Selenium-rich sleep-aiding probiotic goat milk powder and preparation method thereof

Abstract

The application discloses a selenium-rich sleep-aiding probiotic goat milk powder and a preparation method thereof, and belongs to the field of fermentation engineering. First, the probiotic characteristics of probiotic strains, selenium conversion rate, gamma-aminobutyric acid (GABA) and extracellular polysaccharide (EPS) yield are compared, and suitable strains are screened and prepared into freeze-dried bacterial powder. The influence of single and compound bacterial powder on goat milk fermentation is studied, and it is determined that the compound probiotic composed of Lactobacillus mucus, Lactobacillus rhamnosus and Lactobacillus paracasei is the optimal combination, and the compound probiotic is used for fermenting goat milk. Further, the sleep-aiding probiotic goat milk powder is prepared through spray drying. The results show that the selenium conversion rate of the goat milk prepared through the fermentation of the compound probiotic is 100%, and the generation of GABA and EPS is significantly promoted. The goat milk powder prepared through spray drying is rich in probiotics, organic selenium, GABA and EPS, and lays a foundation for the development of functional probiotic goat milk powder.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of fermentation engineering, in particular to a selenium-rich sleep-aiding probiotic goat milk powder prepared by spray drying after fermentation of goat milk by probiotics and a preparation method thereof. BACKGROUND

[0002] Selenium (Se) is a chemical element similar to sulfur, which was first discovered in 1817 by the Swedish chemist Berzelius in the production of sulfur. Selenium is an essential nutrient for plants and has many significant benefits for the health of mammals. A lack of selenium will reduce the activity of glutathione peroxidase in the human body. The content of selenium in the body is very important. If the content is too low, it will damage the immune system and increase the risk of diseases such as Keshan disease, Kashin-Beck disease, and cancer. However, too much selenium will cause selenium poisoning. The International Food and Agriculture Organization (FAO) has stipulated that the daily intake of selenium, an essential trace element in the human body, should be 30-55 μg. Compared with inorganic selenium, organic selenium is more easily absorbed by the human body and has less toxicity. Organic selenium can be prepared by converting inorganic selenium by microorganisms.

[0003] Sleep disorders are a global epidemic affecting 45% of the world's population, and most of the sleep disorder patients are insomnia patients, which greatly affects the normal life of human beings. One of the pathogenesis of insomnia is that GABA (gamma-aminobutyric acid) occurs in the brain edge system. Lactobacillus brevis L-32 isolated from kimchi by SUNG H H et al. can produce GABA through biological transformation, and it has been proved by mouse tests that it can be used as a new type of sleep nutritional supplement. As a GRAS microorganism, probiotics have the ability to produce GABA. It is necessary to screen high-yield GABA probiotic strains and apply them to the development of GABA functional dairy products.

[0004] Goat milk has a stronger buffering capacity than cow milk, and it provides faster acidification in the early stages of fermentation, which is beneficial for the growth of certain probiotic bacteria. Varga et al. observed the survival of Lactobacillus acidophilus LA-5 and Bifidobacterium BB-12 in fermented goat milk for 6 weeks, which were log 6.53 ± 0.40 and log 6.80 ± 0.16 cfu / mL, respectively. Goat milk has a relatively high concentration of fat globules with small particle size, which may contribute to the improved freeze stability of goat dairy products. Since food proteins can cause allergic reactions in some people, allergic reactions to cow milk cheese proteins and whey proteins can occur. However, some reports suggest that people generally have fewer allergic reactions to goat milk due to the protein diversity in goat milk. Consumer preference for fermented goat dairy products has prompted scientists to gradually explore the therapeutic potential of fermented goat milk. Internationally, goat milk is mainly used for cheese production, and fermented goat milk and other products rich in probiotics.

[0005] Goat milk is a characteristic resource in Shaanxi Province, and the production of goat milk and its products ranks first in China. In the past, the products were mainly infant formula goat milk powder, goat milk powder for processing and large packaging, but the development of functional goat milk powder was rare. Selenium is an essential trace element for the human body, and it is usually supplemented by adding inorganic selenium, sodium selenite, to food. Compared with inorganic selenium, organic selenium is more easily absorbed by the human body and has less toxicity. Therefore, the conversion of inorganic selenium to organic selenium by microorganisms is increasingly attracting attention. The commonly used microorganisms are yeast and lactic acid bacteria. Functional probiotics can generate gamma-aminobutyric acid and extracellular polysaccharide, which have functions such as promoting sleep and antioxidant activity. However, there is no research on the development of fermented goat milk powder containing organic selenium, GABA and EPS by functional probiotic fermentation. SUMMARY

[0006] The purpose of the present application is to provide a selenium-rich sleep-aiding probiotic goat milk powder and a preparation method thereof to solve the problems existing in the prior art. By fermenting goat milk with functional probiotics, the conversion rate of inorganic selenium to organic selenium in goat milk can reach 100%, and gamma-aminobutyric acid and extracellular polysaccharide are also generated. The selenium-rich sleep-aiding probiotic goat milk powder obtained by spray drying has functions such as promoting sleep and antioxidant activity.

[0007] To achieve the above-mentioned purpose, the present application provides the following solutions:

[0008] This invention provides a selenium-enriched probiotic for sleep aid, comprising *Limosilactobacillus fermentum* KD6, *Lactaseibacillus rhamnosus* KD5, and / or *Lactaseibacillus paracasei* KD11. The *Limosilactobacillus fermentum* KD6 has the accession number CCTCC NO: M20231642, with an accession date of September 7, 2023; the *Lactaseibacillus rhamnosus* KD5 has the accession number CCTCC NO: M20231641, with an accession date of September 7, 2023; and the *Lactaseibacillus paracasei* KD11 has the accession number CCTCC NO: M2024605, with an accession date of April 1, 2024.

[0009] The above three strains are all deposited at the China Center for Type Culture Collection, located at Wuhan University, Wuhan, China.

