| Royal jelly is also known as royal jelly and bee milk.In recent years,more and more studies have shown that royal jelly has a variety of biological activities,such as anti-tumor,anti-oxidation,memory enhancement,immune regulation,antibacterial,repair of tissue damage and so on.At present,most scholars mainly study the overall efficacy of royal jelly,but there are few reports on the specific efficacy of some components.Protein is an essential nutrient for biological growth and development.Royal jelly protein is the main component of royal jelly,and royal jelly plays an important role in health care and biological functions,such as anti-tumor,anti-oxidation,antibacterial and so on.It may be closely related to royal jelly protein.Therefore,this experimental studied the royal jelly protein extracted under different conditions and explored the anti-tumor effect of its digestion products in vitro,which provided experimental basis for the development of royal jelly protein in the field of food and medicine.1.Royal jelly protein was extracted by six methods(water extraction and acid precipitation,water extraction and ammonia precipitation,alkali extraction and acid precipitation,alkali extraction and ammonia precipitation,salt extraction and acid precipitation,salt extraction and ammonia precipitation).The composition of protein subunits in each experimental group was studied by SDS-PAGE gel electrophoresis.The amino acid content of protein in each experimental group was determined by amino acid analyzer.SDS-PAGE electrophoresis showed that the protein extracted by water extraction and acid precipitation had clear three bands in 52 kDa,and the protein extracted by water extraction and ammonia precipitation had clear two bands in 52 kDa.The protein extracted by alkali extraction acid precipitation and alkali extraction ammonia precipitation had five clear bands at 31 kDa,52-72 kDa and 102 ? 150 kDa,and the distribution of the two subunits was basically the same.The protein extracted by salt extraction and acid precipitation & salt extraction and ammonia precipitation had clear four bands at 52-76 kDa respectively,and the distribution of the two subunits was basically the same.All the proteins extracted by the six methods contained 52 kDa main protein,and the content of the main protein extracted by water extraction and acid precipitation was the highest.The results of amino acid content determination showed that the total amino acid content,essential amino acid content and non-essential amino acid content of the six extraction methods were higher than those of the acid precipitation method and the ammonia precipitation method.The total content of amino acids and the content of non-essential amino acidsextracted by water extraction and acid precipitation were the highest.The protein E / N was extracted by alkali extraction and acid precipitation,and the content of E / T was the highest.The five amino acids with high content in the protein extracted by the six methods were glutamic acid,threonine,arginine,aspartic acid and valine in turn.Because the royal jelly protein extracted by alkali extraction and acid precipitation was the most abundant and the content of amino acid was moderate,it was screened as the experimental protein.2.The extraction process of alkali-soluble protein from royal jelly was optimized by single factor and response surface design experiment.On the basis of single factor experiment,the optimum extraction process of royal jelly protein was determined by four factors and three levels response surface experiment.The results showed that when the temperature was 38.59 ?,the ratio of material to liquid was 1:8,the pH value was 10.15 and the ultrasonic time was 84.27 min,the extraction rate of royal jelly protein was the highest(73.78%).Liquid chromatography-mass spectrometry(LC-MS)was used to analyze qualitatively the royal jelly protein.The results showed that the royal jelly protein contained 63 proteins and 403 peptides.3.The simulated product of royal jelly protein was prepared according to the digestion simulation experiment in vitro.The hydrolysis degree of the hydrolysate was 28.8 ±1.42%,the digestibility was 72.32±3.3%.SDS-PAGE gel analysis showed that all royal jelly proteins were hydrolyzed into small peptides with molecular weight less than 10~15 kDa.The effect of royal jelly protein on the morphology of human gastric cancer cell line SGC-7901 was observed by inverted microscope,and the inhibition rate of royal jelly protein on gastric cancer cell line was detected by MTT assay.Colony formation assay was used to determine the inhibitory effect on colony formation ability of gastric cancer cells.Flow cytometry was used to determine the effect of royal jelly on the cell cycle of gastric cancer.The results showed that the digested products of royal jelly protein in vitro could shrink the morphology of human gastric cancer cells,decrease the cell density and the colony forming ability of human gastric cancer cells(P < 0.05),and inhibit the proliferation of human gastric cancer cell line SGC-7901.The inhibitory rates were 54.58 ±3.48% and62.84 ±1.98% at 24 h and 48 h,respectively,when the concentration of digestive products in vitro was 0.2mg/mL(P < 0.01).Compared with the blank control group,the digested products of royal jelly protein in vitro inhibited the proliferation of human gastric cancer cell line SGC-7901 in a dose-dependent manner and decreased the number of S phase cells,resulting in apoptosis.The expression of p53 protein in gastriccancer cells was increased and the expression of PARP-1 protein was decreased after induced by royal jelly protein digestion products in vitro(P < 0.01),and the expression of p53 protein was increased(P < 0.05),while the expression of p53 protein was decreased(P < 0.05).4.Royal jelly protein oral liquid was prepared by digesting simulated products of royal jelly protein in vitro with sucrose and citric acid.The optimum formula of oral liquid was determined by three factors and three levels orthogonal experiment: The content of royal jelly protein lyophilized powder,sucrose and citric acid were 30%,8%,0.2% respectively.The sensory,physical,chemical and microbial indexes of the oral liquid were in accordance with the national standard.The inhibition rate of SGC-7901 on human gastric cancer cells was detected by MTT assay.It was found that the inhibition rate was 17.97 ±2.31% in a concentration and time dependent manner. |
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