Changes And Mechanism Of Physiologically Active Substances In Monascus Fermented Barley

Nowadays,the idea of healthy life by coarse cereals consumption has become popularized and internationalized.“The characterization and purification technique of special functional nutrients in coarse cereal”,“the development of nutrition and flavor riched coarse cereal”,as well as“the improvement of food product quality and biological function by microbial fermentation”have become research hotspots in the field of coarse grain industry at home and abroad.Coix lachryma-jobi L.is known as“the king of grass family”in the world because of its high nutrition and medicinal values.It has been one of the best food and medicine since ancient times.In this paper,aiming at the technical bottleneck of industrial chain extension due to low byproducts utilization rate and lack of high added value products in coix seed processing,the fermentation and metabolism transformation of broken coix seed was carried out by the probiotic strain,in order to achieve the enrichment and superposition of multiple active components.The transcriptomics and proteomics techniques were used to analyze the formation pathway and quantitative change mechanism of the main active components with high increment.The edible safety and functional activity of fermentation products were evaluated by in vitro and in vivo experimental models,providing a scientific basis for the research and development of high-activity functional products.The main results are given below:1.Among the 16 species resources of coix seed,white coix seed from Qinglong has the highest quality which was rich in protein(13.42 g/100 g)and coixol(7.46 mg/100 g).The protein,crude fat and reducing sugar of broken coix seed were consistent with polished coix seed and coarse coix seed,and its starch(71.30 g/100 g)and crude polysaccharide(60.30/100g)were significantly higher than that of polished coix seed and bran(p<0.05).It also has many other active components,such as coixenolide(4.00 mg/g),coixol(2.83 mg/100 g),rutin(1.20mg/g)et al..Therefore,as a good medium,it can be used for fermentation to enrich target active products.2.The optimum conditions of solid-state fermentation were to add broken coix seed7.22 g/100 mL and H₂O(50:17,V:V),adjust the initial p H to 6.5,sterilize at 121?for 6 min,then to inoculate 9%Monascus purpureus on the broken coix seed medium at 28±1?for 10days.The contents of four characteristic active components in fermentation product were significantly increased,such as coixol(6.50 mg/100 g,4.06 times),coixenolide(17.80 mg/g,4.45 times),and newly increased lovastatin(167.90 mg/100 g)and Monascus pigments(1058U/g).It was noteworthy that the increase of lipophilic components in fermentation product was very significant(p<0.01),which was 2.05 times higher than that in the raw material group,and the extraction rate reached 8.63%.It also contained?-tocopherol(17.88?g/g),?-tocotrienol(72.53?g/g),?-oryzanol(655.01?g/g),?-sitosterol(53.32?g/g)and other active components.In particular,?-tocopherol,one unknown component and?-oryzanol were increased by 162.55,34.30 and 24.99 times,respectively.The unknown component was identified by GC-MS/MS as 3?-hydroxy-androst-5-en-17-one,which was a steroidal hormone compound with high physiological activity and was used for a new type of dietary supplement3.According to the strain dependence of enrichment active components,transcriptomics and proteomics were used to analyze the metabolic pathways and enrichment mechanisms of enrichment active components:The enrichment reasons of tocopherol and its tocotrienol would involve 3 metabolic pathways and the expressions of 12 key functional proteins(enzymes).In Phenylalanine,tyrosine and tryptophan biosynthesis,the protein expressions of 3-dehydroquinate synthase[EC:4.2.3.4],3-dehydroquinate dehydratase I[EC:4.2.1.10],shikimate dehydrogenase[EC:1.1.1.25],Shikimiatekinase[EC:2.7.1.71]and3-phosphoshikimate1-carboxyvinyltransferase[EC:2.5.1.19]were up-regulated to promote shikimic acid enrichment,which was the key of the formation of homogentisic acid.In the tyrosine metabolism,the protein expression of aldehyde dehydrogenase(NAD(P)+)[EC:1.2.1.5]was up-regulated,which promoted the enrichment of homogentisic acid.The protein expressions of homogentisate 1,2-dioxygenase[EC:1.13.11.5]and fumarylacetoacetase[EC:3.7.1.2]were down-regulated to prevent the conversion of homogentisic acid to acetoacetate,and to ensure adequate substrates for tocopherol synthesis.In phenylalanine,tyrosine and tryptophan biosynthesis,the gene expression of 4-hydroxyphenylpyruvate dioxygenase[EC:1.13.11.27]was up-regulated to promote the formation of homogentisic acid from4-hydroxyphenylpyruvate in shikimic acid pathway.Homogentisate phytyltransferase[EC2.5.1.115]catalyzed homogentisic acid(HGA)and isopentenyl pyrophosphate(Pr DP)to form2-methyl-6-phyto-benzoquinone(MPBQ),one of the precursors for forming tocols.Tocopherol cyclase[EC:5.5.1.24]catalyzed the production of?-tocopherol and its tocotrienol from MPBQ precursors.Tocopherol O-methyltransferase[EC:2.1.1.95]promoted the conversion of?-tocopherol and?-tocotrienol to?-tocopherol or its tocotrienol in the fermentation system.The enrichment reasons of?-oryzanol and 3?-hydroxy-androst-5-en-17-one were involved in the steroid biosynthesis pathway and were closely related to the formation and accumulation of steroid compounds.Steroids biosynthesis in coix seed system fermented by M.purpureus were influenced by the genes expressions of sterol 14alpha-demethylase[EC:1.14.14.154],CYP51A[CYP51G1],sterol desaturase[EC:1.14.19.20],7?-sterol5-desaturase[ERG 3],Delta24(24(1))-sterol reductase[ERG 4],and Delta7-sterol5(6)-desaturase[STE 1].They were associated with high accumulations of7-dehydrocholesterol,stigmasterol,campesterol,and sitosterol,and to provide sufficient substrates for esterification of?-oryzanol and sterone.The high protein expressions of sterol24-C-methyltransferase[EC:2.1.1.41]and sterol 14alpha-demethylase[EC:1.14.14.1541.14.15.36]also promoted the high accumulation of phytosterols at the end of the metabolic pathway.The essential oil from fermented coix seed(FCS-O),produced by Supercritical CO₂extraction technology(the extraction yield reached 12.8%),had good physicochemical qualities and abundant active components with physiological function.In particular,?-tocotrienol,?-oryzanol,coixenolide and oleic acid concentrations reached 72.83?g/g,745.96?g/g,9.65 mg/g and 316.58 mg/100 g DW,respectively.The FCS-O exhibited higher antioxidant capability in inhibiting the oxidation of susceptible polyunsaturated fatty acids(EPA/DHA)and better anti-lipid oxidation and peroxidation capabilities.For example,compared to blank control,the concentrations of 7-ketocholestreol and peroxide decreased by12.99 and 2.72 times,only 8.42 g/mL and 16.16 mmol/kg at 168 h of oxidation(p<0.01).FCS-O also had an anti-proliferation effect on human laryngeal carcinoma cell line Hep2(IC₅₀=2.13 mg/mL),but had no cytotoxic effect on normal monkey kidney cell line CV-1.The anticancer activity of Hep2 cells was significantly increased compared with that before fermentation.The IC₅₀of FCS-O in inhibiting HEp2 cell decreased by 42%.In addition,the FCS-O has been confirmed to be a very safe edible oil,with no acute toxicity(LD50>10g/kg bw,considered actually non-toxic)and no induced mutagenicity,cytotoxicity or genotoxicity.These results serve as a good safety reference for future application of the oil from fermented coix seed.The development and utilization of this kind of oil will be beneficial as a food,food ingredient,nutritional supplement,or natural food antioxidant to promote good health function.