In our iGEM journey, we use first principles to return to the most basic conditions of things, split them into elements for deconstruction analysis, and find the best path to achieve the goal.
This part solves two problems:
1. How to improve the yield of bromelain;
2. Find a suitable application for bromelain.
The main problem is divided into two sub-problems for analysis according to the idea of synthetic biology:
1. What problems exist in the industrial production of bromelain in factories that can be solved by synthetic
biology;
2. In which areas can the bromelain products produced by synthetic biology be better applied?
Our question at this stage is for this connection between people and science: how can we as scientists best identify concerns with factories and users and incorporate their values into the solutions we propose?
The initial development of our project was greatly facilitated by open conversations with factory staff—they
taught us how to have conversations, often difficult conversations, on the profit issues that already existed
between factories and synthetic biology. These conversations inform the way we listen to and engage with
people throughout project development in a thoughtful and meaningful way. These conversations ensure that our
projects stem from careful consideration of three key things:
1. In what form synthetic organisms can enter the production process of current factories;
2. How synthetic biology can improve product characteristics in current factories;
3. Values that emerge from the factory's response.
Cangzhou Xiasheng Enzyme Biotechnology Co., Ltd. was established in 2002. It is a wholly-owned subsidiary of Xiasheng Group. It is a scientific and technological enterprise in Hebei Province of the People's Republic of China.
Our discussions with them have been very helpful in constructing factory-fitted synthetic bio-industrial solutions in our current efforts to address bromelain production. We knew we wanted to develop a synthetic biology solution, but our conversation helped us re-ask the question: Can synthetic biology reduce production costs to improve bottom line? They encourage us to focus on the core of industrial issues: the control of costs and profits while meeting sustainable development.
Their bromelain unit price - 27.5-30.2CNY/kg.
Shandong Kangqin Biotechnology Co., Ltd. (People's Republic of China) is a secondary distributor of bromelain.
In order to develop meaningful integrative human practice, they suggest that we focus on a very important enzyme product property: enzyme activity.
Their enzyme activity - 1000000U/g.
Therefore, we decided to incorporate the directed evolution of bromelain into our development process as early as possible.
In addition, in our communication with them, we learned that their source factory is in Nanning, Guangxi, and this information prompted us to find the next factory (their source factory): Nanning Dongheng Huadao Biotechnology Co., Ltd.
One advantage of the synthetic biology approach is Sustainable Development. In our communication with them, they suggested that we need to consider sustainable development, as a traditional biological enzyme purification enterprise, they found their own sustainable development method, which can optimize their shipping cycle, 100kg bromelain Orders are shipped in approximately 3 days.
Their sustainable way - Bromelain from inedible pineapple peels.
This poses new challenges to the design of synthetic biology methods. On the premise that traditional methods can already achieve sustainable development, can synthetic biology do better in sustainable development?
At the same time, as a leading company in the industry, they also shared their advantages with us: enzyme purification technology. Therefore, we also included enzyme purification as part of the overall design and development of the solution.
Ningxia Xiasheng Industrial Group Co., Ltd. (hereinafter referred to as Xiasheng Group Company) was founded in 1996. It is a high-tech private joint-stock enterprise integrating the research and development, production and sales of biological enzyme preparations. An enterprise that realizes the industrialized production of cellulase by technology. The company is the executive director unit of China Fermentation Industry Association, and was rated as "National Key Production Enterprise of Enzyme Preparation Industry" by China Fermentation Industry Association, and took the lead in passing ISO9001:2000 quality management system certification in the industry.
This group is also the parent company of Cangzhou Xiasheng Enzyme Biotechnology Co., Ltd., which we have communicated with for a long time. The staff of this company recommended us to communicate with Ningxia Xiasheng Industrial Group.
We learned about the People's Republic of China quality standards for bromelain in our communication with the group.
This quality standard specifies the number of colonies, the number of coliforms, the number of Escherichia coli in detail. This led us to consider replacing E. coli with other safer chassis early in the development of the project. And hope to use probiotics as a chassis to express bromelain in the future.
We use first principles to split and classify the application scenarios of bromelain, and conduct comprehensive human practice for each application field to help us understand the problem.
