Bifidobacterium breve, fermented ovine colostrum, preparation method thereof and application for enhancing immunity

Abstract

The present application provides a Bifidobacterium breve. The strain has high yield of acetoin and has milk clotting function. The present application provides a fermenting agent comprising the Bifidobacterium breve. The preparation method of the fermenting agent is also provided. The present application also provides a fermented ovine colostrum, which contains a plurality of functional polypeptides. The present application also provides a preparation method of the fermented ovine colostrum. The method can simultaneously ensure high retention rate of nutrients in the ovine colostrum. The present application also provides a polypeptide. The polypeptide can inhibit angiotensin converting enzyme activity, improve vascular endothelial function or inhibit platelet aggregation, and has cardiovascular protection ability. The present application also provides a polypeptide composition. The present application also provides application of the Bifidobacterium breve, the polypeptide and / or the fermented ovine colostrum in the field of food or medicine for helping to enhance immunity.

Description

Technical Field

[0001] This invention relates to the field of probiotic fermentation and functional dairy products, specifically to a Bifidobacterium breve, sheep colostrum prepared using Bifidobacterium breve, a method for preparing sheep colostrum fermented with Bifidobacterium breve, and the application of Bifidobacterium breve in improving the body's immune function and protecting cardiovascular health. Background Technology

[0002] Cardiovascular and cerebrovascular diseases are prevalent chronic diseases worldwide, with atherosclerosis being one of their typical pathological manifestations. Meanwhile, the integrity of the intestinal mucosal immune barrier is not only closely related to the body's immune homeostasis but also directly affects metabolic health. Unhealthy lifestyles such as high-fat diets easily induce intestinal immune dysregulation, leading to impaired intestinal barrier function. Once the intestinal barrier is damaged, endotoxins enter the bloodstream, exacerbating systemic inflammatory responses and ultimately promoting the occurrence and progression of atherosclerosis. Therefore, a clear and close pathological link exists between immune dysfunction and cardiovascular and cerebrovascular diseases. Consequently, there is an urgent need to develop functional products that can simultaneously regulate the body's immune function and protect the cardiovascular and cerebrovascular systems.

[0003] Colostrum, the milk secreted by ewes shortly after giving birth, is rich in various heat-sensitive bioactive substances, including immunoglobulins (IgG, IgA, IgM), lactoferrin, insulin-like growth factor 1 (IGF-1), fibroblast growth factor (FGF), and nerve growth factor (NGF). These bioactive substances provide an important material basis for the application of colostrum in immune regulation and cardiovascular protection. However, the bioactive components in colostrum are mostly heat-sensitive proteins or peptides, which are easily degraded and inactivated during processing and fermentation due to factors such as temperature, microbial protease action, and acidic environments. This results in a low retention rate of bioactive components, severely limiting its functional development and application.

[0004] Probiotic fermentation is an important means of enhancing the functionality of dairy products. Suitable probiotic strains can not only improve product flavor and generate active metabolites, preventing the degradation of active ingredients, but also repair the intestinal mucosal immune barrier, reduce the damage of inflammatory responses to the cardiovascular system, and create a synergistic effect with the functionality of sheep colostrum. However, current research shows that while some strains can improve product characteristics after fermentation, they do not effectively protect the core heat-sensitive active ingredients in sheep colostrum, such as IGF-1, lactoferrin, and immunoglobulins, thus failing to fully realize their immunomodulatory and cardiovascular protective effects. Therefore, screening for high-performance probiotic strains, establishing the optimal process for probiotic fermentation of sheep colostrum, achieving high retention of core active ingredients in sheep colostrum, and verifying their synergistic effects in immunomodulation and cardiovascular protection have become urgent technical challenges in the development of functional sheep dairy products. Summary of the Invention

[0005] The first objective of this invention is to provide a strain of Bifidobacterium breve HX-BB128.

[0006] A second objective of this invention is to provide a fermentation agent comprising Bifidobacterium breve HX-BB128.

[0007] A third objective of this invention is to provide a method for preparing the aforementioned fermenting agent.

[0008] The fourth objective of this invention is to provide a fermented sheep colostrum obtained by fermenting sheep colostrum with the aforementioned Bifidobacterium breve HX-BB128.

[0009] The fifth objective of this invention is to provide a method for preparing the fermented sheep colostrum.

[0010] The sixth objective of this invention is to provide a VLGPVRGPFP polypeptide.

[0011] The seventh object of the present invention is to provide a polypeptide composition containing LLTTDVEK, VLGPVRGPFP.

[0012] The eighth object of the present invention is to provide an application of the aforementioned Bifidobacterium breve HX-BB128, fermented sheep colostrum, and polypeptide.

[0013] This invention is achieved through the following technical solution: A strain of *Bifidobacterium breve*, HX-BB128, with accession number CGMCC NO. 37265, was deposited on January 4, 2026, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China (Institute of Microbiology, Chinese Academy of Sciences). The strain was viable at the time of deposit.

[0014] A starter culture comprising the aforementioned Bifidobacterium breve HX-BB128; or This includes the aforementioned Bifidobacterium breve HX-BB128, Streptococcus salivarius thermophilus subsp. and Lactobacillus delbrueckii bulgaricus subsp. The volume ratio of Bifidobacterium breve HX-BB128 bacterial suspension, Streptococcus salivarius thermophilus bacterial suspension, and Lactobacillus delbrueckii bulgaricus bacterial suspension was (1~2):1:1; The viable count of the *Bifidobacterium breve* HX-BB128 bacterial suspension was 1-1.2 × 10⁻⁶. 9 CFU / mL; The viable count of *Streptococcus thermophilus* subsp. *salivarius* in the bacterial suspension was 1-1.2 × 10⁻⁶. 9CFU / mL; The viable count of *Lactobacillus delbrueckii* subsp. bulgaricus in the culture was 1-1.2 × 10⁻⁶. 9 CFU / mL.

