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. 2019 Jun 11;17(6):348.
doi: 10.3390/md17060348.

Effect of Chlorella Pyrenoidosa Protein Hydrolysate-Calcium Chelate on Calcium Absorption Metabolism and Gut Microbiota Composition in Low-Calcium Diet-Fed Rats

Pengpeng Hua  1   2 , Yu Xiong  3 , Zhiying Yu  4 , Bin Liu  5   6 , Lina Zhao  7
Affiliations

Effect of Chlorella Pyrenoidosa Protein Hydrolysate-Calcium Chelate on Calcium Absorption Metabolism and Gut Microbiota Composition in Low-Calcium Diet-Fed Rats

Pengpeng Hua et al. Mar Drugs. .

Abstract

In our current investigation, we evaluated the effect of Chlorella pyrenoidosa protein hydrolysate (CPPH) and Chlorella pyrenoidosa protein hydrolysate-calcium chelate (CPPH-Ca) on calcium absorption and gut microbiota composition, as well as their in vivo regulatory mechanism in SD rats fed low-calcium diets. Potent major compounds in CPPH were characterized by HPLC-MS/MS, and the calcium-binding mechanism was investigated through ultraviolet and infrared spectroscopy. Using high-throughput next-generation 16S rRNA gene sequencing, we analyzed the composition of gut microbiota in rats. Our study showed that HCPPH-Ca increased the levels of body weight gain, serum Ca, bone activity, bone mineral density (BMD) and bone mineral content (BMC), while decreased serum alkaline phosphatase (ALP) and inhibited the morphological changes of bone. HCPPH-Ca up-regulated the gene expressions of transient receptor potential cation V5 (TRPV5), TRPV6, calcium-binding protein-D9k (CaBP-D9k) and a calcium pump (plasma membrane Ca-ATPase, PMCA1b). It also improved the abundances of Firmicutes and Lactobacillus. Bifidobacterium and Sutterella were both positively correlated with calcium absorption. Collectively, these findings illustrate the potential of HCPPH-Ca as an effective calcium supplement.

