High-activity probiotic freeze-dried yoghurt based on cold stress and calcium synergy technology and preparation method of high-activity probiotic freeze-dried yoghurt

Abstract

The invention discloses high-activity probiotic freeze-dried yoghurt based on a cold stress and calcium synergistic technology and a preparation method of the high-activity probiotic freeze-dried yoghurt. The method comprises the following steps: adding fructo-oligosaccharide, anhydrous cream and whey protein into fresh milk, shearing, homogenizing, pasteurizing, inoculating 3% of a leavening agent, and fermenting; the fermented milk is cooled in three stages, the final temperature is 4 DEG C, and the pH is maintained to be 4.3 + / -0.1 in the whole process; casein phosphopeptides, calcium lactate, magnesium lactate and a mixed freeze-drying protective agent are added, sublimation drying and desorption drying are carried out after pre-freezing is carried out for 4 h, and the vacuum degree is smaller than or equal to 10 Pa. According to the method, thallus stress protein is activated through gradient cooling and cold stress, the calcium absorption rate is improved by combining casein phosphopeptide-calcium lactate compounding, and low saccharification is realized by adopting fructo-oligosaccharide, erythritol and the like as freeze-drying protective agents. The viable count of the final product is more than or equal to 109 CFU / g, the bioavailability of calcium is obviously improved, the total sugar content is less than or equal to 5g / 100g, and the product has the functions of high-activity probiotics and efficient calcium supplement.

Description

Technical Field

[0001] The present invention belongs to the technical field of food processing, and specifically relates to a method for preparing low-sugar freeze-dried yogurt, which improves the survival rate of probiotics through cold stress pretreatment and utilizes casein phosphopeptide (CPP) to cooperate with organic calcium to improve calcium bioavailability. Background Art

[0002] Ice crystal formation during the freeze-drying process and the thermal effects of the secondary drying stage lead to lipid peroxidation and protein denaturation in probiotic cell membranes, compromising their survival. Existing solutions often rely on high concentrations of sugars (such as trehalose and sucrose) as cryoprotectants. While these can inhibit ice crystal growth through glass transition, high-sugar formulations fundamentally conflict with the current trend towards healthy consumption of low sugar and low calories. The solubility of traditional inorganic calcium (calcium carbonate and calcium phosphate) decreases dramatically in acidic milk matrices with a pH ≤ 4.6, leading to flocculation and precipitation in the product and a gritty texture. Furthermore, excess undissolved calcium salts can interfere with the probiotic cell membrane potential, impacting their survival after freeze-drying. Against this backdrop, developing a novel preparation method that can overcome freeze-drying damage mechanisms and coordinate the interactions of functional ingredients has become a pressing technical challenge in the field of functional freeze-dried dairy products. Summary of the Invention

[0003] The purpose of the present invention is to solve the problems of low survival rate of probiotics and low solubility rate of inorganic calcium in existing dairy products, and to provide a high-activity probiotic freeze-dried yogurt based on cold stress and calcium synergistic technology and its preparation method, and to use oligofructose / erythritol freeze-drying protective agent to achieve low saccharification, and the number of viable bacteria in the final product is ≥10 9 CFU / g, calcium bioavailability increased by more than 30%, total sugar content ≤5g / 100g, it has both highly active probiotics and efficient calcium supplementation functions.

[0004] This invention addresses the technical needs of retaining active ingredients and synergizing nutrients during low-temperature processing. It proposes a novel freeze-dried yogurt block preparation process, innovatively integrating the synergistic mechanism of microbial stress induction and nutrient carriers to provide a technical solution for the development of freeze-dried dairy products with high activity, low saccharification, and high calcium utilization. Through an innovative cold stress pretreatment process and the use of casein phosphopeptides and calcium synergistic fortification strategies, this invention establishes a low-sugar, high-efficiency protection system, providing a key technical path for the development of highly active probiotic calcium-fortified foods.

[0005] This invention utilizes casein phosphopeptides fortified with calcium lactate, resulting in high solubility in acidic dairy products. Casein phosphopeptides contain serine, and the phosphate residues of serine can chelate with calcium to form a soluble, stable complex, limiting the precipitation of calcium phosphate salts in the small intestine and promoting calcium absorption. Oligofructose, erythritol, and inulin replace traditional sugar protectants such as sucrose and glucose, creating a low-sugar (total sugar ≤ 5%), high-prebiotic formula that balances functionality and health needs. Cold stress induces the expression of cold shock proteins in the bacteria and improves the fatty acid composition of the cell membrane, significantly enhancing cell membrane stability and further increasing the freeze-drying survival rate.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] A method for preparing highly active probiotic freeze-dried yogurt based on cold stress and calcium synergistic technology, the method comprising:

[0008] (1) Fermentation:

[0009] Fructooligosaccharide, anhydrous butter and whey protein are added to fresh milk, and after shearing, homogenization and pasteurization, 3% starter culture is inoculated and fermented;

[0010] (2) Cold stress pretreatment:

[0011] The fermented milk was cooled in three stages to a final temperature of 4°C, and the pH was maintained at 4.3 ± 0.1 throughout the process;

[0012] (3) Mixing and freeze-drying:

[0013] Add casein phosphopeptide, calcium lactate, magnesium lactate and mixed lyophilization protective agent, pre-freeze for 4 hours, then perform sublimation drying and desorption drying, with a vacuum degree of ≤10 Pa;

[0014] (4) Packaging:

[0015] Aluminum foil is sealed with nitrogen.

