The development of transgenic crops to improve human health by advanced utilization of seed storage proteins

Seed storage proteins are a major component of mature seeds. They are utilized as protein sources in foods. We designed seed storage proteins containing bioactive peptides based on their three-dimensional structures. Furthermore, to create crops with enhanced food qualities, we developed transgenic crops producing seed storage proteins with bioactive peptides. This strategy promises to prevent lifestyle-related diseases by simple daily food consumption. In this review, we discuss a strategy to develop transgenic crops to improve human health by advanced utilization of seed storage proteins.     

Seed Pro-Nutra Care: A tool for characterization of seed storage proteins and database of bioactive peptides having potential health benefits

Seed storage proteins, the major food proteins, possess unique physicochemical characteristics which determine their nutritional importance and influence their utilization by humans. Here, we describe a database driven tool named Seed Pro-Nutra Care which comprises a systematic compendium of seed storage proteins and their bioactive peptides influencing several vital organ systems for maintenance of health. Seed Pro-Nutra Careis an integrated resource on seed storage protein. This resource help in the (I) Characterization of proteins whether they belong to seed storage protein group or not. (II) Identification the bioactive peptides with their sequences using peptide name (III) Determination of physico chemical properties of seed storage proteins. (IV) Epitope identification and mapping (V) Allergenicity prediction and characterization. Seed Pro-Nutra Care is a compilation of data on bioactive peptides present in seed storage proteins from our own collections and other published and unpublished sources. The database provides an information resource of a variety of seed related biological information and its use for nutritional and biomedical application.

Availability

http://www.gbpuat-cbsh.ac.in/departments/bi/database/seed_pro_nutra_care/     

Characterization and production of multifunctional cationic peptides derived from rice proteins

Food proteins have been identified as a source of bioactive peptides. These peptides are inactive within the sequence of the parent protein and must be released during gastrointestinal digestion, fermentation, or food processing. Of bioactive peptides, multifunctional cationic peptides are more useful than other peptides that have specific activity in promotion of health and/or the treatment of diseases. We have identified and characterized cationic peptides from rice enzymes and proteins that possess multiple functions, including antimicrobial, endotoxin-neutralizing, arginine gingipain-inhibitory, and/or angiogenic activities. In particular, we have elucidated the contribution of cationic amino acids (arginine and lysine) in the peptides to their bioactivities. Further, we have discussed the critical parameters, particularly proteinase preparations and fractionation or purification, in the enzymatic hydrolysis process for producing bioactive peptides from food proteins. Using an ampholyte-free isoelectric focusing (autofocusing) technique as a tool for fractionation, we successfully prepared fractions containing cationic peptides with multiple functions.     

High-level production of lactostatin, a hypocholesterolemic peptide, in transgenic rice using soybean A1aB1b as carrier

Hypercholesterolemia, a form of cardiovascular disease, is one of the leading causes of deaths worldwide. Lactostatin (Ile-Ile-Ala-Glu-Lys), derived from β-lactoglobulin in cow’s milk, is a bioactive peptide with hypocholesterolemic activity higher than sitosterol, a known anti-hypercholesterolemic drug. Here, we successfully developed a transgenic rice accumulating a much higher level of lactostatin by inserting 29 IIAEK sequences into the structurally flexible (nonconserved) regions of soybean seed storage protein, A1aB1b, and introducing it into LGC-1 (low glutelin content mutant 1) as host variety. A1aB1b containing 29 lactostatins was expressed in the endosperm of rice seed cells by using seed specific promoters and sorted into novel compartments distinct from normal PB-I (ER-derived protein body) and PB-II (protein storage vacuoles). Transgenic rice seeds accumulated approximately 2 mg of lactostatins/g of dry seeds, which is relatively high compared with previous reports. Our findings suggest that the introduction of a high copy number of bioactive peptide into seed storage proteins as carrier is one of the effective means in producing higher amounts of bioactive peptides in rice.     

Mass spectrometry for nutritional peptidomics: How to analyze food bioactives and their health effects

We describe nutritional peptidomics for discovery and validation of bioactive food peptide and their health effects. Understanding nature and bioactivity of nutritional peptides means comprehending an important level of environmental regulation of the human genome, because diet is the environmental factor with the most profound life-long influence on health. We approach the theme from three angles, namely the analysis, the discovery and the biology perspective. Food peptides derive from parent food proteins via in vitro hydrolysis (processing) or in vivo digestion by various unspecific and specific proteases, as opposed to the tryptic peptides typically generated in biomarker proteomics. A food bioactive peptide may be rare or unique in terms of sequence and modification, and many food genomes are less well annotated than e.g. the human genome. Bioactive peptides can be discovered either empirically or by prediction: we explain both the classical hydrolysis strategy and the bioinformatics-driven reversed genome engineering. In order to exert bioactivity, food peptides must be either ingested and then reach the intestine in their intact form or be liberated in situ from their parent proteins to act locally, that is in the gut, or even systemically, i.e. through the blood stream. This article is part of a Special Section entitled: Understanding genome regulation and genetic diversity by mass spectrometry.     

