| Forage is rich inβ-carotene,which plays an important role in improving the health of dairy cows and the vitamin A content in milk.Studies have found that the loss ofβ-carotene during ensiling is up to 70%,but there were some silages with lowβ-carotene loss,and the reason has not been understood.In this paper,alfalfa was used as experimental material.The purpose was to isolate,screen,and identify squalene,a silage fermentation product with the potential of inhibitingβ-carotene loss,verify its inhibitory effect onβ-carotene loss during ensiling,trace its source,analyze its mechanism of inhibiting the loss ofβ-carotene and the effect of protectingβ-carotene from lactic acid and volatile fatty acids,and evaluate the effect of it combined with common additives on improving the fermentation quality of alfalfa silage and inhibitingβ-carotene loss.The results of this study will provide new ideas for the study of reducingβ-carotene loss during ensiling,and provide theoretical basis and technical support for the research and additives development to inhibitβ-carotene loss.1 Screening,identification,and verification of silage fermentation products inhibitingβ-carotene loss1.1 Screening and characteristic analysis of silage with lowβ-carotene lossTwo-factor randomized block trials of temperature[low temperature(3-20°C,AT),25°C(25CK),30°C(30CK)and 35°C(35CK)]and silage time(10,20,40 and 70 days)were designed,three replicates in each treatment.The results showed that the p H value of each treatment group was higher than 4.8,the content of lactic acid was lower than 80 g/kg DM,the content of butyric acid was 3.16-18.2 g/kg DM,and the fermentation quality was poor.Compared with other treatment groups,the silage stored at 30°C and ensiled for 70 d had the leastβ-carotene loss(P<0.05),which might produce silage fermentation products that inhibitedβ-carotene loss.1.2 Isolation and identification of squalene,a fermentation product of silage inhibiting the loss ofβ-caroteneDesigned for 70-day silage at low temperature(3-20°C,CCK),25°C(25CK),30°C(30CK)and 35°C(35CK)treatment groups,each treatment group includes 6 separate groups.The results showed that:a comparative analysis of silage chromatograms,five substances with high similarity(>80%)existed and had the potential to inhibitβ-carotene loss,which were identified as caproic acid,tyrosine,farnesol,geranylgeraniol,and squalene seperately.Among them,the relative content(28.6%)and similarity of squalene(93.1%)were the highest,which is suitable for the follow-up experiment research object.1.3 Verification of the effect of squalene in inhibitingβ-carotene loss during ensilingThe additive single factor[no additive control group(CON),0.5%squalene treatment group(J1),1%squalene treatment group(J2),and 2%squalene treatment group(J3)]was designed,three replicates in each treatment group and stored at room temperature(20-38°C)for 70 days.The results showed that squalene could not inhibit the loss ofβ-carotene at a low dosage of 0.5%and 1%,and inhibited the loss ofβ-carotene at a dosage of 2%.However,the p H value of all squalene supplemented groups were higher than 5.3,and the contents of butyric acid and ammonia nitrogen exceeded 10 g/kg DM and 200 g/kg N,respectively,indicated poor fermentation quality.2 Tracing the source of squalene and exploring the mechanism of inhibitingβ-carotene loss2.1 Regulation of squalene content in alfalfa silage and the effect of inhibitingβ-carotene lossThe additive single factor[no additive control group(CON),2%molasses treatment group(ML),and 1%sucrose treatment group(SC)]was designed,three replicates in each treatment group and stored at low temperature(3-20°C).The results showed that the squalene content of the control group and the ML treatment group increased significantly after ensiling for 70 days(P<0.05).The squalene content in the ML treatment group reached 50.4 mg/kg DM,and the effect of reducingβ-carotene loss was the best.Although both the control group and the ML treatment group had high levels of squalene,the plant lipoxygenase of the control group was higher than that of the ML treatment group(P>0.05),and the butyric acid content and the number of aerobic bacteria in the control group were more than that of the ML treatment group(P<0.05).Therefore,the loss ofβ-carotene in the control group was significantly higher than that in the ML treatment group.Compared with the control group,the content of squalene and lactic acid in the SC