CN115039887A - Probiotics composition with functions of muscle and bone enhancement - Google Patents

Probiotics composition with functions of muscle and bone enhancement Download PDF

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CN115039887A
CN115039887A CN202210971149.9A CN202210971149A CN115039887A CN 115039887 A CN115039887 A CN 115039887A CN 202210971149 A CN202210971149 A CN 202210971149A CN 115039887 A CN115039887 A CN 115039887A
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freeze
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张雪松
李华文
范雅彬
付鹏飞
梁立贺
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Yuanmin Biotechnology Shandong Co ltd
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Abstract

The invention discloses a probiotic flora composition with functions of muscle and bone enhancement and belongs to the technical field of probiotic flora compositions, wherein the probiotic flora composition comprises the following components in parts by weight: 85-90 parts of load bacterial powder, 4-6 parts of auxiliary materials and 6-9 parts of modified calcium; the preparation method of the load bacterial powder comprises the following steps: preparing seed liquid, preparing fermentation liquor, preparing a freeze-drying protective agent, drying and loading; the preparation method of the probiotic flora composition with the muscle and bone increasing and protecting functions comprises the steps of uniformly mixing the load bacteria powder, the auxiliary material and the modified calcium, and then carrying out vacuum freeze drying to obtain the probiotic flora composition with the muscle and bone increasing and protecting functions; the probiotic composition disclosed by the invention can improve the effects of muscle and bone enhancement, avoid the dependence on probiotics caused by long-term use, ensure that calcium and the probiotics can be used together, and improve the number of viable bacteria in the probiotic composition.

Description

Probiotics composition with functions of muscle and bone enhancement
Technical Field
The invention relates to a probiotic flora composition, in particular to a probiotic flora composition with functions of muscle and bone enhancement.
Background
The human microflora consists of microorganisms that live in the human body and the molecular products of these microorganisms, the organisms in the microflora including bacteria, archaea and unicellular eukaryotes, which are collectively referred to as the microflora. In the human body, the vast majority of microbial populations are present in the gastrointestinal tract, which is composed of 8000 different species of microorganisms. Recent studies have shown that changes in microbiota are associated with several chronic clinical conditions, including obesity and diabetes, heart disease and alzheimer's disease, and through studies on host-microbe interactions, it has been determined that changes in microbiota have effects on the human body, such as regulating nutrient absorption, and that molecular products of microbes and bacteria cross the endothelial barrier into the systemic circulation, and thus, the health-care function of probiotics is also increasingly being emphasized.
The existing health food or health medicine for enhancing muscle function and protecting bones mainly supplements amino acid and calcium, but the excessive supplementation of the amino acid can increase the toxic and side effect of liver and kidney, and the excessive supplementation of the calcium can increase the risk of kidney stone.
The amino acid produced by the fermentation of the probiotics has the functions of reducing muscle protein loss and enhancing muscle function, and the probiotics can also play a role in promoting intestinal peristalsis, thereby improving the absorption of calcium and protecting bones, but the probiotics have the following problems when used for enhancing muscles and protecting bones: the long-term use of the probiotic product for muscle enhancement and bone protection can cause the intestinal tract to gradually lose the function of self-reproduction probiotics, so that the dependence on the probiotics is formed, and once the use is stopped, the intestinal tract function is abnormal, and diarrhea is caused; the probiotics can only promote the absorption of calcium and cannot achieve the purpose of calcium supplement, in order to protect bones, calcium needs to be supplemented additionally, but the calcium is directly added into the probiotics, and organic acid generated by fermentation of the probiotics can cause the precipitation of the calcium, so that the absorption of the calcium is influenced; in order to prolong the preservation time of probiotics and realize normal-temperature preservation, the probiotics are generally prepared into tablets or powder by the technologies of microcapsule embedding, freeze-crystal drying and the like, but high-temperature drying is needed in the process of preparing the tablets or powder, and the probiotics are sensitive to temperature and humidity, so that the viable count of the probiotics is reduced.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides the probiotic flora composition with the functions of muscle and bone enhancement and improvement, which can improve the muscle and bone enhancement effect, avoid the dependence on probiotics caused by long-term use, ensure that calcium and the probiotics can be used together, and improve the viable count in the probiotic flora composition.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
the probiotic flora composition with the functions of muscle and bone enhancement comprises the following components in parts by weight: 85-90 parts of load bacterial powder, 4-6 parts of auxiliary materials and 6-9 parts of modified calcium;
the preparation method of the load bacterial powder comprises the following steps: preparing seed liquid, preparing fermentation liquor, preparing a freeze-drying protective agent, drying and loading;
the method for preparing the seed liquid comprises the following steps: respectively marking and activating lactobacillus plantarum, lactobacillus rhamnosus, lactobacillus paracasei, lactobacillus acidophilus and bifidobacterium longum which are frozen and stored on a solid LB plate, then respectively selecting single bacterial colonies from the LB plate and inoculating the single bacterial colonies into 250mL conical flasks filled with 100mL of liquid culture medium, putting the 250mL conical flasks into a shaking table for shaking table culture, controlling the temperature of the shaking table culture to be 36-38 ℃, the rotating speed to be 220 plus 250rpm, and the time to be 14-15h, and respectively obtaining lactobacillus plantarum seed liquid, lactobacillus rhamnosus seed liquid, lactobacillus paracasei seed liquid, lactobacillus acidophilus seed liquid and bifidobacterium longum seed liquid after the shaking table culture is finished;