[0016] Furthermore, in step (1), the oligofructose content added to the fresh milk is 6% to 12% (w / w), the anhydrous butter content is 0.8% to 2.5% (w / w), and the whey protein content is 1% to 2.5% (w / w).

[0017] Furthermore, in step (1), the fermentation temperature is 37-42°C, the fermentation time is 6-8 h, and the fermentation endpoint is pH 4.5-4.7.

[0018] Furthermore, in step (1), the starter comprises Lactobacillus bulgaricus MN-ZLW-003 (deposit number CGMCC No.3818, deposit date 2010.5.7), Streptococcus thermophilus MN-ZLW-001 (deposit number CGMCC No.3816, deposit date 2010.5.7) and other lactic acid bacteria; the other lactic acid bacteria comprise Lactobacillus delbrueckii subsp. bulgaricus SP1.1 (deposit number CGMCC No.16586, deposit date 2018.10.15), Lactobacillus casei subsp. casei YRL577-1-M (deposit number CGMCC No.17217, deposit date 2019.01.18) and Lactobacillus paracasei zlw-J23ANL (deposit number CGMCC No. 10653, deposited on March 23, 2015); the viable counts of Lactobacillus bulgaricus, Streptococcus thermophilus, and other lactic acid bacteria are 2:2:1. The bacterial species of the starter were isolated by the inventors from natural fermentation products.

[0019] Furthermore, in step (2), the gradient cooling process is from 37~42℃ to 20~25℃, the cooling time is 30~90min, and it is kept for 3~5h; then it is cooled to 15~10℃, the cooling time is 10~30min, and it is kept for 4~6h; finally it is cooled to 4℃, the cooling time is 5~20min, and it is kept for 5~7h.

[0020] Furthermore, in step (3), the amount of casein phosphopeptide added is 0.05% to 0.1% (w / w), the amount of calcium lactate added is 0.8% to 1% (w / w), and the amount of magnesium lactate added is 0.1% to 0.2% (w / w).

[0021] Furthermore, in step (3), the mixed lyoprotectant comprises component A and component B, component A is 5% to 10% (w / w) of oligofructose and / or 3% to 8% (w / w) of erythritol, and component B is one or more of 1% to 4% (w / w) of inulin, 0.03% to 0.05% (w / w) of β-cyclodextrin, or 0.1% to 0.3% (w / w) of microcrystalline cellulose.

[0022] Furthermore, in step (3), the pre-freezing temperature is -30~-25°C.

[0023] Furthermore, in step (3), the temperature is gradually increased in the sublimation drying stage, first from -30 to -25 °C by 10 °C, with a total time of 10 to 13 h; then from -20 to -15 °C to 20 to 25 °C, with a total time of 20 to 25 h; and the temperature is maintained at 15 to 20 °C in the desorption drying stage, with a total time of 10 to 15 h.

[0024] A highly active probiotic freeze-dried yogurt prepared by the above preparation method.

[0025] The beneficial effects of the present invention over the prior art are as follows: the present invention induces probiotics to express cold shock proteins through the synergistic effect of gradient cooling and cold stress, thereby improving the freeze-dried survival rate of probiotics, and the number of viable bacteria in the product is ≥10 9 CFU / g. Casein phosphopeptides and calcium lactate are added to chelate calcium ions, forming a soluble complex and improving calcium bioavailability. Fructooligosaccharides, erythritol, and inulin are used as low-sugar protective agents to achieve a total sugar content of ≤5%. This product combines highly active probiotics with efficient calcium supplementation, providing an efficient technical solution for the development of functional dairy products with high activity, high calcium efficiency, and low glycation properties. DETAILED DESCRIPTION

[0026] The technical solution of the present invention is further described below with reference to the embodiments, but is not limited thereto. Any modification or equivalent replacement of the technical solution of the present invention that does not depart from the spirit and scope of the technical solution of the present invention should be included in the scope of protection of the present invention.

[0027] Specific embodiment 1: A method for preparing highly active probiotic freeze-dried yogurt based on cold stress and calcium synergistic technology comprises the following steps:

[0028] (1) Fermentation

[0029] Add 6%-12% (w / w) fructooligosaccharide, 0.8%-2.5% (w / w) anhydrous butter, and 1%-2.5% (w / w) whey protein to fresh milk. Place the mixture in a shearing machine at 3000 rpm for 3-5 minutes. After shearing, allow to stand for 5 minutes to ensure uniform shearing. Heat the base liquid to 50-60°C and homogenize at a pressure of 5-10 MPa. Pasteurize the homogenized milk at 60-65°C for 30 minutes. Cool the pasteurized milk to 37-42°C. Then, add the prepared starter culture at a 3% dosage to the fermentation vessel. After inoculation, stir thoroughly and maintain the fermentation temperature at 37-42°C for 6-8 hours. Stop fermentation when the pH drops to 4.5-4.7 and the milk is well coagulated. The bacterial strains of the starter were isolated by the inventors from natural fermentation products and preserved in the China General Microorganism Culture Collection Center (CGMCC), including Lactobacillus delbrueckii subsp. bulgaricus (preservation number CGMCC No.3818, preservation date 2010.5.7), Streptococcus thermophilus (preservation number CGMCC No.3816, preservation date 2010.5.7), Lactobacillus delbrueckii subsp. bulgaricus (preservation number CGMCC No.16586, preservation date 2018.10.15), Lactobacillus casei subsp. casei (preservation number CGMCC No. One or more of the following: Lactobacillus paracasei (CGMCC No. 17217, deposited on January 18, 2019) and Lactobacillus paracasei (CGMCC No. 10653, deposited on March 23, 2015), must contain Lactobacillus bulgaricus and Streptococcus thermophilus, and the ratio of Lactobacillus bulgaricus, Streptococcus thermophilus and other lactic acid bacteria is 2:2:1.

