Charu Gupta* and Dhan Prakash
Amity Institute for Herbal Research and Studies, Amity University UP, Noida, India
*Corresponding author e‐mail: charumicro@gmail.com
Bacteria, yeast, and microalgae can act as catalysts for the production of food ingredients, enzymes, and nutraceuticals (Hugenholtz and Smid 2002). With the current trend towards natural ingredients, there is renewed interest in microbial flavors, colors, and bio‐processing using enzymes. Microbial production of substances such as organic acids, enzymes, proteins, vitamins, antibiotics, and hydrocolloids also remains important (Dufossé 2009). Lactic acid bacteria, in particular Lactococcus lactis, have been demonstrated to be ideal cell factories for the production of these important nutraceuticals. Developments in the genetic engineering of food‐grade micro‐organisms mean that the production of certain nutraceuticals can be enhanced or newly induced through overexpression and/or disruption of relevant metabolic genes (Bronzwaer 2008).
Microbes are used in the production of food flavors, carotenoids, flavonoids and terpenoids, enzymes, organic acids for use in food, viable probiotic cells, bacteriocins as food preservatives, amino acids and their derivatives for use in foods, nutraceuticals and medications, microbial polysaccharides for use in food, production of xylitol and other polyols, prebiotic oligosaccharides, polyunsaturated fatty acids, microalgae as sources of food ingredients and nutraceuticals, and production of vitamins.
Recently scientists have found that bacteria can also be a rich source of terpenes, an important ingredient in drugs and food additives. New research at Brown University, published in the Proceedings of the National Academy of Sciences, shows that the genetic capacity of bacteria to make terpenes is widespread (www.biotecnika.org/content/december‐2014/bacteria‐found‐be‐rich‐source‐terpenes‐important‐ingredient‐drugs‐food‐additiv?page=0,1). Using a specialized technique to sift through genomic databases for a variety of bacteria, the researchers found 262 gene sequences that likely code for terpene synthases, an enzyme that catalyzes the production of terpenes. The researchers then used several of those enzymes to isolate 13 previously unidentified bacterial terpenes. Genetically engineered Streptomyces bacterium is used as a bio‐refinery to generate the terpene products.
Micro‐organisms can be used as an adjunct therapy for diseases like protein energy malnutrition (PEM), anemia, diarrhea, cancer, obesity, ulcerative colitis, Crohn’s disease, irritable bowel syndrome, and gluten therapy resistant celiac.
Free radicals play an important role in the origin of numerous diseases, including lifestyle diseases and physiological diseases like high blood pressure, cancer, diabetes, cardiovascular, neurodegenerative, etc. Free radicals act by catalyzing the various toxic oxidative reactions that lead to the formation of toxic lipid peroxides. They also inhibit the enzymes of respiratory chain of mitochondria and damage DNA and proteins which is lethal for the cell. It is therefore important to search for newer alternatives to allopathic medicines with reduced or no side effects.
Antioxidants are biological molecules with the ability to protect vital metabolites from harmful oxidation. Due to this fascinating role, their beneficial effects on human health are of paramount importance. Traditional approaches using solvent‐based extraction from food/non‐food sources and chemical synthesis are often expensive, time‐consuming, and detrimental to the environment. There are numerous medicinal plants that have been reported to possess strong antioxidant activity along with their free radical scavenging activity. Besides plants, various microbes including bacteria and fungi also possess powerful antioxidant activity. Some of the microbes belong to the probiotic group that has the potential to protect the body from dangerous free radicals. Thus various micro‐organisms can be used as natural sources of antioxidants to develop unique functional foods for scavenging free radicals, thereby preventing many diseases (Gupta et al. 2013a). With the advent of metabolic engineering tools, the successful reconstitution of heterologous pathways in Escherichia coli and other micro‐organisms provides a more exciting and amenable alternative to meet the increasing demand for natural antioxidants (Lin et al. 2014).
