TWI814022B - Preparation method of pig lung hydrolyzate and its use for treating or/and preventing metabolic-related diseases - Google Patents

Abstract

The present invention discloses a second use of a porcine lung hydrolyzate. Specifically, the porcine lung hydrolyzate obtained through the process disclosed in the present invention can effectively reduce fat accumulation, reduce fat area, reduce glycated hemoglobin, and reduce insulin resistance. , reduce glucose tolerance, and reduce fat accumulation in liver tissue. Therefore, when an effective dose of the porcine lung hydrolyzate disclosed in the present invention is administered to an individual, it can effectively prevent metabolic syndrome or symptoms caused by a high-oil and high-fat diet. diseases, it can also achieve the effect of treating or improving metabolic-related diseases.

Description

Preparation method of porcine lung hydrolyzate and its use in treating or/and preventing metabolic related diseases

The present invention relates to the reuse of pig slaughter waste, and in particular, to a preparation method of pig lung hydrolyzate and its use for treating or/and preventing metabolic-related diseases.

According to statistics, more than 8 million pigs are slaughtered in Taiwan each year, which means that 8 million sets of offal are produced. However, due to changes in Chinese people's eating habits, the number of people who like to eat pig offal has gradually decreased, resulting in most pig offal being turned into pork. Slaughter waste.

In addition to direct incineration, the current methods of processing pig offal are also processed as feed or compost. However, the economic value of the output is extremely low, which reduces the willingness of manufacturers to reuse pig offal waste, leaving pig offal waste. The amount of waste output continues to increase, and for slaughterhouses, the cost of waste treatment will increase, and the process of waste treatment will also have the possibility of polluting the environment.

Although it is currently proposed to process pig lungs through extraction methods to obtain heparin sodium and low molecular weight heparin, or to obtain collagen through enzyme hydrolysis of pig lungs, however, due to the high processing cost and difficulty of processing technology, it is difficult to be promoted and applied.

The main purpose of the present invention is to provide a method for preparing a porcine lung hydrolyzate, which can reduce the complexity and cost of the process, and can effectively improve the functionality and applicability of the porcine lung hydrolyzate, so as to increase the waste of pig viscera. The reuse rate and economic value of materials, and the effect of reducing environmental pollution.

Another object of the present invention is to provide a second use of a porcine lung hydrolyzate. The porcine lung hydrolyzate obtained through the preparation method disclosed in the present invention has physiological activities, including reducing fat accumulation, reducing fat cell size, and reducing glycation. Hemoglobin, reduce insulin resistance, reduce glucose tolerance, reduce fat accumulation in liver tissue, so as to prevent or treat metabolic-related diseases.

Therefore, in order to achieve the above object, the present invention discloses a preparation method of porcine lung hydrolyzate, which is carried out through a two-stage process of first extraction and then hydrolysis. Specifically, the preparation method of the porcine lung hydrolyzate disclosed in the present invention includes the following steps:

Step a: Take a pig lung and extract it with acetic acid to obtain an extraction product, wherein the extraction product contains collagen;

Step b: hydrolyze the extraction product with a protein hydrolyzing enzyme to obtain the porcine lung hydrolyzate.

In one embodiment of the invention, the proteolytic enzyme is flavor protease.

The porcine lung hydrolyzate obtained through the above preparation method can reduce fat accumulation, reduce fat area, reduce glycated hemoglobin, reduce insulin resistance, reduce glucose tolerance, and reduce liver tissue fat accumulation. Therefore, through the administration of an effective amount The porcine lung hydrolyzate disclosed in the present invention or a component containing the same can effectively prevent metabolic syndrome or diseases caused by high-oil and high-fat diets, and can also treat or improve metabolic-related diseases.

In embodiments of the present invention, the porcine lung hydrolyzate obtained above can be used to prepare a composition, and the composition can be used to treat diseases caused by obesity, diabetes, and visceral fat accumulation, or to prevent metabolic-related diseases. .

In one embodiment of the present invention, the diseases caused by visceral fat accumulation are fatty liver, abdominal obesity-related diseases, cardiovascular diseases, etc.