[0010] Preferably, the mass ratio of the fermenting *Lactobacillus mucinus* KD6, the *Lactobacillus rhamnosus* KD5, and the *Lactobacillus paracasei* KD11 is 1:(1-3):(1-3).

[0011] The present invention also provides a selenium-enriched sleep-aiding probiotic goat milk powder, comprising the aforementioned probiotics.

[0012] Preferably, the selenium content is 143 μg / kg to 278 μg / kg, and the live probiotic count is 0.23 × 10⁻⁶. 8 CFU / g ~ 4.08 × 10⁻⁶ 8 CFU / g, γ-aminobutyric acid (GABA) content is 3.472-4.448 g / kg, and extracellular polysaccharide content is 2.640 g / kg to 3.792 g / kg.

[0013] The present invention also provides a method for preparing selenium-enriched sleep-aiding probiotic goat milk powder, including the step of fermenting goat milk using the probiotics.

[0014] Preferably, the preparation method includes the following steps:

[0015] Prepare sterilized goat milk from fresh goat milk or reconstituted goat milk by adding sodium selenite;

[0016] Probiotics were inoculated into sterilized goat milk and fermented at a constant temperature to produce selenium-enriched sleep-aiding probiotic fermented goat milk.

[0017] Selenium-enriched sleep-aiding probiotics are fermented in goat milk and then dried to produce selenium-enriched sleep-aiding probiotic goat milk powder.

[0018] Preferably, the sterilized goat milk is prepared under the following conditions: 90-95℃ for 5-10 minutes, and the selenium salt is sodium selenite.

[0019] Preferably, the amount of sodium selenite added is 17.5 μg / kg to 35 μg / kg.

[0020] Preferably, the fermentation conditions are as follows: inoculum size of 0.005%-0.025%, fermentation temperature of 33-41℃, fermentation time of 17-25h, and the conversion rate of sodium selenite in the prepared selenium-enriched sleep-aiding probiotic fermented goat milk is 100%; before spray drying, the content of γ-aminobutyric acid is 0.33g / L-0.61g / L, the content of extracellular polysaccharides is 0.32g / L-0.47g / L, and the goat milk powder prepared by spray drying is concentrated about 8 times.

[0021] The drying process includes freeze-drying, vacuum low-temperature spray drying, and high-temperature spray drying. More preferably, the vacuum freeze-drying conditions are: pre-freezing selenium-enriched sleep-aiding probiotic fermented goat milk at -20℃ for 4-6 hours, then freeze-drying it in a vacuum freeze dryer for 20-24 hours, with a vacuum degree of 4-6 Pa; the vacuum low-temperature spray drying conditions are: inlet air temperature of 60℃-80℃, peristaltic pump opening of 30%-50%, and vacuum negative pressure of 0.03-0.04 MPa; the high-temperature spray drying conditions are: inlet air temperature of 140-160℃ and outlet air temperature of 75-95℃.

[0022] The present invention also provides the application of the probiotics in the preparation of selenium-enriched sleep-aiding goat dairy products.

[0023] The present invention discloses the following technical effects:

[0024] This invention, through screening and optimization, found that the viable cell count of single-strain fermentation of goat milk using *Lactobacillus mucilaginosus* KD5, *Lactobacillus rhamnosus* KD6, or *Lactobacillus paracasei* KD11 was significantly higher than that of the compound bacterial powder. However, the selenium conversion rate, GABA, and EPS yield of goat milk fermented with the three-strain compound bacterial powder were significantly higher than those of the single-strain powder. Therefore, considering all factors, a KD5+KD6+KD11 bacterial powder in a ratio of 1:1:1 was determined as the starter culture. The inoculum amount (0.005%-0.025%)... With increasing fermentation temperature (33℃-41℃), the water-holding capacity, sensory evaluation, and yields of γ-aminobutyric acid (GABA) and extracellular polysaccharides in fermented goat milk initially increased and then decreased. With prolonged fermentation time (17h-25h), the yields of GABA and extracellular polysaccharides in fermented goat milk increased, while water-holding capacity and sensory evaluation still showed an initial increase followed by a decrease. The prepared goat milk exhibited a significantly increased GABA yield, reaching 0.938 g / L, and an extracellular polysaccharide yield of 400 mg / L. A large amount of GABA and extracellular polysaccharides can be generated through compound probiotics, and inorganic selenium can be converted into organic selenium. GABA and extracellular polysaccharides have functions such as promoting sleep and antioxidation. Organic selenium is more easily absorbed and has lower toxicity than inorganic selenium. Therefore, this invention provides a selenium-enriched, sleep-aiding fermented goat milk and goat milk powder, offering a new direction for the development of the goat dairy industry in Shaanxi Province. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 The effect of sodium selenite on the selenium conversion rate and pH of probiotic-fermented sheep milk; A: conversion rate, B: pH value;

[0027] Figure 2 The effect of probiotic strains on GABA production during sheep milk fermentation;

[0028] Figure 3 The effect of probiotic strains on EPS production from goat milk fermentation;

[0029] Figure 4 The number of live bacteria in single and compound probiotic powders;

[0030] Figure 5 The effects of single and compound probiotic powders on the number of live bacteria in goat milk;

[0031] Figure 6 The effects of single and compound bacterial cultures on the acidity and pH of fermented sheep milk; A: pH value, B: acidity;

[0032] Figure 7 The effects of single and compound probiotic powders on the water-holding capacity of fermented goat milk;

[0033] Figure 8 The effects of single and compound probiotic powders on the selenium conversion rate of fermented sheep milk;

[0034] Figure 9 The effects of single and compound probiotic powders on GABA production in goat milk;

[0035] Figure 10 The effects of single and compound probiotic powders on the yield of goat milk EPS;

[0036] Figure 11 The effect of inoculum size on pH and acidity of fermented goat milk; A: pH value, B: acidity;

[0037] Figure 12 The effect of inoculum size on water-holding capacity, GABA, sensory evaluation and EPS of probiotic fermented goat milk;