The antioxidant activity of organic extracts of eight fungal species, Ganoderma lucidum, Ganoderma applanatum, Meripilus giganteus, Laetiporus sulphureus, Flammulina velutipes, Coriolus versicolor, Pleurotus ostreatus, and Panus tigrinus, was evaluated for free radical (DPPH• and OH•) scavenging capacity. The highest DPPH scavenging activity was found in the methanol extract of G. applanatum (12.5 μg/mL; 82.80%) and the chloroform extract of G. lucidum (510.2 g/mL, 69.12%). The same extract also showed the highest LP inhibition (91.83%, 85.09%) at 500 μg/mL, while the methanol extracts of G. applanatum and L. sulphureus showed the highest scavenging effect on OH• radicals (68.47%, 57.06%, respectively) at 400 μg/mL. The antioxidative potencies correlated generally with the total phenol content (0.19–9.98 mg/g) (Karaman et al. 2010).
In another study, probiotic strain Lactobacillus plantarum that was used as a starter culture in the production of dried fermented meat products was assessed for its antioxidant activity against peroxide radicals by Nedelcheva et al. (2010). It was found that the inclusion of this culture provided the desired fermentation process in the raw sausage mass and reduced pathogenic flora. The application of starter cultures with antioxidant activity preserved the color of the meat products and delivered substances with antioxidant activity as well (Nedelcheva et al. 2010). Similarly, Miang, a kind of traditional fermented tea leaves containing several kinds of Lactobacilli spp., was investigated for its antioxidant activity. The study suggested that both L. fermentum FTL2311 and L. fermentum FTL10BR strains could liberate certain substances that possessed antioxidant activity expressed as trolox equivalent antioxidant capacity (TEAC) and equivalent concentration (EC) values for free radical scavenging and reducing mechanisms, respectively (Klayraung and Okonogi 2009).
There has been a growing interest in the use of natural ingredients, particularly in the food industry. Ingredients like colors can also be derived from natural sources such as microbes that produce various pigments like carotenoids, melanins, flavins, quinones, prodigiosins and, more specifically, monascins, violacein or indigo (Gupta et al. 2011). There are various examples of bacteria, fungi, and yeast that are colored and can be used to produce natural colors. The major advantage of using microbes for color production over plant sources is that they can be mass multiplied because of their high growth rate. The other advantages of producing pigments from micro‐organisms include independence from weather conditions and geographic variability, easy and fast growth and colors of different shades can be obtained by growing on cheap substrates.
Before extracting colors from microbes, they are checked for safety and efficacy. Microbial colors should be non‐toxic as they play a significant role as food colorants. Fungal cell production offers reliable scalable technology.
Bacteria such as Serratia marcescens, S. rubidaea, Pseudomonas magneslorubra, Vibrio psychroerythrous, V. gazogenes, Bacillus sp., Alteromonas rubra, Rugamonas rubra, and gram‐positive actinomycetes, such as Streptoverticillium rubrireticuli and Streptomyces longisporus, produce colored secondary metabolites. The actinomycete Streptomyces coelicolor A3 produces a closely related linear tripyrrole, undecyl‐prodigiosin, and a cyclic derivative, butylmeta‐ cycloheptyl‐prodiginine, in a 2:1 ratio (Harris et al. 2004).
The red pigment of S. marcescens is known as prodigiosine, a non‐diffusible pigment attached to the inner membrane (Khanafari et al. 2006). Prodigiosin is a multifaceted secondary metabolite produced by S. marcescens, S. rubidaea, V. psychroerythrous, V. gazogenes, A. rubra, Lugomonas rubra and gram‐positive actinomycetes such as Streptoverticillium rubrireticuli and Streptomyces longisporus (Khanafari et al. 2006). This pigment possesses antifungal, immunosuppressive, antiproliferative, and anticancer activity (Pandey et al. 2007).
Color‐producing fungi include Penicillium, Blakeslea trispora, Xanthophyllomyces dendrorhous, Phycomyces blakesleeanus car S mutant, Ashbye gossypii, Eremothecium ashbyii, Epicoccum sp., and genetically modified fungus Fusarium sporotrichioides. Similarly, some examples of color‐producing yeast are Xanthophyllomyces dendrorhous and Rhodotorula. They produce astaxanthin pigment that imparts an orange‐red color and Rhodotorula produces torulene, torularhodin, and carotene pigment (Dufossé 2009). Color‐producing algae include Dunaliella salina, D. bardwil (produces β‐carotene), Haematococcus lacustris, and H. pluvialis compactin resistant mutant (produces astaxanthin and canthaxanthin) (Chattopadhyay et al. 2008; Dufossé 2009).
