Preparation method of nanoscale wall-broken selenium-enriched yeast

Abstract

The invention discloses a preparation method of nanoscale wall-broken selenium-enriched yeast, and relates to the technical field of food biologication.The preparation method comprises the following steps that selenium-enriched yeast suspension liquid is prepared; the selenium-enriched yeast suspension is subjected to mild local pore pretreatment, and local pores are formed in cell walls; a mixed solution containing amphiphilic molecules is added into the treated suspension liquid, a reaction is carried out under the low-strength shearing condition to form nanosheets, and the amphiphilic molecules comprise beta-cyclodextrin or a derivative thereof; and carrying out stabilizing treatment on the obtained product, and drying to obtain the nano-scale wall-broken selenium-enriched yeast. According to the invention, the nano-scale wall breaking with relatively ordered structure of the cell wall is realized, and the release and absorption efficiency of the selenium component in the gastrointestinal tract can be obviously improved; the wall breaking process is mild, low in temperature and low in shearing, the structural integrity and biological activity of selenium active ingredients are effectively protected, and the retention rate of the active ingredients is remarkably superior to that of a traditional violent wall breaking method.

Description

Technical Field

[0001] This invention relates to the field of food biotechnology, and in particular to a method for preparing nanoscale cell wall-broken selenium-enriched yeast. Background Technology

[0002] Selenium-enriched yeast is currently recognized as a high-bioavailability organic selenium source. The selenium in it mainly exists in organic forms such as selenomethionine and selenocysteine. It has the advantages of low toxicity, high biosafety, and relatively good absorption and utilization rate, and has been widely used in functional foods, dietary supplements, animal feed additives, and the pharmaceutical and health care fields.

[0003] However, the yeast cell wall is composed of a complex, multi-layered network structure formed by various macromolecules such as glucan, mannan, chitin, and proteins, exhibiting high mechanical strength and density. This natural barrier results in low digestibility and absorption efficiency of selenium-enriched yeast in intact cell form within the human gastrointestinal tract. A large amount of organic selenium is still excreted in feces as intact cells or larger fragments, failing to fully realize its nutritional value.

[0004] Existing cell wall disruption technologies mainly include mechanical high-pressure homogenization, nanoscale milling, ultrasonic treatment, compound enzymatic hydrolysis, and weak alkali autolysis, among other methods. These methods can disrupt cell walls to some extent and increase the release rate of selenium. However, they generally have the following drawbacks: First, the cell wall disruption process is relatively violent and random, and the cell wall is often randomly broken into irregular fragments, making it difficult to stably obtain nanoscale particles, and the product particle size distribution is wide and uneven. Secondly, high-intensity mechanical shearing, local high temperature, or strong acid and alkali environment can easily cause selenium active ingredients (especially selenomethionine) to undergo oxidation, degradation, or conformational changes, significantly reducing the nutritional value and bioactivity of the final product. Third, existing methods have high energy consumption, demanding equipment requirements, and complex processes, which are not conducive to large-scale industrial production and application in high-end functional foods.

[0005] Therefore, there is an urgent need for a new method that can achieve nanoscale cell wall disruption with relatively ordered structure under mild and low-damage conditions, so as to significantly improve the bioavailability of selenium, maximize the protection of selenium active ingredients, and improve the sensory properties of products. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a method for preparing nanoscale cell-wall-broken selenium-enriched yeast, the specific technical solution of which is as follows: A method for preparing nanoscale cell-wall-broken selenium-enriched yeast includes the following steps: S1. Prepare a selenium-enriched yeast suspension; S2. The selenium-enriched yeast suspension is subjected to a mild local porosification pretreatment to form local pores on the cell wall; S3. At 28~40℃, a mixture containing amphiphilic molecules is added to the suspension treated in step S2, and the mixture is reacted under low shear conditions for 1.5~14 hours to form nanosheets, wherein the amphiphilic molecules include β-cyclodextrin or its derivatives. S4. The product obtained in step S3 is stabilized and dried to obtain nano-sized cell-wall-broken selenium-enriched yeast.