[0030] (2) Cold stress pretreatment

[0031] When the pH drops to 4.5-4.7, stop natural fermentation and cool the fermented milk from 37-42°C to 20-25°C for 30-90 minutes and hold for 3-5 hours. Then, cool it to 15-10°C for 10-30 minutes and hold for 4-6 hours. Finally, cool it to 4°C for 5-20 minutes and hold for 5-7 hours. Maintain the pH at 4.3 ± 0.1 throughout the process, adjusting it with NaHCO3 as needed to avoid over-acidification and damage to the bacteria.

[0032] (3) Mixing and freeze-drying

[0033] 0.05%–0.1% (w / w) casein phosphopeptide, 0.8%–1% (w / w) calcium lactate, and 0.1%–0.2% (w / w) magnesium lactate were passed through a 300-mesh sieve and mixed into the slurry. A lyoprotectant was added to the slurry, and the mixture was mixed under vacuum at 600 rpm for 5–10 min to obtain a uniformly dispersed slurry. The slurry was poured into a mold and rapidly frozen at a pre-freezing temperature of -30–-25°C for 3 h. After pre-freezing, the mold was removed and placed in a freeze dryer for another 1 h. Vacuum drying was then performed at a vacuum pressure of ≤10 Pa. The sublimation drying stage employed a gradient temperature increase: first, from -30–-25°C, increasing by 10°C for 10–13 h; then, from -20–-15°C to 20–25°C for 20–25 h. The desorption drying stage was maintained at a temperature of 15–20°C for 10–15 h. After freeze-drying, the product is immediately sealed in an aluminum foil bag flushed with nitrogen. The mixed freeze-drying protective agent comprises 5% to 10% (w / w) fructooligosaccharides, 3% to 8% (w / w) erythritol, 1% to 4% (w / w) inulin, 0.03% to 0.05% (w / w) beta-cyclodextrin, and 0.1% to 0.3% (w / w) microcrystalline cellulose.

[0034] The starter strain separation method of the present invention comprises a dilution coating method and a plate streaking method.

[0035] 1. Sample Pretreatment

[0036] (1) Sample processing

[0037] Liquid sample: Take 1 mL and add it to 9 mL sterile saline (0.85% NaCl), shake and mix (10 -1 dilution).

[0038] Solid or semi-solid samples: Take 1 g of sample and add it to 9 mL of sterile saline. Homogenize with a sterile mortar and pestle, let it stand for 10 min, and take the supernatant (10 -1 dilution).

[0039] (2) Gradient dilution

[0040] Take 10-1 1 mL of diluent was added to 9 mL of sterile saline and mixed to obtain 10 -2 diluent.

[0041] Repeat the above steps to prepare 10 -3 , 10 -4 , 10 -5 , 10 -6 Serial dilutions. The specific dilution gradient is adjusted according to the bacterial content of the sample.

[0042] 2. Separation by dilution coating method

[0043] Take different dilutions (such as 10 -4 , 10 -5 , 10 -6 Add 100 μL of each sample dilution to the center of a sterile MRS agar plate containing 0.5% CaCO₃. Use a sterile spreader to evenly spread the entire surface of the plate. Invert the plate and incubate at 37°C in an anaerobic environment for 24–72 hours.

[0044] 3. Plate streak purification

[0045] Pick a single colony surrounded by a clear, molten calcium ring on the plate. Mark out the four zones: Use a sterile inoculating loop to pick up a single colony and mark the first zone (approximately 1 / 4 of the plate) on a fresh plate containing the culture medium. Using a sterile inoculating loop, mark out the second zone (approximately 1 / 2 of the plate) from the end of the first zone. Repeat for the third and fourth zones. Incubate the plate in an inverted, anaerobic environment at 37°C for 24-48 hours. A single colony should appear in the fourth zone.

[0046] 4. Preservation of pure strains

[0047] The pure strain was inoculated into MRS liquid medium, cultured at 37°C for 24 hours, and then stored at -80°C for a long term with 20% glycerol.

[0048] Example 1

[0049] A highly active probiotic freeze-dried yogurt based on cold stress and calcium synergistic technology and a preparation method thereof comprises the following steps:

[0050] (1) Fermentation

[0051] 8% (w / w) fructooligosaccharide, 1.5% (w / w) anhydrous butter, and 2% (w / w) whey protein were added to fresh milk. The mixture was placed in a shearing machine at a speed of 3000 rpm for 5 minutes. After shearing, the mixture was allowed to stand for 5 minutes to ensure uniform shearing. The raw base liquid was heated to 50°C and homogenized at a pressure of 10 MPa. The homogenized emulsion was pasteurized at 62°C for 30 minutes. The pasteurized milk was cooled to 37°C, and the prepared production starter was added to the fermentation vessel at a dosage of 3%. After inoculation, the mixture was stirred thoroughly and fermented at 37°C for 8 hours. Fermentation was stopped when the pH dropped to 4.5 and the coagulation was well established. The starter strains were isolated by the inventors from natural fermentation products and preserved in the China General Microorganism Culture Collection (CGMCC), including Lactobacillus bulgaricus MN-ZLW-003 (preservation number CGMCC No. 3818, preservation date 2010.5.7), Streptococcus thermophilus MN-ZLW-001 (preservation number CGMCC No. 3816, preservation date 2010.5.7) and Lactobacillus paracasei zlw-J23ANL (preservation number CGMCC No. 10653), with the viable cell count of 2:2:1.

