table 2.

[0055] Table 2: Sensory Scoring Criteria for Almond-Flavored Fermented Milk

[0056] From the appendix Figure 2 It can be seen that commercially available yogurt a has the best flavor and texture (94.50±1.32), but its ACE inhibition rate is 3.78±1.02%, and it has virtually no blood pressure lowering effect. Using Z6 for fermentation (b) significantly improved the ACE inhibition rate to 27.72±3.52%. Fermentation using Z6 and commercially available fermented milk containing Streptococcus thermophilus and Lactobacillus acidophilus (c) did not show significant changes in texture, but its ACE inhibition rate decreased. Adding almond pulp and continuing fermentation with Z6 increased the ACE inhibition rate of d to 42.55±3.09%. When commercially available fermented milk was used alone to ferment almond-flavored milk, the ACE inhibition rate of e was not significant, but the sensory score significantly improved to 92.67±3.06. Finally, when Z6 and commercially available fermented milk were used together to ferment almond-flavored milk, the ACE inhibition rate of f reached the highest at 52.16±4.46%, with a sensory score of 88.50±1.80. The results indicate that using Z6 as the fermentation strain can better improve the blood pressure-lowering effect, while also increasing the acidity of the milk and altering its flavor. Co-fermentation with commercial strains improves these flavor changes. Adding almonds can both increase the ACE inhibition rate of the flavored milk and provide the unique nutty aroma of almonds, compensating for any lack of flavor. Although increasing the amount of almonds may further improve the ACE inhibition rate, considering the standards for flavored milk raw materials and economic costs, the almond pulp content is fixed at 20% by mass.

[0057] Example 6: Single-factor experiment (1) Effect of different sugar contents on almond fermented milk The effects of sucrose additions of 5%, 6%, 7%, 8%, and 9% on the quality of almond fermented milk and its ACE inhibition were studied under the conditions of 14 h fermentation time, 3% mixed inoculum, 6:1 mixed culture ratio, and 37 ℃.

[0058] (2) Effect of different mixed culture ratios on almond fermented milk The effects of mixed culture ratios of 2:1, 4:1, 6:1, 8:1, and 10:1 on the quality of almond fermented milk and its ACE inhibition were investigated under the conditions of 7% sucrose addition, 14 h fermentation time, 3% mixed culture inoculum, and 37 ℃.

[0059] (3) Effects of different fermentation times on almond fermented milk The effects of 7% sucrose addition, 3% mixed inoculum, a mixed inoculum ratio of 6:1, and a temperature of 37 ℃ on the quality of almond fermented milk and its ACE inhibition were investigated at fermentation times of 10, 12, 14, 16, and 18 h.

[0060] (4) Effect of different mixed inoculum amounts on almond fermented milk The effects of 7% sucrose addition, 14 h fermentation time, 6:1 mixed culture ratio, and 37 ℃ on the quality of almond fermented milk and its ACE inhibition effect were studied.

[0061] (5) Effects of different temperatures on almond fermented milk The effects of temperatures of 31, 34, 37, 40, and 43 °C on the quality of almond fermented milk and its ACE inhibition were investigated under the conditions of 7% sucrose addition, 14 h fermentation time, 3% mixed inoculum amount, and a mixed inoculum ratio of 6:1.

[0062] (6) Results of the influence of different single factors on the optimal formula of almond fermented milk 1. Effects of different sucrose addition amounts on ACE inhibition rate and quality of almond fermented milk See appendix Figure 3 As shown, with other factors constant, the ACE inhibition rate increases with increasing sugar content, reaching its maximum at 7% sucrose (50.45±2.93%), before significantly decreasing and stabilizing. The sensory score of the fermented milk initially increases and then decreases with sugar content, reaching its highest at 7% (90±2.00). Sugar content significantly affects both the ACE inhibition rate and flavor / taste. Sucrose, as a carbon source for the bacteria, can increase sweetness and improve the taste of the fermented milk, while also increasing solids and stabilizing the system. Appropriate sucrose can shorten fermentation time and improve fermentation efficiency. However, excessively high sugar concentrations may lead to increased osmotic pressure and viscosity of the fermentation broth, affecting oxygen transfer and bacterial mixing, thus reducing fermentation efficiency. The figure shows that when the sugar content exceeds 7%, it not only inhibits lactic acid bacteria fermentation and reduces the yield of proteases from hydrolyzed proteins, but also affects the flavor of almonds, reduces the acidity of the fermented milk, alters the flavor, and makes it overly sweet. Therefore, 7% is determined to be the optimal amount of sucrose to be added.