[0038] Figure 13 The effect of fermentation temperature on the pH and acidity of fermented goat milk; A: pH value, B: acidity;

[0039] Figure 14 The effect of fermentation temperature on water-holding capacity, GABA, sensory evaluation and EPS of probiotic fermented goat milk;

[0040] Figure 15 The effects of fermentation time on pH, acidity (A), water-holding capacity, GABA, sensory evaluation, and EPS (B) of probiotic-fermented goat milk were investigated. Detailed Implementation

[0041] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0042] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0043] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0044] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0045] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0046] Example 1

[0047] 1. Strains and raw materials

[0048] The six probiotic strains used in this study were: Limosilactobacillus fermentum KD6, Lacticaseibacillus rhamnosus KD5, Lacticaseibacillus paracasei KD11, Limosilactobacillus fermentum 38, Lactiplantibacillus plantarum 7830, and Lacticaseibacillus rhamnosus L20.

[0049] The goat milk powder was purchased from Xi'an Baiyue Goat Milk Group.

[0050] Lacticaseibacillus rhamnosus KD5 was isolated from kefir grains. After repeated isolation on MRS agar medium to obtain pure colonies of a single strain, it was identified by mass spectrometry using a fully automated microbial mass spectrometer (VITEK MS, bioMérieux, France) and by biochemical identification using a fully automated microbial identification system (VITEK 2, bioMérieux, France). The strain KD5 was identified as Lacticaseibacillus rhamnosus by comparison with the Launchpad software of the VITEK-MS Mass Spectrometer Research Library (RUO) and the AES8.01 database.

[0051] Limosilactobacillus fermentum KD6 was isolated from kefir grains. After fermenting sheep milk, the bacteria were serially diluted and streaked on MRS agar plates to obtain lactic acid bacteria. After repeated primary and secondary screening, a strain with strong γ-aminobutyric acid (GABA) production was obtained. The strain was characterized by homology analysis of a partial 16S rDNA gene fragment. The obtained sequence was compared and analyzed with the base sequences in the NCBI database, confirming that the strain was Limosilactobacillus fermentum. The strain was named Limosilactobacillus fermentum KD6.

[0052] Isolation and Identification of *Lactaseibacillus paracasei* KD11: After crushing fermented goat milk containing kefir grains, the milk was serially diluted and streaked onto MRS agar for isolation. Following repeated purification, a single lactic acid bacterium was obtained. The strain was identified using 16S rDNA. After amplification and purification of the target fragment, the strain was characterized based on homology analysis of partial 16S rDNA gene fragments. The obtained sequence was compared and analyzed with base sequences in the NCBI database, confirming the strain as *Lactaseibacillus paracasei*, named *Lactaseibacillus paracasei* KD11. Its base pair sequence is as follows:

[0053] GACGGCTCGCTCCCTAAAAGGGTTACGCCACCGGCTTCGGGTGTTACAAACTCTCA

[0054] TGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCGTGCTGATCC

[0055] GCGATTACTAGCGATTCCGACTTCGTGTAGGCGAGTTGCAGCCTACAGTCCGAACTGAG

[0056] AATGGCTTTAAGAGATTAGCTTGACCTCGCGGTCTCGCAACTCGTTGTACCATCCATTGT

[0057] AGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCT

[0058] CCGGTTTGTCACCGGCAGTCTTACTAGAGTGCCCAACTAAATGCTGGCAACTAGTCATA

[0059] AGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAAC

[0060] CATGCACCACCTGTCATTTTGCCCCCGAAGGGGAAACCTGATCTCTCAGGTGATCAAAA

[0061] GATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGC

[0062] TTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGG

[0063] AATGCTTAATGCGTTAGCTGCGGCACTGAAGGGCGGAAACCCTCCAACACCTAGCATTC

[0064] ATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGCC

[0065] TCAGCGTCAGTTACAGACCAGACAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTAC

[0066] GCATTTCACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTTTCCCAGTTT

[0067] CCGATGCGCTTCCTCGGTTAAGCCGAGGGCTTTCACATCAGACTTAAAAAACCGCCTGC

[0068] GCTCGCTTTACGCCCAATAAATCCGGATAACGCTTGCCACCTACGTATTACCGCGGCTGC

[0069] TGGCACGTAGTTAGCCGTGGCTTTCTGGTTGGATACCGTCACGCCGACAACAGTTACTCT

[0070] GCCGACCATTCTTCTCCAACAACAGAGTTTTACGACCCGAAAGCCTTCTTCACTCACGC

[0071] GGCGTTGCTCCATCAGACTTGCGTCCATTGTGGAAGATTCCCTACTGCTGCCTCCCGTAG

[0072] GAGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATCAACCTCTCAGTTCGGCTACGTAT

[0073] CATCGCCTTGGTGAGCCATTACCTCACCAACTAGCTAATACGCCGCGGGTCCATCCAAAA

[0074] GCGATAGCTTACGCCATCTTTCAGCCAAGAACCATGCGGTTCTTGGATCTATGCGGTATT

[0075] AGCATCTGTTTCCAAATGTTATCCCCACTTAAGGGCAGGTTACCCACGTGTTACTCACC

[0076] CGTCCGCCACTCGTTCCATGTTGAATCTCGGTGCAAGCACCGATCATCAACGAGAACTC

[0077] GTTCGACT.

[0078] The preservation number of *Lactobacillus fermentatus* KD6 is CCTCC NO: M20231642, and the preservation date is September 7, 2023; the preservation number of *Lactobacillus rhamnosus* KD5 is CCTCC NO: M20231641, and the preservation date is September 7, 2023; the preservation number of *Lactobacillus paracasei* KD11 is CCTCC NO: M2024605, and the preservation date is April 1, 2024. The preservation institution for all three strains is the China Center for Type Culture Collection, located at Wuhan University, Wuhan, China.

