Preparation method and application of nanoscale selenium-enriched yeast

Abstract

The invention relates to the technical field of biology, and particularly discloses a preparation method and application of nanoscale selenium-enriched yeast. According to the preparation method, photocatalysis, ball milling, ultrasound and other technical means are integrated in the same device for the first time, and the preparation method comprises the following specific steps: preparing a yeast suspension, carrying out ultrasonic ball milling treatment with photocatalysis, and repeating the ultrasonic ball milling treatment with photocatalysis for 2-3 times, the step of separately operating photocatalysis, ball milling and ultrasound is omitted, multiple times of sample transfer and raw material loss caused in the transfer process are avoided, respective advantages of the three technical means are fully played, the technical effect of synergistic interaction is achieved, the production period is effectively shortened, and the production cost is reduced. And by matching with a specific process and parameter limitation, the damage of nutrient substances is effectively reduced, so that the nano-scale selenium-enriched yeast with high wall breaking rate, high nanocrystallization degree and high nutrient content is obtained.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a preparation method and application of nano-selenium-enriched yeast. Background Art

[0002] Selenium (Se) is an essential trace nutrient for the human body. Studies have shown that, unlike toxic inorganic selenium, organic selenium (such as selenomethionine, selenocystine and selenocysteine) is the most nutritious and safe form of selenium. Selenium-enriched yeast is currently the most efficient and safest nutritional selenium supplement in China. The selenium content in selenium-enriched yeast reaches 1000-2500mg / kg (i.e. 1000-2500ppm), and it also contains many active ingredients that have important physiological effects on the human body, such as high-quality protein, nucleic acid, yeast polysaccharide and B vitamins. Selenium is added to the growth process of this type of yeast, and is organically combined with proteins and polysaccharides in the yeast, so that it can be more efficiently absorbed and utilized by the human body.

[0003] However, since the yeast wall is relatively thick (0.1-0.3 μm), in order to promote the dissolution of the effective ingredients in the yeast cells, the yeast cells need to be broken. Common cell wall breaking methods include mechanical method, ultrasonic method, pyrolysis method, chemical method and biological method. Existing studies have found that a single cell wall breaking method can usually only break the yeast cell wall, and cannot be ultra-fine to the ultra-fine level. The particle size of the selenium-enriched yeast suspension is large and uneven, which makes the digestive system insufficiently absorbed, resulting in the loss of effective ingredients, and the cell wall breaking rate is difficult to reach 80%. Although the biological method has the advantages of low energy consumption and mild conditions, the reagents used are relatively expensive and not widely used.

[0004] To this end, researchers began to explore "a combination of multiple methods" to break the cell wall. For example, there is an existing method for breaking the cell wall of nano-selenium-enriched yeast, which adopts the method of "first photocatalytic treatment, and then combined with multiple "ball milling + ultrasound" treatments" to fully release the cell wall polysaccharides and other nutrients in the selenium-enriched yeast, and obtain selenium-enriched yeast with a high cell wall breaking rate and nano-scale degree. However, this treatment method is cumbersome to operate and has a long production cycle, which is not conducive to the mass production of nano-selenium-enriched yeast. Summary of the invention

[0005] In order to improve the production efficiency of nano-scale selenium-enriched yeast, the present application provides a preparation method and application of nano-scale selenium-enriched yeast. For the first time, technical means such as photocatalysis, ball milling, and ultrasound are integrated into the same set of devices. Photocatalytic treatment is carried out simultaneously during the ultrasonic ball milling process, eliminating the steps of separately operating photocatalysis, ball milling, and ultrasound, avoiding multiple transfers of samples and the loss of raw materials caused during the transfer process, giving full play to the respective advantages of the three technical means, achieving a synergistic technical effect, and thus effectively shortening the production cycle. Specific process and parameter limitations are used to effectively reduce the destruction of nutrients, thereby obtaining nano-scale selenium-enriched yeast with a high wall breaking rate, a high degree of nano-sizing, and a high nutritional content.

