Preparation method and application of fermented milk rich in protein selenium

Abstract

The invention provides a preparation method and application of fermented milk rich in protein selenium. By performing vigor screening on the seeds, the selenium-rich fermentation effect can be pre-judged in advance, the success rate of subsequent selenium-rich fermentation is guaranteed, the appearance and taste of the product are ensured, and waste of manpower, materials and time is avoided. By establishing a redox potential (ORP) regulation and control method, the conversion capacity of inorganic selenium in the selenium-rich fermentation process can be improved, the proportion content of protein selenium in thalli is increased, and the stability of the viable count in the product preservation process is improved. The prepared selenium-rich fermented milk rich in protein selenium has the effects of improving constipation, nutritional obesity and ulcerative colitis, resisting fatigue, improving anorexia, regulating anxiety and depression states, improving immunity and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of biological fermentation, and in particular relates to a preparation method and application of fermented milk rich in protein and selenium. Background Art

[0002] Sodium selenite is a food nutrient fortifier permitted by Chinese laws and regulations for supplementing selenium, and can be used in fermented dairy products. However, when high concentrations of sodium selenite are added to dairy products for fermentation, red elemental selenium precipitates will be produced, which will cause the appearance and color of the fermented milk to deteriorate and the taste to contain metallic odors, affecting the quality of the product. Summary of the invention

[0003] The present invention aims to solve the problem that high-concentration sodium selenite turns red when added to fermented milk, affecting the appearance and taste of the product. A method for preparing fermented milk rich in protein selenium is provided.

[0004] At the same time, the present invention has developed a selenium-rich fermented milk rich in protein and selenium, which has the effects of improving constipation, improving nutritional obesity, improving ulcerative colitis, resisting fatigue, improving anorexia, regulating anxiety and depression, and enhancing immunity.

[0005] The fermentation strain used in the present invention is Bifidobacterium longum subspecies longum DD98, which is preserved in the General Microbiological Center of China Microbiological Culture Collection Administration, with a preservation number of CGMCC No.16573.

[0006] The specific technical solutions are as follows:

[0007] A method for preparing fermented milk rich in protein and selenium comprises the following steps:

[0008] Step A: Select seeds suitable for fermentation;

[0009] Step B: Preparation of fermentation medium:

[0010] Skim milk powder 40-200g / L, glucose 10-50g / L, oligofructose 1-5g / L, ferrous gluconate 3-20mg / L, magnesium gluconate 5-30mg / L, sterilized at 115℃ for 30min;

[0011] Step C: Preparation of feed medium:

[0012] Skim milk powder 40-200g / L, glucose 10-50g / L, oligofructose 1-5g / L, sodium selenite 1-50mg / L, sterilize at 115℃ for 30min;

[0013] Step D: Fermentation Control Method

[0014] Seed inoculation: Take qualified Bifidobacterium longum DD98 seeds, activate them with MRS liquid medium, and culture them until the number of viable bacteria is about 1-8×10 8 CFU / mL, the cultured Bifidobacterium longum DD98 seed solution was inoculated into the fermentation tank at an inoculum volume ratio of 3-8%, and the fermentation culture was started;

[0015] The present invention further adopts the above technical features, and its advantage is that the present invention adopts the method of screening the vitality of seeds, and the vitality of seeds directly determines the fermentation effect in the fermentation tank. Since the strains need to be fermented in a high concentration of sodium selenite environment, too high a concentration of sodium selenite will affect the vitality of the strains. Therefore, it is necessary to confirm the vitality of the seeds in a high concentration of sodium selenite environment in advance at the seed stage. The present invention uses the β-galactosidase activity of Bifidobacterium longum as a vitality indicator to ensure that the seeds can still maintain high vitality in a high sodium selenite environment.

[0016] Growth expansion and feed culture: After seed inoculation, culture for 3-5 hours. When the number of viable bacteria in the fermentation liquid reaches 1-8×10 8 CFU / mL, start to add an equal volume of fresh sterile fermentation medium at a uniform rate, and the feeding time is 0.5-1h;

[0017] Specifically, the remaining 1 / 2 fermentation volume of fermentation liquid is added to the tank, and the feeding time is controlled to be 0.5-1h. The feeding speed is related to the feeding time. If the feeding time is short, the feeding speed will also be faster, but it is required to feed at a uniform speed. Therefore, the feeding speed range here is determined by the feeding time range later.

[0018] Conversion and ORP control: After the feeding is completed, the conversion culture is continued for 2-4 hours; the ORP of the fermentation liquid shows a downward trend during this stage; when the ORP of the fermentation liquid is too low (below -350mV), carbon dioxide needs to be introduced into the fermentation tank to increase the ORP, ensuring that the ORP of the fermentation liquid is controlled between -350 and -250mV. After the conversion stage is over, the preparation of selenium-enriched fermented milk is completed.

[0019] The present invention further adopts the above technical features, which has the advantage of regulating the redox potential (ORP) during the selenium-enriched fermentation process. Based on long-term selenium-enriched fermentation experience, it is found that in Bifidobacterium longum DD98, the ratio of bacterial protein selenium / total selenium is closely related to the change of ORP during the fermentation process. By controlling the ORP within a certain range, the efficient conversion of protein selenium in Bifidobacterium longum DD98 can be achieved, and the residual inorganic selenium can be reduced.

