| Consumption of vinegar-soaked soybean and peanut is a traditional dietary treatment in China,and these foods have a variety of benefits for the human body.The plant physiology studies reveal that the protein storage vacuoles in cells are acidified during seed germination,and endogenous enzymes such as acidic proteases are activated,inducing the hydrolysis of storage proteins to supply nutrients and energy for plant growth.The activity of endogenous proteases in plant seeds such as peanut,soybean,and sesame has been systematically studied in our laboratory,and it has been confirmed that the endogenous proteases in sesame seeds show the highest activity.Therefore,this paper takes the plant physiological phenomenon that protein storage vesicles are acidified during seed germination as a scientific basis,takes vinegar-soaked peanuts and soybeans as a practical prototype,and selects acetic acid solutionsoaked peeled sesame as a research system based on the compositional characteristics and good antibacterial effect of acetic acid solution,and explores the changes of various components and cellular physiological activities during the soaking process,so as to create a new type of sesamebased food.By using endogenous enzymes,the comprehensive utilizations of sesame proteins and lipids were preliminarily studied.The major results in this study are as follows:Firstly,the soaking model system of peeled sesame seeds was established,that is,a soaking system with a material/liquid mass ratio of 1/3.The effects of acetic acid concentration(0%～2.5%),soaking temperature(25℃～60℃),and soaking time(12 h,1～7 d)on the degradation and release of sesame proteins were investigated.By comprehensively checking the stability and flavor of soaking system,the soaking condition(2.0% acetic acid solution,soaking at 25°C for 7 days)was chosen.In this condition,approximately 60% of the protein components in peeled seeds was released into the soak,and the protein components in soak had a high trichloroacetic acid-nitrogen solubility index(TCA-NSI)value of 89%,indicating the feasibility and efficiency of the soaking treatment by using acetic acid.Secondly,the soaking system was optimized,that is,reducing the amount of liquid as much as possible,with a principle that the liquid could immerse the seeds.At this time,the material/liquid mass ratio was 1/1.5,then the soaking treatment was conducted at 25°C for 7days with 1%～3% acetic acid solution.The results showed that soaking by 2.0% acetic acid solution could release the most protein components into soak,and the protein components in soak had the highest TCA-NSI value.During soaking by 2.0% acetic acid solution,the seeds could be acidified to p H 4.5 within 1 day,and the p H was maintained afterwards,which was perfectly consistent with the optimal p H 4.5 of sesame endogenous proteases.After 7 days soaking,approximately 37% of protein components in peeled seeds released into soak,and the protein components in soak had a high TCA-NSI value of 91%.The residual protein components in seeds had a TCA-NSI value of 57%.By soaking heat-denatured seeds with 2.0%acetic acid solution,it was confirmed that the degradation and release of protein components into soak was predominantly induced by the endogenous proteases.Thirdly,the main components and their related enzymes in the system(soaking at 25°C for 7 days with 2.0% acetic acid solution and a material/liquid mass ratio of 1:1.5)were systematically examined.Tricine-SDS-PAGE and no-enzyme LC-MS/MS results showed that the oil body membrane proteins were fully degraded,11 S globulins were greatly degraded,whereas 2S albumins showed great resistance to endogenous proteases.The TCA-NSI assay,amino acid analysis and SEC-HPLC results showed that in the whole soaking system(i.e.,soak+ soaked seeds),the protein components were composed of 31% free amino acids,39% TCA soluble small peptides and 30% peptides and proteins with high molecular weights,indicating that the sesame proteins were greatly degraded.The lipase activity assay and lipid degradation analysis showed that lipases showed an optima p H range of 4～5,which were dominated by 14 GDSL esterases/lipases.After soaking in above condition,the free fatty acids in sesame oil greatly increased from 0.10% to 28.74%.The phytate-degrading enzymes and phytate content analysis showed that phytate-degrading enzymes could degrade phytate at p H 4.5,and approximately 60% of phytate could be degraded by soaking in above condition.Fourthly,the microstructure change of protein storage vacuole and oil body in sesame seed cell was examined,and the soaking system was further studied via proteomics and metabolomics.The transmission electron microscopy results showed that after 1 d soaking,every protein storage vacuole was disrupted into several protein particles,and a small number of oil body coalesced into large oil droplets.At this time,the cell still kept orderly microstructure to some extent.After 2 days soaking,the cell fully lost its orderly microstructure.After 7 days soaking,just a smaller number of protein particles and an irregular oil phase remained in cell,and it was found that there were some “wounds” appeared on cell wall.The proteomics results showed that the proteins related with stress were up-regulated,whereas the ones related with normal physiological processes of plant cell were down-regulated.Therefore,the soaking treatment could be divided into two stages based on the theory of plant physiology:1)cell stress stage under acid and hypoxia stress conditions,2)disordered biochemical reaction stage-cell death stage,and the two stages were bounded by the endogenous proteases-induced disruption of protein storage vacuole.The metabolomics results showed that the functional metabolites such as spermidine,phenylethylamine,spermine,and resveratrol significantly increased,while harmful metabolites such as urea and triethanolamine significantly decreased,indicating that the endogenous enzymes related to the synthesis and degradation of these metabolites also played a role,which is consistent with the statement of “reducing toxicity and increasing efficiency” in vinegar processing of traditional Chinese medicine.Finally,based on the basic idea of sesame oil processing and the activity of endogenous protease,a new sesame processing technology was designed: firstly,the peeled seeds were dried at low temperature and cold-pressed to obtain sesame oil,and then the highly active endogenous proteases retained in the cold-pressed seeds were used to achieve efficient protein degradation through acetic acid soaking,so as to achieve the comprehensive processing and utilization of sesame proteins and lipids.In this process,sesame protein could reach a release rate of 53%,and the TCA-NSI of the released protein components was approximately 80%.As for the acetic acid soaking system of peeled sesame seeds,it can actually be used as a pre-digested functional food: endogenous enzymes pre-hydrolyze proteins and triglycerides,synthesize some functional metabolites,and degrade phytate and some harmful metabolites.Therefore,it is recommended to “eat and drink” as a way of consuming this food.As for coldpress followed by soaking in acetic acid solution(the process needs to be further improved),after producing sesame oil by cold-press,sesame protein hydrolysate can be obtained by soaking in acetic acid solution. |
