Different Effects of Lard and Vegetable Blend Oil on Intestinal Microorganisms, Enzyme Activity and Blood Routine in Mice

the Abstract: The intake of moderate oils and fats is necessary to maintain the body’s energy balance, and the fatty acid composition of different oils and fats varies in their nutrition and function. The study aimed to investigate the effects of lard and vegetable blend oil on gut microbiota, intestinal enzyme activities, and blood routine. Kunming mice were assigned to the three groups: (1) Control group (CK) was gavage administration with distilled water, (2) Plant oil group (ZWY) was gavage administration with edible vegetable blend oil, (3) Lard group (DWY) was gavage administration with lard. After 42 days, microbiological, digestive enzymes, and blood routine were performed. Compared with the CK group, Escherichia coli , Lactobacilli and Bifidobacteria were significantly decreased ( p < 0.05), the activities of protease, cellulase, amylase, and xylanase were markedly reduced ( p < 0.05), the hemoglobin was significantly increased ( p < 0.05) in the ZWY group and DWY groups, and the hematocrit was increased in the ZWY group ( p < 0.05), while other routine blood indices were increased ( p > 0.05). Compared to the ZWY group, the activity of cellulase and amylase were significantly increased ( p < 0.05), the intestinal microorganism and the routine blood indexes had no significant difference in the DWY group. Lard and vegetable blend oil diet affected the composition of the intestinal microorganisms, and the functions of digestive enzymes. Meanwhile, the levels of digestive enzymes may be correlated with the intestinal microbiota.

The gut microbiota that inhabits the gastrointestinal tract is a complex microbial community, and plays an important role in maintaining human health and micro-ecological balance 9 . The specificity of gut microbiota in response to the internal and external environment of the host, particularly the effect of dietary pattern on the structure of intestinal microorganisms 10 . Researches 11,12 indicated that the composition and content of edible oils strongly affect intestinal microorganisms composition and function. HFD caused intestinal microorganisms dysbiosis, which in turn led to a spectrum of diseases. In healthy people, the majority of fatty acids consumed in the diet are accessible to the bloodstream through the efficient process of digestion and absorption 13 . Therefore, blood routine could be applied on early diagnosis of disease and the nutritional evaluation of diet.
In a preliminary experiment 14 , we confirmed that proper intake of vegetable blend oil contributed to promoting the growth of prebiotics and increasing enzyme activities. Therefore, this research aimed to investigate the different effects of lard mainly SFA or vegetable blend oil mainly UFA on blood routine, gut enzyme activities, and intestinal microorganisms through the in vivo animal experiment. To explore the correlation between different oils and health, as well as to provide the scientific basis for a food guide.

Animals
Eighteen specific pathogen-free SPF Kunming mice half male and half female, 20 2 g were obtained from Slakes experimental Co., Ltd license number: SCXK Xiang 2019-0009 . The mice were raised under stable conditions temperature 23-25 , relative humidity 50-70 in the laboratory animal center of Hunan University of Chinese Medicine. The process of animal experiments was conducted under animal protocols approved by the Animal Ethics and Welfare Committee of the Hunan University of Chinese Medicine.

Diets
The compositions of diet are corn starch 30 , soybean meal 29 , wheat 26 , salt 1 , bone meal 1 , lysine 1 , and water 12 , which provided by the laboratory animal center of the Hunan University of Chinese Medicine. Lard and vegetable blend oil brand: Arowana Golden Ratio Edible Blended Oil, manufacturer: Yihai Jiali Yueyang grain and oil industry Co., Ltd, license number: SC10243060200170 were purchased in Wal-Mart, Changsha, Hunan. Vegetable blend oil is composed of soybean oil 49.0 , corn oil 9.0 , rapeseed oil 23.5 , sunflower seed oil 14.0 , peanut oil 0.5 , sesame oil 0.6 , flaxseed oil 0.4 , and rice oil 3.0 . In our experiment, the ratio of SFAs and UFAs for vegetable blend oil is 1:6.2, and lard is 1:0.95.

Animal groups
After the adaptation period, Kunming mice were randomly divided into three groups n 6 per group : 1 Control group CK was treated with distilled water, 2 Plant oil group ZWY was treated with edible vegetable blend oil, 3 lard group DWY was treated with lard. Dose gavage was 0.2 mL twice a day for 42 consecutive days. As shown in Fig. 1, experimental design and general conditions of the animals.

Extraction of intestinal contents
After 42 days, the intestinal contents jejunum to the rectum of all the mice in each group were collected in a sterile environment and stored at 4 .

