Lactobacillus rhamnosus Lr-Ca011, postbiotics, calcium products for promoting calcium absorption and their nanoemulsion homogenization preparation method

Abstract

The present invention provides a Lactobacillus rhamnosus Lr-Ca011 for promoting calcium absorption, a postbiotic, a calcium product and a method for preparing the same by nanoemulsification homogenization, belonging to the technical field of probiotics. The Lactobacillus rhamnosus Lr-Ca011 of the present invention is deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, with the deposit number of CGMCC No. 32037, and can promote calcium absorption. The present invention also ferments the Lactobacillus rhamnosus Lr-Ca011 to prepare a postbiotic, which also has the effect of promoting calcium absorption. In addition, the present invention mixes and disperses an aqueous phase and an oil phase containing the postbiotic to obtain a coarse emulsion, and then adopts a nanoemulsification homogenization method to obtain a calcium product. This nanoemulsification homogenization method enables the coarse emulsion to form more stable nanoparticles, can effectively control the release of functional components, makes the calcium product have the characteristic of high absorption rate, and improves the bioavailability of calcium.

Description

Technical Field

[0001] The present invention relates to the technical field of probiotics, and specifically relates to a Lactobacillus rhamnosus Lr-Ca011, postbiotics, calcium products for promoting calcium absorption, and a nanoemulsion homogenization preparation method thereof. Background Art

[0002] Calcium is an important nutrient in the human body and the most abundant inorganic element in the human body, with a total amount exceeding 1 kilogram. Most of the calcium (about 99%) is deposited in the bones in a solid state, and throughout our lives, it is constantly metabolized, dynamically changing the shape and hardness of the bones; a small part of the calcium (1%) exists in the blood and soft tissue cells in a free state, regulating various functions that maintain life activities, such as maintaining brain development, heart beating, muscle function, etc. Calcium deficiency can lead to a series of functional disorders in the body. Every year, various diseases caused by "calcium deficiency" will bring heavy economic pressure and mental burden to the patient's family and society. Therefore, how to improve the body's absorption of calcium is not only related to personal health but also a major issue related to the health of the whole people.

[0003] Currently, common methods to improve calcium absorption in the human body are as follows: (1) consuming calcium-rich products, such as black sesame seeds, laver, black beans, black fungus, etc.; (2) consuming foods rich in vitamin D and lysine, such as egg yolks, pig livers, beans, etc.; (3) enabling the human skin to produce vitamin D by sun exposure. Vitamin D is also called the "sunshine vitamin". As long as the skin on the face and hands is exposed and receives 15 minutes of sunlight exposure every day, the produced vitamin D can play its role and promote calcium absorption in the human body; (4) exercise can increase the blood circulation speed of the body, accelerate the delivery of calcium in the blood to the bones, promote bone growth, increase bone mass, and also stimulate the body's demand for calcium. With the development of science and technology, many studies have confirmed that supplementing probiotics can also play a role in promoting calcium absorption. However, different probiotic strains have significant differences in the effect of calcium absorption, and even for the same strain of bacteria, using different processes to make raw materials or end products will also lead to significant differences in their promoting effects.

[0004] Nanoparticles are particles with a diameter less than 1 μm. Functional factors can be embedded inside the nanoparticles or adsorbed on the surface and delivered into the body. Nanoparticles have the advantages of small particle size and strong adhesion. They can not only enter and exit cells through a variety of different pathways (such as: (1) endocytosis, enhancing the ability of functional factors to cross intestinal epithelial cells; (2) uptake of particles through microfold cells (M cells) in Peyers patches in the ileum; (3) tight junctions (TJs) between epithelial cells also form a natural epithelial barrier, affecting the permeability of functional factors; (4) transported into and out of cells through transporters, affecting the accumulation of functional factors in cells), but also increase the contact time between functional factors and intestinal mucosa, thereby increasing the absorption of functional factors and improving the bioavailability of oral preparations. Of course, nanoparticles are not the smaller the better. Extremely small particles are sometimes toxic to cells because they are easily absorbed by cells. When the particles are not small enough, the ability to promote the absorption of functional factors is not strong enough.

[0005] Therefore, providing a Lactobacillus rhamnosus that promotes calcium absorption and preparing postbiotics, calcium products, etc. through appropriate nanoemulsion homogenization technology poses a challenge to current research. Summary of the Invention

[0006] In view of the above problems, the present invention provides a Lactobacillus rhamnosus Lr-Ca011 that promotes calcium absorption, a postbiotic, a calcium product, and a method for preparing the same by nanoemulsion homogenization.