[0063] 2. Effects of different mixed culture ratios on ACE inhibition rate and quality of almond fermented milk See appendix Figure 4 As shown, to improve the ACE inhibition rate of flavored milk, mixed fermentation was carried out using Lactobacillus Z6 as the main strain and strains from commercial fermented milk as a supplement. When the ratio increased from 2:1 to 6:1, the ACE inhibition rate was the lowest at only 28.48±3.74%, with little change in sensory scores, insufficient milky aroma, and weak acidity. When the ratio reached 8:1, both indicators rebounded to 38.11±3.12% and 88.33±3.51%, respectively. However, the ACE inhibition rate remained low at this point, indicating that other factors limited the improvement in quality.

[0064] 3. Effects of different time points on ACE inhibition rate and quality of almond fermented milk See appendix Figure 5 As shown, fermentation time has a significant impact on fermented milk, and sensory scores and ACE inhibition rates show a certain correlation. At 12 h of fermentation, the sensory score (90.0±2.0) and ACE inhibition rate (46.42±0.65%) reach their maximum, indicating that Z6 is in the initial stage of logarithmic growth. Both indicators tend to stabilize at the end of logarithmic growth at 18 h. This suggests that Z6 produces ACE-inhibiting peptides optimally during the early stage of logarithmic growth in fermented milk. Excessive fermentation time may further degrade hydrolysates with ACE-inhibiting activity. Studies show that lower molecular weight ACE-inhibiting peptides are unstable and are further decomposed into small oligopeptides or amino acids, with the peptide's active domain being disrupted, thus reducing inhibitory activity. Therefore, a fermentation time of 12 h was chosen.

[0065] 4. Effects of different inoculum amounts on ACE inhibition rate and quality of almond fermented milk See appendix Figure 6 As shown, the inoculum size also significantly affects sensory scores and ACE inhibition rates. At an inoculum size of 1%, the ACE inhibition rate reaches 40.58±1.45%, but the flavor is poor and the taste is too sweet. At an inoculum size of 3%, the sensory score reaches 89.67±2.08, and the ACE inhibition rate is 40.99±0.86%. Subsequently, as the inoculum size increases, the ACE inhibition rate begins to decline, corresponding to the effect of excessive Z6 due to prolonged fermentation time. Considering all factors, an inoculum size of 3% is the most suitable. The sharp drop in ACE inhibition rate at an inoculum size of 4% may be due to an excessively large inoculum size, causing the probiotics to grow too quickly within a given time, resulting in competitive inhibition.

[0066] 5. Effects of different temperatures on ACE inhibition rate and quality of almond fermented milk See appendix Figure 7 As shown, the effect of temperature on the growth rate of lactic acid bacteria caused changes in the indicators of fermented milk. At 31 ℃ and 34 ℃, *Streptococcus thermophilus* did not grow easily; *Lactobacillus* grew slowly at 40 ℃ and 43 ℃. The optimal growth temperature for *Lactobacillus* Z6 was 35 ℃, but at this temperature, the growth rate was too fast, and the ACE inhibition rate could not reach its maximum. When the temperature reached 37 ℃, the sensory score was 91.33±2.08, and the ACE inhibition rate was 51.69±1.85%, reaching the maximum value of each single-factor indicator. This shows that suitable temperature has the greatest positive effect on the indicators.

[0067] The optimal fermentation scheme obtained from single-factor experiments was: sugar content 7%, mixed culture ratio 8:1, fermentation time 12 h, inoculum size 3%, and temperature 37 ℃.

[0068] Example 7: Physicochemical Indicators and Microbiological Detection of Almond-Flavored Fermented Milk According to GB 19302—2010 "National Food Safety Standard for Fermented Milk", the main factors affecting the nutritional value and safety of almond-flavored fermented milk were tested.

[0069] (1) Determination of acidity and pH value Test method: Refer to GB 5009.239—2016 "National Food Safety Standard - Determination of Acidity in Food".

[0070] Acidity was determined using a direct titration method, with the following formula:

[0071] X1, the acidity of the sample. ° T (mL / 100g, calculated as the number of milliliters of 0.1 mol / L NaOH consumed per 100 g sample); c1, molar concentration of NaOH standard solution, mol / L; V1, volume of NaOH standard solution consumed during titration, mL; V0, volume of NaOH standard solution consumed in the blank test, mL; 100, 100 g sample; m1, mass of sample, g; 0.1, molar concentration of NaOH as defined by acidity theory, mol / L.

[0072] pH values ​​were measured using a pH meter.

