CN107549817B - Moringa oleifera natural organic calcium and preparation method thereof - Google Patents
Moringa oleifera natural organic calcium and preparation method thereof Download PDFInfo
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Abstract
The invention discloses moringa natural organic calcium and a preparation method thereof, and the moringa natural organic calcium takes moringa leaves as a main raw material, and is fermented in stages by mixed microorganisms, wherein the mixed microorganisms are lactic acid bacteria and saccharomycetes, and the moringa ingredients can be degraded in the fermentation process so as to extract natural water-soluble calcium in the moringa; macromolecular proteins and saccharides in the moringa oleifera are further degraded by utilizing symbiotic action and fermentation characteristics of different microbial fermentations to obtain polypeptides, amino acids, organic acids and the like, carboxyl, amino and the like on a polypeptide chain can be combined with calcium ions, the occurrence form of moringa oleifera calcium is changed, the small intestine absorption rate is increased, the bioavailability is improved, and the bone density is increased. The freeze drying technology retains the biological activities of polypeptide, amino acid and the like to the maximum extent, and the moringa oleifera freeze-dried powder is mixed with the auxiliary materials of natural sweet orange powder, glycoprotein, xylitol, soybean lecithin and the like to prepare the moringa oleifera organic calcium product.
Description
Technical Field
The invention belongs to the technical field of calcium preparations, and particularly relates to moringa oleifera natural organic calcium and a preparation method thereof.
Background
Calcium is one of the most abundant minerals in human body, called vital metal by people, is an essential element in life, has important effect on maintaining and regulating physiological functions of human skeleton, muscle, cell, circulation, immunity and other systems, and is mainly manifested by skeletal diseases, such as rickets in children and osteoporosis in climacteric women and old people. Calcium nutrition enhancers in the current market can be divided into three categories: inorganic calcium nutrition enhancer, biological calcium nutrition enhancer and organic calcium nutrition enhancer.
The inorganic calcium nutrition enhancer mainly refers to inorganic calcium salt processed from calcium carbonate ore, such as calcium carbonate, calcium hydroxy phosphate, calcium chloride, calcium hydroxide, calcium hydrogen phosphate, etc. Calcium carbonate and calcium oxide are alkaline and can be absorbed only after reacting with a large amount of gastric acid in the stomach to generate ionized calcium, so that the stomach is difficult to secrete enough gastric acid to ionize the ionized calcium in limited time, and is more difficult for children, old people and people suffering from gastric diseases, and the normal gastrointestinal function of a human body can be influenced after long-term administration. Calcium hydroxy phosphate is an insoluble inorganic salt, has limited solubility to gastric acid, and high phosphorus inhibits the absorption and utilization of calcium. Calcium chloride has the best water solubility, but is basically eliminated due to large side effects such as acute toxicity and the like. The biological calcium nutrition enhancer is inorganic calcium salt prepared from animal bone or shell, and Margarita. Heavy metal ions can be enriched in animal bones, so that the heavy metal in the biological calcium nutrition enhancer exceeds the standard, the standard of food hygiene is difficult to achieve, and the biological calcium nutrition enhancer cannot be taken for a long time. The organic calcium nutrition enhancer is mainly calcium lactate, calcium acetate, calcium gluconate, calcium citrate, etc. The organic calcium nutrition enhancer has good solubility, small irritation to stomach and intestine and high sanitary safety, but has the defects of low calcium content, toxic and side effects of different degrees and the like.
At present, most calcium preparations are prepared by chemical methods, and the calcium preparations have various defects, such as low human body absorption rate, influence on body health caused by-products, high production cost of the calcium preparations and the like. The market urgently needs a high-efficiency, safe and green calcium supplement product, and the moringa leaves are a new resource food rich in calcium and protein, and have certain market potential in developing natural plant organic calcium.
Moringa oleifera (Moringa oleifera, or drumstick tree, Horseradish tree), which is a perennial plant of Moringa in Moringaceae, is a fast-growing tree species native to southern foot of Himalayan mountain in North India, has strong drought resistance, and is favored to be grown in sandy soil. At present, 14 varieties are known in total, but only two varieties of Moringa oleifera and Moringa africana are supplied for edible cultivation. The moringa oleifera is praised as a magic tree due to its rich nutritive and medicinal values. The root, stem, leaf, flower and seed of the moringa oleifera are high in protein, calcium, potassium, magnesium, fiber, vitamin, fat and cholesterol, and comprehensive in nutrient content, wherein calcium and protein in the moringa oleifera leaf are respectively 4 times and 2 times of those of milk, potassium is 3 times of that of banana, iron is 3 times of that of spinach, vitamin C is 7 times of that of orange, and carotene is 4 times of that of carrot. The moringa oleifera has various physiological activities, and has the activities of resisting oxidation, resisting tumors, reducing blood sugar, reducing blood fat, overcoming insomnia, treating anemia, resisting viruses, inhibiting bacteria and the like. However, although the moringa oleifera has so many excellent effects, the moringa oleifera is still not fully applied at present, and especially in the aspect of activation and utilization of plant calcium, the moringa oleifera cannot exert the maximum effect all the time. Calcium in the moringa oleifera is mainly insoluble calcium phytate and calcium oxalate, the content of ionized calcium is low, the absorptivity of the moringa oleifera is low after the moringa oleifera is directly eaten, and the insoluble calcium phytate and the insoluble calcium oxalate are deposited in a body to easily form calculi, so that certain health risks exist.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides moringa natural organic calcium and a preparation method thereof. The moringa natural organic calcium provided by the invention has high solubility, is less influenced by acidity and temperature, and can ensure the dissolution state of calcium and promote intestinal wall absorption in neutral and alkaline environments of small intestine.
