Amino acid requirements in humans: with a special emphasis on the metabolic availability of amino acids

Due to advances made in the development of stable isotope based carbon oxidation methods, the determination of amino acid requirements in humans has been an active area of research for the past 2 decades. The indicator amino acid oxidation (IAAO) method developed in our laboratory for humans has been systematically applied to determine almost all indispensable amino acid requirements in adult humans. Nutritional application of experimentally derived amino acid requirement estimates depends upon the capacity of food proteins to meet the amino acid requirements in humans. Therefore, there is a need to know the proportion of dietary amino acids which are bioavailable, or metabolically available to the body for protein synthesis following digestion and absorption. Although this concept is widely applied in animal nutrition, it has not been applied to human nutrition due to lack of data. We developed a new in vivo method in growing pigs to identify the metabolic availability of amino acids in foods using the IAAO concept. This metabolic availability method has recently been adapted for use in humans. As this newly developed IAAO based method to determine metabolic availability of amino acids in foods is suitable for rapid and routine analysis in humans, it is a major step forward in defining the protein quality of food sources and integrating amino acid requirement data with dietary amino acid availability of foods.     

Utilisation of Chlorella vulgaris cell biomass for the production of enzymatic protein hydrolysates

Studies on enzymatic hydrolysis of cell proteins in green microalgae Chlorella vulgaris 87/1 are described. Different proteases can be used for production of hydrolysates from ethanol extracted algae. The influence of reaction parameters on hydrolysis of extracted biomass with pancreatin was considered, and the composition of hydrolysates (Cv-PH) was investigated in relation to the starting materials. Significant changes in the degree of hydrolysis were observed only during the first 2h and it remained constant throughout the process. An enzyme-substrate ratio of 30-45 units/g algae, an algae concentration of 10-15% and pH values of 7.5-8.0 could be recommended. Differences in the chromatographic patterns of Cv-PH and a hot-extract from Chlorella biomass were observed. Adequate amounts of essential amino acids (44.7%) in relation to the reference pattern of FAO for human nutrition were found, except for sulfur amino acids. Cv-PH could be considered as a potential ingredient in the food industry.     

COMMERCIAL DEVELOPMENTS IN MICROALGAL BIOTECHNOLOGY

A number of important advances have occurred in microalgal biotechnology in recent years that are slowly moving the field into new areas. New products are being developed for use in the mass commercial markets as opposed to the "health food" markets. These include algal-derived long-chained polyunsaturated fatty acids, mainly docosahexaenoic acid, for use as supplements in human nutrition and animals. Large-scale production of algal fatty acids is possible through the use of heterotrophic algae and the adaptation of classical fermentation systems providing consistent biomass under highly controlled conditions that result in a very high quality product. New products have also been developed for use in the development of pharmaceutical and research products. These include stable-isotope biochemicals produced by algae in closed-system photobioreactors and extremely bright fluorescent pigments. Cryopreservation has also had a tremendous impact on the ability of strains to be maintained for long periods of time at low cost and maintenance while preserving genetic stability.     

Updates on industrial production of amino acids using <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

l-Amino acids find various applications in biotechnology. l-Glutamic acid and its salts are used as flavor enhancers. Other l-amino acids are used as food or feed additives, in parenteral nutrition or as building blocks for the chemical and pharmaceutical industries. l-amino acids are synthesized from precursors of central carbon metabolism. Based on the knowledge of the biochemical pathways microbial fermentation processes of food, feed and pharma amino acids have been developed. Production strains of Corynebacterium glutamicum, which has been used safely for more than 50 years in food biotechnology, and Escherichia coli are constantly improved using metabolic engineering approaches. Research towards new processes is ongoing. Fermentative production of l-amino acids in the million-ton-scale has shaped modern biotechnology and its markets continue to grow steadily. This review focusses on recent achievements in strain development for amino acid production including the use of CRISPRi/dCas9, genome-reduced strains, biosensors and synthetic pathways to enable utilization of alternative carbon sources.     

