Studies On Carotenoid Production From Food Waste By Phaffia Rhodozyma Based On Flow Cytometry And Cell Sorting

Food waste(FW),consisting of catering waste,is wasted globally at an annual volume of about 1.3 billion tons.Its traditional incineration,landfill and composting will cause environmental pollution and serious waste of resources.FW rich in nutrients is an excellent medium for microbial growth.Therefore,the value-added of FW based on microorganisms as an environmentally friendly and technically-economically feasible strategy for producing valuable products has attracted considerable attention.Astaxanthin synthesized by Phaffia rhodozyma is an orange-red carotenoid,which is widely used in medicine,food,aquaculture and cosmetics for its enormous antioxidant activity and coloring properties.However,the astaxanthin content of P.rhodozyma is low,the raw material prices of carbon sources(soluble sugars,etc.)and nitrogen sources(yeast powder,etc.)fluctuate greatly,and the energy consumption of fermentation at 17～22 °C is high,resulting in high fermentation cost,which seriously restricts the large-scale production and application of astaxanthin.Based on the above problems,this paper used mutagenesis and flow cytometry and cell sorting to sort P.rhodozyma with high carotenoid production and studied the feasibility of FW used by P.rhodozyma,and explored the mechanism of P.rhodozyma directly using FW.And the feed potential application of its fermented products was studied.Firstly,P.rhodozyma were screened by UV mutagenesis and flow cytometry,and a strain that could stably produce high carotenoid at 25 ℃ was obtained.The strain obtained carotenoid production(32.89 mg/L)and content(6.68 mg/g)in YPD medium were significantly increased by 31.59% and 32.28% compared with the original strain,respectively.Based on the screening strains,the feasibility of using FW as a fermentation substrate for P.rhodozyma was explored.The results showed that FW could be directly fermented as a substrate by P.rhodozyma without hydrolysis treatment.The effects of four fermentation strategies on the growth and metabolism of P.rhodozyma were compared.The direct fermentation(DF)obtained 129.50 mg/L carotenoid production and 1.08 mg/L/h productivity at a substrate concentration of 10% FW and 22 °C,which were 61.25% and 61.19% higher than that of separate hydrolysis and fermentation(SHF),respectively,and the carotenoid production and productivity were close to simultaneous saccharification fermentation(SSF)(155.53 mg/L,1.30 mg/L/h)and 1 h pre-hydrolysis SSF(PSSF)(157.34 mg/L,1.31 mg/L/h),indicating that the DF strategy of FW was feasible.The mechanism of DF of FW by P.rhodozyma was further explored.Studies had shown that 31.59% of the rice starch in FW was partially converted into small molecules such as dextrin under high temperature sterilization conditions.P.rhodozyma produced amylase under the induction to further hydrolyze the small molecules to produce sugar and promote carotenoid production.The amount of sugar produced by hydrolysis of amylase increased by 90.28%compared with that of 0 h,and the carotenoid production was 8.87 mg/L.In addition,metal elements(411.50 mg/L K,1404.42 mg/L Na,447.68 mg/L Ca,2.55 mg/L Zn,1.17 mg/L Mn,3.71 mg/L Al,6.63 mg/L Fe,68.48 mg/L Mg and 0.35 mg/L Cu)and 9.05% soybean oil in FW could promote the growth and accumulation of carotenoids of P.rhodozyma.The addition of metal elements and soybean oil significantly increased the biomass(0.46 g/L,3.11 g/L)by36.17% and 29.05%,respectively,compared with the control group.This indicated that the three nutrient components in FW were beneficial to the growth and carotenoid accumulation of carotenoids of P.rhodozyma.Based on the screening strains and DF strategy,the substrate concentration of FW was optimized.The optimal substrate concentration was 20%,and the carotenoid productivity was1.49 mg/L/h.It was found that the high concentration of substrate could inhibit the growth and accumulation of carotenoids of yeast to some extent.The fed-batch fermentation of FW found that dry FW inhibited the synthesis of carotenoids of P.rhodozyma,while the feeding of wet FW could promote the accumulation of carotenoids.The optimal feeding strategy was 10%FW+5% wet FW(12 h)+ 5% wet FW(36 h),and the carotenoid production reached 192.62mg/L,which was significantly increased by 21% compared with batch culture.The dried fermented product contained 13.47% starch,8.46% lipid,36.56% protein,25.59%neutral detergent fiber and 13.08% acid detergent fiber and other nutrients,and the heavy metal content all met the feed standards.The fermentation of P.rhodozyma significantly increased the protein and amino acid content of FW.The analysis of essential amino acids/total amino acids(44.88%)and essential amino acids/non-essential amino acids(115.62%)showed that the fermentation product was a protein source with better quality,and the amino acid score and chemical score indicated that the first limiting amino acid of the fermentation product was lysine,while the essential amino acid index evaluation(0.98)of other essential amino acid components(except lysine)indicated that the fermentation product supplemented with lysine met the standard of high-quality protein source,and its trace element content was lower than that of common feed.Finally,1 kg of FW was fermented by P.rhodozyma to obtain 373 g of fermentation products,784 mg of carotenoids and 111 mg of astaxanthin.