食物蛋白酶解物中的生物活性肽

本文讨论了食物蛋白质酶解物中的生物活性肽及其药理作用     

Potential Applications of Food Derived Bioactive Peptides in Management of Health

Recently the rise in noncommunicable diseases and side effects of drugs has promoted the research in food components with biologically active molecules. These bioactive components are vital in reducing and regulating the onset of such chronic degenerative diseases. Many food derived peptides are biologically active fragments encrypted within the primary protein sequence in nascent (inactive) form, hence also called ‘cryptides’. These bioactive peptides range in size from 2 to 50 amino acids. They function beyond their basic nutritional benefits. Upon oral administration, these peptides play various roles such as opiate like, antioxidative, immunomodulatory, antihypertensive, hypocholesterolemic, mineral binding, antiobesity and antimicrobial. Both animal and plant proteins are rich sources of bioactive peptides having specific physiological and biochemical functions. Digestion of proteins in vivo or in vitro produces free amino acids and peptides which enter circulatory system and exert systemic effect. Bioactive peptides can be produced in vivo through gastrointestinal digestion whereas in vitro through chemical processing of food proteins with acid, alkali, heat and enzymatic hydrolysis either by digestion or fermentation. Protein hydrolysates being rich source of bioactive peptides can serve as an alternative to intact protein and elemental formula in the development of functional foods.     

Putting microbes to work: dairy fermentation, cell factories and bioactive peptides. Part I: overview

A variety of milk-derived biologically active peptides have been shown to exert both functional and physiological roles in vitro and in vivo, and because of this are of particular interest for food science and nutrition applications. Biological activities associated with such peptides include immunomodulatory, antibacterial, anti-hypertensive and opioid-like properties. Milk proteins are recognized as a primary source of bioactive peptides, which can be encrypted within the amino acid sequence of dairy proteins, requiring proteolysis for release and activation. Fermentation of milk proteins using the proteolytic systems of lactic acid bacteria (LAB) is an attractive approach for generation of functional foods enriched in bioactive peptides given the low cost and positive nutritional image associated with fermented milk drinks and yoghurt. In this review, we discuss the exploitation of such fermentation towards the development of functional foods conferring specific health benefits to the consumer beyond basic nutrition. In particular, in Part I, we focus on the release of encrypted bioactive peptides from a range of food protein sources, as well as the use of LAB as cell factories for the de novo generation of bioactivities.     

Plant seeds as bioreactors for recombinant protein production

Plants are attractive expression systems for the economic production of recombinant proteins. Among the different plant-based systems, plant seed is the leading platform and holds several advantages such as high protein yields and stable storage of target proteins. Significant advances in using seeds as bioreactors have occurred in the past decade, which include the first commercialized plant-derived recombinant protein. Here we review the current progress on seeds as bioreactors, with focus on the different food crops as production platforms and comprehensive strategies in optimizing recombinant protein production in seeds.     

Putting microbes to work: dairy fermentation, cell factories and bioactive peptides. Part II: bioactive peptide functions

A variety of milk-derived biologically active peptides have been shown to exert both functional and physiological roles in vitro and in vivo, and because of this are of particular interest for food science and nutrition applications. Biological activities associated with such peptides include immunomodulatory, antibacterial, anti-hypertensive and opioid-like properties. Milk proteins are recognized as a primary source of bioactive peptides, which can be encrypted within the amino acid sequence of dairy proteins, requiring proteolysis for release and activation. Fermentation of milk proteins using the proteolytic systems of lactic acid bacteria is an attractive approach for generation of functional foods enriched in bioactive peptides given the low cost and positive nutritional image associated with fermented milk drinks and yoghurt. In Part II of this review, we focus on examples of milk-derived bioactive peptides and their associated health benefits, to illustrate the potential of this area for the design and improvement of future functional foods.     

Enrichment of cereal protein lysine content by altered tRNA(lys) coding during protein synthesis

The world's major crops are deficient in lysine and several other amino acids essential for human and animal nutrition. Increasing the content of these amino acids in cereals, our major source of dietary energy, can help feed a global population whose reliance upon dietary protein is growing faster than crop yields. Here we document the heritable expression in rice, the world's major cereal crop, of tRNA(lys) species that introduce lysine at alternative codons during protein synthesis, resulting in a significant enrichment of the lysine content of proteins in rice seeds without changing the types or quantities of the seed storage proteins.     

