| Every year, we could obtained a great number of Oreochromis sp.(Tilapia)byproducts during aquatic products processing. Tilapia byproducts are diverse nutrients,especially rich protein, high-quality and efficient source of animal protein, which is high-quality animal protein source. But now the utilization of Tilapia byproducts still stay at the lagging stage. Most of them are abandoned and only a small part are processed into low-value products such as fish meal. This not only results in waste of resources but also causes pollution of the natural environment. How to use these valuable resources effectively has become the focus of the world’s scientists.In recent years, along with in-depth study on the feeding of active peptides, we found more and more physiological functions, which showed great potential and broad prospects.As the important members of the feeding active peptides, antimicrobial lipopeptides and nutritional small peptides are considered as the most promising alternatives to antibiotics.However, due to complicated production process, high costs and large pollution, their industrialization progress has gone slowly. If we combine these two production process, we could simplify the process, reduce costs significantly and achieve zero emissions production, which breaks through the efficient industrial production technology bottleneck.In regards to the above problems, this study used Tilapia byproducts as the main nutrient medium, added bran and bagasse as ventilation material and inoculated Bacillus natto NT-6 to conduct solid state fermentation to produce composite feeding of active peptides which contained antimicrobial lipopeptides and small peptides; in order to enhance the yield of the antimicrobial lipopeptides, fermentation substrate and conditions were optimized; throughout the fermentation process, dynamic changes of the parameter index were explored, based on this, the fermentation dynamic models were established; the substrate was digested further and the hydrolysis conditions were optimized to obtain active peptides. The polypeptides obtained were isolated and purified, then put through mass spectrometry. The molecular weight and amino acid sequence were determined.Finally the parameter indexes of the final product were determined to evaluate its quality.The contents and results are as follows:1. The effects of Tilapia byproducts to bran ratio, amount of bagasse, the initial moisture content of the substrate, initial p H, inoculation volume, fermentation temperatureand time on the yield of antimicrobial lipopeptide were explored, by using LC-MS. The components and yield of antimicrobial lipopeptide were detected, based on single factor experiments. The fermentation conditions were further optimized through response surface method and the optimal fermentation parameters showed as follows: Tilapia byproducts to bran ratio at 3.48:1; initial moisture content of 69.34%; fermentation time of 61.69 h and fermentation temperature of 28.97 ℃. Under the above conditions, the yield of antimicrobial lipopeptides reached 7.32 g/kg. Through this optimization process, the antimicrobial lipopeptides production was significantly improved.2. The dynamic changes of the various physical indicators(p H, temperature,moisture), yield of antimicrobial lipopeptides, degree of hydrolysis, protein, cell growth and enzyme activity were monitored throughout the fermentation process by using Origin Software. The experimental data of cell growth, protein consumption and lipopeptide generation were fitted, and based on this, appropriate fermentation kinetic models were established. The results showed that R2 of fitting equation were all above 0.98 and the fitting degree was excellent. This study provided theoretical basis for improving this technological application to the actual production.3. The fermentation substrate was digested further. Taking the degree of hydrolysis as index, the effects of temperature, time, p H and the ratio of water on the endogenous protein enzymatic hydrolysis extent were investigated. Single factor experiments showed that all these four factors had affected the degree of hydrolysis significantly. Through the response surface design, the optimal enzymatic process was obtained: temperature of 48.56 ℃, p H7.43, the ratio of water at 1.07: 1, time of 8.29 h, the degree of hydrolysis reached 37.56%,to achieve the maximum yield of active small peptides.4. In this experiment, the active ingredients in the final product were analyzed comprehensively, including the active small peptides, antimicrobial lipopeptides and other technical indicators were also detected. On one hand, the product polypeptide from Tilapia byproducts digested proteins was isolated through ultrafiltration. Firstly, the DPPH radical scavenging activity of each part was detected. The 1000 Da part, had the highest activity(56.85%). This part was then further purified through Sephadex G-25 gel chromatography to obtain five elution peaks; TPH-1, TPH-2, TPH-3, TPH-4 and TPH-5 respectively. The most active component TPH-4(70.58%) was detected by LC-MS/MS method. The results showed that TPH-4 contained two kinds of small peptides of molecular weights 566.47 and679.52, and amino acid sequence correspondingly were Leu-Asn-Asp and Leu-Met-Leu-Leu respectively. On the other hand, the composition and content of antimicrobial lipopeptides in the dried final product were detected by using LC-MSmethod. The results showed that antimicrobial lipopeptides in the final product mainly contained surfactin(995.20,1008.20,1058.20) and iturin(1044.30,1071.20). In addition,there was a small amount of fengycin(1493.00). The yield of antimicrobial lipopeptides was 6.93 g/kg. A crude protein content of up to 32.13 g/100g(dry weight), oligopeptide content of 12.09% and degree of hydrolysis reached 37.32% was also detected. The number of probiotic Bacillus natto was 9.0×108cfu/g(dry weight). Through the fermentation process, protein digestibility, protein solubility and essential amino acids content were significantly higher than before. Which means, product quality has been significantly improved. The final product has both rich antimicrobial lipopeptides and small active peptides, it’s hopeful to become high-quality feed additives into production. |
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