能量标签技术及其在红树林生态系统能流研究中的应用

传统上认为红树林输出的有机质产生巨大的能流,支持了巨大的河口和近岸水域生态系统的次级生产。但能量标签技术的研究结果却显示红树林输出的有机质的作用并没有如此巨大。用红树碎屑难消化特性来解释此现象,此外数学模型模拟分析发现潮汐的稀释作用也可以解释这种现象。但这两者都不能解释,在其他初级生产者稀少时,红树材输出的有机质可以被大量利用的现象。在有红树林的河口和近海岸水域生态系统中,藻类等非红树初级生产者具有比红树植物更高的初级生产力,而且更容易被动物获得和消化。可以认为是藻类等巨大初级生产力的竞争作用导致红树初级生产在消费者组织中很难被发现,如此上面提到的难题就能得到很好的解决。此外能量标签技术检测出的是红树的初级生产在消费者组织中的相对比率,不是绝对数量值,从此角度看,能量标签技术的结果与传统观点不是矛盾而是互相补充的关系。由此推测红树的初级生产应该还是被消费者所利用,只是它们在消费者初级营养来源组成中占的比例并不大,但其绝对数量并不少。这与传统观点认为的红树的初级生产被大量利用,支撑了具有巨大的次级生产稍有不同。此外,能量标签技术在红树林生态系统中的适用性尚未检验;计算食物组成的数学工具不是很完善;实验设计上考虑的不够全面;对定量研究有一定的影响。     

Amino acid uptake profiling of wild type and recombinant Streptomyces lividans TK24 batch fermentations

Streptomyces lividans is considered an interesting host for the secretory production of heterologous proteins. To obtain a good secretion yield of heterologous proteins, the availability of suitable nitrogen sources in the medium is required. Often, undefined mixtures of amino acids are used to improve protein yields. However, the understanding of amino acid utilization as well as their contribution to the heterologous protein synthesis is poor.In this paper, amino acid utilization by wild type and recombinant S. lividans TK24 growing on a minimal medium supplemented with casamino acids is profiled by intensive analysis of the exometabolome (metabolic footprint) as a function of time. Dynamics of biomass, substrates, by-products and heterologous protein are characterized, analyzed and compared. As an exemplary protein mouse Tumor Necrosis Factor Alpha (mTNF-α) is considered.Results unveil preferential glutamate and aspartate assimilation, together with glucose and ammonium, but the associated high biomass growth rate is unfavorable for protein production. Excretion of organic acids as well as alanine is observed. Pyruvate and alanine overflow point at an imbalance between carbon and nitrogen catabolism and biosynthetic fluxes. Lactate secretion is probably related to clump formation. Heterologous protein production induces a slowdown in growth, denser clump formation and a shift in metabolism, as reflected in the altered substrate requirements and overflow pattern. Besides glutamate and aspartate, most amino acids are catabolized, however, their exact contribution in heterologous protein production could not be seized from macroscopic quantities.The metabolic footprints presented in this paper provide a first insight into the impact and relevance of amino acids on biomass growth and protein production. Type and availability of substrates together with biomass growth rate and morphology affect the protein secretion efficiency and should be optimally controlled, e.g., by appropriate medium formulation and substrate dosing. Overflow metabolism as well as high biomass growth rates must be avoided because they reduce protein yields. Further investigation of the intracellular metabolic fluxes should be conducted to fully unravel and identify ways to relieve the metabolic burden of plasmid maintenance and heterologous protein production and to prevent overflow.     

Role of essential fatty acids in trophometabolic interactions in the freshwater ecosystems (a review)

Polyunsaturated fatty acids (PUFA) of omega3 family are of crucial physiological importance for the most animals and they are an essential and deficient component of human nutrition. These compounds are most effectively synthesized by some groups of algae, hence, aquatic ecosystems are considered to be the main source of these PUFA for human nutrition. Factors controlling the content and distribution of omega3 PUFA in freshwater organisms of basic trophic levels and determined PUFA final production in freshwater ecosystems are considered in the review. PUFA biosynthesis is known to be tightly related to basic fatty acid metabolic pathways. Hence, fatty composition and the PUFA content of major freshwater hydrobiont groups, including bacteria, algae, invertebrates and vertebrates, and environmental and population age effects are described. The peculiarities of PUFA transfer between organisms of various trophic levels are discussed. The essential omega3 PUFA is one of the important parameter of food quality of aquatic consumers and they can determine the rate of energy and matter transfer between producers and primary consumers and, as a result, in a whole freshwater food chain. Analysis of PUFA content and its regulation in biomass of various fish populations indicates that freshwater ecosystems are of the same value in respect of PUFA sources as marine ecosystems. Despite the great practical importance, the studies focused on production and whole pools of omega3 PUFA in different freshwater ecosystems are still scarce and need to be continued.     