[0020] The step A comprises: seed vitality screening and judgment:

[0021] Step A1: Take ≥10 test tubes filled with sterile MRS liquid culture medium, add sodium selenite with a mass concentration of 10-50 mg / L, and filter sterilize. Take out the glycerol storage tube of Bifidobacterium longum DD98 from the refrigerator, inoculate each test tube with a volume ratio of 8-10% of Bifidobacterium longum DD98 liquid, and after anaerobic culture at 37°C for 16-18 hours, sample and measure the specific activity of the crude enzyme solution of β-galactosidase in each test tube, and perform statistics;

[0022] Step A2: When the specific activity of the crude β-galactosidase solution in the test tube is ≥1.5U / mg, it is recorded as "qualified". The specific activity of the crude β-galactosidase solution in all test tubes is measured. When the qualified rate is ≥90%, the seeds can be considered suitable for fermentation of selenium-enriched fermented milk.

[0023] The present invention adopts Bifidobacterium longum DD98, which can absorb sodium selenite (inorganic selenium) into the bacteria, convert and synthesize selenium-rich protein, thereby forming protein selenium, avoiding the conversion of inorganic selenium into red elemental selenium precipitate. The process can ensure that the appearance color of selenium-enriched fermented milk is normal, milky white or slightly yellow, without generating red elemental selenium precipitate and no peculiar smell.

[0024] The present invention further adopts the above technical features, and its advantage lies in that a matching selenium-enriched fermentation process control method is established. By regulating the redox potential (ORP), the proportion of protein selenium in the product can be increased, and the product has higher bioavailability.

[0025] During the fermentation process, Bifidobacterium longum DD98 can utilize sodium selenite (inorganic selenium) to synthesize selenium-rich proteins in the body. The selenium integrated in the protein is called protein selenium, which has higher bioavailability and safety compared with inorganic selenium. The present invention can significantly increase the content of protein selenium converted in fermented milk by establishing a method for regulating ORP process parameters, so that sodium selenite (inorganic selenium) is basically completely converted. Therefore, the prepared selenium-rich fermented milk has better bioavailability and safety, and can increase the stable storage time of fermented milk.

[0026] The present invention also provides the use of fermented milk rich in protein and selenium in effectively improving constipation symptoms, promoting intestinal peristalsis, and regulating intestinal flora.

[0027] The present invention also provides the use of fermented milk rich in protein and selenium in alleviating obesity, improving inflammation in the body caused by nutritional obesity, and improving body immunity.

[0028] The present invention also provides the use of fermented milk rich in protein and selenium in alleviating the symptoms of ulcerative colitis, reducing oxidative stress and repairing the intestinal barrier.

[0029] The present invention also provides the use of fermented milk rich in protein and selenium in significantly improving sports endurance and having a significant anti-fatigue effect.

[0030] The present invention also provides the use of fermented milk rich in protein and selenium in significantly increasing the food intake of young anorexia mice and promoting weight gain.

[0031] The present invention also provides the use of fermented milk rich in protein and selenium in improving anxious and depressive states, effectively regulating emotions, and increasing the content of neurotransmitters and brain-derived neurotrophic factors in the body.

[0032] The present invention also provides the use of fermented milk rich in protein and selenium in improving immunity, promoting lymphocyte proliferation, increasing the phagocytic ability of macrophages, and enhancing the activity of NK cells.

[0033] Beneficial effects:

[0034] 1. By screening the vitality of seeds, the effect of selenium-enriched fermentation can be predicted in advance, which is conducive to ensuring the success rate of subsequent selenium-enriched fermentation, ensuring the appearance and taste of the product, and avoiding waste of manpower, materials and time.

[0035] 2. By establishing an oxidation-reduction potential (ORP) control method, the conversion capacity of inorganic selenium in the selenium-enriched fermentation process can be improved, the proportion of protein selenium in the bacteria can be increased, and it is beneficial to improve the stability of the number of live bacteria during product preservation.

[0036] 3. This selenium-rich fermented milk can effectively improve constipation symptoms, promote intestinal peristalsis, and regulate intestinal flora.

[0037] 4. It can effectively relieve obesity, improve inflammation in the body caused by nutritional obesity, and enhance the body's immunity.

[0038] 5. This selenium-rich fermented milk can effectively relieve the symptoms of ulcerative colitis, reduce oxidative stress, and repair the intestinal barrier.

[0039] 6. This selenium-rich fermented milk can significantly improve athletic endurance and has a significant anti-fatigue effect.

[0040] 7. This selenium-enriched fermented milk can significantly increase the food intake of young anorexic mice and is beneficial to weight gain.

[0041] 8. The selenium-enriched fermented milk can significantly improve the anxious and depressive state, effectively regulate the mood, and increase the content of neurotransmitters and brain-derived neurotrophic factor in the body.

[0042] 9. This selenium-rich fermented milk can significantly enhance immunity, promote lymphocyte proliferation, improve the phagocytic ability of macrophages, and enhance the activity of NK cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 This is an appearance diagram of the milky white fermented milk obtained by the present invention.

[0044] Figure 2 It is a curve chart of protein selenium / total selenium in bacteria and ORP.

[0045] Figure 3 It is a curve chart of the number of live bacteria in fermented milk and the storage time.

[0046] Figure 4 Changes in intestinal propulsion rate of mice in each group.

[0047] Figure 5 is the water content of feces of mice in each group.

[0048] Figure 6 It has the appearance of dark red fermented milk.