[0015] The method for preparing the fermenting agent includes the following steps: Strawberry Bifida HX-BB128, or streaked with Streptococcus salivarius thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, and single bacteria were picked and placed into liquid MRS medium. After anaerobic culture at 37°C and two rounds of activation, the bacteria were centrifuged to obtain probiotic sludge. Mix the probiotic slurry with the freeze-drying protectant evenly, and freeze-dry to obtain the final product.

[0016] The MRS culture medium comprises the following components by weight percentage: yeast peptone 1.5-3.0%, yeast extract 1.5-3.0%, trehalose 2.0-3.0%, sodium acetate 0.3-0.5%, dipotassium hydrogen phosphate 0.1-0.2%, diammonium hydrogen citrate 0.1-0.2%, magnesium sulfate heptahydrate 0.02-0.03%, manganese sulfate monohydrate 0.005-0.010%, calcium chloride 0.005-0.010%, L-cysteine ​​hydrochloride 0.005-0.010%, and water as balance; The freeze-drying protectant comprises the following components by weight percentage: xylooligosaccharide 10-20%, skim milk powder 10-15%, maltodextrin 2-6%, monosodium glutamate 1.0-2.5%, sodium ascorbate 0.1-0.2%, and water as the balance.

[0017] A fermented sheep colostrum, wherein the fermented sheep colostrum is obtained by fermenting sheep colostrum powder with the aforementioned fermenting agent.

[0018] The preparation method of the sheep colostrum powder includes the following steps: inactivating sheep colostrum at 65°C for 30 minutes, centrifuging or filtering to remove impurities, and then vacuum freeze-drying at -40°C or low-temperature spray drying at 40-50°C until the moisture content is no more than 3%.

[0019] The method for preparing fermented sheep colostrum includes the following steps: The strain was inoculated into MRS medium and incubated at 37℃ for 12-16 hours, followed by two generations of activation to obtain a viable count of 1-1.2 × 10⁻⁶. 9 CFU / mL activated bacterial solution; The emulsion containing 6wt%-7wt% sheep colostrum powder and 0.8wt%-1.2wt% sucrose was sterilized at 65℃ for 30-40 minutes and then cooled to 37℃ to obtain a sterile fermentation substrate. The activated bacterial solution was inoculated into the fermentation substrate at an inoculation rate of 2% (v / v), and fermented at 37°C for 12-24 hours until the fermentation substrate curdled, thus obtaining fermented sheep colostrum.

[0020] A polypeptide was isolated from sheep colostrum after fermentation by the aforementioned Bifidobacterium breve HX-BB128. The polypeptide sequence is VLGPVRGPFP.

[0021] A polypeptide composition comprising a polypeptide with the sequence LTLTDVEK and a polypeptide with the sequence VLGPVRGPFP.

[0022] The use of the aforementioned Bifidobacterium breve HX-BB128 in the preparation of health products or pharmaceuticals that help enhance immunity.

[0023] The use of one of the aforementioned polypeptides in the preparation of health products or pharmaceuticals that help enhance immunity.

[0024] The use of the fermented sheep colostrum described above in the preparation of health products or pharmaceuticals that help enhance immune function.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows: The *Bifidobacterium breve* strain provided by this invention exhibits significant advantages in strain specificity. The *Bifidobacterium breve* strain HX-BB128 screened in this invention is derived from sheep colostrum, is well-suited for sheep colostrum fermentation systems, and possesses high acetoin production, curdling, and antioxidant functions. After fermentation with this *Bifidobacterium breve*, the dual key polypeptides LTLTDVEK and VLGPVRGPFP can be isolated from sheep colostrum.

[0026] The fermentation method provided by this invention achieves high retention of active ingredients. When fermenting sheep colostrum using *Bifidobacterium breve* HX-BB128, the active peptides produced by its metabolism can form a complex system with the heat-sensitive active ingredients in sheep colostrum, reducing the damage to the spatial structure of active ingredients caused by protease hydrolysis and acidic environment, and significantly improving the retention rates of immunoglobulins, lactoferrin, growth factors, etc. In the fermented sheep colostrum prepared by the fermentation method provided by this invention, the IgG retention rate is as high as over 95%, the IGF-1 retention rate reaches 92.67%, and the NGF retention rate reaches 96%.

[0027] The fermentation method provided by this invention enables the efficient enrichment of key peptides. During the fermentation process, Bifidobacterium breve HX-BB128 can be directionally metabolized, thereby achieving a thousand-fold increase in the abundance of LTLTDVEK and the generation of new VLGPVRGPF peptides.

[0028] The polypeptides LTLTDVEK and VLGPVRGPFP provided by this invention can produce a synergistic effect, thereby achieving the dual effects of immune regulation and cardiovascular protection.

[0029] The fermented sheep colostrum provided by this invention can achieve multi-dimensional synergistic intervention in intestinal mucosal barrier repair, immune homeostasis maintenance, lipid metabolism regulation, and vascular inflammation inhibition. Its effects in improving immunity and protecting the cardiovascular system are significantly better than those of single probiotics, unfermented sheep colostrum, and simple mixtures of the two. Attached Figure Description

[0030] Figure 1 The mass spectrum of the peptide LTLTDVEK is shown. Figure 2 The mass spectrum of the peptide VLGPVRGPFP is shown. Figure 3 The changes in body weight of mice in different groups are shown; Figure 4 The relative mRNA expression of cytokines (TNF-α and IL-1β) in different groups of mice is shown; Figure 5 The mRNA expression of tight junction proteins (ZO-1 and Occludin) in different groups of mice is shown; Figure 6 The expression of the CD68 gene mRNA in the aortic root tissue of different groups of mice is shown. Detailed Implementation