[0079] 2. Experimental Methods

[0080] 2.1 Strain activation

[0081] Six lyophilized probiotics were inoculated into MRS broth medium and incubated at 37°C for 24 hours to complete one activation. Then, they were inoculated into fresh MRS broth medium at a 5% inoculation rate (v / v) and incubated at 37°C for 24 hours. The above operation was repeated twice to activate three generations for later use.

[0082] 2.2 Screening of selenium-enriched probiotics

[0083] The activated third-generation strains were inoculated at a 5% inoculation rate into goat milk with a gradient of sodium selenite concentrations from 0 to 100 μg / mL. The mixtures were cultured at 37°C for 24 h. The color of the fermented milk was observed, and its pH value and conversion rate were measured. The effects of different selenium addition amounts on the color and selenium conversion rate of the selenium-enriched probiotic fermented milk were investigated. Based on the measured indicators, suitable selenium-enriched strains for goat milk fermentation were screened.

[0084] 2.3 Screening of γ-aminobutyric acid-producing probiotics

[0085] The activated strains were inoculated into goat milk at a 5% inoculation rate. After fermentation at 37°C for 24 hours, the whey was separated, and the GABA content was determined by Berthlot colorimetric method.

[0086] 2.4 Screening of probiotics producing extracellular polysaccharides

[0087] The activated strains for three generations were inoculated into goat milk at a 5% inoculum, fermented at 37°C for 24 hours, and the whey was separated to prepare EPS. The collected precipitate (crude EPS) was dissolved in deionized water, freeze-dried, and weighed. Strains with higher EPS production were screened.

[0088] 2.5 Preparation of Probiotic Powder

[0089] The selected functional probiotics were inoculated into MRS broth medium and cultured at 37℃ for 24 hours. The cells were collected by centrifugation (8000r, 10min, 4℃), washed with sterile physiological saline, centrifuged again, and washed once more. Then, an equal amount of freeze-drying protectant was added to the bacterial sludge, pre-frozen at -20℃ for 5 hours, and then freeze-dried in a freeze dryer for 24 hours to obtain probiotic powder.

[0090] 2.6 Preparation of Probiotic Fermented Goat Milk

[0091] Goat milk powder was prepared into 12.5% ​​(m / v) reconstituted milk using purified water, sterilized at 90℃ for 15 minutes and cooled, probiotics were inoculated into the reconstituted goat milk and shaken well, and fermented at a constant temperature of 37℃ until pH 4.5, at which point fermentation was stopped to obtain fermented goat milk.

[0092] In addition, the effects of probiotic powder and its compound powder on sheep milk fermentation were studied, and the viable count, selenium conversion rate, γ-aminobutyric acid production, extracellular polysaccharide production, water holding capacity, sensory evaluation, acidity, and pH were measured.

[0093] (1) Viable bacteria count determination

[0094] Determination of viable bacteria count in fermented milk: Refer to the "Introduction to the Method of Counting Viable Bacteria by Dilution Plating Plate" by Gong Junhui et al.

[0095] Viable cell count determination of bacterial powder: Under aseptic conditions, take 0.1g of target bacterial powder and add 0.9% sterile physiological saline to 10mL to dilute to 10 times the original solution. -2 The concentration was determined, and then diluted and plated for counting in the same manner as above. Finally, the number of viable bacteria per unit (CFU / g) was calculated.

[0096] (2) Selenium conversion rate determination

[0097] The selenium content was determined according to the method described by Chen Yongbo et al. in "Research Progress in Selenium Detection Technology". Sodium selenite was used as the standard substance, and a standard curve for sodium selenite was obtained. The standard curve equation was y = 0.0475x + 0.2802 for selenium concentrations in the range of 10-50 μg / mL, and the regression coefficient R0 was [missing value]. 2 The value reached 0.9955. Within the range of 0-10 μg / mL, the standard curve equation was y = 0.0758x + 0.1407, and the regression equation coefficient R0 was [value missing]. 2 The absorbance value reached 0.9945, indicating a good linear relationship between absorbance and sodium selenite concentration. Within this linear range, it can be used to determine the inorganic selenium content. The difference between the residual inorganic selenium content and the total selenium added is the organic selenium yield, and the selenium conversion rate can be obtained by the ratio of organic selenium to total selenium.

[0098] (3) Gamma-aminobutyric acid determination

[0099] The γ-aminobutyric acid (GABA) content was determined according to the method described by Yang Shengyuan et al. in "Study on Colorimetric Quantitative Method for Glutamate Decarboxylase Activity Determination". The GABA concentration was within the range of 0-1.0 μg / mL. The standard curve equation was y = 2.0686x + 0.0804, and the regression coefficient R0 was [missing value]. 2 The absorbance reached 0.9968, indicating a good linear relationship between absorbance and γ-aminobutyric acid (GABA) concentration. Within this linear range, it can be used for the determination of GABA concentration.

[0100] (4) Extracellular polysaccharide assay

[0101] Following the steps outlined in Li Yilin's "Preparation, Structural Identification and Application Research of Extracellular Polysaccharides from Lactic Acid Bacteria", crude EPS was collected and converted into yield per unit culture medium (mg / L).

[0102] (5) Water-holding capacity measurement

[0103] Referring to Huang Wenqi et al.'s "Effects of Tremella polysaccharide on Immunity and Intestinal Mucosal Barrier Function in Probiotic-Fermented Camel Milk in Mice", the following formula was used for calculation:

[0104] Water holding capacity (%) = m / M × 100%

[0105] In the formula: m—mass of the precipitate (g); M—mass of the fermented milk (g).

[0106] (6) Sensory evaluation of fermented goat milk

[0107] Following Zhang Qiuhong's method of "Optimization and Isolation Purification of ACE Inhibitory Peptides Produced by Lactobacillus plantarum Fermentation in Goat Milk", the fermented goat milk was evaluated by more than 5 people after tasting, and the results were scored out of 100.

[0108] (7) Acidity determination: Refer to GB 5009.239-2016 and use the phenolphthalein indicator method for determination.

