Preparation method and application of sturgeon slime rich in strontium product

Abstract

The present application relates to the technical field of food processing, and particularly relates to a preparation method and application of a sturgeon mucus strontium-rich product. The preparation method of the sturgeon mucus strontium-rich product comprises the following steps: mixing sturgeon mucus and a strontium-containing raw material, and obtaining a fermentation product after fermentation by a fermentation strain. The method uses sturgeon mucus and strontium as raw materials, and a sturgeon mucus strontium-rich product is prepared through microbial fermentation. The method can efficiently convert inorganic strontium into organic strontium, improve the bioavailability of strontium, and prepare a product rich in organic strontium, and rich in nutrients such as amino acids, minerals and probiotics. The product has multiple biological activities such as antioxidant and bone cell proliferation promotion, and realizes high-value utilization of sturgeon processing by-products.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of food processing, and particularly relates to a preparation method and application of a sturgeon mucus strontium-rich product. BACKGROUND

[0002] Strontium (Sr) is ubiquitous in nature and human tissues, is one of the essential trace nutrients for the human body, is similar in chemical properties to calcium, is related to human bone mineral metabolism, and is a normal component of human bones and teeth. In the medical field, strontium has the effects of promoting bone health, preventing cardiovascular diseases, and preventing dental caries, and also has the functions of anti-cancer, anti-diabetes, promoting angiogenesis, and protecting the heart. The human body mainly obtains strontium from drinking water and food, such as cereals, nuts, leafy vegetables, and dairy products. With the important role of strontium in the human body being continuously explored, strontium-rich products such as strontium-containing drugs and strontium-rich mineral water have also been further developed.

[0003] Although strontium-containing drugs have been widely used, they are a kind of non-natural artificial synthetic compounds and cannot be actively metabolized by the human body, but are passively excreted out of the body, so their effect and safety are not as good as natural Sr. It is necessary to supplement Sr through diet, and it is best to supplement it in the form of a natural Sr compound. Therefore, it is of great significance to develop strontium-rich food. SUMMARY

[0004] The present application provides a preparation method and application of a sturgeon mucus strontium-rich product.

[0005] Sturgeon is a common fish resource. At present, in addition to the main product caviar, the fish meat, fish bones and fish skin of sturgeon have been widely used to produce collagen peptides, chondroitin sulfate and other products. However, the by-product sturgeon mucus has not been effectively utilized. It is found in the research and development process that the microbial fermentation of sturgeon mucus and strontium-containing raw materials can produce strontium-rich products rich in strontium amino acids and other organic strontium, which have various biological activities such as antioxidant and bone cell proliferation promotion.

[0006] Specifically, the present application provides the following technical solutions:

[0007] The present application provides a preparation method of a sturgeon mucus strontium-rich product, which comprises: mixing sturgeon mucus and strontium-containing raw materials, and then fermenting the mixture by a fermentation strain to obtain a fermentation product.

[0008] In the above method, the fermentation strain is Bacillus coagulans.

[0009] Preferably, the fermentation strain is Bacillus coagulans L-H7.

[0010] The Bacillus coagulans L-H7 has been preserved in the China General Microbiological Culture Collection Center (address: No. 1, Beichen West Road, No. 3, Beijing Chaoyang District, Microbiology Institute of Chinese Academy of Sciences, postcode: 100101) on October 11, 2019, and the preservation number is CGMCC No. 18662. The strain has been disclosed in the patent application CN110724651A.

[0011] In the above method, the strontium contained in the strontium-containing raw material is inorganic strontium.

[0012] Preferably, the strontium-containing raw material is one or more selected from strontium salt, strontium hydroxide, and strontium oxide.

[0013] In some embodiments of the present application, the strontium-containing raw material is strontium chloride.

[0014] In the above method, the sturgeon mucus is the mucus on the surface of the sturgeon.

[0015] Preferably, in the fermentation, the mass ratio of sturgeon mucus to strontium in the strontium-containing raw material is (0.1-0.4):(0.004-0.01).

[0016] Further preferably, in the fermentation, the mass ratio of sturgeon mucus to strontium in the strontium-containing raw material is (0.15-0.3):(0.005-0.009).

[0017] Preferably, in the fermentation, the inoculation amount of the fermentation strain is 1×10 3 -1×10 5 CFU / mL of fermentation substrate. More preferably, 1×10 3 -1×10 4 CFU / mL of fermentation substrate. The fermentation substrate is a mixture of sturgeon mucus and strontium-containing raw material.