[0031] The range descriptions used herein, such as numerical ranges and proportional ranges, include all possible sub-ranges and single numerical values ​​within that range. For example, the range descriptions of "1 to 6" or "1 to 6" cover all sub-ranges (such as 1 to 3, 2 to 5, etc.) and single numbers (such as 1, 2, 3, 4, 5, 6) between 1 and 6. Unless otherwise specified, the terms "including" and "contains" as used herein mean "including but not limited to"; relational terms such as "first" and "second" are used only to distinguish different entities or operations and do not imply an actual order or relationship; "and / or" indicates that multiple situations can exist individually or simultaneously; expressions such as "at least one," "multiple," and "at least one" refer to any combination of the corresponding objects, including combinations of single or multiple objects. The proportional relationships mentioned herein, such as mass ratios and molar ratios, should be understood as the correspondence between the first and second terms of a proportional formula, according to the order of description. The raw materials, reagents, instruments, and equipment used herein can all be obtained through commercial purchase or prepared using existing methods.

[0032] Example 1 This embodiment provides a method for preparing sheep colostrum powder. The method employs a complete process of "cold chain transportation - sterilization (65℃, 30 min) - low-temperature drying (vacuum freeze drying and / or low-temperature spray drying)," with the specific steps as follows: 1. Pretreatment of sheep colostrum: Cold chain transportation and storage: Collect sheep colostrum from healthy ewes within 24-72 hours after giving birth, place the collected sheep colostrum in a 2℃-4℃ cold chain transport box and transport it to the factory, with a transportation time not exceeding 4 hours; after arriving at the factory, store the sheep colostrum in a -20℃ cold storage for no more than 7 days, and avoid repeated freeze-thaw cycles.

[0033] 2. Sterilization: Remove the refrigerated sheep colostrum, allow it to return to room temperature, and thoroughly mix. Then, pasteurize at a constant temperature of 65℃ for 30 minutes. Continuous gentle stirring is maintained throughout the sterilization process to prevent localized overheating that could denature or inactivate active components such as lactoferrin, immunoglobulins, and growth factors in the sheep colostrum. After sterilization, discontinue heating the sheep colostrum and rapidly cool it to below 25℃.

[0034] 3. Low-temperature drying: After removing impurities from the sterilized sheep colostrum, it can be dried using vacuum freeze-drying or low-temperature spray drying methods, depending on production needs, to prepare sheep colostrum powder. The powder is dried until the moisture content is ≤3%. 4. Post-processing: The dried sheep colostrum powder is granulated through a 50-80 mesh sieve to remove lumps. Then, it is packaged and sealed in a sterile environment and stored in a cool, dry place to complete the preparation of sheep colostrum powder.

[0035] Example 2 This embodiment provides a preliminary screening method for bacterial strains exhibiting acetoin production, curdling function, and antioxidant properties. The specific steps are as follows: 1. The screening process for acetoin-producing strains is as follows: The creatine colorimetric method was used to screen strains capable of synthesizing acetoin. A 50 mL creatine mixture was prepared by dissolving 1 g of 1-naphthol, 0.1 g of creatine, and 4 g of NaOH. The strain was then inoculated into a test tube containing 0.5 mL of the creatine mixture. The strain that rapidly turned the creatine mixture red was the one capable of producing acetoin.

[0036] 2. The screening process for strains exhibiting curdling behavior is as follows: Prepare bacterial suspensions using the bacteria listed in Table 1 (after culture, the viable cell count is 1×10⁻⁶). 9(Approximately CFU / mL), and then inoculate the bacterial solution into 12% (w / v) sheep colostrum culture medium at an inoculation rate of 2% (v / v) (sheep colostrum culture medium preparation: 12% (w / v) sheep colostrum powder, with the remainder being water), and incubate at a constant temperature of 37℃ for 12~24h. Observe the culture medium visually to see if curdling occurs, and record the degree of curdling.

[0037] Table 1. Screening of strains exhibiting acetoin production and curdling function Note: —: indicates no red phenomenon; +: indicates slight red phenomenon, low-yield β-match; ++: indicates moderate red phenomenon, medium-yield β-match; +++: indicates strong red phenomenon, high-yield β-match.

[0038] According to the results in Table 1, the strains that produce high levels of acetoin and have curdling function were selected as Lactococcus faecium N41S1, Lactococcus lactis subsp. lactis N45S17, Bifidobacterium breve HX-BB128, Streptococcus salivarius subsp. thermophilus, and Lactobacillus delbrueckii subsp. bulgaricus.

[0039] 3. The screening process for strains with antioxidant properties is as follows: Antioxidant performance evaluation: The bacteria listed in Table 2 were used to prepare bacterial suspensions (the viable count of the bacterial suspension after culture was 1×10⁻⁶). 9 The colostrum (approximately CFU / mL) was inoculated at a rate of 2% (v / v) into 12% (w / v) sheep colostrum culture medium and incubated at 37℃ for 12-24 hours. The antioxidant capacity of the fermented sheep colostrum before and after fermentation was determined using the Total Antioxidant Capacity (T-AOC) assay kit (FRAP method) and the DPPH free radical scavenging capacity (Total Antioxidant Capacity DPPH method).

[0040] Table 2 Antioxidant properties of sheep colostrum after fermentation by different strains Table 2 shows that fermented sheep colostrum obtained by fermenting sheep colostrum with Bifidobacterium breve HX-BB128, Streptococcus salivarius subsp. thermophilus, and Lactobacillus delbrueckii subsp. bulgaricus exhibited increased T-AOC and DPPH free radical scavenging rates. In particular, HX-BB128 showed the best antioxidant effect.