[0052] (2) Cold stress pretreatment

[0053] When the pH drops to 4.5, stop the natural fermentation and cool the fermented milk from 37°C to 25°C for 30 minutes and hold for 4 hours. Then, cool the fermented milk from 25°C to 10°C for 10 minutes and hold for 5 hours. Finally, cool the fermented milk from 10°C to 4°C for 5 minutes and hold for 5 hours. Maintain the pH at 4.3 ± 0.1 throughout the process, adjusting the temperature with NaHCO3 as needed to avoid over-acidification and damage to the bacteria.

[0054] (3) Mixing and freeze-drying

[0055] 0.08% (w / w) casein phosphopeptide, 1% (w / w) calcium lactate, and 0.15% (w / w) magnesium lactate were passed through a 300-mesh sieve and mixed into the slurry. A lyoprotectant was added to the slurry, and the mixture was mixed under vacuum at 600 rpm for 10 minutes to obtain a uniformly dispersed slurry. The slurry was poured into a mold and rapidly frozen at -30°C for 3 hours. After pre-freezing, the mold was removed and placed in a freeze dryer for another hour. Vacuum drying was then performed at a vacuum pressure of ≤10 Pa. The sublimation drying stage employed a gradient temperature increase: first, increasing the temperature from -30°C by 10°C over 30 minutes and maintaining it at 10°C for 10 hours; then, increasing the temperature from -20°C to 25°C over 30 minutes and maintaining it at 25°C for 20 hours. The desorption drying stage was maintained at 20°C for 10 hours. After lyophilization, the slurry was immediately sealed in aluminum foil bags flushed with nitrogen. The freeze-drying protective agent comprises 8% (w / w) oligofructose, 5% (w / w) erythritol, 2% (w / w) inulin, 0.04% (w / w) beta-cyclodextrin and 0.2% (w / w) microcrystalline cellulose.

[0056] Example 2

[0057] A highly active probiotic freeze-dried yogurt based on cold stress and calcium synergistic technology and a preparation method thereof comprises the following steps:

[0058] (1) Add 6% (w / w) oligofructose, 2% (w / w) anhydrous butter, and 1.5% (w / w) whey protein to fresh milk. Place the prepared liquid in a shearing machine at a shearing speed of 3000 rpm and a shearing time of 5 min. After shearing, let it stand for 5 min to ensure uniform shearing. Heat the raw base liquid to 60°C and homogenize at a pressure of 10 MPa. Pasteurize the homogenized emulsion at a sterilization temperature of 62°C for 30 min. Cool the sterilized milk to 42°C, then add the prepared production starter at a dosage of 3% to the fermentation container, stir thoroughly after inoculation, and ferment at a temperature of 42°C for 8 h. Stop fermentation when the pH drops to 4.7 and the coagulation state is good. The starter strains were isolated by the inventors from natural fermentation products and preserved in the China General Microorganism Culture Collection (CGMCC), including Lactobacillus bulgaricus MN-ZLW-003 (preservation number CGMCC No. 3818, preservation date 2010.5.7), Streptococcus thermophilus MN-ZLW-001 (preservation number CGMCC No. 3816, preservation date 2010.5.7) and Lactobacillus casei subspecies casei YRL577-1-M (preservation number CGMCC No. 17217, preservation date 2019.01.18), with the viable cell count of 2:2:1.

[0059] (2) Cold stress pretreatment

[0060] When the pH drops to 4.7, stop natural fermentation and cool the fermented milk from 42°C to 25°C for 40 minutes, holding it for 4 hours. Then, cool the fermented milk from 25°C to 10°C for 20 minutes, holding it for 4 hours. Finally, cool the fermented milk from 10°C to 4°C for 10 minutes, holding it for 5 hours. Maintain the pH at 4.3 ± 0.1 throughout the process, adjusting it with NaHCO3 as needed to avoid over-acidification and damage to the bacteria.

[0061] (3) Mixing and freeze-drying

[0062] 0.1% (w / w) casein phosphopeptide, 1% (w / w) calcium lactate, and 0.2% (w / w) magnesium lactate were passed through a 300-mesh sieve and mixed into the slurry. A lyoprotectant was added to the slurry, and the mixture was mixed under vacuum at 600 rpm for 5–10 minutes to obtain a uniformly dispersed slurry. The slurry was poured into a mold and rapidly frozen at -30°C for 3 hours. After pre-freezing, the mold was removed and placed in a freeze dryer for another 1 hour. Vacuum drying was then performed at a vacuum pressure of ≤10 Pa. The sublimation drying stage was performed using a gradient temperature increase: first, from -30°C, increasing by 10°C for 10 hours, and then from -20°C to 25°C for 20 hours. The desorption drying stage was maintained at 20°C for 10 hours. After lyophilization, the slurry was immediately sealed in aluminum foil bags flushed with nitrogen. The freeze-drying protective agent contains 10% (w / w) oligofructose, 3% (w / w) erythritol, 1% (w / w) inulin, 0.05% (w / w) β-cyclodextrin and 0.1% (w / w) microcrystalline cellulose.

