Sheep colostrum composition with sleep improving function and application thereof

Abstract

The invention discloses a sheep colostrum composition capable of improving sleep and application of the sheep colostrum composition, and belongs to the technical field of microbial fermentation and functional food. The core of the invention lies in that a composite leavening agent is compounded by using the casei paracasei GP66 and the streptococcus salivarius subsp. Thermophilus HX-ST36, and then the sheep colostrum of concentrated whey protein, galactooligosaccharide and acerola cherry powder is subjected to synergistic fermentation. According to the fermentation process, IgG in sheep colostrum can be efficiently reserved, and the content of small molecule peptide and gamma-aminobutyric acid in a fermentation system is remarkably increased. Meanwhile, the invention also generates a novel pentapeptide, the pentapeptide has a sleep aiding effect, and the amino acid sequence of the pentapeptide is Arg-Phe-Leu-Trp-Pro (RFLWP). Animal experiments prove that the traditional Chinese medicine composition can play a role in improving sleep by regulating a neurotransmitter system. The invention provides a new way of a product for improving sleep, which is natural, safe and clear in mechanism.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation and sheep colostrum product development. It describes the preparation of a fermented sheep colostrum composition by fermenting fresh sheep colostrum using a compound bacterial strain fermenting agent. The core of this invention lies in using a compound fermenting agent of *Lactobacillus paracasei* GP66 and *Streptococcus salivarius* subsp. *thermophilus* HX-ST36 to ferment low-temperature sterilized fresh sheep colostrum, followed by freeze-drying to obtain a sheep colostrum composition that improves sleep. Background Technology

[0002] Sleep is a fundamental physiological process for human survival, crucial for maintaining cognitive function, emotional stability, metabolic balance, and immune defense. However, with the accelerated pace and increased stress of modern society, sleep disorders have become a global public health problem. Epidemiological surveys show that more than one-third of adults suffer from insomnia, manifesting in various forms such as difficulty falling asleep, sleep maintenance disorders, early awakening, and feeling unrefreshed after waking. The causes of insomnia are complex and diverse. Modern medicine considers it the result of the interaction of biological, psychological, and social factors, including anxiety and depression caused by long-term mental stress, irregular sleep patterns and lighting environments, as well as the effects of chronic diseases and medications. From the perspective of traditional Chinese medicine, insomnia is mostly classified under the category of "insomnia," with its core pathogenesis often related to "liver qi stagnation, deficiency of both heart and spleen, and yin deficiency with fire excess." This leads to yang qi failing to enter yin, resulting in an imbalance of yin and yang. The pathological product "blood stasis" further hinders the harmony of qi and blood, exacerbating restlessness.

[0003] Currently, interventions for insomnia mainly fall into two categories: drug therapy and probiotic therapy. Regarding drug therapy, while chemical sedatives can take effect quickly, long-term use requires attention to their inhibitory effects on the central nervous system and the potential risk of dependence. Traditional Chinese medicine (TCM) treatment, based on syndrome differentiation, uses a combination of multiple herbs, demonstrating the advantage of holistic regulation; however, its material basis and molecular mechanisms need further clarification, and the quality and uniformity control of compound preparations remains a challenge. With the rise of the "gut-brain axis" theory, non-pharmacological interventions have gained attention. For example, supplementing with specific probiotics to regulate the gut microbiota and indirectly promote the intestinal synthesis of neuroactive substances such as gamma-aminobutyric acid (GABA) provides a new approach to improving sleep. However, this pathway is relatively indirect, has a relatively long onset period, and its effectiveness is easily affected by the individual's baseline gut microbiota. Furthermore, while melatonin supplements are suitable for regulating sleep rhythms, their effectiveness may be limited for insomnia caused by non-rhythmic factors.

[0004] Sheep colostrum contains immunoglobulins and various growth factors, and it has functions such as regulating immunity and repairing the intestines. However, although traditional physical processing methods (such as low-temperature spray drying and freeze drying) can preserve nutrients well, they may still cause varying degrees of inactivation or functional loss for some heat-sensitive or structurally complex bioactive substances. This limits the exploration and efficient utilization of the deeper functional potential of sheep colostrum.

