Effects Of Bacillus Natto Sp.fermentation On Nutritional Characteristics Of Moringa Oleifera Leaves

Moringa oleifera,native to India,is commonly known as its rich nutrition and medicinal value of all parts of the organization.Moringa oleifera leaf is rich in protein,minerals,vitamins,and other nutrients,but also contains GABA,flavonoids and polyphenols and other physiologically functional plant compounds,has a high nutritional value.However,due to the combination of protein and polyphenols in Moringa oleifera leaves,the digestion and absorption of protein by the human body will be affected.Therefore,this study uses dried Moringa oleifera leaves as raw material,adopts fermentation,a classic used food processing method,and uses high-yield protease Bacillus natto sp.to ferment Moringa oleifera leaves in order to improve the nutritional value of Moringa leaf,and to lay the foundation for the development of Moringa leaf related products.Firstly,three protease-producing strains were isolated from commercial enzyme capsule using casein plate method,and both are Bacillus subtilis identified by 16S rDNA molecular identification.Then,they were verified by the fibrin plate method,the results showed that the strain NK1 had the ability of thrombolysis,indicating that the strain was Bacillus natto sp..The physiological and biochemical characteristics of strain NK1 were studied,the results showed that strain NK1 had good ability of high temperature resistance,the survival rate was92%at a constant temperature of 90°C in water bath for 1 h,and it was still tolerable in 12%sodium chloride solution for 4 h.In addition,strain NK1 can also be metabolized to produce cellulases and amylases.Secondly,the dried Moringa oleifera leaves were pretreated to enable the dissolution of nutrients that can be used by NK1 such as reducing sugars and amino acids.The results showed that the optimum pretreatment of Moringa oleifera leaves were as follows:ratio of material to liquid 1:20?w/v?,handled in a constant temperature water bath at 50°C for 2.5 h.At this point,the content of reducing sugar in the supernatant was 1.48±0.06 g/100 m L,the content of flavonoids was 0.22±0.03 g/100 m L,and the content of amino nitrogen was 0.13±0.05 g/100 mL.Thirdly,the pretreated slurry was used as a fermentation substrate,and the process conditions such as slurry pH,the type and amount of added carbon sourc,liquid volume and fermentation time were optimized.The results showed that the best fermentation conditions for Bacillus natto sp.in pretreatment slurry were as follows:the pH of the slurry was 7.0,3%maltose was added,the volume was 50 mL/250 mL,and the fermentation time was 60 h.At this point,the protease enzyme activity in the fermentation broth reached 43000 U/g,and the protein dissolution rate in the fermentation broth was increased by 19.30%compared to the pretreatment supernatant.In addition,the changes of key nutrients in the supernatant were also studied.The results showed that free amino acid content in the supernatant after fermentation increased significantly.The amount of GABA dissolution was 22 mg/100 mL,and its content was 42.90times higher than that of the pretreatment supernatant.The molecular weight of bioactive peptides was generally in the range of 200²000,the molecular weight of peptides in this range in the fermentation pernatant of Moringa oleifera leaves was 36.87%,which was15.46%higher than that of the pretreatment supernatant.After fermentation,the content of Mg increases from 32.11 mg/L to 67.56 mg/L,the content of Fe increases from 0.11 mg/L to1.07 mg/L.and the content of Zn increased from 0.63 mg/L to 0.92 mg/L,other minerals did not change much.The contents of V_B1,V_B6,and V_B9 were reduced by 58.49%,94.23%,and77.78%,respectively.The contents of V_B2 and V_B3 were increased by 50.00%and 16.51%,respectively.Vitamin B which are unstable to light,heat,and pH are susceptible to loss in this process.Since Moringa leaf is rich in flavonoids,so the antioxidant capacity of soluble substances before and after fermentation was comprehensively determined.The antioxidant capacity was measured by T-AOC method and ORAC method,respectively.The T-AOC method was used to determine the total antioxidant capacity of the sample solution,which was 76.63 U/mL before fermentation and 73.51 U/mL after fermentation.The ORAC method was used to measure the oxygen radical absorption capacity of the sample solution,it was1472 mg TE/100 mL before fermentation and 1335 mg TE/100 m L after fermentation.The results of the two chemical methods all indicate that the fermentation has little effect on the antioxidant capacity of the supernatant.In addition,the antioxidant capacity of the supernatant in cells was evaluated using the HT-29 cell as a model.The results of experiments on inhibiting H₂O₂ oxidative damage cells showed that the samples before and after fermentation could inhibit the oxidative damage caused by H₂O₂,and the cell viability could be increased by about 42%.Cellular antioxidant activity assay?CAA?results showed that both of the supernatant had good antioxidant capacity,and the antioxidant activity could reach more than 68%.