Studies have shown that microbes have the potential to produce some essential amino acids. Metabolic engineering constantly improves the productivity of amino acid‐producing strains, mainly Corynebacterium glutamicum and Escherichia coli strains. Classic mutagenesis and screening have been accelerated by combination with intracellular metabolite sensing. Synthetic biology approaches have allowed access to new carbon sources to realize a flexible feedstock concept. Moreover, new pathways for amino acid production as well as fermentative production of non‐native compounds derived from amino acids or their metabolic precursors have been developed. These include dipeptides, α,ω‐diamines, α,ω‐diacids, keto acids, acetylated amino acids, and ω‐amino acids (Wendisch 2014).
There are numerous examples of bacteria that can produce different types of amino acids. Mycosporines and mycosporine‐like amino acids (MAAs), including shinorine (mycosporine‐glycine‐serine) and porphyra‐334 (mycosporine‐glycine‐threonine), are UV‐absorbing compounds produced by cyanobacteria, fungi, and marine micro‐ and macroalgae. MAAs have the ability to protect these organisms from damage by environmental UV radiation. Its structure elucidation revealed that the novel MAA is mycosporine‐glycine‐alanine, which substitutes l‐alanine for the l‐serine of shinorine (Miyamoto et al. 2014).
In another recent study, it was found that a microbial‐like pathway contributes to phenylalanine biosynthesis in plants via a cytosolic tyrosine: phenyl‐pyruvate aminotransferase (Yoo et al. 2013). Phenylalanine is a vital component of proteins in all living organisms and in plants is a precursor for thousands of additional metabolites. Animals are incapable of synthesizing phenylalanine and must obtain it directly or indirectly from plants. While plants can synthesize phenylalanine in plastids through arogenate, the contribution of an alternative pathway via phenylpyruvate, as it occurs in most microbes, has not been demonstrated. This study established that plants also utilize the microbial‐like phenylpyruvate pathway to produce phenylalanine, and flux through this route is increased when the entry point to the arogenate pathway is limiting. It was found that the plant phenylpyruvate pathway utilizes a cytosolic aminotransferase that links the co‐ordinated catabolism of tyrosine to serve as the amino donor, thus interconnecting the extraplastidial metabolism of these amino acids. This discovery unlocks another level of complexity in the plant aromatic amino acid regulatory network, unveiling new targets for metabolic engineering.
The bacteria produce amino acids but they also help in reducing D‐amino acid toxicity with the help of their enzyme “racemases.” It is known that D‐amino acids are toxic for life on earth, yet they are formed constantly due to geochemical racemization and bacterial growth (the cell walls of which contain D‐amino acids). Studies have shown that D‐amino acids are recycled by some bacteria, which use D‐amino acids as a source of nitrogen by running enzymatic racemization in reverse. Consequently, when soils are inundated with racemic amino acids, resident bacteria consume D‐ as well as L‐enantiomers, either simultaneously or sequentially depending on the level of their racemase activity. Bacteria thus protect life on earth by keeping environments free of D‐amino acid (Zhang and Sun 2014).
All bacteria are capable of reverse racemization. Specifically, when soils are inundated with LD‐alanine, LD‐aspartic acid, LD‐glutamic acid, and LD‐leucine, resident bacteria absorb L‐ and D‐enantiomers in equal or nearly equal rates. In the case of alanine, the conversion of D‐enantiomers appears to be enabled by constitutive alanine racemases that are ordinarily anabolic in function (i.e., cell wall synthesis). In the case of the other three amino acids, exposure to L‐enantiomers appears to induce catabolic racemases. As a result, as soon as L‐enantiomers are exhausted, the organisms could consume D‐enantiomers (Zhang and Sun 2014).
Humans and other organisms have essential requirements for a range of different vitamins to assist in metabolic pathways, and as such, a plentiful dietary intake of vitamins is linked with improved health. Vitamins are essential micronutrients. Their key role is to enter the biochemical processes as co‐enzymes. The B‐group or B‐complex vitamins include thiamin (B1), riboflavin (B2), niacin (B3), pyridoxine (B6), pantothenic acid (B5), biotin (B7 or H), folate (B11–B9 or M) and cobalamin (B12). These molecules are water soluble and play an important role in metabolism, particularly the cellular metabolism of carbohydrates (thiamin), proteins and fats (riboflavin and pyridoxine). B‐group vitamins, normally present in many foods, can be easily removed or destroyed during cooking and food processing, so their deficiency is rather common in the human population. For this reason, several countries have adopted laws requiring the fortification of certain foods with specific vitamins and minerals (Burgess et al. 2009; LeBlanc et al. 2010).
