Protease improves key indicators of fermented soybean meal
Soybean meal is a by-product obtained after soybean oil is extracted from soybeans. It has a high content of protein and essential amino acids, and its composition is reasonable and balanced. It is a high-quality plant protein raw material in animal husbandry. However, since soybean meal contains a variety of anti-nutritional factors, it reduces the digestion, absorption and utilisation of nutrients by livestock, and even has a negative impact on the growth, development and health of animals. Fermented soybean meal is made from high-quality soybean meal as the raw material. It uses a combination of microbial fermentation and proteolytic enzyme hydrolysis to remove many of the anti-nutritional factors in the soybean meal. At the same time, the fermentation process produces some flavour substances such as mellow and lactic acid, which improves the nutritional value and palatability of the soybean meal.
1 Effect of the ratio of ingredients to water, temperature and fermentation time on the content of small peptides in fermented soybean meal
Fermented soybean meal uses biological techniques such as microbial fermentation and enzyme engineering. It uses soybean meal as the raw material, and adds yeast, lactic acid bacteria, Bacillus subtilis and protease during the solid-state fermentation process to reduce the content of crude fibre, non-starch polysaccharides and antigenic proteins in soybean meal, thereby improving the quality of soybean meal[6]. In this experiment, the effects of the material-to-water ratio, temperature and fermentation time on the fermentation of soybean meal were studied using a 33 orthogonal experiment.
Table 1: Orthogonal test factor level table
| Test number: | Water-to-material ratio (A) | Temperature (B) | Fermentation time (C) |
| 1 | 01:00.4 | 33℃ | 48h |
| 2 | 01:00.6 | 37℃ | 60h |
| 3 | 01:00.8 | 41℃ | 72h |
| Test number: | Water-to-material ratio (A) | Temperature (B) | Fermentation time (C) | Small peptide content (%) |
| 1 | 1 | 1 | 1 | 11.194 |
| 2 | 2 | 2 | 2 | 18.261 |
| 3 | 3 | 3 | 3 | 16.048 |
| 4 | 1 | 2 | 3 | 15.967 |
| 5 | 2 | 3 | 1 | 12.935 |
| 6 | 3 | 1 | 2 | 19.571 |
| 7 | 1 | 3 | 2 | 12.898 |
| 8 | 2 | 1 | 3 | 18.565 |
| 9 | 3 | 2 | 1 | 15.968 |
| K1 | 40.059 | 49.33 | 40.097 | |
| K2 | 49.761 | 50.169 | 50.73 | |
| K3 | 51.587 | 41.881 | 50.58 | |
| R | 11.528 | 8.288 | 10.483 | |
| k1 | 13.353 | 16.443 | 13.366 | |
| k2 | 16.587 | 16.722 | 16.91 | |
| k3 | 17.196 | 13.96 | 16.86 | |
| r | 3.84 | 2.763 | 3.494 | |
| optimal level | A3 | B2 | C2 | |
| influencing factors | A>C>B | |||
The results showed that the optimal combination was A3B2C2, and the influence of the three factors on the fermentation product was: ratio of ingredients to water > fermentation time > temperature. Since the results did not show a significant effect of the water-to-material ratios of 1:0.6 and 1:0.8, there was no significant difference between the fermentation temperatures of 33 °C and 37 °C, and the fermentation times of 60 h and 72 h, the A2B2C2 conditions were adopted as cost-effective and suitable for large-scale production control, i.e. a water-to-material ratio of 1:0.6, a fermentation temperature of 37 °C, and a fermentation time of 60 h.
2Application of protease in the fermentation of soybean meal
Antinutritional factors in soybean meal are divided into two categories: heat-stable antinutritional factors, including mainly soybean antigenic proteins (globulin and β-conglycinin) and soybean oligosaccharides (mainly raffinose and stachyose); and heat-sensitive antinutritional factors, including trypsin inhibitor, urease and lectins[7]. Heat-sensitive anti-nutritional factors can be inactivated by heating, but heat-stable anti-nutritional factors such as globulins, β-conglycinin, raffinose and stachyose are not easily destroyed during general feed processing[8]. In this experiment, the application of Anji acid protease AP-10, alkaline protease AP-20 and their combinations in fermented soybean meal was studied under the conditions of a feed-to-water ratio of 1:0.6, a reaction temperature of 37°C and a fermentation time of 60h.