In the form of a literature review, we have extensively investigated the application of bromelain in the medical field and compared the feasibility of these applications.
| Cardiovascular diseases | Coronary heart disease (heart attack), vascular disease, cerebrovascular disease (stroke), increased blood pressure, angina pectoris, coagulation and fibrinolytic disorders Treatment - dissolves atherosclerotic plaque. | |
|---|---|---|
| Regulates blood coagulation homeostasis: procoagulant at low concentrations and anticoagulant at high concentrations. | ||
| Antibacterial | Inhibit bacterial toxin secretion. | |
| Increase antibiotic absorption and activity. | The mechanism is unclear, and the application potential is currently small. | |
| Anti-parasitic. | Commonly used by veterinarians, few human-related studies. | |
| Antifungal infection: Candida albicans, Fusarium verticillium, Fusarium oxysporum and Fusarium proliferators. | agronomically important. | |
| immune regulation | Bromelain can participate in the gene expression control of immune response in multiple molecular signaling pathways, and its control has dual roles. | |
| Inhibits lipopolysaccharide-induced inflammatory response. | ||
| As an immunostimulatory factor. | Increased proliferation of T cells, in addition to T cell co-stimulatory activity of splenocytes and purified B cells. | |
| Anti-cancer | Has molecular anticancer activity. | Inactive in clinical trials, the cause is unknown, and the mechanism is unknown. |
| allergy | Bromelain protects against allergy through proteolytic degradation of CD44. | |
|---|---|---|
| Chronic sinusitis, osteoarthritis | Postoperative anti-edema effects that have been clinically studied may be effective for this. | |
| Debridement and wound healing | One of the components of bromelain is escharase, which can effectively eliminate eschar. | |
| COVID-19 |
1. Bromelain interacts with sarscov2 by targeting ACE2. 2. Control the symptoms of the disease. 【Anti-inflammatory and anticoagulant ability】 |
Studies have shown that bromelain can remove the spike protein and hemagglutinin protein interaction targets of Semliki Forest virus, Sindbis virus, mouse gastrointestinal coronavirus, hemagglutinating encephalomyelitis virus, and H1N1 influenza virus. |
Studies have shown that chitosan nanofibers containing bromelain (2% and 4% w/v) were prepared by electrospinning. The physicochemical properties of the synthesized nanofibers were evaluated. Chitosan-2%w/v bromelain has better physicochemical properties and release properties, and lower cytotoxicity. In experimental animal models, chitosan-2% w/v bromelain nanofibers were more effective in healing burn skin than chitosan nanofibers alone.[1]
The combination of bromelain and dexamethasone sodium phosphate induces massive activation of mesenchymal stem cells, increases hyaluronic acid and collagen production, and releases anti-inflammatory cytokines. The results of an in vitro study showed that the combined use of bromelain and dexamethasone sodium phosphate stimulated the pro-wound-healing activity and regenerative properties of mesenchymal stem cells better than bromelain and dexamethasone alone.[2]
Studies have shown that bromelain is more effective in removing caries than papain. The mean value of residual carious dentin thickness obtained in group II (bromelain) was 36.74 μm, which was much lower than the mean value obtained by group I (papain) of 73.84 μm. The mean time obtained for group II (bromelain) was 335.30 seconds, which was almost equal to the mean time for group I (papain) of 352.33 seconds.[3]
We compared the burgeoning bromelain to the fairly established papain in a literature review.
Meat tenderizer: It is a cysteyl protease that is often used in the food processing industry to make meat tenderizers. Papain can degrade the proteases of myofibrils and connective tissues, decompose actomyosin and collagen bands into smaller molecular polypeptides and amino acids, and break the tendon and muscle filaments of meat, causing The taste is smoother and fresher; at the same time, the effect of the tenderizer is more obvious during the processing of meat food, and papain has high thermal stability and will not lose its activity at higher temperatures, so It is often used in the processing of meat tenderizers.
Beer: The use of papain to add to beer can make the quality of beer better, mainly because the protein in beer will be fully hydrolyzed after encountering papain, and some of the formed complexes will be hydrolyzed at the same time. Produce more cysteine Peptides and amino acids of aminoacyl proteases that keep beer highly clear during freezing. At the same time, it can also improve the taste of the beer and make the quality of the beer better.
Biscuit loosening agent: adding papain to biscuits and pastries can destroy the strength of flour, make the biscuits more fluffy, make the biscuits have better appearance and oiliness, and reduce the defective products in the production process of the biscuits probability, improve production efficiency. In addition, with papain, the use of sugar and oil can be reduced, and it is widely used in the production of various cakes and biscuits.
Seasoning sauce: papain can decompose brewer's yeast, and then extract the yeast extract with rich aroma, which can be made into seasoning sauce after seasoning, which improves the utilization value of yeast.
Coagulation of casein (but some websites have also retrieved that papain can be used for the coagulation of casein, but it is not its main use, and there is no detailed production route), increase the PDI value and NSI value of soy cake and soy flour to produce products containing The breakfast products of soybean flour (more detailed literature on how to increase this article was not found) The casein currently produced in China is mainly casein hydrochloride, but the application of casein hydrochloride is limited due to its low acidity.
Both are mild enzyme preparations, the specificity is much lower than other enzyme preparations, and can act on a variety of substrates; at the same time, the stability is high, and the unit enzyme activity retained in various parts of the body is high.