[0109] (8) pH measurement: pH was measured directly at room temperature using a pHs-3c acidity meter.

[0110] 2.7 Single-factor experiment on fermentation conditions

[0111] The probiotic powder was inoculated into reconstituted goat milk, and the effects of fermentation time (17h, 19h, 21h, 23h, 25h), fermentation temperature (33℃, 35℃, 37℃, 39℃, 41℃), and inoculation amount (0.005%, 0.010%, 0.015%, 0.020%, 0.025%) on probiotic-fermented goat milk were studied. The selenium addition amount was in accordance with the national standard (GB14880-2012) for the addition range of 140μg / kg to 280μg / kg in prepared milk powder (excluding infant milk powder). The calculated selenium addition amount in liquid milk was 17.5μg / kg to 35μg / kg. Within this addition range, no inorganic selenium was detected in the fermented goat milk, that is, the selenium conversion rate was 100%, and the inorganic selenium was completely converted into organic selenium. Therefore, the selenium conversion rate was not measured in subsequent studies. The pH value, acidity, sensory evaluation, water-holding capacity, selenium enrichment capacity, γ-aminobutyric acid (GABA) yield, and extracellular polysaccharide yield of fermented milk were measured. The basic experimental conditions were a fermentation time of 18 h, a fermentation temperature of 37 °C, and an inoculum size of 0.005%. The optimal conditions for each single factor were obtained, and then combined to form the optimal fermentation conditions.

[0112] 2.8 Preparation of Selenium-Enriched Sleep-Aiding Probiotic Goat Milk Powder

[0113] The selenium-enriched sleep-aiding probiotic fermented goat milk prepared in section 2.7 was processed using vacuum freeze-drying, high-temperature spray drying, and vacuum low-temperature spray drying, respectively. Vacuum freeze-drying was the preferred method, with the following conditions: the selenium-enriched sleep-aiding probiotic fermented goat milk was pre-frozen at -20℃ for 4-6 hours, and then freeze-dried in a vacuum freeze dryer for 20-24 hours at a vacuum degree of 4-6 Pa. The high-temperature spray drying conditions were: inlet air temperature of 140-160℃ and outlet air temperature of 75-95℃. The vacuum low-temperature spray drying conditions were: inlet air temperature of 60℃-80℃, peristaltic pump opening of 30%-50%, and vacuum negative pressure of 0.03-0.04 MPa. The viable bacteria count, survival rate, and GABA content of the dried selenium-enriched sleep-aiding probiotic goat milk powder were determined.

[0114] 3. Results and Analysis

[0115] 3.1 Effects of probiotic strains on selenium enrichment and pH in fermented goat milk

[0116] The results are shown in Table 1.

[0117] Table 1. Effect of sodium selenite addition on the color of probiotic-fermented goat milk.

[0118]

[0119] Table 1 shows that when the sodium selenite concentration is less than 20 μg / mL, fermented goat milk does not turn red. However, with the increase of sodium selenite concentration, KD11 and KD5 both showed a small amount of red, giving the goat milk a pinkish appearance. When the sodium selenite concentration exceeds 80 μg / mL, the fermented milk of KD11 and KD5 turns dark red. The red substance that makes the probiotic fermentation broth appear is nano-selenium from organic selenium. The higher the yield of nano-selenium, the redder the color of the fermentation broth. When the inoculum size is constant, the darker the color of the fermentation broth, the higher the conversion rate of selenium by the strain.

[0120] The conversion rate and pH value of goat milk are shown in [reference needed]. Figure 1 In sections A and B, the overall selenium conversion rate decreased with increasing sodium selenite concentration. However, strains 38, KD11, 7830, and L20 showed a slight increase when sodium selenite concentration was below 30 μg / mL; when the sodium selenite concentration exceeded their tolerance level, the conversion rate often experienced a sharp decline. Specifically, from... Figure 1As shown in Figure A, the selenium conversion rate of strain KD5 ranges from 21.11% to 74.18%, with the highest value at 10 μg / mL and the lowest value at 90 μg / mL. The selenium conversion rate of strain KD6 ranges from 22.23% to 66.23%. When the concentration is less than 40 μg / mL, the conversion rate gradually increases, reaching the highest value at 40 μg / mL. This indicates that the sodium selenite tolerance limit of strain KD6 is around 40 μg / mL. This may be due to the different tolerance and utilization abilities of probiotics to selenium at different growth stages, with the lowest conversion rate at 100 μg / mL. The selenium conversion rate of strain KD11 ranged from 30.72% to 88.86%, with the highest value at 10 μg / mL and the lowest value at 50 μg / mL, gradually stabilizing thereafter. This indicates that strain KD11 has good tolerance at this concentration, ensuring that its conversion rate is not affected. The selenium conversion rate of strain 38 ranged from 24.92% to 68.83%, with the highest value at 40 μg / mL and the lowest value at 80 μg / mL. The selenium conversion rate of strain 7830 ranged from 33.63% to 89.35%, with the highest value at 10 μg / mL and the lowest value at 70 μg / mL, stabilizing at around 40%. The selenium conversion rate of strain L20 ranged from 23.84% to 66.12%. When the concentration was less than 30 μg / mL, the selenium conversion rate gradually increased, reaching the highest value at 30 μg / mL and the lowest value at 70 μg / mL.

[0121] Depend on Figure 1 As shown in Figure B, the pH of selenium-enriched probiotic fermented milk increases with the concentration of sodium selenite. This means that the higher the concentration of sodium selenite, the greater the impact on the proliferation of probiotics in goat milk. This is because sodium selenite has a certain degree of toxicity. When its concentration is too high, the probiotics themselves will also be inhibited by inorganic selenium during the process of converting inorganic selenium into organic selenium.

