阅读说明：本技术 聚球藻源富钙纳米多聚磷酸体补钙剂及其制备方法 (Calcium supplement of calcium-rich nano poly-phosphate from synechococcus and preparation method thereof ) 是由 吴浩浩 曾名湧 杨益盛 冯一农 于 2020-05-27 设计创作，主要内容包括：本发明公开了一种聚球藻源富钙纳米多聚磷酸体(PPB-Ca)补钙剂及其制备方法,其制备方法,包括下列步骤：采用钙强化medium A培养基(钙浓度为300-1500 mg/L)培养聚球藻至对数后期,收集藻体,现用或冷冻保藏；将藻体置于沸水中抽提5-30 min,离心后取上清,获得藻体水提物；将藻体水提物进行葡聚糖凝胶(Sephadex G50/G75/G100/G150)柱层析,收集排阻峰组分；对排阻峰组分进行超滤(截留分子量3-10 KDa)浓缩后,冻干,即得PPB-Ca样品。本发明中PPB-Ca是粒径约为30-120 nm的球形微粒,在小鼠体外结扎肠段中能够以内吞作用的方式被高效吸收和转运,在大鼠口服药物代谢动力学和骨质疏松模型中也表现出良好的生物可利用性,尤其在对抗植酸对钙吸收的抑制作用方面效果显著,故PPB-Ca是一种理想的微藻源补钙剂,可应用于素食习惯导致的钙缺乏人群的营养强化和营养补充。(The invention discloses a calcium supplementing agent of calcium-rich nano polyphosphate (PPB-Ca) of synechococcus source and a preparation method thereof, wherein the preparation method comprises the following steps: culturing synechococcus pluvialis by adopting a calcium-enhanced medium A culture medium (the calcium concentration is 300-; extracting algae in boiling water for 5-30 min, centrifuging, and collecting supernatant to obtain algae water extract; subjecting the algae water extract to Sephadex G50/G75/G100/G150 column chromatography, and collecting the components with exclusion peak; and (3) performing ultrafiltration (the cut-off molecular weight is 3-10 KDa) on the exclusion peak components, concentrating, and freeze-drying to obtain the PPB-Ca sample. The PPB-Ca is spherical particles with the particle size of about 30-120nm, can be efficiently absorbed and transported in an endocytosis mode in a mouse in-vitro ligated intestinal segment, has good bioavailability in a rat oral pharmacokinetics and osteoporosis model, and particularly has a remarkable effect on resisting the inhibition effect of phytic acid on calcium absorption, so the PPB-Ca is an ideal microalgae source calcium supplement and can be applied to the nutrition strengthening and nutrition supplement of people with calcium deficiency caused by vegetarian habits.)

1. The application of synechococcus in the production of a novel calcium nutrient supplement or fortifier based on nano polyphosphate body.

2. A calcium supplement agent containing Nanocladium synechococcus source calcium is characterized by comprising calcium-rich polyphosphate nanoparticles (PPB-Ca).

3. The method for preparing the nannochloropsis source nano calcium supplement of claim 2, comprising the steps of:

(1) and (3) culturing synechococcus by adopting a calcium-enhanced medium A culture medium until the alga body is collected at the later logarithmic phase.

(2) The algae body water extract is obtained by a hot water extraction method, namely, the algae body is put into boiling water for extraction and then cooled to room temperature, and supernatant is collected after centrifugation to obtain the algae body water extract.

(3) Separating the algae body water extract by using sephadex column chromatography, and collecting exclusion peak components;

(4) and (4) performing ultrafiltration concentration on the exclusion peak component in the step (3), and freeze-drying to obtain the PPB-Ca sample.

4. The method for preparing the nannochloropsis source nanometer calcium supplement of claim 3, wherein the concentration of calcium in the calcium-enriched medium A culture medium of step (1) is 300-1500 mg/L.

5. The method for preparing the nannochloropsis source nano calcium supplement of claim 3, wherein the hot water extraction time of step (2) is 5-30 min.