Keywords: Chlorella pyrenoidosa protein hydrolysate (CPPH); Chlorella pyrenoidosa protein hydrolysate-calcium chelate (CPPH-Ca); calcium absorption; gene expression; gut microbiota.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Biochemical parameters in the serum of Ca-deficient rats after oral gavage with different Ca. Serum Ca (A), Serum P (B), Serum alkaline phosphatase (Serum ALP) (C). Note: control, normal group; model, low calcium group; HCaCO3, high dosage of CaCO3 group; HGCa, high dosage of calcium gluconate group; LCPPH + LCaCO3, low dosage of CPPH supplemented with low dosage of CaCO3 group; MCPPH + MCaCO3, model dosage of CPPH supplemented with model dosage of CaCO3 group; HCPPH + HCaCO3, high dosage of CPPH supplemented with high dosage of CaCO3 group; LCPPH-Ca, low dosage of CPPH-Ca group; MCPPH-Ca, model dosage of CPPH-Ca group; HCPPH-Ca, high dosage of CPPH-Ca group. Data are expressed as the mean ± SD (n = 10). One-way ANOVA with Tukey’s test. Different letters indicate significant effect with p < 0.05.
Figure 2
Figure 2
Weight index, length and diameter of femurs and tibias of Ca-deficient rats after treatment with different Ca. Femur weight index (A), Femur length (B), Femur diameter (C), Tibia weight index (D), Tibia length (E), Tibia diameter (F). Note: control, normal group; model, low calcium group; HCaCO3, high dosage of CaCO3 group; HGCa, high dosage of calcium gluconate group; LCPPH + LCaCO3, low dosage of CPPH supplemented with low dosage of CaCO3 group; MCPPH + MCaCO3, model dosage of CPPH supplemented with model dosage of CaCO3 group; HCPPH + HCaCO3, high dosage of CPPH supplemented with high dosage of CaCO3 group; LCPPH-Ca, low dosage of CPPH-Ca group; MCPPH-Ca, model dosage of CPPH-Ca group; HCPPH-Ca, high dosage of CPPH-Ca group. Data are expressed as the mean ± SD (n = 10). One-way ANOVA with Tukey’s test. Different letters indicate significant effect with p < 0.05.
Figure 3
Figure 3
Femurs bone mineral content (BMC) and bone mineral density (BMD) of Ca-deficient rats after oral gavage with different Ca in the experimental period. Proximal BMC (A), Central BMC (B), Distal BMC (C), Proximal BMD (D), Centrality BMD (E), Distal BMD (F). Note: control, normal group; model, low calcium group; HCaCO3, high dosage of CaCO3 group; HGCa, high dosage of calcium gluconate group; LCPPH + LCaCO3, low dosage of CPPH supplemented with low dosage of CaCO3 group; MCPPH + MCaCO3, model dosage of CPPH supplemented with model dosage of CaCO3 group; HCPPH + HCaCO3, high dosage of CPPH supplemented with high dosage of CaCO3 group; LCPPH-Ca, low dosage of CPPH-Ca group; MCPPH-Ca, model dosage of CPPH-Ca group; HCPPH-Ca, high dosage of CPPH-Ca group. Data are expressed as the mean ± SD (n = 10). One-way ANOVA with Tukey’s test. Different letters indicate significant effect with p < 0.05.
Figure 4
Figure 4
Histopathological analysis of rat kidney tissues in different groups at 100× magnification. control (A), model (B), HCaCO3 (C), HGCa (D), LCPPH + LCaCO3 (E), MCPPH + MCaCO3 (F), HCPPH + HCaCO3 (G), LCPPH-Ca (H), MCPPH-Ca (I), HCPPH-Ca (J).
Figure 5
Figure 5
Apparent calcium absorption rate (ACAR) and calcium accumulation rate (CAR) of Ca-deficient rats after oral gavage with different Ca. ACAR (A), CAR (B). Note: control, normal group; model, low calcium group; HCaCO3, high dosage of CaCO3 group; HGCa, high dosage of calcium gluconate group; LCPPH + LCaCO3, low dosage of CPPH supplemented with low dosage of CaCO3 group; MCPPH + MCaCO3, model dosage of CPPH supplemented with model dosage of CaCO3 group; HCPPH + HCaCO3, high dosage of CPPH supplemented with high dosage of CaCO3 group; LCPPH-Ca, low dosage of CPPH-Ca group; MCPPH-Ca, model dosage of CPPH-Ca group; HCPPH-Ca, high dosage of CPPH-Ca group. Data are expressed as the mean ± SD (n = 10). One-way ANOVA with Tukey’s test. Different letters indicate significant effect with p < 0.05.
Figure 6
Figure 6
mRNA expression levels of genes involved in calcium-promoting mechanism as determined using real-time PCR. Transient receptor potential cation V6 (TRPV6) (A), Transient receptor potential cation V5 (TRPV5) (B), calcium-binding protein-D9k (CaBP-D9K) (C), plasma membrane Ca-ATPase (PMCA1b) (D). Note: control, normal group; model, low calcium group; HCaCO3, high dosage of CaCO3 group; HGCa, high dosage of calcium gluconate group; LCPPH + LCaCO3, low dosage of CPPH supplemented with low dosage of CaCO3 group; MCPPH + MCaCO3, model dosage of CPPH supplemented with model dosage of CaCO3 group; HCPPH + HCaCO3, high dosage of CPPH supplemented with high dosage of CaCO3 group; LCPPH-Ca, low dosage of CPPH-Ca group; MCPPH-Ca, model dosage of CPPH-Ca group; HCPPH-Ca, high dosage of CPPH-Ca group. Data are expressed as the mean ± SD (n = 10). One-way ANOVA with Tukey’s test. Different letters indicate significant effect with p < 0.05.
Figure 7
Figure 7
Changes in the bacterial composition of rat intestinal contents according to different genera. Composition of gut microbiota at the genus level. Note: control, normal group; model, low calcium group; HCaCO3, high dosage of CaCO3 group; HGCa, high dosage of calcium gluconate group; LCPPH + LCaCO3, low dosage of CPPH supplemented with low dosage of CaCO3 group; MCPPH + MCaCO3, model dosage of CPPH supplemented with model dosage of CaCO3 group; HCPPH + HCaCO3, high dosage of CPPH supplemented with high dosage of CaCO3 group; LCPPH-Ca, low dosage of CPPH-Ca group; MCPPH-Ca, model dosage of CPPH-Ca group; HCPPH-Ca, high dosage of CPPH-Ca group. T-test was used to calculate significant differences between group.
Figure 8
Figure 8
Heatmap of Spearman’s correlation between caecal microbiota of significant differences and biochemical indexes. The depth of the color corresponds the extent of relevance between caecal microbiota and biochemical indexes. (FDR adjusted p < 0.05).
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