A review of fish-derived antioxidant and antimicrobial peptides: their production, assessment, and applications

Fishes are rich sources of structurally diverse bioactive compounds. In recent years, much attention has been paid to the existence of peptides with biological activities and proteins derived from foods that might have beneficial effects for humans. Antioxidant and antimicrobial peptides isolated from fish sources may be used as functional ingredients in food formulations to promote consumer health and improve the shelf life of food products. This paper presents an overview of the antioxidant and antimicrobial peptides derived from various fishes. In addition, we discuss the extraction of fish proteins, enzymatic production, and the techniques used to isolate and characterize these compounds. Furthermore, we review the methods used to assay the bioactivities and their applications in food and nutraceuticals.     

In silico analysis of the large and small subunits of cereal RuBisCO as precursors of cryptic bioactive peptides

Ribulose-1,5-bisphosphate carboxylase (RuBisCO) is a plant metabolic enzyme and the most abundant protein on earth, but remains largely underutilized in the food system. Bioinformatics analysis was conducted to evaluate the prospects of RuBisCO from widely cultivated cereals (rice, barley, wheat, oat, sorghum, corn) as sources of bioactive peptides, and results were compared to commonly consumed proteins. The large and small RuBisCO subunits were found to contain several bioactive peptides with biological functions relevant to the management and treatment of diabetes, cardiovascular and neurodegenerative diseases, specifically inhibition of angiotensin converting enzyme and dipeptidyl peptidase-IV, antioxidative property and activation of ubiquitin-mediated proteolysis. Due to high sequence homology, there was negligible difference in occurrence frequency of bioactive peptides within large RuBisCO subunits unlike small subunits, which produced more diverse profiles of bioactive peptides among the cereals. The cereal RuBisCO displayed similar or better prospects as other food proteins except milk proteins, thereby providing cheaper and sustainable precursors of bioactive peptides. Simulated enzymatic hydrolysis of RuBisCO subunits indicated that thermolysin and papain had preferred cleavage patterns for releasing the cryptic peptides compared to gastrointestinal proteases. These findings will contribute towards utilization of RuBisCO as alternative sources of peptide-based nutraceuticals for human health promotion.     

The starch granule associated proteomes of commercially purified starch reference materials from rice and maize

Commercially available reference materials are integral components of many experimental protocols, as it is critical to compare one's results to those derived from well-characterized standards. Most reference materials are well defined, with all their components being cataloged. However, certain reference materials, such as commercially prepared starch samples, can have undefined components, potentially limiting their usefulness as standards. The proteome of commercially prepared starch has not been documented, and to that end, we initiated a mass spectrometry-based survey of the proteins associated with starch granules in commercially prepared rice and maize starch samples. We performed direct trypsin treatments of starch samples and sequenced both the water-soluble peptides liberated into the aqueous supernatant and the peptides released from the starch granule surface by isopropanol solvent washing. We discovered that the majority of proteins, in both rice and maize samples, were involved in either carbohydrate metabolism or storage. We also documented proteins that are markers for seed maturity and for starch mobilization.     

Industrial-scale manufacturing of pharmaceutical-grade bioactive peptides

Recent studies have shown that most peptide sequences encrypted in food proteins confer bioactive properties after release by enzymatic hydrolysis. Such bioactivities, which include antithrombotic, antihypertensive, immunomodulatory and antioxidant properties, are among the traits that are of biological significance in therapeutic products. Bioactive peptides could therefore serve as potential therapeutic agents. Moreover, research has shown that peptide therapeutics are toxicologically safe, and present less side effects when compared to small molecule drugs. However, the major conventional methods i.e. the synthetic and biotechnological methods used in the production of peptide therapeutics are relatively expensive. The lack of commercially-viable processes for large-scale production of peptide therapeutics has therefore been a major hindrance to the application of peptides as therapeutic aids. This paper therefore discusses the plausibility of manufacturing pharmaceutical-grade bioactive peptides from food proteins; the challenges and some implementable strategies for overcoming those challenges.     

植物种子贮藏蛋白质及其细胞内转运与加工

高等植物种子成熟过程中贮存大量的贮藏蛋白质作为种子发芽和初期生长的重要营养来源。根据溶解性不同,种子贮藏蛋白质可分为白蛋白、球蛋白、醇溶蛋白和谷蛋白4类。在种子胚发育过程中,醇溶蛋白在粗面内质网合成后形成蛋白质聚集体,直接出芽形成蛋白体并贮存其中。白蛋白、球蛋白和谷蛋白在粗面内质网以分子量较大的前体形式合成后,根据各自的分选信号进入特定的运输囊泡,经由受体依赖型运输/聚集体形式运输转运至蛋白质贮藏型液泡中,然后经过液泡加工酶等的剪切转换为成熟型贮藏蛋白质并贮存其中。蛋白质的合成、分选、转运和加工等过程影响种子蛋白质的品质及含量。该文对种子贮藏蛋白质的分类和运输、加工以及这些过程对种子蛋白质品质和含量的影响进行了概述。     