[0007] To achieve the above invention object, the present invention provides the following technical solutions:

[0008] The present invention provides a Lactobacillus rhamnosus Lr-Ca011 that promotes calcium absorption, classified and named as Lactobacillus rhamnosus Lacticaseibacillus rhamnosus , deposited in the China General Microbiological Culture Collection Center on September 23, 2024, with the deposit number CGMCC No. 32037.

[0009] The present invention also provides the application of the above Lactobacillus rhamnosus Lr-Ca011 in the preparation of foods or health products that promote calcium absorption.

[0010] Preferably, the food is a postbiotic and the health product is a calcium product.

[0011] The present invention also provides a postbiotic prepared by fermenting the above Lactobacillus rhamnosus Lr-Ca011.

[0012] Preferably, the postbiotic includes the following raw materials: Lactobacillus rhamnosus Lr-Ca011, quinoa, potato, pumpkin seed kernels, wolfberries, glucose monohydrate, and purified water.

[0013] The present invention also provides a method for preparing the above postbiotics, comprising: mixing quinoa, potato, pumpkin seed kernels, wolfberries, anhydrous glucose and purified water according to a weight ratio of (15-43):(5-35):(10-30):(8-12):(10-30):(200-250), inoculating Lactobacillus rhamnosus Lr-Ca011 for fermentation, subjecting to high-pressure homogenization, drying and pulverizing to obtain the postbiotics.

[0014] Preferably, the pressure of the high-pressure homogenization is 1000-1500 bar, the time is 5-15 min, and the number of times is 1-4 times.

[0015] The present invention also provides a calcium product prepared from the above postbiotics, comprising the following raw materials in parts by weight: 40-50 parts of oil, 25-35 parts of calcium citrate, 3-7 parts of stabilizer, 2-5 parts of postbiotics, 1-3 parts of emulsifier and 10-20 parts of purified water.

[0016] Preferably, the oil is one or more of conjugated linoleic acid glyceride, medium-chain triglyceride, caprylic capric triglyceride, cocoa butter, anhydrous butter, walnut oil, linseed oil; the emulsifier is one or more of whey protein, casein, soy protein isolate, pasteurized egg yolk liquid, lecithin; the stabilizer is one or more of pectin, L-arabinic gum, carrageenan, xanthan gum, gelatin, konjac powder.

[0017] The present invention also provides a method for preparing the above calcium product by nano-emulsification homogenization, comprising: mixing oil, emulsifier, postbiotics and calcium citrate to obtain an oil phase; mixing the stabilizer and purified water to obtain a water phase; mixing and dispersing the oil phase and the water phase, and subjecting to high-pressure homogenization to obtain the calcium product; the pressure of the high-pressure homogenization is 100-160 MPa, the time is 10-20 min, and the number of times is 1-4 times.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention provides a Lactobacillus rhamnosus Lr-Ca011, which is deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, and the deposit number is CGMCC No. 32037, which can promote calcium absorption.

[0020] The present invention also provides a postbiotic fermented by Lactobacillus rhamnosus Lr-Ca011, which has a promoting effect on calcium absorption. Compared with other Lactobacillus rhamnosus (such as CICC ® 6162, CICC ® 6158, CICC ® 22173, CICC ® 6139, CICC® 6241, CICC ® Postbiotics prepared by fermentation such as 6135, etc. The postbiotics of the present invention have a better effect of promoting calcium absorption.

[0021] The present invention uses nanoemulsion homogenization technology to prepare calcium products. After mixing the aqueous phase and the oil phase containing the postbiotic of Lactobacillus rhamnosus Lr-Ca011 evenly, a coarse emulsion is dispersed, and then the droplets in the coarse emulsion are further fully broken into more stable nanoparticles by high pressure to form a nanoemulsion. This can greatly reduce the use of surfactants, effectively reduce the particle size of the emulsion, enhance the stability of the emulsion system, and at the same time produce a pleasant taste. Due to its small particle size, the nanoemulsion can effectively prevent the separation of the emulsion, has the function of quickly transporting bioactive components into the cell membrane, can well overcome the disadvantages of poor water solubility, low stability and easy oxidation, effectively control the release of functional components, make the calcium product have the characteristic of high absorption rate, and improve the biological utilization rate of calcium. The test results show that the calcium absorption promotion effect of the calcium product of the present invention is 3.18 times that of calcium carbonate tablets with the same calcium content.