6. The method for preparing the NanoCa Synechococcus source calcium supplement of claim 3, wherein the Sephadex G50/G75/G100/G150 is used as the Sephadex gel in step (3).

7. The method for preparing the nannochloropsis source nano-calcium supplement of claim 3, wherein the cut-off molecular weight of the ultrafiltration membrane of step (4) is in the range of 3-10 KDa.

8. The Nanocalcium calcium supplement derived from Synechococcus according to any one of claims 3 to 7.

Technical Field

The invention relates to a preparation method and application of a calcium supplementing agent of calcium-rich nano polyphosphate (PPB-Ca) from synechococcus.

Background

Calcium is the most abundant essential mineral element in human body, has various physiological effects in bone and tooth structure, blood coagulation, nerve transmission, electromyography, internal and external secretion, etc., and enjoys the reputation of "life element". The dietary recommended calcium intake for individuals over 4 years of age is 1000 to 1300 mg/d depending on age and physiological condition. It has been reported that by 2011, about 35 million people worldwide still face the risk of calcium deficiency due to insufficient calcium intake (684 ± 211 mg/d), especially in africa and asia. In addition to insufficient calcium intake, calcium malabsorption caused by dietary calcium absorption inhibitors (especially phytic acid) is also a significant cause of calcium deficiency. Calcium deficiency leads to secondary hyperparathyroidism and is closely related to the pathogenesis and low bone mass of osteoporosis. Therefore, the development of a calcium nutrition enhancer capable of resisting inhibitors such as phytic acid is particularly urgent. The nano calcium has good solubility (namely, can form stable colloidal solution), and is relatively resistant to the adverse effects of inhibitors such as phytic acid and the like compared with ionized calcium, so the nano calcium is an ideal calcium supplement.

Polyphosphate is a non-toxic macromolecular polymer suitable for biomineralization to form mineral nanoparticles under mild conditions as a 'soft template'. The biomineralization synthesized nanoparticles have better colloid stability, so the biomineralization synthesized nanoparticles are very suitable for the production of nano mineral nutritional supplements or fortifiers. Polyphosphate is synthesized in biological cells by polyphosphate kinase catalysis, and is usually present in the cells in the form of polyphosphate body (PPB), which is an inclusion body with a nanometer or submicron scale (15-200 nm). The cationic species in the PPB is mainly calcium, so the PPB has the potential of being developed into a natural nano calcium nutritional supplement or enhancer as a carrier of mineral nutritional element calcium, but no relevant research report exists at home and abroad at present.

Synechococcus 7002 is a unicellular cyanobacterium having various excellent characteristics suitable for biotechnological and food industry applications, for example, being capable of growing in various ways including photoautotrophic, heterotrophic, and mixed culture, having broad salinity and high light tolerance, very short doubling time (< 4 h), not being found to secrete any cyanobacterial toxin or having related homologous genes, and being capable of photosynthetic fermentation using inexpensive seawater as a culture medium without encroaching on cultivated land, food, fresh water, fossil energy, and the like. The PPB is present in the synechococcus 7002 in the form of nanoparticles. Therefore, Synechococcus 7002 is an ideal cell factory for the production of novel nano-PPB mineral nutritional supplements or fortifiers.

Disclosure of Invention

The invention aims to provide a novel Synechococcus source PPB-Ca calcium supplement and a preparation method thereof.

The technical problem to be solved by the invention is as follows: a nanometer calcium supplement is prepared from Synechococcus 7002, and contains PPB-Ca.

The technical scheme adopted for realizing the purpose of the invention is as follows:

culturing synechococcus 7002 by using a calcium-enhanced medium A culture medium to the late logarithmic phase, and collecting algal bodies; extracting with hot water to obtain algae water extract, separating with Sephadex column chromatography, and collecting the components with exclusion peak; and (4) carrying out ultrafiltration concentration on the exclusion peak component, and freeze-drying to obtain the PPB-Ca sample.

The preparation method of the synechococcus source PPB-Ca calcium supplement specifically comprises the following steps:

(1) culturing synechococcus with calcium-enriched medium A (calcium concentration is 300-1500 mg/L) until late logarithmic phase, and collecting the algae.