[0041] Example 3 In this embodiment, secondary screening of bacterial strains was conducted to identify probiotics with good fermentation performance. The specific screening process is as follows: The strains screened in Example 2 (Bifidobacterium breve HX-BB128, Streptococcus salivarius subsp. thermophilus, and Lactobacillus delbrueckii subsp. bulgaricus) were used to ferment sheep colostrum, and relevant indicators of the fermented sheep colostrum were measured. The specific fermentation steps are as follows: Single probiotic activation: Glycerol tubes of the selected strains were streaked for isolation. Single bacteria were picked and inoculated into MRS medium and incubated at 37℃ for 12-16 h. After two generations of activation, viable counts of 1-1.2 × 10⁻⁶ were obtained. 9 CFU / mL bacterial suspension; Preparation and sterilization of sheep colostrum substrate: The emulsion containing 6wt%-7wt% sheep colostrum powder and 0.8wt%-1.2wt% sucrose (i.e. sheep colostrum fermentation substrate) is sterilized at a constant temperature of 65℃ for 30 minutes. After sterilization, it is quickly cooled to 37℃ for later use. Sheep colostrum was fermented using a single-strain solution (containing only Bifidobacterium breve strain HX-BB128) and a compound-strain solution. The preparation method of the compound bacterial solution is as follows: Take the activated single probiotic solution (live count 1-1.2 × 10⁻⁶) 9 Multiple groups of compound probiotic solutions were prepared by mixing CFU / mL according to volume ratio. The formulas of each group are as follows. After mixing, the solutions were thoroughly mixed and set aside. Compound bacterial solution 1: Bifidobacterium breve HX-BB128: Streptococcus salivarius subsp. thermophilus = (1-2):1 (v / v); Compound bacterial solution 2: Bifidobacterium breve HX-BB128: Lactobacillus delbrueckii subsp. bulgaricus = (1-2):1 (v / v); Compound bacterial solution 3: Streptococcus salivarius thermophilus subsp.: Lactobacillus delbrueckii subsp. bulgaricus = (1-2):1 (v / v); Compound bacterial solution 4: Streptococcus salivarius thermophilus subsp.: Lactobacillus delbrueckii subsp. bulgaricus: Bifidobacterium breve HX-BB128 = (1:1:1) ~ (2:1:1) (v / v); Fermentation group setup (single-strain fermentation group + multi-strain fermentation group) Using sterilized sheep colostrum as the fermentation substrate, single probiotic solutions and compound probiotic solutions were inoculated and fermented, respectively. A blank control group, a single-strain fermentation group, and a compound-strain fermentation group were set up. Inoculation and fermentation conditions were uniform across all groups. The specific group divisions are as follows: 1. Blank control group No probiotic culture was inoculated. Only the sterilized sheep colostrum matrix was taken and placed in a constant temperature environment of 37°C for static culture. The culture time was the same as that of the fermentation group.

[0042] 2. Single-strain fermentation group Three experimental groups were set up. Each group was inoculated with bacterial solution HX-BB128, bacterial solution, and bacterial solution into sheep colostrum matrix at an inoculation amount of 2% (v / v), and fermented at a constant temperature of 37℃ for 24 hours.

[0043] 3. Compound microbial fermentation group Four experimental groups were set up. Each group was inoculated with compound bacterial solution 1 to compound bacterial solution 4 at an inoculation amount of 2% (v / v) into the sheep colostrum matrix and fermented at a constant temperature of 37℃ for 24 hours.

[0044] Sheep colostrum is rich in various bioactive substances, including immunoglobulins (IgG, IgA, IgM), lactoferrin, insulin-like growth factor-1 (IGF-1), fibroblast growth factor (FGF), and nerve growth factor (NGF). Among these, IgG and IgM are core immunoglobulins; lactoferrin possesses antibacterial, anti-inflammatory, and immune cell activation functions; and NGF participates in maintaining immune homeostasis by regulating immune cell differentiation and function. Furthermore, IGF-1 can activate the eNOS pathway in endothelial cells, promoting NO production and improving vasodilation, while simultaneously inhibiting excessive proliferation of vascular smooth muscle cells and macrophage infiltration, thus slowing the progression of atherosclerosis. FGF and NGF stimulate angiogenesis and have neurotrophic effects, supporting neuronal survival and regeneration. However, these components are all heat-sensitive proteins or peptides, which are easily degraded and inactivated by proteases produced by lactic acid bacteria and the acidic environment during prolonged fermentation, resulting in low retention rates. Therefore, to screen for dominant fermentation strains with high retention rates of bioactive components, corresponding ELISA kits can be used to detect the levels of immunoglobulins, lactoferrin, and secretory factors in different groups.

[0045] The fermented sheep colostrum prepared by fermentation of single probiotic culture and compound probiotic culture was tested using IgG and IgM kits. The test results are shown in Table 3.

[0046] Table 3 Comparison of immunoglobulin levels in different groups Immunoglobulins (IgG and IgM) in sheep colostrum are heat-sensitive proteins that are easily degraded and inactivated under conventional fermentation conditions, resulting in low retention rates.

[0047] As shown in Table 3, the colostrum fermented with either the single-strain Bifidobacterium breve HX-BB128 group or the compound bacterial solution 4 contained higher levels of IgG and IgM, and the immunoglobulin retention rate after fermentation was high, exceeding 95%. Therefore, the present invention, by using Bifidobacterium breve HX-BB128 to ferment colostrum, can significantly reduce the degradation of immunoglobulins in colostrum and greatly improve their retention rate.

[0048] To further verify the effectiveness of Bifidobacterium breve HX-BB128, the lactoferrin and secretory factor content of sheep colostrum obtained by fermentation with HX-BB128 and compound bacteria 2 (without HX-BB128) and compound bacteria 4 (containing HX-BB128) were measured. The results are shown in Table 4. Specifically, the contents of secretory factors and lactoferrin in the fermented sheep colostrum of each group were detected using sheep insulin-like growth factor 1 (IGF-1) ELISA kit, sheep basic fibroblast growth factor (FGF) assay kit, sheep nerve growth factor (NGF) ELISA kit, and lactoferrin (LTF) ELISA kit, respectively.