[0022] The Lactobacillus rhamnosus Lr-Ca011 of the present invention is classified and named as Lactobacillus rhamnosus Lacticaseibacillus rhamnosus , deposited in the General Microbiological Culture Collection Center of the China Committee for Culture Collection of Microorganisms, with the deposit number CGMCC No.32037, the deposit date is September 23, 2024, and the deposit address is the Institute of Microbiology, Chinese Academy of Sciences. Detailed implementation mode

[0023] The present invention provides a Lactobacillus rhamnosus Lr-Ca011 that promotes calcium absorption, which is classified and named as Lactobacillus rhamnosus Lacticaseibacillus rhamnosus , deposited in the General Microbial Center of the China Committee for Culture Collection of Microorganisms, the deposit date is September 23, 2024, and the deposit number is CGMCC No.32037.

[0024] The Lactobacillus rhamnosus Lr-Ca011 of the present invention is derived from the intestinal tract of healthy adults.

[0025] The Lactobacillus rhamnosus Lr-Ca011 of the present invention can effectively improve the absorption rate and retention rate of calcium in the human body, and has a very significant effect on promoting human bone health.

[0026] The present invention also provides the application of the above-mentioned Lactobacillus rhamnosus Lr-Ca011 in the preparation of foods or health products that promote calcium absorption.

[0027] The food of the present invention is preferably postbiotics, and the health product is preferably a calcium product.

[0028] The present invention also provides a postbiotic containing the above-mentioned Lactobacillus rhamnosus Lr-Ca011.

[0029] The postbiotic of the present invention comprises the following raw materials: Lactobacillus rhamnosus Lr-Ca011, quinoa, potato, pumpkin seed kernel, wolfberry fruit, glucose monohydrate and purified water.

[0030] The present invention also provides a preparation method of the above-mentioned postbiotic, comprising: mixing quinoa, potato, pumpkin seed kernel, wolfberry fruit, glucose monohydrate and purified water according to a weight ratio of (15-43):(5-35):(10-30):(8-12):(10-30):(200-250), inoculating Lactobacillus rhamnosus Lr-Ca011 for fermentation, subjecting to high-pressure homogenization, drying and pulverizing to obtain the postbiotic.

[0031] The pressure of the high-pressure homogenization in the present invention is preferably 1000-1500 bar, more preferably 1200 bar, the time is preferably 5-15 min, more preferably 10 min, and the number of times is preferably 1-4 times, more preferably 2 times.

[0032] The quinoa of the present invention is rich in protein and has a balanced proportion, contains all natural amino acids, and is also rich in minerals, vitamins and various functional components (such as flavonoids, polyphenols, quercetin, rutin, etc.). It is a whole-nutrition food that can meet the basic nutritional needs of humans and is known as the "nutritional gold". Among them, the high content of lysine can promote the absorption of calcium. Potatoes are rich in B vitamins, a large number of trace elements, high-quality protein, amino acids, fat and high-quality starch and other nutrients. Among them, the protein composition is closest to animal protein, and the protein and vitamin C contents are both 10 times that of apples, with strong antioxidant properties. It can not only provide nutrition for the growth of probiotics, promote the proliferation of probiotics, but also protect the strains during freeze-drying and improve the survival rate of the strains. Pumpkin seed kernel is a plant seed kernel mainly composed of fat and protein and contains various nutrients. It contains not only 17 kinds of amino acids, but also various minerals such as zinc, selenium, manganese, copper, iron, calcium, phosphorus, magnesium, potassium and sodium. The elements with relatively high contents among these are Zn, Fe, K and Ca, which can promote the growth, development and intellectual growth of the human body and enhance the immune function. One of the main active components in wolfberry fruit is wolfberry polysaccharide, which has a prebiotic-like effect, can quickly promote the proliferation of probiotics, induce the production of short-chain fatty acids. The monosaccharides that make up wolfberry polysaccharide mainly include glucose, rhamnose, galactose, arabinose, mannose, fucose and xylose. Among them, the contents of glucose, galactose and arabinose are relatively high. In addition, there are also sucrose, raffinose, ethyl-β-D-glucopyranoside, 6-phosphogluconic acid, etc.

[0033] When preparing postbiotics with Lactobacillus rhamnosus Lr-Ca011 of the present invention, a new type of culture medium is used for sufficient fermentation. The culture medium not only contains a variety of nutrients such as rich high-quality proteins, amino acids, vitamins, minerals, lipids, and carbohydrates, which can provide sufficient nutrition for the growth and metabolism of Lr-Ca011, but also contains prebiotic-like natural components, which can promote the massive proliferation of Lr-Ca011. In addition, the culture medium contains a variety of functional sugars, and without the need to add additional cryoprotectants, it can reduce the irreversible damage caused by ice crystals to the bacterial cells during freeze-drying, enabling the strain to be safely in a dormant state and protecting the activity of the strain. The obtained bacterial powder is rich in active Lr-Ca011 and its metabolites, and is beneficial to promoting calcium absorption after being consumed by the human body.