the method for preparing the fermentation liquor comprises the following steps: respectively inoculating lactobacillus plantarum seed liquid, lactobacillus rhamnosus seed liquid, lactobacillus paracasei seed liquid, lactobacillus acidophilus seed liquid and bifidobacterium longum seed liquid in the same MRS liquid culture medium in sequence, controlling the inoculation amount of lactobacillus plantarum to be 1.5-2%, the inoculation amount of lactobacillus rhamnosus to be 1-1.5%, the inoculation amount of lactobacillus paracasei to be 1.5-2%, the inoculation amount of lactobacillus acidophilus to be 0.5-1% and the inoculation amount of bifidobacterium longum to be 1-2%, performing stirring fermentation culture after the inoculation is completed, controlling the temperature of the stirring fermentation culture to be 36-38 ℃, controlling the rotating speed to be 320-plus 350rpm, controlling the ventilation volume to be 1.5-2vvm, and controlling the time to be 17-18h, and obtaining fermentation liquor after the stirring fermentation culture is completed;
the MRS liquid culture medium consists of the following components: 6-7g/L tryptone, 5-6g/L glucose, 4-6g/L beef extract, 3-5g/L yeast extract, 1.5-2g/L sodium citrate, 0.5-1g/L Tween 80, 0.2-0.5g/L manganese sulfate, 0.1-0.15g/L rhamnolipid and the balance of deionized water;
the method for preparing the freeze-drying protective agent comprises the following steps: mixing sucrose, beta-glucan, algal polysaccharide, sodium chloride, maltodextrin and skim milk powder, then carrying out ball milling, controlling the ball-material ratio during ball milling to be 8-10:1, the ball milling rotation speed to be 350-plus-380 rpm, the ball milling time to be 30-35min, obtaining a primary freeze-drying protective agent after ball milling is finished, uniformly mixing the primary freeze-drying protective agent, carrageenan, polyglycerol fatty acid ester and pregelatinized starch, then carrying out low-frequency ultrasonic treatment, controlling the frequency of the low-frequency ultrasonic treatment to be 22-25kHz, controlling the power to be 380-plus-400W, and obtaining the freeze-drying protective agent after the low-frequency ultrasonic treatment is finished;
in the preparation of the freeze-drying protective agent, the weight ratio of sucrose, beta-glucan, algal polysaccharide, sodium chloride, maltodextrin and skimmed milk powder is 10-12:3-5:2-4:0.5-1:1-3: 2.5-4;
in the preparation of the freeze-drying protective agent, the weight ratio of the primary freeze-drying protective agent to the carrageenan to the polyglycerol fatty acid ester to the pregelatinized starch is 18-20:4-6:1-3: 2-4;
the drying method comprises the following steps: carrying out centrifugal separation on the fermentation liquor, controlling the rotation speed of 2800 plus 3000rpm and the time of 5-6min during the centrifugal separation, taking a solid phase obtained after the centrifugal separation as bacterial sludge, uniformly mixing the bacterial sludge and a freeze-drying protective agent according to the weight ratio of 90-92:6-8, carrying out vacuum freeze-drying treatment, controlling the temperature of the vacuum freeze-drying treatment to be-35 ℃ to-32 ℃, the vacuum degree to be 800 plus 900Pa and the treatment time to be 50-55min, and obtaining bacterial powder after the vacuum freeze-drying treatment;
the load method comprises the following steps: mixing resistant dextrin, ferric pyrophosphate, tricalcium phosphate and polyvinylpyrrolidone, then performing microwave oscillation, controlling the intensity of the microwave oscillation to be 60-80W, controlling the time of the microwave oscillation to be 20-25min, obtaining a carrier after the microwave oscillation is finished, adding the carrier and the bacterial powder into a reaction container, and stirring at the stirring speed of 80-100rpm for 20-25min at the temperature of 12-15 ℃ to obtain load bacterial powder;
in the load, the weight ratio of the resistant dextrin to the ferric pyrophosphate to the tricalcium phosphate to the polyvinylpyrrolidone is 8-10:3-5:1-2: 2-4;
in the load, the weight ratio of the bacterial powder to the carrier is 7-8: 10;
the total amount of probiotics in the load bacterium powder is 2.2 multiplied by 10 10 -2.5×10 10 cfu/g；
The auxiliary materials comprise the following components in parts by weight: 10-13 parts of sodium carboxymethylcellulose, 3-5 parts of sodium caseinate, 1-3 parts of fatty acid sucrose polyester and 2-5 parts of glucose;
the preparation method of the modified calcium comprises the following specific steps: adding calcium gluconate, calcium acetate and calcium lactate into a ball mill for ball milling, controlling the ball-material ratio during ball milling to be 15-18:1, the ball milling rotation speed to be 300-320rpm, the ball milling time to be 35-40min, obtaining mixed calcium after the ball milling is finished, uniformly mixing the mixed calcium, low-ester pectin, corn starch, maltose, propylene glycol fatty acid ester and deionized water, then carrying out vacuum spray drying, controlling the vacuum degree in the vacuum spray drying process to be 500-600Pa, the inlet temperature to be 130-135 ℃, the outlet temperature to be 65-70 ℃, obtaining primary modified calcium after the vacuum spray drying is finished, carrying out liquid nitrogen spraying on the primary modified calcium, controlling the outlet pressure of the liquid nitrogen spraying to be 0.4-0.5MPa, the spraying speed to be 3-3.5m/s, the working frequency to be 35-40Hz and the spraying time to be 12-15s, after the liquid nitrogen spraying is finished, naturally recovering to room temperature to obtain modified calcium;
in the preparation of the modified calcium, the weight ratio of calcium gluconate to calcium acetate to calcium lactate is 7-8:3-5: 2-4;
in the preparation of the modified calcium, the weight ratio of the mixed calcium to the low-ester pectin to the corn starch to the maltose to the propylene glycol fatty acid ester to the deionized water is 18-20:5-7:4-6:1-2:0.2-0.5: 48-50.
The preparation method of the probiotic flora composition with the muscle and bone strengthening function comprises the steps of uniformly mixing the load bacteria powder, the auxiliary material and the modified calcium, then carrying out vacuum freeze drying, controlling the temperature of the vacuum freeze drying treatment to be-35-30 ℃, the vacuum degree to be 700-800Pa, the treatment time to be 40-45min, and finally obtaining the probiotic flora composition with the muscle and bone strengthening function after the vacuum freeze drying is finished.
