Toxicological Evaluation Study Of Capsococcal Protein

The production of "gas protein" with natural gas as the substrate can be traced back to the 1960s.To meet the nutritional needs of animals and alleviate protein supply constraints,a variety of bacterial proteins have been developed as alternative protein ingredients for animal protein feeds.In recent years,the feed industry in our country has also begun to pay attention to the introduction and development of this type of bacterial protein feed.Capsococcal protein is an important single-cell protein,but it is produced by a bacterial fermentation process,the finished product contains various components from the culture medium and bacterial metabolites,in addition to the bacterial protein.Therefore,it is necessary to evaluate its safety before approval for marketing.In this study,a systematic toxicological safety evaluation of capsococcal protein was conducted to provide the necessary toxicological information for the approval and marketing of this bacterial protein as an alternative protein feed ingredient.This study not only contributes to the development and application of new animal alternative protein feed ingredients,but also has important significance for the sustainable development of animal husbandry and human and animal health.1.Acute toxicity study of capsococcal proteinForty Wistar rats(180-220 g)and ICR mice(18-22 g)were randomly assigned to treated and control groups,with equal numbers of males and females.The test substance were given orally by the maximum limited method and the control group was given an equal volume of solvent control.The animals were observed continuously for 14 days.During the observation period,clinical signs and death were recorded,and their body weight were respectively measured before administration and on day 7,day 14.On the 15th day,surviving rats and mice were euthanized for visual pathological examination to observe skin,mucosa,natural pores,and organ tissues,with gross results recorded.The results showed that no significant abnormalities were observed in all rats and mice after drug administration.The subjects showed no adverse effects on body weight gain,feeding,drinking,and defecation,and no toxic reactions were observed.Autopsy of all organ tissues and administration sites at the end of the trial showed no significant abnormalities.The results showed that the LD50 of this capsococcal protein was higher than 10 g/kg b.w.in rats and mice.According to the WTO acute toxicity classification standard of exogenous chemicals,capsococcal protein is actually non-toxic.2.Subchronic toxicity study of capsococcal proteinEighty Wistar rats(70-110 g),comprising equal numbers of males and females,were randomly divided into four groups of 20 rats each after adaptive feeding.According to the guidelines of the 90-day subchronic toxicity test and the LD50 of the test substance,the four groups received capsococcal protein by medicated feed at levels of 5%,2.5%,1%,and 0%,respectively,for three months.General clinical observations were conducted daily during the trial,and body weight,food intake,and water consumption were measured weekly.Ten rats in each group were randomly selected at the middle(45 d)and end(91 d)of the test for hematological and biochemical tests after blood collection.The main organs were removed to calculate organ coefficients and perform histopathological examination.Clinical observations showed no abnormal mortality and any adverse clinical symptoms related to the test substance.There were significant differences in weight gain between the treatment groups and the control group(p<0.05,p<0.01).Statistical analysis showed a positive correlation between the dose of the subjects and the body weight of the animals,which was associated with 65%protein content of the subject.The mean feed intake of rats in the medium and high dose groups and the mean water intake of male rats in the high dose group were highly significant compared with the control group(p<0.01).It is a routine physiological change for animals to increase their feed and water requirements due to weight gain.The hematological test results showed differences in individual parameters at the middle and end of administration compared with the control group(p<0.05,p<0.01).However,they all were within the normal reference values and were normal physiological changes.The blood biochemical test results of each dose group were not significantly different from the control group(p≥0.05).The organ weights and organ coefficients of individual rats were different compared to the control group(p<0.05,p<0.01).No treatment-related changes were found at the clinicopathological and histopathological examination.This difference was not related to the toxicity of the test substances.The test results showed that no significant harmful effects were observed in rats fed continuously for 90 d at a concentration of 5%in the diet of the high-dose group of subjects.The mean daily intakes of the subjects in the high dose group were 3281 