Optimization Of Fermentation Process Of Yellow Wine Lees And Its Utilization In Lactating Dairy Cows

Soybean meal is heavily insufficient in China.It is urgent to develop unconventional protein feed resources.As a by-product of the yellow wine industry,yellow wine lees(YWL)have high protein content with low price.However,fresh YWL is difficult to preserve due to its high moisture content,and there exists alcohol residue that cannot be directly fed to animals.Meanwhile,amino acid profiles are not balanced in fresh YWL.Therefore,this study was conducted to improve the feeding value of YWL through the method of microbial solid-state fermentation and explore its application in dairy cow diet.Firstly,the solid-state fermentation conditions of fermenting YWL were optimized to increase its protein content and balance its amino acid profile.Based on the in vitro rumen fermentation test and the evaluation of antioxidant activity,feeding trial was conducted to investigate lactation performance and nitrogen(N)utilization in lactating dairy cows receiving yellow wine lees(YWL)as a partial replacement for soybean meal(SBM).The results obtained will provide a technical basis for the utilization of fermented YWL in animal feeding.1.Optimization of the conditions for solid-state fermentation of YWLIn this part,microbial solid-state fermentation was employed to upgrade the feeding value of YWL.The whole genome was resequenced for Bacillus subtilis and Candida utilis used in this study,and the metabolic gene discovery was analyzed by KEGG pathway enrichment.Compared with the reference Bacillus and yeast,subtilis and had the most abundant amino acid synthesis and metabolic pathways,and Candida utilis was the most abundant in carbohydrate metabolism we well as amino acid synthesis and metabolic pathways,suggesting that they could enhance the feed value of fermented products in solid state fermentation.First,C.utilis KF01 and B.subtilis KF02 were inoculated separately or together in solid medium to compare their effects,and the combination of both strains were chosen for the following studies.Subsequently,the optimizing condition of microbial solid-state fermentation was performed.The fermentation conditions including inoculation ratio,fermentation temperature,water content of the culture medium,fermentation time,and number of turning over the fermentation medium were optimized by single factor experiments.It was found that the fermentation temperature and medium had larger influence on the fermentation results.The water content and the inoculation ratio of the strain were then subjected to a 3×3 orthogonal design to optimize the fermentation conditions to obtain the optimum crude protein and polypeptide yield.The optimal conditions obtained were as follows:the ratio of water to total solid medium was 53:100(v/w),the temperature was 30 ℃,the inoculation ratio of Candida utilis and Bacillus subtilis was 2:1,and the fermentation time was 44 h.The number of times of turning over was 2 times.The content of crude protein and polypeptide of the fermented YWS was 14.5 and 40.9%,respectively,higher than that of the unfermented product2.Evaluation of in vitro nutritional value and antioxidant capacity of fermented YWL2.1 In vitro nutritional evaluation of fermented YWLThe amino acid content of the fermented YWL increased by 26.1%compared with the unfermented YWL.The increase in essential amino acids(31.2%)was even greater than in non-essential AA(21.1%).In particular,the levels of lysine and methionine(the two most restricted amino acids in dairy diets)were highly enriched.The in vitro fermentation test showed that the fermented YWL increased the rumen microbial protein concentration and had a higher crude protein digestibility.2.2 The antioxidation of fermented YWLThe total antioxidant capacity and free radical scavenging ability were used to evaluate the antioxidant properties of the YWL before and after fermentation.The fermented YWL significantly improved the reducing ability,DPPH free radical scavenging ability,reducing power,superoxide radical scavenging ability,and hydroxyl radical scavenging ability.The correlation analysis showed that the increase of antioxidant performance of the YWL during fermentation was related to the conversion of polyphenols and production of small molecular proteins and peptides.A part of small molecule proteins were isolated and identified by 2D protein electrophoresis,revealing the mechanism involved in improving the antioxidant capacity of the fermented YWL.2.3 Effect of YWL replacing SBM on in vitro rumen fermentationIn vitro rumen fermentation test was conducted to