Compared with the prior art, the invention has the beneficial effects that:
(1) the probiotic flora composition with the muscle and bone increasing and protecting functions disclosed by the invention has the advantages that the bacteria powder is loaded, the primary freeze-drying protective agent, the carrageenan, the polyglycerol fatty acid ester and the pre-gelatinized starch are uniformly mixed and then subjected to the low-frequency ultrasonic treatment step, and the primary modified calcium is sprayed with liquid nitrogen in the preparation of the modified calcium, so that the absorption of calcium by probiotics can be promoted, the muscle and bone increasing and protecting effects of the probiotics are improved, the tests are carried out on SPF male SD rats, and after the probiotic flora composition with the muscle and bone increasing and protecting functions is continuously fed for 30 days, the average bone density is 187.1-191.4mg/cm 2 Average gastrocnemius muscle weight/body weight is 5.47-5.52 mg/g;
(2) according to the probiotic flora composition with the muscle and bone increasing and protecting functions, the bacteria powder is loaded, the primary freeze-drying protective agent, the carrageenan, the polyglycerol fatty acid ester and the pre-gelatinized starch are uniformly mixed and then subjected to the low-frequency ultrasonic treatment step, and the primary modified calcium is sprayed with liquid nitrogen in the preparation of the modified calcium, so that the regulation effect of the probiotics can be better exerted, dependence on the probiotics caused by long-term use is avoided, the probiotic flora composition is tested on SPF male SD rats, after the probiotic flora composition with the muscle and bone increasing and protecting functions is continuously fed for 30d, the feeding is stopped for 15d, and no diarrhea occurs;
(3) the probiotic flora composition with the functions of muscle and bone enhancement provided by the invention can ensure that calcium and probiotics can be used together without mutual influence by loading the bacterial powder, uniformly mixing the primary freeze-drying protective agent, carrageenan, polyglycerol fatty acid ester and pregelatinized starch, then carrying out low-frequency ultrasonic treatment, and spraying liquid nitrogen on the primary modified calcium in the preparation of the modified calcium, wherein the total quantity of the probiotics in the probiotic flora composition prepared by the invention is 1.9 multiplied by 10 10 -2.1×10 10 cfu/g, the total amount of probiotics is 1.5 multiplied by 10 after being preserved in the dark for 10 days at the temperature of 2 DEG C 10 -1.6×10 10 cfu/g, the probiotic flora composition prepared by the invention is prepared into a 10% probiotic water solution, and then the probiotic water solution is placed at 25 ℃ and is kept still for 1d without precipitation.
Detailed Description
In order to more clearly understand the technical features, objects, and effects of the present invention, specific embodiments of the present invention will now be described.
Example 1
The probiotic flora composition with the functions of muscle and bone enhancement comprises the following components in parts by weight: 85 parts of load bacterial powder, 4 parts of auxiliary materials and 6 parts of modified calcium;
the preparation method of the load bacterial powder comprises the following steps: preparing seed liquid, preparing fermentation liquor, preparing a freeze-drying protective agent, drying and loading;
the method for preparing the seed liquid comprises the following steps: respectively scribing and activating lactobacillus plantarum, lactobacillus rhamnosus, lactobacillus paracasei, lactobacillus acidophilus and bifidobacterium longum which are frozen and stored on a solid LB plate, then respectively selecting single bacterial colonies from the LB plate and inoculating the single bacterial colonies into 250mL conical flasks filled with 100mL of liquid culture medium, putting the 250mL conical flasks into a shaking table for shaking table culture, controlling the temperature of the shaking table culture to be 36 ℃, the rotating speed to be 220rpm, and the time to be 14h, and respectively obtaining lactobacillus plantarum seed liquid, lactobacillus rhamnosus seed liquid, lactobacillus paracasei seed liquid, lactobacillus acidophilus seed liquid and bifidobacterium longum seed liquid after the shaking table culture is finished;
the method for preparing the fermentation liquor comprises the following steps: respectively inoculating lactobacillus plantarum seed liquid, lactobacillus rhamnosus seed liquid, lactobacillus paracasei seed liquid, lactobacillus acidophilus seed liquid and bifidobacterium longum seed liquid in the same MRS liquid culture medium in sequence, controlling the inoculation amount of lactobacillus plantarum to be 1.5%, the inoculation amount of lactobacillus rhamnosus to be 1%, the inoculation amount of lactobacillus paracasei to be 1.5%, the inoculation amount of lactobacillus acidophilus to be 0.5% and the inoculation amount of bifidobacterium longum to be 1%, carrying out stirring fermentation culture after inoculation is finished, controlling the temperature of the stirring fermentation culture to be 36 ℃, the rotating speed to be 320rpm, the ventilation volume to be 1.5vvm and the time to be 17h, and obtaining fermentation liquor after the stirring fermentation culture is finished;
the MRS liquid culture medium consists of the following components: 6g/L tryptone, 5g/L glucose, 4g/L beef extract, 3g/L yeast extract, 1.5g/L sodium citrate, 0.5g/L Tween 80, 0.2g/L manganese sulfate, 0.1g/L rhamnolipid and the balance of deionized water;