[0122] 3.2 Effects of probiotic strains on the production of γ-aminobutyric acid (GABA) from goat milk fermentation

[0123] like Figure 2 As shown, without the addition of monosodium glutamate (MSG), all six probiotic strains produced high concentrations of GABA in goat milk. This is likely due to the high activity of glutamate decarboxylase in the probiotics, which utilizes the amino acids in goat milk to convert them into GABA. After fermentation at 37℃ for 24 hours, the GABA yield in goat milk ranged from 0.068 to 0.390 g / L; among them, KD5, KD6, and KD11 showed significantly higher GABA yields in goat milk than other strains (P < 0.05).

[0124] 3.3 Effects of probiotic strains on EPS production from goat milk fermentation

[0125] like Figure 3As shown, all six probiotic strains were able to convert and produce EPS in goat milk, but the EPS production capacity varied significantly among the strains. For goat milk, the EPS production ranking was: KD6 > KD11 > KD5 > 38 > L20 > 7830. Strain KD6 had the highest EPS production at 474 mg / L, while strain 7830 had the lowest at 332 mg / L. The high EPS production of the six probiotic strains in this invention may be because goat milk is richer in nutrients than sugar-producing media, making it more suitable as a fermentation substrate for EPS production by probiotics.

[0126] Taking into account the probiotic characteristics, selenium conversion rate, GABA and EPS yield of each strain, KD5, KD6 and KD11 were selected for subsequent powder preparation.

[0127] 3.4 Research and Development of Functional Probiotic Fermented Goat Milk

[0128] 3.4.1 Preparation of Single and Compound Probiotic Powders

[0129] Freeze-drying was used to prepare freeze-dried probiotic powders of three strains of bacteria. Four different compound bacterial powders were then prepared by blending the powders: KD5+KD11, KD5+KD6, KD6+KD11, and KD5+KD6+KD11 (all compound bacterial powders were prepared at a mass ratio of 1:1 or 1:1:1 for each bacterial powder). Viable cell counts were determined for both single and compound bacterial powders: Under aseptic conditions, 0.1 g of the target bacterial powder was added to 10 mL of 0.9% sterile physiological saline to dilute it to 10% of the original solution. -2 The concentration was determined, and then diluted using the same method as above before plating and counting. Finally, the number of viable cells per unit (CFU / g) was calculated, and the results were as follows: Figure 4 As shown in the figure. The results showed that the viable count of both single and compound probiotic powders was 1×10⁻⁶. 11 The CFU / g level was above 8.8, with KD6 showing the highest viable count (P < 0.05) at 8.8 × 10⁻⁶. 11 CFU / g.

[0130] 3.4.2 Effects of single and compound bacterial cultures on sheep milk fermentation

[0131] This study investigated the effects of probiotic powder and its compound powder on sheep milk fermentation, measuring viable cell count, selenium conversion rate, γ-aminobutyric acid (GABA) production, extracellular polysaccharide production, water-holding capacity, acidity, and pH. Results are shown below. Figures 5 to 10 .

[0132] Viable bacteria were counted using the dilution plating method. Figure 5 The results show that the viable bacteria count in the fermented milk produced by the starter culture was 1×10⁻⁶. 9 The CFU / mL level was above 3.13 × 10⁻⁶, with the highest live bacteria count in KD5 mature goat milk reaching 3.13 × 10⁻⁶. 9CFU / mL; for goat milk viable bacteria count range: 1.87-2.54×10⁻⁶ 9 CFU / mL; KD6 had the highest viable count (P < 0.05).

[0133] As shown in 6A, the pH of fermented milk with starter culture gradually decreases as fermentation time increases; the fermentation endpoint of fermented goat milk generally occurs around 18 hours, with the three-strain fermented milk fermenting the fastest and reaching the fermentation endpoint first.

[0134] Depend on Figure 6 According to B, the acidity of fermented milk with starter culture gradually increases with the increase of fermentation time, and the difference between different strains is small; for goat milk, the fermentation endpoint generally occurs around 16 hours, among which the fermentation speed of three-strain fermented milk is the fastest.

[0135] Depend on Figure 7 The water-holding capacity of fermented goat milk ranged from 21.03% to 26.15%, with KD5+KD11 fermented goat milk exhibiting the highest water-holding capacity (P < 0.05), reaching 27.61% after ripening. The water-holding capacity of fermented goat milk after ripening showed an increasing trend, which may be due to the interaction between whey protein and casein during refrigeration, forming a stable colloidal structure that enhances water-holding capacity.

[0136] Since adding sodium selenite exceeding 20 μg / mL will cause the fermented milk to turn red, the addition amount of sodium selenite was chosen to be 10 μg / mL. Goat milk was cultured to the fermentation endpoint, and the selenium conversion rate was measured as follows: Figure 8 As shown in the figure, the selenium conversion rate of goat milk ranged from 58.79% to 86.85%, with KD11 exhibiting the highest conversion rate. Fermentation time may also contribute to the differences in selenium conversion rate.

[0137] Depend on Figure 9 It can be seen that the GABA production of goat milk ranges from 0.307 to 0.588 g / L, and the GABA production is the highest in the three-strain compound fermentation (P < 0.05).

[0138] Depend on Figure 10 It is known that the EPS yield of goat milk ranges from 330 to 394 mg / L, with the highest EPS yield in three-strain fermented milk. Probiotics can synthesize homopolysaccharides and heteropolysaccharides depending on the substrate composition. In terms of EPS yield, lactic acid bacteria have a stronger ability to synthesize homopolysaccharides, with yields often exceeding 1 g / L, while the yield of heteropolysaccharides is only 40-200 mg / L. Probiotics in goat milk often mainly synthesize homopolysaccharides. However, since this invention uses multi-strain combination fermentation, it is possible to synthesize a variety of different polysaccharide types, including homopolysaccharides, heteropolysaccharides, and neutral heteropolysaccharides.

[0139] By comparing the selenium enrichment capacity, γ-aminobutyric acid (GABA) production, and extracellular polysaccharide production of probiotic powder and its compound powder in fermenting goat milk, and combining the live bacteria count, acidity, pH, and water-holding capacity of fermented goat milk, it was determined that the fermentation agent with a ratio of 1:1:1 of KD5+KD6+KD11 probiotic powder compound was the most effective and was used for subsequent fermentation process optimization.