Where humans have vitamin deficiencies, supplementation with non‐food vitamins is a standard remedy. Addition of vitamins to animal feeds also promotes healthy livestock. Microbes possess the simplicity and flexibility needed for economic vitamin production. Many micro‐organisms can be genetically modified to overexpress enzymes in the synthesis pathway of various vitamins. With the ability to grow on cheap feedstocks and produce an improved yield compared to chemical synthesis, microbial production of vitamins also boasts a much lower cost in terms of the energy input necessary. Maintaining microbial fermentation conditions is significantly less expensive than the different conditions needed for a variety of reaction steps in the chemical process.
The adaptability of lactic acid bacteria (LAB) to the fermentation process, their biosynthetic capacity and metabolic versatility are some of the principal features that facilitate the application of LAB in foods for producing, releasing and/or increasing specific beneficial compounds. Among these, vitamin production by LAB has recently gained attention. The proper selection and exploitation of nutraceutical‐producing LAB is an interesting strategy to produce novel fermented foods with increased nutritional and/or health‐promoting properties. Fermented milks or bread with high levels of B‐group vitamins (such as folate and riboflavin) can be produced by LAB‐promoted biosynthesis (Arena et al. 2014).
Vitamin production by lactobacilli is becoming a focus point to obtain novel fermented foods through nutraceutical‐producing LAB (Capozzi et al. 2012; LeBlanc et al. 2011). Several lactobacilli have the genes involved in riboflavin biosynthesis, although the genetic capability to biosynthesize riboflavin is species and/or strain specific. In fact, the rib operon has been shown to have interruptions in some bacteria, for example L. plantarum WCFS1, resulting in the inability to produce riboflavin (Capozzi et al. 2011).
Conversely, L. plantarum NCDO 1752, L. plantarum JDMI and others strains of L. plantarum recently isolated from cereals‐derived products (Burgess et al. 2006; Capozzi et al. 2011) can grow in the absence of riboflavin since they are capable of synthesizing this protein. The riboflavin operon in all lactobacilli studied displays the same gene organization coding for a riboflavin‐specific deaminase and reductase (ribG), a riboflavin synthase α subunit (ribB), a bifunctional enzyme which also catalyzes the formation of 3,4‐dihydroxy‐2‐butanone 4‐phosphate from ribulose 5‐phosphate (ribA), and a riboflavin synthase β subunit (ribH). There are still challenges that face microbial production of vitamins that are difficult to address. For example, in engineered microbes that can express every enzyme necessary to produce the desired vitamin, allosteric inhibition from the vitamin or intermediates in the synthesis pathway can hamper the yield. Also, particularly in the case of riboflavin production, very little of the vitamin is secreted by the microbes, meaning that the cultured cells must be lyzed in order to obtain the vitamin and replaced with a new culture (i.e., difficulties in feasibility of continuous culture).
Global population increase in the past few decades has intensified protein malnutrition especially in the developing world where agricultural industry is not reliable. Promising biotechnological methods have been established to alleviate the world’s protein deficit.
Single cell protein (SCP) production from lignocellulose biomass is part of the upcoming technology aimed at providing protein supplement for both human food and animal feeds. Micro‐organisms such as algae, fungi, yeast, and bacteria are involved in the bioconversion of low‐cost feed stocks such as lignocelluloses to produce biomass rich in proteins and amino acids. Even the cladodes of Opuntia ficus indica (cactus pear) have potential for SCP production in arid and semi‐arid regions (Gabriel et al. 2014). Various bacteria, molds, yeasts, and algae have been employed for the production of SCP. The bacteria include Brevibacterium, Methylophilus methylitrophus, Achromobacter delvaevate, Acinetobacter calcoacenticus, Aeromonas hydrophilla, Bacillus megaterium, B. subtilis, Lactobacillus sp., Cellulomonas sp., Methylomonas methylotrophus, Pseudomonas fluorescens, Rhodopseudomonas capsulata, Flavobacterium sp., Thermomonospora fusca and others. Some of the algae used are Chlorella pyrenoidosa, C. sorokiana, Chondrus crispus, Scenedesmus acutus, Porphyrium sp. and Spirulina maxima (Mahasneh 1997). The filamentous fungi that have been used include Chaetomium celluloliticum, Fusarium graminearum, Aspergillus fumigatus, A. niger, A. oryzae, Cephalosporium cichhorniae, Penicillium cyclopium, Rhizopus chinensis, Scytalidium acidophilum, Tricoderma viridae, T. alba, and Paecilomyces varioti. Yeasts such as Candida utilis (Torula yeast), C. lipolytica, C. tropicalis, C. novellas, C. intermedia, and Saccharomyces cerevisiae are among the various organisms that have been used for the production of SCP (Becker 2007). The micro‐organisms to be cultured should be non‐pathogenic, must have good nutritional values, must be usable as food and feed, should not contain toxic compounds, and production costs should be low. For example, Quorn, a leading brand of mycoprotein food product in the UK and Ireland, is extracted from a fungus, Fusarium venenatum. It is high in protein and dietary fiber and low in saturated fat and salt.