treatment group significantly reduced(P<0.05),the p H value and the number of aerobic bacteria increased(P>0.05).Therefore,the fermentation quality was poorer,and the loss ofβ-carotene could not be reduced.2.2 Traceability to the source of squalene that inhibitsβ-carotene lossThe 16S r DNA high-throughput sequencing analysis of all the silage treatment groups in section 1.1 showed that anaerobic and facultative anaerobic bacteria such as Lactococcus,Klebsiella,Citrobacter,Enterobactern,and Leuconostoc could produce squalene synthase and increased the squalene content in alfalfa silage.Lactococcus bacteria were the main source of squalene in alfalfa silage.Lactococcus lactis subsp.hordniae was traced as the source of squalene.2.3 The mechanism of squalene inhibitingβ-carotene lossThe experiment was divided into three parts:(1)Set different concentrations ofβ-carotene solution,including 1%β-carotene solution group(A),5%β-carotene solution group(B)and 10%β-carotene solution group(C),detected the fluorescence intensity;(2)Set different concentrations ofβ-carotene-squalene mixed solutions on the basis of part 1,including 5%squalene solution group(X),10%squalene solution group(Y)and 20%squalene solution group(Z),detected the fluorescence intensity;(3)Set up different concentrations ofβ-carotene,squalene and silage fermentation products mixed solutions on the basis of part 2,including(1)1%,2%,4%,8%lactic acid concentration group;(2)1%,2%,3%acetic acid concentration group;(3)0.1%,0.2%,0.3%,0.5%propionic acid concentration group;(4)0.1%,0.2%,0.3%,0.5%butyric acid concentration group;(5)a concentration group(8%lactic acid,0.5%acetic acid,0.5%propionic acid,0.5%butyric acid mixed addition);(6)b concentration group(8%acetic acid,0.5%lactic acid,0.5%propionic acid,0.5%butyric acid mixed addition);(7)c concentration group(8%propionic acid,0.5%acetic acid,0.5%lactic acid,0.5%butyric acid mixed addition);(8)d concentration group(8%butyric acid,0.5%acetic acid,0.5%propionic acid,0.5%lactic acid mixed addition),and detected the fluorescence intensity.The results showed that squalene could protectβ-carotene from being damaged by lactic acid and volatile fatty acids and inhibited its loss.The effect of squalene in protectingβ-carotene was affected by the concentration ofβ-carotene and the concentration of lactic acid and butyric acid;the effect of squalene in protectingβ-carotene from acetic acid and butyric acid was better than that of lactic acid and propionic acid.Taken together,squalene had the best effect in protectingβ-carotene from acetic acid.3 Creation of additives to inhibitβ-carotene lossTwo-factor randomized block trials of additives[no additive control group(CON),1%squalene treatment group(SQ),2%molasses treatment group(ML),0.4%propionic acid treatment group(PA),1×10~5cfu/g FW Lactobacillus plantarum YM3 and 0.2%cellulase treatment group(LPEN),1%squalene and 2%molasses treatment group(SQTM),1%squalene and 0.4%propionic acid treatment group(SQPA)and 1×10~5cfu/g FW Lactobacillus plantarum YM3,0.2%cellulase and 1%squalene mixed treatment group(SQLPEN)]and silage time(3,15,45 and 90 d)were designed,three replicates in each treatment group and stored at room temperature(20-38°C).The results showed that the addition of 1%squalene to silage alone and its combination with molasses could not reduce the loss ofβ-carotene in alfalfa silage.On ensiling for 3,15,and 45 d,compared with the control group,the p H value and ammonia nitrogen content of the SQLPEN added group significantly decreased(P<0.05),the lactic acid content significantly increased(P<0.05),and the loss ofβ-carotene significantly decreased(P<0.05).In summary,the condition(30°C silage for 70 days)of ensiled alfalfa with lowβ-carotene loss was found out,and squalene was screened and identified.Adding squalene did not improve the fermentation quality of alfalfa silage.Only adding high doses of squalene(2%)could reduce the loss ofβ-carotene.The addition of molasses could increase the content of squalene.The Lactococcus genus bacteria was the main source of squalene in alfalfa silage.Lactococcus lactis subsp.hordniae produced squalene and had been isolated.The mechanism of squalene protectingβ-carotene from being destroyed by lactic acid and volatile fatty acids was clarified.The additive was created to improve the fermentation and nutritional quality of alfalfa silage and reduce the loss ofβ-carotene. |
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