[0049] Figure 7 Comparison diagram of the milky white selenium-enriched fermented milk of Example 2 (left) and the dark red selenium-enriched fermented milk of Comparative Example 1 (right). DETAILED DESCRIPTION

[0050] The preferred embodiments of the present invention are further described in detail below in conjunction with the accompanying drawings:

[0051] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent companies unless otherwise specified.

[0052] The fermentation strain used in the present invention is Bifidobacterium longum subspecies longum DD98, which is preserved in the General Microbiological Center of China Microbiological Culture Collection Administration, with a preservation number of CGMCC No.16573.

[0053] The following is the preparation process of selenium-enriched fermented milk:

[0054] (1) Seed vitality screening

[0055] Take ≥10 test tubes filled with sterile MRS liquid culture medium, add sodium selenite with a mass concentration of 10-50 mg / L, and filter sterilize. Take out the glycerol preservation tube of Bifidobacterium longum DD98 from the refrigerator, inoculate each test tube with 8-10% (volume ratio) of Bifidobacterium longum DD98 bacterial liquid, and after anaerobic culture at 37° C. for 16-18 hours, sample and measure the specific activity of the crude enzyme liquid of β-galactosidase in each test tube, and make statistics.

[0056] When the specific activity of the crude β-galactosidase solution in the test tube is ≥1.5U / mg, it is recorded as "qualified". The specific activity of the crude β-galactosidase solution in all test tubes is measured. When the qualified rate is ≥90%, the seeds can be considered suitable for fermentation of selenium-enriched fermented milk.

[0057] (2) Selenium-enriched fermentation process:

[0058] Preparation of fermentation medium:

[0059] Skim milk powder 40-200g / L, glucose 10-50g / L, fructooligosaccharide 1-5g / L, ferrous gluconate 3-20mg / L, magnesium gluconate 5-30mg / L, sterilize at 115℃ for 30min.

[0060] Preparation of feed medium:

[0061] Skim milk powder 40-200g / L, glucose 10-50g / L, oligofructose 1-5g / L, sodium selenite 1-50mg / L, sterilize at 115℃ for 30min.

[0062] Fermentation control method:

[0063] (a) Seed inoculation

[0064] Take qualified Bifidobacterium longum DD98 seeds, activate them with MRS liquid medium, and culture them until the viable count is about 1-8×10 8 CFU / mL. The cultured Bifidobacterium longum DD98 seed solution was inoculated into the fermentation tank at an inoculation rate of 3-8% (volume ratio) to start fermentation. During the entire fermentation process, the temperature was controlled at 37-42°C and the pH was controlled at 5.5-6.5.

[0065] (b) Growth expansion and fed-batch culture

[0066] After the seeds are inoculated, they are cultured for 3-5 hours. When the number of viable bacteria in the fermentation liquid reaches 1-8×10 8 CFU / mL, start to add an equal volume of fresh sterile fermentation medium at a uniform rate, and the feeding time is 0.5-1h.

[0067] (c) Conversion and ORP regulation

[0068] After the feeding is completed, the conversion culture is continued for 2-4 hours. The ORP of the fermentation liquid shows a downward trend during this stage. When the ORP of the fermentation liquid is too low, carbon dioxide needs to be introduced into the fermentation tank to increase the ORP, ensuring that the ORP of the fermentation liquid is controlled between -350 and -250mV. After the conversion stage is over, the preparation of selenium-enriched fermented milk is completed.

[0069] Example 1

[0070] Vitality screening and judgment of selenium-enriched fermented milk working seeds:

[0071] Take 12 test tubes filled with sterile MRS liquid culture medium, add sodium selenite with a mass concentration of 10 mg / L, and filter sterilize. Take out the glycerol preservation tube of Bifidobacterium longum DD98 from the refrigerator, inoculate each test tube with 8% (volume ratio) of Bifidobacterium longum DD98 bacterial liquid, and after anaerobic culture at 37°C for 16 hours, sample and measure the specific activity of β-galactosidase in each test tube. The results are as follows: 11 tubes have a specific activity of ≥1.5U / mg, which are recorded as "qualified", and the qualified rate is 91.7%. The seeds are suitable for fermentation of selenium-enriched fermented milk. The test results are shown in Table 1.

[0072] Table 1

[0073]

[0074]

[0075] Example 2

[0076] Preparation of fermentation medium:

[0077] Skim milk powder 200g / L, glucose 50g / L, fructooligosaccharide 5g / L, ferrous gluconate 20mg / L, magnesium gluconate 30mg / L, sterilize at 115℃ for 30min.

[0078] Preparation of feed medium:

[0079] Skim milk powder 200g / L, glucose 50g / L, oligofructose 5g / L, sodium selenite 50mg / L, sterilize at 115℃ for 30min.

[0080] The qualified Bifidobacterium longum DD98 seeds in Example 1 were activated with MRS liquid medium and cultured until the viable count was 8×10 8 CFU / mL. The cultured Bifidobacterium longum DD98 seed solution was inoculated into the fermentation tank at an inoculation rate of 8% (volume ratio) to start fermentation. During the entire fermentation process, the temperature was controlled at 37°C and the pH was controlled at 5.5.

[0081] After the seeds were inoculated, they were cultured for 5 h. When the number of viable bacteria in the fermentation liquid reached 8 × 10 8 CFU / mL, start to add an equal volume of fresh sterile fermentation medium at a uniform rate, and the feeding time is 1h.