Linking nectar amino acids to fitness in female butterflies

Nectar of butterfly-pollinated flowers contains generally higher levels of amino acids than does nectar of flowers pollinated by most other animal types. One proposed explanation is that these amino acids promote butterfly fitness, although the evidence has been equivocal. In a new study, Mevi-Schütz and Erhardt showed that nectar amino acids enhanced fecundity in the butterfly Araschnia levana, but only when the larval diet was poor. Their results support the hypothesis that butterflies are agents of selection for higher nectar amino acid production, suggest that the larval food plant has a key role in the evolution of the flower-butterfly mutualism, and demonstrate that the importance, to butterfly reproduction, of different nutrient sources varies with butterfly nutritional state.     

四碳有机酸生物合成的代谢工程研究进展

四碳有机酸作为重要的平台化学品,广泛应用于食品、化工、农业、医药和生物材料等领域。与传统的石化法相比,利用微生物发酵生产四碳有机酸具有反应条件温和、过程绿色环保等优势,具有广泛的应用前景。文中总结了四碳有机酸的生物合成途径和代谢机制,着重讨论了天然菌株生产四碳有机酸以及基于菌种选育和代谢工程改造策略提高四碳有机酸合成能力的研究进展及挑战,为四碳有机酸的高效生物合成提供广阔的参考方向。     

Enzyme research and applications in biotechnological intensification of biogas production

Biogas technology provides an alternative source of energy to fossil fuels in many parts of the world. Using local resources such as agricultural crop remains, municipal solid wastes, market wastes and animal waste, energy (biogas), and manure are derived by anaerobic digestion. The hydrolysis process, where the complex insoluble organic materials are hydrolysed by extracellular enzymes, is a rate-limiting step for anaerobic digestion of high-solid organic solid wastes. Biomass pretreatment and hydrolysis are areas in need of drastic improvement for economic production of biogas from complex organic matter such as lignocellulosic material and sewage sludge. Despite development of pretreatment techniques, sugar release from complex biomass still remains an expensive and slow step, perhaps the most critical in the overall process. This paper gives an updated review of the biotechnological advances to improve biogas production by microbial enzymatic hydrolysis of different complex organic matter for converting them into fermentable structures. A number of authors have reported significant improvement in biogas production when crude and commercial enzymes are used in the pretreatment of complex organic matter. There have been studies on the improvement of biogas production from lignocellulolytic materials, one of the largest and renewable sources of energy on earth, after pretreatment with cellulases and cellulase-producing microorganisms. Lipids (characterised as oil, grease, fat, and free long chain fatty acids, LCFA) are a major organic compound in wastewater generated from the food processing industries and have been considered very difficult to convert into biogas. Improved methane yield has been reported in the literature when these lipid-rich wastewaters are pretreated with lipases and lipase-producing microorganisms. The enzymatic treatment of mixed sludge by added enzymes prior to anaerobic digestion has been shown to result in improved degradation of the sludge and an increase in methane production. Strategies for enzyme dosing to enhance anaerobic digestion of the different complex organic rich materials have been investigated. This review also highlights the various challenges and opportunities that exist to improve enzymatic hydrolysis of complex organic matter for biogas production. The arguments in favor of enzymes to pretreat complex biomass are compelling. The high cost of commercial enzyme production, however, still limits application of enzymatic hydrolysis in full-scale biogas production plants, although production of low-cost enzymes and genetic engineering are addressing this issue.     

Microbial production of organic acids: expanding the markets

Microbial production of organic acids is a promising approach for obtaining building-block chemicals from renewable carbon sources. Although some acids have been produced for some time and in-depth knowledge of these microbial production processes has been gained, further microbial production processes seem to be feasible, but large-scale production has not yet been possible. Citric, lactic and succinic acid production exemplify three processes in different stages of industrial development. Although the questions being addressed by current research on these processes are diverging, a comparison is helpful for understanding microbial organic acid production in general. In this article, through analysis of the current advances in production of these acids, we present guidelines for future developments in this fast-moving field.     