Determination of microorganisms in intestinal con-
tents Bacteria: beef extract-peptone agar medium; E. coli: eosin-methylene blue agar medium; Lactobacillus spp.: deMan Rogosa Sharpe agar medium; Bifidobacteria spp.: Bifidobacteria agar medium. The number of gut microorganisms was measured by the plate count method. Under sterile conditions, a certain amount of intestinal content was weighed into sterile water bottles containing glass beads. After shaking at 120 rpm for 30 min, tenfold dilutions were made and cultures were inoculated by the smearing. Total numbers of bacteria and E. coli were detected after being cultured at 37 for 24 h, the numbers of Lactobacillus and Bifidobacteria were determined after being anaerobically cultured at 37 for 48 h. Each dilution was repeated three times, averaged and the number of colonies per gram of gut contents was calculated.

Analysis of enzyme activity in intestinal contents
Under sterile conditions, a certain amount of the intestinal contents were taken hereafter, placed in triangular bottles containing sterile water, and heated in a water bath at 40 for 30 min to completely release the enzymes from the intestinal contents. Then the contents were centrifuged at 3000 rpm for 10 min and the enzyme activities in the supernatant were analyzed by UV spectrophotometer. The activities of amylase, xylanase, and cellulase were determined by the DNS colorimeter method and the protease activity was determined by the Feline-phenol method 15 .

Routine blood examination
Blood samples 1.5-2.0 mL were taken from mice. The blood was injected into EDTA-K2 anticoagulant tubes and mixed evenly. Blood samples were sent to the laboratory of the First Affiliated Hospital of Hunan University of Chinese Medicine for testing within 2 h.

Statistical analysis
Data were expressed as mean standard deviation mean SD . Statistical analysis was performed by one-way ANOVA followed by Tukey s test using SPSS v23.0 IBM Corp., Armonk, NY, USA . Differences between groups were considered statistically significant at p 0.05.

Observation of mice performance
All animals had similar body weights when the experiments started. After 42 days of the experiment, the weight of all groups had no significant difference Fig. 2 . There were no deaths and abnormalities throughout the experiment.

Effects of different edible oil intake on the intestinal microorganisms of mice
The intestinal microorganisms play an important role in human energy regulation, nutrient absorption, and immunity 16 . However, the intestinal microorganisms composition and relative abundance usually change in response to the external environment, particularly the dietary pattern 17 . From Fig. 3, it appears that the intestinal microorganisms of mice were altered to various degrees after intake of plant oil and lard. Compared with the CK group, Escherichia coli, Lactobacilli, and Bifidobacteria were significantly decreased in the ZWY group and DWY group p 0.05 . Compared to the ZWY group, the number of Lactobacilli and Bifidobacteria were decreased in the lard group p 0.05 . The above results suggested that lard and plant oil intake had a large effect on the intestinal microorganisms of mice. Among them, the effect of lard intake on intestinal microorganisms is more significant, which inhibits the growth of probiotics such as Lactobacillus and Bifidobacterium, induced intestinal microorganisms dysbiosis.

Effects of different edible oil intake on the intestinal
enzymatic activity of mice Most nutritional components are too complex for immediate use and must be broken down into simpler compounds, which can then be absorbed by the body 18 . This  digestive process is catalyzed by enzymes that are either endogenous or produced by the host s microbial population. As shown in Fig. 4, the activities of amylase, protease, xylanase, and cellulase in the ZWY group and the DWY group were significantly lower than in the CK group p 0.05 . The activities of amylase and cellulase in the DWY group were significantly higher than the ZWY group p 0.05 , and there was no significant difference in the activities of protease and xylanase between these two groups p 0.05 . Intestinal enzyme activity affects the body s absorption of nutrients and intestinal vitality. Edible oil intake would reduce the activities of those four intestinal enzymes, and plant oil was more serious than lard in terms of reduced amylase and cellulase activities.