[0005] The invention patent with publication number CN 115590889 A, entitled "A Bifidobacterium breve for regulating biological circadian rhythms and its application", describes how a specific probiotic strain can directly regulate the expression of circadian rhythm genes in the host (animal) brain and increase serotonin and melatonin levels, while improving gut microbiota and related metabolites (such as GABA), thereby improving cognitive and memory deficits caused by sleep deprivation.

[0006] In the invention patent with publication number CN117502513A, entitled "A Sleep-Aid Fermented Milk and Its Preparation Method", the off-flavor of Chinese medicinal materials (sour jujube seed and poria cocos) is removed by ultrasonic treatment, and then added to the raw milk along with exogenous functional ingredients (tea theanine), and then fermented to produce a pleasant-tasting sleep-aid fermented milk.

[0007] The invention patent CN118652798B, entitled "A Sleep-Aiding Probiotic Goat Milk Powder and Its Preparation Method", describes a method of directly fermenting goat milk with Lactobacillus myxitis (KD6) to naturally produce the known sleep-aiding neurotransmitter GABA without the need for additional precursor substances, ultimately producing a functional goat milk powder that is rich in both GABA and probiotics.

[0008] Therefore, there is an urgent need in this field for an innovative solution that can integrate the natural nutrition and precise sleep-aiding function of sheep colostrum, while overcoming the limitations of exogenous added ingredients. Applying specific probiotic fermentation technology to sheep colostrum rich in active proteins holds promise for the targeted release of novel peptides with direct neuromodulation functions, producing entirely new therapeutic effects.

[0009] This invention aims to ferment sheep colostrum using specific probiotics. Through fermentation and hydrolysis, peptides of varying sizes and sequences are produced, transforming the active ingredient from "exogenous addition" to "endogenous creation." The goal is to develop a fermented sheep colostrum product that improves sleep. Summary of the Invention

[0010] The first objective of this invention is to provide a polypeptide that improves sleep.

[0011] A second objective of this invention is to provide a method for preparing the aforementioned polypeptide.

[0012] A third objective of this invention is to provide a sheep colostrum composition, which is obtained by fermenting sheep colostrum with Lactobacillus paracasei GP66 and Streptococcus salivarius subsp. thermophilus HX-ST36, concentrated lactoglobulin, galactooligosaccharides, and acerola cherry powder.

[0013] The fourth objective of this invention is to provide a method for preparing a sheep colostrum composition.

[0014] A fifth object of the present invention is to provide an application of the said polypeptide.

[0015] A sixth object of the present invention is to provide an application of the said colostrum composition.

[0016] A novel pentapeptide with sleep-improving activity, produced by the co-fermentation of the compound fermentation agent and specific raw materials, has the amino acid sequence Arg-Phe-Leu-Trp-Pro (RFLWP).

[0017] A fourth objective of this invention is to provide a sheep colostrum composition product containing the aforementioned pentapeptide RFLWP and having a sleep-promoting function.

[0018] The fifth object of the present invention is to provide a method for preparing the aforementioned sheep colostrum composition.

[0019] A sixth object of the present invention is to provide the use of the said colostrum composition or the said pentapeptide RFLWP in the preparation of a sleep-improving functional food.

[0020] This invention is achieved through the following technical solution: A polypeptide having the sequence Arg-Phe-Leu-Trp-Pro.

[0021] The method for preparing the polypeptide includes the following steps: By weight percentage, 2% concentrated whey protein, 0.6-1.2% galactooligosaccharides, and 0.3-0.7% acerola cherry powder are added to sheep colostrum. After sterilization, a compound fermentation agent including Lactobacillus paracasei GP66 and Streptococcus salivarius subsp. thermophilus HX-ST36 is inoculated, fermented, inactivated, and freeze-dried to obtain sheep colostrum composition. After reconstituted and centrifuged, the supernatant was ultrafiltered to retain small peptides with a molecular weight of less than 10 kDa. The peptides were then separated by cation exchange chromatography and reversed-phase high-performance liquid chromatography to obtain the final product.

[0022] The sterilization temperature is 60℃ and the time is 30 minutes; The fermentation temperature was 37°C, and the time was 24 hours. The inactivation temperature is 60℃ and the time is 30 minutes; The freeze-drying temperature is -60 to -80℃.