氨基酸生产和海洋生物的氨基酸资源开发

氨基酸在医药、食品、饲料等领域有着极为重要和广泛的用途,世界上氨基酸总需求量以５～１０％递增,市场竞争十分激烈。生物资源提取、化学合成、生物合成和综合法是生产氨基酸的４种技术,目前的发展趋势为生物合成和综合法,特别是将现代生物工程技术应用于氨基酸生产。另外,氨基酸生产领域另一个新的倾向是海洋生物氨基酸资源的开发和应用,尤其是海洋生物所产生的特殊氨基酸、肽及其衍生物的开发,同时,综合利用海产品加工后的废弃物来生产氨基酸也受到重视。     

Enzyme research and applications in biotechnological intensification of biogas production

Biogas technology provides an alternative source of energy to fossil fuels in many parts of the world. Using local resources such as agricultural crop remains, municipal solid wastes, market wastes and animal waste, energy (biogas), and manure are derived by anaerobic digestion. The hydrolysis process, where the complex insoluble organic materials are hydrolysed by extracellular enzymes, is a rate-limiting step for anaerobic digestion of high-solid organic solid wastes. Biomass pretreatment and hydrolysis are areas in need of drastic improvement for economic production of biogas from complex organic matter such as lignocellulosic material and sewage sludge. Despite development of pretreatment techniques, sugar release from complex biomass still remains an expensive and slow step, perhaps the most critical in the overall process. This paper gives an updated review of the biotechnological advances to improve biogas production by microbial enzymatic hydrolysis of different complex organic matter for converting them into fermentable structures. A number of authors have reported significant improvement in biogas production when crude and commercial enzymes are used in the pretreatment of complex organic matter. There have been studies on the improvement of biogas production from lignocellulolytic materials, one of the largest and renewable sources of energy on earth, after pretreatment with cellulases and cellulase-producing microorganisms. Lipids (characterised as oil, grease, fat, and free long chain fatty acids, LCFA) are a major organic compound in wastewater generated from the food processing industries and have been considered very difficult to convert into biogas. Improved methane yield has been reported in the literature when these lipid-rich wastewaters are pretreated with lipases and lipase-producing microorganisms. The enzymatic treatment of mixed sludge by added enzymes prior to anaerobic digestion has been shown to result in improved degradation of the sludge and an increase in methane production. Strategies for enzyme dosing to enhance anaerobic digestion of the different complex organic rich materials have been investigated. This review also highlights the various challenges and opportunities that exist to improve enzymatic hydrolysis of complex organic matter for biogas production. The arguments in favor of enzymes to pretreat complex biomass are compelling. The high cost of commercial enzyme production, however, still limits application of enzymatic hydrolysis in full-scale biogas production plants, although production of low-cost enzymes and genetic engineering are addressing this issue.     

植物吸收利用有机氮营养研究进展

植物矿质营养学问世以后,人们一直认为无机氮是植物吸收氮素的主要形态.随着研究手段的改进和研究内容的不断深入,现已证实许多没有菌根的维管植物都可以直接吸收可溶性有机氮,特别是小分子的氨基酸.由此引起了人们对植物有机营养、植物营养方式多样化问题的重视.研究表明：氨基酸可以通过多种方式释放到土壤溶液中,土壤中的氨基酸主要来源于微生物、动植物及其代谢产物等.土壤氨基酸含量受土壤温湿度、所施的有机肥料、生长的植物种类及其生长发育时期的影响.植物对氨基酸的吸收是一个主动吸收过程,受载体调节,并与能量状况有关,同时受介质中pH和温度的影响.但是有关植物吸收氨基酸的机理及其生态过程还需进行深入的研究.     

Variation in nitrogen source utilisation by Pisolithus isolates maintained in axenic culture

The influence of various inorganic and organic nitrogen sources on biomass production by 17 isolates of Australian Pisolithus spp. was investigated before and after a 3-year maintenance period in axenic culture. While some isolates produced similar or higher amounts of biomass on NH₄ ⁺ or certain amino acids after the maintenance period, there was a general trend to reduced biomass production on these substrates. Biomass production by most isolates on bovine serum albumin increased significantly after maintenance. The data are discussed in relation to the use of axenic culture growth experiments for investigations of inter- and intraspecific physiological variation in ectomycorrhizal fungi. Accepted: 22 February 2001     

Comprehensive screening of Arabidopsis mutants suggests the lysine histidine transporter 1 to be involved in plant uptake of amino acids