[0006] In a first aspect, the present application provides a method for preparing nano-selenium-enriched yeast, comprising the following steps:

[0007] a. Mixing selenium-enriched yeast with water to prepare a yeast suspension with a concentration of 5-12wt%;

[0008] b. Add zinc oxide to the yeast suspension, mix well, and place in a ball mill with ultrasonic function, put in an impact ball, place a light source 20±2 cm above the ball mill, control the speed of the ball mill to 80-100 r / min, and illuminate for 10-15 min with the cover open to obtain a photocatalytic ball mill material;

[0009] c. Close the ball mill cover, and continue to perform ultrasonic ball milling on the photocatalytic ball mill material at the same time, control the ball mill speed to 400-600r / min, control the ultrasonic power to 200-400w, and the ultrasonic ball milling time to 30-60min to obtain ultrasonic ball mill material;

[0010] d. Repeat steps b and c 2-3 times, collect the last ultrasonic ball milling material, collect the suspension after multiple centrifugation to remove the photocatalyst, and freeze-dry it into a powdery product to obtain the nano-selenium-enriched yeast.

[0011] Furthermore, in step c, trehalose is added to the yeast suspension during the ball milling process, and the mass ratio of the trehalose to the selenium-enriched yeast is 1:200-250.

[0012] Furthermore, the trehalose is added after the yeast suspension has completed the first light treatment.

[0013] Furthermore, in step c, the temperature of the ball mill is controlled at ≤55°C.

[0014] Furthermore, in step b, the mass ratio of the zinc oxide to the selenium-enriched yeast is 1:100-150.

[0015] Furthermore, the zinc oxide is nano zinc oxide.

[0016] Furthermore, in step b, the material of the impact balls for ball milling is zirconium oxide, and the particle size is 0.5-1 cm.

[0017] Furthermore, in step b, the ball-to-material ratio of the ball milling is 2-5:1.

[0018] In a second aspect, the present application provides a nano-scale selenium-enriched yeast, which is prepared by the above-mentioned preparation method.

[0019] In summary, this application has the following beneficial effects:

[0020] 1. This application uses a ball mill with ultrasonic function, which can realize intermittent photocatalytic treatment while ultrasonic ball milling, can avoid the steps of photocatalysis, ball milling, and ultrasonic treatment, avoid the multiple transfer of samples and the loss of raw materials caused during the transfer process, and effectively shorten the wall breaking treatment time of selenium-enriched yeast. In the process of grinding selenium-enriched yeast, zinc oxide is synchronously ground to increase its specific surface area, and the surface activity is effectively stimulated to produce more active oxygen. The activated zinc oxide will be evenly dispersed on the side of selenium-enriched yeast with grinding, thereby effectively improving the efficiency of breaking the covalent bonds of selenium-enriched yeast on the cell surface. At this time, the cell wall is easily mechanically damaged due to the breakage of covalent bonds, thereby effectively improving the wall breaking rate. Since selenium-enriched yeast is easy to destroy its nutrients after long-term photocatalysis after wall breaking, repeating steps b and c realizes intermittent illumination, which helps the covalent bond to be broken step by step on the one hand, thereby ensuring that the selenium-enriched yeast wall is broken more evenly and thoroughly; on the other hand, it can avoid the reaction for a long time and cause the nutrients in the yeast to be destroyed; thus obtaining a nano-selenium-enriched yeast with a high wall breaking rate, a high degree of nanoization, and high nutritional content.

[0021] 2. Based on the fact that selenium-enriched yeast breaks down in sequence during the grinding process, if the nutrients of the selenium-enriched yeast that breaks down first flow out and continue to be ground and illuminated for a long time, the nutrients are prone to deterioration. This application can effectively reduce the destruction of nutrients that break out of the wall by mechanical grinding and photocatalysis by adding an appropriate amount of trehalose. However, trehalose has a certain anti-ultraviolet effect. If it is added in advance before ball milling, it is easy to form a unique protective film on the cell surface, which will increase the difficulty of breaking the wall of selenium-enriched yeast to a certain extent. This application has found through research that if it is added after the selenium-enriched yeast completes the first light treatment, the selenium-enriched yeast prepared in this way can better protect the nutrients from damage while maintaining a high wall-breaking rate.

