British Journal of clinical pharmacology (2017) 83 152 – 162 152
Milk-derived bioactive peptides andtheirhealth promoting effects: a potential role in atherosclerosis
Correspondence Professor Desmond Fitzgerald, UCD Conway Institute, University College Dublin Belfifield, Dublin 4, Ireland. Tel.: +3531 716 6734; E-mail: des.fifitzgerald@ucd.ie
Received 17 December 2015; revised 15 April 2016; accepted 23 April 2016
Simone Marcone1,3, Orina Belton2 and Desmond J. Fitzgerald1
Correspondence Professor Desmond Fitzgerald, UCD Conway Institute, University College Dublin Belfifield, Dublin 4, Ireland. Tel.: +3531 716 6734; E-mail: des.fifitzgerald@ucd.ie
Received 17 December 2015; revised 15 April 2016; accepted 23 April 2016
Simone Marcone1,3, Orina Belton2 and Desmond J. Fitzgerald1
1School of Medicine and Medical Science, 2School of Biomolecular and Biomedical Science, UCD Conway Institute, University College Dublin, Dublin,and 3Food for Health Ieland, UCD Conway Institute, University College Dublin, Dublin, Ireland
Keywords atherosclerosis, bioactive peptides, cardiovascular disease, milk proteins, immunomodulation, inflflammation Bioactive peptides derived from milk proteins are food components that, in addition to their nutritional value, retain many biological properties and have therapeutic effects in several health disorders, including cardiovascular disease. Amongst these, atherosclerosis is the underlying cause of heart attack and strokes. It is a progressive dyslipidaemic and inflflammatory disease where accumulation of oxidized lipids and inflflammatory cells leads to the formation of an atherosclerotic plaque in the vessel wall. Milk-derived bioactive peptides can be released during gastrointestinal digestion, food processing or by enzymatic and bacterial fermentation and are considered to promote diverse benefificial effects such as lipid lowering, antihypertensive, immnomodulating, anti-inflflammatory and antithrombotic effects. In this review, an overview of the diverse biological effects of these compounds is given, particularly focusing on their benefificial properties on cardiovascular disease and proposing novel mechanisms of action responsible for their bioactivity. Attempts to prevent cardiovascular diseases target modififications of several risk factors such as high blood pressure, obesity, high blood concentrations of lipids or insulin resistance. Milk-derived bioactive peptides are a source of health-enhancing components and the potential health benefifit of these compounds has a growing commercial potential. Consequently, they have been incorporated as ingredients in functional foods, as dietary supplements and as pharmaceuticals to promote health and reduce risk of chronic diseases.
I. Preface
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I. Preface
In the past half century, many research results have pointed out that milk contains quite a lot of bioactive components, including lactoferrin, lysozyme, immunoglobulin, growth factor, hormon, and small molecular peptides produced after hydrolysis. In commercial and scientific research, nisin can be produced by enzymatic hydrolysis or microbial fermentation. Its main sources are various caseins (α -, β -, γ -, and κ - casein) or whey proteins in milk. With the increasing number of bioactive peptides revealed, the development and application of such materials have become the focus of the market. Nisin can be hydrolyzed twice in the digestive tract after being eaten by human body, and then its activity can be destroyed or increased. Therefore, the efficacy of nisin still needs animal experiments or clinical verification of human body. For example, kvlpqpq peptide produced by microbial fermentation of casein will be hydrolyzed by human body to produce kvlpvp after eating, and greatly enhance the activity of inhibiting vasoconstrictor converting enzyme, which shows that the development and research of lactopeptide is quite complex.
At present, scientists and commercial companies have developed a large number of nisin. Research shows that the biological activities of nisin include antithrombotic, antihypertensive, anti-inflammatory, antioxidant, antibacterial, anti obesity, etc., and each nisin can have more than 2-3 biological activities, which is suitable for the management of diseases with multiple causes, such as cardiovascular, digestive, endocrine, immune, and spiritual Diseases of the meridians. At present, many bioactivities of nisin are related to blood pressure, cholesterol, inflammation of blood vessels, thrombosis, and antioxidation. It is quite suitable for the development of cardiovascular health care, in order to reduce the hardness of artery wall, improve the activity of endothelial cells, and maintain the health of circulatory system.