2.1 Research on the application of acid protease in fermented soybean meal
This paper studies the application of Anji Acid Protease AP-10 in fermented soybean meal at different concentration gradients. The results show that with the increase of enzyme addition, the content of small peptides in fermented soybean meal gradually increases, and its small peptide content is greater than 12%, crude protein content is greater than 50%, and alkali-soluble protein is about 70%. All indicators meet and exceed the average industry indicators for fermented soybean meal.
| Enzyme addition | Small peptide content | Crude protein | Alkaline protein |
| 0 | 3. 75% | 46. 38% | 57.58% |
| 0.50% | 12. 42% | 50.04% | 68. 73% |
| 0.80% | 12. 74% | 50.55% | 69.09% |
| 1.00% | 13.36% | 50.86% | 70.43% |
| 1.20% | 13. 93% | 51.33% | 71.54% |
| 1. 5% | 14.01% | 51.55% | 72.02% |
Figure 1: Application of the acid protease AP-10 in fermented soybean meal Index
2.2 Study on the application of alkaline protease in fermented soybean meal
This paper studies the application of Anji Alkaline Protease AP-20 in fermented soybean meal at different concentration gradients. The results show that with the increase of enzyme addition, the content of small peptides in fermented soybean meal gradually increases, and its small peptide content is greater than 16%, crude protein content is greater than 50%, and alkali-soluble protein is greater than 70%. It is suitable for producing high-small-peptide fermented soybean meal with high acid-soluble protein content.
| Enzyme addition | Small peptide content | Crude protein | alkaline protein |
| 0.00% | 3. 75% | 46.38% | 57.58% |
| 0. 5% | 16.20% | 51.13% | 70.93% |
| 0. 8% | 17.11% | 52.47% | 75.53% |
| 1. 0% | 18. 26% | 52.71% | 77.88% |
| 1. 2% | 20.43% | 53.05% | 78.06% |
| 1. 5% 6 | 20.93% | 52.58% | 78.52% |
Figure 2: Application of alkaline protease AP-20 in fermented soybean meal Index
2.3 Application research of protease composition in fermented soybean meal
After enzymolysis with acidic protease AP-10, the viscosity of the fermented soybean meal material is low, making it easy to dry. The product has good palatability and a small peptide content of 12-15%, which can meet the mass production requirements for fermented soybean meal. After enzymatic hydrolysis with Anqi alkaline protease AP-20, the fermented soybean meal has a small peptide content of up to 16-21%, which is suitable for the production and application of high-quality fermented soybean meal with high small peptide content. This paper combines the product characteristics of AP-10 and AP-20 and applies them in combination to the preparation of fermented soybean meal. Under the same product index requirements, the amount of enzyme preparation can be reduced, which can effectively reduce the production cost of fermented soybean meal.
| AP-10+AP-20 | Small peptide content | Crude protein | Alkaline protein |
| 0.8%+0 | 9.00% | 49.31% | 74.62% |
| 0.7%+0.1% | 10.70% | 50.19% | 72.234 |
| 0.6%+0.2% | 12. 394 | 50.76% | 71.94% |
| 0.5%+0.3% | 13.19% | 51.28% | 71.82% |
| 0.4%+0.4% | 13.75% | 51.72% | 88. 99% |
| 0.3%+0.5% | 14.77% | 51.83% | 79.21% |
| 0.2%+0. 6% | 0. 1518 | 0. 5206 | 0. 7545 |
| 0.1%+0.7% | 16.08% | 52.90% | 75.57% |
| 0+0.8% | 16.47% | 53.02% | 76.00% |
Figure 3: Indicators of protease composition in fermented soybean meal
3, Discussion of the application effect of protease in fermented soybean meal
3.1 Discussion of the distribution of acid-soluble protein and alkali-soluble protein peptides in fermented soybean meal after protease application
The fermented soybean meal after enzymolysis with the acidic protease AP-10 has an appropriate content of small peptides, with more than 95% of the acid-soluble protein ≤180Da, and the alkali-soluble protein distributed between 5000-10000Da and below 180Da. Fermented soybean meal after enzymatic hydrolysis with alkaline protease AP-20 has a high content of small peptides, with more than 95% of acid-soluble proteins ≤180Da, and very few alkaline-soluble proteins greater than 5000Da, which are basically evenly distributed below 2000Da.
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