[0022] 3. In the process of ball milling, friction will generate heat, and ultrasonic treatment will also increase the temperature of the sample, but overheating will destroy the nutrients in the selenium-enriched yeast. The ball mill used in this application is also equipped with a temperature control system to reduce the destruction of nutrients by effectively controlling the grinding temperature.

[0023] 4. The present application uses a specific amount of nano zinc oxide to promote the effective photocatalytic reaction while reducing the damage of light to the nutrients in the yeast. Nano zinc oxide itself is non-toxic and safe and reliable. It can also effectively inhibit the growth of bacteria and maintain the stability of nutrients in the selenium-enriched yeast after the yeast cell wall is broken.

[0024] 5. This application uses zirconium oxide as the impact ball material for ball milling. Like zinc oxide, zirconium oxide is also a semiconductor material, which also has a certain photocatalytic effect. Through the combined use of two semiconductor materials, during the illumination and collision process, it can quickly transfer photogenerated electrons, effectively reduce the recombination between photogenerated electrons and photogenerated holes, increase the content of active oxygen, and greatly improve the efficiency of photocatalysis, thereby effectively improving the wall breaking efficiency of selenium-enriched yeast. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a microscopic image of the selenium-enriched yeast after the first light treatment in Example 1 of the present invention (observed under a 20× objective lens)

[0026] Figure 2 This is the hydrated particle size distribution diagram of the selenium-enriched yeast after the first light treatment in Example 1 of the present invention.

[0027] Figure 3 This is a microscopic image of the selenium-enriched yeast in Example 1 of the present invention after completing one "ball milling with photocatalysis-ultrasonic ball milling" treatment (observed under a 20× objective lens)

[0028] Figure 4 This is the hydrated particle size distribution diagram of the selenium-enriched yeast in Example 1 of the present invention after completing one "ball milling with photocatalysis-ultrasonic ball milling" treatment

[0029] Figure 5 This is a microscopic image of the selenium-enriched yeast in Example 1 of the present invention after completing two "ball milling with photocatalysis-ultrasonic ball milling" treatments (observed under a 20× objective lens)

[0030] Figure 6 This is the hydrated particle size distribution diagram of the selenium-enriched yeast in Example 1 of the present invention after completing two "ball milling with photocatalysis-ultrasonic ball milling" treatments

[0031] Figure 7 This is a scanning electron microscope image (SEM image) of the selenium-enriched yeast raw material in Example 1 of the present invention.

[0032] Figure 8 This is a scanning electron microscope image (SEM image) of the selenium-enriched yeast after cell wall breaking in Example 1 of the present invention. DETAILED DESCRIPTION

[0033] Preparation examples of raw materials and / or intermediates

[0034] The raw materials and auxiliary materials used in the present invention can be purchased from the market. Among them, selenium-enriched yeast was purchased from Angel Yeast Co., Ltd., with a selenium content of 2000ppm; trehalose was purchased from Shanghai Yuanye Biotechnology Co., Ltd., with an effective ingredient of 98% food grade.

[0035] Preparation example of nano ZnO powder

[0036] Prepare 50mL of Zn(NO3)2 solution and NaOH solution with a concentration of 0.2mol / L, mix the NaOH solution with the Zn(NO3)2 solution at room temperature and stirring, continue stirring for 20min, transfer to a centrifuge tube and centrifuge for 5min at a speed of 6000rpm, discard the supernatant, wash the precipitate once with distilled water, discard the supernatant after centrifugation, disperse the precipitate in 80mL of H2O2 solution with a concentration of 1mol / L, heat to 75℃ under sufficient stirring, stir and react for 1h, centrifuge for 10min at a speed of 8000rpm, wash the precipitate once with distilled water and anhydrous ethanol respectively, dry at 80℃ for 30min to obtain ZnO precursor. Then place it in a crucible, heat treat it at 400℃ for 2h, take it out after cooling to room temperature, and obtain nano ZnO powder after grinding and dispersion.

[0037] The present application is further described in detail below in conjunction with the accompanying drawings, embodiments and comparative examples.