the method for preparing the freeze-drying protective agent comprises the following steps: mixing sucrose, beta-glucan, algal polysaccharide, sodium chloride, maltodextrin and skim milk powder, then carrying out ball milling, controlling the ball-material ratio during ball milling to be 8:1, controlling the ball milling rotation speed to be 350rpm, carrying out ball milling for 30min, obtaining a primary freeze-drying protective agent after ball milling is finished, uniformly mixing the primary freeze-drying protective agent, carrageenan, polyglycerol fatty acid ester and pregelatinized starch, then carrying out low-frequency ultrasonic treatment, controlling the frequency of the low-frequency ultrasonic treatment to be 22kHz, controlling the power to be 380W, and finishing the low-frequency ultrasonic treatment to obtain the freeze-drying protective agent;
wherein the weight ratio of the sucrose, the beta-glucan, the algal polysaccharide, the sodium chloride, the maltodextrin and the skimmed milk powder is 10:3:2:0.5:1: 2.5;
wherein the weight ratio of the primary freeze-drying protective agent to the carrageenan to the polyglycerol fatty acid ester to the pregelatinized starch is 18:4:1: 2;
the drying method comprises the following steps: carrying out centrifugal separation on fermentation liquor, controlling the rotating speed during centrifugal separation to be 2800rpm, controlling the time to be 5min, taking a solid phase obtained after centrifugal separation as bacterial sludge, uniformly mixing the bacterial sludge and a freeze-drying protective agent in a weight ratio of 90:6, carrying out vacuum freeze-drying treatment, controlling the temperature of the vacuum freeze-drying treatment to be-35 ℃, controlling the vacuum degree to be 800Pa, controlling the treatment time to be 50min, and obtaining bacterial powder after the vacuum freeze-drying treatment;
the load method comprises the following steps: mixing resistant dextrin, ferric pyrophosphate, tricalcium phosphate and polyvinylpyrrolidone, then performing microwave oscillation, controlling the intensity of the microwave oscillation to be 60W, controlling the time of the microwave oscillation to be 20min, obtaining a carrier after the microwave oscillation is finished, adding the carrier and bacterial powder into a reaction container, and stirring at the stirring speed of 80rpm at 12 ℃ for 20min to obtain loaded bacterial powder;
wherein the weight ratio of the resistant dextrin to the ferric pyrophosphate to the tricalcium phosphate to the polyvinylpyrrolidone is 8:3:1: 2;
wherein the weight ratio of the bacterial powder to the carrier is 7: 10;
the total amount of probiotics in the load bacterium powder is 2.2 multiplied by 10 10 cfu/g；
The auxiliary materials comprise the following components in parts by weight: 10 parts of sodium carboxymethylcellulose, 3 parts of sodium caseinate, 1 part of fatty acid sucrose polyester and 2 parts of glucose;
the preparation method of the modified calcium comprises the following specific steps: adding calcium gluconate, calcium acetate and calcium lactate into a ball mill for ball milling, controlling the ball-to-material ratio during ball milling to be 15:1, controlling the ball milling rotation speed to be 300rpm, controlling the ball milling time to be 35min, obtaining mixed calcium after ball milling, uniformly mixing the mixed calcium, low-ester pectin, corn starch, maltose, propylene glycol fatty acid ester and deionized water, then carrying out vacuum spray drying, controlling the vacuum degree in the vacuum spray drying process to be 500Pa, the inlet temperature to be 130 ℃, the outlet temperature to be 65 ℃, obtaining primary modified calcium after vacuum spray drying, carrying out liquid nitrogen spraying on the primary modified calcium, controlling the outlet pressure of liquid nitrogen spraying to be 0.4MPa, the spraying speed to be 3m/s, the working frequency to be 35Hz, the spraying time to be 12s, and naturally recovering to the room temperature after liquid nitrogen spraying is finished to obtain the modified calcium;
wherein the weight ratio of the calcium gluconate to the calcium acetate to the calcium lactate is 7:3: 2;
wherein the weight ratio of the mixed calcium to the low-ester pectin to the corn starch to the maltose to the propylene glycol fatty acid ester to the deionized water is 18:5:4:1:0.2: 48.
The preparation method of the probiotic flora composition with the muscle and bone strengthening function comprises the steps of uniformly mixing the load bacteria powder, the auxiliary material and the modified calcium, then carrying out vacuum freeze drying, controlling the temperature of the vacuum freeze drying treatment to be-35 ℃, the vacuum degree to be 700Pa, and the treatment time to be 40min, and finally obtaining the probiotic flora composition with the muscle and bone strengthening function after the vacuum freeze drying is finished.
Example 2
The probiotic flora composition with the functions of muscle and bone enhancement comprises the following components in parts by weight: 87 parts of load bacterial powder, 5 parts of auxiliary materials and 8 parts of modified calcium;
the preparation method of the load bacterial powder comprises the following steps: preparing seed liquid, preparing fermentation liquor, preparing a freeze-drying protective agent, drying and loading;
the method for preparing the seed liquid comprises the following steps: respectively scribing and activating lactobacillus plantarum, lactobacillus rhamnosus, lactobacillus paracasei, lactobacillus acidophilus and bifidobacterium longum which are frozen and stored on a solid LB plate, then respectively selecting single bacterial colonies from the LB plate and inoculating the single bacterial colonies into a 250mL conical flask filled with 100mL of liquid culture medium, putting the 250mL conical flask into a shaking table for shaking table culture, controlling the temperature of the shaking table culture to be 37 ℃, the rotating speed to be 230rpm and the time to be 14.5h, and respectively obtaining lactobacillus plantarum seed liquid, lactobacillus rhamnosus seed liquid, lactobacillus paracasei seed liquid, lactobacillus acidophilus seed liquid and bifidobacterium longum seed liquid after the shaking table culture is finished;
