Research background
Apostichopus japonicus has been widely farmed in East Asia in recent years due to its high nutritional and economic value. However, due to the rapid expansion of the scale of breeding, the high density of breeding and the deterioration of the environment, it has led to the occurrence of serious diseases dominated by Vibrio, as well as the large number of pests such as ciliates, which has caused huge economic losses to the breeding industry. Therefore, improving the immunity and disease resistance of ginseng is the focus of current research.
Nutritional immunology is a new way for aquatic animals to regulate the immune response and enhance disease resistance. Using probiotics, Bacillus subtilis fermented fishmeal and scallop skirts, polyunsaturated fatty acids, polysaccharides from plants and fungi as dietary supplements may improve animal health by boosting non-specific immune systems and promote animal growth by increasing digestion and absorption of nutrients.
Fishmeal has a high protein content and good quality, and is considered the most important raw material in aquatic feed. However, with the rapid development of the aquaculture industry, the gap between fishmeal supply and demand is getting bigger and bigger, and the price of fishmeal has risen. Finding a good and cheap alternative to fishmeal is a top priority for agrifeed research and development. Recent studies have shown that adding an appropriate proportion of fishmeal substitutes to feed (fermented soybean meal, chicken feather protein hydrolysate, pumpkin seed cake, yeast fermented rapeseed meal, spirulina powder, blood meal, etc.) does not affect the animal's terminal weight, body weight gain, or specific growth rate.
Bacillus subtilis has been used as a probiotic to improve the growth performance, non-specific immunity and disease resistance of Ginseng. However, the effect of probiotic fermented feeds on the immune capacity of ginseng has not been reported so far.
Experimental methods
Isolation and identification of candidate probiotics: Three strains of candidate probiotics were isolated from the water and bottom mud of ginseng cultivar, namely cellulase strain (strain XW15), lipase strain (strain ZF3) and protease strain (strain DB1). The results of 16S rDNA sequencing showed that strains XW15, ZF3, and DB1 were Bacillus licheniformis (99% similarity), Bacillus subtilis (99%) and Bacillus subtilis (99% similarity), respectively. They were cellulase strain (strain XW15), lipase strain (strain ZF3), and protease strain (strain DB1). The results of 16S rDNA sequencing showed that strains XW15, ZF3, and DB1 were Bacillus licheniformis (99% similarity), Bacillus subtilis (99%) and Bacillus subtilis (99% similarity), respectively. None of the three candidate probiotics are pathogenic, and the strains XW15 and DB1 inhibit the growth of alternate Pseudomonas, so these three candidate probiotics are used for fermentation of fishmeal and scallop skirts.
Feed preparation: The candidate probiotics (3 strains of candidate probiotics are mixed in equal amounts, the total number is 1×109 CFU/ml) is fermented for 24, 48 and 72 h on the skirt of fishmeal and scallop under 30◦C conditions, respectively, and the optimal fermentation time is determined according to the content of free amino acids and small peptides in the raw materials after fermentation, and then the raw materials are prepared under this condition to prepare experimental feed. Three feeds are prepared: CK (unfermented fishmeal), FF (fermented fishmeal) and FSM (fermented scallop skirt) with the following recipe:
Culture experiments: The experiment set up a total of 3 experimental groups: control group (CK), fermented fishmeal group (FF) and fermented scallop skirt group (FSM), 3 replicates per group, 20 ginseng per replicate, the initial weight of ginseng was 9.3±0.05 g, and the experimental cycle was 60 days. Samples are collected at the end of the breeding experiment for subsequent analysis.
Experimental results
1. Changes in feed composition after probiotic fermentation
Probiotic fermentation significantly alters the composition of nutrients in fishmeal and scallop skirts. The content of free amino acids in fishmeal increased significantly after 72 h of fermentation (Figure A), while the content of small peptides increased significantly after fermentation for 48 and 72 h (Figure C). The significant increase in the free amino acid content in the skirt of scallops was significantly increased after fermentation 48 and 72 h (Figure B), while the content of small peptides increased significantly after fermentation 24, 48 and 72 h (Figure D). The free amino acids and small peptide content in the skirt of fishmeal and scallops increased significantly after 72 h of fermentation, so feed was prepared from fishmeal and scallop skirts that were fermented for 72 h.
2. Effect of fermented feed on growth performance
There were no significant differences in body weight gain (Figure A) and specific growth rates (P> 0.05) between the fermented fish meal, fermented scallop skirt group and the control group (P > 0.05), indicating that fermented fish meal and fermented scallop skirt did not have a significant effect on the growth performance of ginseng. This may be because all feeds contain enough nutrients to support the growth of ginseng.
3. Effect on the number of cells in the body cavity, respiratory burst activity and phagocytic activity
Compared with the control group, there was no significant change in the number of ginseng lumen cells in the fermented fish meal group, while the number of ginseng lumen cells in the fermented scallop skirt group increased significantly (Figure A). There was no significant change in the respiratory activity of ginseng in the fermented fish meal group, while the respiratory activity of ginseng in the fermented scallop skirt group was significantly improved (Figure B). There was no significant change in the phagocytic activity of ginseng in the fermented fishmeal group and the fermented scallop skirt group (Figure B). It can be seen that the skirt of probiotic fermented scallops can improve the number of cells in the body cavity of ginseng and the activity of respiratory explosions.
4. Effect on the activity of immune and antioxidant-related enzymes
Enzyme activities of immune-related enzymes such as alkaline phosphatase (APK), acid phosphatase (ACP), and lysozyme (LZM) were significantly elevated in the fermented fishmeal group and fermented scallop skirt group (FigureSA-C). Total antioxidant capacity (T-AOC) enzyme activity was significantly reduced in the fermented fishmeal group and fermented scallop skirt group (Figure D). Superoxide dismutase (SOD) activity was significantly reduced in the fermented fishmeal group (Figure E), while catalase (CAT) activity was significantly increased in the fermented scallop skirt group (Figure F).
5. Effects on immunity and antioxidant-related gene expression
The expression levels of immune and antioxidant-related genes in the intestines (A, D, G), respiratory tract (B, E, H) and body wall (C, F, I) were studied. The results showed that the expression level of LZM in the intestine (Figure A) and body wall (Figure C) of the fermented scallop skirt group increased significantly, while the expression level of LZM in the respiratory tree (Fig. B) of the fermented fishmeal group and the fermented scallop skirt group decreased significantly. The expression level of CAT in the fermented fishmeal group and fermented scallop skirt group in the ginseng intestine (Figure D) and the respiratory tree (Figure E) increased significantly, while the expression level of CAT in the body wall (Figure F) decreased significantly. The expression level of SOD in the intestine (Figure G) and respiratory tree (Figure H) of the fermented scallop skirt group decreased significantly, while the expression level of SOD in the body wall (Figure I) increased significantly.
conclusion
In this study, there were no significant differences in body weight gain and specific growth rates between the fermented scallop skirt group and the control group, suggesting that the fermented scallop skirt may be a potential alternative to fishmeal cultured with ginseng. Fermented fish meal and scallop skirts enhance the non-specific immune and antioxidant activity of ginseng. The results of this study lay the foundation for further understanding the relationship between probiotics and non-specific immunity of ginseng and the potential of fermented scallop skirts to replace fishmeal in ginseng culture.
Source: Aquatic Animal Health Assessment