[0140] 3.4.3 Effects of fermentation conditions on probiotic fermentation of goat milk

[0141] Based on the amount of selenium added in milk powder, the maximum amount of selenium added in goat milk is calculated to be 0.08 μg / mL. No inorganic selenium was detected in fermented goat milk, meaning the selenium conversion rate was 100%. The selenium enrichment capacity of each strain will not be discussed separately thereafter.

[0142] Depend on Figure 11 It can be seen that as the inoculum size increases, the rate of pH decrease in fermented milk increases significantly, the acidity of fermented milk increases significantly, and the fermentation time gradually shortens; when the inoculum size increases from 0.005% to 0.025%, the fermentation time of goat milk gradually decreases from 19h to 15h.

[0143] Subsequent parameters, including water-holding capacity, γ-aminobutyric acid (GABA) yield, and extracellular polysaccharide yield, were measured at their respective fermentation endpoints. Sensory evaluation of the fermented milk was conducted after fermentation and subsequent refrigeration and ripening. The results are as follows: Figure 12As shown, the water-holding capacity of fermented goat milk initially increased and then decreased with increasing inoculum amount. This may be because, with increasing inoculum amount, the efficiency of probiotics in utilizing casein in goat milk increases, resulting in enhanced coagulation. However, when the inoculum amount is too large, the bacteria produce acid too quickly, reaching the fermentation endpoint in a shorter time, leading to insufficient coagulation of the fermented milk. The water-holding capacity of goat milk reached its maximum value of 27.71% when the inoculum amount was 0.015%. The GABA production in fermented goat milk initially increased and then decreased with increasing inoculum amount. This may be because, with increasing inoculum amount, the bacteria grow faster within the same time frame, increasing GABA production. However, when the inoculum amount is too large, the bacteria produce acid too quickly, leading to reaching the fermentation endpoint in a shorter time. Furthermore, excessively high bacterial concentrations prevented effective binding to the substrate, and competition between strains inhibited GAD conversion efficiency. Simultaneously, the limited nutrients in goat milk were insufficient to support the metabolic activities of all bacteria, ultimately leading to reduced GABA production. The GABA production in fermented goat milk generally reached its maximum of 0.384 g / L at an inoculum size of 0.010%. Sensory scores for fermented goat milk initially increased and then decreased with increasing inoculum size; the highest sensory score of 67 was achieved at an inoculum size of 0.010%. EPS production in fermented goat milk initially increased and then decreased with increasing inoculum size, possibly because the bacteria grew faster within the same timeframe with higher inoculum sizes, increasing EPS production. However, excessively high inoculum sizes resulted in rapid acid production, reaching the fermentation endpoint in a shorter time, leading to reduced EPS production. The EPS production in fermented goat milk reached its maximum of 421 mg / L at an inoculum size of 0.010%. Considering all indicators, an inoculum size of 0.010% was recommended for fermented goat milk.

[0144] Depend on Figure 13 It can be seen that as the temperature increases, the rate of pH decrease in fermented milk increases significantly, the acidity of fermented milk increases significantly, and the fermentation time gradually shortens; when the temperature increases from 33℃ to 41℃, the fermentation time of goat milk gradually decreases from 26h to 17h.

[0145] Subsequent parameters, including water-holding capacity, γ-aminobutyric acid (GABA) yield, and extracellular polysaccharide yield, were measured at their respective fermentation endpoints. Sensory evaluation of the fermented milk was conducted after fermentation and subsequent refrigeration and ripening. The results are as follows: Figure 14As shown in the figure, the water-holding capacity of fermented goat milk initially increased and then decreased with increasing temperature. This may be because higher temperatures lead to more vigorous probiotic metabolism, resulting in enhanced curdling, but excessively high temperatures can cause insufficient curdling. The water-holding capacity of goat milk reached its maximum of 26.64% at 39℃. The GABA yield of fermented goat milk initially increased and then decreased with increasing temperature, possibly because higher temperatures allow for faster bacterial growth within the same time frame, increasing GABA production. However, excessively high temperatures cause rapid acid production by the bacteria, reaching the fermentation endpoint in a shorter time, thus reducing the time for bacterial conversion to GABA. The GABA yield of goat milk reached its maximum of 0.407 g / L at 39℃. The sensory score of fermented goat milk initially increased and then decreased with increasing temperature; the sensory score reached its highest point of 66 at 39℃. The EPS yield of fermented goat milk initially increased and then decreased with increasing temperature, possibly because higher temperatures shortened the time for bacterial conversion to EPS. As fermentation progresses, various nutrients in the fermented milk are gradually depleted, and competition among bacterial strains intensifies, leading to a decrease in the rate of EPS synthesis. Simultaneously, higher temperatures also affect the stable accumulation of EPS in the fermented milk, resulting in reduced EPS yield. The EPS yield of fermented goat milk reaches its maximum value of 389 mg / L at 39℃. Considering all factors, the optimal fermentation temperature for goat milk is 39℃.

[0146] The effects of inoculum size and fermentation temperature on different goat milk fermentations have been studied above. Subsequently, goat milk fermentation was conducted using the optimal inoculum size and fermentation temperature, and the effect of different fermentation times on probiotic powder fermentation of goat milk was investigated. The results are as follows: Figure 15 As shown. By Figure 15 As shown in section A, the pH of fermented goat milk gradually decreases with increasing fermentation time; and the rate of decrease slows down, possibly because as fermentation time increases, the reduction of nutrients and the accumulation of acid lead to a decrease in the rate of bacterial growth and metabolism. Conversely, the acidity of fermented goat milk gradually increases with increasing fermentation time. Figure 15As shown in Figure B, the water-holding capacity of fermented goat milk initially increased and then stabilized with increasing fermentation time. This may be because, with increasing fermentation time, probiotics fully fermented in the goat milk. However, as fermentation exceeded the endpoint, over-fermentation led to whey separation, affecting the quality of the fermented milk. The water-holding capacity of goat milk reached its maximum of 26.63% after 23 hours of fermentation. The GABA production of goat milk increased with increasing fermentation time, reaching a maximum of 0.605 g / L at 25 hours. The sensory score of fermented goat milk initially increased and then decreased with increasing fermentation time. Before 19 hours, fermentation was incomplete, and curdling was not yet complete. After 23 hours, over-fermentation resulted in an overly sour taste, leading to a decrease in the sensory score. The sensory score of fermented goat milk reached its highest point of 69 points at 21 hours. The EPS production of fermented goat milk gradually increased with increasing fermentation time, possibly because the bacteria had sufficient time to convert and accumulate EPS. The EPS production of fermented goat milk reached its maximum of 474 mg / L at 25 hours. Taking into account various indicators, the optimal fermentation time for goat milk is 19 hours.