Physico-chemical,Sensory and Toxicity Characteristics of Dipeptidyl Peptidase-IV Inhibitory Peptides from Rice Bran-derived Globulin Using Computational Approaches

Rice processing industry released an enormous amount of the rice bran which is underutilized. Rice bran contains various proteins that can be used for the production of bioactive peptides. These bioactive peptides might be suitable ingredients for the development of functional food products. The objective of this study was to explore the potential of rice bran-derived globulin proteins as a suitable precursor of bioactive peptides with especially reference to dipeptidyl peptidase IV (DPP-IV) inhibitory peptides. The various computational approaches (BLAST, BIOPEP, PeptideRanker, PepDraw, Pepcalc, and ToxinPred) were used to predict the potential of the globulin proteins. Ficain protease majorly released the DPP-IV inhibitory peptides from rice bran-derived globulin proteins as compared with other proteases used in this study. Furthermore, primary structure, physico-chemical, sensory, and allergic characteristics of the theoretically release bioactive DPP-IV inhibitory peptides were also studied. The result of this study provides a theoretical basis for the development of rice bran globulin proteins as a suitable source for the generation of bio-functional ingredients for glycaemic management and further demonstrates the usefulness of computational approaches.     

The Medicago truncatula small protein proteome and peptidome

The small protein and native peptide component of plant tissues is a neglected area of proteomic studies. We have used fractionation techniques for denatured and nondenatured protein preparations combined with 2-D LC tandem mass spectrometry to examine the sequences of small proteins and peptides in four tissues of the model legume, Medicago truncatula: the root tip and root of germinating seedlings, nitrogen fixing nodules, and young leaves. The isolation and fractionation strategies successfully enriched the small protein and native peptide content of the samples. Eighty-one small M. truncatula proteins and native peptides were identified. Most samples were dominated by ribosomal and histone proteins, and leaf samples possessed photosynthesis-related proteins. Secreted proteins such as lipid transfer proteins were common to several tissues. Twenty-four hours after germination, the roots and root tip tissues possessed several "seed-specific" and late-embryogenesis proteins. We conclude that these proteins are present in cells prior to germination and that they are subsequently used as a nutritional source for the young tissues. Native UV absorbing peptides were detected in very low molecular weight fractions and sequenced. Each peptide shared C-terminal residues and showed homology to the seed storage protein legumin. The strategies used here would be suitable for combining bioassays and mass spectrometry to identify bioactive peptides in the M. truncatula peptidome.     

Bioactive peptides derived from food proteins preventing lifestyle-related diseases

Many kinds of bioactive peptides which might prevent lifestyle-related diseases are released from food proteins after enzymatic digestion. Inhibitory peptides for angiotensin I-converting enzyme (ACE) having anti-hypertensive effect have been isolated from enzymatic digests of various food proteins. LKPNM, which was isolated from the thermolysin digest of dried bonito was activated 8-fold by ACE itself and showed a prolonged effect after oral administration. Two vasorelaxing peptides, ovokinin and ovokinin(2-7), showing antihypertensive effect after oral administration were obtained from ovalbumin digests. We found that low molecular weight peptides derived from food proteins lowered serum cholesterol without increasing excretion of cholesterol and bile acids. An immunostimulating peptide isolated from an enzymatic digest of soybean protein prevented alopecia induced by cancer chemotherapy.     

The possible roles of food-derived bioactive peptides in reducing the risk of cardiovascular disease

Vascular diseases such as atherosclerosis, stroke or myocardial infarction are a significant public health problem worldwide. Attempts to prevent vascular diseases often imply modifications and improvement of causative risk factors such as high blood pressure, obesity, an unfavorable profile of blood lipids or insulin resistance. In addition to numerous preventive and therapeutic drug regimens, there has been increased focus on identifying dietary compounds that may contribute to cardiovascular health in recent years. Food-derived bioactive peptides represent one such source of health-enhancing components. They can be released during gastrointestinal digestion or food processing from a multitude of plant and animal proteins, especially milk, soy or fish proteins. Biologically active peptides are considered to promote diverse activities, including opiate-like, mineral binding, immunomodulatory, antimicrobial, antioxidant, antithrombotic, hypocholesterolemic and antihypertensive actions. By modulating and improving physiological functions, bioactive peptides may provide new therapeutic applications for the prevention or treatment of chronic diseases. As components of functional foods or nutraceuticals with certain health claims, bioactive peptides are of commercial interest as well. The current review centers on bioactive peptides with properties relevant to cardiovascular health.