[0007] Preferably, step S1 specifically includes the following sub-steps: S11. A high-selenium-tolerant brewer's yeast strain is used, with molasses or a mixture of glucose and maltose as the carbon source for selenium-enriched fermentation; S12. Sodium selenite is added in multiple batches during fermentation to ensure that the organic selenium content reaches ≥96%; S13. After fermentation, collect the yeast sludge and wash it 2-3 times with pure water. Adjust the solids concentration to 18-26% (w / v) to obtain a selenium-enriched yeast suspension.

[0008] Preferably, the mild local porosimetry pretreatment in step S2 includes a weak alkaline chelation treatment and / or a mild enzymatic hydrolysis treatment, as well as a short-term heat stress treatment.

[0009] Preferably, the weakly alkaline chelation treatment specifically includes: adjusting the selenium-enriched yeast suspension obtained in step S1 to pH 8.5~9.5, adding EDTA or its salt chelating agent, the concentration of which after addition is 0.3~2.0g / L, and treating at a temperature of 50~65℃ for 60~180 minutes.

[0010] Preferably, the mild enzymatic hydrolysis treatment specifically includes: adjusting the selenium-enriched yeast suspension to pH 5.5~6.8, adding a complex protease or a neutral / acidic protease, with the amount of enzyme added being 200~800 U / g of the dry yeast weight, and treating at a temperature of 45~55℃ for 1.5~5.0 hours.

[0011] Preferably, the short-term heat stress treatment specifically includes: heating the selenium-enriched yeast suspension, which has undergone weak alkaline chelation treatment and / or mild enzymatic hydrolysis treatment, to 68~75°C, holding it at that temperature for 5~30 minutes, and then rapidly cooling it to below 40°C.

[0012] Preferably, in step S3, the mixture containing amphiphilic molecules is an aqueous solution of β-cyclodextrin or hydroxypropyl-β-cyclodextrin, with a concentration of 0.9% to 4.2% (w / v).

[0013] Preferably, the mixture containing the amphiphilic molecule further contains an auxiliary amphiphilic molecule, which is selected from at least one of sodium caprylate, sodium decanoate, sucrose fatty acid ester and soybean lecithin.

[0014] Preferably, the low-intensity shear condition in step S3 is selected from at least one of the following: a) Mechanical stirring, with a stirring speed of 200~600 rpm; b) A circulating pump combined with a static mixer controls the liquid shear rate at 4~20m / s; c) Low-power ultrasonic treatment, with ultrasonic power of 50~500 W and duty cycle of 30%~70%; The temperature during the low-intensity shearing process is controlled to not exceed 40°C.

[0015] Preferably, step S4 specifically includes the following sub-steps: S41. Slowly heat the suspension after the reaction in step S3 to 50~60℃ and keep it at that temperature for 20~90 minutes; S42. Use a ceramic membrane with a pore size of 0.1~0.5μm or high-speed centrifugation to remove residual intact cells or larger particles; S43. Add a complex antioxidant to the system, wherein the complex antioxidant includes vitamin C and at least one natural plant-derived antioxidant selected from phytic acid, tea polyphenols, and rosemary extract; S44. The suspension after adding the protective agent is spray-dried or freeze-dried to obtain nano-sized cell wall broken selenium-enriched yeast powder, wherein the inlet air temperature of spray drying is 140~170℃ and the outlet air temperature is 80~95℃.

[0016] The method for preparing nanoscale cell-wall-broken selenium-enriched yeast provided by this invention has the following beneficial effects: 1. Cell wall disruption at the nanoscale with a relatively ordered structure was achieved, and the products mainly exhibited a layered nanosheet structure with a thickness of nanometers and a regular morphology, which is beneficial to significantly improve the release and absorption efficiency of selenium in the gastrointestinal tract. 2. The cell wall disruption process is gentle, low-temperature, and low-shear, which effectively protects the structural integrity and bioactivity of selenium active ingredients (especially selenomethionine), and the retention rate of active ingredients is significantly better than that of traditional violent cell wall disruption methods. 3. Energy consumption is significantly reduced, eliminating the need to rely on high-cost, high-energy-consuming equipment such as ultra-high pressure homogenizers. The process conditions are mild, making it easier to achieve large-scale, continuous production. 4. The obtained nano-sized cell wall broken selenium-enriched yeast powder has improved sensory properties, significantly reduced the bitterness unique to yeast, and improved the palatability of the product. 5. The product has an ordered structure, providing a better material basis for subsequent applications in high-end functional foods, precision nutrition, and controlled-release formulations, and has a broader market application prospect. Detailed Implementation

[0017] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be described in detail below. The description in this part is only exemplary and explanatory, and should not be used to limit the scope of protection of the present invention in any way.

