Levels of protein and the limiting amino acids in the diet severely affects the larval growth. The results have shown that application of B. subtilis protease had no significant effects on FW, ADG, ADFI and FCR of the 3rd instar silkworm. It may be because the silkworms were weighed and grouped in the beginning of the experiment, which caused some stress on the silkworm and affected the function of the protease. Another cause may be the period of 3rd instar larvae was too short to show the outcomes of feeding, so the growth performance of the silkworm in the test group was not significantly different from that of the control group. At the 4th and 5th instars, FW, ADG, ADFI and FCR of the test group were extremely significantly higher than those of the control group, indicating that the addition of B. subtilis protease could significantly improve FW, ADG, ADFI and FCR of the 4th and 5th instar silkworms. Nutritional tests were mostly carried out by rearing larvae on artificial diets, and the effects of supplementation of specific substance on larval growth, development, and survival were carefully examined(29). Beneficial effect of protease was studied for different animals. A study proposed that exogenous protease could increase the ADFI and ADG of broilers, but the FCR was not affected (19), similarly it was reported that the supplementation of exogenous protease resulted a decrease in FCR and an increase in weight gain (30) It was also observed that the addition of protease in the growing-finishing period led to a decrease in ADG of pigs (31). Reasons for the difference in results were most likely because of different compositions of diets, dose concentrations and the sources of protease.
It was reported that adding protease in a low protein or high protein diet could improve feed efficiency, they investigated that broiler feed supplemented with a protease could improve the CPD and EED (32). Similarly, our present study found that B. subtilis protease could significantly improve CPD, FCR and CSCR. In our test, an increase in the apparent digestibility of crude protein and crude lipid may be one of the reasons for the improvement in growth and production performance. The addition of exogenous protease could increase the hydrolysis of dietary protein, thereby making more peptides and amino acids available for utilization in the small intestine (33, 34). The digestion and absorption of crude protein in feed directly affects the synthesis of silk protein. Therefore, increasing CPD will increase the ability of silkworm to synthesize silk, which can improvement the feed efficiency. This study showed that protease can significantly increase the EED and CSCR of the test group as compared to the control group. However, the results of this study showed that silkworm CFD of the test group was significantly higher than that of the control group. It was reported that probiotics can enhance the enzymatic activity which in turn can enhance the nutritional parameters of silkworm (35).
The results of this experiment showed that the number of dead worm cocoon in the control group was higher than that in the test group. The reason may be that the protease increased the CPD and increased the absorbable amino acids in the mulberry leaves. It was found that amino acids could reduce the total larval duration and mortality of larvae and pupae(36). It was found that during metamorphosis in silkworm, changes in protease activity in the midgut were observed. Especially remarkable increase in the enzyme activity was seen before emergence (37). Since enzymes serves as a reaction catalyst of any biological system. So, the activities of digestive enzymes in silkworm like, alkaline protease and alkaline phosphatase can help in silkworm breeding program for improvement of cocoon characters (38). The addition of B. subtilis protease in this experiment increased the total number of defective cocoons. Since the test group was added with B. subtilis protease, the survival rate of the silkworm in the experimental group was improved, so the density of the test group was higher than that of the control group, and more silkworm excrement was produced than the control group. The poor environment was the main cause of defective cocoons.
The ability of silkworm to synthesize silk protein directly affects the yield of silk. The quality of silkworm cocoon is evaluated by measuring the CN, CSW and CSR. Under the same PW conditions, the heavier the cocoon shell, the higher the CSR. The CSW is an index of cocoon silk quantity. In B. mori, particularly, up to 65% of digested nitrogen is utilized for silk production during the last instar and the level of dietary protein and limiting amino acids in the diet strongly affects larval growth and silk production. In this study, the results of cocoon quality showed that B. subtilis protease could significantly increase the APW, ACSW, CN and ACW. This may be related to the increased CPD by B. subtilis protease. In the test group, only the CSR was slightly lower than that of the control group. Because some of the defective cocoon cannot be weighed in the test group, not to be included in the calculation, so the CSR of the control group was slightly higher, but the data of ACSW and the ACW could also indicate that the addition of B. subtilis protease could improve the silkworm cocoon quality. It could increase the yield of silk, which is very important for the silk industry.
The results showed that the silkworm had a relatively high degree of digestion and utilization of crude protein and crude lipid components in mulberry leaves. The results were related to the crude protein providing the development and growth of the individual and the accumulation of protein to provide the raw material for the subsequent cocoon. Since the main component of silk is protein, silkworm absorbs the crude protein in mulberry leaves, and some of it is deposited into the silk gland through biochemical reaction in the body, which was used for later cocooning. Insect adipocytes can store a great amount of lipid reserves as cytoplasmic lipid droplets and lipid metabolism is essential for growth and reproduction and provides energy needed during extended non-feeding periods (39). Silkworm spinning is a process of converting amino acids in feed into silk glands and excreting them. The amino acid content in feed and the ability to synthesize silk fibroin will affect the amount of silk. Development of silk gland mainly occur in the 5th instar stage, at this time, the nutrients in the feed directly affect the synthesis of silk. Therefore, the appropriate addition of some physiologically active substances in the 5th instar stage will increase the silkworm's ability to synthesize silk, which is beneficial to improve the feed efficiency. However, the results of this experiment showed that the addition of B. subtilis protease had no significant effect on the development of silk gland in silkworm.
The digestive enzymes of the midgut control the digestion and absorption of various nutrients in the feed. Results showed that the addition of B. subtilis protease could increase the protease activity of the midgut in the silkworm. It may be because the added protease can induce the secretion of enzymes in the midgut, or that the added protease remains in the intestine. One study reported that the activity of natural protease changes during developmental stages of the silkworm, it decreases after the mature larva period, reaches a peak before emergence, and decreases markedly thereafter. But there was no great change in the protein concentration in the midgut except for the adult stage (37). However results of our study showed that feeding B. subtilis protease caused an increase in the protease activity which indicated that the silkworm had higher efficiency in protein absorption. After 3 days of silkworm at 5th instar, the amino acids in the body need to be transported to the silk gland to synthesize silk protein (40, 41). Therefore, after feeding B. subtilis protease, the silkworm increased the CPD and CSCR.
In conclusion B. subtilis protease can improve the growth performance and FCR of silkworm, and improve its economic benefits. Therefore, B. subtilis protease can be used as a feed additive for the sericulture industry.