Using intestinal probiotics to promote calcium absorption
Calcium is a mineral necessary for the body to maintain healthy bones. Bone loss can lead to metabolic bone diseases, such as osteoporosis. This situation often occurs in the elderly population. Since the calcium intake of the Chinese elderly usually cannot meet the Nutrition Society's recommended standards, calcium deficiency is more common for the Chinese elderly. According to reports, the amount of calcium absorbed by the body depends on the amount of soluble calcium in the duodenum and proximal jejunum. Due to the precipitation of calcium ions in the intestines with antagonists such as oxalate, phytate, and cellulose, calcium's solubility may be reduced. In China, because vegetables account for a large proportion of the traditional diet, the rich oxalate in vegetables will prevent the elderly from absorbing calcium.
Recently, people have attracted great interest in enhancing calcium solubility and subsequent calcium absorption by food substances (especially peptides). Combining calcium with peptides to prevent calcium precipitation can effectively increase calcium absorption in the body. So far, Calcium-binding peptides have been found and selected in various food sources, including egg yolks, cow cheese protein, whey, soybeans, wheat germ, tilapia, Alaskan pollock, and shrimp processing by-products. One of the most widely known is the casein phosphopeptides (CPPs) in milk. CPPs can promote calcium absorption by chelating calcium with phosphoserine residues. Some peptides lacking phosphoserine residues also enhance intestinal calcium absorption by binding calcium to Asp and Glu residues.
Inspired by short peptides that can bind calcium, we reviewed and designed a peptide (CaAP) containing a series of short peptides that can promote calcium absorption. We let probiotics express and secrete this peptide in the intestine to promote calcium absorption in the intestine. By introducing LuxI/LuxR quorum sensing system, bacteria can first gain population advantage by expressing antimicrobial peptides, thereby colonizing and forming flora in the intestine. When the number of bacteria reaches a certain level, the antimicrobial peptide secretion system will be turned off and the secretion of CaAPs will be turned on. In this case, the antimicrobial peptides will not be secreted out but will inhibit itself so that the number of flora can be maintained in a stable range. Meanwhile, CaAPs will be continuously expressed and secreted, combined with calcium ions in the intestine, and promote the absorption of calcium ions by epithelial cells.
Through this design, we hope to develop a stable and sustainable endogenous drug delivery method that can truly improve the quality of life and health of human beings, especially the elderly. This project can also promote the progress of SDG Goal3: Good health and well-being.
Calcium Absorption Peptide (CaAP)
Casein phosphopeptides are phosphorylated casein of digest milk, so protein kinases are required. However, given that protein kinases play an essential role in signal transduction, and they may not be able to fold and function correctly in the chassis, it is challenging to phosphorylate peptides in bacteria. Therefore, we choose short peptides without phosphoserine residues to promote calcium absorption.
Through literature search and database screening, currently selected peptides are GPAGPHGPVG, FDHIVY, YQEPVIAPKL[1], NDEELNK[2], DHTKE[3]. Experiments have determined these peptides can promote higher calcium absorption.
GPAGPHGPVG, FDHIVY, and YQEPVIAPKL
GPAGPHGPVG, FDHIVY, and YQEPVIAPKL are derived from tilapia collagen hydrolysate. In terms of safety, the MTT test showed that among the three short peptides, YQEPVIAPKL at six tested concentrations had no cytotoxicity. In the concentration range of 25 to 500μg/mL, neither GPAGPHGPVG nor FDHIVY affects cell viability.
Use the o-cresol-catechol colorimetric method to verify their calcium chelating activity (Figure 5). The calcium chelating activity of GPAGPHGPVG, FDHIVY, and YQEPVIAPKL respectively reached 18.80±0.49, 35.73±0.74, 28.4±0.94 mg/g, indicating that the FDFDY peptide with the shortest sequence has more calcium chelating sites. For these peptides, the composition and the peptide sequence may play an essential role in the calcium chelating activity. Except for the binding site of casein phosphopeptide (CPP) “Ser(p)-Ser(p)-Ser(p)-Glu-Glu,” the most likely calcium-binding sites are Asp(D) and Glu(E )carboxyl. Besides, due to the δ-N of the imidazole ring of His(H) and the ε-amino nitrogen of Lys(K), necessary amino acids such as His(H) and Lys(K) have a significant contribution to the calcium-binding activity of the peptide.
The results further showed that compared with adding calcium and phosphorus, adding GPAGPHGPVG, FDHIVY, and YQEPVIAPKL respectively increased calcium transport by 89±9, 202±12, and 130±7%. As the transport time increases, the difference in calcium transport induced by each peptide becomes more evident. FDHIVY has the most significant effect on calcium transport, followed by YQEPVIAPKL and then GPAGPHGPVG. This result is consistent with their calcium chelating activity. Previous findings indicate that peptide chelating activity is necessary to promote calcium absorption in the Caco-2 cell monolayer.
Calcium absorption peptides with high biological activity are believed to affect calcium absorption by interacting with membranes and opening specific calcium channels. (10) The most suitable peptide structure can bind calcium for transport and release ions quickly. (9,42) calcium may be absorbed after being released from the peptide, or it may be absorbed in the form of calcium peptide chelate.
NDEELNK
Asn-Asp-Glu-Glu-Leu-Asn-Lys (NDEELNK) hepeptide is derived from the trypsin hydrolysate of sea cucumber eggs. This peptide can spontaneously bind calcium at a stoichiometric ratio of 1:1, and the calcium-binding site may involve the carboxyl oxygen and amino nitrogen atoms of two glutamic acids and one aspartic acid residues in the NDEELNK peptide. Besides, it is reported in the literature that the DEELNK-calcium complex undergoes decomposition and self-aggregation in a smaller-sized grid during the digestive process of the gastrointestinal tract, which may help to absorb calcium across the Caco-2 cell monolayer.
DHTKE
Asp-His-Thr-Lys-Glu (DHTKE) pentapeptide comes from the hydrolysis of egg white. This peptide can spontaneously bind calcium at a stoichiometric ratio of 1:1, and the calcium-binding site corresponds to the carboxyl oxygen, amino nitrogen, and imidazole nitrogen atoms () of the DHTKE peptide. Besides, Caco-2 cells were used in vitro to study the DHTKE-calcium complex's effect on improving calcium absorption. Compared with the control without calcium, the results show that the DHTKE-calcium complex can promote the penetration of calcium into the cytoplasm and further increase the Caco-2 cell monolayer's calcium absorption by more than seven times.
Kill Switch
https://2020.igem.org/Team:Fudan/Design/kill_switch
However, for the Chinese population, the bottlenecks are as follows:
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However, the currently popular calcium supplement method on the market is either to adjust diet or to consume a lot of calcium supplementing pills. For the elderly, they suffer from poor memory or neglect the importance of calcium pills, often forgetting to consume the supplement; for children, they often refuse to take calcium pills or to maintain some diet high in calcium, thus requiring the "liquid calcium" method to ensure sufficient calcium intake. Ho可以 继续大段内容
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再来例子 Market Analysis
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