A team investigated the effects of single and combined proteolytic hydrolysis on poultry by-products myofibrillar protein and collagen. The aim is to create more value for all the components of these complex by-products. A rational method was used to select the proteases that showed the highest activity against the major protein-rich components in mechanical chicken bone residue (MDCR), and the activities of 18 proteases on chicken, turkey tendon and MDCR were evaluated. preliminary screening. And ultimately help make the most of and increase the value of poultry by-products.[4]
Oranges are a widely used fruit all over the world because they contain Cit s 2, a major allergen, and are prone to allergic reactions. There is a team that lowers Cit s 2 levels by mixing fresh orange juice and pineapple. The level of Cit s 2 in orange juice digested with pineapple extract was determined by quantitative enzyme-linked immunosorbent assay, and it was found that the level of Cit s 2 decreased with the prolongation of the reaction temperature and time. 40-fold dilution of pineapple extract and 0.1 mg/mL of bromelain and treatment at 37 °C for 30 min helped to reduce residual Cit s 2 levels below the critical value of 15%, respectively. After clinical research, it is expected that the protease will have a wide range of applications in the hypoallergenic food industry.[5]
One study evaluated the biological properties of bromelain-hydrolyzed brown rice protein peptides (eb-RPH) in relation to flavor profiles. Evaluation of flavor profiles using the BIOPEP database showed that their umami peptides (ESDVVSDL, GSGVGGAK, and SSVGSAG) had higher frequencies and lower Q-values (938.75 to 282.22), suggesting that these peptides can be used as good-tasting health care products Reinforcing ingredients. Isolated brown rice protein hydrolyzate (<1 kDa) has potential as a functional food ingredient in nutraceutical and beverage products with good taste and health benefits.[6]
Bromelain in pineapple stems was covalently immobilized on different supports to select more efficient biocatalysts for the treatment of labile proteins in white wine. Both immobilized forms of bromelain showed better substrate affinity and longer half-life relative to the free enzyme. By immobilizing bromelain on chitosan microspheres, a food-safe and promising biocatalyst was produced, which can catalyze the decomposition of unstable proteins in wine and improve the quality of wine.[7]
Natural latex (NRL) allergy is caused by extractable latex proteins in dipping products. This is a major concern for consumers sensitive to allergic extractable protein (EP) in products such as NRL gloves. The aim of this study was to develop an economical method to reduce EP in NRL finished products. Different glove samples were treated with a 5% solution of each partially purified enzyme for 2 h at 60°C. Residues in treated samples were quantified using a modified Lowry analysis method (ASTM D5712-10). A 54% (±11%) reduction in EP was found, and these results clearly demonstrate that the selected natural protease contributes significantly to the reduction in total EP in the finished NRL.[8]
The biggest advantage of using synthetic biology to produce bromelain is that it can make the production cycle of bromelain more flexible instead of being limited by the supply of pineapple peels. In the case of sufficient supply of raw materials, although the method of synthetic biology production cannot be particularly fast, if you have continuous orders that can be produced continuously, the output and product quality of each time are relatively more controllable.
From the perspective of factory value and application value, the proteolytic ability of bromelain is a very prominent performance indicator. Bromelain after screening and directed evolution can be expressed by the method of synthetic biology to optimize the above indicators.
Sustainable production is one of the core values of our project. Therefore, when we screened the application scenarios of incorporating bromelain, we focused on the safety and sustainability of the solution.
In the traditional food engineering field, compared with papain, bromelain has no unique effect on protein food tenderization and the competing products are quite mature.
In the casein industry, bromelain is difficult to replace by other enzymes. However, due to product properties and industrial scale, the application of pineapple protein is limited.
Analyzing the progress of research and development and application of bromelain in the medical direction, most of the existing researches focus on the treatment of a certain disease or a certain type of congenital disease, and many studies have been carried out to the clinical trial stage.
First of all, the prospects of the drug perspective are very small. Due to the time constraints of the project and the policy and process issues of drug approval, it is difficult for us to make a decent finished product at the end. Therefore, if we want to do healthcare, our focus should preferably be on health products, medical testing, and equipment.
Second, health care products. At present, there are food products related to bromelain in the foreign market [Quercetin Lungs Compound Dihydroquercetin Imported Lung Health Products Nutritional Genuine 500mg 120 Capsules], the role is limited to promoting digestion, etc., and our previous work selected stem pineapple The instability of proteases, with their optimum pH at 6-7, and bromelain under stressful conditions is likely to limit their health benefits and pharmacological applications. The degree of completion within 1 year is still very low.
Finally, the current production of bromelain has stability maintenance issues. Several current studies have focused on stabilization by immobilizing bromelain. We can study effective drug delivery vehicles containing bromelain - nanoparticles of silica, gold or chitosan covered with bromelain to carry antibiotics or other drugs, and finish.