[0049] Table 4. Effects of secretory factors and lactoferrin content in colostrum from different groups As shown in Table 4, compared with the blank control group, the fermentation group containing *Bifidobacterium breve* HX-BB128 significantly improved the retention rates of IGF-1, FGF, NGF, and lactoferrin in sheep colostrum. Specifically, after fermentation with HX-BB128 alone, the retention rates of IGF-1 reached 92.67%, NGF 96%, FGF 91.71%, and lactoferrin 88.89%. In contrast, the retention rates of the above active ingredients in the combined bacterial culture group without HX-BB128 were significantly lower than those in the single-strain and combined bacterial culture groups containing HX-BB128. These results indicate that *Bifidobacterium breve* HX-BB128 is a key strain for improving the retention rates of active ingredients in sheep colostrum. Its fermentation effectively reduces the degradation of IGF-1, FGF, NGF, and lactoferrin, significantly improving their retention rates and providing an important material basis for the functional activity of fermented sheep colostrum.

[0050] After identification, strain HX-BB128 was found to be a short-lived Bifidobacterium, derived from sheep colostrum samples.

[0051] The 16S sequence of the Bifidobacterium breve HX-BB128 is as follows:

[0052] Example 4 After fermentation with compound bacterial solution 4, the peptide differences between the blank control group (unfermented) and the sheep colostrum fermented with compound bacterial solution 4 were compared.

[0053] S1: Single probiotic activation: Glycerol tubes containing *Streptococcus thermophilus* subsp. *salicylic acid*, *Lactobacillus delbrueckii* subsp. *bulgaricus*, and *Bifidobacterium breve* HX-BB128 were streaked to isolate the bacteria. Single bacteria were picked and inoculated into MRS medium, and incubated at 37°C for 12-16 h. After two generations of activation, viable counts of 1-1.2 × 10⁻⁶ were obtained. 9 CFU / mL probiotic solution; S2: Preparation and sterilization of sheep colostrum substrate: Place the emulsion containing 6wt%-7wt% sheep colostrum powder and 0.8wt%-1.2wt% sucrose (i.e. sheep colostrum fermentation substrate) at a constant temperature of 65℃ for 30 minutes for sterilization. After sterilization, quickly cool to 37℃ for later use. S3: Compound Probiotic Fermentation: Preparation of compound bacterial solution: Take the activated single probiotic solution from step S1 and mix it with other probiotic solutions in a volume ratio to obtain compound probiotic solution 4. Compound probiotic solution 4: Streptococcus salivarius thermophilus subsp.: Lactobacillus delbrueckii subsp. bulgaricus: Bifidobacterium breve HX-BB128 = 1:1:1~2:1:1 (v / v). Mix thoroughly after mixing and set aside.

[0054] Using sterilized sheep colostrum as the fermentation substrate, the following groups were inoculated and fermented.

[0055] 1. Blank control group No probiotic culture was inoculated. Only the sterilized sheep colostrum matrix was taken and placed in a constant temperature environment of 37°C for static culture. The culture time was the same as that of the fermentation group.

[0056] 4. Compound microbial fermentation group Compound bacterial solution 4: Streptococcus salivarius thermophilus subsp.: Lactobacillus delbrueckii subsp. bulgaricus: Bifidobacterium breve HX-BB128 = (1:1:1) ~ (2:1:1) (v / v).

[0057] The compound bacterial solution 4 was inoculated into the sheep colostrum matrix at an inoculation rate of 2% (v / v) and fermented at a constant temperature of 37℃ for 24 hours.

[0058] Peptides were extracted from fermented sheep colostrum and analyzed by LC-MS / MS. A database search was conducted to screen for peptides with an abundance significantly greater than 2 and small molecule peptides with a molecular weight less than 1 kDa, as shown in Table 5.

[0059] Table 5 Peptide Identification Table Notably, new peptides appeared after fermentation. The top 5 most abundant new peptides are shown in Table 6.

[0060] Table 6. Abundant novel peptides in fermented sheep colostrum The active peptides produced by fermentation (such as VRGPFPILV, LTLTDVEK, VLGPVRGPFP, etc.) can stabilize and retain growth factors such as IGF-1, NGF, and FGF, as well as immune active substances such as lactoferrin and immunoglobulins, by forming peptide-protein complexes, scavenging reactive oxygen species (ROS), and inhibiting protease hydrolysis, thus preventing them from being degraded and inactivated during processing and storage.

[0061] Furthermore, several peptides with clear immunomodulatory and cardiovascular protective activities were identified from fermented sheep colostrum. Among them, peptides such as VRGPFPILV, QDKIHP, LTLTDVEK, AIHPRKE / ALHPRKE, and VRGPFPIL can activate macrophages, enhance NK cell activity, promote the secretion of anti-inflammatory factors, or improve humoral immunity, thus exerting protective and anti-inflammatory effects on the intestinal mucosal barrier. ACE assays showed that peptides such as LTLTDVEK, FPKYPVEP, and VLGPVRGPFP can inhibit angiotensin-converting enzyme (ACE) activity, improve vascular endothelial function, or inhibit platelet aggregation. Therefore, peptides such as LTLTDVEK, FPKYPVEP, and VLGPVRGPFP possess potential for cardiovascular protection, including lowering blood pressure and preventing thrombosis. Among these, peptides such as LTLTDVEK and VLGPVRGPFP possess dual immunomodulatory and cardiovascular protective activities, simultaneously achieving the combined effects of secretory factor protection, immune enhancement, and cardiovascular protection, which is the key material basis for the multifunctional effects of fermented sheep colostrum.

[0062] The mass spectra of LTLTDVEK and VLGPVRGPFP are as follows: Figure 1-2 As shown.