[0038] Example 1

[0039] This embodiment discloses a method for preparing nano-selenium-enriched yeast, comprising the following steps:

[0040] a. Weigh 10 g of selenium-enriched yeast powder and mix it with water to prepare a yeast suspension with a concentration of 10 wt%;

[0041] b. Add 80 mg of the nano ZnO powder obtained in the preparation example to the yeast suspension, mix well and place in a ball mill with ultrasonic function, put in impact balls, use zirconium oxide as the impact balls, the ball-to-material ratio is 3:1, the ball particle size is 0.5-1 cm, place the light source 20±2 cm above the ball mill, control the ball mill speed to about 90 r / min (allowed to fluctuate within the range of 80-100 r / min), and expose to light for 10 min with the cover opened to obtain photocatalytic ball mill material.

[0042] c. Close the ball mill cover, and continue to perform ultrasonic ball milling on the photocatalytic ball mill material at the same time, control the ball mill speed to 500r / min, the ball milling temperature to 50°C, the ultrasonic power to 300w, and the ultrasonic ball milling time to 60min to obtain the ultrasonic ball mill material;

[0043] d. Repeat steps b and c twice, collect the last ultrasonic ball milling material, and centrifuge at 800r / min for 1min because the density of nano ZnO is greater than that of the yeast wall-breaking liquid. Take the upper suspension, repeat the centrifugation for 3 times to remove the nano ZnO as much as possible, collect the upper suspension after the last centrifugation, and freeze-dry it into a powdery product to obtain nano-selenium-enriched yeast, in which the nano ZnO can be recycled and reused.

[0044] Embodiment 2-6

[0045] In Examples 2-6, based on the method of Example 1, the process parameters are adjusted. For specific adjustments, see Table 1 below.

[0046] Table 1 Process parameters corresponding to Examples 1-6

[0047] Example 1 Example 2 Example 3 Example 4 Example 5 Example 6 Selenium-enriched yeast / g 10 5 12 10 10 10 Selenium-enriched yeast concentration / wt% 10 5 12 10 10 10 Zinc oxide / mg 80 50 80 80 80 80 Ball mill speed / r / min 500 400 600 500 500 500 Ultrasonic power / w 300 200 400 300 300 300 Ultrasonic ball milling time / min 60 40 30 40 54 60 Particle size / cm 0.5-1 0.5-1 0.5-1 0.5-1 0.5-1 0.5-1 Ball to Material Ratio 3:1 3:1 3:1 3:1 3:1 3:1 Ball milling temperature / ℃ 50 50 50 50 50 50 Lighting time / min 10 15 10 10 13 13 Repeat times / times 2 2 3 3 2 2 Total wall breaking time / min 140 110 120 150 134 146

[0048] Note: Total wall breaking time = total ball milling time with photocatalysis + total ultrasonic ball milling time. In this embodiment, the photocatalysis is carried out during the low-speed ball milling process. Therefore, when calculating the total wall breaking time, only the total ball milling time needs to be included.

[0049] Embodiment 7-8

[0050] In Example 7-8, the amount of nano ZnO powder added is adjusted based on the method of Example 1. In Example 7, the amount of nano ZnO powder added is 50 mg; and in Example 8, the amount of nano ZnO powder added is 125 mg.

[0051] Examples 9-13

[0052] In Examples 9-13, based on the method of Example 1, trehalose is further added during the ball milling process, specifically after the yeast suspension completes the first light treatment (when step b is completed for the first time). The amount of trehalose added in Example 9 is 35 mg, the amount of trehalose added in Example 10 is 40 mg, the amount of trehalose added in Example 11 is 45 mg, the amount of trehalose added in Example 12 is 50 mg, and the amount of trehalose added in Example 13 is 55 mg.

[0053] Examples 14-15

[0054] In Examples 14 and 15, the time of adding trehalose is adjusted based on the method of Example 12. In Example 14, trehalose is added to the yeast suspension at the same time as the nano ZnO powder is added; and in Example 15, trehalose is added after the yeast suspension has completed the second light treatment (when step b is completed for the second time).

[0055] Examples 16-18

[0056] In Examples 16-18, the ball milling temperature is adjusted based on the method of Example 12. The ball milling temperature of Example 16 is controlled at 40°C, the ball milling temperature of Example 17 is controlled at 55°C, and the ball milling temperature of Example 18 is controlled at 60°C.