[0073] (2) Determination of viscosity and water holding capacity Viscosity determination: Take an appropriate amount of yogurt at 4 ℃ and put it into a beaker. Insert the NDJ-5S No. 3 rotor and temperature probe into the yogurt and equilibrate for 5 min. Measure the sample at a speed of 30 r / min. The measurement time should be controlled within 30 s. The rotor should be carefully cleaned before and after the test. Set up 3 parallel samples for each sample. Adjust the measurement results according to the viscometer instruction manual.

[0074] Determination of water-holding capacity: Accurately weigh 20 g of yogurt sample and pour it into a funnel lined with qualitative filter paper. Place the funnel on an Erlenmeyer flask and let it stand at room temperature for 120 min. Collect the filtrate and weigh it. Calculate the difference in weight between the flask and the sample. Set up 3 parallel samples for each sample. The formula is as follows:

[0075] (3) Determination of crude peptide yield Based on Wang Jiaolin's method, we made some modifications.

[0076] Take an appropriate volume of fermented milk and centrifuge at 5000 ×g for 10 min. Take 2.5 mL of the supernatant and add an equal volume of 0.1 g / mL TCA. Let stand for 30 min and centrifuge at 4 ℃ and 2000 ×g for 20 min. Take 2 mL of the supernatant and add 8 mL of biuret reagent. Let stand at 25 ℃ for 30 min and measure the OD value at 540 nm. Calculate the peptide content in the sample solution by referring to the casein standard curve.

[0077]

[0078] ρ , the mass concentration of peptide in the sample, mg / mL; V, the total volume of the sample, mL; m, the mass of the sample, mg.

[0079] (4) Determination of soluble protein content The soluble protein content was determined using the Coomassie Brilliant Blue staining method. A standard curve was plotted with absorbance on the ordinate and bovine serum protein concentration (mg / mL) on the abscissa. 1 mL of fermented milk sample was mixed with 5 mL of Coomassie Brilliant Blue G250 solution, incubated at room temperature for 2 min, and the absorbance was measured at 595 nm. The formula for calculating soluble protein content (mg / g) is as follows:

[0080] In the formula: p ' represents the protein concentration in the final diluted sample solution, mg / mL; V' represents the final diluted sample solution volume, mL; m' represents the mass of the sample used for the fermented milk sample, g.

[0081] (5) Microbial limits The viable bacterial count was determined according to GB / T 4789.35—2023, "National Food Safety Standard - Microbiological Examination of Food - Determination of Total Colony Count", for the count of Lactobacillus and Streptococcus thermophilus.

[0082] Coliform bacteria GB / T 4789.3—2016 "National Food Safety Standard for Microbiological Examination of Food: Coliform Count" Plate Count Method.

[0083] (6) Results 1. Physicochemical Indicators The prepared almond-flavored fermented milk met all the requirements of the "National Food Safety Standard for Fermented Milk". Compared with commercially available yogurt, the almond-flavored fermented milk had better acidity and viscosity, measuring 107.43±2.92%, respectively. °The temperature (T) was 2713.67±108.21 mPa·s, and the water-holding capacity was 62.38±4.23%, which was relatively lower. Z6's excellent acid-producing properties resulted in fermented milk with a higher acidity than previously prepared almond, coix seed, and pecan yogurts. Simultaneously, the increased protein hydrolysates after fermentation led to an increase in small-molecule solubles, significantly improving the crude peptide yield and soluble protein content, reaching 21.27±0.90% and 3.17±0.15 mg / g, respectively. The addition of sucrose and almonds reduced acidity, resulting in a moderately sweet and sour taste. The addition of Z6 provided better control over acidity and produced extracellular polysaccharides, increasing yogurt consistency and enhancing gel stability.

[0084] Table 3: Physicochemical Indicators of Almond-Flavored Fermented Milk

[0085] 2. Determination of microbiological indicators The selected dilution gradient is 10. -7 The number of lactobacilli counted on MRS and MC plates was 5.0 × 10⁻⁶. 8 CFU / mL, thermophilic streptococcus count was 4.5 × 10⁻⁶. 8 CFU / mL. In VRBA medium, *E. coli* utilizes lactose as a carbon source for fermentation, producing acidic metabolites that turn the medium from purple to red. The results of mixing plates after diluting the fermented milk at three different gradients showed no red turn, indicating that *E. coli* was not detected. Z6 has previously been shown to have significant inhibitory activity against *E. coli*, demonstrating its ability to inhibit *E. coli* both in the flavored milk itself and after consumption. Table 4: Microbiological Indicators of Almond-Flavored Fermented Milk