[0063] Example 5 This embodiment provides a method for preparing a probiotic agent, the specific steps of which are as follows: Probiotic fermentation culture: After streaking Bifidobacterium breve HX-BB128, single bacteria were picked and placed into liquid MRS, and anaerobic cultured at 37°C. After two rounds of activation, the bacteria were centrifuged to obtain probiotic sludge. The culture medium comprises the following components by weight percentage: yeast peptone 1.5-3.0%, yeast extract 1.5-3.0%, trehalose 2.0-3.0%, sodium acetate 0.3-0.5%, dipotassium hydrogen phosphate 0.1-0.2%, diammonium hydrogen citrate 0.1-0.2%, magnesium sulfate heptahydrate 0.02-0.03%, manganese sulfate monohydrate 0.005-0.010%, calcium chloride 0.005-0.010%, L-cysteine ​​hydrochloride 0.005-0.010%, and distilled water as the balance; Preparation of probiotic freeze-dried powder: The probiotic slurry is mixed evenly with a freeze-drying protectant and freeze-dried at -40°C to -80°C to obtain probiotic freeze-dried powder; The freeze-drying protectant comprises the following components by weight percentage: xylooligosaccharide 10-20%, skim milk powder 10-15%, maltodextrin 2-6%, monosodium glutamate 1.0-2.5%, sodium ascorbate 0.1-0.2%, and water as the balance.

[0064] The probiotics are *Bifidobacterium breve* HX-BB128 or *Bifidobacterium breve* HX-BB128, *Streptococcus salivarius* subsp. *thermophilus*, and *Lactobacillus delbrueckii* subsp. *bulgaricus*, with a bacterial volume ratio of (1~2):1:1 (v / v). The viable count in the bacterial solution of the three bacteria is 1-1.2 × 10⁻⁶. 9 CFU / mL.

[0065] Example 6 This embodiment provides a method for preparing fermented sheep colostrum freeze-dried powder, the specific steps of which are as follows: After fermentation, the sheep colostrum is inactivated at a constant temperature of 65℃ for 30 minutes. After inactivation, impurities in the system are removed. Then, depending on production needs, either vacuum freeze-drying at -40℃ or low-temperature spray drying at 40-50℃ is used for drying until it becomes dry powder. The moisture content of the resulting sheep colostrum powder is controlled to be no more than 3%, thus obtaining fermented sheep colostrum freeze-dried powder.

[0066] Example 7 animal experiments This embodiment verifies the regulatory effect of sheep colostrum obtained by fermentation of Bifidobacterium breve HX-BB128 on the intestinal mucosal immune barrier of mice and its protective effect on the cardiovascular system, and clarifies its functional advantages compared with unfermented sheep colostrum, providing animal experimental evidence for the application of fermented sheep colostrum in the fields of immunity and cardiovascular health.

[0067] Experimental Design Fifty male SPF-grade C57BL / 6J mice, aged 6-8 weeks and weighing 18-22g, were selected. The mice were housed in an SPF-grade environment with a temperature of 25±2℃ and a humidity of 50±5%, with a 12-hour light-dark cycle and free access to food and water. The experiment was conducted after one week of acclimatization.

[0068] Sixty mice that underwent adaptive feeding were randomly divided into six groups of ten mice each using a random number table: blank control group (CK group), probiotic group (Y group), sheep colostrum group (R group), probiotic + sheep colostrum compound group (YR group), fermented sheep colostrum group (FR group), and model control group (M group). Each group received gavage intervention for two consecutive weeks, with a gavage volume of 0.2 mL.

[0069] Model construction: Starting from week 1 of intervention, mice in each group were fed a high-fat diet for 8 consecutive weeks with free access to food and water to construct a high-fat induced intestinal immune disorder and susceptibility to atherosclerosis model.

[0070] The fermented sheep colostrum powder is prepared by fermenting sheep colostrum with Bifidobacterium breve HX-BB128 according to the process described in Example 5 of this invention, and is prepared into a suspension with sterile physiological saline before use.

[0071] Unfermented sheep colostrum powder: Sheep colostrum raw materials from the same batch as fermented sheep colostrum, prepared according to the process described in Example 1, without probiotic fermentation treatment; prepared into a suspension with sterile physiological saline before use.

[0072] Probiotic powder: Bifidobacterium breve HX-BB128 powder, live bacteria count ≥1×10 10 CFU / g, prepared as a bacterial suspension with sterile physiological saline before use.

[0073] Sterile saline: Sterile saline was used as a blank control.

[0074] The relevant experiments were conducted according to the following groups: Blank control group (CK group): fed with normal feed throughout the course of feeding, and administered an equal volume of physiological saline by gavage daily; Model control group (M group): fed a high-fat diet (containing 2% cholesterol, 10% lard, and 88% basal diet, used to construct a model of susceptibility to atherosclerosis and intestinal immune disorder; the basal diet was SPF grade mouse standard diet), and was given an equal volume of physiological saline by gavage daily; Probiotic group (Group Y): fed with high-fat diet, and administered 1 mL / kg of Bifidobacterium breve suspension by gavage daily; Sheep colostrum group (R group): fed with high-fat diet, and administered 1.0 g / kg of sheep colostrum powder suspension by gavage daily; Probiotics + sheep colostrum compound group (YR group): fed with high-fat feed, and administered daily by gavage a compound suspension prepared by uniformly mixing Bifidobacterium breve powder and sheep colostrum powder at a mass ratio of 1:1, with a dosage of 1.0 g / kg (of which Bifidobacterium breve powder and sheep colostrum powder each accounted for 0.5 g / kg). Fermented sheep colostrum group (FR group): fed with high-fat diet, and administered 1.0g / kg of fermented sheep colostrum powder suspension by gavage daily; Indicator Testing 1. Changes in mouse body weight and organ index: Mice were weighed weekly, and data were recorded. Liver, spleen, and thymus were collected from each group of mice, weighed, and their organ indices were calculated. Organ index (mg / g) = organ weight × 1000 / mouse fasting body weight 2. Serum immune factor detection: The levels of secretory immunoglobulin G, TNF-α, IL-1β and IL-10 in serum were detected by ELISA, strictly following the instructions of the kit.