[0023] The eluent used in the cation exchange chromatography had a pH of 6.0. The elution buffer used in the cation exchange chromatography was 0-1.0M NaCl.

[0024] The reversed-phase high-performance liquid chromatography used a C18 column; The eluent used in the reversed-phase high-performance liquid chromatography was 0.1% trifluoroacetic acid-acetonitrile water eluent. The elution conditions used in the reversed-phase high-performance liquid chromatography were 10%B (0 min-5 min), 25%B (5-25 min), 25%-40%B (25-30 min), 40%B (30-40 min), 40-80%B (40-45 min), 80%B (45-50 min), and 80-10%B (50-60 min).

[0025] A sheep colostrum composition comprising the aforementioned polypeptide is obtained from sheep colostrum containing, by weight percentage, 2% whey protein concentrate, 0.6-1.2% galactooligosaccharides, and 0.3-0.7% acerola cherry powder, fermented with Lactobacillus paracasei GP66 and Streptococcus salivarius subsp. thermophilus HX-ST36.

[0026] The preparation method of the aforementioned sheep colostrum composition includes the following steps: S1 involves activating and culturing Lactobacillus paracasei GP66 and Streptococcus salivarius subsp. thermophilus HX-ST36 to prepare a compound fermentation agent; S2 adds 2% concentrated whey protein, 0.6-1.2% galactooligosaccharides, and 0.3-0.7% acerola cherry powder to sheep colostrum, mixes it, sterilizes it at 60℃ for 30 minutes, and then cools it.

[0027] S3 is inoculated with the compound fermentation agent, fermented at 37°C for 24 hours, inactivated at 60°C for 30 minutes, freeze-dried at -60 to 80°C and pulverized to obtain the final product.

[0028] In S1, the ratio of viable bacteria of Lactobacillus paracasei GP66 and Streptococcus salivarius subsp. thermophilus HX-ST36 is 1:1 to 1:2.

[0029] The aforementioned polypeptides are used in the preparation of functional foods, health products, or drugs that improve sleep; or in the preparation of functional foods, health products, or drugs that relieve anxiety and / or regulate neurotransmitters.

[0030] The aforementioned sheep colostrum composition is used in the preparation of functional foods, health products, or drugs that prolong sleep time; or in the preparation of functional foods, health products, or drugs that increase the levels of γ-aminobutyric acid and 5-hydroxytryptamine in the hypothalamus.

[0031] Compared with the prior art, the present invention has the following outstanding advantages: This invention provides a novel pentapeptide RFLWP obtained from fermented sheep colostrum, which has sleep-improving activity. Its amino acid sequence is Arg-Phe-Leu-Trp-Pro.

[0032] This invention provides a method for preparing a sheep colostrum composition, which uses a composite fermentation agent composed of *Lactobacillus paracasei* GP66 and *Streptococcus thermophilus* subsp. *salivarius* HX-ST36, and optimizes the proportion of raw materials (sheep colostrum, whey protein concentrate, galactooligosaccharides, and acerola cherry powder) for synergistic fermentation. This method not only highly hydrolyzes proteins to release target peptides but also enriches GABA and maximizes the preservation of the inherent immune activity of sheep colostrum.

[0033] The fermented sheep colostrum composition and its pentapeptide RFLWP provided by this invention have been shown in animal experiments to significantly prolong sleep time in sleep-deprived mice. Its mechanism of action is clear: it effectively increases the levels of key inhibitory neurotransmitters GABA and 5-HT in the hypothalamus, indicating that it improves sleep function by directly regulating the central neurotransmitter system.

[0034] The fermented sheep colostrum composition prepared by this invention not only achieves precise sleep aid function, but also possesses the inherent immune support potential of sheep colostrum and the comprehensive nutritional value of abundant peptides, GABA, etc. produced by fermentation, thus achieving a combination of functionality, nutrition, and naturalness. Detailed Implementation

[0035] Example 1 In vitro screening of strains This embodiment uses *Lactobacillus paracasei* GP66 (patent publication number CN120059998A, published, accession number CGMCC No. 32952) and *Streptococcus thermophilus* subsp. HX-ST36 (patent publication number CN120082475A, published, accession number CGMCC No. 32955) for fermentation.