[0018] This embodiment provides a method for preparing nanoscale cell wall-broken selenium-enriched yeast, comprising the following steps: S1. Prepare selenium-enriched yeast suspension.

[0019] S2. The selenium-enriched yeast suspension was subjected to a mild local porosification pretreatment to form local pores with an average pore size of about 18~85nm on the cell wall.

[0020] S3. At 28~40℃, a mixture containing amphiphilic molecules is added to the suspension treated in step S2, and the mixture is reacted under low shear conditions for 1.5~14 hours to form layered nanosheets, wherein the amphiphilic molecules include β-cyclodextrin or its derivatives.

[0021] S4. The product obtained in step S3 is stabilized and dried to obtain nano-sized cell-wall-broken selenium-enriched yeast.

[0022] This embodiment provides a method for preparing nanoscale cell wall-breaking selenium-enriched yeast. Its core lies in achieving gentle and orderly nanoscale destruction of the cell wall through a novel technical path of "firstly locally weakening to create nanoscale pores / weak points, and then using amphiphilic molecules to mediate ordered peeling".

[0023] Specifically: First, a certain number of nanoscale pores and weak regions are selectively created on the cell wall surface and between layers through a mild local porosimetry pretreatment, without damaging the overall structural integrity of the cell wall, thus providing "entry points" and "slip channels" for subsequent molecular insertion.

[0024] Subsequently, an amphiphilic molecular system centered on β-cyclodextrin or its derivatives was introduced under low temperature and low shear conditions. These molecules utilize the preferential recognition and inclusion ability of the hydrophobic lumen of β-cyclodextrin on locally exposed hydrophobic regions of the cell wall (lipid residues near pores, hydrophobic regions of proteins, etc.), gradually inserting themselves into the multilayer network structure of the cell wall like "molecular wedges," and gradually expanding and sliding along the natural interlayer interfaces of the cell wall with the synergistic effect of the auxiliary amphiphilic molecules.

[0025] Under sustained low-intensity shear force, the cell wall no longer breaks randomly, but tends to peel off in a relatively ordered layered manner along the pre-formed weak interlayer surfaces, eventually forming a layered nanosheet structure with a thickness on the nanometer scale and a width of hundreds of nanometers, rather than disordered small fragments.

[0026] The entire process is conducted under low and mild conditions, supplemented by antioxidant protection measures, which effectively avoids the problem of oxidative degradation of active ingredients that is common in traditional high-intensity cell wall breaking methods.

[0027] The ordered nano-exfoliation mechanism employed in this embodiment is not limited to a specific pretreatment method or a single type of amphiphilic molecule, but is based on the general technical logic of "local porosification providing an entry point + amphiphilic molecule interface-guided insertion + gentle shearing-driven interlayer exfoliation". Therefore, those skilled in the art, without departing from the core concept of the invention, can reasonably select and adjust the pretreatment combination method, the type and ratio of amphiphilic molecules, the specific implementation form of low-intensity shearing (mechanical, fluid, acoustic, etc.), and subsequent stabilization methods according to the specific compositional characteristics of the yeast cell wall, the target particle size requirements, and the production equipment conditions, thereby obtaining nanoscale cell wall-breaking selenium-enriched yeast products with different particle size distributions and different layered characteristics.

[0028] Furthermore, the layered nanosheet structure obtained by this ordered exfoliation has a large specific surface area, adjustable interlayer spacing, and a relatively complete spatial conformation, providing a good structural basis for subsequent functional applications (such as controlled release encapsulation of active ingredients, targeted delivery, and preparation of composite functional materials).