In another embodiment of the present invention, the metabolism-related disease is caused by hyperlipidemia and/or hyperglycemia.

The present invention discloses a second use of a porcine lung hydrolyzate. Specifically, the porcine lung hydrolyzate obtained through the process disclosed in the present invention can effectively reduce fat accumulation, reduce fat area, reduce glycated hemoglobin, and reduce insulin resistance. , reduce glucose tolerance, and reduce fat accumulation in liver tissue. Therefore, when an effective dose of the porcine lung hydrolyzate disclosed in the present invention is administered to an individual, it can effectively prevent metabolic syndrome or symptoms caused by a high-oil and high-fat diet. diseases, it can also achieve the effect of treating or improving metabolic-related diseases.

Furthermore, the pig lung hydrolyzate disclosed in the present invention is prepared through a two-stage process, which means that the pig lungs are first extracted with acetic acid or a solution containing acetic acid, such as 50% edible vinegar, and then The extracted product is hydrolyzed with enzymes, and the hydrolytic enzyme used is a proteolytic enzyme, such as flavor protease or other commonly used or well-known ones in the technical field to which the present invention belongs.

The "metabolic syndrome" referred to in this invention refers to abnormalities in blood pressure, blood lipids, blood sugar, waist circumference and other indicators, which will increase an individual's risk of suffering from diseases such as diabetes, heart disease, hypertension, stroke, obesity, etc., and if it can Improving metabolic syndrome, such as lowering blood sugar, slowing down insulin resistance, and lowering blood lipids, can reduce an individual's risk of suffering from the above diseases, which means to achieve the effect of preventing individuals from suffering from the above diseases.

The "metabolism-related diseases" referred to in the present invention refer to diseases caused by metabolic syndrome, such as diabetes, heart disease, hypertension, stroke, obesity or complications of the above diseases.

In the following examples, the present invention uses mice as test animals, but it is not limited to the fact that the porcine lung hydrolyzate disclosed in the present invention can only be used in mice, and the dosage that is intended to be used in other animals can be determined according to the technical field to which the present invention belongs. To convert based on common knowledge, such as referring to the formula provided on the US FDA website or the table provided by the Taiwan International Life Sciences Association, for example, the 60 kg adult dose is 1, and when converted to mice, it must be multiplied by 9.01 times, and converted to Rats must be multiplied by 6.25 times.

The data in the following examples were analyzed using the One-Way ANOVA method and post hoc testing was performed using Duncan's multiple-range test.

Example 1: Preparation of porcine lung hydrolyzate

Take pig lungs, first extract their collagen with 50% acetic acid, then hydrolyze the extracted collagen with flavorzyme enzyme, heat it to 90ºC for about 5 minutes to inactivate the enzyme, and then freeze it After drying, prepare porcine lung hydrolyzate powder.

The total protein content of pig lung hydrolyzate powder was analyzed and quantified. The instructions for total protein content analysis are as follows: first measure the dry weight of the test sample, then take different volumes of hydrolyzate and concentrate it to 50 ml (including 2 grams of dry weight). After performing the centrifugation procedure (10000xg, 15 minutes), take 1 mL of the supernatant (dry matter weight 0.04g) for filtering, and use the LC column for analysis, which means pouring 500 mL DDW (deuterium depleted water) into the column Sephadex In LH-20 (Amersham Pharmasia Biotech, Tokyo, Japan), the flow rate was 1 mL/min to replace 20% ethanol in the column, and then 30% methanol was pumped into the column. Pour the above supernatant into the column at a flow rate of 0.5 mL/min or 0.8 mL/min, elute with 30% methanol at a flow rate of 0.5 ml/min, and measure using an online spectrophotometer with a wavelength of 280 nm. The results are shown in Figure 1 Show.

It can be seen from the results in Figure 1 that the porcine lung hydrolyzate disclosed in the present invention was analyzed with an LC column, and its main peaks were at 117, 249, 284, and 337 minutes.