[0063] Example 3

[0064] A highly active probiotic freeze-dried yogurt based on cold stress and calcium synergistic technology and a preparation method thereof comprises the following steps:

[0065] (1) Fermentation

[0066] Fresh milk was mixed with 6% (w / w) fructooligosaccharide, 2.5% (w / w) anhydrous butter, and 2.5% (w / w) whey protein. The mixture was placed in a shearing machine at a speed of 3000 rpm for 5 minutes. After shearing, the mixture was allowed to stand for 5 minutes to ensure uniform shearing. The base liquid was heated to 50°C and homogenized at a pressure of 8 MPa. The homogenized emulsion was pasteurized at 62°C for 30 minutes. The pasteurized milk was cooled to 42°C, and the prepared starter culture was added to the fermentation vessel at a rate of 3%. After inoculation, the culture was stirred thoroughly and fermented at a temperature of 42°C. Fermentation was terminated when the pH dropped to 4.5 and the coagulation was satisfactory. The starter strains were isolated by the inventors from natural fermentation products and preserved in the China General Microorganism Culture Collection (CGMCC), including Lactobacillus bulgaricus MN-ZLW-003 (deposit number CGMCC No. 3818, deposit date 2010.5.7), Streptococcus thermophilus MN-ZLW-001 (deposit number CGMCC No. 3816, deposit date 2010.5.7) and Lactobacillus delbrueckii subsp. bulgaricus SP1.1 (deposit number CGMCC No. 16586, deposit date 2018.10.15), with the viable cell count being 2:2:1.

[0067] (2) Cold stress pretreatment

[0068] When the pH drops to 4.5, stop the natural fermentation and cool the fermented milk from 42°C to 25°C for 50 minutes and hold for 4 hours. Then, cool the fermented milk from 25°C to 10°C for 30 minutes and hold for 5 hours. Finally, cool the fermented milk from 10°C to 4°C for 15 minutes and hold for 6 hours. Maintain the pH at 4.3 ± 0.1 throughout the process, adjusting the temperature with NaHCO3 as needed to avoid over-acidification and damage to the bacteria.

[0069] (3) Mixing and freeze-drying

[0070] 0.05% (w / w) casein phosphopeptide, 1% (w / w) calcium lactate, and 0.1% (w / w) magnesium lactate were passed through a 300-mesh sieve and mixed into the slurry. A lyoprotectant was added to the slurry, and the mixture was mixed under vacuum at 600 rpm for 5 minutes to obtain a uniformly dispersed slurry. The slurry was poured into a mold and rapidly frozen at -30°C for 3 hours. After pre-freezing, the mold was removed and placed in a freeze dryer for another 1 hour. Vacuum drying was then performed at a vacuum pressure of ≤10 Pa. The sublimation drying stage was performed using a gradient temperature increase: first, from -30°C, increasing by 10°C for 10 hours, and then from -20°C to 25°C for 20 hours. The desorption drying stage was maintained at 20°C for 10 hours. After lyophilization, the slurry was immediately sealed in aluminum foil bags flushed with nitrogen. The freeze-drying protective agent contains 5% (w / w) oligofructose, 8% (w / w) erythritol, 4% (w / w) inulin, 0.03% (w / w) β-cyclodextrin and 0.15% (w / w) microcrystalline cellulose.

[0071] Example 4

[0072] A highly active probiotic freeze-dried yogurt based on cold stress and calcium synergistic technology and a preparation method thereof comprises the following steps:

[0073] (1) Fermentation

[0074] Fresh milk was added with 12% (w / w) fructooligosaccharide, 0.8% (w / w) anhydrous butter, and 1% (w / w) whey protein. The mixture was placed in a shearing machine at a speed of 3000 rpm for 3 minutes. After shearing, the mixture was allowed to stand for 5 minutes to ensure uniform shearing. The base liquid was heated to 50°C and homogenized at a pressure of 8 MPa. The homogenized emulsion was pasteurized at 62°C for 30 minutes. The pasteurized milk was cooled to 40°C, and the prepared production starter was added to the fermentation vessel at a rate of 3%. After inoculation, the mixture was stirred thoroughly and fermented at 40°C for 7 hours. Fermentation was stopped when the pH dropped to 4.6 and the coagulation was complete. The starter strains were isolated by the inventors from natural fermentation products and preserved in the China General Microorganism Culture Collection (CGMCC), including Lactobacillus bulgaricus MN-ZLW-003 (preservation number CGMCC No. 3818, preservation date 2010.5.7), Streptococcus thermophilus MN-ZLW-001 (preservation number CGMCC No. 3816, preservation date 2010.5.7) and Lactobacillus paracasei zlw-J23ANL (preservation number CGMCC No. 10653), with the viable cell count of 2:2:1.

[0075] (2) Cold stress pretreatment

[0076] When the pH drops to 4.6, stop the natural fermentation and cool the fermented milk from 37°C to 23°C for 20 minutes and hold for 3.5 hours. Then, cool the fermented milk from 25°C to 10°C for 10 minutes and hold for 4.5 hours. Finally, cool the fermented milk from 10°C to 4°C for 8 minutes and hold for 5 hours. Maintain the pH at 4.3 ± 0.1 throughout the process, adjusting the temperature with NaHCO3 as needed to avoid over-acidification and damage to the bacteria.