[0034] The present invention also provides a calcium product containing the above postbiotics, comprising the following raw materials in parts by weight: 40-50 parts of oil, 25-35 parts of calcium citrate, 3-7 parts of stabilizer, 2-5 parts of postbiotics, 1-3 parts of emulsifier, and 10-20 parts of purified water.

[0035] The oil in the present invention is preferably one or more of conjugated linoleic acid glyceride, medium-chain triglyceride, caprylic capric triglyceride, cocoa butter, anhydrous butter, walnut oil, and linseed oil, more preferably medium-chain triglyceride; the emulsifier is one or more of whey protein, casein, soy protein isolate, pasteurized egg yolk liquid, and lecithin, more preferably whey protein; the stabilizer is preferably one or more of pectin, L-arabinic gum, carrageenan, xanthan gum, gelatin, and konjac powder, more preferably xanthan gum.

[0036] The present invention also provides a method for preparing the calcium product by nanoemulsion homogenization, comprising: mixing the oil, emulsifier, postbiotics, and calcium citrate to obtain an oil phase; mixing the stabilizer and purified water to obtain a water phase; mixing and dispersing the oil phase and the water phase, and then performing high-pressure homogenization to obtain the calcium product; the pressure of the high-pressure homogenization is 100-160 MPa, the time is 10-20 min, and the number of times is 1-4 times. The pressure of the high-pressure homogenization is preferably 130 MPa, the time is preferably 13 min, and the number of times is preferably 3 times.

[0037] The nanoemulsion homogenization technology of the present invention first prepares an aqueous phase and an oil phase in a certain proportion and mixes them evenly. The preliminary dispersion is carried out by a high-speed disperser to obtain a coarse emulsion, and then the droplets in the coarse emulsion are further broken into more stable nanoparticles by high pressure to form a nanoemulsion. This can greatly reduce the use of surfactants, effectively reduce the particle size of the emulsion, enhance the stability of the emulsion system, and at the same time produce a pleasant taste. Due to its small particle size, the nanoemulsion can effectively prevent the separation of the emulsion, has the function of quickly transporting bioactive components into the cell membrane, can well overcome the disadvantages of poor water solubility, low stability and easy oxidation, effectively control the release of functional components, make calcium products have the characteristics of high absorption rate, and improve the biological utilization rate of calcium.

[0038] In the present invention, unless otherwise specified, all raw material components are commercially available products well-known to those skilled in the art.

[0039] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the scope of protection of the present invention.

[0040] In the following embodiments, unless otherwise specified, all are conventional methods.

[0041] The materials, reagents, etc. used in the following embodiments can be obtained from commercial sources unless otherwise specified.

[0042] Examples 1-6

[0043] Preparation of Lactobacillus rhamnosus Lr-Ca011 bacterial powder

[0044] 1. Select quinoa, pumpkin seeds that are dry, free of mildew and insect damage, wash and drain; select fresh, non-germinated potatoes, peel and cut into pieces; select qualified goji berries and monohydrate glucose.

[0045] 2. Liquid material 1: Weigh several parts of quinoa, potatoes, pumpkin seeds, goji berries, and monohydrate glucose, with the weight parts shown in Table 1, mix them evenly, place them in a wall breaker, add 235 parts by weight of purified water, break the wall at 35000 r / min for 3 min, filter with an 80-mesh filter, take the filtrate, sterilize at 121 °C for 20 min, and cool to 35 °C to obtain.

[0046] 3. Bacterial liquid: Take out the freeze-dried preservation tube of Lactobacillus rhamnosus Lr-Ca011, let it stand at room temperature for 1 h, suck 0.6 mL of normal saline, add it to the freeze-dried preservation tube, shake until the contents are dissolved, suck 10 μL, spread it on the MRS solid medium, culture at 35 °C for 36 h, pick 2 loops of colonies with an inoculation loop, inoculate them onto 10 mL of MRS liquid medium, culture at 35 °C for 36 h, and adjust the bacterial liquid concentration to 5×10 9 CFU / mL, thus obtaining it.

[0047] 4. Add it to Feed Liquid 1 according to the addition amount of 10% (volume ratio) of the bacterial liquid, mix evenly, culture at 35 °C for 72 h, then freeze-dry with a vacuum freeze dryer, crush the obtained product, pass through an 80-mesh sieve, and the collected powder is the bacterial powder.