Example 2: Animal testing

Take a plurality of C57BL/6J male mice, randomly group them, and establish a high-fat feed-induced metabolic disease mouse model, in which:

Group 1 consisted of normal mice and were given normal feed;

The second group is a high-fat feed-induced metabolic disease model mouse. Each mouse was fed a high-fat feed with 45% energy source being fat until ten weeks old. The pig lung hydrolyzate disclosed in the present invention was not administered. ;

The third group is a porcine lung hydrolyzate low-dose group. Each mouse is fed with 45% of high-fat feed containing fat as its energy source until ten weeks old. The pigs disclosed in the present invention are administered via tube feeding every day. Lung hydrolyzate, dose 50 mg/kg;

The 4th group is a medium-dose group of porcine lung hydrolyzate. Each mouse is fed with 45% high-fat feed containing fat as its energy source until ten weeks old, and then the pigs disclosed in the present invention are administered via tube feeding every day. Lung hydrolyzate, dose 100 mg/kg;

The fifth group is a high-dose porcine lung hydrolyzate group. Each mouse is fed with 45% high-fat feed containing fat as its energy source until it reaches ten weeks of age. The pigs disclosed in the present invention are administered via tube feeding every day. Lung hydrolyzate, dose 200 mg/kg;

Group 6 is the positive control group. Each mouse was fed with high-fat feed containing 45% of its energy source as fat until ten weeks old, and then administered commercially available green tea extract (Hanxin Technology) via tube feeding every day. , Taiwan), the dose is 61.5 mg/kg.

The above-mentioned groups of mice were raised under their own conditions for 16 weeks, and the weight of the mice in each group was measured at each week. The results are shown in Table 1. The test was terminated when the mice in each group were 26 weeks old, sacrificed, and blood and tissue samples were collected. , for subsequent analysis.

From the results in Table 1 below, it can be seen that the administration of high-fat and high-sugar diet can indeed cause the weight of the mice in the second group to increase significantly compared with the mice in the first group. That is to say, the administration of high-fat and high-sugar diet can indeed build metabolism. disease mouse model; compared with the weight of mice in the second group, administration of the porcine lung hydrolyzate disclosed in the present invention can reduce the weight gain of mice with metabolic diseases, thereby effectively preventing or improving obesity. Or the effect on metabolism-related diseases, and as the dosage increases, the effect of reducing body weight also increases, indicating that the porcine lung hydrolyzate disclosed in the present invention can slow down weight gain, prevent or improve metabolism-related diseases at high doses. The effect of disease is better.

Table 1: Body weight measurement results and changes of mice in each group during the test period Weight(g) Test weeks Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Week 0 22.1±1.21 22.0±0.67 22.6±0.80 21.8±0.53 22.4±1.28 21.9±1.08 Week 1 21.8±1.16 22.2±0.69 22.6±1.22 21.9±1.67 22.5±1.64 21.8±1.33 Week 2 24.4±1.08 27.3±0.96 28.4±0.65 28.0±0.87 28.3±1.57 25.1±1.38 Week 3 24.3±0.83 28.9±1.17 29.6±1.16 28.8±1.29 29.0±1.40 26.6±1.02 Week 4 24.9±1.81 30.6±1.54 31.7±1.24 30.7±1.72 31.1±1.63 27.9±1.38 Week 5 24.7±1.35 31.4±1.61 32.5±3.18 31.7±2.05 32.8±2.22 28.7±1.64 Week 6 25.5±1.61 33.2±2.30 35.8±2.21 32.8±3.46 35.6±2.64 30.1±2.14 Week 7 25.7±1.98 35.5±2.38 36.9±2.28 34.4±3.25 36.7±2.83 30,6±2.09 Week 8 26.2±2.11 38.0±2.57 38.7±2.51 36.4±3.19 38.2±3.23 32.5±1.98 Week 9 26.7±2.06 39.8±2.65 40.4±2.50 37.4±3.52 39.2±3.33 33.5±1.84 Week 10 27.2±1,87 41.6±2.61 41.5±2.62 38.5±3.55 40.3±3.81 34.8±1.97 Week 11 28.2±2.20 43.6±2.58 42.5±2.83 39.1±3.73 41.1±3.67 35.6±1.84 Week 12 28.6±2.74 44..8±2.42 43.5±2.67 39.9±3.53 42.4±3.72 36.3±1.54 Week 13 29.1±2.85 45.9±2.04 44.4±2.57 41.5±3.31 43.0±3.40 37.8±1.59 Week 14 30.5±2.54 46.7±1.89 45.3±2.35 42.1±3.21 43.7±2.90 38.7±2.05 Week 15 31.0±2.71 48.0±1.63 46.0±1.64 42.8±2.83 43.9±2.40 40.4±1.72 Week 16 30.4±3.07 48.6±1.32 46.4±1.76 43.4±2.99 43.8±2.32 40.4±2.14 weight gain 8.3±3.28 26.6±1.37 23.8±2.27 21.6±2.75 21.4±1.73 18.5±2.07