The technical scheme of the invention is as follows: a moringa oleifera natural organic calcium is obtained by performing staged gradient fermentation on moringa oleifera leaf powder by mixed microorganisms; the mixed microorganism is lactobacillus and yeast.
Preferably, the mixed microorganism comprises lactobacillus acidophilus, lactobacillus johnsonii and saccharomyces cerevisiae I, and the weight ratio of the three strains is 3-6: 3-6: 2 to 4.
Preferably, the mixed microorganism is lactobacillus acidophilus, lactobacillus johnsonii, lactobacillus reuteri and yeast I, and the weight ratio of the four strains is 3-5: 3-5: 2-4: 2 to 4.
The second aspect of the invention also provides a preparation method of the moringa natural organic calcium, the moringa leaves are pretreated and then uniformly mixed with water, autoclaved, then added with strains of mixed microorganisms and glycogen for staged gradient fermentation, and after the fermentation is finished, the supernatant is centrifuged, concentrated in vacuum, frozen and dried or spray-dried to obtain the moringa organic calcium.
Further, the moringa oleifera leaf pretreatment process comprises the following steps: selecting tender fresh moringa leaves, deactivating enzyme at 105-110 ℃ for 10-15 min, and drying at 60-70 ℃; and (3) crushing the dried moringa leaves by using a grinding machine or a powdering machine, and sieving by using a sieve of 50-70 meshes for later use.
Further, after pretreatment, the moringa leaves are mixed with water according to the proportion of 1: 15-20, and then uniformly stirring at 75-85 ℃.
Further, adding the domesticated strain into the moringa oleifera liquid according to a proportion, wherein the total weight of the domesticated strain wet thalli accounts for 0.25-0.35% of the weight of the moringa oleifera fermentation liquid; and adding lactose accounting for 8.0-12.0% of the weight of the moringa oleifera leaf dry powder, wherein the added lactose is used as a carbon source for microbial growth.
Further, the acclimatization process of the lactic acid bacteria is as follows: transferring lactic acid bacteria into an MRS broth liquid culture medium for static culture, wherein the culture temperature is 30-40 ℃, the culture time is 48-72 hours, taking out the culture medium after the culture medium is turbid, shaking up the culture medium, transferring the culture medium into a next generation of MRS broth moringa oleifera culture medium, and the ratio of MRS broth culture medium powder to moringa oleifera is 3-6: 1, after finishing the culture, performing gradient acclimation on the lactic acid bacteria by adopting a culture medium with the ratio of MRS broth powder to moringa leaf powder being 4:1, 3:1, 2:1, 1:2, 1:3, 1:4, 1:5 and 0:1, culturing by adopting a moringa culture medium after acclimation for ten generations, and storing in a refrigerator at 2-4 ℃ for later use;
the domestication process of the yeast is as follows: transferring the saccharomyces cerevisiae 1 into a potato glucose liquid culture medium for shaking culture, wherein the shaking culture temperature is 25-35 ℃, the culture revolution is 100-150rpm, the culture is performed for 48-72h, the culture medium is taken out after precipitation, the culture medium is uniformly shaken and then transferred into a next generation potato glucose broth moringa oleifera culture medium, and the ratio of broth culture medium powder to moringa oleifera is 3-6: after culturing for 3 days at the temperature of 1.28-30 ℃, performing gradient acclimation on the saccharomyces cerevisiae 1 by adopting a culture medium with the ratio of potato glucose powder to moringa oleifera leaf powder being 4:1, 3:1, 2:1, 1:2, 1:3, 1:4, 1:5 and 0:1, culturing for ten generations by adopting a moringa oleifera culture medium, and storing in a refrigerator at the temperature of 2-4 ℃ for later use.
Further, the staged gradient fermentation operates as follows: firstly carrying out anaerobic fermentation for 20-24 h at the temperature of 36-38 ℃, reducing the pH value to 4.0-4.5, then carrying out aerobic fermentation for 24-36 h at the temperature of 26-30 ℃, and finally carrying out fermentation for 18-24 h at the temperature of 36-38 ℃ (anaerobic culture), and finishing fermentation when the pH value reaches 3.75-3.85.
Further, after the fermentation is finished, centrifuging for 20min at 4000r/min, centrifuging to obtain a supernatant, and performing water bath vacuum concentration on the supernatant at 50-55 ℃ and under 0.55-0.8 Mpa.
The moringa calcium product prepared by the method is green powder, has the faint scent of moringa and sweet orange, is slightly sweet and slightly astringent, and has good instant solubility and stability; per 100g of sample: the total calcium content is 2050-2315 mg, the chelated calcium content is 1658-1730 mg, the total protein content is 17.0-18.2 g, the polypeptide content is 8.3-10.1 g, the free amino acid content is 1391-1504 mg, and the nutritional calcium supplement food has high nutritional and calcium supplement values.
The feeding experiment of rats shows that the moringa oleifera organic calcium product has no obvious side effect on the rats and has better safety. The moringa oleifera organic calcium product can improve the quality, the length and the diameter of the thighbone of a rat and obviously increase the bone calcium content. Calcium absorption rate measurement results show that the calcium absorption rate of the moringa oleifera organic calcium product is significantly higher than that of a calcium chloride control group with the same calcium level. The moringa oleifera organic active calcium can be used as an effective calcium nutrition enhancer.