New apps are often more interesting, and breakthroughs in new apps can lead to greater benefits. The treatment of protein food scraps with bromelain for recycling as feed has high sustainable development. We decided to continue researching the security of this direction and implement it.
According to Professor Cui Hongjie's description, there are more and more studies on bromelain at home and abroad. Bromelain is also a feed additive (cat and dog) allowed by the state. If the mechanism of action of bromelain can be fully understood, its market demand should also be great.）
In order to have a deeper understanding and evaluation of the application of bromelain in the field of feed, we discussed online with Professor Cui Hongjie of Fujian A&F University. According to Professor Cui Hongjie's description, there are more and more studies on bromelain at home and abroad, and bromelain is also a feed additive (for cats and dogs) allowed by the state. If the mechanism of action of bromelain can be fully understood, its market demand should also be great.
At present, there is no clear evidence to prove the bacteriostatic effect of bromelain, but some reasonable assumptions can be made about its bacteriostatic principle from the results of some in vitro experiments. Specific amino acids similar to the cationic antimicrobial peptide bromelain interfere with the cell membrane and cell wall structure of Gram-negative and Gram-positive bacteria.
In terms of Safety, according to the National Standard of Feed Hygiene of the People's Republic of China, we have revised the project implementation ideas.
In order to optimize the safety issue of chassis residues, we used the probiotic Bacillus subtilis as the chassis. Our original idea was to directly ferment the protein feed without inactivating the chassis during purification. Let Bacillus subtilis enter the digestive system of livestock as a probiotic.
There were disagreements within our team in evaluating the feasibility of the above ideas. Therefore, we showed our ideas to Shao Jianchun, associate professor of Fujian A&F University, and hoped to get his evaluation of this plan. According to Shao Jianchun, genetically modified probiotics are used as a chassis for synthetic biology production, and the products must be inactivated to meet the national standards of the People's Republic of China.
Therefore, we choose to extinguish the chassis during the separation and purification process to meet product safety standards.
[1] Bayat S, Amiri N, Pishavar E, Kalalinia F, Movaffagh J, Hashemi M. Bromelain-loaded chitosan nanofibers prepared by electrospinning method for burn wound healing in animal models. Life Sci. 2019 Jul 15;229:57-66. doi:10.1016/j.lfs.2019.05.028. Epub 2019 May 11. PMID: 31085247.
[2] Ghensi P, Cucchi A, Bonaccorso A, Ferroni L, Gardin C, Mortellaro C, Zavan B.In Vitro Effect of Bromelain on the Regenerative Properties of Mesenchymal Stem Cells. J Craniofac Surg. 2019 Jun;30(4):1064-1067. doi:10.1097/SCS.0000000000004862. PMID: 30358745.
[3] Reddy VK, Nagar P, Reddy S, Ragulakollu R, Tirupathi SP, Ravi R, Purumadla U.Bromelain vs Papain Gel for Caries Removal in Primary Teeth. J Contemp Dent Pract. 2019 Nov 1;20(11):1345-1349. PMID: 31892689.
[4] Lindberg D, Kristoffersen KA, Wubshet SG, Hunnes LMG, Dalsnes M, Dankel KR,Høst V, Afseth NK. Exploring Effects of Protease Choice and Protease Combinations in Enzymatic Protein Hydrolysis of Poultry By-Products. Molecules.2021 Aug 31;26(17):5280. doi: 10.3390/molecules26175280. PMID: 34500712; PMCID:PMC8434180.]
[5] Kiyota K, Yoshimitsu M, Kajimura K, Yamano T. Reduction of Orange AllergenCit s 2 Levels in Fresh Orange Juice with Pineapple Bromelain EnzymaticTreatment. Shokuhin Eiseigaku Zasshi. 2020;61(1):17-21. doi:10.3358/shokueishi.61.17. PMID: 32336714.
[6] Selamassakul O, Laohakunjit N, Kerdchoechuen O, Yang L, Maier CS. Bioactive peptides from brown rice protein hydrolyzed by bromelain: Relationship between biofunctional activities and flavor characteristics. J Food Sci. 2020 Mar;85(3):707-717. doi: 10.1111/1750-3841.15052. Epub 2020 Feb 11. PMID:32043604.
[7] Laohakunjit N, Selamassakul O, Kerdchoechuen O. Seafood-like flavour obtained from the enzymatic hydrolysis of the protein by-products of seaweed (Gracilariasp.). Food Chem. 2014 Sep 1;158:162-70. doi: 10.1016/j.foodchem.2014.02.101.Epub 2014 Mar 1. PMID: 24731327.
[8] Perera ALHA, Perera BGK. Development of an Economical Method to Reduce the Extractable Latex Protein Levels in Finished Dipped Rubber Products. Biomed Res Int. 2017;2017:9573021. doi: 10.1155/2017/9573021. Epub 2017 Jun 19. PMID: 28706952; PMCID: PMC5494553.