Example 3: Glucose tolerance test

For the oral glucose tolerance test, mice in each group were first fasted for 16 hours, and then oral glucose (2.0 g/kg) was administered to the mice for the test. About 0.5 μL of blood was collected from the tail vein of mice at 120 minutes to obtain the blood glucose at each blood collection point in the fasting and glucose tolerance tests. The blood glucose was detected with a commercially available blood glucose machine (I-SENS, Inc, Korea) to obtain the blood glucose levels in the fasting and glucose tolerance tests. The blood glucose values at each blood collection point are shown in Table 2 below.

From the results in Table 2, it can be seen that in the data measured by the mice in the second group, the area under the curve is significantly higher than that of the mice in the first group, indicating that the mice in the second group have insulin resistance; and compared with the mice in the second group In terms of the area under the curve of mice, the mice in the 3rd to 5th groups who were administered the porcine lung hydrolyzate disclosed by the present invention all decreased significantly, and as the dosage increased, the decreasing effect became better, indicating that the administration of this invention The pig lung hydrolyzate disclosed in the invention can effectively slow down insulin resistance, and has a better slowing effect at high doses. In other words, the pig lung hydrolyzate disclosed in the invention has the effect of preventing or improving obesity, metabolic syndrome or diabetes. .

Table 2: Results of oral glucose tolerance test of mice in each group Blood sugar (mg/dL) Blood collection point Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 0 minutes 149.2±41.8 217.1±37.2 174.9±18.7 152.3±13.8 156.5±21.4 157.6±23.5 30 minutes 267.1±41.0 535.5±53.6 354.8±84.4 317.4±63.5 316.4±52.2 289.0±41.8 60 minutes 188.2±22.1 415.1±90.2 274.0±42.4 239.9±30.9 242.6±34.7 238.2±34.7 90 minutes 162.8±18.0 369.5±59.0 261.7±44.7 208.6±31.8 225.1±24.9 221.7±42.9 120 minutes 157.9±18.8 341.4±66.2 245.8±49.4 196.8±30.5 204.5±12.1 204.3±28.4 Area under the curve (AUG, x 10 ³ ) 23.1±2.3 48.0±6.2 33.0±3.9 28.2±2.7 28.9±2.7 27.9±1.9

Example 3: Serum biochemical value detection

Mice in each group were sacrificed after the experiment, and whole blood was collected respectively. Serum was collected after centrifugation. The serum triglycerides (sTG), serum total cholesterol (sTC) and glycated hemoglobin (HbA1c) of the mice in each group were analyzed. , the results are shown in Table 3 below.

From the results in Table 3, it can be seen that the serum triglycerides, serum total cholesterol and glycated hemoglobin of the mice in the second group are significantly higher than those of the mice in the first group, indicating that the blood lipids and blood sugar of the mice in the second group are increased for a long time. phenomenon, there is a risk of suffering from metabolic diseases, such as cardiovascular disease and diabetes; and compared with the mice in the 2nd group, the serum triglycerides, serum total cholesterol and Glycated hemoglobin was significantly lower than that of the mice in the second group, indicating that administration of the pig lung hydrolyzate disclosed in the present invention can effectively slow down the increase in blood lipids and blood sugar levels in obese individuals, and with the administration of the pig lung hydrolyzate disclosed in the present invention The higher the dose of lung hydrolyzate, the better the effect of slowing down the increase in blood lipids and blood sugar in obese individuals.