the method for preparing the fermentation liquor comprises the following steps: respectively inoculating lactobacillus plantarum seed liquid, lactobacillus rhamnosus seed liquid, lactobacillus paracasei seed liquid, lactobacillus acidophilus seed liquid and bifidobacterium longum seed liquid in the same MRS liquid culture medium in sequence, controlling the inoculation amount of lactobacillus plantarum to be 1.7%, the inoculation amount of lactobacillus rhamnosus to be 1.2%, the inoculation amount of lactobacillus paracasei to be 1.7%, the inoculation amount of lactobacillus acidophilus to be 0.7% and the inoculation amount of bifidobacterium longum to be 1.5%, performing stirring fermentation culture after inoculation is completed, controlling the temperature of the stirring fermentation culture to be 37 ℃, controlling the rotating speed to be 330rpm, controlling the ventilation quantity to be 1.7vvm, controlling the time to be 17.5h, and obtaining fermentation liquor after the stirring fermentation culture is completed;
the MRS liquid culture medium consists of the following components: 6.5g/L tryptone, 5.5g/L glucose, 5g/L beef extract, 4g/L yeast extract, 1.7g/L sodium citrate, 0.7g/L Tween 80, 0.3g/L manganese sulfate, 0.12g/L rhamnolipid and the balance of deionized water;
the method for preparing the freeze-drying protective agent comprises the following steps: mixing sucrose, beta-glucan, algal polysaccharide, sodium chloride, maltodextrin and skim milk powder, then carrying out ball milling, controlling the ball-material ratio during ball milling to be 9:1, the ball milling rotation speed to be 360rpm, the ball milling time to be 32min, obtaining a primary freeze-drying protective agent after ball milling is finished, uniformly mixing the primary freeze-drying protective agent, carrageenan, polyglycerol fatty acid ester and pregelatinized starch, then carrying out low-frequency ultrasonic treatment, controlling the frequency of the low-frequency ultrasonic treatment to be 23kHz, controlling the power to be 390W, and finishing the low-frequency ultrasonic treatment to obtain the freeze-drying protective agent;
wherein the weight ratio of the sucrose, the beta-glucan, the algal polysaccharide, the sodium chloride, the maltodextrin and the skimmed milk powder is 11:4:3:0.7:2: 3;
wherein the weight ratio of the primary freeze-drying protective agent to the carrageenan to the polyglycerol fatty acid ester to the pregelatinized starch is 19:5:2: 3;
the drying method comprises the following steps: carrying out centrifugal separation on fermentation liquor, controlling the rotation speed during centrifugal separation to be 2900rpm, controlling the time to be 5.5min, taking a solid phase obtained after centrifugal separation as bacterial sludge, uniformly mixing the bacterial sludge and a freeze-drying protective agent in a weight ratio of 91:7, carrying out vacuum freeze-drying treatment, controlling the temperature of the vacuum freeze-drying treatment to be-33 ℃, controlling the vacuum degree to be 850Pa, controlling the treatment time to be 52min, and obtaining bacterial powder after the vacuum freeze-drying is finished;
the load method comprises the following steps: mixing resistant dextrin, ferric pyrophosphate, tricalcium phosphate and polyvinylpyrrolidone, then performing microwave oscillation, controlling the intensity of the microwave oscillation to be 70W, controlling the time of the microwave oscillation to be 22min, obtaining a carrier after the microwave oscillation is finished, adding the carrier and the bacterial powder into a reaction container, and stirring at the stirring speed of 90rpm at the temperature of 13 ℃ for 22min to obtain loaded bacterial powder;
wherein the weight ratio of the resistant dextrin to the ferric pyrophosphate to the tricalcium phosphate to the polyvinylpyrrolidone is 9:4:1.5: 3;
wherein the weight ratio of the bacterial powder to the carrier is 7.5: 10;
the total amount of probiotics in the load bacterium powder is 2.3 multiplied by 10 10 cfu/g；
The auxiliary materials comprise the following components in parts by weight: 12 parts of sodium carboxymethylcellulose, 4 parts of sodium caseinate, 2 parts of fatty acid sucrose polyester and 3 parts of glucose;
the preparation method of the modified calcium comprises the following specific steps: adding calcium gluconate, calcium acetate and calcium lactate into a ball mill for ball milling, controlling the ball-to-material ratio during ball milling to be 16:1, controlling the ball milling rotation speed to be 310rpm, controlling the ball milling time to be 37min, obtaining mixed calcium after ball milling, uniformly mixing the mixed calcium, low-ester pectin, corn starch, maltose, propylene glycol fatty acid ester and deionized water, then performing vacuum spray drying, controlling the vacuum degree in the vacuum spray drying process to be 550Pa, the inlet temperature to be 132 ℃, the outlet temperature to be 67 ℃, obtaining primary modified calcium after vacuum spray drying, spraying liquid nitrogen to the primary modified calcium, controlling the outlet pressure of liquid nitrogen spraying to be 0.45MPa, the spraying speed to be 3.2m/s, the working frequency to be 37Hz, the spraying time to be 13s, and naturally recovering to the room temperature after the liquid nitrogen spraying is finished to obtain the modified calcium;
wherein the weight ratio of the calcium gluconate to the calcium acetate to the calcium lactate is 7.5:4: 3;
wherein the weight ratio of the mixed calcium to the low-ester pectin to the corn starch to the maltose to the propylene glycol fatty acid ester to the deionized water is 19:6:5:1.5:0.3: 49.
The preparation method of the probiotic flora composition with the muscle and bone increasing and protecting functions comprises the steps of uniformly mixing the load bacteria powder, the auxiliary material and the modified calcium, then carrying out vacuum freeze drying, controlling the temperature of vacuum freeze drying treatment to be-32 ℃, the vacuum degree to be 750Pa, treating for 42min, and finally carrying out vacuum freeze drying to obtain the probiotic flora composition with the muscle and bone increasing and protecting functions.