[0057] Examples 19-22

[0058] In Examples 19-22, the impact balls are adjusted based on the method of Example 12. In Example 19, a zirconium oxide impact ball with a ball diameter of 1.5-2 cm is used; in Example 20, an agate bead impact ball with a ball diameter of 0.5-1 cm is used; in Example 21, the ball-to-material ratio is adjusted to 2:1; in Example 22, the ball-to-material ratio is adjusted to 5:1.

[0059] Comparative Example 1

[0060] This comparative example discloses a method for preparing nano-selenium-enriched yeast, comprising the following steps:

[0061] a. Weigh 10 g of selenium-enriched yeast powder and dissolve it in water to prepare a yeast suspension with a concentration of 10 wt %; add 80 mg of the nano ZnO powder obtained in the preparation example, stir evenly at 1200 r / min, place in a dark box, continue to stir at the same speed and irradiate with light for 30 min, the distance between the light source and the liquid surface of the suspension is 20 cm, and then centrifuge at 800 r / min for 1 min to obtain a photocatalytic selenium-enriched yeast suspension;

[0062] b. Pour the selenium-enriched yeast suspension into a ball mill and grind it. Use zirconium oxide as the impact ball, the ball-to-material ratio is 3:1, the ball particle size is 0.5-1cm, the rotation speed is 500rpm, and the ball mill is taken out after 60 minutes to obtain the ball mill material;

[0063] c. Ultrasonic treatment of the ball mill was performed using an ultrasonic cell disruptor, with the ultrasonic power controlled at 300w and the ultrasonic time at 15min to obtain an ultrasonic material;

[0064] d. Repeat steps b and c twice, i.e. "ball milling-ultrasound-ball milling-ultrasound", and collect the last ultrasonic material. The total wall breaking time = photocatalytic time 30 min + ball milling time 120 min + ultrasonic time 30 min = 180 min, and freeze-dry it into a powdery product to obtain nano-selenium-enriched yeast.

[0065] Comparative Example 2

[0066] This comparative example discloses a method for preparing nano-selenium-enriched yeast, comprising the following steps:

[0067] a. Weigh 10 g of selenium-enriched yeast powder, dissolve it in water, and prepare a yeast suspension with a concentration of 10 wt %; add 200 mg of the nano ZnO powder obtained in the preparation example, stir evenly at 1200 r / min, and place in a dark box, continue to stir at the same speed while irradiating with light for 30 min, with the distance between the light source and the liquid surface of the suspension being 20 cm, and then centrifuge at 800 r / min for 1 min to obtain a photocatalytic selenium-enriched yeast suspension;

[0068] b. Pour the selenium-enriched yeast suspension into a ball mill and grind it. Use zirconium oxide as the impact ball, the ball-to-material ratio is 3:1, the ball particle size is 0.5-1cm, the rotation speed is 500rpm, and the ball mill is taken out after 60 minutes to obtain the ball mill material;

[0069] c. Ultrasonic treatment of the ball mill was performed using an ultrasonic cell disruptor, with the ultrasonic power controlled at 300w and the ultrasonic time at 10min to obtain an ultrasonic material;

[0070] d. Repeat steps b and c for 4 times, i.e. "ball milling-ultrasound-ball milling-ultrasound-ball milling-ultrasound-ball milling-ultrasound", collect the last ultrasonic material, the total wall breaking time = photocatalytic time 30 min + total ball milling time 240 min + total ultrasonic time 40 min = 310 min, freeze-dry into a powdery product to obtain nano-selenium-enriched yeast.

[0071] Comparative Example 3

[0072] This comparative example discloses a method for preparing nano-selenium-enriched yeast, comprising the following steps:

[0073] a. Weigh 10 g of selenium-enriched yeast powder and mix it with water to prepare a yeast suspension with a concentration of 10 wt%;

[0074] b. Add 80 mg of the nano ZnO powder obtained in the preparation example to the yeast suspension, mix well and place in a ball mill with ultrasonic function, put in impact balls, use zirconium oxide as the impact ball, the ball-to-material ratio is 3:1, the ball particle size is 0.5-1 cm, place the light source 20±2 cm above the ball mill, control the ball mill speed to 100 r / min, and illuminate for 20 min with the cover open to obtain photocatalytic ball mill material.