[0082] After the feeding is completed, the conversion culture is continued for 4 hours. The ORP of the fermentation liquid shows a downward trend during this stage. When the ORP of the fermentation liquid is too low, carbon dioxide needs to be introduced into the fermentation tank to increase the ORP to ensure that the ORP of the fermentation liquid is controlled at -250mV. After the conversion stage is over, the preparation of selenium-enriched fermented milk is completed.

[0083] Take the prepared selenium-enriched fermented milk, centrifuge at high speed and discard the supernatant, observe the color of the bacterial sludge, then wash it with sterile water 3 times, freeze-dry the bacterial sludge, and determine the ratio of protein selenium / total selenium in the bacteria. At the same time, take the selenium-enriched fermented milk and directly determine the total selenium, inorganic selenium content, and viable bacteria count in the fermented milk. The determination results are shown in Table 2:

[0084] Table 2

[0085]

[0086] Milky white fermented milk looks like this Figure 1 shown.

[0087] Example 3

[0088] Preparation of fermentation medium:

[0089] Skim milk powder 40g / L, glucose 10g / L, fructooligosaccharide 1g / L, ferrous gluconate 3mg / L, magnesium gluconate 5mg / L, sterilize at 115℃ for 30min.

[0090] Preparation of feed medium:

[0091] Skim milk powder 40g / L, glucose 100g / L, oligofructose 1g / L, sodium selenite 1mg / L, sterilize at 115℃ for 30min.

[0092] Fermentation control method:

[0093] The qualified Bifidobacterium longum DD98 seeds in Example 1 were activated with MRS liquid medium and cultured until the viable count was about 1×10 8 CFU / mL. The cultured Bifidobacterium longum DD98 seed solution was inoculated into the fermentation tank at an inoculation rate of 3% (volume ratio) to start fermentation. During the entire fermentation process, the temperature was controlled at 42°C and the pH was controlled at 6.5.

[0094] After the seeds were inoculated, they were cultured for 3 h. When the number of viable bacteria in the fermentation liquid reached 1×10 8 CFU / mL, start to add an equal volume of fresh sterile fermentation medium at a uniform rate, and the feeding time is 0.5h.

[0095] After the feeding is completed, the conversion culture is continued for 2 hours. The ORP of the fermentation liquid shows a downward trend during this stage. When the ORP of the fermentation liquid is too low, carbon dioxide needs to be introduced into the fermentation tank to increase the ORP to ensure that the ORP of the fermentation liquid is controlled at -350mV. After the conversion stage is over, the preparation of selenium-enriched fermented milk is completed.

[0096] Take the prepared selenium-enriched fermented milk, centrifuge at high speed and discard the supernatant, observe the color of the bacterial sludge, then wash it with sterile water 3 times, freeze-dry the bacterial sludge, and determine the ratio of protein selenium / total selenium in the bacteria. At the same time, take the selenium-enriched fermented milk and directly determine the total selenium, inorganic selenium content, and viable bacteria count in the fermented milk. The determination results are shown in Table 3:

[0097] Table 3

[0098]

[0099] Example 4

[0100] Effect of ORP regulation on the ratio of protein and selenium in bacteria during transformation:

[0101] The selenium-enriched fermentation process is the same as that in Example 2, except that: in the conversion stage after the feeding is completed, different redox potentials (ORP) are artificially adjusted to control the ORP in the fermentation system to be -400mV, -350mV, -300mV, -250mV, and -150mV, respectively. After the fermentation is completed, the bacteria are obtained by the same treatment as in Example 2, and the protein selenium / total selenium in the bacteria are measured respectively. The results are shown in the figure below. It can be seen that when the ORP is controlled to -350mV, -300mV, and -250mV during the conversion process, the protein selenium / total selenium in the bacteria can be maintained at more than 80%. Figure 2 shown.

[0102] Embodiment 5:

[0103] Effect of ORP regulation during the conversion process on the long-term storage stability of the number of live bacteria in fermented milk:

[0104] The selenium-enriched fermentation process is the same as that in Example 2, except that: in the conversion stage after the feeding is completed, different redox potentials (ORPs) are artificially adjusted to control the ORP in the fermentation system to be -400mV, -350mV, -300mV, -250mV, and -150mV, respectively. After the fermentation is completed, several samples of selenium-enriched fermented milk are obtained and stored in a refrigerator at 4°C. The changes in the number of viable bacteria in the selenium-enriched fermented milk over time are observed, and the results are shown in the figure below. According to the long-term storage results, when the ORP is controlled to -350mV, -300mV, and -250mV during the conversion process, the number of viable bacteria in the fermented milk can be maintained at 1×10 after 360 days of storage. 9CFU / mL or more. Under these three conditions, the proportion of protein selenium in the bacteria is higher, which is more conducive to the long-term stable preservation of the bacteria.

[0105] Embodiment 6:

[0106] Effects of selenium-enriched fermented milk on constipated mice:

[0107] The mice were induced to have slow-transmission constipation by continuous intragastric administration of 50 mg / kg loperamide hydrochloride for 7 days. The specific method of constipation model construction can be found in the article "Experimental study on slow-transmission constipation model of mice induced by loperamide hydrochloride". A normal group (NC), a model group (M), and an intervention group (Se-DD98) were set up respectively. The intervention period was 21 days. The intervention group was intervened by the selenium-enriched fermented milk prepared in Example 2. The treatment of each group was as follows: the M group continued to be intragastricly administered with a daily dose of 50 mg / kg loperamide hydrochloride + 0.8 mL / d of normal saline, and the intervention group was intragastricly administered with a daily dose of 50 mg / kg loperamide hydrochloride + 0.8 mL / d of Se-DD98. The daily intragastric volume of normal saline in the NC group was consistent with that of the M group and the intervention group.