[0018] Preferably, the temperature of the fermentation is 35-37℃, and the time of the fermentation is 20-40h.

[0019] In the above method, the fermentation product is subjected to solid-liquid separation, and the precipitate is collected and freeze-dried.

[0020] Preferably, the solid-liquid separation adopts the method of centrifugation.

[0021] Preferably, after collecting the precipitate, a step of washing the precipitate to remove strontium attached to the surface of the cells is further included.

[0022] The present application also provides a sturgeon mucus strontium-rich product prepared by the above method.

[0023] Preferably, the content of organic strontium in the sturgeon mucus strontium-rich product is 2-20 mg / kg.

[0024] Preferably, the content of amino acid in the sturgeon mucus strontium-rich product is not less than 30%, preferably not less than 40%, and more preferably 40-60%.

[0025] The present application provides the following application of the above-mentioned sturgeon mucus strontium-rich product:

[0026] (1) application in the preparation of food, medicine or feed;

[0027] (2) application in the preparation of antioxidant products;

[0028] (3) application in the preparation of products with the effect of promoting bone cell proliferation and / or anti-osteoporosis.

[0029] Preferably, the food, medicine or feed has antioxidant and bone cell proliferation promoting effects.

[0030] The beneficial effects of the present application at least include: the preparation method of the sturgeon mucus strontium-rich product provided by the present application uses sturgeon mucus and strontium as raw materials, and the sturgeon mucus strontium-rich product is prepared by microbial fermentation. This method can efficiently convert inorganic strontium into organic strontium, improve the bioavailability of strontium, and prepare a product rich in organic strontium, and rich in nutrients such as amino acids, minerals and probiotics. The product has antioxidant, bone cell proliferation promoting and other biological activities, and realizes high-value utilization of sturgeon processing by-products. DETAILED DESCRIPTION

[0031] In order to make the purpose, technical scheme and advantages of the present application clearer, the technical scheme in the present application will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0032] Embodiment 1

[0033] The present embodiment provides a preparation method of a sturgeon mucus strontium-rich product, comprising the following steps:

[0034] (1) Collection and preparation of sturgeon mucus: rinse the fish skin with ultrapure water, scrape and collect the mucus, and freeze-dry for standby.

[0035] (2) Enrichment treatment: Strontium chloride was prepared into an aqueous solution with a concentration of 50 mg / L and sterilized for later use; the sterile strontium chloride solution was added to 150 mL of sturgeon mucus with a concentration of 1 g / L at a volume ratio of 2:1, and after stirring and mixing evenly, a bacterial suspension of Bacillus coagulans L-H7 (1×10⁻⁶) was added at an inoculation rate of 2.6% (volume ratio). 5 (CFU / mL). After incubation at 37°C for 27 hours, the bacterial culture was collected for later use.

[0036] (3) After centrifuging the bacterial culture at 10,000 rpm for 15 min, collect the precipitate, wash it three times with physiological saline to remove the strontium attached to the cell surface, and then freeze-dry it to obtain the strontium-rich sturgeon mucus product.

[0037] Example 2

[0038] This embodiment provides a method for preparing a strontium-rich product from sturgeon mucus, including the following steps:

[0039] (1) Collection and preparation of sturgeon mucus: Rinse the fish skin with ultrapure water, scrape and collect the mucus, and freeze-dry it for later use.

[0040] (2) Enrichment treatment: Strontium chloride was prepared into an aqueous solution with a concentration of 70 mg / L and sterilized for later use; the sterile strontium chloride solution was added to 150 mL of sturgeon mucus with a concentration of 2 g / L at a volume ratio of 1:1, and after stirring and mixing evenly, a bacterial suspension of Bacillus coagulans L-H7 (1×10⁻⁶) was added at an inoculation rate of 4% (volume ratio). 5 (CFU / mL). After incubation at 37°C for 36 hours, the bacterial culture was collected for later use.

[0041] (3) After centrifuging the bacterial culture at 10,000 rpm for 15 min, collect the precipitate, wash it three times with physiological saline to remove the strontium attached to the cell surface, and then freeze-dry it to obtain the strontium-rich sturgeon mucus product.

[0042] Comparative Example 1

[0043] This comparative example provides a method for preparing a sturgeon mucus-rich product, which differs from the method in Example 1 only in that Bacillus coagulans L-H7 is replaced with Lactobacillus plantarum (a commercially available strain, CICC No. 24986).

[0044] Comparative Example 2

[0045] This comparative example provides a method for preparing a sturgeon mucus-rich product, which differs from the method in Example 1 only in that Bacillus coagulans L-H7 is replaced with Staphylococcus aureus (a commercially available strain, CICC No. 25173).