According to the third aspect of the invention, the invention also provides a nutritional product containing the moringa oleifera organic calcium, wherein the nutritional product comprises 45-50% of moringa oleifera fermentation liquid dry powder, 10-15% of natural sweet orange powder, 1.3-1.5% of aspartame, 25-30% of xylitol and 8.5-10% of soybean lecithin by weight. The nutritional product can be made into buccal tablet, granule or solution.
One of the characteristics of the invention is that the invention selects the compound fermentation of lactic acid bacteria and yeast, and the lactic acid bacteria (Lactobacillus acidophilus, Lactobacillus johnsonii and Lactobacillus reuteri) produce organic acids such as lactic acid through fermentation, so that the peppery taste and astringent taste of the moringa oleifera can be reduced, and the taste is improved, especially the Lactobacillus reuteri, so that the effect of improving the taste and flavor of the product is optimal; meanwhile, the lactic acid can be combined with calcium to form milk calcium, so that the calcium absorption efficiency of a human body is promoted; the yeast has the function of degrading phytic acid and oxalic acid, can release calcium ions combined with oxalic acid and phytic acid in plants, and increases the content of water-soluble calcium. The saccharomyces cerevisiae can grow in the pH value range of 3.0-7.5, the optimum pH value is 4.5-5.0, and the lactobacillus generates organic acid to reduce the pH value of the environment of the moringa fermentation liquid and promote the growth and the propagation of the saccharomyces cerevisiae. The conversion of the moringa calcium is improved and the bioavailability is improved by utilizing the symbiotic effect of different microbial fermentations.
The lactobacillus and the saccharomycetes can decompose macromolecular substances into micromolecular substances which are easy to utilize in cell metabolism, meanwhile, the protein content in the moringa leaves is rich and can reach 27.1-31.4%, macromolecular protein can be degraded into micromolecular protein polypeptide in the microbial fermentation process, and free amino and carboxyl in polypeptide molecules are combined with water-soluble calcium ions to form a polypeptide calcium chelate; meanwhile, the moringa polypeptide is partially degraded to generate amino acid, the amino acid is combined with calcium to form an amino acid calcium chelate, and both the polypeptide calcium and the amino acid calcium can increase the bioavailability and the bone deposition rate of calcium ions and increase the calcium supplement effect. Ca2+The calcium chelate and the molecular calcium are absorbed and utilized by human bodies mainly in intestinal tracts, do not form precipitates and are directly absorbed by the small intestines under the alkaline environment of the small intestines, have better stability and can be used as a calcium supplement with higher activity.
In addition, a small amount of lactose is added in the fermentation process as a carbon source starter for the rapid growth of microorganisms, the generation of lactic acid is promoted, calcium lactate is formed, and the growth of mixed bacteria can be avoided in a rapid acidic environment.
The second characteristic of the invention is that the invention adopts mixed microorganism to ferment in a grading and gradient way, the first stage of the grading fermentation is: fermenting for 20-24 h at 36-38 ℃ (pH value is reduced to 4.0-4.5, anaerobic culture), wherein the purpose of fermentation at this stage is to ensure that three lactic acid bacteria grow and reproduce rapidly under anaerobic condition and sugar sources such as lactose, etc., reduce environmental pH value, generate a large amount of calcium lactate, and decompose substances such as moringa oleifera polysaccharide, macromolecular protein, etc.; in the second stage, fermentation is carried out for 24-36 h (aerobic culture) at 28-30 ℃, and the fermentation is mainly beneficial to yeast growth and the degradation of substances such as phytic acid and the like under the condition that the pH value is 4.0-4.5, so that calcium ions are released, meanwhile, the macromolecular components of the moringa oleifera are further degraded, and beneficial metabolites are generated; and finally, fermenting for 18-24 h at 36-38 ℃ (anaerobic culture), and ending fermentation when the final pH value reaches 3.75-3.85, so that the acidity of the solution environment is further reduced, and the dissolution of nutrient components in the moringa oleifera and the generation of beneficial metabolites such as polypeptide, amino acid and the like are increased.
The invention is characterized in that most calcium preparations in the prior art are solid, the disintegration and the solubility of the tablet in the gastrointestinal tract have important influence on the drug absorption, most calcium preparations need gastric acid action, such as calcium carbonate and calcium hydrophosphate, so the calcium preparations are not suitable for people lacking gastric acid or insufficient gastric acid secretion, and the moringa natural calcium provided by the invention has good instant solubility and can release calcium ions within a neutral pH value range, so the moringa natural calcium is excreted into the small intestine by the stomach 2h after being taken and is absorbed by the organism, thereby all calcium elements in the calcium preparations can be absorbed to the maximum extent. The chelated calcium content of the moringa organic calcium product provided by the invention can reach 73.4-75.7%, and pharmacokinetics shows that the moringa organic calcium product has high bioavailability, is generally higher than calcium supplement products on the market, and can effectively improve osteoporosis of middle-aged and elderly people and increase bone density.
In a simulated gastric juice environment, the calcium chelation rate of the moringa oleifera is reduced to 50.46 percent and is rapidly reduced to 8.72 percent within 15 min; when the simulated gastric digestion time is prolonged to 2h, the decline rate of the chelation rate gradually slows down, and finally declines to 2.49%, at which time Ca is present2+Mainly in free form. This is probably due to the lower pH in the stomach, H+High concentration of H with extremely high content+With Ca2+Compete for most of the binding sites on moringa oleifera polypeptideAnd the structure of polypeptide chelated calcium is destroyed, resulting in rapid reduction of chelation rate. In the process of simulating intestinal digestion, the chelating rate of the fishbone gelatin calcium peptide chelate in simulated intestinal fluid is (48.15 +/-1.77)%, and the chelating rate of the chelate in intestinal electrolyte is (55.58 +/-0.68)%. This is due to the increased pH after entering the intestine, the H in the environment+Gradually decrease in concentration of Ca2+And the chelating ability of the polypeptide is enhanced, and finally the calcium peptide chelating rate is increased, so that the absorptivity of calcium in moringa oleifera organic calcium is higher than that of ionized calcium and calcium carbonate in a common calcium product.