[0075] c. Control the ball mill speed to 500 r / min, the ball milling temperature to 50°C, the ultrasonic power to 300 w, and the ultrasonic ball milling time to 120 min to obtain ultrasonic ball milled material; then centrifuge at 800 r / min for 1 min, take the upper suspended material, repeat the centrifugation 3 times to remove the nano ZnO powder as much as possible, collect the upper suspended material after the last centrifugation, the total wall breaking time = the total photocatalytic time of 20 min + the total ultrasonic ball milling time of 120 min = 140 min, freeze-dry into a powdery finished product, and obtain nano-selenium-enriched yeast.

[0076] Performance test

[0077] 1. Determination of selenium-enriched yeast morphology

[0078] The photocatalytic ball-milled material after the first light treatment in Example 1, the ultrasonic material after the first "ball milling with photocatalysis-ultrasound" treatment, and the ultrasonic material after the second "ball milling with photocatalysis-ultrasound" treatment were centrifuged at 800 r / min for 1 min, and the upper suspended material was taken. The centrifugation was repeated 3 times to remove the nano-ZnO powder as much as possible, and the upper suspended material after the last centrifugation was collected as the sample to be tested.

[0079] The above-mentioned samples were placed in a cell culture dish and their morphological changes were observed using a biological optical microscope. Figure 1 , Figure 3 , Figure 5 It should be noted that the size of intact selenium-enriched yeast cells is relatively large, belonging to the micron level, and they have strong scattering of light, and appear brighter under the microscope. However, the size of fragments of selenium-enriched yeast cells is relatively small, belonging to the nanometer level, and the scattering of light is significantly weakened, and they appear darker under the microscope. In order to obtain an image with accurate exposure, Figure 1 , Figure 3 , Figure 5 All the images were obtained under automatic exposure parameters. Due to the difference in exposure time, the background brightness was different. The hydrodynamic sizes of the three samples were measured using a dynamic laser scattering particle size analyzer (DLS). The results are shown in Figure 2 , Figure 4 , Figure 6 The morphology of the selenium-enriched yeast of Example 1 before and after cell wall breaking was observed using a scanning electron microscope (SEM). Figure 7 , Figure 8 .

[0080] Depend on Figure 1-6It can be seen that after the selenium-enriched yeast has undergone one "10-minute low-speed ball milling and illumination", the selenium-enriched yeast cells still maintain their structural integrity, but some covalent bonds on the cell wall surface have been oxidized and broken; after completing one "ball milling with photocatalysis-ultrasonic ball milling" treatment, more than 50% of the selenium-enriched yeast cells have been completely broken; after completing two "ball milling with photocatalysis-ultrasonic ball milling" treatments, under the microscope, no complete selenium-enriched yeast cells can be observed, the cell wall breaking rate of selenium-enriched yeast cells is close to 100%, and the degree of nano-crystallization reaches 96% (size is less than 1μm). Figure 7-8 It can be seen that the unbroken selenium-enriched yeast of Example 1 has a good appearance and a size between 3-5 μm; the size of the broken selenium-enriched yeast is concentrated between 100-300 nm, D50 is 0.23 μm, cell fragments with a size less than 200 nm account for about 42%, and cell fragments with a size less than 300 nm account for about 79%, with a high degree of nano-crystallization.

[0081] 2. Evaluation of the cell wall breaking effect of selenium-enriched yeast

[0082] The hydrodynamic size of the nano-selenium-enriched yeast samples after the wall-breaking treatment obtained in Examples 1-22 and Comparative Examples 1-3 was measured by a dynamic light scattering particle size analyzer (DLS), and was expressed as the median particle size D50. The D50 value means that 50% of the particle size is less than or equal to this value. The wall-breaking rate and nano-degree of each sample were determined based on the DLS percentage content data. The wall-breaking rate refers to the proportion of cell fragments with a particle size of less than 3 μm to the total number of particles; the nano-degree refers to the proportion of cell fragments with a particle size of less than 1 μm to the total number of yeast cell particles. The wall-breaking effects of Examples 1-22 and Comparative Examples 1-3 are shown in Table 2 below.