Plant nitrogen (N) uptake is a key process in the global N cycle and is usually considered a "bottleneck" for biomass production in land ecosystems. Earlier, mineral N was considered the only form available to plants. Recent studies have questioned this dogma and shown that plants may access organic N sources such as amino acids. The actual mechanism enabling plants to access amino acid N is still unknown. However, a recent study suggested the Lysine Histidine Transporter 1 (LHT1) to be involved in root amino acid uptake. In this study, we isolated mutants defective in root amino acid uptake by screening Arabidopsis (Arabidopsis thaliana) seeds from ethyl methanesulfonate-treated plants and seeds from amino acid transporter T-DNA knockout mutants for resistance against the toxic D-enantiomer of alanine (Ala). Both ethyl methanesulfonate and T-DNA knockout plants identified as D-Ala resistant were found to be mutated in the LHT1 gene. LHT1 mutants displayed impaired capacity for uptake of a range of amino acids from solutions, displayed impaired growth when N was supplied in organic forms, and acquired substantially lower amounts of amino acids than wild-type plants from solid growth media. LHT1 mutants grown on mineral N did not display a phenotype until at the stage of flowering, when premature senescence of old leaf pairs occurred, suggesting that LHT1 may fulfill an important function at this developmental stage. Based on the broad and unbiased screening of mutants resistant to D-Ala, we suggest that LHT1 is an important mediator of root uptake of amino acids. This provides a molecular background for plant acquisition of organic N from the soil.     

Synthesis of biologically active lipids by fungus Mucor lusitanicus 306D grown on media with various composition

Growth and lipogenesis of fungus Mucor lusitanicus 306 D producing gamma-linolenic acid was studied under various regimes of nitrogen and carbon nutrition. Media containing food industry wastes such as maize extract, molasses, and protein hydrolysate were used. Content of gamma-linolenic acid was higher when using carbohydrates such as glucose and molasses as carbon sources and urea as a nitrogen source. At high glucose concentration (100 g/l), fed batch cultivation provided high content of gamma-linolenic acid in lipids (1 g/l). After extraction of lipids, fungus biomass contained 42% proteins with all essential amino acids. Defatted biomass was shown to be effectively assimilated by minks.     

Influences of fish on food web structure and function in mountain lakes

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Fermentation for future food systems: Precision fermentation can complement the scope and applications of traditional fermentation

Modern biotechnology holds great potential for expanding the scope of fermentation to create novel foods and improve the sustainability of food production.

The growing human population and global warming pose an impending threat for global food security (Linder, 2019). This has prompted a critical re‐examination of the food supply chain from producers to consumers in order to increase the overall efficiency of food production, storage and transport. Much research in plant science consequently aims to increase production with new, high‐yield crop, fruit and vegetable varieties better adapted to changing climatic conditions. Yet, there is also much room for improving food safety by minimising food losses and recycling waste, valorising by‐products, improving nutritional value and increasing storage time. This is where fermentation comes in as a cost‐efficient, versatile and proven technology that extends the shelf life of food products and enhances their nutritional content. Moreover, there is enormous potential in fermentation to further increase efficiency and product range and even create new food products from non‐food biomass.

… there is enormous potential in fermentation to further increase efficiency and product range and even create new food products from non‐food biomass.

In a broader sense, fermentation can be defined as the cultivation of microorganisms such as bacteria, yeasts and fungi to break down complex molecules into simpler ones, notably organic acids, alcohols or esters. In a practical sense, it is one of the oldest food processing technologies to increase storage life along with cooking, smoking or air‐drying: fermentation was already fully industrialised for producing beer and bread millennia ago in ancient Mesopotamia and Egypt. It is also an elegant and simple technology as these microorganisms do most of the work without much human involvement.Louis Pasteur’s discovery that microorganisms cause fermentation laid the basis for further improvement of the technology from traditional spontaneous fermentation to the use of defined starter cultures. Fermentation is now widely used to produce alcoholic beverages, bread and pastry, dairy products, pickled vegetables, soy sauce and so on. More recent advances based on genomics and synthetic biology include precision and biomass fermentation to produce specific compounds for the food and chemical industry or medicinal use. This is not the limit though: when combined with genomics, fermentation has even greater potential for creating novel foods and other products.     

The human muscle nicotinic acetylcholine receptor alpha-subunit exist as two isoforms: a novel exon.

Analysis of acetylcholine receptor clones isolated from a human leg muscle cDNA library, revealed that the alpha-subunit existed as two isoforms. A novel exon, coding for 25 amino acids, was located in the human genomic DNA sequence; its insertion into the alpha-subunit gives the new isoform of 462 amino acids. In addition, mRNAs for the two isoforms were found in equal proportions in poly(A)+ RNA obtained from three further sources including partially denervated and innervated human muscle and the rhabdomyosarcoma cell line TE671. Both protein isoforms can be expressed in E. coli. No evidence of a sequence related to that of the new exon was found in cDNA derived from poly(A)+ RNA isolated from fetal calf or embryonic chick muscle or Torpedo marmorata electric organ.