Compared with the prior art, the invention has the following beneficial effects: the organic calcium provided by the invention has high solubility, is less influenced by acidity and temperature, can ensure the dissolution state of calcium in neutral and alkaline environments of small intestine, promotes intestinal wall absorption, and reduces iron absorption obstruction, generally speaking, the calcium can obstruct human body from absorbing other mineral substances, while the moringa natural calcium can reduce the obstruction of human body to iron absorption, and can effectively overcome the side effects brought by other calcium nutrition enhancers. The product has reasonable calcium-phosphorus ratio and contains components for promoting calcium absorption of human body.
The method provided by the invention has the advantages of reasonable process, high production efficiency, good sensory quality of the product and stable property; the moringa oleifera organic calcium product obtained by processing contains a large amount of plant organic calcium, and also contains a large amount of proteins, polypeptides, amino acids, vitamins and mineral elements necessary for human bodies, can effectively supplement calcium deficiency of the human bodies, improve osteoporosis, eliminate fatigue and improve sleep, is a nutritional and healthy green food, and has wide market prospect.
Drawings
FIG. 1 is a flow chart of the processing of natural organic calcium moringa oleifera products;
FIG. 2 shows the effect of different sugar sources on the calcium content of moringa oleifera fermentation dry powder;
fig. 3 is a pharmacokinetic profile.
Detailed Description
The technical solution of the present invention will be described in further detail with reference to specific examples, but the present invention is not limited to the following technical solutions.
The strains adopted in the following examples are respectively derived from China center for culture Collection of microorganisms and Guangdong province center for culture Collection of microorganisms.
Domestication of lactic acid bacteria: respectively transferring lactobacillus acidophilus, lactobacillus johnsonii and lactobacillus reuteri into an MRS broth liquid culture medium for standing culture, wherein the culture temperature range is preferably 30-40 ℃, more preferably 36-38 ℃, the culture time is preferably 48-72h, taking out after the culture medium is turbid, transferring the culture medium into a next generation MRS broth moringa culture medium (adding moringa leaf dry powder into a solid broth culture medium to replace partial culture medium powder), the ratio of the broth culture medium powder to moringa is 5:1, simultaneously adding glucose accounting for 20-30% of the weight of moringa powder, inoculating a lactic acid bacteria solution grown in a previous generation (full MRS culture medium), the adding amount of the lactic acid bacteria solution is 2-4% (volume ratio) of the MRS broth moringa culture medium, after culturing for 3d at 36-38 ℃, adding the lactic acid bacteria solution into the next generation MRS broth culture medium according to the volume ratio of 2-4% (volume ratio of the broth powder to the moringa leaf powder in the culture medium is 4:1), domesticating at the same temperature, time and mode, sequentially domesticating the three lactic acid bacteria in a gradient manner (the ratio of MRS broth powder to moringa leaf powder in the culture medium is 4:1, 3:1, 2:1, 1:2, 1:3, 1:4, 1:5 and 0:1), culturing for ten generations by adopting a moringa culture medium (a mixture of moringa powder and glucose), and storing in a refrigerator at 2-4 ℃ for later use.
Domestication of yeast: the saccharomyces cerevisiae 1 is transferred into a potato glucose liquid culture medium for shaking culture, the temperature range of the shaking culture is preferably 25-35 ℃, more preferably 28-30 ℃, the rotation range of the shaking culture is preferably 100-150rpm, more preferably 110-120rpm, the time of the shaking culture is preferably 48-72h, the saccharomyces cerevisiae 1 is taken out after the culture medium is precipitated, the culture medium is evenly shaken and then transferred into a next generation potato glucose broth moringa culture medium (solid potato broth culture medium is added with moringa oleifera leaf dry powder to replace part of culture medium powder), the ratio of the broth culture medium powder to the moringa oleifera is 5:1, glucose accounting for 20-30% of the weight of the moringa oleifera powder is simultaneously added, then a previous generation lactobacillus (bacterial liquid whole potato and glucose broth culture medium is inoculated) is inoculated, the inoculation amount is 2-4% (volume ratio) of the potato glucose broth moringa oleifera culture medium, after culturing for 3 days at 28-30 ℃, adding the mixture into a next generation culture medium according to the volume ratio of 2-4% (the ratio of potato glucose broth powder to moringa oleifera leaf powder in the culture medium is 4:1), sequentially carrying out gradient acclimation on saccharomyces cerevisiae 1 according to the acclimation temperature, acclimation time and acclimation mode, wherein the ratio of potato glucose broth powder to moringa oleifera leaf powder in the culture medium is 4:1, 3:1, 2:1, 1:2, 1:3, 1:4, 1:5 and 0:1, culturing for ten generations by adopting a moringa oleifera culture medium (a mixture of moringa oleifera powder and glucose), and storing in a refrigerator at 2-4 ℃ for later use.