[0048] Table 1 Raw material configuration of Feed Liquid 1 in Examples 1-6 (parts by weight)

[0049]

[0050] Test Example 1

[0051] Calcium absorption experiment test

[0052] 1. Experimental animals: SPF-grade healthy male ICR mice, weighing 40 g - 50 g.

[0053] 2. Randomly divide the mice into 13 groups, with 10 mice in each group, denoted as Group A - Group M. Groups B - M are all given intraperitoneal injection of D-galactose at 120 mg / (kg·d) for 56 consecutive days to establish an aging osteoporosis mouse model, and Group A is given intraperitoneal injection of the same dose of normal saline.

[0054] With the increase of age, the intestinal absorption rate of calcium usually shows a gradually decreasing trend, leading to the appearance of osteoporosis symptoms. D-galactose-induced male mouse osteoporosis is a relatively practical animal model for studying senile osteoporosis. After continuous injection of D-galactose, excessive oxygen free radicals are produced in male mice, which damage collagen, resulting in oxidative damage of various cells, affecting the normal metabolism of osteocytes, causing mineral loss, and accelerating the aging of male mice.

[0055] 3. After the modeling is completed, gavage the mice with drugs, the gavage volume is 1 mL / 100 g bw, for 28 consecutive days. The drug administration situation is shown in Table 2. All gavage samples are dissolved with sterile normal saline, and the gavage inorganic calcium source is CaCl2. During the drug administration period, the mouse feed is standard feed and the drinking water is deionized water.

[0056] Table 2 Mouse grouping and drug administration situation

[0057]

[0058] 4. On the 15th day of administration, all the mice were transferred to a dedicated metabolic cage. On the 26th, 27th, and 28th days, the feces and urine of each test mouse were collected. The calcium contents in the ingested feed, collected feces, and urine were determined by the flame atomic absorption spectrometry method in "GB 5009.92-2016 National Food Safety Standard - Determination of Calcium in Foods", and calculated according to the following formulas to obtain the average values of calcium absorption rate (%) and retention rate (%). The results are shown in Table 3.

[0059] (1) Intake calcium = Calcium amount in feed + Calcium amount by gavage;

[0060] (2) Absorbed calcium = Intake calcium amount - Fecal calcium amount;

[0061] (3) Calcium absorption rate = Absorbed calcium / Intake calcium × 100%;

[0062] (4) Retained calcium = Intake calcium amount - Fecal calcium amount - Urine calcium amount;

[0063] (5) Retention rate = Retained calcium / Intake calcium.

[0064] Table 3 Calcium absorption and retention in mice

[0065]

[0066] As can be seen from Table 3, compared with group A, the calcium absorption rate and retention rate of mice in group B were significantly reduced, indicating that the senile mouse model was successfully established and the intestinal calcium absorption ability was significantly decreased. Compared with group B, the calcium absorption rate and retention rate of mice in groups C - H increased to varying degrees, indicating that the Lactobacillus rhamnosus Lr-Ca011 powder had a promoting effect on calcium absorption. Due to the differences in the culture medium formulations, the effects of the powder on promoting calcium absorption were different. Among them, the calcium absorption rate and retention rate of mice in group H were the highest, indicating that the powder in Example 6 had the best effect on promoting calcium absorption. Therefore, Example 6 was selected as the optimal formulation of the culture medium, and the obtained Lactobacillus rhamnosus Lr-Ca011 powder could promote calcium absorption.

[0067] Examples 7 - 11

[0068] Preparation of postbiotics

[0069] 1. Select quinoa and pumpkin seeds that are dry, free from mildew and insect damage, wash and drain; select fresh and non-germinated potatoes, peel and cut into pieces; select qualified wolfberries and glucose monohydrate.

[0070] 2. Liquid material 1: Weigh 33 parts by weight of quinoa, 20 parts by weight of potato, 17 parts by weight of pumpkin seed kernels, 10 parts by weight of wolfberries, and 20 parts by weight of glucose monohydrate. Mix them evenly, place them in a wall breaker, add 235 parts by weight of purified water, break the wall at 35000 r / min for 3 minutes, filter with an 80-mesh filter screen, take the filtrate, sterilize it at 121 °C for 20 minutes, and cool it to 35 °C to obtain it.

[0071] 3. Bacterial liquid: Take out the freeze-dried preservation tube of Lactobacillus rhamnosus Lr-Ca011, let it stand at room temperature for 1 hour, suck 0.6 mL of physiological saline, add it to the freeze-dried preservation tube, shake until the content dissolves, suck 10 μL, spread it on the MRS solid medium, culture it at 35 °C for 36 hours, pick 2 loops of colonies with an inoculation loop, inoculate them into 10 mL of MRS liquid medium, culture it at 35 °C for 36 hours, and adjust the bacterial liquid concentration to 5×10 9 CFU / mL to obtain it.