Example 3
The probiotic flora composition with the functions of muscle and bone enhancement comprises the following components in parts by weight: 90 parts of load bacterial powder, 6 parts of auxiliary materials and 9 parts of modified calcium;
the preparation method of the load bacterial powder comprises the following steps: preparing seed liquid, preparing fermentation liquor, preparing a freeze-drying protective agent, drying and loading;
the method for preparing the seed liquid comprises the following steps: respectively scribing and activating lactobacillus plantarum, lactobacillus rhamnosus, lactobacillus paracasei, lactobacillus acidophilus and bifidobacterium longum which are frozen and stored on a solid LB plate, then respectively selecting single bacterial colonies from the LB plate and inoculating the single bacterial colonies into 250mL conical flasks filled with 100mL of liquid culture medium, putting the 250mL conical flasks into a shaking table for shaking table culture, controlling the temperature of the shaking table culture to be 38 ℃, the rotating speed to be 250rpm and the time to be 15 hours, and respectively obtaining lactobacillus plantarum seed liquid, lactobacillus rhamnosus seed liquid, lactobacillus paracasei seed liquid, lactobacillus acidophilus seed liquid and bifidobacterium longum seed liquid after the shaking table culture is finished;
the method for preparing the fermentation liquor comprises the following steps: respectively inoculating lactobacillus plantarum seed liquid, lactobacillus rhamnosus seed liquid, lactobacillus paracasei seed liquid, lactobacillus acidophilus seed liquid and bifidobacterium longum seed liquid in the same MRS liquid culture medium in sequence, controlling the inoculation amount of lactobacillus plantarum to be 2%, the inoculation amount of lactobacillus rhamnosus to be 1.5%, the inoculation amount of lactobacillus paracasei to be 2%, the inoculation amount of lactobacillus acidophilus to be 1% and the inoculation amount of bifidobacterium longum to be 2%, performing stirring fermentation culture after inoculation is finished, controlling the temperature of the stirring fermentation culture to be 38 ℃, the rotating speed to be 350rpm, the ventilation volume to be 2vvm, the time to be 18h, and obtaining fermentation liquor after the stirring fermentation culture is finished;
the MRS liquid culture medium consists of the following components: 7g/L tryptone, 6g/L glucose, 6g/L beef extract, 5g/L yeast extract, 2g/L sodium citrate, 1g/L Tween 80, 0.5g/L manganese sulfate, 0.15g/L rhamnolipid and the balance of deionized water;
the method for preparing the freeze-drying protective agent comprises the following steps: mixing sucrose, beta-glucan, algal polysaccharide, sodium chloride, maltodextrin and skim milk powder, then carrying out ball milling, controlling the ball-material ratio during ball milling to be 10:1, controlling the ball milling rotation speed to be 380rpm, carrying out ball milling for 35min, obtaining a primary freeze-drying protective agent after ball milling is finished, uniformly mixing the primary freeze-drying protective agent, carrageenan, polyglycerol fatty acid ester and pregelatinized starch, then carrying out low-frequency ultrasonic treatment, controlling the frequency of the low-frequency ultrasonic treatment to be 25kHz, controlling the power to be 400W, and finishing the low-frequency ultrasonic treatment to obtain the freeze-drying protective agent;
wherein the weight ratio of the sucrose, the beta-glucan, the algal polysaccharide, the sodium chloride, the maltodextrin and the skimmed milk powder is 12:5:4:1:3: 4;
wherein the weight ratio of the primary freeze-drying protective agent to the carrageenan to the polyglycerol fatty acid ester to the pregelatinized starch is 20:6:3: 4;
the drying method comprises the following steps: carrying out centrifugal separation on fermentation liquor, controlling the rotating speed during the centrifugal separation to be 3000rpm, controlling the time to be 6min, taking a solid phase obtained after the centrifugal separation as bacterial sludge, uniformly mixing the bacterial sludge and a freeze-drying protective agent in a weight ratio of 92:8, carrying out vacuum freeze-drying treatment, controlling the temperature of the vacuum freeze-drying treatment to be-32 ℃, controlling the vacuum degree to be 900Pa, controlling the treatment time to be 55min, and obtaining bacterial powder after the vacuum freeze-drying is finished;
the load method comprises the following steps: mixing resistant dextrin, ferric pyrophosphate, tricalcium phosphate and polyvinylpyrrolidone, then performing microwave oscillation, controlling the intensity of the microwave oscillation to be 80W, controlling the time of the microwave oscillation to be 25min, obtaining a carrier after the microwave oscillation is finished, adding the carrier and the bacterial powder into a reaction container, and stirring at the stirring speed of 100rpm at 15 ℃ for 25min to obtain loaded bacterial powder;
wherein the weight ratio of the resistant dextrin to the ferric pyrophosphate to the tricalcium phosphate to the polyvinylpyrrolidone is 10:5:2: 4;
wherein the weight ratio of the bacterial powder to the carrier is 8: 10;
the total amount of probiotics in the load bacterium powder is 2.5 multiplied by 10 10 cfu/g；
The auxiliary materials comprise the following components in parts by weight: 13 parts of sodium carboxymethylcellulose, 5 parts of sodium caseinate, 3 parts of fatty acid sucrose polyester and 5 parts of glucose;
the preparation method of the modified calcium comprises the following specific steps: adding calcium gluconate, calcium acetate and calcium lactate into a ball mill for ball milling, controlling the ball-to-material ratio during ball milling to be 18:1, controlling the ball milling rotation speed to be 320rpm, controlling the ball milling time to be 40min, obtaining mixed calcium after ball milling, uniformly mixing the mixed calcium, low-ester pectin, corn starch, maltose, propylene glycol fatty acid ester and deionized water, then performing vacuum spray drying, controlling the vacuum degree in the vacuum spray drying process to be 600Pa, the inlet temperature to be 135 ℃, the outlet temperature to be 70 ℃, obtaining primary modified calcium after vacuum spray drying, spraying liquid nitrogen to the primary modified calcium, controlling the outlet pressure of liquid nitrogen spraying to be 0.5MPa, the spraying speed to be 3.5m/s, the working frequency to be 40Hz, the spraying time to be 15s, and naturally recovering to the room temperature after the liquid nitrogen spraying is finished to obtain the modified calcium;
wherein the weight ratio of the calcium gluconate to the calcium acetate to the calcium lactate is 8:5: 4;
wherein the weight ratio of the mixed calcium to the low-ester pectin to the corn starch to the maltose to the propylene glycol fatty acid ester to the deionized water is 20:7:6:2:0.5: 50.
The preparation method of the probiotic composition with the muscle and bone increasing and protecting functions comprises the steps of uniformly mixing the load bacteria powder, the auxiliary material and the modified calcium, then carrying out vacuum freeze drying, controlling the temperature of the vacuum freeze drying treatment to be-30 ℃, the vacuum degree to be 800Pa, the treatment time to be 45min, and finally carrying out vacuum freeze drying to obtain the probiotic composition with the muscle and bone increasing and protecting functions.
Comparative example 1
The technical scheme of the embodiment 1 is adopted, and the difference is that: the preparation of the load bacteria powder omits a loading step, namely, the bacteria powder obtained in the drying step is directly used for preparing the probiotic flora composition with the functions of muscle and bone enhancement instead of the load bacteria powder in the preparation of the load bacteria powder.