[0046] Comparative Example 3

[0047] This comparative example provides a method for preparing a sturgeon mucus-rich product, which differs from the method in Example 1 only in that Bacillus coagulans L-H7 is replaced with Lactobacillus plantarum L-ZS9 (this strain has been deposited at the China General Microbiological Culture Collection Center (address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, postcode 100101), with accession number CGMCC No. 11669, and has been disclosed in patent application CN111374278A).

[0048] Comparative Example 4

[0049] This comparative example provides a method for preparing a strontium-rich product from sturgeon mucus, which differs from the method in Example 1 only in that sturgeon mucus is replaced with carp mucus.

[0050] Comparative Example 5

[0051] This comparative example provides a method for preparing a strontium-rich product from sturgeon mucus, which differs from the method in Example 1 only in that sturgeon mucus is replaced with catfish mucus.

[0052] Comparative Example 6

[0053] This comparative example provides a method for preparing a strontium-rich product from sturgeon mucus, which differs from the method in Example 1 only in that sturgeon mucus is replaced with silver carp mucus.

[0054] Experimental Example 1

[0055] 1. Determination of Strontium Tolerance of Strains

[0056] The strontium resistance of the strains used in the enrichment steps of Examples 1, 2, and Comparative Examples 1-3 was tested, and the specific methods are as follows:

[0057] Different amounts of strontium chloride were added to the modified MRS medium to prepare culture solutions with strontium chloride concentrations of 0, 3, 6, 12, 24, and 48 μg / mL. These solutions were then inoculated at a 2.6% inoculum rate into *Bacillus coagulans* L-H7, *Lactobacillus plantarum* CICC No. 24986, *Staphylococcus aureus* CICC No. 25173, and *Lactobacillus plantarum* L-ZS9, respectively. The solutions were incubated at 37℃ for 48 h, and the growth and survival of each strain were measured. The proliferation rate of each strain under different strontium chloride concentrations was calculated using the formula: Proliferation rate = (Treatment A - Initial A) / Initial A * 100%, where Treatment A is the absorbance value at 600 nm after 48 h of incubation for each treatment group, and Initial A is the absorbance value at 600 nm immediately after inoculation. The results are shown in Table 1.

[0058] Table 1

[0059]

[0060] 2. Determination of Strontium Content in Sturgeon Mucus-Rich Products

[0061] The strontium content in the strontium-rich products obtained in each embodiment and comparative example was determined using the following methods:

[0062] Weigh 0.1 g (accurate to 0.0001 g) of sample powder and place it in the digestion vessel. Add 7 mL of concentrated nitric acid and perform microwave digestion. After complete digestion, transfer the digest to a 50 mL volumetric flask and dilute to volume. A blank control was also included. The strontium content was determined using inductively coupled plasma-atomic emission spectrometry (ICP-AES). The results are shown in Table 2.

[0063] Table 2

[0064]

[0065]

[0066] The results showed that the strontium content of the strontium-enriched products prepared by the methods of Examples 1 and 2 was significantly higher than that of Comparative Examples 1-6. This indicates that the methods of Examples 1 and 2 can more effectively enrich strontium.

[0067] 3. Analysis of the degree of organicification of strontium

[0068] The degree of strontium organication in the strontium-rich products obtained in each embodiment and comparative example was determined using the following methods:

[0069] The lyophilized samples were placed in dialysis bags and dialyzed for 48 hours. The content of organostrontium in the dialysis samples was determined by HG-AAS method. The results are shown in Table 3.

[0070] Table 3

[0071] Group Organic strontium content (mg / kg) Example 1 10 Example 2 6.3 Comparative Example 1 4.8 Comparative Example 2 2.7 Comparative Example 3 4.9 Comparative Example 4 1.5 Comparative Example 5 2.2 Comparative Example 6 0.9

[0072] The results showed that the organic strontium content of the strontium-rich products prepared by the methods of Examples 1 and 2 was significantly higher than that of Comparative Examples 1-6, indicating that the methods of Examples 1 and 2 can efficiently convert inorganic strontium into organic strontium.

[0073] 4. Determination of amino acid distribution and content

[0074] The amino acid distribution and content of the strontium-enriched products prepared in each example and comparative example were determined. The determination of 16 amino acids (excluding tryptophan and cysteine) in the samples was performed according to GB 5009.124-2016 "Determination of Amino Acids in Food". Tryptophan in the samples was determined by high-performance liquid chromatography (HPLC); cysteine ​​in the samples was determined using an amino acid analyzer. The results are shown in Table 4.