In a recent study, researchers demonstrated the production of SCP from different varieties of orange peel using Aspergillus niger and Saccharomyces cerevisiae. The bioconversion of fruit wastes into valuable products like SCP has the ability to solve the worldwide food protein deficiency by obtaining an economical product for food and feed. In addition, using wastes as a substrate for the production of high nutritious product may also alleviate environmental pollution to some extent (Azam et al. 2014).
In another study, the conversion of the enzymatic hydrolyzate of shellfish chitin to SCP was investigated. The end product of chitin hydrolysis by Penicillium ochrochloron chitinase was mainly N‐acetyl‐D‐glucosamine; its further utilization as a substrate for SCP production using Yarrowia lipolytica NCIM 3450 was studied. The 2% chitin hydrolyzate was found to be optimal for SCP production. Fish diets were formulated to replace fishmeal partially by SCP from Yarrowia lipolytica using chitin hydrolyzate in diets of Lepidocephalus thermalis. The result indicated that a 50% yeast SCP diet gave a better growth response in Lepidocephalus thermalis than other formulations (Patil and Jadhav 2014).
The biopreservation of foods using bacteriocinogenic LAB from foods is an innovative approach. Biopreservation or biocontrol refers to the use of natural or controlled microbiota or its antibacterial products to extend the shelf‐life and enhance the safety of foods (Stiles 1996). Since LAB occur naturally in many food systems and have a long history of safe use in fermented foods, and are thus classed as Generally Regarded As Safe (GRAS), they have great potential for extended use in biopreservation.
One such class is defined as bacteriocins. Bacteriocins are heat‐stable ribosomally synthesized antimicrobial peptides produced by various bacteria, including food‐grade LAB. These antimicrobial peptides have huge potential as both food preservatives and next‐generation antibiotics targeting multiple drug‐resistant pathogens (Perez et al. 2014). LAB bacteriocins are inherently tolerant to high thermal stress and are known for their activity over a wide pH range. These antimicrobial peptides are also colorless, odorless, and tasteless, which further enhances their potential usefulness. Bacteriocins have a fast‐acting mechanism, which forms pores in the target membrane of bacteria, even at extremely low concentrations. They are also easily degraded by proteolytic enzymes due to their proteinaceous nature. Therefore, bacteriocin fragments do not live long in the human body or in the environment, which minimizes the opportunity of target strains to interact with the degraded antibiotic fragments; this is the common starting point in the development of antibiotic resistance. The most significant advantage of bacteriocins over conventional antibiotics is their primary metabolite nature since they have relatively simple biosynthetic mechanisms compared with conventional antibiotics, which are secondary metabolites. This fact makes them easily amenable through bioengineering to increase either their activity or specificity towards target micro‐organisms (Perez et al. 2014).
Besides bacteriocins, LAB also produce a wide variety of other active antagonistic metabolites such as organic acids (lactic, acetic, formic, propionic, butyric, hydroxyl‐phenyl‐lactic, and phenyllactic), diverse antagonistic compounds (carbon dioxide, ethanol, hydrogen peroxide, fatty acids, acetoin, diacetyl, reuterin, reutericyclin), antifungal compounds (propionate, phenyl‐lactate, hydroxyphenyl‐lactate, cyclic dipeptides, phenyllactic acid, and 3‐hydroxy fatty acids), and bacteriocins (such as nisin, pediocins, lacticins, enterocins and many others) (Oliveira et al. 2014). Other bacterial groups (especially those from the genus Bacillus) are also attracting attention because of the diversity of antimicrobial peptides they produce, some of which could also be exploited as biopreservatives.
