Identification Of Phlorotannins From Sargassum And Its Effect On Rumen Unsaturated Fatty Acid Biohydrogenation In Vitro

The increase in the content of Saturated Fatty Acid（SFA）in ruminant products often led to the occurrence of cardiovascular diseases,which was caused by the biohydrogenation of feed Unsaturated Fatty Acid（UFA）in the animal rumen,so how to inhibit biohydrogenation has become the focus of current research.It has been found that tannin can inhibit fatty acid hydrogenation in animal body and regulate fatty acid composition in animal products.Phlorotannins（PT）,also known as phloroglucinol tannin,has the basic structure of phloroglucinol,which only exists in Brown algae.Studies have shown that phlorotannins have positive effects on improving animal performance and regulating microbial flora.Based on this,we predicted that phlorotannins could regulate rumen microorganisms to inhibit hydrogenation.Therefore,in this paper,phlorotannins were extracted from Sargassum to clarify its composition,and the effects of phlorotannins on rumen fermentation and rumen fatty acid hydrogenation were determined by in vitro method.（1）The crushed Sargassum was extracted by extraction method,and the phlorotannins extracts were obtained.The crude extracts were purified by Sephadex LH-20 glucan gel,and the phlorotannin extracts were obtained by lyophilization.After that,the extracts were separated by ultra-high performance liquid chromatography tandem mass spectrometry（UPLC-MS/MS）,phloroglucinol was used as the standard substance for quantitative analysis,and other substances were matched by non-target search database,so as to achieve qualitative and quantitative analysis of the extracts.The results showed that there were five peaks in the extract chromatogram,and the residence time（RT）were 0.93,1.74,2.71,12.26,34.15,respectively.According to the database matching,the substances corresponding to the five peaks were:Phloroglucinol and 2,4,5-Trimethoxybenzoic Acid,P-Hydroxybenzoic Acid,Zinniol,Allopregnanolone.Moreover,the content of Phloroglucinol in the extract was 91.26%.（2）The effects of different supplemental levels（125,250,500 μgPT·mL-1）of phlorotannins extract on rumen fermentation and main rumen bacteria content of dairy cows were investigated in vitro,and the optimal supplemental level was selected.The results showed that the supplementation of 120,250 and 500 μgPT·ml^-1 phlorotannins extract of brown algae significantly decreased rumen pH and NH3-N concentration（P<0.05）and significantly increased the numbers of Prevotella ruminicola in the rumen（P<0.05）;The supplementation level of 500 μgPT·ml^-1 significantly increased Ruminococcus flavefaciens（P<0.05）,propionic acid content was significantly increased（P<0.05）,promoted the transformation of rumen fermentation type,and significantly decreased the contents of isobutyric acid and isovaleric acid（P<0.05）;The optimum supplemental level of phlorotannins was 125 μg·ml^-1.（3）The effects of phlorotannins extract on rumen fermentation,microflora and UFA were investigated in vitro,and the microbial pathway of phlorotannins extract regulating biohydrogenation was explored.The results showed that 1.7%phlorotannins were added to the diet could significantly increase the digestibility,USFA,MUFA and PUFA contents（P<0.05）,decreased NH3-N concentration,SFA and C18:0 content（P<0.05）.At the same time,the addition of phlorotannins significantly increased the relative abundance of Firmicutes,Proteobacteria and Fibrobacterota at the phylum level（P<0.05）and the relative abundance of Prevotellaceae_UCG-001,Prevotellaceae_UCG-003 and P-251-O5 at the generic level（P<0.05）.Conclusions:Phloroglucinol and 2,4,5-Trimethoxybenzoic Acid,P-Hydroxybenzoic Acid,Zinniol,Allopregnanolone are the main components of phlorotannins extract obtained from Sargassum sargassum.The content of phloroglucinol was 91.26%.The addition of phlorotannins extract can improve the digestibility of feed in vitro,inhibit protein decomposition,cause incomplete biohydrogenation by regulating rumen flora,increase the content of UFA and reduce the content of SFA.