[0083] 3. Determination of effective nutrient content after selenium-enriched yeast cell wall breaking

[0084] 3.1 Determination of organic selenium content

[0085] The total Se content was determined according to (GB 5009.268-2016, Determination of multiple elements in food, Method 1, ICP-MS method).

[0086] 3.2 Determination of protein content

[0087] Protein was determined using the Kjeldahl method (GB 5009.5-2016, Determination of protein in food, Method 1, Kjeldahl method)

[0088] 3.3 Determination of vitamin B content

[0089] The determination of vitamin B1 was carried out by high performance liquid chromatography (GB 5009.84-2016, Determination of vitamin B1 in foods, Method 1, High Performance Liquid Chromatography); the determination of vitamin B2 was carried out by high performance liquid chromatography (GB 5009.85-2016, Determination of vitamin B2 in foods, Method 1, High Performance Liquid Chromatography).

[0090] The results of the determination of the effective content of nutrients in the nano-selenium-enriched yeast of Examples 1-22 and Comparative Examples 1-3 are shown in Table 2 below.

[0091] Table 2 Measurement results of Examples 1-22 and Comparative Examples 1-3

[0092]

[0093] Combined with Table 2, by comparing the test results of Examples 1-6 with those of Comparative Examples 1-2, it can be obtained that the total wall breaking time of the selenium-enriched yeast of the present application is about 100-150 min, while the total wall breaking time of Comparative Example 1 is 180 min, and the total wall breaking time of Comparative Example 2 is 310 min. It can be seen that the method of synchronous photocatalytic ball milling and synchronous ultrasonic ball milling used in the present application can avoid the steps of photocatalysis, ball milling, and ultrasonic treatment respectively and multiple sample transfers on the basis of ensuring a high wall breaking rate, a high degree of nano-crystallization, and a high effective nutrient content, thereby effectively shortening the wall breaking time of selenium-enriched yeast and improving the production efficiency of nano-selenium-enriched yeast.

[0094] The present application further compares the test results of Example 1 with those of Comparative Example 3, and it can be concluded that the present application adopts a treatment method of multiple step-by-step photocatalysis to have a higher wall-breaking rate, nano-ization degree and effective nutrient release rate. This is based on the fact that selenium-enriched yeast is easily destroyed by long-term photocatalysis after the wall is broken. The use of multiple step-by-step illumination, on the one hand, helps to gradually break the covalent bonds, thereby ensuring that the selenium-enriched yeast wall is broken more evenly and thoroughly; on the other hand, it can avoid the destruction of nutrients in the yeast due to long-term reaction; thereby obtaining nano-selenium-enriched yeast with a high wall-breaking rate, high nano-ization degree and high nutritional content.

[0095] Comparing the test results of Example 1 with those of Examples 7-8, it can be obtained that the mass ratio of nano zinc oxide to selenium-enriched yeast in Example 7 is 1:200. At this time, the cell wall breaking rate and nano-degree of selenium-enriched yeast cells are low, and the content of effective nutrients obtained is low. It can be seen that the content of the above-mentioned nano zinc oxide is relatively small in the process of the present application. The mass ratio of nano zinc oxide to selenium-enriched yeast in Example 8 is 1:80. At this time, the cell wall breaking rate and nano-degree of selenium-enriched yeast cells are high, but the corresponding content of effective nutrients is low. This is due to the excessive oxidation and decomposition of effective nutrients. It can be seen that the content of the above-mentioned nano zinc oxide is relatively high in the process of the present application. Therefore, the amount of nano zinc oxide in the present application needs to be appropriate, and it is preferably limited to 1:100-150. In addition, nano zinc oxide can also effectively inhibit the growth of miscellaneous bacteria, and the selection of nano zinc oxide can also maintain the stability of nutrients after the selenium-enriched yeast cell wall is broken.