[0029] The method for preparing nanoscale cell-wall-broken selenium-enriched yeast provided in this embodiment has the following beneficial effects: 1. Cell wall disruption at the nanoscale with a relatively ordered structure was achieved, and the products mainly exhibited a layered nanosheet structure with a thickness of nanometers and a regular morphology, which is beneficial to significantly improve the release and absorption efficiency of selenium in the gastrointestinal tract. 2. The cell wall disruption process is gentle, low-temperature, and low-shear, which effectively protects the structural integrity and bioactivity of selenium active ingredients (especially selenomethionine), and the retention rate of active ingredients is significantly better than that of traditional violent cell wall disruption methods. 3. Energy consumption is significantly reduced, eliminating the need to rely on high-cost, high-energy-consuming equipment such as ultra-high pressure homogenizers. The process conditions are mild, making it easier to achieve large-scale, continuous production. 4. The obtained nano-sized cell wall broken selenium-enriched yeast powder has improved sensory properties, significantly reduced the bitterness unique to yeast, and improved the palatability of the product. 5. The product has an ordered structure, providing a better material basis for subsequent applications in high-end functional foods, precision nutrition, and controlled-release formulations, and has a broader market application prospect.

[0030] Furthermore, step S1 specifically includes the following sub-steps: S11. A high-selenium-tolerant brewer's yeast strain is used, with molasses or a mixture of glucose and maltose as the carbon source for selenium-enriched fermentation.

[0031] S12. During the fermentation process, sodium selenite is added in multiple batches to ensure that the proportion of organic selenium reaches ≥96%.

[0032] S13. After fermentation, collect the yeast sludge and wash it 2-3 times with pure water. Adjust the solids concentration to 18-26% (w / v) to obtain a selenium-enriched yeast suspension.

[0033] Specifically, the core of this step is to use a high-selenium-tolerant Saccharomyces cerevisiae strain to control fermentation, so that inorganic selenium (sodium selenite) can be efficiently converted into organic selenium (mainly selenomethionine) and obtain a suitable biomass, providing high-quality raw materials with low inorganic selenium residue for subsequent gentle cell wall breaking.

[0034] Fermentation uses molasses or a mixture of glucose and maltose as the carbon source. Sodium selenite is added as the selenium source in 6-10 additions, with the total selenium concentration (dry basis) controlled at 1800-3500 mg / kg and the selenium uptake rate maintained at 0.40-0.60 mg Se / g dry weight·h. The fermentation endpoint is achieved by controlling the yeast dry weight at 18-28 g / L and the organic selenium content at ≥96%.

[0035] The method for detecting the proportion of organic selenium can first use ICP-MS or atomic fluorescence spectrometry to determine the total selenium content, and then use HPLC-ICP-MS or dialysis-anion exchange chromatography to separate inorganic selenium (Se(IV) and Se(VI)), and calculate the proportion of organic selenium by subtracting inorganic selenium from total selenium.

[0036] After fermentation, the solids concentration can be adjusted by centrifuging or plate and frame filtration to collect yeast sludge, washing it 2-3 times with pure water, and then determining the solids content by gravimetric method (drying at 105℃ to constant weight) or rapid volume-dry weight conversion method, and adjusting it to 18-26% (w / v).

[0037] By using batch feeding and precise rate control, the organic selenium content is consistently ≥96%, which significantly increases the proportion of available selenium in the raw materials and reduces the potential risk of inorganic selenium toxicity in subsequent products. At the same time, the appropriate biomass concentration facilitates uniform treatment of the subsequent suspension.

[0038] Furthermore, the mild local pore-forming pretreatment in step S2 includes a weak alkaline chelation treatment and / or a mild enzymatic hydrolysis treatment, as well as a short-term heat stress treatment.

[0039] Furthermore, the weakly alkaline chelation treatment specifically includes: adjusting the selenium-enriched yeast suspension obtained in step S1 to pH 8.5~9.5, adding EDTA or its salt chelating agent at a concentration of 0.3~2.0 g / L, and treating at a temperature of 50~65℃ for 60~180 minutes.

[0040] Furthermore, the mild enzymatic hydrolysis treatment specifically includes: adjusting the selenium-enriched yeast suspension to pH 5.5~6.8, adding a complex protease or a neutral / acidic protease, with the amount of enzyme added being 200~800 U / g of the dry yeast weight, and treating at a temperature of 45~55℃ for 1.5~5.0 hours.

[0041] Furthermore, the short-term heat stress treatment specifically includes: heating the selenium-enriched yeast suspension, which has undergone weak alkaline chelation treatment and / or mild enzymatic hydrolysis treatment, to 68~75℃, holding it at this temperature for 5~30 minutes, and then rapidly cooling it to below 40℃.