Example 1 fermentation with Lactobacillus acidophilus, Lactobacillus johnsonii, Lactobacillus reuteri and Saccharomyces cerevisiae I
(1) Drying: and (3) selecting tender fresh moringa leaves, deactivating enzyme at 105-110 ℃ for 10-15 min, and drying at 65 ℃.
(2) Grinding: pulverizing the dried moringa leaves by using a pulverizer or a powder grinding machine, and sieving by using a 60-mesh sieve for later use.
(3) Adding water and mixing uniformly: according to the formula, the moringa oleifera powder: 1 part of water: (15-20) adding warm water (75-85 ℃) and uniformly stirring.
(4) And (3) high-pressure sterilization: and (3) filling the moringa aqueous solution into a suitable glass or stainless steel vessel, and transferring the vessel to an autoclave for sterilization. And sterilizing at 121 ℃ for 15-20 min, and allowing the sterilization pot to be opened after the temperature of the sterilization pot is reduced to 50 ℃, so that the loss of the moringa sample due to bottle spraying caused by deposition of moringa powder and rapid reduction of air pressure and temperature at the bottom of the container is avoided.
(5) Strains (domesticated), sugar source addition: adding lactobacillus and yeast domesticated by moringa oleifera into the moringa oleifera liquid in proportion, wherein the addition proportion of the three strains is lactobacillus acidophilus: lactobacillus johnsonii: lactobacillus reuteri: the proportion of the saccharomyces cerevisiae I is 3:3:2:2, the addition amount of the strain is 0.15-0.25% (the weight of the domesticated wet thalli accounts for the proportion weight of the moringa oleifera fermentation liquid); and (3) selecting and adding 8.0-12.0% of lactose (accounting for the weight percentage of the moringa oleifera leaf dry powder), wherein the added lactose can be used as a carbon source for the growth of microorganisms.
(6) Gradient temperature fermentation: and (3) finishing fermentation by adopting a gradient temperature fermentation mode of 37 ℃ (24h, anaerobic) to 28 ℃ (36h, aerobic) to 37 ℃ (24h, anaerobic) when the final pH value reaches 3.75-3.85.
(7) Centrifuging: centrifuging for 20min under 4000r/min, centrifuging to obtain supernatant, and using residue for development of other products.
(8) And (3) vacuum concentration: concentrating the supernatant in water bath at 50-55 deg.C and 0.75Mpa to one third of the original liquid, transferring to stainless steel plate, and quick freezing at-80 deg.C.
(9) And (3) freeze drying: transferring the quick-frozen iron plate into a freeze dryer, freeze-drying the supernatant sample under the conditions of-50 to-40 ℃ and 10 to 20Pa of vacuum degree, crushing the freeze-dried sample, and sieving the crushed freeze-dried sample with a 60-mesh sieve for later use.
(10) Adding auxiliary materials: selecting natural sweet orange powder, aspartame and xylitol as auxiliary materials, and uniformly mixing the auxiliary materials with the moringa oleifera fermentation dry powder to obtain the moringa oleifera organic calcium product. The formula proportion of the product is 45 percent of moringa fermentation broth dry powder, 15 percent of natural sweet orange powder, 1.5 percent of glycoprotein, 30 percent of xylitol and 8.5 percent of soybean lecithin.
(11) And (3) sterilization: the powder material treated in the previous step is sent into a baking oven and sterilized for 30-40min at the temperature of 120-.
(12) Packaging: vacuum packaging the sterilized powder by an automatic packaging machine to obtain a final product; the process flow is shown in FIG. 1.
In addition, the addition of different sugar sources can provide carbon sources for the rapid growth of microorganisms, but the influence of different sugar sources on the production and metabolism of microorganisms is different, as shown in FIG. 2. As shown in FIG. 2, the highest calcium content in the moringa oleifera fermentation product prepared by adding lactose to ferment moringa oleifera can reach 4977mg/100 g. The calcium content of the moringa fermentation products of different sugar sources is in the order of lactose, glucose, sucrose, maltose and fructose.
Example 2 continuous anaerobic fermentation
The fermentation mode in the embodiment 1 is changed from staged fermentation to fermentation at 37 ℃ for 72h under anaerobic condition, the fermentation is finished when the pH value is 3.75-3.85, other conditions are not changed, the total solid matter content and the calcium content of the final product are respectively 89.1% and 83.3% of the staged fermentation content, and the yield and the product quality are reduced; the fermentation mode in the example 1 is changed from staged fermentation to fermentation at 37 ℃ for 72h under aerobic condition, the pH value is difficult to reduce, the pH value in the whole fermentation process is reduced from 5.67 to 5.11, meanwhile, the taste and flavor of the product are obviously reduced, and the unpleasant odor is generated, and the yield and the calcium content of the product are 82.33 percent and 93.78 percent of the staged fermentation respectively. Ending the fermentation, keeping other conditions unchanged, and reducing the yield and the product quality, wherein the final solid content and the calcium content of the product are respectively 89.1 percent and 83.3 percent of the fermentation content in stages; the fermentation mode in the example 1 is changed from staged fermentation to 30 ℃ fermentation for 72h under anaerobic condition, other conditions are not changed, the total solid matter content and the calcium content of the final product are 77.16% and 63.53% of the staged fermentation content respectively, and the yield and the product quality are reduced.