[0036] The specific screening process is as follows: Nine bacterial strains were selected from the laboratory: *Lactobacillus paracasei* GP66, *Lactobacillus acidophilus* N35-27, *Lactobacillus paracasei* N132, *Streptococcus salivarius* subsp. *thermophilus* N83c15-2, *Lactobacillus delbrueckii* subsp. *bulgaricus* N101M3, *Streptococcus salivarius* subsp. *thermophilus* N108M2, *Streptococcus salivarius* subsp. *thermophilus* N108M4, *Lactococcus faecium* N110M5, and *Streptococcus salivarius* subsp. *thermophilus* HX-ST36. Cryopreserved glycerol tubes were streaked onto MRS plates and incubated at 37°C for 24-48 hours. After single colonies grew on the plates, the culture was passaged three times and then aseptically transferred to broth MRS medium and sheep colostrum liquid medium, respectively, and incubated at 37°C for 24-48 hours.

[0037] After cultivation, the viable cell counts of each strain in the two culture media were determined (pour plate count method), as well as the pH value, acidity (°T, Girner degree, sodium hydroxide titration method), and γ-aminobutyric acid (GABA, high performance liquid chromatography) content after fermentation in sheep colostrum liquid medium. The results are shown in Table 1. Strains GP66 and HX-ST36 showed the best growth adaptability in sheep colostrum medium and had strong acid production capacity. In addition, GP66 also had a high GABA production capacity. Therefore, GP66 and HX-ST36 were selected as the core strains for the starter culture.

[0038] Table 1. Strain screening

[0039] Example 2 Combination of fermentation raw material composition To screen for raw materials that synergistically enhance sheep colostrum fermentation, 2% (w / v) of the following raw materials were added to sheep colostrum liquid culture medium: whey protein isolate, whey protein concentrate, galactooligosaccharides, isomaltooligosaccharides, inulin, acerola cherry powder, wolfberry powder, and carrot powder. The medium was then sterilized at 60°C for 30 min. GP66 and HX-ST36 were inoculated into the culture medium containing the raw materials. A blank control was set up with an uninoculated culture medium containing the raw materials. The OD difference (ΔOD600nm) after 24 hours of culture was calculated using the blank control as a base to compare the synergistic growth effect of different raw materials with GP66 and HX-ST36. The results are shown in Table 2.

[0040] Table 2. Screening of raw materials for synergistic effect

[0041] Based on the data analysis shown in Table 2, the following screening conclusions can be drawn: concentrated whey protein, galactooligosaccharides, and acerola cherry powder all exhibited the best synergistic growth-promoting effects on both GP66 and HX-ST36 strains, indicating that these three raw materials can most effectively provide nutrients or growth stimulation for the proliferation of the strains. In contrast, wolfberry powder and carrot powder had relatively weaker growth-promoting effects on the two strains. Therefore, concentrated whey protein, galactooligosaccharides, and acerola cherry powder were selected as synergistic enhancing ingredients for subsequent verification of the fermentation process of the compound formulation.

[0042] Example 3 Preparation of the composition To further enhance the synergistic effect of the composition, this example uses GP66 and HX-ST36 to ferment a culture medium containing sheep colostrum, whey protein concentrate, galactooligosaccharides, and acerola cherry powder, comparing the changes in composition before and after fermentation. The specific steps are as follows: 3.1 Evaluation of Synergistic Effect of Compositions S1: Preparation of Compound Fermentation Agent After streaking glycerol tubes containing GP66 and HX-ST36 separately, single bacteria were picked and cultured in MRS liquid medium. Then, 2% bacterial suspension was inoculated into MRS liquid medium and fermented at 37°C and pH 6.8 for 24 hours. After fermentation, the cultures were centrifuged at 6000 r / min for 10 min, the supernatant was discarded, and the following freeze-drying protectants were added to the precipitate: 10% skim milk powder, 5% trehalose, 2% glycerol, 1% ascorbic acid, and the remainder water. These were mixed thoroughly in a 1:2 ratio and then freeze-dried and pulverized at -80°C to obtain GP66 and HX-ST36 bacterial powders. GP66 and HX-ST36 were then mixed thoroughly at a viable cell ratio of 1:1-2.