[0108] After 21 days of intervention, the intestinal propulsion rate, fecal water content, and changes in intestinal flora in feces of mice in each group were measured. The results are shown in Table 4. Figure 4 and Figure 5 As shown: Figure 4 This is a graph of changes in intestinal propulsion rate, which reflects the peristaltic propulsion rate of the small intestine for the contents. The higher the intestinal propulsion rate, the faster the intestinal peristalsis rate, the more lubricated it is and the less likely it is to cause constipation. Figure 5 This is a stool moisture content chart, which reflects the moisture content in the stool. The lower the moisture content, the drier and harder the stool, and the higher the degree of constipation.

[0109] Table 4 Changes in intestinal microbial flora in each group

[0110]

[0111] The test results show that selenium-enriched fermented milk can effectively improve the intestinal motility of constipated mice, improve the water content of feces, and relieve constipation symptoms. At the same time, it can reduce the content of enterococci and Escherichia coli in the intestines of constipated mice, increase the content of bifidobacteria and lactobacilli, and effectively improve and regulate the intestinal flora.

[0112] Embodiment 7:

[0113] Effects of selenium-enriched fermented milk on nutritional obesity:

[0114] The mouse obesity model was established by feeding with a high-fat diet. For the specific modeling method, please refer to the article "Effects of electroacupuncture on the structure and composition of intestinal flora in nutritionally obese mice". A normal group (NC), a model group (M), and an intervention group (Se-DD98) were set up respectively. After the model was successfully established, the intervention period was 14 days, and the intervention group was intervened with the selenium-enriched fermented milk prepared in Example 2. The treatment of each group was as follows: the NC group was fed with a normal diet and gavaged with 1 mL / d of normal saline; the M group was fed with a high-fat diet and gavaged with 1 mL / d of normal saline; the intervention group was fed with a high-fat diet and gavaged with 1 mL / d of Se-DD98.

[0115] After 14 days of intervention, the changes in the indicators of mice in each group were measured as shown in Table 5:

[0116] Table 5

[0117] Test items Normal group Obesity model group Intervention Group Weight (g) 24.13 34.05 28.74 Lee's Index 3.04 3.57 3.18 Fat percentage (%) 1.95 5.67 2.31 TNF-α(ng / L) 53.45 58.66 53.97 IL-1β (ng / L) 76.68 79.82 76.93 IL-6 (ng / L) 163.27 168.41 164.59 IgA (ng / L) 1.81 1.78 1.82 IgG (ng / L) 2.49 2.48 3.01

[0118] The higher the levels of TNF-α, IL-1β, and IL-6, the higher the level of inflammation. IgA and IgG are antibodies in the serum, and the higher their levels, the stronger the body's immunity.

[0119] The test results show that selenium-rich fermented milk can effectively improve the body weight of nutritionally obese mice, reduce the Lee's index and fat coefficient of mice, improve the inflammation in the body caused by nutritional obesity, and enhance the body's immunity.

[0120] Embodiment 8:

[0121] Effect of selenium-enriched fermented milk on ulcerative colitis:

[0122] Dextran sulfate sodium (DDS) was used to induce the establishment of a mouse ulcerative colitis (UC) model. For the specific modeling method, please refer to the article "Study on the pharmacodynamic effects and mechanisms of vitexin in alleviating ulcerative colitis in mice". A normal group (NC), a model group (M), and an intervention group (Se-DD98) were set up respectively. After the model was successfully established, the intervention period was 7 days, and the intervention group was intervened with the selenium-enriched fermented milk prepared in Example 2. The treatment of each group was as follows: the NC group and the M group were gavaged with 1 mL / d of normal saline; the intervention group was gavaged with 1 mL / d of Se-DD98.

[0123] After 7 days of intervention, the changes in the indicators of mice in each group were measured as shown in Table 6:

[0124] Table 6

[0125]

[0126]

[0127] The malondialdehyde content can reflect the level of lipid peroxidation in the body. The higher the content, the greater the degree of oxidative stress in the body. The activities of superoxide dismutase, catalase, and glutathione reductase in the body reflect the antioxidant capacity.

[0128]

[0129] Reduced expression of intestinal tight junction proteins will increase intestinal permeability and lead to intestinal barrier damage.

[0130] The above test results show that selenium-rich fermented milk can effectively improve the symptoms of ulcerative colitis, alleviate weight loss, bloody stools, shortened colon, enlarged spleen, etc. caused by ulcerative colitis, and effectively improve the disease activity index score and colon pathology score of mice. At the same time, it can reduce the level of oxidative stress, increase the activity of antioxidant enzymes, and repair the intestinal barrier.

[0131] Embodiment 9:

[0132] The effect of selenium-rich fermented milk in fighting fatigue:

[0133] The mice were subjected to fatigue rotarod test and exhaustive swimming test to test the anti-fatigue effect of selenium-enriched fermented milk on mice. For the specific methods of fatigue rotarod test and exhaustive swimming test, please refer to the article "Study on the Anti-fatigue Effect and Mechanism of Hyperoside on Mice". In this experiment, the mice were divided into a fatigue control group and an experimental group. The experimental intervention period was 14 days, and the experimental group was intervened with the selenium-enriched fermented milk prepared in Example 2. The experimental group was gavaged with 1 mL of selenium-enriched fermented milk every day, and the control group was gavaged with 1 mL of normal saline every day.