EXAMPLE 3 preparation of organic calcium by Strain modification
The moringa natural organic calcium fermentation liquor prepared by adopting the four strains and the staged fermentation has the calcium content of (4972 +/-116) mg/100g after being freeze-dried or spray-dried, is domesticated by adopting the same method for saccharomyces cerevisiae 1, saccharomyces cerevisiae 2, lactobacillus bulgaricus, lactobacillus thermophilus, lactobacillus bulgaricus, lactobacillus casei, lactobacillus acidophilus, lactobacillus johnsonii, lactobacillus reuteri, bifidobacterium longum, lactobacillus fermentum and lactobacillus rhamnosus, is inoculated with the same amount of strains for fermentation at the optimal temperature (30 ℃ for saccharomycetes and 37 ℃) and has the same fermentation conditions, and the calcium content of the supernatant after the centrifugation of the fermentation liquor is respectively (4171 +/-97), (3822 +/-221), (3211 +/-103), (3166 +/-217), (3301 +/-117), (3455 +/-88), (9 +/-106), (3819 +/-179) after being spray-dried, (3779 + -211), (3008 + -164), (3471 + -119), (3551 + -103) mg/100g, that is, the efficiency of preparing moringa organic calcium by saccharomyces cerevisiae 1 in the yeast is significantly higher than that by saccharomyces cerevisiae 2, and the three bacteria with the highest extraction efficiency of moringa organic calcium in the 10 lactic acid bacteria are lactobacillus johnsonii, lactobacillus reuteri and lactobacillus acidophilus respectively.
EXAMPLE 4 preparation of organic calcium by Strain modification
Fermenting under the optimal fermentation condition, researching the influence of different strains and composition modes thereof on the calcium content in the moringa oleifera fermentation liquor, and designing an experimental group as follows: adding 0.15% of Moringa oleifera liquid into Saccharomyces cerevisiae 1, and performing aerobic fermentation at 30 deg.C for 84 hr; secondly, the step of: lactobacillus acidophilus: lactobacillus johnsonii: lactobacillus reuteri 3:3:2, with the addition of 0.15%, anaerobic fermentation at 37 ℃ for 84h (c): lactobacillus acidophilus: lactobacillus johnsonii: lactobacillus reuteri: the saccharomyces cerevisiae I is 3:3:2:2, the addition amount is 0.15%, and the anaerobic fermentation is carried out for 84h at 37 ℃: lactobacillus acidophilus: lactobacillus johnsonii: lactobacillus reuteri: yeast I is 3:3:2:2, the adding amount is 0.15%, the ratio of staged fermentation (37 ℃ (24h, anaerobic) -28 ℃ (36h, aerobic) -37 ℃ (24h, anaerobic), lactobacillus johnsonii to saccharomyces cerevisiae I is 5:5:3, the adding amount is 0.15%, the sensory scores of samples after centrifugation and spray drying of supernatant of five groups of fermentation liquor of staged gradient fermentation 37 ℃ (24h, anaerobic) -28 ℃ (36h, aerobic) -37 ℃ (24h, anaerobic), are 71.2, 78.9, 83.3, 86.6 and 79.1, the calcium contents are respectively (4151 +/-77), (3944 +/-136), (4233 +/-103), (4985 +/-174), (4544 +/-125) mg/100g, the sensory scores and the calcium contents of staged mixed strain fermentation powder are the highest, and the product and the sensory quality are better.
Example 5 preparation of a nutraceutical containing organic calcium
Example 1 after preparation of the organic calcium, adjuvants are added: selecting natural sweet orange powder, aspartame and xylitol as auxiliary materials, and uniformly mixing the auxiliary materials with the moringa oleifera fermentation dry powder to obtain the moringa oleifera organic calcium product. The formula proportion of the product is 45 percent of moringa fermentation broth dry powder, 15 percent of natural sweet orange powder, 1.5 percent of glycoprotein, 30 percent of xylitol and 8.5 percent of soybean lecithin.
The powder material treated in the previous step is sent into a baking oven and sterilized for 30-40min at the temperature of 120-. And (5) carrying out vacuum packaging on the sterilized powder through an automatic packaging machine to obtain a final product.
Effect verification experiment: (1) a certain product on the market and a moringa natural organic calcium product are selected to carry out rat pharmacokinetic analysis, a specific experiment is that 20 SD rats are taken, female and male halves are taken, after adaptive feeding is carried out for 7d, the SD rats are randomly divided into 2 groups according to body weight and sex, different calcium agents are converted into calcium elements, namely, each kilogram of animals weighs 10 calcium elements (mg/kg) each time, the weight of each group is 10, 9 time phase points are set, the calcium dose is 200 mg/kg., fasting is carried out for 12 hours before the experiment, water is not forbidden, gastric perfusion administration is carried out, 0, 15, 30, 60, 90, 120, 180, 240 and 300min after the administration, eyeball blood is taken, serum is centrifugally separated, the serum is sealed and stored in a refrigerator, and the calcium ion concentration in the blood is detected by using a calcium kit, and the result is shown in a graph 3 and a table 1.
TABLE 1 pharmacokinetic parameters
As can be seen from Table 1, the drug-time curve area of the moringa natural organic calcium is significantly higher than that of a calcium product on the market under the same dosage, and the relative bioavailability of the moringa natural organic calcium is 110.26% of that of the calcium product on the market, namely the bioavailability and the activity of the moringa natural organic calcium are higher.