[0043] S2: Screening for the optimal starter culture To screen the optimal compound fermentation agent and the synergistic ratio of raw materials, a multi-factor design was adopted: Based on fresh sheep colostrum with 2% concentrated whey protein, three addition levels of galactooligosaccharides (0.6%, 0.9%, 1.2%) and acerola cherry powder (0.3%, 0.5%, 0.7%) were set, resulting in nine experimental groups. Simultaneously, a fermentation group C1 containing only 2% concentrated whey protein and sheep colostrum, and a pure sheep colostrum fermentation group C2 were established as controls. After culturing all groups with the compound fermentation agent (3.5-5 × 10¹¹ CFU / L) for 24 hours, the synergistic promoting effect of different raw material combinations on cell growth was evaluated by measuring ΔOD600nm (with group C1 as the control), and pH was measured to reflect acid production. The optimal ratio was screened, and the results are shown in Table 3.

[0044] Table 3. Effects of different ratios on the growth of the compound fermentation agent.

[0045] Based on the data analysis in Table 3, group 5 (0.9% galactooligosaccharides and 0.5% acerola cherry powder) showed the best synergistic promoting effect, with the highest ΔOD600nm and the lowest endpoint pH, indicating the most vigorous cell growth and acid production metabolism. This ratio was significantly better than the control group (C1 group) which only added whey protein. Therefore, the optimal synergistic combination was determined to be 2% whey protein concentrate, 0.9% galactooligosaccharides, and 0.5% acerola cherry powder for subsequent fermentation verification.

[0046] 3.2 Preparation of the composition Composition 1: Fresh sheep colostrum is mixed with 2% concentrated whey protein, 0.9% galactooligosaccharides, and 0.5% acerola cherry powder, sterilized at 60℃ for 30 minutes, freeze-dried at -60 to 80℃ and pulverized to obtain the final product.

[0047] Composition 2: The optimal synergistic combination of inoculated compound fermentation agent (3.5-5 × 10¹¹ CFU / L) was fermented at 37℃ for 24 hours. After fermentation, the inactivation was carried out at 60℃ for 30 min, followed by freeze-drying and pulverization at -60 to 80℃ to obtain Composition 2. Composition 1 or 2 was added to physiological saline at a weight (g) to volume (mL) ratio of 1:9, and its viable cell count, pH and acidity, protein content (Kjeldahl method), IgG retention rate (ELISA), soluble peptide content (o-phthalaldehyde OPO colorimetric method), and γ-aminobutyric acid (GABA) were evaluated.

[0048] The results, as shown in Table 4, demonstrate that the composition achieved highly efficient multiple synergistic transformations after fermentation with a specific compound fermenting agent: probiotics proliferated significantly and produced acid, effectively hydrolyzing proteins into small peptides and fully preserving the core immune-active components of sheep colostrum. The fermentation process also resulted in high GABA production, further enhancing the product's sleep-aiding potential. This fermentation process simultaneously achieves three core objectives: efficient peptide production, preservation of natural immune activity, and GABA enrichment, providing a solid material foundation for the composition's sleep-aiding efficacy.

[0049] Table 4. Composition and Changes After Fermentation

[0050] 3.3 Liquid chromatography-mass spectrometry (LC-MS) analysis of peptides In the analysis of peptide profiles in fermented sheep colostrum by liquid chromatography-mass spectrometry (LC-MS / MS), a small molecule pentapeptide with potential biological activity (RFLWP) was identified, with the amino acid sequence Arg-Phe-Leu-Trp-Pro.

[0051] Example 4 Extraction, identification and in vitro functional verification of polypeptides (RFLWP) 4.1 Extraction and purification Raw materials: Fermented sheep colostrum powder (composition 2) prepared in Example 3.2 or supernatant of fermentation broth.

[0052] Preliminary separation: After reconstitution / centrifugation of the sample, the supernatant is ultrafiltered (molecular weight cutoff <10kDa) to collect the fraction rich in small peptides.

[0053] Chromatographic purification: Cation exchange chromatography was used, and elution was performed with buffer solutions of pH 6.0 and 0-1.0M NaCl gradient. The fraction containing the target peptide was collected based on the UV absorption peak.