Cardiovascular disease is a complex disease with multiple causes. Because of its complex causes, it is impossible to achieve effective management by single oriented treatment or prevention. At present, it is only used for antihypertensive, triglyceride, cholesterol and blood glucose lowering drugs, which can not effectively manage and control the pathological deterioration such as vascular inflammation and sclerosis. Through reviewing the literature since 1986, our team explored the possibility that nisin can be widely used for cardiovascular disease prevention and regulation.
2、 Bioactivity of active lactopeptide
Nisin can simultaneously affect a variety of risk factors related to cardiovascular diseases, such as blood pressure, thrombus, inflammation, and lipid metabolism, providing a possibility of improving the cardiovascular system as a whole. At present, it is known that many kinds of lactopeptides produced by human digestive tract, enzyme hydrolysis and microbial fermentation are released from milk protein through protease hydrolysis. The functions of these peptides in antihypertensive, insulin secretion regulation and blood glucose control, antithrombotic, anti-inflammatory, blood lipid concentration regulation, immune regulation, anti-inflammatory are further verified by scientific research (Table 1, figure 2), which are briefly described below.
2.1 antihypertensive
Hypertension is the most easily detected and controlled risk factor in cardiovascular patients. At present, ACE inhibitor is often used to control hypertension patients' blood pressure. Vasoconstrictor converting enzyme can convert vasoconstrictor-i into vasoconstrictor-ii, or inhibit bradykinin activity which can relax blood vessels and cause blood pressure rise. At present, ACE inhibitor can effectively reduce blood pressure, but it still has side effects such as hypotension, dry cough and renal function damage. In addition to drugs, peptides from milk, fish, plants and other sources can also inhibit ACE activity. Among them, many peptides from casein and whey protein have been reported to have ACE inhibitor activity. For example, valpro pro and ile pro, which are produced by fermentation of milk protein, have good effect on regulating blood pressure through the activity test of spontaneously hypertensive rats and human clinical test. More importantly, as antihypertensive peptides do not cause excessive blood pressure reduction in individuals with normal blood pressure, it can avoid the risk of hypotension caused by ACE inhibitors. In addition to the inhibition of ACE activity, nisin can also promote the production of vasodilator components by increasing prostaglandins, nitric oxide, carbon monoxide in serum, so as to reduce blood pressure. In Table 1, many lactopeptides with antihypertensive activity have been sorted out, but whether these lactopeptides produced through enzymatic hydrolysis or microbial fermentation will be further hydrolyzed by the digestive tract in human body and affect their activity still needs more animal experiments or human clinical studies to confirm.
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2.2 anti thrombosis
In the process of atherosclerotic atherosclerosis, platelet activation and aggregation will lead to thrombosis in the blood vessels, which will cause blood flow obstruction and lead to myocardial infarction, stroke and other complications, which will cause a threat to life. The current antithrombotic drugs are mainly used to inhibit platelet function and promote fibrinolysis. Many peptides produced by the hydrolysis of milk protein or kappa casein showed the activity of inhibiting platelet aggregation, so as to achieve the purpose of antithrombotic. Among them, kdqdk, taqvtstev and qvtstev, which are produced by the hydrolysis of κ - casein, showed the effect of inhibiting platelet aggregation and thrombosis in vitro. In addition, the krds peptide produced by lactoferrin hydrolysis is similar to the known RGDS sequence of antiplatelet agglutination peptide, and the two peptides can inhibit the release of serotonin in platelets and avoid thrombosis, but their mechanisms are different. The active peptide derived from the two proteins not only has the function of antithrombotic, but also has no cytotoxicity. Compared with the current antithrombotic drugs with serious side effects such as bleeding, the lactopeptide material with antithrombotic activity but no side effects is quite suitable for developing as functional food and providing to consumers with high risk of thrombosis.
