Streptozotocin (STZ, Sigma Chemical St. Louis, MO, USA), chloral hydrate, sterilized saline water, citric acid, and sodium citrate (Sinopharm Chemical Reagent Co. Ltd., Shanghai, China) were purchased.

Three proteins, namely, SP, LP, and SPP were added as 23% (w/w) in SP ON formula, LP ON formula, and SPP ON formula, respectively. The proteins of LP and SPP were equally added (11.5% w/w of each) in the LCSSP ON formula. The rest of the formula was made up of 5% fish oil powder, 15% olive oil powder, 12% corn starch, 17.5% cassava starch, 9% oligosaccharide fructose, 5% isomaltose, 10% vanilla, 3.5% various vitamin, and mineral elements (Table 1). All raw materials for the formula were food-grade and purchased from Henan Jianjiu Industrial Co. (Zhengzhou, Henan, China). Formula tableting was done using a tablet machine (Mini PRESS-IISF, India).

The methods and standards used to determine the component of the formula were listed in Table 2.

All data were analyzed as the mean ± SD and levels were compared among groups using one-way ANOVA. Statistical analysis was done using the Statistical Program for the Social Sciences Version 23.0 (SPSS, Chicago, IL, USA). Line chart and histogram were prepared using origin pro-2019 (Wellesley Hills, MA, USA). Differences were taken as significantly different when p < 0.05 and were considered very significant when p < 0.01.

The formulations of four kinds of the ON formula were analyzed according to the methods in Table 2 and listed in Table 4. The nutrient compositions of the four ON formula were analyzed in SPG, LPG, LCSSPG, and SPPG. The protein contents were 19.9, 20.1, 21.3, and 21.8 g/100 g, respectively. Carbohydrate contents were 43, 42.9, 43.6, and 42.1 g/100 g, respectively. Fat contents were 19.1, 19.3, 19.5, and 19.6 g/100g, respectively. The total energy of four ON formula was lower than 1870.5KJ. The fat-supplied energy was less than 35%, carbohydrate-supplied energy was less than 45%, and the proportion of protein-supplied energy was less than 23% in all self-made oral formula.

The compositions of the four ON formula were similar and protein type was the only way to distinguish them. The differences among those three proteins were, mainly, amino acid composition. Eighteen kinds of amino acids were detected in SP which included 1.8 g of methionine, 6.1 g of lysine 1.2 g of cysteine, 4.0 g of threonine, and 4.6 g/16 g N of alanine on average and so on (23). Stefan et al. demonstrated that the essential amino acids content of SP and LP was 27% and 43%, respectively (24). SPP, as a novel high-quality raw material for complementary medicine food and the mass ratio of essential or non-essential amino acids, was.77, which was higher than the FAO-WHO requirements (>0.6) (25).

Relatively lower essential amino acids content led to better protein metabolism according to the results. Nonetheless, the amino acid mixtures cannot reach identical results comparing with protein consumption (26). Plasma amino acid, closely related to muscle protein synthesis, was increased after protein intake (24, 25).

Diet can also lower the rate of developing diabetes in those with impaired glucose tolerance (7, 27, 28). Proper amounts of vitamins and minerals which include zinc, chromium, magnesium, vitamin B group, and inositol were added to alleviate glucose and lipid metabolism disorder in T2DM mice (28). An amount of 10 g/100 g dietary fiber was supplemented in the ON formula to prolong gastric emptying time and delay glucose absorption. A series of systematic reviews and meta-analyses suggested that the T2DM rate was decreased 15-30% when there is a 25–29 g dietary fiber intake according to a 2019 review report (29). Tapioca starch was added to the ON formula to stable postprandial BLG (30).

Fish oil- and olive oil-supplemented diets provided better glycaemic and lipid metabolic control in both human and animal experiments comparing with a normal diet. Eight grams of olive oil per day has a 5% T2DM risk-reducing effect according to a 22-year follow-up study (31). David et al. showed that an HFD, supplemented with fish oil for 30 weeks lowered the effects of liver cholesterol, cholesterol ester, and triacylglycerol in C57BL/6J mice compared with mice fed an HFD supplemented with lard (32). In this present study, the self-made ON formula combined all the features above.

The TP, ALB, and GLO levels in serum were analyzed to explore whether ON formula supplemented with specifically designed proteins had an effect on protein metabolism (Table 5). The TP, ALB, and ALB/GLO levels were decreased in BG when compared with CG. The concentration of TP in LCSSPG, SPPG, and SPG was decreased to 31, 39, and 39.5 mmol/L, respectively, which nearly reached normal levels after 4 weeks. The ALB level was significantly increased from 15.0 to 22 and 22.5 in SPPG and SPG, respectively. The TP, ALB, and GLO levels were increased in PG (p < 0.05) while no statistically significant difference in the ratio of ALB/GLO level was observed.

A decrease in the levels of TP, ALB, and GLO was verified both in T2DM humans and mice which may relate to the factor of insulin resistance in pancreatic β-cells (33). As expected, specially added proteins such as SPP and SP can release functional amino acids which can repair amino acid stimulated protein synthesis to further alleviate whole-body protein metabolism. Taken together, the results demonstrated that ON formula containing suitable protein such as SP or SPP at a proportion of 23 g/100 g could bring back protein levels to normal in plasma comparing with normal diet and even better than available ON formula products.

