Optimizing the scale-up production of fermented astragalus and its benefits to the performance and egg quality of laying hens

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TL;DR

This study optimized the large-scale solid-state fermentation of astragalus using Lactobacillus pentosus Stm, achieving a lactic acid bacteria count of 206 × 10⁸ cfu/g and lactic acid content of 15.0% at the plant scale (1 ton). When fed to laying hens at 1% of the diet, fermented astragalus significantly reduced the feed-to-egg ratio and egg cholesterol content while increasing the egg-laying rate. These benefits were attributed to improved intestinal health via enrichment of beneficial bacteria such as Prevotellaceae UCG-001 and Enterococcus, supporting fermented astragalus as a promising feed additive for poultry.

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**Background:** Astragalus membranaceus is a traditional Chinese medicine with documented health benefits, but direct addition to feed can cause side effects such as anti-nutritional factors and strong odor. Fermentation is a promising method to enhance bioactive compounds and reduce adverse effects. While previous studies have shown that fermented astragalus (FA) improves performance in poultry, few have examined the underlying mechanisms, particularly the role of intestinal microbiota, and none have systematically optimized and scaled up FA production for industrial application.

**Methods:** Thirteen lactic acid bacteria (LAB) strains were screened for solid-state fermentation (SSF) of astragalus based on LAB count and lactic acid content. Lactobacillus pentosus Stm was selected as the optimal strain. Single-factor optimization was performed at the lab scale (250 g) for bran addition, feed-water ratio, inoculation ratio, glucose addition, and enzyme addition (xylanase, cellulase, pectinase), with an orthogonal test for enzyme optimization. The process was scaled to 10 kg bags for temperature (30–42°C) and time (48–120 h) optimization, then to 1 ton at plant scale. Bioactive compounds (flavonoids, saponins, polysaccharides) and bacterial community composition were monitored during plant-scale fermentation. A feeding experiment was conducted with 384 laying hens (Nonda-5, 42 weeks) randomly assigned to three groups (4 replicates of 32 hens each): control (CK, normal feed), FA (1% FA + 99% normal feed), and UA (1% unfermented astragalus + 99% normal feed) for 28 days. Performance (egg weight, feed-to-egg ratio, laying rate, bad egg rate), egg quality (protein, lipid, cholesterol, Haugh unit, shell strength), serum immunity (IgA, IgM, IgY, IL-2, IL-6, TNF-α), and cecal microbiota (16S rRNA amplicon sequencing) were assessed.

**Key Results:** After optimization and scale-up to 1 ton, LAB count reached 206 × 10⁸ cfu/g and lactic acid content reached 15.0%, representing increases of over 18-fold and 12-fold, respectively, compared to pre-optimization levels. Total flavonoids reached 6.6%, saponins reached 4.3%, and astragalus polysaccharides peaked at 24.3% at 84 h. Potential pathogens decreased from 35.5% in raw material to below 4.5% after fermentation, while Lactobacillus became dominant at 84.6% abundance. In the feeding trial, the FA group showed a significantly reduced feed-to-egg ratio compared to CK and UA groups (p < 0.05), a significantly higher egg-laying rate, and a significantly lower percentage of bad eggs. Egg cholesterol content was significantly reduced in the FA group for both whole egg and egg yolk (p < 0.05). Protein and lipid content were not significantly affected. The liver index was significantly higher in both FA and UA groups compared to CK (p < 0.05). Serum IgA and IgM were significantly higher in the UA group than CK (p < 0.05), while IgY was significantly lower in the FA group (p < 0.05). IL-2, IL-6, and TNF-α showed no significant differences. Intestinal microbiota analysis revealed significantly higher Chao1 richness and Shannon diversity in the FA group (p < 0.05). PCoA showed significant separation between groups (ANOSIM R = 0.3137, p = 0.001). LEfSe analysis identified Prevotellaceae UCG-001, Prevotellaceae NK3B31, and Enterococcus as core bacteria enriched in the FA group, while Desulfovibrio and Negativibacillus were enriched in the UA group.

**Clinical Implications:** This study provides a systematic, scalable method for producing fermented astragalus with high LAB counts and bioactive compound content. The findings demonstrate that dietary supplementation with 1% FA can improve laying hen performance and egg quality, particularly by reducing the feed-to-egg ratio and egg cholesterol content. The mechanism appears to involve modulation of the intestinal microbiota, with enrichment of beneficial genera (Prevotellaceae UCG-001, Enterococcus) that produce short-chain fatty acids and may reduce serum cholesterol. These results support FA as a promising, safe feed additive for the poultry industry, with potential for commercial-scale production. The reduced egg cholesterol content also offers a potential marketing advantage for low-cholesterol egg products.