[0077] (3) Mixing and freeze-drying

[0078] 0.1% (w / w) casein phosphopeptide, 1% (w / w) calcium lactate, and 0.2% (w / w) magnesium lactate were passed through a 300-mesh sieve and mixed into the slurry. A lyoprotectant was added to the slurry, and the mixture was mixed under vacuum at 600 rpm for 10 minutes to obtain a uniformly dispersed slurry. The slurry was poured into a mold and rapidly frozen at -28°C for 3 hours. After pre-freezing, the mold was removed and placed in a freeze dryer for another 1 hour. Vacuum drying was then performed at a vacuum pressure of ≤10 Pa. The sublimation drying stage was performed using a gradient temperature increase: first, from -28°C, increasing by 10°C for 10 hours, and then from -18°C to 23°C for 20 hours. The desorption drying stage was maintained at 18°C ​​for 10 hours. After lyophilization, the slurry was immediately sealed in aluminum foil bags flushed with nitrogen. The freeze-drying protective agent contains 10% (w / w) oligofructose, 3% (w / w) erythritol, 4% (w / w) inulin, 0.03% (w / w) beta-cyclodextrin and 0.2% (w / w) microcrystalline cellulose.

[0079] Example 5

[0080] A highly active probiotic freeze-dried yogurt based on cold stress and calcium synergistic technology and a preparation method thereof comprises the following steps:

[0081] (1) Fermentation

[0082] Fresh milk was added with 12% (w / w) fructooligosaccharide, 0.8% (w / w) anhydrous butter, and 1% (w / w) whey protein. The mixture was placed in a shearing machine at a speed of 3000 rpm for 3 minutes. After shearing, the mixture was allowed to stand for 5 minutes to ensure uniform shearing. The raw base liquid was heated to 60°C and homogenized at a pressure of 8 MPa. The homogenized emulsion was pasteurized at 65°C for 30 minutes. The pasteurized milk was cooled to 38°C, and the prepared production starter was added to the fermentation vessel at a dosage of 3%. After inoculation, the mixture was stirred thoroughly and fermented at 38°C for 8 hours. Fermentation was stopped when the pH dropped to 4.7 and the coagulation was well established. The starter strains were isolated by the inventors from natural fermentation products and preserved in the China General Microorganism Culture Collection (CGMCC), including Lactobacillus bulgaricus MN-ZLW-003 (preservation number CGMCC No. 3818, preservation date 2010.5.7), Streptococcus thermophilus MN-ZLW-001 (preservation number CGMCC No. 3816, preservation date 2010.5.7) and Lactobacillus paracasei zlw-J23ANL (preservation number CGMCC No. 10653), with the viable cell count of 2:2:1.

[0083] (2) Cold stress pretreatment

[0084] When the pH drops to 4.7, stop the natural fermentation and cool the fermented milk from 37°C to 25°C for 25 minutes and hold for 3 hours. Then, cool the fermented milk from 25°C to 10°C for 20 minutes and hold for 4.5 hours. Finally, cool the fermented milk from 10°C to 4°C for 6 minutes and hold for 5.5 hours. Maintain the pH at 4.3 ± 0.1 throughout the process, adjusting the temperature with NaHCO3 as needed to avoid over-acidification and damage to the bacteria.

[0085] (3) Mixing and freeze-drying

[0086] 0.05% (w / w) casein phosphopeptide, 1% (w / w) calcium lactate, and 0.1% (w / w) magnesium lactate were passed through a 300-mesh sieve and mixed into the slurry. A lyoprotectant was added to the slurry, and the mixture was mixed under vacuum at 600 rpm for 7 minutes to obtain a uniformly dispersed slurry. The slurry was poured into a mold and pre-frozen at -28°C for 3 hours. After pre-freezing, the mold was removed and placed in a freeze dryer for another 1 hour. Vacuum drying was then performed at a vacuum pressure of ≤10 Pa. The sublimation drying stage was performed using a gradient temperature increase: first, from -28°C, increasing by 10°C for 10 hours, and then from -18°C to 23°C for 20 hours. The desorption drying stage was maintained at 18°C ​​for 10 hours. After lyophilization, the slurry was immediately sealed in aluminum foil bags flushed with nitrogen. The freeze-drying protective agent contains 5% (w / w) oligofructose, 8% (w / w) erythritol, 1% (w / w) inulin, 0.035% (w / w) β-cyclodextrin and 0.2% (w / w) microcrystalline cellulose.

[0087] Comparative Example 1

[0088] The same as Example 1, except that the fermentation was carried out at a constant temperature of 37°C until the pH value reached 4.5, and then the temperature was directly lowered to 4°C.

[0089] Comparative Example 2

[0090] The same as Example 1, except that sucrose replaces the lyoprotectant.

[0091] Comparative Example 3

[0092] The same as Example 1, except that casein phosphopeptide was not added.

[0093] Comparative Example 4

[0094] The method is the same as Example 1, except that there is no gradient temperature increase during the freeze-drying process.

[0095] Comparative Example 5

[0096] The same as Example 1, except that no lyoprotectant, casein phosphopeptide, calcium lactate and magnesium lactate were added, the temperature was directly lowered to 4°C after fermentation at a constant temperature of 37°C to pH 4.5, and there was no gradient temperature increase during the freeze-drying process.

[0097] Under cold stress, the cold shock proteins CSPA / CSPB in probiotics bind to RNA, preventing the formation of RNA secondary structures at low temperatures, promoting ribosome assembly to maintain protein synthesis, and activating downstream cold-adaptation genes such as the fatty acid desaturase gene, desA. Their expression levels are significantly upregulated with gradual cooling. Furthermore, cold stress promotes the synthesis of unsaturated fatty acids by inducing the desA gene, providing substrates for cyclopropane fatty acid (CFA) production. Furthermore, acid stress activates the cfa gene, increasing cyclopropanation efficiency and the CFA / SFA ratio. This enhances membrane flexibility within rigidity, balances membrane fluidity at low temperatures, and reduces ice crystal damage. The synergistic effect of these two factors can improve the freeze-dried probiotic survival rate, while also ensuring calcium absorption and enzyme activity by enhancing membrane integrity, thereby ensuring bacterial recovery and preserving functional activity.

