Protective Effect Of Lactobacillus Casei Fermentation Of Pomegranate Peel, Chebulo And Their Combination On Carassius Infected With Aeromonas Hydrophila

The rapid development of aquaculture in recent years has increased company revenues,but has also created a number of challenges,due to intensive feeding conditions or inadequate feeding techniques,etc.,resulting in frequent bacterial and viral diseases.Aeromonas hydrophila is a strongly virulent strain of bacteria that can cause morbidity in aquatic animals and cause great losses to aquaculture.At present,most of the diseases caused by Aeromonas hydrophila in China are treated by applying antibiotics and chemicals,but excessive application can easily lead to pollution,increased resistance and increased mortality of fish,so the development of environmentally friendly and healthy antibiotic alternative drugs is the focus of research and development.Lactobacillus fermentative herbal medicine is able to break down the active substances of herbal medicines to improve their efficacy and reduce adverse reactions,and more and more studies have shown that fermented herbal medicines can significantly improve the productivity of animals by regulating their intestinal microorganisms,and can also enhance their immune system,thus achieving a good therapeutic effect.Therefore,lactic acid bacteria fermentation of Chinese medicine has a broad application value.This experiment is divided into two main parts:Experiment 1: In vitro inhibition of Lactobacillus fermenting pomegranate peel,chebulo and combination against Aeromonas hydrophilaIn this study,Lactobacillus casei(ATCC393,LC)was selected to ferment pomegranate peel and chebulo and the combination of two Chinese herbal medicines through its acid-producing property,which stimulated the active ingredients in the Chinese medicines andincreased their efficacy,hus in vitro inhibition of Aeromonas hydrophlia TPS-30.The results showed that LC fermented pomegranate peels were more effectuai than the LC fermented ones.The results showed that the fermented product was prepared by mixing the fermentation solution with LC fermented pomegranate peel,LC fermented horehound and LC fermented pomegranate peel and horehound all had inhibitory effects on A.hydrophila,however,A.hydrophila were most inhibited by fermentation products,followed by the LC+pomegranate peel and LC+chebulo groups,and the fermentation products from the combination of LC+pomegranate peel and chebulo had the strongest inhibitory effect on the growth of A.hydrophila.The inhibition of the growth curve of A.hydrophila was most obvious in the LC+pomegranate peel and LC+chebulo group.Secondly,when the effect of each group on the biofilm of Aeromonas hydrophila was examined at different concentrations,the combination of LC+pomegranate peel and chebulo also showed some inhibition on the biofilm formation of A.hydrophila.In conclusion,the combination of LC+pomegranate peel and chebulo had the most significant inhibitory ability on A.hydrophila.Experiment 2: Evaluation of the immune effect of fermentation of pomegranate peel and chebulo by Lactobacillus casei and the combinationIn order to evaluate the immune effect of Lactobacillus casei fermented Chinese herbal medicines,crucian carp were selected as the test animals and divided into 7 groups: PBS group,pomegranate peel group,chebulo group,pomegranate peel and chebulo combination group,LC fermented pomegranate peel group,LC fermented chebulo group,LC fermented pomegranate peel and chebulo combination group.The Chinese medicine aqueous decoction and fermentation products were coated with the base diet to feed the crucian carp continuously for28 d by oral immunization,and the tissues and sera of each group were collected at 0d before,7d,14 d,21d and 28 d after immunization to detect their relevant immune indexes.The serum indices of LZM,C3,C4,AKP,ACP,SOD and IgM of crucian carp were measured by ELISA.The results showed that all indicators in the serum increased after 28 d of continuous feeding,and higher levels of IgM could bedetected in the serum after the immunization of crucian carp by oral immunization,and the LC+co-administration group was significantly higher than the LC+chebulo group and LC+pomegranate peel group.Detection of expression levels of immune-related genes IL-1β,IL-10,TNF-α and TGF-β in various tissues of Carassius auratus using q RT-PCR.The results showed that the expression levels of IL-1β,IL-10,TNF-α and TGF-β in the gut,spleen and kidney showed an increasing trend in experimental group,especially the expression of each gene in the intestine was the most obvious,indicating that the lactic acid bacteria fermented the Chinese medicine and then entered the intestine by orally intothe intestinal tract can stimulate the relevant cytokines in the intestinal tract of fish,thusimproving the immunity of crucian carp.The results of the attack test conducted at the end of feeding showed that all the fish in the PBS group died.The immune protection rates of the pomegranate peel group,chebulo group and the two Chinese herbal medicines combined group were 28.47%,25.93% and 31.72% respectively,and the immune protection rates of the LC+pomegranate peel group,LC+chebulo group and LC+combined group were 46.34%,42.45% and 40.52% respectively,indicating that the protection effect by The protection effect after fermentation by lactic acid bacteria was significantly better than that of the aqueous decoction group,and the effect of the combination of the two herbs was higher than that of the single herbs.In conclusion,in this experiment,two herbal medicines,pomegranate peel and chebulo,were selected for single and combined use and fermented with Lactobacillus casei.The in vitro test can verify that both the aqueous decoction of the herbal medicines and the fermented products of Lactobacillus casei can inhibit the growth of Aeromonas hydrophila,but the fermented products of Lactobacillus casei are more effective.The in vivo test can prove that the fermented product can effectively improve the relevant immune indexes and the immunity of the organism in the serum of crucian carp as a feed additive.This trial will provide a foundation for the development and use of a new generation of green,low toxicity surrogate antibiotics.