The effects of ON formula consumption on lipid metabolism were shown in Figure 2. Statistical differences were conducted among seven groups which were shown in Figure 1. The TC, TG, LDL-C were increased significantly after 8 weeks high-fat diet (HFD) combined with STZ intraperitoneal injection. The HDL-C level was significant decreased in the BG comparing with CG (p < 0.01). The serum TC level was significantly reduced after consumption of SP, SPP, LCSSPP, PG, and LP comparing with BG (Figure 2A). The TG level was significantly decreased after administration of SP, SPP, LCSPP, and LP comparing with BG (Figure 2B). In the study, it was found that SP, SPP, LCSSP, and LP ON formula had a great role in decreasing the serum TG and TC level in HFD induced mice. The HDL-C levels were increased significantly on LCSSPPG, SPPG, SPG, and PG comparing with the BG group indicating that LCSSPP, SPP, and SP oral formula may lower the risk of HDL-C loss in HFD-induced mice (Figure 2C). The level of serum LDL-C was greatly lowered after consumption of PG, SPPG, LCSSPG, and SPG while there was no significant difference in the LPG group which needed further analysis (Figure 2D). These data indicated that PG, SPPG, LCSSPG, and SPG had a good supporting role in decreasing the serum LDL-C level in HFD induced mice.

This study proved that the ON formula could significantly reduce serum TC, TG, HDL-C, and LDL-C levels in T2DM mice comparing with a normal diet. The results are consistent with previous research reported by Sung et al. (33). The proteins used in the ON formula are highly responsible for those changes in lipid metabolism. Sun He et al. found the anti-obesity activity of a silkworm pupa peptide in an HFD feeding in rats by inhibiting the differentiation of preadipocytes and adipogenesis (33). Rizaldy et al. verified the decreased weight and adiposity of LP in male obese rats due to decreased hepatic lipidosis compared to control, partly through downregulation of lipogenic and upregulation of β-oxidation transcripts in the liver (34). Based on literature reviews, a diet with soy protein containing isoflavones reduced low-density lipoprotein (LDL) (35), but without clear effects on TG or high-density lipoprotein (HDL). The results demonstrated similar results which alleviate lipid metabolism by high-quality protein addition.

The BLG and fasting BLG changes during the experiment are shown in Figure 3, respectively. The FBG and BG were enhanced from 7 to 10 wks and peaked in the tenth week. The FBG has a great reduction with the value of 46.7, 24.1, 21.8, and 33.9% in SPG, LCSSPG, SPPG, and PG group, respectively, after 5 wks of treatment. Continuous elevation of FBG and BLG in BG was observed, which is strong evidence for T2DM modeling making. The FBG level was decreased to 16.8 to 8.9, 12.8, 13.2, and 11 in SPG, LCSSPG, SPPG, and PG groups at the end of the experiment. Furthermore, there were no wide fluctuations in BLG levels at the end of treatment, which needed to be further analyzed.

The study, in vivo, indicated that the administration of the ON formula to the HFD-fed mice combined with an intraperitoneal injection of STZ produced a remarkable reduction of BLG and FBG after 5 weeks of treatment with SPPG, SPG, and LCSSPG. This was true except for the LPG and PG since they showed a low reduction on these parameters. The main proteins used in the formulations played an important role in the hypoglycaemic effect. Further, another study has shown that soybean supplementation would be helpful to control BLG and serum lipid in patients with diabetes (35). The soybean fiber contains compounds with high viscosity like pectin, galactomannans, and arabinogalactans that delay gastric emptying and glucose absorption. Novels peptides from silkworm pupae were found by Yu et al. (36) with high α-glucosidase-inhibiting activity so that this protein can retard glucose absorption and suppressing postprandial hyperglycaemia.

The body weight changes after the 15-week feeding in the experiment are shown in Figure 4. Body weight gain rate was similar at the beginning of feeding in the HFD induced group (LPG, SPG, LCSSPG, SPPG, BG, and PG) and their body weight doubled after 8 weeks of HFD feeding. Increased thirst, frequent urination, and unexplained weight loss were the most common symptoms of patients with T2DM (37). The body weight decreased sharply (3.26 to 5.33 g per mouse) after T2DM models were successfully established in the 10th week in SPG, SPPG, LPG, CG, BG, and LCSSPG with no obvious differences among groups, which was consistent with the above mentioned. Body weight increased after 2 weeks of treatment and increased onward but slightly decreased (about 0.8–1.2 g per mice) after 5 weeks of treatment in all groups except CG and LG. Body weight loss was stopped after 2 weeks of treatment and increased onward until the end of treatment in LPG. The body weight loss in BG was continued at the end of the experiment which claimed the successful development of T2DM mice.

Body weight, on behalf of whole-body nutrient metabolism index with losses over 10% could lead to metabolism dysfunction (38). Thus, the degree of weight stability and restoration was taken as a key factor in reversing T2DM symptoms. The body weight declined to the minimum value after 2 weeks of treatment and then plateaued and increased. The body weight gain occurred after a 2-week interval, which was similar to the previous study by Qingbo et al. (25). Over 10% of body weight loss was observed among all groups in the 2-week treatment of minimum weight. Therefore, a suitable amount of ON intervention in time is necessary to inhibit the deterioration of body weight loss. An obvious difference in body weight loss among groups was observed according to statistical analysis. The body weight gain was more rapid in the group fed with ON tablets compared with BG and PG. In the current study, at least a 3-week consumption of ON formula could help to reverse T2DM induced body weight loss according.