It can be seen from this that the porcine lung hydrolyzate disclosed in the present invention can effectively slow down long-term blood sugar content and blood lipid concentration, and can achieve the effect of improving or preventing metabolic related diseases, such as cardiovascular disease, hypertension, heart disease, diabetes or the above. Complications of disease.

Table 3: Serum biochemical values of mice in each group mg/dL Project Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 Serum triglycerides 56.5±2.67 87.9±17.0 74.0±39.6 61.0±34.0 57.0±22.9 77.5±37.2 serum total cholesterol 130.5±24.0 175.0±11.1 138.0±29.1 119.5±26.0 122.0±10.1 134.0±22.1 Glycated hemoglobin 6.4±6.4 9.7±9.4 7.9±7.9 7.7±7.7 7.3±7.3 6.8±6.8

Example 4: Detection of liver biochemical indicators

The mice in each group were sacrificed after the end of the experiment, and the liver tissues were taken respectively. After weighing, about 0.1 g of liver tissue was taken, and homogenized solution (3 mL 1.15% KCl, 2 mL Tris-EDTA pH 8.9, 1.5 mL acetic acid) was used. After homogenizing the liver tissue, centrifuge it for 10 minutes (4500 × g), take the supernatant, and then detect and analyze the triglyceride (hTG) and total cholesterol (hTC) of each mouse liver tissue. The results are shown in Table 4 below.

From the results in Table 4, it can be seen that the triglyceride and total cholesterol contents in the liver tissue of the mice in the 2nd group were significantly higher than those in the 1st group of mice, indicating that the liver tissue of the mice in the 2nd group accumulated a large amount of fat and may suffer from metabolic diseases. The risk of diseases, such as fatty liver; and compared with the mice in the second group, the triglycerides and total cholesterol in the liver tissue of the mice in the third to fifth groups were significantly lower than those in the second group. It is shown that administration of the porcine lung hydrolyzate disclosed in the present invention can effectively slow down the accumulation of fat in liver tissue, and as the dosage of the porcine lung hydrolyzate disclosed in the present invention increases, the effect of reducing fat accumulation in liver tissue increases. The better.

It can be seen from the results of this example that under a high-fat and high-oil diet, the porcine lung hydrolyzate disclosed in the present invention can effectively prevent or improve fat accumulation in liver tissue, thereby achieving the effect of improving or preventing metabolism-related diseases, such as fatty liver, Cirrhosis or complications of the above diseases.

Table 4: Liver biochemical indicators of mice in each group mg/dL Project Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 liver triglycerides 25.3±6.5 39.8±7.6 28.3±8.9 24.8±7.9 24.6±3.2 21.1±7.7 total liver cholesterol 0.9±0.1 1.1±0.3 0.8±0.1 0.7±0.1 0.7±0.1 0.7±0.1 Liver weight (g) 1.0±0.1 1.7±0.2 1.4±0.2 1.2±0.2 1.2±0.2 1.0±0.1

Example 5: Detection of adipose tissue

After the mice in each group were sacrificed, abdominal fat, perirenal fat, and mesenteric fat were collected and weighed. The results are shown in Table 5 below. Furthermore, the abdominal fat, perirenal fat and mesenteric fat of mice in each group were paraffin-embedded and sectioned, stained with hematoxylin and eosin, observed using an optical microscope, photographed and recorded, and labeled with adipocytes using Cytation 5. The area of each cell was analyzed. After observing three fields of view, the average size of 50 cells was selected for further statistical analysis. The results are shown in Figures 2 and 3.