[0134] After 14 days of intervention, the changes in the indicators of mice in each group were measured as shown in Table 7:

[0135] Table 7

[0136]

[0137] ATP is the body's main energy source. Changes in ATP content in the body can directly reflect the body's energy supply status and exercise capacity.

[0138] The results show that selenium-rich fermented milk can significantly improve the exercise endurance of mice and has a significant anti-fatigue effect.

[0139] Embodiment 10:

[0140] Effect of selenium-enriched fermented milk on improving anorexia:

[0141] The anorexia model of young mice was established by feeding with special feed. For the specific modeling method, please refer to the article "Effects of Simo Decoction on Anorexia Model of Young Mice". A normal group (NC), a model group (M), and an intervention group (Se-DD98) were set up respectively. After the model was successfully established, the intervention period was 14 days, and the intervention group was intervened with the selenium-enriched fermented milk prepared in Example 2. The treatment of each group was as follows: the NC group was fed with a normal diet and gavaged with 1 mL / d of normal saline; the M group continued to be fed with special feed and gavaged with 1 mL / d of normal saline; the intervention group was fed with special feed and gavaged with 1 mL / d of Se-DD98.

[0142] After 14 days of intervention, the changes in the indicators of mice in each group were measured as shown in Table 8:

[0143] Table 8

[0144]

[0145] The results show that selenium-enriched fermented milk can significantly increase the food intake of young anorexic mice and is beneficial to the weight gain of mice.

[0146] Embodiment 11:

[0147] Effect of selenium-enriched fermented milk on improving anxiety-related depression:

[0148] An anxious depression model was established in rats. For the specific modeling method, please refer to the article "Effects of water extracts of Albizzia julibrissin and Semen Ziziphi Spinosae on animal models of anxious depression based on single-drug combination". A normal group (NC), a model group (M), and an intervention group (Se-DD98) were set up respectively. After the model was successfully established, the intervention period was 21 days, and the intervention group was intervened with the selenium-enriched fermented milk prepared in Example 2. The treatment of each group was as follows: the NC group and the M group were gavaged with 1 mL / d of normal saline; the intervention group was gavaged with 1 mL / d of Se-DD98.

[0149] After 21 days of intervention, the animals in each group were subjected to an open field test, and the changes in the indicators of the animals in each group were measured as shown in Table 9:

[0150] Table 9

[0151]

[0152] The levels of brain-derived neurotrophic factor and 5-hydroxytryptamine in depressed animals are significantly reduced.

[0153] The results show that selenium-enriched fermented milk can significantly improve the behavioral state of anxious and depressed animals, effectively regulate their emotions, and increase the levels of neurotransmitters and brain-derived neurotrophic factor in the body.

[0154] Embodiment 12:

[0155] Effect of selenium-enriched fermented milk on improving immunity:

[0156] Hydrocortisone was used to establish a mouse model of low immunity. For the specific modeling method, please refer to the article "Compound propolis extract improves the immunity of mice". A normal group (NC), a model group (M), and an intervention group (Se-DD98) were set up respectively. After the model was successfully established, the intervention period was 21 days, and the intervention group was intervened with the selenium-enriched fermented milk prepared in Example 2. The treatment of each group was as follows: the NC group and the M group were gavaged with a dose of 1 mL / d of normal saline; the intervention group was gavaged with a dose of 1 mL / d of Se-DD98.

[0157] After 21 days of intervention, the changes in the indicators of the animals in each group were measured as shown in Table 10:

[0158] Table 10

[0159]

[0160] The results show that selenium-enriched fermented milk can significantly enhance the immunity of immunocompromised mice, promote lymphocyte proliferation, improve the phagocytic ability of macrophages, and enhance the activity of NK cells.

[0161] Comparative Example 1

[0162] Take 12 test tubes filled with sterile MRS liquid culture medium, add sodium selenite with a mass concentration of 10 mg / L, and filter sterilize. Take out the glycerol preservation tube of Bifidobacterium longum DD98 from the refrigerator, inoculate each test tube with 8% (volume ratio) of Bifidobacterium longum DD98 bacterial liquid, and after anaerobic culture at 37°C for 16 hours, sample and measure the specific activity of β-galactosidase in each test tube, among which 8 tubes have a specific activity of ≥1.5U / mg, and the qualified rate is 66.7%. As shown in Table 11.

[0163] Table 11

[0164] project quantity Qualified quantity 8 Total number of tests 12 Pass rate 66.7％

[0165] The seeds were used to prepare selenium-enriched fermented milk, and the preparation process was the same as in Example 2. Then, relevant indicators were tested, as shown in Table 12:

[0166] Table 12

[0167] Test items result Total selenium content of selenium-enriched fermented milk 22880μg / L Content of inorganic selenium in selenium-enriched fermented milk 5836μg / L Number of live bacteria in selenium-enriched fermented milk <![CDATA[1.6×10 8 CFU / mL]]> Appearance and taste of selenium-enriched fermented milk Dark red with metallic smell Protein selenium / total selenium in bacteria 56.3％

[0168] Dark red fermented milk looks like Figure 6 shown.