[0042] The aforementioned process parameters collectively define a mild localized pore-forming pretreatment. The principle is to selectively disrupt the outer cell wall connections (protein cross-linking, hydrogen bonds) using low-intensity chemical / enzymatic / thermal methods, creating localized pores / weak regions with an average pore size of 18~85nm. This provides low-energy-barrier channels for subsequent insertion of amphiphilic molecules, achieving a pretreatment effect of "minimum damage and maximum guidance".

[0043] The weakly alkaline chelation treatment specifically involves adjusting the pH to 8.5–9.5, adding EDTA or its salt chelating agents (concentration 0.3–2.0 g / L), maintaining a temperature of 50–65℃, and treating for 60–180 minutes. This primarily chelates Mg. 2+ Ca 2+ Plasma loosens the wall layer.

[0044] The mild enzymatic hydrolysis treatment is as follows: adjust the pH to 5.5~6.8, add a complex protease or neutral / acidic protease (enzyme addition amount 200~800 U / g dry yeast), temperature 45~55℃, treat for 1.5~5.0 hours, and specifically degrade the outer mannoprotein.

[0045] The short-term thermal stress treatment is as follows: the temperature is raised to 68~75℃, held for 5~30 minutes, and then rapidly cooled to below 40℃, utilizing the difference in thermal expansion of the wall layers to generate local microcracks or nanoscale pores.

[0046] The three methods can be flexibly combined (such as weak base followed by enzymatic hydrolysis, or enzymatic hydrolysis followed by direct thermal stress), and the average pore size can be verified by BET nitrogen adsorption or cryo-transmission electron microscopy (Cryo-TEM).

[0047] Compared with traditional strong enzymatic hydrolysis or long-term acid-base treatment, this pretreatment causes minimal damage, with very low loss of selenium active ingredients at this stage. At the same time, it efficiently creates the "nano-entry point" required for subsequent orderly exfoliation, significantly improving the overall process's gentleness and protectiveness.

[0048] Further, in step S3, the mixture containing amphiphilic molecules is an aqueous solution of β-cyclodextrin or hydroxypropyl-β-cyclodextrin, with a concentration of 0.9% to 4.2% (w / v).

[0049] Furthermore, the mixture containing the amphiphilic molecule also contains an auxiliary amphiphilic molecule, which is selected from at least one of sodium caprylate, sodium decanoate, sucrose fatty acid ester and soybean lecithin.

[0050] Furthermore, the low-intensity shear condition in step S3 is selected from at least one of the following: a) Mechanical stirring, with a stirring speed of 200~600 rpm.

[0051] b) A circulating pump combined with a static mixer controls the liquid shear rate at 4~20m / s.

[0052] c) Low-power ultrasonic treatment, with ultrasonic power of 50~500 W and duty cycle of 30%~70%.

[0053] The temperature during low-intensity shearing processes should not exceed 40℃.

[0054] Specifically, the above process parameters and steps further define the ordered nano-exfoliation stage, the core of which is that the hydrophobic cavity of β-cyclodextrin (or its derivatives) preferentially encapsulates the locally exposed hydrophobic region of the cell wall, and assists amphiphilic molecules as "molecular wedges" to synergistically open the interlayer, driving the cell wall to exfoliate orderly along the interlayer under low-intensity shear, forming layered nanosheets.

[0055] The main amphiphilic molecules include: aqueous solutions of β-cyclodextrin or hydroxypropyl-β-cyclodextrin, at a concentration of 0.9% to 4.2% (w / v).

[0056] The auxiliary amphiphilic molecules (1 to 2 types can be selected) include: sodium caprylate or sodium decanoate 0.05% to 0.45%, sucrose fatty acid ester 0.08% to 0.6%, and soybean lecithin 0.1% to 0.7%.

[0057] The addition method is as follows: the pre-dissolved mixture is slowly added dropwise or intermittently in 10 to 20 portions, with the addition process taking 40 to 120 minutes.

[0058] Low-strength shear conditions are: a) Mechanical stirring at 200~600 rpm for 5~14 hours; b) Circulating pump + static mixer, shear rate 4~20 m / s, continuous for 3~9 hours; c) Low-power ultrasound (power 50~500 W, duty cycle 30%~70%), lasting 1.5~5 hours.