[0075] Table 4

[0076]

[0077]

[0078] The results showed that the distribution and content of amino acids changed significantly in the strontium-rich products prepared by different methods. However, based on the total amino acid content, it can be seen that the total amino acid content of the strontium-rich products prepared by the methods of Example 1 and Example 2 was significantly higher than that of Comparative Examples 1-6. It can be considered that the strontium-rich products prepared by Example 1 and Example 2 can greatly meet the recommended intake of FAO / WHO and are very high-quality protein resources.

[0079] 5. Nutritional index evaluation of sturgeon mucus-rich products

[0080] The nutritional indicators, such as probiotic activity, flavor index, and mineral content, of the strontium-enriched products prepared in each embodiment and comparative example were tested. The specific methods are as follows:

[0081] (1) Detection of bacterial viability: Dissolve 1 mg of strontium-enriched product in physiological saline, take 1 mL of the liquid and count the viable bacteria using the pouring method. After incubation at 37°C for 48 h, record the total number of colonies on the plate.

[0082] (2) Mineral content detection: The determination of calcium, iron, potassium, magnesium, sodium and phosphorus in the sample was carried out in accordance with GB 5009.268-2016 "Determination of multiple elements in food" (Second method).

[0083] The results of the viable bacteria count test are shown in Table 5.

[0084] Table 5

[0085] Group Viable cell number (lg(CFU / mL)) Example 1 8.15±0.02 Example 2 8.04±0.04 Comparative Example 1 6.17±0.02 Comparative Example 2 7.29±0.05 Comparative Example 3 7.03±0.01 Comparative Example 4 6.49±0.02 Comparative Example 5 7.03±0.03 Comparative Example 6 6.17±0.01

[0086] The results of viable bacterial counts for each sample showed that the viable bacterial counts in the strontium-rich products prepared by the methods of Examples 1 and 2 were significantly higher than those in Comparative Examples 1-6, indicating that the methods of Examples 1 and 2 can effectively maintain the viability of the strains and provide nutrients for the proliferation of the strains, thus promoting their growth.

[0087] The results of mineral content testing are shown in Table 6.

[0088] Table 6

[0089] Group Calcium (mg / g) Potassium (mg / g) Iron (mg / g) Phosphorus (mg / g) Sodium (mg / g) Magnesium (mg / g) Example 1 21.75±0.01 4.71±0.01 0.00407±0.01 11.34±0.02 0.21±0.02 5.55±0.02 Example 2 19.21±0.05 3.80±0.03 0.0025±0.05 9.49±0.07 0.29±0.01 4.04±0.01 Comparative Example 1 17.04±0.01 3.71±0.03 0.001±0.04 4.36±0.05 0.40±0.03 3.06±0.05 Comparative Example 2 14.31±0.02 2.39±0.01 0.0005±0.05 4.36±0.05 0.39±0.03 3.42±0.03 Comparative Example 3 18.11±0.01 3.04±0.13 0.004±0.03 5.55±0.05 0.17±0.01 4.12±0.07 Comparative Example 4 17.65±0.01 2.97±0.02 0.00305±0.01 4.77±0.03 0.43±0.01 4.38±0.03 Comparative Example 5 19.08±0.03 4.22±0.01 0.0034±0.02 5.92±0.02 0.35±0.02 3.91±0.04 Comparative Example 6 18.88±0.04 4.01±0.02 0.001±0.05 6.03±0.05 0.18±0.05 3.22±0.01

[0090] The results of mineral element content of each sample showed that the ratio of calcium, phosphorus and magnesium in the strontium-rich products prepared by the methods of Example 1 and Example 2 was about 4:2:1, which is more conducive to human digestion and absorption and meets the dietary calcium, phosphorus and magnesium ratio proposed by the Chinese Nutrition Society.

[0091] 6. Efficacy determination of sturgeon mucus-rich products

[0092] The antioxidant activity and the effect on osteoblast proliferation of the strontium-rich products prepared in each embodiment and comparative example were tested. The specific methods are as follows:

[0093] (1) Antioxidant activity detection: A certain amount of sample was added, followed by 3 mL of 0.1 mM DPPH (dissolved in 95% ethanol). After shaking and mixing, the mixture was reacted at room temperature in the dark for 30 min. The supernatant was collected by centrifugation, and the absorbance was measured at 517 nm. 200 μg / L of 2,6-di-tert-butyl-p-cresol was used as a positive control. The results were calculated using the following formula:

[0094] DPPH free radical scavenging rate = (1-A) 样品 / A 空白 )×100%.