[0169] Comparison of the milky white selenium-enriched fermented milk of Example 2 (left) and the dark red selenium-enriched fermented milk of Comparative Example 1 (right) Figure 7As shown. Normal fermented milk is milky white. The dark red color of fermented milk is because sodium selenite is not completely converted into protein selenium in the bacteria, but remains in the fermented milk liquid in the form of red elemental selenium, causing the fermented milk to turn dark red. The elemental selenium remaining in the fermented milk will not only cause the appearance of the fermented milk to turn dark red, but also have a metallic odor, affecting the appearance and taste. At the same time, the protein selenium in the bacteria is safer than elemental selenium, so the residual elemental selenium will affect the quality of the product.

[0170] According to the experimental data of Comparative Example 1 and Example 2, it can be seen that the process of screening the vitality of seeds before selenium-enriched fermentation is very important and necessary. The qualified rate of seeds will directly affect the appearance, quality and taste of the final fermented milk. At the same time, qualified seeds will increase the proportion of protein selenium in the bacteria, which is beneficial to improve the safety and bioavailability of the product.

[0171] Comparative Example 2

[0172] The qualified long bifidobacterium DD98 seeds in Example 1 were used to prepare selenium-enriched fermented milk. The fermentation preparation process was the same as that in Example 2, except that during the conversion culture process after the feeding was completed, the ORP in the fermenter was in a natural state, and no artificial intervention was performed on the ORP of the fermenter. The relevant results of the determination after the fermentation were completed are shown in Table 13:

[0173] Table 13

[0174]

[0175] According to the experimental data of Comparative Example 2 and Example 2, it can be seen that in the conversion process of selenium-enriched fermentation, appropriate ORP artificial regulation is a very important process. If reasonable ORP artificial regulation is not performed, it will lead to high inorganic selenium residues in the product and low protein selenium conversion efficiency, resulting in a slight metallic odor in the product, which seriously affects the product quality.

[0176] Comparative Example 3

[0177] The qualified long bifidobacterium DD98 seeds in Example 1 were used to prepare selenium-enriched fermented milk. The fermentation preparation process was the same as that in Example 2, except that the fermentation medium did not contain ferrous gluconate and magnesium gluconate. The specific fermentation medium formula was: skim milk powder 200g / L, glucose 50g / L, oligofructose 5g / L, sterilized at 115°C for 30min. The relevant results after the fermentation were measured as shown in Table 14:

[0178] Table 14

[0179]

[0180] According to the experimental data of Comparative Example 3 and Example 2, it can be seen that the ferrous gluconate and magnesium gluconate components in the fermentation culture play an important role in the selenium-enriched fermentation process. If these two substances are missing in the culture medium, the bacterial growth will be affected, the number of viable bacteria will decrease, the inorganic selenium residue in the product is high, the conversion efficiency of protein selenium is low, and the product has a slight metallic odor, which seriously affects the product quality.

[0181] Comparative Example 4

[0182] The qualified Bifidobacterium longum DD98 seeds in Example 1 were used to prepare selenium-enriched fermented milk. The fermentation preparation process was the same as that in Example 2, except that: after the seeds were inoculated and cultured for 4 hours, the number of viable bacteria in the fermentation liquid was 1×10 7 CFU / mL, the feed was started. After the fermentation was completed, the relevant results were shown in Table 15:

[0183] Table 15

[0184]

[0185] According to the experimental data of Comparative Example 4 and Example 2, it can be seen that the selection of a reasonable feeding time point is very important. If the feeding is started when the number of viable bacteria in the fermentation broth has not grown to a predetermined number, the number of viable bacteria in the final product will be low, the residual amount of inorganic selenium in the product will be high, the conversion efficiency of protein selenium will be low, the product will appear dark red and have a slight metallic odor, which will seriously affect the appearance and quality of the product.

[0186] Comparative Example 5

[0187] The qualified long bifidobacterium DD98 seeds in Example 1 were used to prepare selenium-enriched fermented milk. The fermentation preparation process was the same as that in Example 2, except that: the seeds were cultured for 5 h after inoculation, and when the number of viable bacteria in the fermentation liquid reached 8×10 8 CFU / mL, quickly add an equal volume of fresh sterile fermentation medium, and complete the feeding in about 10 minutes. After the fermentation is completed, the relevant results are shown in Table 16:

[0188] Table 16

[0189]

[0190] According to the experimental data of Comparative Example 5 and Example 2, it can be seen that a suitable feeding rate is very important for the selenium-enriched fermentation process. If the feeding rate is too fast, the inorganic selenium residue in the product will be high, the conversion efficiency of protein selenium will be low, the product will appear dark red and have a slight metallic odor, which seriously affects the appearance and quality of the product.

[0191] Comparative Example 6

[0192] The qualified long bifidobacterium DD98 seeds in Example 1 were used to prepare selenium-enriched fermented milk. The fermentation preparation process was the same as that in Example 2, except that after the feeding was completed, no conversion culture was performed and the fermentation was terminated directly. The relevant results after the fermentation were measured are shown in Table 17:

[0193] Table 17

[0194]

[0195]

[0196] According to the experimental data of Comparative Example 6 and Example 2, it can be seen that the conversion and cultivation process after the feeding is completed is one of the important steps to achieve efficient selenium-enriched fermentation. If the conversion and cultivation is not carried out after the feeding is completed, the residual amount of inorganic selenium in the product will be greatly increased, the conversion efficiency of protein selenium will be extremely low, the product will appear dark red and have a distinct metallic odor, which seriously affects the appearance and quality of the product.