As can be seen from FIG. 3, the blood calcium concentration of rats reaches the peak at 15min, and the peak value of the moringa oleifera organic calcium product is higher than that of a certain product on the market, which is related to the fact that the calcium part in the moringa oleifera calcium exists in the form of calcium lactate and the absorption rate is faster; however, after the peak is reached, the blood calcium concentration begins to decrease, the blood calcium concentration of a certain calcium product on the market is greatly reduced, the blood calcium concentration is always lower than that of a moringa natural organic calcium product, meanwhile, the blood calcium concentration later stage of the moringa organic calcium is in a slow stage, which is related to the existence of the moringa organic calcium in a chelating manner, polypeptide calcium and amino acid calcium directly enter cells in an integral manner in the small intestine, and then calcium ions are decomposed to enter blood under the action of cell sap, so that calcium in the body and the blood calcium reach a dynamic balance, and a slow release effect is achieved.
(2) Selecting a certain product on the market and food-grade calcium chloride, designing low, medium and high dosage groups of moringa oleifera natural organic calcium products and a blank control group to carry out animal experiments on rats, and researching calcium metabolism experiments and index changes of thighbones of the rats. The specific experiment comprises randomly dividing 60 tested animals into 56 groups according to body constitution, setting low, medium and high dosage groups of moringa oleifera organic calcium samples, and respectively intragastrically administering different dosage level samples (Ca)2+Dosage levels were 50, 100, 200mg/kg), respectively; simultaneously setting a calcium chloride control group and a calcium product control group in the market (both are combined with peppery food)Calcium levels were the same in the calcium-rich group), and the same volume of physiological saline was perfused into the stomach of the placebo group. All rats ingested low calcium feed (calcium content 0.1%), drunk deionized water, and gavage continuously for 4 weeks, and body mass changes and feed intake were recorded. Calcium metabolism experiment: carrying out 3d calcium metabolism experiment after 4 weeks, transferring the animals into a metabolism cage, respectively collecting feces and urine of each tested animal for 72h, and accurately recording the feed intake of each tested animal; the calcium content in the feed, feces and urine was measured, and the apparent absorption rate and retention rate of calcium were calculated, with the specific results shown in table 2. Measuring femoral indexes: stripping the thighbone of the rat, and weighing the thighbone after removing muscles and fascia; measuring the length and width of the bone and measuring the content of bone calcium. The calcium content in the feed, feces, urine and thighbone is measured by the atomic absorption method of GB/T5009.92-2003 'determination of calcium in food', and the detection results are shown in Table 3.
The body mass is one of the apparent indexes of the health of the organism, and the abnormal increase and decrease of the body mass indicate that the health condition of the rat is possibly abnormal. The diet, defecation and activity of the rats were normal during the experimental period. No obvious abnormality is observed in the observation of each organ in the dissection process, which preliminarily shows that the moringa oleifera organic calcium has no side effect on rats and has better safety.
TABLE 2 calcium metabolism index in rats
Note: the difference of the same row shoulder mark letters shows obvious difference (P <0.05)
The absorption and utilization of calcium in the organism generally includes 3 processes of ingestion, absorption and excretion. The absorption and retention rates of calcium reflect the absorption of calcium in the gastrointestinal tract, and the extent to which the absorbed calcium is stored and used by the body to maintain normal physiological functions. As shown in Table 2, the calcium absorption and retention rates of the blank control group were significantly higher than those of the remaining sample groups (P <0.05) due to the low intake and the high deficiency. With the gradual increase of the organic calcium intake of the moringa oleifera, the absorption rate and the storage rate tend to decrease. Meanwhile, the calcium absorption rate and the storage rate of the high-dose group are higher than those of a calcium chloride control group and a certain calcium product on the market, and the calcium absorption rate reaches a significant level (P is more than 0.05).
TABLE 3 Effect of different calcium sources on femoral index
As shown in Table 3, there was a tendency for the quality of the femurs of the rats in each group to increase, but there was no significant difference (P > 0.05); there is no significant difference in the indexes of femur length and diameter. Compared with a blank control group, the bone calcium content of a rat fed with the low-dose moringa natural organic calcium is obviously increased (P is less than 0.05), the bone calcium content of a medium-dose moringa natural organic calcium and calcium chloride control group and a calcium supplement product on the market is obviously increased (P is less than 0.01), namely, the increase of the calcium intake and the increase of the calcium content of thighbone are in a dose-dependent relationship, the bone calcium content of the rat fed with the moringa natural organic calcium is obviously higher than that of a calcium low-dose group and is not lower than that of the calcium chloride control group with a corresponding dose, and the absorption rate of calcium is not lower than that of the calcium chloride control group, so that the moringa natural organic calcium has the effect of improving the bone density, and the calcium supplement effect is better than that of a calcium supplement product on the market, and the moringa.
Claims (7)
1. The moringa natural organic calcium is characterized in that the organic calcium is obtained by performing gradient fermentation on moringa leaf powder by stages through mixed microorganisms; the mixed microorganism is lactobacillus and yeast;
the staged gradient fermentation operation is as follows: firstly, carrying out anaerobic fermentation for 20-24 h at the temperature of 36-38 ℃, reducing the pH value to 4.0-4.5, then carrying out aerobic fermentation for 24-36 h at the temperature of 26-30 ℃, finally carrying out anaerobic fermentation for 18-24 h at the temperature of 36-38 ℃, and finishing fermentation when the pH value reaches 3.75-3.85; centrifuging for 20min at 4000r/min after fermentation is finished, centrifuging to obtain supernatant, and concentrating the supernatant in water bath vacuum at 50-55 ℃ and 0.55-0.8 Mpa;
the mixed microorganism is Lactobacillus acidophilus, Lactobacillus johnsonii and Saccharomyces cerevisiae I, and the weight ratio of the three strains is 3-6: 3-6: 2-4; or
The mixed microorganism is lactobacillus acidophilus, lactobacillus johnsonii, lactobacillus reuteri and saccharomyces cerevisiae I, and the weight ratio of the four strains is 3-5: 3-5: 2-4: 2 to 4.