[0075] 3. Relative expression of cytokine mRNA in mouse colon tissue: The relative expression levels of TNF-α and IL-10 and the relative expression levels of tight junction proteins (ZO-1 and Occludin) mRNA in mouse colon tissue were detected by RT-PCR.

[0076] 4. Detection of atherosclerotic plaques: Aortic root tissue fixed with 4% paraformaldehyde was taken, dehydrated, cleared, embedded in paraffin, serially sectioned, stained with Oil Red O, and the plaque formation was observed under an optical microscope. ImageJ software was used to calculate the percentage of plaque area to the total lumen area of ​​the blood vessel. 5. Blood lipid index detection: The levels of total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and oxidized low-density lipoprotein (ox-LDL) in serum were detected using a fully automated biochemical analyzer and reagent kits; 6. The mRNA expression level of the CD68 gene in the aortic root tissue was detected by real-time quantitative PCR.

[0077] Results Analysis 1. Changes in mouse body weight and organ index The establishment of a mouse model of intestinal immune dysregulation and susceptibility to atherosclerosis was evaluated based on changes in mouse body weight and organ indices. It was observed that the body weight of the model group mice showed a decreasing trend, while from day 7 onwards, the body weight loss in the experimental group mice improved, showing an increasing trend. Figure 3 ).

[0078] Table 7. Effects of different raw materials on organ indices in mice. Table 7 shows that the spleen and thymus are important immune organs in mice. After induction with a high-fat diet, the spleen and thymus indices of mice decreased significantly. After intervention with probiotics and sheep colostrum, the spleen and thymus indices increased significantly compared with the model group.

[0079] 2. Changes in immune factors Table 8. Effects of different raw materials on changes in serum immune factors. IgG is a key substance in humoral immunity and a crucial component of mucosal immunity. TNF-α and IL-1β are cytokines that play crucial roles in the immune system, regulating immunity and other aspects. Compared with the CK group, the M group showed significantly decreased levels of IgG, TNF-α, and IL-1β, and significantly increased levels of IL-10, indicating the successful establishment of the experimental model and the establishment of an immune dysregulation state. The abnormally elevated IL-10 may be related to an imbalance between pro-inflammatory and anti-inflammatory factors. As shown in Table 8, the probiotic-colostrum compound composition and fermented colostrum described in this invention can significantly improve the decrease in IgG, TNF-α, and IL-1β and the abnormal increase in IL-10 in the immune dysregulation model, effectively restoring the body's immune balance. Among them, the fermented colostrum group showed the best effect in terms of IgG recovery and IL-10 correction.

[0080] 3. The relative expression of cytokine mRNA in mouse colon tissue was determined by... Figure 4 As can be seen, compared with the blank control group (CK group), the mRNA expression levels of pro-inflammatory factors TNF-α and IL-1β in the M group were significantly increased, indicating that the model group was in a state of obvious inflammatory activation. After intervention with different raw materials, the mRNA expression of TNF-α and IL-1β in the colon of mice in each group was significantly downregulated compared with the M group, with the downregulation in the YR group and FR group being more significant. Overall, they were closer to the normal expression levels of the CK group, indicating that compound intervention and fermentation process optimization can more effectively inhibit the transcriptional activation of inflammatory factors, thereby improving the body's inflammatory imbalance.

[0081] Depend on Figure 5 As can be seen, compared with the CK group, the mRNA expression levels of tight junction proteins ZO-1 and Occludin in the M group were significantly reduced, indicating that the intestinal barrier function of the model group was severely impaired. After intervention with different raw materials, the mRNA expression of ZO-1 and Occludin in each group was significantly restored compared with the M group, with the restoration of the YR and FR groups being more significant. Overall, they were closer to the normal expression levels of the CK group, indicating that compound intervention and fermentation process optimization can more effectively restore the structure and function of intestinal tight junctions, thereby improving the integrity of the intestinal barrier and providing important support for the body's immune homeostasis and metabolic health.

[0082] 4. Percentage of aortic plaque area and serum lipid levels Table 9. Effects of each group of raw materials on the proportion of aortic plaque area and serum lipid levels in mice. As shown in Table 9, compared with the model group, the probiotic and sheep colostrum compound composition and fermented sheep colostrum described in this invention can significantly reduce the plaque area. In particular, the fermented sheep colostrum group (FR group) can significantly reduce its plaque area.

[0083] The high-fat diet-induced model control group showed significant dyslipidemia and a sharp increase in ox-LDL, a core pathogenic factor of atherosclerosis. The probiotic group, sheep colostrum group, probiotic + sheep colostrum combination group, and fermented sheep colostrum group all effectively improved dyslipidemia. Among them, the probiotic + sheep colostrum combination group and the fermented sheep colostrum group showed more significant effects in reducing TC, LDL-C, and ox-LDL levels, significantly better than the single-ingredient intervention groups. This indicates that through synergistic effects or optimized fermentation processes, these two methods can more effectively regulate cholesterol metabolism and reduce lipid peroxidation damage, demonstrating greater application potential in preventing and improving high-fat diet-induced atherosclerosis, improving cardiovascular and cerebrovascular problems, and enhancing immunity.

[0084] CD68 is a specific marker of macrophages. In atherosclerotic plaques, macrophage infiltration and activation are key steps in disease progression. The greater the number of macrophages within the plaque, the higher the expression level of CD68, indicating a more severe inflammatory response and a more unstable plaque.