[0059] The temperature should be controlled at ≤40℃ throughout the process. Nitrogen gas or a trace amount of vitamin C (0.03%~0.15%) can be added for protection.

[0060] This stage achieves a transformation from "random crushing" to "ordered layered exfoliation," resulting in products with regular morphology, large specific surface area, and full exposure of activity. At the same time, low temperature and low shear maximize the retention of sensitive components such as selenomethionine, and the activity retention rate is significantly higher than that of traditional methods.

[0061] Furthermore, step S4 specifically includes the following sub-steps: S41. Slowly heat the suspension after the reaction in step S3 to 50~60℃ and keep it at that temperature for 20~90 minutes.

[0062] S42. Use a ceramic membrane with a pore size of 0.1~0.5μm or high-speed centrifugation to remove residual intact cells or larger particles.

[0063] S43. Add a complex antioxidant to the system, wherein the complex antioxidant includes vitamin C and at least one natural plant-derived antioxidant selected from phytic acid, tea polyphenols, and rosemary extract.

[0064] S44. The suspension after adding the protective agent is spray-dried or freeze-dried to obtain nano-sized cell wall broken selenium-enriched yeast powder, wherein the inlet air temperature of spray drying is 140~170℃ and the outlet air temperature is 80~95℃.

[0065] Specifically, the principle of this step is to stabilize the nanosheet structure by gently raising the temperature, precisely remove residual intact cells by removing impurities, use multiple mechanisms of compound antioxidants to synergistically protect the selenium component, and use spray / freeze drying to quickly form and stabilize it in the long term.

[0066] in: S41: Slowly heat to 50~60℃ and keep warm for 20~90 minutes to initially stabilize the stripping system.

[0067] S42: Use 0.1~0.5 μm pore size ceramic membrane filtration or high-speed centrifugation to remove residual intact cells and larger particles.

[0068] S43: Contains a complex antioxidant protectant, including 0.05%~0.25% vitamin C and at least one selected from phytic acid, tea polyphenols, and rosemary extract (total concentration 0.1%~0.4%).

[0069] S44: Spray drying (inlet air temperature 140~170℃, outlet air temperature 80~95℃) or freeze drying to obtain a light beige ultrafine powder.

[0070] This step minimizes the loss of activity during the drying process, resulting in a product with high purity, good flowability, significantly reduced bitterness, and strong stability. It provides high-quality, sensory-friendly nano-grade cell-wall broken selenium-enriched yeast powder for high-end functional food applications.

[0071] Specific embodiments are provided below. These embodiments are intended to enable those skilled in the art to more fully understand the present invention, but do not limit the present invention in any way.

[0072] Example Selenium-enriched fermentation was conducted in a bioreactor using a selenium-tolerant *Saccharomyces cerevisiae* strain. The fermentation medium consisted of molasses and a glucose-maltose mixture as the carbon source. Sodium selenite solution was added in eight separate additions during fermentation, with a total selenium addition of approximately 2850 mg / kg (dry basis). The fermentation temperature was 30℃, the pH was maintained at 5.5, the stirring speed was 300 rpm, and the aeration rate was 1.0 vvm. The final dry weight of the fermentation was approximately 24.6 g / L.

[0073] After fermentation, the yeast sludge was collected using a high-speed disc centrifuge, washed three times with pure water, resuspended in pure water, and the solids concentration was adjusted to 22% (w / v) to obtain 1 L of selenium-enriched yeast suspension (stored in a 2 L glass reaction flask for later use).

[0074] Place 1 L of suspension in a jacketed 2 L glass reactor, adjust the pH to 9.0 (using 1 mol / L NaOH solution), add 1.0 g of EDTA-Na2, heat to 58℃, and stir at a constant temperature (250 rpm) for 120 min.

[0075] After the treatment was completed, the temperature was rapidly increased to 71°C and held for 15 minutes to induce short-term thermal stress. Then, cooling water was circulated to reduce the temperature to 35°C.

[0076] Prepare a mixture of amphiphilic molecules in advance: 25.0 g of hydroxypropyl-β-cyclodextrin, 3.0 g of sucrose fatty acid ester, and 4.0 g of soybean lecithin, dissolved in 200 mL of pure water.