[0096] Comparing the test results of Example 1 with those of Example 9-15, it can be obtained that the present application can effectively reduce the nutrients that are broken out by mechanical grinding and photocatalysis by adding an appropriate amount of trehalose. Based on the fact that trehalose has a certain anti-ultraviolet effect, referring to the test results of Example 14, the wall breaking rate and nano-degree of its selenium-enriched yeast are significantly reduced. It can be seen that if it is added in advance before ball milling, it is easy to form a unique protective film on the cell surface, which will increase the wall breaking difficulty of selenium-enriched yeast to a certain extent. In addition, referring to the test results of Example 15, the effective nutrient content of its selenium-enriched yeast is reduced. It can be seen that after the selenium-enriched yeast completes the first light treatment, it is added, and the selenium-enriched yeast thus prepared can better protect the nutrients from destruction while maintaining a high wall breaking rate. Wherein the present application further limits the mass ratio of trehalose to selenium-enriched yeast to 1:200-250, so as to ensure that nano-scale selenium-enriched yeast has a higher wall breaking rate, nano-degree and effective nutrient content.

[0097] By comparing the test results of Example 1 with those of Examples 16-18, it can be concluded that when preparing nano-selenium-enriched yeast in the present application, the ball milling temperature will also affect the performance of the product to a certain extent. This is based on the fact that frictional heat will be generated during the ball milling process, and ultrasonic treatment will also increase the temperature of the sample, but overheating will destroy the nutrients of the selenium-enriched yeast. For this reason, the present application effectively controls the grinding temperature, preferably controlling the temperature to ≤55°C, to reduce the destruction of nutrients.

[0098] By comparing the test results of Example 1 with those of Examples 19-22, it can be concluded that the size, material and amount of the impact ball in the ball milling process of the present application will also affect the performance of the product to a certain extent. Among them, the present application preferably uses zirconium oxide impact balls with a particle size of 0.5-1 cm. Zirconium oxide, as a material for impact balls for ball milling, also has a certain photocatalytic effect and can work together with zinc oxide under light conditions to effectively improve the wall breaking efficiency of selenium-enriched yeast.

[0099] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

Claims

1. A method for preparing nano-selenium-enriched yeast, characterized in that: The following steps are involved: a. Mixing selenium-enriched yeast with water to prepare a yeast suspension with a concentration of 5-12wt%; b. Add zinc oxide to the yeast suspension, mix well, and place in a ball mill with ultrasonic function, put in an impact ball, place a light source 20±2 cm above the ball mill, control the speed of the ball mill to 80-100 r / min, and illuminate for 10-15 min with the cover open to obtain a photocatalytic ball mill material; c. Close the ball mill cover, and continue to perform ultrasonic ball milling on the photocatalytic ball mill material at the same time, control the ball mill speed to 400-600r / min, control the ultrasonic power to 200-400w, and the ultrasonic ball milling time to 30-60min to obtain ultrasonic ball mill material; d. Repeat steps b and c 2-3 times, collect the last ultrasonic ball milling material, collect the suspension after multiple centrifugation to remove the photocatalyst, and freeze-dry it into a powdery product to obtain the nano-selenium-enriched yeast.

2. The method for preparing a nano-selenium-enriched yeast according to claim 1, characterized in that: In step c, trehalose is further added to the yeast suspension during the ball milling process, and the mass ratio of the trehalose to the selenium-enriched yeast is 1:200-250.

3. The method for preparing a nano-selenium-enriched yeast according to claim 2, characterized in that: The trehalose is added after the yeast suspension has completed the first light treatment.

4. The method for preparing a nano-selenium-enriched yeast according to claim 1, characterized in that: In step c, the temperature of the ball mill is controlled at ≤55°C.

5. The method for preparing a nano-selenium-enriched yeast according to claim 1, characterized in that: In step b, the mass ratio of the zinc oxide to the selenium-enriched yeast is 1:100-150.

6. The method for preparing a nano-selenium-enriched yeast according to claim 5, characterized in that: The zinc oxide is nano zinc oxide.

7. The method for preparing a nano-selenium-enriched yeast according to claim 1, characterized in that: In step b, the material of the impact balls for ball milling is zirconium oxide, and the particle size is 0.5-1 cm.

8. The method for preparing nano-selenium-enriched yeast according to claim 7, characterized in that: In step b, the ball-to-material ratio of the ball milling is 2-5:

1.

9. A nano-selenium-enriched yeast, characterized in that: The method is prepared by any one of claims 1 to 8.

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