mg/kg b.w.and 3138 mg/kg b.w.for male and female rats,respectively,discounted from feed intake and animal body weight.Therefore,the maximum no-effect dose of 5%capsococcal protein fed to rats for 90 d was at least 3138 mg/kg b.w.3.Genotoxicity study for capsococcal protein3.1 Salmonella typhimurium revert mutation testA group of qualified Salmonella typhimurium histidine-deficient strains(TA97,TA98,TA100,TA102,TA1535)were selected for the test.The exogenous metabolic activation system was used to induce rat liver microsomal enzyme(S9)with PCBs.The highest dose of test substance was determined to be 10 mg/plate according to the pre-test results and diluted 2-fold to 0.625 mg/plate.At the same time,a solvent control group(sterile water)and a positive control group were set,with three parallel in each group.A replicate were conducted to record the number of spontaneous revertant colonies in each group.The results showed that the mean number of revertant colonies per plate of the five tested strains treated with the test substance was within twice that of the solvent control.No dose-response relationship was observed,with or without the metabolic activation system(S9),indicating that capsococcal protein was not mutagenic to Salmonella typhimurium.3.2 Mouse bone marrow cell micronucleus testSixty clean-grade ICR mice(25-30 g)were randomly divided into five groups of 12 males and females each.Following the regulations of the mammalian erythrocyte micronucleus test and considering the LD50 of the test substance,the high,medium,and low dose groups were 10,000 mg/kg b.w.,5,000 mg/kg b.w.,and 2,500 mg/kg b.w.,respectively.Each group included a solvent control(CMC 20 mL/kg b.w.)and a positive control(CP 40 mg/kg b.w.).Oral administration of capsococcal protein was carried out twice at 30 h,and bone marrow samples were collected and filmed 6 h after the second dose to calculate the micronucleus rate.The results demonstrated that the ratio of polychromatic erythrocytes to mature erythrocytes(PCE/RBC)remained within the normal range in all groups.Furthermore,the rate(‰)of polychromatic erythrocytes micronucleated in the bone marrow of mice in all dose groups of capsococcal protein was not significantly different from that of the negative control group(P>0.05)but was significantly different from that of the positive control group(P<0.01).These findings suggest that capsococcal protein is not genotoxic to mouse bone marrow cells.3.3 Sperm malformation test in miceSixty male ICR mice(18-20 g)were randomly assigned to five groups of 12 mice each.The high,medium,and low dose groups were 10,000 mg/kg b.w.,5,000 mg/kg b.w.,and 2,500 mg/kg b.w.,respectively.In addition,a solvent control group(CMC 20 mL/kg b.w.)and a positive control group(CP 40 mg/kg b.w.)were included.The test substance was orally administered for five consecutive days,and the animals were killed by dislocation of the neck on day 35.Bilateral epididymal films were taken to count the number of malformations in 1,000 spermatozoa per animal and to calculate the malformation rate.The results revealed that the rate of malformed sperm in the negative control group was within the quality control range.Furthermore,the sperm malformation rate in each dose group of the test substance was not significantly different from the negative control group(P>0.05)and significantly different from the positive control group(P<0.01).Therefore,it can be concluded that capsococcal protein was not genotoxic to mouse germ cells.4.Passive skin allergy study for capsococcal protein240 Wistar rats were randomly divided into 8 groups of 30 rats each(half male and half female)The low,medium,and high dose groups received 250 mg/kg b.w.,500 mg/kg b.w.,and 10,000 mg/kg b.w.,respectively,based on the dose design principle of the passive allergy test.The positive control group received bovine serum albumin(15 mg/kg b.w.),while the negative control group received 0.9%saline(3.0 mL/kg b.w.).The antiserum prepared in advance in each dose group was diluted with 0.9%normal saline into three concentrations of 1:2,1:8 and 1:32.Passive sensitization was induced by intradermal injection of 0.1 mL of antiserum of each corresponding group in a pre-depilated area(3×4 cm2)on the back of the animals.After 24 and 48 h of passive sensitization,each group was stimulated by subcutaneous injection of the same stimulating antigen as the sensitizing dose,while 1.0%evans blue stain 1.0 mL was injected intravenously.The extent of the passive skin allergy test reaction was determined according to the extent of local skin blue staining.The results showed that the doses of capsococcal protein administered in the range of 250 to 1,000 mg/kg body weight was negative for the passive skin allergy test in rats.The results of the toxicological evaluation tests showed that the maximum no-effect dose of capsococcal protein fed to rats for 90 d was at least 3138 mg/kg b.w.,with no genotoxicity and no allergic reactions.It is safe as an alternative protein ingredient for animal protein feed.