(2) Extracting with hot water for 5-30 min, cooling to room temperature, centrifuging, and collecting supernatant.

(3) Separating the algae body water extract by using Sephadex G50/G75/G100/G150 column chromatography, and collecting exclusion peak components;

(4) and (4) performing ultrafiltration concentration on the exclusion peak component in the step (3), and freeze-drying to obtain the PPB-Ca sample.

Preferably, in the step (1) of the present invention, the calcium concentration in the calcium-fortified medium A is 0.81g/L, and the biomass of Synechococcus and the yield of PPB-Ca are at optimum levels when the medium is cultured to the late logarithmic phase.

Furthermore, the optimal range of the hot water extraction time in the step (2) of the invention is 10-15 min. The time is short, and the extraction of PPB is not sufficient; if the time is too long, the long-chain structure of the polyphosphate can be damaged to a certain extent, and the yield of the PPB-Ca is reduced.

Further, the best specification model of the Sephadex gel of the step (3) of the invention is Sephadex G100. The effect of removing small molecular impurities by Sephadex G50/G75 is slightly poor, and the experimental efficiency of Sephadex G150 is slightly low.

Preferably, the ultrafiltration membrane of step (4) of the present invention has a molecular weight cut-off in the range of 3 to 10 kDa. The ultrafiltration membrane with the molecular weight cutoff of less than 3KDa has lower concentration efficiency; the ultrafiltration membrane with the cut-off molecular weight of more than 10 KDa can allow part of PPB with smaller particle size to pass through, and the yield of PPB-Ca is reduced.

The invention has the beneficial effects that:

the PPB-Ca is spherical particles with the particle size of about 30-120nm, can be efficiently absorbed and transported in an endocytosis mode in a mouse in-vitro ligated intestinal segment, has good bioavailability in a rat oral pharmacokinetics and osteoporosis model, and particularly has a remarkable effect on resisting the inhibition effect of phytic acid on calcium absorption, so the PPB-Ca is an ideal microalgae source calcium supplement and can be applied to the nutrition strengthening and nutrition supplement of people with calcium deficiency caused by vegetarian habits.

Drawings

The following describes the embodiments and advantages of the present invention in further detail with reference to the accompanying drawings.

FIG. 1 structural characterization of PPB-Ca. a) Dynamic light scattering count rate; b) a polydispersity index; c) a hydrated particle size distribution; d) a Zeta potential; e) a transmission electron microscope image; f) scanning an electron microscope image; g) energy spectrum.

FIG. 2 shows the effect of the intestinal segment of the mouse ligated in vitro on the absorption and transport of PPB-Ca. a) The effect of transmembrane transport of PPB-Ca in the intestinal section of the mouse ligated in vitro; b) effect of endocytosis inhibitors on transmembrane transport of PPB-Ca in vitro ligated intestinal segments in mice. The different superscript letters (a-e) represent statistically significant differences: (P<0.05). Note: 2-APB: 2-aminoethyl dibenzoborate.

FIG. 3 shows pharmacokinetic profiles of rats orally administered with different calcium sources.

FIG. 4 effect of different types of sources of gavage on levels of a) serum calcium, b) serum phosphorus, c) serum Osteocalcin (OCN) and d) bone-derived alkaline phosphatase (BALP) in osteoporotic rats. The different superscript letters (a-d) represent statistically significant differences: (P<0.05）。

FIG. 5 quantitative analysis of a) Bone Mineral Content (BMC) and b) Bone Mineral Density (BMD) of osteoporotic rats orally administered with different calcium sources. The different superscript letters (a-d) represent statistically significant differences: (P<0.05）。

Fig. 6 Scanning Electron Microscopy (SEM) observation of rat femurs, each set of pictures defining the metabolic state of bone by surface topography: a) blank control group; b) a calcium ion group; c) calcium ion + phytic acid group; d) PPB-Ca group; e) PB-Ca + phytic acid group; f) calcium carbonate group.

Detailed Description