investigate the rumen gas production and fermentation parameters of the simulated total mixed diet with SBM replaced partially by YWL or fermented YWL.Associate effect between different combinations was also evaluated.The optimum ratio of YWL and fermented YWL replacing soybean meal was obtained.When the ratio of YWL or fermented YWL to SBM was at 1:1(w/w),the rumen fermentation parameters were the closest to the control group with SBM as the main protein source diet),with a stronger positive associative effect.3.Lactation performance and N utilization in lactating dairy cows receiving YWL as a replacement for SBMA feeding trial was conducted to investigate lactation performance and N utilization in lactating dairy cows receiving YWL as a partial replacement for SBM.Fifteen lactating Chinese Holstein Dairycows(days in milk:190± 15.2 d,body weight:692±70 kg)were selected for the 3×3 replicated Latin square test(repeated = 5).The three diets were as follows(DM basis):(1)a diet containing 18%SBM(Control),and(2)a diet containing unfermented YWL partially replacing SBM(UM),(3)A diet(FM)containing a portion of fermented YWL instead of SBM.Each trial pernod lasted for 20 days,the first 15 days were used to adapt to the diet,and the last 5 days were used for sampling.During the sampling period,milk production was recorded and milk compositions were analyzed.Rumen fermentation,nitrogen utilization,blood variables and heat stress resistance of lactating dairy cows were also investigated.3.1 Effects on lactation performance and blood variablesDry matter intake was higher in control and FM groups than in UM(P = 0.04),with no difference between control and FM(P>0.05).Compared with control and FM,the yield of milk(P = 0.02),milk protein(P = 0.02),and energy-corrected milk(P =0.05)was lower in UM,with no difference between the control and FM(P>0.05).No statistical differences were found in milk fat,protein,lactose and total solids among three treatments(P>0.05).Milk urea nitrogen,somatic cell count,feed efficiency and nitrogen efficiency were also not affected by dietary treatment(P>0.05).Income over cost in FM tended to be higher than in control(P = 0.06),with no difference between UM and control(P>0.05).Blood urea nitrogen concentration in UM was lower than in FM and control(P = 0.03).Overall,the dietary treatment did not adversely affect the blood variables of the cow(P>0.05).3.2 Effect on rumen fermentation,and nitrogen metabolismTotal volatile fatty acid,acetic acid and microbial protein concentrations were significantly lower in the rumen fluid of the UM than in control(P<0.05),with no difference between control and FM(P>0.05).Dietary treatment did not change the rumen fermentation mode(P>0.05).The rumen rapid(P = 0.38)and slow degradation fraction(P = 0.58)of dietary protein were not affected by the diet;crude protein degradation rate constant of the UM group were lower compared with control and FM(P = 0.04)with no difference between the control and FM(P>0.05)Compared with the UM group,cows fed fermented YWL and control diets had higher nitrogen intake and higher rumen organic matter degradation rate,resulting in increased microbial protein synthesis and metabolizable protein production in dairy cows,and hence improved milk performance.In general,partial replacement of soybean meal with fermented YWL has no adverse effect on nitrogen utilization and metabolism of lactating dairy cows3.3 Effect of on heat stress of dairy cowsCompared to the control with SBM,feeding of unfermented and fermented YWL tended to have lower rectal temperature(P = 0.06)and had lower respiratory rate(P ＝0.05)of cows under heat stress.At the same time,feeding of YWL improved the level of blood superoxide dismutase,glutathione cobalt oxide and total antioxidant capacity(P<0.05).Through hemocyte analysis of cow blood,it was found that unfermented and fermented YWL reduced the number of inflammatory cells(leukocytes,neutrophils)in dairy cows under heat stress,suggesting that it may reduce the risk of inflammation while facing hot weather.In summary,in this study,a novel feed of fermented YWL with high protein and amino acid content and antioxidant activity were prepared by solid-state fermentation of Bacillus subtilis and Candida utilis.In combination of the results in dairy cows feeding trial with the in vitro simulation test,it is indicated that fermented YWL can replace part of the soybean meal in dairy cow diets withour adverse effect on lactation performance,rumen fermentation and nitrogen use efficiency,but feeding of YWL can reduce feed cost,and alleviate heat stress in dairy cows under high temperature conditions.