[0197] Comparative Example 7

[0198] The qualified long bifidobacterium DD98 seeds in Example 1 were used to prepare selenium-enriched fermented milk. The fermentation preparation process was the same as that in Example 2, except that: in the conversion stage, when the ORP of the fermentation liquid was too low, a trace amount of hydrogen peroxide was added to the fermentation tank to increase the ORP, so that the ORP of the fermentation liquid was controlled at -250mV. The relevant results after the fermentation were measured are shown in Table 18:

[0199] Table 18

[0200]

[0201] According to the experimental data of Comparative Example 7 and Example 2, it can be seen that the use of carbon dioxide to increase the ORP in the fermentation tank is an important step in achieving selenium-enriched fermentation. If other oxidants are used to increase the ORP, it is easy to inhibit the growth of strictly anaerobic Bifidobacterium longum, significantly reduce the number of viable bacteria in the tank, and affect the number of viable bacteria in selenium-enriched fermented milk. At the same time, a low number of viable bacteria is not conducive to the efficient conversion of inorganic selenium into protein selenium, resulting in a low bioavailability of selenium in selenium-enriched fermented milk.

[0202] The above contents are further detailed descriptions of the present invention in combination with specific preferred embodiments, and it cannot be determined that the specific implementation of the present invention is limited to these descriptions. For ordinary technicians in the technical field to which the present invention belongs, several simple deductions or substitutions can be made without departing from the concept of the present invention, which should be regarded as falling within the protection scope of the present invention.

Claims

1. A method for preparing fermented milk rich in protein selenium, characterized in that: It includes the following steps: Step A: Select seeds suitable for fermentation; Step B: preparing fermentation medium; Step C: preparing feed medium; Step D: Fermentation regulation.

2. The preparation method according to claim 1, characterized in that In step B, skim milk powder 40-200 g / L, glucose 10-50 g / L, oligofructose 1-5 g / L, ferrous gluconate 3-20 mg / L, magnesium gluconate 5-30 mg / L, sterilize at 115°C for 30 min.

3. The preparation method according to claim 1, characterized in that: In step C, skim milk powder 40-200 g / L, glucose 10-50 g / L, oligofructose 1-5 g / L, sodium selenite 1-50 mg / L, sterilized at 115°C for 30 min.

4. The preparation method according to claim 1, characterized in that: Step D includes the following steps: Seed inoculation: Take qualified fermented seeds, activate them with MRS liquid medium, and culture them until the number of viable bacteria is about 1-8×10 8 CFU / mL, the cultured fermentation seed liquid is inoculated into the fermentation tank according to a certain inoculation amount to start fermentation culture; Growth expansion and feed culture: After seed inoculation, culture for 3-5 hours. When the number of viable bacteria in the fermentation liquid reaches 1-8×10 8 CFU / mL, start to add an equal volume of fresh sterile fermentation medium at a uniform rate, and the feeding time is 0.5-1h; Conversion and ORP regulation: When the feeding is completed, continue the conversion culture for 2-4 hours; ensure that the ORP of the fermentation broth is controlled at -350 to -250 mV; after the conversion phase is completed, the preparation is completed.

5. The preparation method according to claim 4, characterized in that: The fermented milk fermentation strain is Bifidobacterium longum subspecies longum DD98, which is preserved in the General Microbiological Center of China Microbiological Culture Collection Administration, with a preservation number of CGMCC No.16573.

6. The preparation method according to claim 1, characterized in that: The step A comprises: seed vitality screening and judgment: Step A1: Take ≥ 10 test tubes filled with sterile MRS liquid culture medium, add sodium selenite, and filter sterilize; add 8-10% by volume of inoculum solution to each test tube, after anaerobic culture, sample and measure the specific activity of crude β-galactosidase solution in each test tube, and perform statistics; Step A2: When the specific activity of the crude β-galactosidase solution in the test tube is measured to be ≥1.5 U / mg, it is recorded as "qualified"; the specific activity of the crude β-galactosidase solution in all test tubes is measured, and when the qualified rate is ≥90%, the seeds can be considered suitable for fermentation of selenium-enriched fermented milk.

7. The preparation method according to claim 6, characterized in that In the step A1, sodium selenite is added at a mass concentration of 10-50 mg / L.

8. The preparation method according to claim 6, characterized in that: The temperature of the anaerobic culture in step A1 is 37° C. and the anaerobic time is 16-18 hours.

9. Use of the protein-selenium-rich fermented milk as claimed in claim 1 in effectively improving constipation symptoms, promoting intestinal peristalsis, and regulating intestinal flora.

10. Use of the protein-selenium-rich fermented milk according to claim 1 in alleviating obesity, improving inflammation in the body caused by nutritional obesity, and improving body immunity.

11. Use of the protein-selenium-rich fermented milk according to claim 1 in alleviating the symptoms of ulcerative colitis, reducing oxidative stress, and repairing the intestinal barrier.

12. Use of the protein-selenium-rich fermented milk according to claim 1 in significantly improving sports endurance and significantly anti-fatigue effects.

13. Use of the protein-selenium-rich fermented milk according to claim 1 in significantly increasing the food intake of young anorexic mice and promoting weight gain.

14. Use of the protein-selenium-rich fermented milk according to claim 1 in improving anxious and depressive states, effectively regulating emotions, and increasing the content of neurotransmitters and brain-derived neurotrophic factor in the body.

15. Use of the protein-selenium-rich fermented milk according to claim 1 in improving immunity, promoting lymphocyte proliferation, enhancing the phagocytic ability of macrophages, and strengthening the activity of NK cells.

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