[0054] Further analysis was performed using reversed-phase high-performance liquid chromatography (RP-HPLC) with a C18 column and gradient elution in a system containing 0.1% trifluoroacetic acid (mobile phase A) and acetonitrile (mobile phase B). The flow rate was 10 mL / min, and the injection volume was 5 mL. The elution conditions (concentration of mobile phase B) were: 10% (0 min-5 min), 25% (5-25 min), 25%-40% (25-30 min), 40% (30-40 min), 40-80% (40-45 min), 80% (45-50 min), and 80-10% (50-60 min). A single peak with the retention time consistent with the RFLWP standard was collected. The substance was obtained by vacuum freeze-drying for subsequent identification and functional verification.

[0055] 4.2 Molecular identification The purified substance was reconstituted with 0.1% formic acid aqueous solution, separated by a C18 capillary column, and analyzed by mass spectrometry, acquiring secondary fragment spectra of the parent ion. The obtained mass spectrometry data were searched in the UniProt database, limiting the species to sheep. The results showed a highly consistent peptide segment in the protein reference sequence of this species. This pentapeptide (RFLWP) is located in the amino acid range of sheep lactoferrin (UniProt accession number: D3G9G3) from amino acid positions 70 to 160.

[0056] The structure of RFLWP is shown below. It is composed of arginine, phenylalanine, leucine, tryptophan, and proline, with the molecular formula C37H51N9O6 and a molecular weight of 717.87. The specific m / z values ​​are as follows: 717.40 (100.0%), 718.40 (40.8%), 719.40 (10.5%), 718.39 (3.3%), and 720.41 (1.1%).

[0057]

[0058] Example 5 Study on the ameliorative effect on sleep deprivation mice 5.1 Laboratory Animals and Grouping Sixty 8-week-old male C57BL / 6J mice were acclimatized for one week and then randomly divided into 6 groups (n=10). Normal control group: No model was established, and the same volume of sterile saline was administered by gavage daily.

[0059] Model control group: Parachlorophenylalanine (PCPA) model was established, and the same volume of sterile saline was administered by gavage daily.

[0060] Positive control group: PCPA modeling, daily gavage administration of 100 mg / kg BW of γ-aminobutyric acid solution.

[0061] Experimental group 1 / 2: PCPA modeling, daily gavage administration of fermented sheep colostrum powder containing RFLWP pentapeptide, with two doses (e.g., 50 mg / kg BW and 100 mg / kg BW).

[0062] Experimental group 3: PCPA modeling, daily gavage administration of 150 mg / kg BW Example 3.2 composition 1.

[0063] 5.2 Establishment and Intervention of Sleep Deprivation Model Except for the normal control group, the other three groups received intraperitoneal injections of p-chlorophenylalanine (PCPA) solution for two consecutive days to establish an insomnia model. After successful model establishment, each group began gavage intervention according to the above design for two weeks.

[0064] 5.3 Detection Indicators and Results To evaluate the direct sleep-promoting effect of the test substance, mice in each group were intraperitoneally injected with sodium pentobarbital at a dose of 50 mg / kg BW after the intervention period. The sleep duration was recorded from the time of injection until the righting reflex disappeared (defined as the mouse being unable to return to its right position spontaneously for more than 60 seconds).

[0065] Table 5. Effects of different interventions on sleep duration in sleep-deprived mice

[0066] As shown in Table 5, the sleep-aiding effect of the peptide-rich fermentation product (experimental group 2) was comparable to that of the positive control GABA, while the sleep-aiding effect of the unfermented, identical raw material composition (experimental group 3) was significantly weaker. This result directly confirms that fermentation is an essential step in activating the activity of raw materials and promoting the generation of key sleep-aiding peptides. Secondly, it can be seen from experimental groups 1 and 2 that the sleep improvement achieved by the fermented composition through peptides is dose-dependent.

[0067] After observation, the neurotransmitter content in the hypothalamus was measured. The hypothalamus was isolated, weighed, and stored in liquid nitrogen for later use. Pre-cooled physiological saline was added at a weight (g) to volume (mL) ratio of 1:9, and tissue homogenate was prepared at 4°C. The homogenate was centrifuged at 3000 r / min for 15 min. The hypothalamic supernatant was taken and the contents of 5-HT (5-hydroxytryptamine) and GABA were determined according to the ELISA kit instructions.