[0072] 4. Add the bacterial liquid to Liquid material 1 according to the addition amount of 10% (volume ratio), mix evenly, culture it at 35 °C for 72 hours, homogenize the obtained product with a high-pressure homogenizer, the homogenization conditions are 1200 bar and the time is 10 minutes, repeat the homogenization, the number of homogenization times is shown in Table 4, then freeze-dry it with a vacuum freeze dryer, pulverize the obtained product with a pulverizer, and pass it through an 80-mesh sieve. The collected powder is the postbiotic.

[0073] Table 4 Homogenization times of postbiotics in different examples

[0074]

[0075] Experimental example 2

[0076] 1. Select mice (SPF-grade healthy male ICR mice, weighing 40 g - 50 g) modeled by the method of Experiment 1, divide them into 5 groups, with 10 mice in each group. Administer drugs to the mice by gavage, the gavage volume is 1 mL / 100 g bw, for 28 consecutive days. The drug administration situation is shown in Table 5. All gavage samples are dissolved with sterile physiological saline, and the inorganic calcium source for gavage is CaCl2. During the drug administration period, the mouse feed is standard feed and the drinking water is deionized water.

[0077] Table 5 Grouping and drug administration situation of mice

[0078]

[0079] 2. On the 15th day of administration, all the mice were transferred to a dedicated metabolic cage. On the 26th, 27th, and 28th days, the feces and urine of each test mouse were collected. The calcium contents in the ingested feed, collected feces, and urine were determined by the flame atomic absorption spectrometry method of "GB 5009.92-2016 National Food Safety Standard - Determination of Calcium in Foods". The average values of calcium absorption rate (%) and retention rate (%) were calculated, and the results are shown in Table 6.

[0080] Table 6 Calcium Absorption and Retention in Mice

[0081]

[0082] From the data in Table 3 and Table 6, it can be seen that compared with Group A, the calcium absorption rate and retention rate of mice in Groups I - L increased to varying degrees, indicating that postbiotics have a promoting effect on calcium absorption. The calcium absorption rate and retention rate of mice in Groups I - K continuously increased, indicating that when the homogenization times ≤ 2 times, the more the homogenization times, the better the effect of promoting calcium absorption. The calcium absorption rate and retention rate of mice in Groups K - M were relatively close, indicating that when the homogenization times > 2 times, further increasing the homogenization times did not significantly increase the effect of promoting calcium absorption. That is, when the homogenization times were 2 times, the effect of postbiotics promoting calcium absorption basically reached saturation. Therefore, 2 times of homogenization was selected as the optimal number of homogenization times for postbiotics.

[0083] Comparative Examples 1 - 6

[0084] Six different Lactobacillus rhamnosus strains were used to prepare postbiotics according to the method of Example 9 (compared with Example 9, only the Lactobacillus rhamnosus strains were different). The six different Lactobacillus rhamnosus strains are shown in Table 7.

[0085] Table 7 Lactobacillus rhamnosus Strains of Comparative Examples 1 - 6

[0086]

[0087] The Lactobacillus rhamnosus strains used in Comparative Examples 1 - 6 were all from the China Center for Industrial Culture Collection.

[0088] Test Example 3

[0089] 1. Mice (SPF - grade healthy male ICR mice, weighing 40 g - 50 g) were modeled by the method of Test 1 and divided into 7 groups with 10 mice in each group. Each group of mice was given gavage administration with a gavage volume of 1 mL / 100 g bw for 28 consecutive days. The administration situation is shown in Table 8. All gavage samples were dissolved in sterile normal saline, and the gavage inorganic calcium source was CaCl2. During the administration period, the mouse feed was standard feed and the drinking water was deionized water.

[0090] Table 8 Grouping and Administration of Mice

[0091]

[0092] 2. On the 15th day of administration, all the mice were transferred to a dedicated metabolic cage. On the 26th, 27th, and 28th days, the feces and urine of each test mouse were collected, and the calcium contents in the ingested feed, collected feces, and urine were determined by the flame atomic absorption spectrometry method of "GB 5009.92-2016 National Food Safety Standard Determination of Calcium in Foods". The average values of calcium absorption rate (%) and retention rate (%) were calculated, and the results are shown in Table 9.