2.3 antioxidant
Oxidative stress is another major cause of vascular inflammation, arteriosclerosis and eventually cardiovascular disease. The production of oxidative stress is mainly due to the production of excessive reactive oxygen species (ROS) or the decline of antioxidant mechanism in vivo, which leads to the destruction of DNA, RNA, protein, lipid and other substances in cells, leading to diseases such as atherosclerosis. However, ROS content is not as low as possible. It has been pointed out that ROS at low concentration can regulate cell signaling pathway, and then control cell protein phosphorylation, ion channel switch, and gene transcription. The main antioxidants in traditional foods are vitamin C, vitamin E, carotenoids, etc. Interestingly, recent in vitro experiments have shown that peptides produced by the hydrolysis of whey protein and casein, such as yfypel, can degrade superoxide dismutants. In addition, the peptide produced by the hydrolysis of casein with corolase enzyme has also been reported to have the activity of scavenging free radicals. In other literatures, 42 peptides containing wyslamasdi showed the best antioxidant activity. At present, scientists speculate that the high activity of this sequence should be related to the amino acids in peptide sequence, but more studies are needed to confirm it.
2.4 anti hyperlipidemia
Patients with hyperlipidemia and hypercholesterolemia are the high risk population of cardiovascular disease, especially prone to atherosclerosis. Therefore, the use of antilipidemic and cholesterol lowering drugs for blood lipid control is a very important part of the health management of these patients. In addition to drugs, more and more health care materials such as soybean protein hydrolysate have also been reported to have the activity of regulating lipids in blood. In addition, more and more studies have pointed out that different peptides of whey protein hydrolysate have the activity of lowering cholesterol in blood, among which iiaek peptide produced by hydrolysis of milk protein is the most famous. Ilaek was released by hydrolysis of β - lactoglobulin protein in milk, and its cholesterol lowering activity was similar to that of β - sitosterol. Another study found that lactostatin, a peptide with cholesterol lowering activity, can regulate cholesterol decomposition by regulating ERK-MAPK signaling pathway; in addition, lactostatin can activate cholesterin 7-hydroxylase (CYP7A1) transcription to promote cholesterol metabolism and decomposition, which can achieve the purpose of reducing blood cholesterol.
2.5 insulin secretion and blood glucose control
Patients with hypertension, hyperlipidemia, and hypercholesterolemia, if accompanied by type 2 diabetes, will significantly increase the risk of other cardiovascular diseases. Type 2 diabetes is mainly due to the gradual loss of body cells' sensitivity to insulin, which gradually worsens into the inability to secrete insulin to control blood sugar, and then induce vascular inflammation. Studies have shown that milk protein or hydrolyzed peptide can regulate insulin secretion and blood glucose control. For example, whey protein and casein can increase insulin secretion, whey protein is better than casein. In the experiment tested by healthy subjects, it was found that the effect of ingesting 20-50 grams of whey protein per day was similar to that of ingesting whey protein hydrolysate. In patients with type 2 diabetes, 18 grams of whey protein a day stimulated insulin production. In animal experiments, it was confirmed that continuous intake of whey protein for up to six weeks can increase the sensitivity of rats to insulin. Its function may come from the high concentration of amino acid produced by hydrolysis of whey protein through the digestive tract, and then induce pancreatic β - cells to secrete insulin. Compared with the above functions, there are few studies on the effect of nisin on insulin sensitivity and blood glucose control, and the mechanism of action is not clear.