Comparative example 2
The technical scheme of the embodiment 1 is adopted, and the difference is that: in the preparation of the load bacterial powder, in the step of preparing the freeze-drying protective agent, the step of uniformly mixing the primary freeze-drying protective agent, the carrageenan, the polyglycerol fatty acid ester and the pre-gelatinized starch and then carrying out low-frequency ultrasonic treatment is omitted, namely, the primary freeze-drying protective agent is used for preparing the probiotic flora composition with the functions of muscle and bone enhancement instead of the freeze-drying protective agent.
Comparative example 3
The technical scheme of the embodiment 1 is adopted, and the difference is that: in the preparation of the modified calcium, the steps of uniformly mixing mixed calcium, low-ester pectin, corn starch, maltose, propylene glycol fatty acid ester and deionized water, then carrying out vacuum spray drying and carrying out liquid nitrogen spraying on the primary modified calcium are omitted, namely the mixed calcium is used for replacing the modified calcium and directly used for preparing the probiotic flora composition with the functions of muscle and bone enhancement.
Test example 1
The probiotic flora compositions having the functions of muscle and bone enhancement according to examples 1 to 3 and comparative examples 1 to 3 were subjected to the following tests, and the test methods and results were as follows:
selecting 140 SPF male SD rats with age of 8 weeks and weight of 200 + -10 g, dividing the rats into 7 groups, numbering A1-A7, and each group comprises 20 rats; feeding all SPF male SD rats into the same animal room, then feeding each group of SPF male SD rats in cages, ensuring that the temperature in the animal room is 24 +/-2 ℃ and the humidity is 60 +/-2%, enabling animals to eat freely, ensuring that the types of feed fed by each group of SPF male SD rats are the same, after 7d adaptive feeding, feeding the groups of SPF male SD rats A1-A6 with the probiotic flora composition with the muscle and bone increasing and protecting functions of the examples 1-3 and the comparative examples 1-3 while continuing feeding with the feed, wherein the corresponding relation is as follows:
Figure 461666DEST_PATH_IMAGE001
the daily consumption of probiotics of each SPF male SD rat is controlled to be 0.2g, the A7 group is fed with the feed without any probiotic group composition, the blank control is used, 5 SPF male SD rats are randomly selected from each group after being continuously fed for 30d, the bone density (BMD) of each SPF male SD rat is respectively measured by using an X-ray determinator, then the SPF male SD rats are killed, the weights of gastrocnemius muscles are measured, and then the average bone density and the average gastrocnemius weight/weight of each group of SPF male SD rats are calculated, and the test results are as follows:
Figure 50911DEST_PATH_IMAGE002
then the feeding of probiotics will be stopped, the remaining SPF-grade male SD rats will continue to be fed for 15d, the diarrhea of each group of SPF-grade male SD rats is observed, and the diarrhea number of each group of SPF-grade male SD rats is recorded, and the results are recorded as follows:
Figure 627385DEST_PATH_IMAGE003
test example 2
The total number of the probiotics in the probiotic colony compositions with the functions of muscle and bone strengthening in examples 1 to 3 and comparative examples 1 to 3 was measured respectively and used as the total number of the probiotics before being stored in the dark, and then after being stored in the dark at a temperature of 2 ℃ for 10 days, the total number of the probiotics in the probiotic colony compositions was measured respectively and used as the total number of the probiotics after being stored in the dark, and the measurement results were as follows:
Figure 943966DEST_PATH_IMAGE004
test example 3
Respectively dissolving the probiotic flora compositions with the muscle and bone strengthening and protecting functions of the examples 1-3 and the comparative examples 1-3 in deionized water to prepare a probiotic water solution with the mass fraction of 10%, standing the probiotic water solution at 25 ℃ for 1d, observing and recording whether precipitates exist, wherein the recorded results are as follows:
Figure 164863DEST_PATH_IMAGE005
all percentages used in the present invention are mass percentages unless otherwise indicated.
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (4)

1. The probiotic flora composition with the functions of muscle and bone enhancement is characterized by comprising the following components in parts by weight: 85-90 parts of load bacterial powder, 4-6 parts of auxiliary materials and 6-9 parts of modified calcium;
the preparation method of the load bacterial powder comprises the following steps: preparing seed liquid, preparing fermentation liquor, preparing a freeze-drying protective agent, drying and loading;
the method for preparing the freeze-drying protective agent comprises the following steps: mixing sucrose, beta-glucan, algal polysaccharide, sodium chloride, maltodextrin and skim milk powder, then carrying out ball milling, controlling the ball-material ratio during ball milling to be 8-10:1, the ball milling rotation speed to be 350-plus-380 rpm, the ball milling time to be 30-35min, obtaining a primary freeze-drying protective agent after ball milling is finished, uniformly mixing the primary freeze-drying protective agent, carrageenan, polyglycerol fatty acid ester and pregelatinized starch, then carrying out low-frequency ultrasonic treatment, controlling the frequency of the low-frequency ultrasonic treatment to be 22-25kHz, controlling the power to be 380-plus-400W, and obtaining the freeze-drying protective agent after the low-frequency ultrasonic treatment is finished;
in the preparation of the freeze-drying protective agent, the weight ratio of sucrose, beta-glucan, algal polysaccharide, sodium chloride, maltodextrin and skimmed milk powder is 10-12:3-5:2-4:0.5-1:1-3: 2.5-4;
in the preparation of the freeze-drying protective agent, the weight ratio of the primary freeze-drying protective agent to the carrageenan to the polyglycerol fatty acid ester to the pregelatinized starch is 18-20:4-6:1-3: 2-4;