[0093] Table 9 Calcium Absorption and Retention in Mice

[0094]

[0095] From the data in Table 9, it can be seen that postbiotics prepared from Lactobacillus casei rhamnosus from different sources have different effects on calcium absorption. Among them, the postbiotics obtained by the preparation method of Example 6 of the present invention can significantly improve the calcium absorption rate and retention rate of mice. Obviously, the Lactobacillus casei rhamnosus Lr-Ca011 of the present invention has a better effect on promoting calcium absorption compared with other similar Lactobacillus casei rhamnosus.

[0096] Examples 12 - 16

[0097] Preparation of Calcium Products

[0098] 1. Select qualified soybean oil, medium-chain triglycerides, xanthan gum, postbiotics, and whey protein; select qualified calcium citrate.

[0099] 2. Coarse emulsion: Weigh 45.5 parts by weight of medium-chain triglycerides, add 1.5 parts by weight of whey protein while stirring, continue stirring for 10 min, then add 3 parts by weight of the postbiotics of Example 9, continue stirring for 10 min, then add 30 parts by weight of calcium citrate, continue stirring for 3 h, and filter through a 100-mesh filter screen. The collected filtrate is the oil phase; add 5 parts by weight of xanthan gum to 15 parts by weight of purified water at 100 °C and stir to dissolve. The resulting solution is the water phase. Mix the oil phase and the water phase evenly, and shear with a high-speed disperser at 8000 rpm for 3 min. The resulting product is the coarse emulsion.

[0100] 3. Homogenize the coarse emulsion with a high-pressure homogenizer at a temperature of 25 °C. The homogenization pressure is shown in Table 10. Homogenize 3 times, 13 min each time. The resulting product is the calcium product.

[0101] Table 10 Homogenization Pressures of Calcium Products in Different Examples

[0102]

[0103] Test Example 4

[0104] The particle sizes of the calcium products in Examples 12 - 16 were measured using a nano particle size and zeta potential analyzer. Each product was measured 3 times, and the average value was calculated. The results are shown in Table 11.

[0105] Table 11 Influence of pressure on nano particle size

[0106]

[0107] As can be seen from Table 11, the pressure has a significant influence on the particle size of the calcium product, but it does not continuously decrease with the increase of pressure. The particle size of the nanoemulsion first decreases and then increases with the continuous increase of pressure. The calcium product in Example 15 has the smallest particle size, that is, when the pressure is 130 MPa, the calcium product reaches the minimum particle size.

[0108] Test Example 5

[0109] Human test

[0110] 1. Volunteers aged 12 - 17 were recruited as subjects to participate in the test. They were randomly divided into 5 groups, denoted as Group A - Group E, with 8 people in each group. Calcium was supplemented in the form of calcium products. The grouping and consumption situation are shown in Table 12. All subjects met the following conditions: willing to participate, in good health, and without liver, kidney diseases and skeletal system diseases that affect bone metabolism.

[0111] Table 12 Subject grouping and sample consumption situation

[0112]

[0113] 2. It was consumed 3 times a day, 1 hour after meals, for 10 consecutive days. The subjects lived in a centralized accommodation and could not consume any food and beverages other than the experimental food provided. The subjects were first given 3 days to adapt to the experimental diet, and then, with a 3 - day cycle for each recipe, they consumed the experimental diet for 7 days. The diet of the subjects for 10 days (including meals, snacks and drinking water) was collected.

[0114] 3. 2 capsules (200 mg) of carmine capsules were consumed before breakfast on the 4th day and the 11th day respectively to mark the feces. Starting from when the feces turned red, the feces of each subject were collected until the red color appeared again in the feces on the 11th day. The feces were collected once every 24 hours.

[0115] 4. The urine of the subjects for 24 hours every day was collected and 1% hydrochloric acid solution was added and mixed evenly.

[0116] 5. The calcium content in diet, collected feces and urine was determined by flame atomic absorption spectrometry according to the "National Food Safety Standard Determination of Calcium in Foods GB 5009.92-2016", and the average values of calcium absorption rate (%) and retention rate (%) were calculated. The results are shown in Table 13.

[0117] Table 13 Calcium Absorption and Retention of Subjects

[0118]

[0119] As can be seen from Table 13, among Group A - Group E, the subjects in Group C had the highest calcium absorption rate and retention rate, indicating that the calcium product in Example 14 had the best effect on promoting calcium absorption. Combining with the experimental results in Table 11, the particle size of Group C was larger than that of Group D, indicating that it was not the smaller the particle size, the better the calcium absorption effect. The calcium product with the optimal particle size had the best effect on promoting calcium absorption. Therefore, Example 14 was selected as the optimal pressure for homogenization of the calcium product, and the obtained calcium product had a high absorption rate of calcium element.