2.6 anti inflammation and immune regulation
Inflammatory reaction can be divided into wound, acute inflammation caused by infection and chronic inflammation spontaneously produced by the body. Among them, long-term chronic inflammation will cause many chronic diseases such as rheumatoid arthritis, atherosclerotic sclerosis, and even cancer. Although it is clear that the relationship between inflammation of circulatory system and cardiovascular disease is known, only aspirin is used in patients with cardiovascular disease in non steroidal anti-inflammatory drugs, and no drug can effectively inhibit the inflammatory response in atherosclerotic sclerosis. In addition to its poor efficacy, the serious side effects of the drug are also considered. It can be seen from the current in vitro experiments that lactosein, such as VPP, lactoferrin and lactoferrin, can be conveyed to anti-inflammatory effect through different mechanisms. VPP can reduce the interaction between leukocytes and vascular endothelial cells, and then reduce the production of inflammatory reaction. The peptide produced by the hydrolysis of casein by corolase can inhibit the inflammatory reaction by regulating macrophages. Lactoferrin is a widespread immunoregulatory protein, which can inhibit the expression of pro-inflammatory cytokines produced by LPS activated NF - κ B in monocytes; in addition, lactoferrin can inhibit the expression of ICAM-1 and cytokines in endothelial cells of large arteries, inhibit the proliferation and movement of endothelial cells in bovine arteries, and maintain normal endothelial cells. In addition, lactoferrin can avoid monocyte maturation and Th1 cell activation which can stimulate inflammatory response. Our team also found that active nisin can inhibit NF-B signaling pathway by inhibiting PPAR - γ pathway, avoid monocyte adhesion to vascular endothelial cells, and induce subsequent inflammatory response and plaque formation.
Based on the previous results of many in vitro experiments, scientists further analyzed the possibility of nisin regulating physiological inflammatory response in animal experiments. First of all, VPP and tripeptide of iPP have been proved to be able to improve enteritis or atherosclerotic sclerosis, mainly by regulating the expression of inflammation related cell hormones and low-density lipoprotein oxidation related genes; whey protein and casein hydrolysate can improve the skin inflammation of NC / Nga mice and arthritis symptoms of rats, respectively. Our team has recently confirmed that nisin can inhibit NF - κ B through PPAR - γ, thus regulating the inflammatory response of human vascular endothelial cells, and avoiding monocyte adhesion to endothelial cells to cause atherosclerosis.
2.7 immune regulation
In addition to anti-inflammatory, many nisin have also been reported to have regulatory activities on different immune cells. For example: 1. A variety of milk proteins such as α - β - κ - casein, whey protein, and casein have been proved to regulate lymphocyte proliferation. 2. 2. The peptide produced by β - casein after fermentation and hydrolysis by lactobacillus can regulate the activity of monocyte and Th1 cell. 3. The peptide with the same sequence as bovine γ - casein fragment, β - tyrphine-7, or β - tyrphine-10 amino acid can promote the proliferation of immune cells, especially Tyr Gly and Tyr Gly Gly fragments. Interestingly, low-dose β - tyrphine-7 and β - tyrphine-10 peptide showed inhibitory activity on the proliferation of immune cells, but high-dose peptide could promote cell division. 4. Lactoferrin and lactoferrin regulate immunity by regulating granulocyte formation, killer cell activation and antibody secretion. In addition, some peptides with ACE inhibitory activity have also been confirmed to enhance bradykinin activity to stimulate macrophages, promote lymphocyte movement and lymphokine secretion and regulate immunity. In conclusion, many different peptides have significant immunomodulatory function, which shows that lactoprotein and derived peptides have high potential in regulating immunity and inhibiting inflammation.
3、 Mechanism of nisin improving arteriosclerosis
At present, several kinds of nisin have been pointed out to have potential application in atherosclerotic management, but there is still no clear explanation for the mechanism of nisin improving arteriosclerosis. If we can further clarify the molecular mechanism of nisin in improving arteriosclerosis, it will benefit the development of cardiovascular health care materials. Arteriosclerosis is a process of slow formation. In the process, it can be found that there will be plaque on the wall of the tube, which is caused by the aggregation of lipids, platelets and immune cells, and then lead to dangerous complications such as myocardial infarction and stroke. In the process of atherosclerotic atherosclerosis, the inflammatory response of vascular endothelial cells is one of the most important risk factors. It is closely related to the risk factors such as hypertension, obesity, hyperlipidemia and diabetes, and causes the inflammation and damage of endothelial cells under the interaction.