Effects of different edible oil intake on the blood rou-
tine of mice Blood routine examination contains three systems including white blood cells, red blood cells, and platelets, and is one of the basic clinical examination items. White blood cells were increased, suggesting inflammation 19,20 . In Fig.  5, the white blood cell count in the ZWY group was a little higher than the CK group and DWY group, but there was no significant difference between the three groups p 0.05 .
Platelets are key in promoting intravascular thrombus formation in infection, a process termed immunothrombo-sis , which contributes to containing pathogens, but also potentially damages the host 21,22 . We can know from Table  1, plant oil and lard intake could increase the platelet count, mean platelet volume, and platelet distribution width p 0.05 , and the DWY group was higher than the ZWY group p 0.05 . It was suggested that lard could promote platelet activity.
The main function of red blood cells is to transport Fig. 4 Effect of edible oil intake on intestinal enzyme activities. Data were expressed as mean SD . Statistical analysis was used by one-way ANOVA followed by Tukey s test *p 0.05 . Fig. 5 Effect of edible oil intake on white blood cells. Data were expressed as mean SD . Statistical analysis was used by one-way ANOVA followed by Tukey s test.
oxygen, and a decrease in red blood cells, hemoglobin, and hematocrit is suggestive of anemia, and an increase is suggestive of polycythemia 23 . The red blood cell hemoglobin content and mean corpuscular hemoglobin concentration had no significant difference within these three groups p 0.05 . Table 2 showed that compared with the CK group, the ZWY group was significantly higher in hematocrit p 0.05 . Meanwhile, the ZWY group and the DWY group in red blood cell count, hematocrit, mean corpuscular volume, and the red cell distribution width were higher than the CK group p 0.05 . Compared with the ZWY group, the DWY group had a lower red blood cell count, hematocrit, mean corpuscular volume, and higher red blood cell distribution width, but there was no significant difference p 0.05 . The result was illustrated in Table 3, compared with the CK group, plant oil and lard intake could increase the hemoglobin content of the mice p 0.05 , but there was no significant difference in the hemoglobin content between the ZWY group and the DWY group p 0.05 .

Correlation between intestinal enzyme activities and
intestinal microorganisms Pearson correlation analysis was performed. Intestinal enzyme activity is closely related to intestinal microorganisms, so the correlation between the two was analyzed. As can be seen in Fig. 6, the total bacteria count was significantly correlated with amylase, cellulase, and xylanase; Escherichia coli and Bifidobacteria showed significant positive correlations with amylase, protease, cellulase, xylanase; Lactobacilli showed a significant positive correlation with amylase, protease, xylanase. These suggested that intake of lard and vegetable blend oil could affect the function of intestinal digestive enzymes. Data were expressed as (mean±SD). Statistical analysis was used by one-way ANOVA followed by Tukey' s test. PLT, platelet; PCT, platelet crit; MPV, mean platelet volume; PDW, platelet distribution width.  Data were expressed as (mean±SD). Statistical analysis was used by oneway ANOVA followed by Tukey' s test. Different letters indicated significant differences at p＜0.05 among groups. HGB, Hemoglobin; MCH, Mean red blood cell hemoglobin content; MCHC, mean corpuscular hemoglobin concentration.