[0098] The calcium absorption-promoting freeze-dried yogurt products containing live bacteria prepared in Examples 1-5 and Comparative Examples 1-5 were sampled and tested:

[0099] (1) Protein expression detection:

[0100] A 5-g solid sample was dissolved in physiological saline and centrifuged at 10,000 rpm for 10 min to collect the cells. The cells were lysed using a lysis buffer containing protease inhibitors, and the protein concentration was accurately determined by the BCA assay. Subsequently, 20 μg of total protein was loaded onto a 12% separating gel for SDS-PAGE electrophoresis, and the protein was transferred to a PVDF membrane. The membrane was then incubated with primary antibodies (CSPA and CSPB) at a 1:1000 dilution, followed by a secondary antibody (HRP)-labeled (HRP) antibody at a 1:5000 dilution. Finally, ECL chemiluminescence was used for color development, and grayscale values ​​were analyzed using ImageJ software. Fold Change of the target protein was calculated using β-actin as an internal reference. The results are shown in Table 1.

[0101] (2) Membrane fatty acid analysis:

[0102] Dissolve 5 g of solid sample in saline, centrifuge at 8000 rpm for 10 min, and discard the supernatant. Wash with 8.5 g / L sterile saline and centrifuge three times (6000 rpm, 4°C, 15 min). Discard the supernatant and weigh 0.5 g of bacterial sludge. Add 1.5 mL of 1 mol / L sodium methoxide in methanol to the resulting sludge, shake vigorously for 1.5 min, and let stand at 4°C for 10 min. Add 1 mL of n-hexane solution, shake for 1 min, let stand for 5 min, and centrifuge at 6000 rpm for 5 min. Aspirate the supernatant, filter through an organic filter, and place the resulting sample in a gas phase flask for fatty acid content determination. GC-MS analysis of cell membrane fatty acid composition and content was performed.

[0103] (3) Calcium ion absorption and transport rate:

[0104] Cells in the logarithmic growth phase were seeded in 12-well Transwell cell culture plates. When the cells formed a dense monolayer, the old culture medium was aspirated and discarded. After washing the upper and lower chambers of the Transwell with calcium- and magnesium-free HBSS buffer, HBSS solution was added to the upper and lower chambers, respectively, and incubated in an incubator for 40 min to remove the residual culture medium. After removing the upper and lower solutions, 0.5 mL of the sample to be absorbed was added to the upper chamber, and 1.5 mL of HBSS buffer was added to the lower chamber. After incubation in the incubator for 2 h, the solution in the lower chamber was removed and the calcium ion concentration (A1) in the solution was determined by atomic absorption spectrometry. The cells in the upper chamber were washed with PBS solution and lysed with 2% SDS solution. The calcium ion concentration (A2) in the cells was determined. The calcium ion concentration in the initial calcium preparation solution was recorded as A0. The calcium transport rate = A1 / A0, and the calcium absorption rate = A2 / A0. The results are shown in Table 2.

[0105] Sensory evaluation of the calcium-absorbing freeze-dried yogurt products containing live bacteria and prepared in Examples 1-5 and Comparative Examples 1-4 was conducted: A sensory evaluation panel of 30 professionally trained personnel evaluated the products based on appearance (smooth, uniform, and smooth without graininess), flavor (yogurt aroma with a moderate sweetness and sourness), texture (no cavitation, breakage, or glassiness), and mouthfeel (crispness and stickiness). The total score was 100, and the average score was used as the final result. The sensory evaluation scoring criteria are shown in Table 3, and the results are shown in Table 4 below.

[0106] The calcium absorption-promoting freeze-dried yogurt products containing live bacteria prepared in Examples 1-5 and Comparative Examples 1-4 were sampled and tested:

[0107] (1) Adhesion: The adhesion of the sample was analyzed using a texture analyzer. After quickly taking the sample out of the packaging bag, 5 mL of distilled water was evenly dripped on the material. After 30 seconds, a full texture test was performed using a physical property analyzer. The tensile force when the probe was raised was used to represent the adhesion of the sample (unit: g).

[0108] (2) Brittleness: After quickly taking out the sample from the packaging bag, perform a compression test using a physical property tester, repeating 10 times. Record the force of material disintegration and the number of peaks generated in each test, and take the average value. The brittleness is expressed as the average value of the number of peaks generated in the test, and the unit is "pieces". The more peaks there are, the better the brittleness of the product.

[0109] (3) Total bacterial count: The total bacterial count in freeze-dried yogurt was determined using the plate count method.

[0110] (4) Survival rate: The total number of colonies in yogurt before and after freeze-drying was determined by plate counting method. Survival rate % = (number of viable bacteria after freeze-drying / number of viable bacteria before freeze-drying) × 100%. The test results are shown in Table 5.

[0111] Table 1 CSPA, CSPB protein expression and CFA / SFA ratio

[0112]

[0113] Table 2 Calcium absorption rate and calcium transport rate

[0114]

[0115] Table 3 Sensory evaluation criteria

[0116]

[0117] Table 4 Sensory evaluation scores

[0118]

[0119] Table 5 Performance test table

[0120]

[0121] Analyzing the data in Tables 1, 2, and 3, we can see that:

[0122] (1) In combination with Examples 1 to 5 and Comparative Examples 1 to 5, a highly active probiotic freeze-dried yogurt based on cold stress and calcium synergistic technology has the characteristics of low sugar, high content of probiotics, and high calcium supplementation, and has good sensory properties.