[0077] At a constant temperature of 35℃, the mixture was added to the reactor in 15 portions (approximately 13 mL each time, with 6-minute intervals), for a total addition time of 90 minutes. After addition, a combined shearing method was used: mechanical stirring at 400 rpm, coupled with intermittent low-power ultrasound (probe-type ultrasonic instrument, power 120 W, duty cycle 50%, 30-second pause every 30 seconds). The total reaction time was 6 hours, with the temperature controlled at 35~38℃, and nitrogen protection was maintained throughout the process.

[0078] After the reaction is complete, slowly raise the temperature to 55℃ (heating rate 2℃ / min) and hold for 50 min.

[0079] A 0.2 μm ceramic membrane filtration system (cross-flow filtration, transmembrane pressure 0.3 MPa) was used to remove residual intact cells and larger particles.

[0080] Add 1.5 g of vitamin C and 2.5 g of tea polyphenols to the filtrate and stir to dissolve.

[0081] The powder was dried using a spray dryer (peristaltic pump feed rate 15 mL / min, inlet air temperature 155℃, outlet air temperature 88℃) to obtain approximately 218 g of light beige nano-sized cell wall broken selenium-enriched yeast powder (yield approximately 99.1%).

[0082] Test method: Particle size distribution: 0.2 g of dry powder was dispersed in 5 mL of 0.1% Tween-80 aqueous solution and ultrasonically dispersed for 3 min. The particle size distribution was then measured using a laser particle size analyzer (wet method, refractive index 1.59, absorptivity 0.01) and the D50 and D90 values ​​were recorded (the average of three parallel measurements was taken).

[0083] Cell wall breakage rate: Take the dry powder, rehydrate and disperse it, dilute it and drop it onto a hemocytometer. Use an optical microscope (400×) to count the total number of intact cells and broken cells in the field of view (count 10 fields of view for each sample, >100 cells). Cell wall breakage rate = (number of broken cells / total number of cells) × 100%.

[0084] Sensory evaluation (bitterness): Ten trained sensory evaluators prepared the samples into a 1% aqueous suspension and conducted blind evaluation (1 = no bitterness, 9 = extremely bitterness), and the average value was taken.

[0085] The test data is shown in Table 1 below: Comparative Example Using the same selenium-enriched yeast suspension as in Example 1 (the fermentation process, collection and washing, and solid concentration adjustment were all the same), 1 L of raw material suspension was obtained (and placed in a 2 L glass container for later use).

[0086] 1 L of suspension was placed in a laboratory high-pressure homogenizer for three-stage high-pressure homogenization: First-level pressure: 80 MPa Second-level pressure: 120 MPa Level 3 pressure: 100 MPa The process is repeated 4 times, and after each cycle, the temperature is cooled to <60℃ through a plate heat exchanger (the actual outlet temperature is controlled at 55~62℃).

[0087] After homogenization, a 0.2 μm ceramic membrane filtration system (cross-flow filtration, transmembrane pressure 0.3 MPa) was used to remove larger residues.

[0088] Add 1.5 g of vitamin C and 2.5 g of tea polyphenols to the filtrate and stir to dissolve.

[0089] The powder was dried using a spray dryer (peristaltic pump feed rate 15 mL / min, inlet air temperature 155℃, outlet air temperature 88℃) to obtain approximately 215 g of light yellow cell wall broken selenium-enriched yeast powder (yield approximately 97.7%).

[0090] The testing method is the same as in the embodiment, and the test data is shown in Table 2 below: This article uses specific examples to illustrate the principles and implementation methods of the present invention. The descriptions of the above embodiments are only for the purpose of helping to understand the method and core ideas of the present invention. The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the technical scope disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be within the scope of protection of the present invention.

Claims

1. A method for preparing nanoscale cell-wall-broken selenium-enriched yeast, characterized in that, Includes the following steps: S1. Prepare a selenium-enriched yeast suspension; S2. The selenium-enriched yeast suspension is subjected to a mild local porosification pretreatment to form local pores on the cell wall; S3. At 28~40℃, a mixture containing amphiphilic molecules is added to the suspension treated in step S2, and the mixture is reacted under low shear conditions for 1.5~14 hours to form nanosheets, wherein the amphiphilic molecules include β-cyclodextrin or its derivatives. S4. The product obtained in step S3 is stabilized and dried to obtain nano-sized cell-wall-broken selenium-enriched yeast.