2. A preparation method of natural moringa organic calcium is characterized in that moringa leaves are pretreated and then uniformly mixed with water, autoclaved, added with strains of mixed microorganisms and glycogen for staged gradient fermentation, centrifuged after the fermentation is finished, and supernatant is obtained, and subjected to vacuum concentration, freeze drying or spray drying to obtain the moringa organic calcium; the mixed microorganism is lactobacillus and yeast;
the moringa oleifera leaf pretreatment process comprises the following steps: selecting tender fresh moringa leaves, deactivating enzyme at 105-110 ℃ for 10-15 min, and drying at 60-70 ℃; crushing the dried moringa leaves by using a grinding machine or a powdering machine, and sieving by using a sieve of 50-70 meshes for later use;
the mixed microorganism comprises lactobacillus acidophilus, lactobacillus johnsonii and saccharomyces cerevisiae I, wherein the weight ratio of the three strains is 3-6: 3-6: 2 to 4, or
The mixed microorganism is lactobacillus acidophilus, lactobacillus johnsonii, lactobacillus reuteri and saccharomyces cerevisiae I, and the weight ratio of the four strains is 3-5: 3-5: 2-4: 2-4;
the staged gradient fermentation operation is as follows: firstly, carrying out anaerobic fermentation for 20-24 h at the temperature of 36-38 ℃, reducing the pH value to 4.0-4.5, then carrying out aerobic fermentation for 24-36 h at the temperature of 26-30 ℃, finally carrying out anaerobic fermentation for 18-24 h at the temperature of 36-38 ℃, and finishing fermentation when the pH value reaches 3.75-3.85; centrifuging for 20min at 4000r/min after fermentation is finished, centrifuging to obtain supernatant, and concentrating the supernatant in water bath vacuum at 50-55 ℃ and 0.55-0.8 Mpa.
3. The method for preparing natural moringa organocalcium according to claim 2, wherein the moringa leaves are pretreated and then mixed with water in a ratio of 1: 15-20, and then uniformly stirring at 75-85 ℃.
4. The method for preparing natural moringa organic calcium as claimed in claim 2, wherein the domesticated strain is added into the moringa liquid in proportion, and the total weight of the wet thalli of the domesticated strain accounts for 0.15-0.25% of the weight of the moringa fermentation liquid; and adding lactose accounting for 8.0-12.0% of the weight of the moringa oleifera leaf dry powder, wherein the added lactose is used as a carbon source for microbial growth.
5. The method for preparing the moringa natural organic calcium as claimed in claim 4, wherein the acclimation process of the lactic acid bacteria is as follows: transferring lactic acid bacteria into an MRS broth liquid culture medium for static culture, wherein the culture temperature is 30-40 ℃, the culture time is 48-72 hours, taking out the culture medium after the culture medium is turbid, shaking up the culture medium, transferring the culture medium into a next generation MRS broth moringa culture medium, and the ratio of MRS broth culture medium powder to moringa oleifera is 3-6: 1, after the culture is finished, performing gradient acclimation on the lactic acid bacteria by adopting a culture medium with the ratio of MRS broth powder to moringa leaf powder being 4:1, 3:1, 2:1, 1:2, 1:3, 1:4, 1:5 and 0:1, culturing for ten generations by adopting a moringa culture medium, and storing in a refrigerator at 2-4 ℃ for later use.
6. The method for preparing natural moringa organic calcium according to claim 2, wherein the domestication process of the yeast is as follows: transferring the saccharomyces cerevisiae 1 into a potato glucose liquid culture medium for shaking culture, wherein the shaking culture temperature is 25-35 ℃, the culture revolution is 100-150rpm, the culture is performed for 48-72h, the culture medium is taken out after precipitation, the culture medium is uniformly shaken and then transferred into a next generation potato glucose broth moringa oleifera culture medium, and the ratio of broth culture medium powder to moringa oleifera is 3-6: after culturing for 3 days at the temperature of 1.28-30 ℃, performing gradient acclimation on the saccharomyces cerevisiae 1 by adopting a culture medium with the ratio of potato glucose powder to moringa oleifera leaf powder being 4:1, 3:1, 2:1, 1:2, 1:3, 1:4, 1:5 and 0:1, culturing for ten generations by adopting a moringa oleifera culture medium, and storing in a refrigerator at the temperature of 2-4 ℃ for later use.
7. A nutritional product containing the moringa natural organic calcium as described in claim 1, wherein the moringa fermentation broth dry powder is 45-50%, the natural sweet orange powder is 10-15%, the aspartame is 1.3-1.5%, the xylitol is 25-30%, and the soybean lecithin is 8.5-10% by weight.
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| CN108771157A (en) * | 2018-06-13 | 2018-11-09 | 海南木辣达生物科技有限公司 | A kind of Moringa leaf vegetables and preparation method thereof |
| CN110024938A (en) * | 2019-04-18 | 2019-07-19 | 云南农业大学 | A kind of biodegrading process of leaf of Moringa mesoxalic acid and application |
| CN110326671A (en) * | 2019-06-05 | 2019-10-15 | 云南省高原特色农业产业研究院 | A kind of Moringa calcium extracts and Moringa high calcium walnut beverage preparation method |
| CN115944061A (en) * | 2022-12-23 | 2023-04-11 | 山东劲牛集团股份有限公司 | Preparation method of quick-acting calcium type fermented high-calcium vegetable powder |
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