From the results in Table 5, Figure 2 and Figure 3, it can be seen that the fat weight and area of various parts of the mice in the 2nd group are higher than those in the 1st group of mice, indicating that the mice in the 2nd group are indeed obese model mice, which means Long-term feeding of high-fat and high-oil feed will indeed cause the mice in group 2 to accumulate visceral fat. Compared with the mice in group 2, the weight and area of fat in each part of the mice in groups 3 to 5 are respectively smaller. The mice in the second group showed a significant decrease, indicating that administration of the porcine lung hydrolyzate system disclosed in the present invention can effectively reduce visceral fat accumulation, and the effect of reducing visceral fat is better than that of drugs.

It can be seen from the above that under a high-fat and high-oil diet, the pig lung hydrolyzate disclosed in the present invention can effectively prevent or improve the accumulation of visceral fat. Therefore, the pig lung hydrolyzate disclosed in the present invention can improve or prevent metabolism-related diseases. effect.

Table 5: Fat weight of different parts of mice in each group Weight(g) Project Group 1 Group 2 Group 3 Group 4 Group 5 Group 6 belly fat 1.16±0.36 2.60±0.37 2.52±0.38 2.01±0.36 1.91±0.43 2.11±0.38 fat around kidneys 0.46±0.19 1.94±0.40 1.46±0.20 1.45±0.21 1.38±0.17 1.27±0.12 mesenteric fat 0.34±0.12 1.77±0.24 1.27±0.31 1.10±0.30 1.13±0.27 0.86±0.19 total fat 1.96±0.65 6.20±1.00 4.65±0.72 4.55±0.87 4.42±0.86 4.65±0.56

without

Figure 1 shows the results of LC column analysis of the porcine lung hydrolyzate disclosed in the present invention, where the flow rate is 0.8 mL/min. Figure 2 shows the results of hematoxylin and eosin staining of adipose tissue sections in different parts of mice in each group. Figure 3 shows the results of scanning the adipose tissue sections of different parts of mice in each group and using analysis software to calculate the fat area of each adipose tissue.

without

Claims (6)

A method of using a pig lung hydrolyzate to prepare a composition for treating obesity, wherein the pig lung hydrolyzate is prepared through the following steps: Step a: Take a pig lung and extract it with acetic acid to obtain a Extraction product, wherein the extraction product contains collagen; step b: hydrolyze the extraction product with a flavor protease, and heat to 90°C for about 5 minutes to obtain the porcine lung hydrolyzate. A method of using a pig lung hydrolyzate to prepare a composition for treating diabetes, wherein the pig lung hydrolyzate is prepared by the following steps: Step a: Take a pig lung and extract it with acetic acid to obtain a Extraction product, wherein the extraction product contains collagen; step b: hydrolyze the extraction product with a flavor protease, and heat to 90°C for about 5 minutes to obtain the porcine lung hydrolyzate. A method of using a pig lung hydrolyzate to prepare a composition for treating diseases caused by visceral fat accumulation, wherein the pig lung hydrolyzate is prepared by the following steps: Step a: Take a pig lung, add an acetic acid Extraction is performed to obtain an extraction product, wherein the extraction product contains collagen; step b: hydrolyze the extraction product with a flavor protease, and heat to 90°C for about 5 minutes to obtain the porcine lung hydrolyzate. The use as described in claim 3, wherein the disease caused by visceral fat accumulation is fatty liver. A method of using porcine lung hydrolyzate to prepare a composition for preventing diseases caused by hyperglycemia, wherein the porcine lung hydrolyzate is prepared by the following steps: Step a: Take a pig lung and extract it with acetic acid to obtain an extraction product, wherein the extraction product contains collagen; Step b: Hydrolyze the extraction product with a flavor protease and heat it to 90°C for about 5 minutes , to obtain the porcine lung hydrolyzate. A method of using a pig lung hydrolyzate to prepare a composition for preventing diseases caused by hyperlipidemia, wherein the pig lung hydrolyzate is prepared by the following steps: Step a: Take a pig lung and extract it with acetic acid , obtaining an extraction product, wherein the extraction product contains collagen; step b: hydrolyze the extraction product with a flavor protease, and heat to 90°C for about 5 minutes to obtain the pig lung hydrolyzate.

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