In the early stage of atherosclerotic sclerosis, the main feature is that monocytes gather on the endothelial cells, causing small wounds and moving the aggregated monocytes to the bottom of the intimal layer. This process can be found that endothelial cells will abnormally increase the expression of cell adhesion proteins (VCAM-1, ICAM-1 and E-selectin) and cell hormones (IL-8 and MCP-1). In addition to the changes in cell level, many pathways involved in atherosclerotic process, such as ppar - γ, JNK, ERK, and STAT3, have been studied. PPAR - γ plays an important role in regulating the early stage of atherosclerotic atherosclerosis, and PPAR - γ activation can inhibit the NF - κ B signaling pathway which can promote the inflammatory response. In the course of studying how nisin achieves anti-inflammatory and anti arteriosclerotic activity, our team found that the fermentation hydrolysate of casein sodium can inhibit NF - κ B activity by activating PPAR - γ. In terms of molecular regulation, we found that hydrolysates can inhibit the expression of pro-inflammatory factors that were previously activated by TNF - α, such as cell attachment proteins (VCAM-1, ICAM-1, e-sel) and chemokines (IL-8 and MCP-1) in endothelial cells, and the function of the hydrolysates can be completely destroyed by PPAR - γ inhibitor (GW9662), indicating that milk fermentation hydrolysates can control the downstream molecular mechanism mainly through PPAR - γ activation Purpose. Our team further separated the peptides from the hydrolysate for analysis. The same as other research teams, we found that the active peptides (molecular weight less than 5 kDa) in the fermentation products can activate PPAR - γ and NF - κ B. However, due to the complexity of peptides in casein sodium hydrolysate, it can not be ruled out whether there are other signaling pathways involved in the early stage of atherosclerosis. In addition, our team also found that milk protein hydrolysate (especially casein hydrolysate) can block the binding of TLR-4 and LPS on monocytes, so that tlp-4 can not activate the downstream JNK and NK - κ B signal pathways, thus inhibiting the expression of pro-inflammatory factors and cell adhesion factors, and avoiding monocyte aggregation in endothelial cells.
To sum up, active nisin is a very suitable health care raw material to resist the formation or deterioration of atherosclerotic sclerosis. For example, the adhesion and movement of monocytes on vascular endothelial cells is a very important step in the early stage of atherosclerosis. In the future, we can further develop appropriate health food by screening a large number of special active nisin that can block this step 。
Four. Conclusion
At present, more and more nisin have shown multiple biological activities, which can be used to develop health food and bring new changes to the management of cardiovascular disease. In patients with cardiovascular disease, atherosclerotic thrombosis can cause serious complications and death. Although the exact cause of atherosclerotic sclerosis still needs more research, the existing evidence shows that the interaction of genetic and environmental factors (such as high-fat diet, smoking, obesity, etc.) is closely related to the occurrence of atherosclerosis. At present, reducing blood lipid or cholesterol can effectively maintain triglyceride and cholesterol in blood, but the effect of these drugs on avoiding thrombus caused by arteriosclerosis is not good, so how to adjust the quality of blood vessels is the best choice. Nisin can inhibit inflammation, hypertension, cholesterol formation and free radicals. Meanwhile, the side effects of this kind of material are far less than that of current drugs, and it is more suitable for cardiovascular disease patients.
At present, several kinds of nisin products have been sold in the health care market, and casein derived nisin has been used as health care materials and may be used as drugs. However, there are still some problems to be overcome: 1. Many of the bioactivities of nisin are confirmed by in vitro experiments. It is impossible to confirm whether the bioactivities of nisin will remain after the second hydrolysis of human digestive tract after consumption. 2. Although nisin is a very safe and suitable natural material for long-term consumption, in the past, scientists seldom focused on its safety assessment, and a large number of potential risks of high dose and long-term use must be excluded before marketing. 3. It is necessary to further understand the bioavailability, pharmacokinetics, and the determination of dosage and frequency of nisin in the development of health food. In the future, we still need to invest more research to confirm its safety and mechanism, and provide products that can really bring health promotion effect to consumers.
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