[0120] Test Example 6

[0121] Calcium Absorption Rate Test of Rats

[0122] 1. Sample preparation: Mix 70 parts by weight of sorbitol and 30 parts by weight of calcium carbonate, and make tablet candies by direct compression. Dissolve the tablet candies in purified water to make a 50% concentration solution, denoted as Solution A.

[0123] 2. Experimental animals: SD rats, female, 10 - 12 weeks old.

[0124] 3. After 7 days of adaptive feeding, 18 rats were divided into 3 experimental groups, named model control group, experimental group 1 and experimental group 2, with 6 rats in each group.

[0125] 4. Osteoporosis modeling was performed on the rats: Anesthetize, prepare the ventral skin by shaving, depilating and disinfecting, make an incision about 2 cm long in the mid - ventral line using a sterile surgical scissor, separate and expose the ovaries, ligate with suture and then remove both ovaries. Replace the uterus into the abdominal cavity, suture the muscle and skin layer by layer, disinfect the wound with iodophor and place it on a heating pad to wait for the animal to wake up and then raise it normally.

[0126] 5. After the modeling surgery, drug administration started the next day. Experimental group 1 was given 1 mL of the calcium product in Example 14 by gavage daily, experimental group 2 was given 2 mL of Solution A by gavage daily, and the model control group was given 1 mL of normal saline by gavage daily for 4 consecutive weeks.

[0127] 6. After the last administration was completed, the rats in each group were euthanized, and the femurs were removed. The muscles and connective tissues around the femurs were removed. After carbonization on the furnace, they were placed in a muffle furnace at 800 °C for ashing for 2 h. After cooling to room temperature, they were placed in a desiccator and the ash weight was weighed with an analytical balance. 0.1 g of bone ash was weighed for each rat, 10 mL of 5% nitric acid solution was added and transferred to a 25-mL colorimetric tube. It was placed at room temperature for 48 h for digestion, and the solution became colorless and transparent. The digestion solution was taken to measure the absorbance, and at the same time, a reagent blank was made. The calcium content in the sample was obtained from the standard curve, and the average value of each group was calculated, reserved to 2 decimal places, and rounded off by the rounding method. The results are shown in Table 14.

[0128] Table 14 Bone calcium absorption of rats in each group

[0129]

[0130] As can be seen from Table 14, the ratio of the improvement rates between Experimental Group 1 and Experimental Group 2 is about 3.18, indicating that the calcium product of Example 14 has a significant effect on promoting bone calcium absorption, and its improvement effect is 3.18 times that of calcium carbonate tablets with the same calcium content.

[0131] The above are only the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A postbiotic prepared by fermentation, characterized in that: The raw materials are: Lactobacillus rhamnosus Lr-Ca011 that promotes calcium absorption, quinoa, potatoes, pumpkin seeds, wolfberries, glucose monohydrate and purified water; the classification of the Lactobacillus rhamnosus Lr-Ca011 is named Lactobacillus rhamnosus (Lacticaseibacillus rhamnosus) , deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, the deposit date is September 23, 2024, and the deposit number is CGMCCNo.32037.

2. A method for preparing the postbiotics according to claim 1, characterized in that: include: Quinoa, potato, pumpkin seed kernel, wolfberry, glucose monohydrate and purified water are mixed in a weight ratio of (15-43):(5-35):(10-30):(8-12):(10-30):(200-250), inoculated with Lactobacillus rhamnosus Lr-Ca011 for fermentation, homogenized under high pressure, dried and crushed to obtain the postbiotics.

3. The preparation method according to claim 2, characterized in that: The high pressure homogenization is performed at a pressure of 1000-1500 bar, for a time of 5-15 minutes, and for 1-4 times.

4. A calcium product prepared from the postbiotics according to claim 1, characterized in that: The calcium product is composed of the following raw materials in parts by weight: 40-50 parts of oil, 25-35 parts of calcium citrate, 3-7 parts of stabilizer, 2-5 parts of postbiotics, 1-3 parts of emulsifier and 10-20 parts of purified water; The fat is medium-chain triglyceride; the emulsifier is whey protein; the stabilizer is xanthan gum.

5. A method for preparing a calcium product by nanoemulsification and homogenization according to claim 4, characterized in that: include: The oil, emulsifier, postbiotics and calcium citrate are mixed to obtain an oil phase; the stabilizer is mixed with purified water to obtain an aqueous phase; the oil phase and the aqueous phase are mixed and dispersed, and then high-pressure homogenized to obtain the calcium product; the pressure of the high-pressure homogenization is 100-160MPa, the time is 10-20min, and the number of times is 1-4 times.

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