[0068] Table 6. Effects of neurotransmitter content in the hypothalamus of mice

[0069] The data in Table 6 show that the fermentation product rich in RFLWP pentapeptide can significantly increase the levels of key inhibitory neurotransmitters GABA and 5-HT in the hypothalamus of sleep-deprived mice in a dose-dependent manner; its increasing effect is comparable to or even better than that of the positive control GABA, and is significantly superior to the unfermented raw material composition of the same material.

Claims

1. A polypeptide, characterized in that: The sequence of the polypeptide is Arg-Phe-Leu-Trp-Pro.

2. The method for preparing the polypeptide according to claim 1, characterized in that: Includes the following steps: By weight percentage, 2% concentrated whey protein, 0.6-1.2% galactooligosaccharides, and 0.3-0.7% acerola cherry powder are added to sheep colostrum. After sterilization, a compound fermentation agent including Lactobacillus paracasei GP66 and Streptococcus salivarius subsp. thermophilus HX-ST36 is inoculated, fermented, inactivated, and freeze-dried to obtain sheep colostrum composition. After reconstituted and centrifuged, the supernatant was ultrafiltered to retain small peptides with a molecular weight of less than 10 kDa. The peptides were then separated by cation exchange chromatography and reversed-phase high-performance liquid chromatography to obtain the final product.

3. The method for preparing the polypeptide as described in claim 2, characterized in that: The sterilization temperature is 60℃ and the time is 30 minutes; The fermentation temperature was 37°C, and the time was 24 hours. The inactivation temperature is 60℃ and the time is 30 minutes; The freeze-drying temperature is -60 to -80℃.

4. The method for preparing the polypeptide according to claim 2, characterized in that: The eluent used in the cation exchange chromatography had a pH of 6.

0. The elution buffer used in the cation exchange chromatography was 0-1.0M NaCl.

5. The method for preparing the polypeptide as described in claim 2, characterized in that: The reversed-phase high-performance liquid chromatography used a C18 column; The eluent used in the reversed-phase high-performance liquid chromatography was 0.1% trifluoroacetic acid-acetonitrile water eluent. The elution conditions used in the reversed-phase high-performance liquid chromatography were 10%B (0 min-5 min), 25%B (5-25 min), 25%-40%B (25-30 min), 40%B (30-40 min), 40-80%B (40-45 min), 80%B (45-50 min), and 80-10%B (50-60 min).

6. A sheep colostrum composition comprising the polypeptide of claim 1, characterized in that: It includes sheep colostrum, whey protein concentrate, galactooligosaccharides, acerola cherry powder, Lactobacillus paracasei GP66, and Streptococcus salivarius thermophilus subsp. HX-ST36.

7. The method for preparing the sheep colostrum composition according to claim 6, characterized in that: Includes the following steps: S1 involves activating and culturing Lactobacillus paracasei GP66 and Streptococcus salivarius subsp. thermophilus HX-ST36 to prepare a compound fermentation agent; S2 adds 2% concentrated whey protein, 0.6-1.2% galactooligosaccharides, and 0.3-0.7% acerola cherry powder to sheep colostrum, mixes it, sterilizes it at 60℃ for 30 minutes, and then cools it. S3 is inoculated with the compound fermentation agent, fermented at 37°C for 24 hours, inactivated at 60°C for 30 minutes, freeze-dried at -60 to 80°C and pulverized to obtain the final product.

8. The method for preparing the sheep colostrum composition according to claim 6, characterized in that: In S1, the ratio of viable bacteria of Lactobacillus paracasei GP66 and Streptococcus salivarius subsp. thermophilus HX-ST36 is 1:1 to 1:

2.

9. The application of the polypeptide as described in claim 1, characterized in that: Applications in the preparation of functional foods, health products, or medicines that improve sleep; or Applications include the preparation of functional foods, health products, or drugs that combat anxiety and / or regulate neurotransmitters.

10. The application of the sheep colostrum composition as described in claim 6, characterized in that: Applications in the preparation of functional foods, health products, or pharmaceuticals that prolong sleep time; or It can be used to prepare functional foods, health products, or drugs that increase the levels of γ-aminobutyric acid and 5-hydroxytryptamine in the hypothalamus.

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