[0147] By combining the optimal conditions for inoculation amount, fermentation temperature, and fermentation time, the suitable fermentation conditions for fermented goat milk were obtained: goat milk inoculation amount 0.010%, fermentation time 19h, and fermentation temperature 39℃.

[0148] The selenium-enriched probiotic fermented goat milk (selenium addition of 35 μg / kg) obtained under the above-mentioned optimal fermentation conditions was dried using the following methods: vacuum freeze-drying (pre-freezing the selenium-enriched probiotic fermented goat milk at -20℃ for 4-6 hours, then freeze-drying in a vacuum freeze dryer for 20-24 hours at a vacuum degree of 4-6 Pa), high-temperature spray drying (inlet air temperature 140-160℃, outlet air temperature 75-95℃), and vacuum low-temperature spray drying (inlet air temperature 60℃-80℃, peristaltic pump opening 30%-50%, vacuum negative pressure 0.03-0.04 MPa). The number of live probiotics, survival rate, and γ-aminobutyric acid content in the prepared selenium-enriched probiotic goat milk powder are shown in Table 2.

[0149] Table 2. Effects of drying method on the preparation of selenium-enriched sleep-aiding probiotic goat milk powder

[0150]

[0151] As shown in Table 2, the viable count of probiotics was higher than 2.0 × 10⁻⁶ in all three drying methods. 7 CFU / g, viable count range of 2.3 × 10⁻⁶ 7 CFU / g - 4.08 × 10 8The CFU / g and survival rate ranged from 3.31% to 60.02%, with GABA content ranging from 3.472 g / kg to 4.448 g / kg, EPS content from 2.640 g / kg to 3.792 g / kg, and inorganic selenium content being 0, indicating that all were converted into organic selenium. The total selenium content ranged from 271 μg / kg to 282 μg / kg, which is close to the theoretical value of 280 μg / kg. High-temperature spray drying had the lowest viable count, survival rate, and GABA content, while vacuum freeze drying had the highest viable count, survival rate, and GABA content. Vacuum low-temperature spray drying had all indicators in between.

[0152] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A selenium-enriched, sleep-aiding compound probiotic, characterized in that, The contents include *Limosilactobacillus fermentum* KD6, *Lacticaseibacillus rhamnosus* KD5, and *Lacticaseibacillus paracasei* KD11, wherein the preservation number of *Limosilactobacillus fermentum* KD6 is CCTCC NO: M20231642, the preservation number of *Lacticaseibacillus rhamnosus* KD5 is CCTCC NO: M20231641, and the preservation number of *Lacticaseibacillus paracasei* KD11 is CCTCC NO: M2024605.

2. The compound probiotic as described in claim 1, characterized in that, The mass ratio of the fermenting *Lactobacillus mucinus* KD6, the *Lactobacillus rhamnosus* KD5, and the *Lactobacillus paracasei* KD11 is 1:(1-3):(1-3).

3. A selenium-enriched probiotic goat milk powder for promoting sleep, characterized in that, Includes the compound probiotics as described in claim 1 or 2.

4. The selenium-enriched sleep-aiding probiotic goat milk powder as described in claim 3, characterized in that, The selenium content ranges from 143 μg / kg to 278 μg / kg, and the live probiotic count is 0.23 × 10⁻⁶. 8 CFU / g ~4.08×10 8 CFU / g, γ-aminobutyric acid is 3.472-4.448 g / kg, and extracellular polysaccharide content is 2.640 g / kg~3.792 g / kg.

5. A method for preparing selenium-enriched sleep-aiding probiotic goat milk powder, characterized in that, The step includes fermenting goat milk using the compound probiotics described in claim 1 or 2.

6. The preparation method according to claim 5, characterized in that, The preparation method includes the following steps: Prepare sterilized goat milk from fresh goat milk or reconstituted goat milk by adding sodium selenite; Probiotics were inoculated into sterilized goat milk and fermented at a constant temperature to produce selenium-enriched sleep-aiding probiotic fermented goat milk. Selenium-enriched sleep-aiding probiotics are fermented in goat milk and then dried to produce selenium-enriched sleep-aiding probiotic goat milk powder.

7. The preparation method according to claim 6, characterized in that, The sterilized goat milk preparation conditions are: 90-95℃ for 5-10 minutes.

8. The preparation method according to claim 6, characterized in that, The amount of sodium selenite added is 17.5 μg / kg-35 μg / kg.

9. The preparation method according to claim 6, characterized in that, The fermentation conditions were as follows: inoculum size of 0.005%-0.025%, fermentation temperature of 33-41℃, and fermentation time of 17-25h. The prepared selenium-enriched sleep-aiding probiotic fermented goat milk had a sodium selenite conversion rate of 100%, a γ-aminobutyric acid content of 0.33g / L-0.61g / L, and an extracellular polysaccharide content of 0.32g / L-0.47g / L. The drying process includes freeze drying, vacuum low-temperature spray drying, and high-temperature spray drying.

10. The application of the compound probiotics as described in claim 1 or 2 in the preparation of selenium-enriched sleep-aiding goat dairy products.

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