[0123] (2) Combining Examples 1 to 5 and Comparative Example 1, the survival rate of the probiotics in Example 1 was higher than that in Comparative Example 1, verifying the core role of stress-induced stress proteins and maintaining the integrity of cell membranes. Cold stress can improve the survival rate of probiotics.

[0124] (3) Combining Examples 1 to 5 and Comparative Example 2, the total sugar content of Examples 1 to 5 is lower than that of Comparative Example 2. Compared with sucrose, the low-sugar lyoprotectant can reduce the stickiness of the product, improve the crispness of the product and the survival rate of probiotics, and better meet the low-calorie demand.

[0125] (4) Combining Examples 1 to 5 and Comparative Example 3, the calcium absorption rate and transport rate of Examples 1 to 5 are higher than those of Comparative Example 3. The addition of casein phosphopeptide and calcium lactate improves the bioavailability of calcium.

[0126] (5) Combining Examples 1 to 5 and Comparative Example 4, the survival rate of probiotics in Examples 1 to 5 is higher than that in Comparative Example 1, verifying the process advantage of gradient heating.

[0127] (6) In combination with Examples 1 to 5 and Comparative Example 5, the total bacterial count and survival rate, calcium absorption rate, transport rate and sensory properties of the probiotics in Examples 1 to 5 were higher than those in Comparative Example 5, which verified the superiority of a high-activity probiotic freeze-dried yogurt based on cold stress and calcium synergistic technology and its preparation method.

Claims

1. A method for preparing highly active probiotic freeze-dried yogurt based on cold stress and calcium synergistic technology, characterized in that: The method is: (1) Fermentation: Fructooligosaccharide, anhydrous butter and whey protein are added to fresh milk, and after shearing, homogenization and pasteurization, 3% starter culture is inoculated and fermented; (2) Cold stress pretreatment: The fermented milk was cooled in three stages to a final temperature of 4°C, and the pH was maintained at 4.3 ± 0.1 throughout the process; (3) Mixing and freeze-drying: Add casein phosphopeptide, calcium lactate, magnesium lactate and mixed lyophilization protective agent, pre-freeze for 4 hours, then perform sublimation drying and desorption drying, with a vacuum degree of ≤10 Pa; (4) Packaging: Aluminum foil is sealed with nitrogen.

2. The method according to claim 1, wherein: In step (1), the oligofructose content added to the fresh milk is 6% to 12% (w / w), the anhydrous butter content is 0.8% to 2.5% (w / w), and the whey protein content is 1% to 2.5% (w / w).

3. The method according to claim 1, wherein: In step (1), the fermentation temperature is 37-42°C, the fermentation time is 6-8 h, and the fermentation end point is pH 4.5-4.

7.

4. The method according to claim 1, wherein: In step (1), the starter includes Lactobacillus bulgaricus MN-ZLW-003 (preservation number CGMCC No.3818, preservation date 2010.5.7), Streptococcus thermophilus MN-ZLW-001 (preservation number CGMCC No.3816, preservation date 2010.5.7) and other lactic acid bacteria; the other lactic acid bacteria include Lactobacillus delbrueckii subsp. bulgaricus SP1.1 (preservation number CGMCC No.16586, preservation date 2018.10.15), Lactobacillus casei subsp. casei YRL577-1-M (preservation number CGMCC No.17217, preservation date 2019.01.18) and Lactobacillus paracasei zlw-J23ANL (preservation number CGMCC No.10653, deposited on March 23, 2015); the viable counts of Lactobacillus bulgaricus, Streptococcus thermophilus and other lactic acid bacteria are 2:2:

1.

5. The method according to claim 1, wherein: In step (2), the gradient cooling process is from 37~42℃ to 20~25℃, the cooling time is 30~90 min, and it is kept for 3~5 h; then it is cooled to 15~10℃, the cooling time is 10~30 min, and it is kept for 4~6 h; finally it is cooled to 4℃, the cooling time is 5~20 min, and it is kept for 5~7 h.

6. The method according to claim 1, wherein: In step (3), the amount of casein phosphopeptide added is 0.05% to 0.1% (w / w), the amount of calcium lactate added is 0.8% to 1% (w / w), and the amount of magnesium lactate added is 0.1% to 0.2% (w / w).

7. The method according to claim 1, wherein: In step (3), the mixed lyophilization protective agent includes component A and component B, component A is 5% to 10% (w / w) of oligofructose and / or 3% to 8% (w / w) of erythritol, and component B is one or more of inulin 1% to 4% (w / w), β-cyclodextrin 0.03% to 0.05% (w / w) or microcrystalline cellulose 0.1% to 0.3% (w / w).

8. The method according to claim 1, wherein: In step (3), the pre-freezing temperature is -30~-25℃.

9. The method according to claim 1, wherein: In step (3), the temperature is gradually increased in the sublimation drying stage, first from -30 to -25 °C by 10 °C, with a total time of 10 to 13 h; then from -20 to -15 °C to 20 to 25 °C, with a total time of 20 to 25 h; the temperature is maintained at 15 to 20 °C in the desorption drying stage, with a total time of 10 to 15 h.

10. A highly active probiotic freeze-dried yogurt prepared by the preparation method according to any one of claims 1 to 9.

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