[0095] (2) Detection of osteoblast proliferation activity: The effect of the samples on the proliferation activity of osteoblast MC3T3-E1 cells was detected using a CCK-8 assay kit. Specifically, after digesting the cultured adherent cells with trypsin, the cells were subjected to a reaction at a ratio of 5 × 10⁻⁶ cells / mL. 5 At a density of 100 μL / well, cell suspensions were seeded into 96-well cell culture plates. Cells were cultured overnight at 37°C with 5% CO2. The culture medium was then replaced with fresh DMEM containing different concentrations of strontium-enriched products, and cultured for another 24 hours. The medium was discarded, and the cells were washed 1-2 times with PBS. DMEM containing 10% CCK-8 solution was then added, and the cells were incubated at 37°C for 3-4 hours. The absorbance at 450 nm was measured and recorded using a microplate reader, and cell viability under different treatments was calculated.

[0096] The results of the antioxidant activity test are shown in Table 7.

[0097] Table 7

[0098] Group DPPH scavenging rate (%) Example 1 55.27％ Example 2 41.09％ Comparative Example 1 29.69％ Comparative Example 2 35.32％ Comparative Example 3 35.55％ Comparative Example 4 39.99％ Comparative Example 5 41.24％ Comparative Example 6 20.15％

[0099] The results showed that the strontium-rich product prepared by the method of Example 1 had the highest DPPH scavenging rate and the highest antioxidant activity. The strontium-rich product prepared by the method of Example 2 also had high antioxidant activity, and its DPPH scavenging rate was significantly higher than that of Comparative Examples 1, 2, 3, and 6, and comparable to that of Comparative Examples 4 and 5.

[0100] The results of the cell proliferation activity assay are shown in Table 8.

[0101] Table 8

[0102] Group Promotion of cell proliferation activity (%) Example 1 60.15％ Example 2 52.04％ Comparative Example 1 21.36％ Comparative Example 2 27.53％ Comparative Example 3 19.77％ Comparative Example 4 30.43％ Comparative Example 5 28.53％ Comparative Example 6 27.16％

[0103] The results showed that the strontium-rich products prepared by the methods of Examples 1 and 2 had significantly higher osteoblast proliferation-promoting activity than those of Comparative Examples 1-6, indicating that the strontium-rich products prepared by the methods of Examples 1 and 2 have a significant effect on promoting osteoblast proliferation and can be developed into new products with osteoporosis-relieving functions.

[0104] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a sturgeon mucus-rich product, characterized in that, The method includes: mixing sturgeon mucus and strontium-containing raw materials, fermenting them with fermentation bacteria to obtain a fermented product; separating the fermented product into solid and liquid components, collecting the precipitate, and freeze-drying it; The fermentation strain is Bacillus coagulans L-H7; The sturgeon mucus is the mucus on the surface of the sturgeon's body.

2. The method for preparing sturgeon mucus-rich products according to claim 1, characterized in that, The strontium-containing raw material is selected from one or more of strontium salts, strontium hydroxide, and strontium oxide.

3. The method for preparing sturgeon mucus-rich products according to claim 1 or 2, characterized in that, In the fermentation process, the mass ratio of sturgeon mucus to strontium in the strontium-containing raw material is (0.1-0.4):(0.004-0.01).

4. The method for preparing sturgeon mucus-rich products according to claim 1 or 2, characterized in that, In the fermentation process, the inoculum size of the fermentation strain is 1×10⁻⁶. 3 -1×10 5 CFU / mL fermentation substrate.

5. The method for preparing the sturgeon mucus-rich product according to claim 3, characterized in that, In the fermentation process, the inoculum size of the fermentation strain is 1×10⁻⁶. 3 -1×10 5 CFU / mL fermentation substrate.

6. The method for preparing strontium-rich sturgeon mucus product according to any one of claims 1, 2, and 5, characterized in that, The fermentation temperature is 35-37℃, and the fermentation time is 20-40 hours.

7. The sturgeon mucus-rich product prepared by the method according to any one of claims 1 to 6.

8. The sturgeon mucus-rich product according to claim 7, characterized in that, The sturgeon mucus-rich product contains 2-20 mg / kg of organic strontium.

9. Any of the following applications of the sturgeon mucus-rich product according to claim 7 or 8: (1) Application in the preparation of drugs or feed; (2) Application in the preparation of antioxidant products; (3) Application in the preparation of products that promote osteoblast proliferation and / or have anti-osteoporosis effects.

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