2. The method for preparing nanoscale cell-wall-broken selenium-enriched yeast according to claim 1, characterized in that, Step S1 specifically includes the following sub-steps: S11. A high-selenium-tolerant brewer's yeast strain is used, with molasses or a mixture of glucose and maltose as the carbon source for selenium-enriched fermentation; S12. Sodium selenite is added in multiple batches during fermentation to ensure that the organic selenium content reaches ≥96%; S13. After fermentation, collect the yeast sludge and wash it 2-3 times with pure water. Adjust the solids concentration to 18-26% (w / v) to obtain a selenium-enriched yeast suspension.

3. The method for preparing nanoscale cell-wall-broken selenium-enriched yeast according to claim 1, characterized in that, The mild local porosimetry pretreatment described in step S2 includes a weak alkaline chelation treatment and / or a mild enzymatic hydrolysis treatment, as well as a short-term heat stress treatment.

4. The method for preparing nanoscale cell-wall-broken selenium-enriched yeast according to claim 3, characterized in that, The weakly alkaline chelation treatment specifically includes: adjusting the selenium-enriched yeast suspension obtained in step S1 to pH 8.5~9.5, adding EDTA or its salt chelating agent, with a concentration of 0.3~2.0 g / L after addition, and treating at a temperature of 50~65℃ for 60~180 minutes.

5. The method for preparing nanoscale cell-wall-broken selenium-enriched yeast according to claim 3, characterized in that, The mild enzymatic hydrolysis treatment specifically includes: adjusting the selenium-enriched yeast suspension to pH 5.5~6.8, adding a complex protease or a neutral / acidic protease, with the amount of enzyme added being 200~800 U / g of the dry yeast weight, and treating at a temperature of 45~55℃ for 1.5~5.0 hours.

6. The method for preparing nanoscale cell-wall-broken selenium-enriched yeast according to any one of claims 3 to 5, characterized in that, The short-term heat stress treatment specifically includes: heating the selenium-enriched yeast suspension, which has undergone weak alkaline chelation treatment and / or mild enzymatic hydrolysis treatment, to 68~75℃, keeping it at that temperature for 5~30 minutes, and then rapidly cooling it to below 40℃.

7. The method for preparing nanoscale cell-wall-broken selenium-enriched yeast according to claim 1, characterized in that, In step S3, the mixture containing amphiphilic molecules is an aqueous solution of β-cyclodextrin or hydroxypropyl-β-cyclodextrin with a concentration of 0.9% to 4.2% (w / v).

8. The method for preparing nanoscale cell-wall-broken selenium-enriched yeast according to claim 7, characterized in that, The mixture containing amphiphilic molecules also contains auxiliary amphiphilic molecules, which are selected from at least one of sodium caprylate, sodium decanoate, sucrose fatty acid esters, and soybean lecithin.

9. The method for preparing nanoscale cell-wall-broken selenium-enriched yeast according to claim 7 or 8, characterized in that, The low-intensity shear condition mentioned in step S3 is selected from at least one of the following: a) Mechanical stirring, with a stirring speed of 200~600 rpm; b) A circulating pump combined with a static mixer controls the liquid shear rate at 4~20m / s; c) Low-power ultrasonic treatment, with ultrasonic power of 50~500 W and duty cycle of 30%~70%; The temperature during the low-intensity shearing process is controlled to not exceed 40°C.

10. The method for preparing nanoscale cell-wall-broken selenium-enriched yeast according to claim 1, characterized in that, Step S4 specifically includes the following sub-steps: S41. Slowly heat the suspension after the reaction in step S3 to 50~60℃ and keep it at that temperature for 20~90 minutes; S42. Use a ceramic membrane with a pore size of 0.1~0.5μm or high-speed centrifugation to remove residual intact cells or larger particles; S43. Add a complex antioxidant to the system, wherein the complex antioxidant includes vitamin C and at least one natural plant-derived antioxidant selected from phytic acid, tea polyphenols, and rosemary extract; S44. The suspension after adding the protective agent is spray-dried or freeze-dried to obtain nano-sized cell wall broken selenium-enriched yeast powder, wherein the inlet air temperature of spray drying is 140~170℃ and the outlet air temperature is 80~95℃.

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