Discussion
Dietary oils and fats are the important macronutrients for humans and in terms of consumption, they are one of the three main classes of food, besides carbohydrates and proteins. They are necessary for energy supplying and maintaining the normal functions of the human body. HF diet easily induces lipid metabolism disorders and oxidative stress injury, which are risk factors for CVDs and metabolic syndrome 24,25 . Besides, the long-term HFD intake causes internal environment change of the body, and part of the flora is inhibited, thereby causing dysbacteriosis 26 . The normal dynamic balance of the intestinal flora is broken, causing the clinical symptom. The type and quality of fatty acids play an important role in human health, and the contents of SFA, MUFA, and PUFA in different oils are not the same 27,28 Fig. 7 . The content of SFA is higher in lard than in plant oil, so lard diet-induced accumulation of body fat, liver and serum lipids, which can increase the risk of obesity, non-alcoholic fatty acid liver disease, and atherosclerosis. The mixed oil diet-induced body fat accumulation, but did not cause lipid accumulation in the liver and serum 29 . In this study, mixed oil SFA: UFA was 1:6.3, lard was 1:0.95. Aimed to investigate the effects of edible oils of different compositions on blood routine, intestinal enzyme activities, and intestinal microorganisms in mice.
The intestinal microorganisms can be altered by the in-  ternal and external environments, and the intestinal microorganisms are associated with pathology, physiology, and various diseases. HFD induces inflammation by increasing endotoxin levels in the intestinal lumen as well as in the plasma by altering the composition of the intestinal microorganisms and increasing its intestinal permeability 30 . Excessive fat intake by the organism, which leads to overload of the mitochondrial electron transport chain, easily causes oxidative stress in the intestinal tissue and changes the microenvironment in the intestine, resulting in a disturbance of the intestinal microorganism 31,32 . Bifidobacterales and Lactobacillales are probiotics of the intestinal tract, which inhibit the growth of pathogenic microorganisms and protect the intestinal mucosa by producing bacteriocins or organic acids. Conversely, reduction of probiotics impairs intestinal homeostasis and the intestinal mucosal barrier, promotes endotoxin production and growth, and leads to intestinal inflammation. In terms of Bifidobacterales, gram-positive bacteria are associated with inflammation development. SFA and high in unsaturated fats diets have negative correlations with the abundance of Bifidobacterales 33 . The abundance of Lactobacillales decreased after intake of HFD 34,35 . A previous study 36 revealed that Lactobacillales have anti-obesity activities, and HFD has harmful effects on Lactobacillales. Our results suggested that Escherichia coli, Lactobacillales, and Bifidobacterales were significantly decreased in the ZWY group and the DWY group. As opposed to passer-by bacteria, most of the gut flora are long-term host microorganisms. The cycle of this experiment was only 42 days, while the impact of edible oil may be long-term chronic, which explains the small differences in intestinal microorganisms diversity and abundance after ingestion of lard and vegetable blended oils.
Enzymes control all metabolic processes in the human system from simple digestion of food to highly complex immune response 37 . Physiological reactions occurring in healthy individuals are disturbed when enzymes are deficient or absent. Bacillus is the main gut microbes producing proteases and xylanase, as well as cellulase producing Bacteroides, Clostridium, prebacteroides. In addition, the gut can also produce digestive enzymes. In this study, we found that the plant oil or lard continued intake reduced the activities of proteases, xylanases, amylases, and cellulases. Correlation analysis by intestinal microorganisms and enzyme activity, we found that the main reason was a decrease in the number of enzyme-producing gut microbes. The reason may be due to the accumulation of acid metabolites produced by intestinal microorganisms associated with intestinal oil metabolism that lower the pH of the gut environment. Plant oils significantly reduced the activities of amylase and cellulase, the reason may be that plant oils contain more PUFA, and PUFA can be transformed into isomerized PUFA trans-fatty acids by Lacto-bacillus, Escherichia, Bacteroides. When the trans-fatty acids accumulate to a certain extent, they may exert opposite effects, inhibiting the growth of amylase and cellulaseproducing gut microbes 38,39 . And the higher amount of dietary carbohydrates in plant oils than that of lard.
A variety of fatty acids exists in the diet of humans, in the bloodstream of humans, and the cells and tissues of humans. It was obvious that within any fatty acid class, different members have different actions and effects. It was reported that a high intake of SFA increases the risks of coagulation, inflammation, and insulin resistance. However, moderate amounts of UFA are good for health and can replace common saturated fatty acids. The blood routine can be sensitive to pathological alterations and related conditions that occur in human function, which has a positive reference value for multiple disease tests. RBC parameters included RBC count, RBC cloth width, the mean volume of RBC, and hematocrit. Research 40 has indicated that elevated HCT levels may be positively associated with cardiovascular risk factors. Thus, the combination of HCT values and cardiovascular risk factors may enable the early diagnosis of cardiovascular diseases CVDs . The erythrocyte count, hematocrit, mean corpuscular volume, and red cell distribution width in the lard and plant oil groups were higher than those in the control group in this study. Hemoglobin is one of the markers for assessment of the occurrence, development, and prognosis of cardiovascular disease, and it can monitor the condition better in combination with other indicators. We found a significant increase in hemoglobin after ingestion of lard and plant oil, which was more pronounced with lard. The parameters of platelets consist of platelet count, platelet specific volume, mean platelet volume, and platelet volume distribution width, where platelet volume distribution width is a relatively specific indicator of platelet activation. Platelet activation is essential for thrombus formation 41 . Through this study, it was found that the platelet count, platelet mean volume, and platelet distribution width was slightly higher in the lard and plant oil groups than in the control group, and higher in the lard group than in the plant oil group. If an abnormally elevated white blood cell count is present, patients may present with conditions such as uremia, pyogenic infections, and leukemia, whereas patients with an abnormally decreased white blood cell count may present with associated conditions such as cirrhosis, anemia, and hyperthyroidism. The impact of edible oil may be long-term chronic. Research has found that a high-fat diet containing lard accelerated lipid and glucose metabolism for 4 weeks 42 . We found that the values of indicators such as hematocrit, hemoglobin, platelet distribution width, and white blood cell count had an elevated trend after the intake of lard and plant oil in mice. Whereas these indicators are closely associated with several CVDs, such as coronary heart disease, hypertension, and slow coronary reflow, suggesting that regardless of the type of fats and oil, a long-term high-fat diet will have a negative impact.

Conclusion
In summary, short-term fats and oil intake affected the composition of the gut microbiota, and the functions of digestive enzymes. Meanwhile, multiple indicators related to CVDs appear abnormal in blood routine, which indicates that long-term excessive intake of saturated fatty acids is detrimental to human health. This study initially explored the effects of different edible oils on intestinal microorganisms and health, and we will focus on the different effects of the length of fatty acid chains and the degree of fatty acid saturation on intestinal microorganisms. Therefore, reasonable and effective fats and oils intake may be a key factor in disease prevention.