the drying method comprises the following steps: carrying out centrifugal separation on the fermentation liquor, controlling the rotation speed of 2800 plus 3000rpm and the time of 5-6min during the centrifugal separation, taking a solid phase obtained after the centrifugal separation as bacterial sludge, uniformly mixing the bacterial sludge and a freeze-drying protective agent according to the weight ratio of 90-92:6-8, carrying out vacuum freeze-drying treatment, controlling the temperature of the vacuum freeze-drying treatment to be-35 ℃ to-32 ℃, the vacuum degree to be 800 plus 900Pa and the treatment time to be 50-55min, and obtaining bacterial powder after the vacuum freeze-drying treatment;
the load method comprises the following steps: mixing resistant dextrin, ferric pyrophosphate, tricalcium phosphate and polyvinylpyrrolidone, then performing microwave oscillation, controlling the intensity of the microwave oscillation to be 60-80W, controlling the time of the microwave oscillation to be 20-25min, obtaining a carrier after the microwave oscillation is finished, adding the carrier and the bacterial powder into a reaction container, and stirring at the stirring speed of 80-100rpm for 20-25min at the temperature of 12-15 ℃ to obtain load bacterial powder;
in the load, the weight ratio of the resistant dextrin to the ferric pyrophosphate to the tricalcium phosphate to the polyvinylpyrrolidone is 8-10:3-5:1-2: 2-4;
in the load, the weight ratio of the bacterial powder to the carrier is 7-8: 10;
the total amount of probiotics in the load bacterium powder is 2.2 multiplied by 10 10 -2.5×10 10 cfu/g；
The auxiliary materials comprise the following components in parts by weight: 10-13 parts of sodium carboxymethylcellulose, 3-5 parts of sodium caseinate, 1-3 parts of fatty acid sucrose polyester and 2-5 parts of glucose;
the preparation method of the modified calcium comprises the following specific steps: adding calcium gluconate, calcium acetate and calcium lactate into a ball mill for ball milling, controlling the ball-to-material ratio during ball milling to be 15-18:1, controlling the ball milling rotation speed to be 300-320rpm, the ball milling time to be 35-40min, obtaining mixed calcium after ball milling, uniformly mixing the mixed calcium, low-ester pectin, corn starch, maltose, propylene glycol fatty acid ester and deionized water, performing vacuum spray drying, controlling the vacuum degree in the vacuum spray drying process to be 500-600Pa, the inlet temperature to be 130-135 ℃, the outlet temperature to be 65-70 ℃, obtaining primary modified calcium after vacuum spray drying, performing liquid nitrogen spraying on the primary modified calcium, controlling the outlet pressure of the liquid nitrogen spraying to be 0.4-0.5MPa, the spraying speed to be 3-3.5m/s, the working frequency to be 35-40Hz, and the spraying time to be 12-15s, after the liquid nitrogen spraying is finished, naturally recovering to room temperature to obtain modified calcium;
in the preparation of the modified calcium, the weight ratio of calcium gluconate to calcium acetate to calcium lactate is 7-8:3-5: 2-4;
in the preparation of the modified calcium, the weight ratio of the mixed calcium to the low-ester pectin to the corn starch to the maltose to the propylene glycol fatty acid ester to the deionized water is 18-20:5-7:4-6:1-2:0.2-0.5: 48-50.
2. The probiotic bacterial group composition with the functions of muscle and bone enhancement according to claim 1, wherein the method for preparing the seed liquid is as follows: respectively marking and activating the lactobacillus plantarum, the lactobacillus rhamnosus, the lactobacillus paracasei, the lactobacillus acidophilus and the bifidobacterium longum which are frozen and stored on a solid LB plate, then respectively selecting a single bacterial colony from the LB plate to inoculate into a 250mL conical flask filled with 100mL of liquid culture medium, putting the 250mL conical flask into a shaking table to carry out shaking table culture, controlling the temperature of the shaking table culture to be 36-38 ℃, the rotating speed to be 220 plus 250rpm, and the time to be 14-15h, and respectively obtaining lactobacillus plantarum seed liquid, lactobacillus rhamnosus seed liquid, lactobacillus paracasei seed liquid, lactobacillus acidophilus seed liquid and bifidobacterium longum seed liquid after the shaking table culture is finished.
3. The probiotic flora composition with muscle and bone strengthening function according to claim 1, wherein the method for preparing the fermentation liquor is as follows: respectively inoculating lactobacillus plantarum seed liquid, lactobacillus rhamnosus seed liquid, lactobacillus paracasei seed liquid, lactobacillus acidophilus seed liquid and bifidobacterium longum seed liquid in the same MRS liquid culture medium in sequence, controlling the inoculation amount of lactobacillus plantarum to be 1.5-2%, the inoculation amount of lactobacillus rhamnosus to be 1-1.5%, the inoculation amount of lactobacillus paracasei to be 1.5-2%, the inoculation amount of lactobacillus acidophilus to be 0.5-1% and the inoculation amount of bifidobacterium longum to be 1-2%, performing stirring fermentation culture after the inoculation is completed, controlling the temperature of the stirring fermentation culture to be 36-38 ℃, controlling the rotating speed to be 320-plus 350rpm, controlling the ventilation volume to be 1.5-2vvm, and controlling the time to be 17-18h, and obtaining fermentation liquor after the stirring fermentation culture is completed;
the MRS liquid culture medium consists of the following components: 6-7g/L tryptone, 5-6g/L glucose, 4-6g/L beef extract, 3-5g/L yeast extract, 1.5-2g/L sodium citrate, 0.5-1g/L Tween 80, 0.2-0.5g/L manganese sulfate, 0.1-0.15g/L rhamnolipid and the balance of deionized water.
4. The probiotic flora composition with the muscle and bone strengthening function as claimed in claim 1, wherein the preparation method of the probiotic flora composition with the muscle and bone strengthening function comprises the steps of uniformly mixing the load bacteria powder, the auxiliary material and the modified calcium, then carrying out vacuum freeze drying, controlling the temperature of the vacuum freeze drying treatment to be-35 ℃ to-30 ℃, controlling the vacuum degree to be 700-800Pa, and controlling the treatment time to be 40-45min, and finally obtaining the probiotic flora composition with the muscle and bone strengthening function after the vacuum freeze drying is finished.
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