[0086] This application describes an almond-flavored fermented milk prepared using a strain of Lactobacillus Z6 and bacteria from commercial fermented milk. The ACE inhibition rate was 52.16±4.46%, and the sensory score was 88.50±1.80. Furthermore, the lactic acid bacteria activity was excellent, with a Lactobacillus count of 5.0×10⁻⁶. 8 CFU / mL, thermophilic streptococcus count was 4.5 × 10⁻⁶. 8 CFU / mL. Optimal process conditions were: sugar content 7%, mixed culture ratio 8:1, fermentation time 12 h, inoculum size 3%, and temperature 37 ℃. Acidity 107.43±2.92. ° The physicochemical properties, including temperature (T), viscosity (2713.67±108.21 mPa.s), and water holding capacity (62.38±4.23%), all meet national safety standards. This research provides an application pathway for the development of Lactobacillus Z6, broadens the market prospects for the utilization of Xinjiang almond resources, and offers a new approach to the research and development of functional fermented milk.

[0087] The above embodiments are only for illustrating the technical concept and features of the present invention in a specific scenario. Their purpose is to enable those who need this technology to understand the content of the present invention and implement it. They do not limit the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A fermented milk with almond flavor, characterized in that, Made from fresh milk, almond pulp, sucrose, pectin, and lactobacillus. Lactobacillus sp. Z6 suspension and commercially fermented milk containing commercial strains of Streptococcus thermophilus and Lactobacillus acidophilus.

2. The almond-flavored fermented milk as described in claim 1, characterized in that, It is prepared using the following steps: (1) After filtering and purifying the fresh milk, it is subjected to standardized treatment and preheated to 60 °C; (2) Mix almond pulp and milk in a 1:4 ratio, add 5% to 9% sucrose and 0.1% to 0.25% pectin; (3) The mixed raw materials are processed by a colloid mill at a pressure of 90-100 bar to ensure that the liquid is uniform and fine; (4) Heat the homogenized liquid to 90-100 °C and sterilize for 10 min. Then, quickly cool the sterilized liquid to 40-50 °C and add lactobacillus. Lactobacillussp Z6 suspension and commercial fermented milk were mixed at a volume ratio of (2~10):1 as a starter culture, stirred evenly, and then bottled. (5) After bottling, inoculate the liquid with 1% to 5% of the mixed starter culture and place it in a constant temperature fermentation chamber at 37 to 42 ℃ for 10 to 18 h. After fermentation, quickly cool it down to 0 to 4 ℃ and perform post-ripening treatment to make the flavor and texture of the fermented milk more stable.

3. The almond-flavored fermented milk as described in claim 2, characterized in that, The almond pulp in step (2) is prepared as follows: after shelling, select almonds free from mold, pests, and obvious damage, and soak them at room temperature for 2-3 hours; add the soaked almonds to a 0.2% NaOH solution and blanch for 30 seconds, then cool, peel, and rinse thoroughly. Add water to the rinsed almonds and pulp them at a 1:10 ratio, then add 100 mg / kg V... C Alternatively, a 0.2% Na2SO3 solution can be used for color protection treatment to prevent oxidation and discoloration. The treated almond pulp should be refrigerated and stored for later use.

4. The almond-flavored fermented milk as described in claim 2, characterized in that, The lactobacillus mentioned Lactobacillussp The number of viable bacteria in the Z6 suspension is not less than 1.0 × 10⁻⁶. 8 CFU / mL, the bacterial count of Streptococcus thermophilus and Lactobacillus acidophilus in commercial fermented milk is 1.0 × 10⁻⁶. 8 CFU / mL.

5. The almond-flavored fermented milk as described in claim 2, characterized in that, The amount of sucrose added in step (2) is 7%.

6. The almond-flavored fermented milk as described in claim 2, characterized in that, In step (4), lactobacillus is added Lactobacillussp Z6 suspension was mixed with commercial fermented milk at a volume ratio of 8:1 as a starter culture.

7. The almond-flavored fermented milk as described in claim 2, characterized in that, In step (5), the inoculum amount of the mixed fermentation agent is 3%, the fermentation temperature is 37 ℃, and the fermentation time is 12 h.

8. The use of almond-flavored fermented milk as described in any one of claims 1 to 7 in the preparation of antihypertensive preparations or health foods; or the use of almond-flavored fermented milk prepared by the above method in the same field.

9. The use as described in claim 8, wherein the antihypertensive preparation includes improving the antihypertensive function of the subject, wherein the antihypertensive function is to enhance the inhibitory activity of angiotensin-converting enzyme (ACE).

10. A product that helps enhance angiotensin-converting enzyme (ACE) inhibitory activity, comprising almond-flavored fermented milk as described in any one of claims 1 to 7 or almond-flavored fermented milk prepared by the method described in the product.

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