[0085] like Figure 6 It was found that, compared with the CK group, the mRNA expression level of CD68 gene in the aortic root tissue of the M group was significantly increased, suggesting that there was significant local macrophage infiltration and inflammatory activation in the high-fat diet-induced atherosclerosis model. After intervention in different groups, the expression of CD68 gene was significantly downregulated compared with the M group, with the downregulation effect being more significant in the YR and FR groups. This indicates that the intervention method described in this invention can effectively reduce local macrophage infiltration in the aorta and alleviate the local inflammatory microenvironment of plaques. Combined with the multidimensional evidence of reduced blood lipids (TC, LDL-C, ox-LDL) to reduce lipid deposition, downregulation of pro-inflammatory factors (TNF-α, IL-1β) to alleviate systemic inflammation, and recovery of tight junction proteins (ZO-1, Occludin) to repair the intestinal barrier and reduce endotoxin entry into the blood, these factors together constitute a complete intervention evidence chain of lipid metabolism regulation - systemic inflammation suppression - local anti-inflammatory repair - intestinal barrier homeostasis. This confirms that this invention can synergistically improve immune and atherosclerosis-related pathologies from multiple levels and has significant application value.

[0086] The above description is merely a specific embodiment of this application and disclosure, enabling those skilled in the art to understand or implement this application and disclosure. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of this application and disclosure. Therefore, this application and disclosure are not to be limited to the embodiments shown herein, but are to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A type of Bifidobacterium breve HX-BB128, characterized in that: The Latin name of the aforementioned Bifidobacterium breve is Bifidobacterium breve, and its accession number is CGMCC NO.37265.

2. A fermentation agent, characterized in that: Including the Bifidobacterium breve HX-BB128 as described in claim 1; or Including *Bifidobacterium breve* HX-BB128, *Streptococcus salivarius* subsp. *thermophilus*, and *Lactobacillus delbrueckii* subsp. *bulgaricus* as described in claim 1; The volume ratio of Bifidobacterium breve HX-BB128 bacterial suspension, Streptococcus salivarius thermophilus bacterial suspension, and Lactobacillus delbrueckii bulgaricus bacterial suspension was (1~2):1:1; The viable count of the *Bifidobacterium breve* HX-BB128 bacterial suspension was 1-1.2 × 10⁻⁶. 9 CFU / mL; The viable count of *Streptococcus thermophilus* subsp. *salivarius* in the bacterial suspension was 1-1.2 × 10⁻⁶. 9 CFU / mL; The viable count of *Lactobacillus delbrueckii* subsp. bulgaricus in the culture was 1-1.2 × 10⁻⁶. 9 CFU / mL.

3. A method for preparing the fermenting agent according to claim 2, characterized in that: Includes the following steps: Strawberry Bifida HX-BB128, or streaked with Streptococcus salivarius thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, and single bacteria were picked and placed into liquid MRS medium. After anaerobic culture at 37°C and two rounds of activation, the bacteria were centrifuged to obtain probiotic sludge. The probiotic slurry is mixed evenly with a freeze-drying protectant and then freeze-dried to obtain the fermentation agent.

4. The method for preparing the fermenting agent as described in claim 3, characterized in that: The MRS culture medium comprises the following components by weight percentage: yeast peptone 1.5-3.0%, yeast extract 1.5-3.0%, trehalose 2.0-3.0%, sodium acetate 0.3-0.5%, dipotassium hydrogen phosphate 0.1-0.2%, diammonium hydrogen citrate 0.1-0.2%, magnesium sulfate heptahydrate 0.02-0.03%, manganese sulfate monohydrate 0.005-0.010%, calcium chloride 0.005-0.010%, L-cysteine ​​hydrochloride 0.005-0.010%, and water as balance; The freeze-drying protectant comprises the following components by weight percentage: xylooligosaccharide 10-20%, skim milk powder 10-15%, maltodextrin 2-6%, monosodium glutamate 1.0-2.5%, sodium ascorbate 0.1-0.2%, and water as the balance.

5. A fermented sheep colostrum, characterized in that: The fermented sheep colostrum is obtained by fermenting sheep colostrum powder with the fermenting agent described in claim 2.

6. The fermented sheep colostrum as described in claim 5, characterized in that: The preparation method of the sheep colostrum powder includes the following steps: inactivating sheep colostrum at 65°C for 30 minutes, centrifuging or filtering to remove impurities, and then vacuum freeze-drying at -40°C or low-temperature spray drying at 40-50°C until the moisture content is no more than 3%.

7. A method for preparing fermented sheep colostrum according to claim 5, characterized in that: Includes the following steps: The strain was inoculated into MRS medium and incubated at 37℃ for 12-16 hours, followed by two generations of activation to obtain a viable count of 1-1.2 × 10⁻⁶. 9 CFU / mL activated bacterial solution; The emulsion containing 6wt%-7wt% sheep colostrum powder and 0.8wt%-1.2wt% sucrose was sterilized at 65℃ for 30-40 minutes and then cooled to 37℃ to obtain a sterile fermentation substrate. The activated bacterial solution was inoculated into the fermentation substrate at an inoculation rate of 2% (v / v), and fermented at 37°C for 12-24 hours until the fermentation substrate curdled, thus obtaining fermented sheep colostrum.

8. A polypeptide, characterized in that: Obtained from the fermented sheep colostrum described in claim 5; The polypeptide sequence is VLGPVRGPFP.

9. A polypeptide composition, characterized in that: The composition comprises a polypeptide with the sequence LTLTDVEK and the polypeptide of claim 8.

10. The use of the *Bifidobacterium breve* HX-BB128 according to claim 1 in the preparation of health products or pharmaceuticals that help enhance immunity; or The use of the polypeptide of claim 8 in the preparation of health products or pharmaceuticals that help enhance immunity; or The use of the fermented sheep colostrum of claim 5 in the preparation of health products or pharmaceuticals that help enhance immunity.

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