Another compound produced by the group of LAB is reutericyclin. This unique tetramic acid is a negatively charged, highly hydrophobic antagonist. Reutericyclin acts as a proton ionophore, resulting in dissipation of the proton motive force (Gänzle 2004). It lacks activity towards yeasts and fungi, but is active on gram‐positive bacteria including Lactobacillus spp., Bacillus subtilis, B. cereus, Enterococcus faecalis, S. aureus, and Listeria innocua. Spore germination of Bacillus species was inhibited by this antimicrobial compound, but the spores remained unaffected under conditions that do not permit germination. As in many other antagonists, inhibition of gram‐negative bacteria (E. coli and Salmonella) is observed under conditions that disrupt the outer membrane, including truncated lipopolysaccharides (LPS), low pH and high salt concentrations. Reutericyclin was shown to be produced in concentrations active against competitors during growth of Lactobacillus reuteri in sourdough. It was proposed that reutericyclin‐producing strains may have applications in the biopreservation of foods (Gänzle 2004).
Cancer is a group of diseases characterized by unregulated growth and spread of abnormal cells, which can result in death if not controlled (Bicas et al. 2008). Sauerkraut can be an important part of diets designed for healing cancer. Sauerkraut is fermented white cabbage. Lacto‐fermented cabbage has a long history of providing benefits for many different health conditions, and now it is proving to be beneficial for cancer. Cabbage, by itself, offers a number of health benefits, but the fermentation process increases the bioavailability of nutrients, rendering sauerkraut even more nutritious than the original cabbage (Lipski 2013).
Epidemiological studies have shown that consumption of cabbage and sauerkraut is connected with significant reduction of breast cancer incidence. Estrogens are considered a major breast cancer risk factor and their metabolism by P450 enzymes substantially contributes to carcinogenic activity. The effect of cabbage and sauerkraut juices of different origin on the expression profile of the estrogen metabolism key enzymes (CYP1A1, CYP1A2, CYP1B1) was investigated in breast cell lines MCF7, MDA‐MB‐231, and MCF10A. The effects of cabbage juices were compared with that exerted by indole‐3‐carbinol (I3C) and 3,3'‐diindolylmethane (DIM). Treatment with cabbage juices or indoles for 72 hours affected the expression of CYP1 family genes in a cell type‐dependent manner. The results supported epidemiological observations and partly explained the mechanism of the chemopreventive activity of white cabbage products (Szaefer et al. 2012a).
In another study, it was shown that the breakdown products of glucosinolates in cabbage may affect both the initiation phase of carcinogenesis, by decreasing the amount of DNA damage and cell mutation, and the promotion phase, by blocking the processes that inhibit programmed cell death and stimulate unregulated cell growth. A study published in 2012 in the journal Nutrition Cancer showed that consumption of cabbage and sauerkraut is connected with significant reduction of breast cancer incidence. This research supported the observation that the consumption of sauerkraut was beneficial for the prevention of breast cancer in women (Licznerska et al. 2013; Szaefer et al. 2012b).
The anticancer enzyme glutaminase is derived from a microbial source. Glutaminase is widely distributed in micro‐organisms including bacteria, yeasts, and fungi. It is a potent antileukemic agent. L‐Glutaminase, in combination with or as an alternative to asparaginase, could be of significance in enzyme therapy for cancer, especially acute lymphocytic leukemia. The enzyme mainly catalyzes the hydrolysis of amido bond of L‐glutamine. In addition, some enzymes also catalyze glutamyl transfer reaction. A highly savory amino acid, L‐glutamic acid and a taste‐enhancing amino acid of infused green tea, theanine can be synthesized by employing a hydrolytic or transfer reaction catalyzed by glutaminase. Therefore, glutaminase is one of the most important flavor‐enhancing enzymes. The detection of glutamine was accomplished by a coupled enzyme system composed of glutaminase plus glutamate oxidase, while the detection of glutamic acid was carried out by a single enzyme: glutamate oxidase.
However, microbial sources are preferred for large‐scale production. Major genera of micro‐organisms reported to produce glutaminase are bacteria species including Acetobacter liquefaciens, Micrococcus luteus, Achromobacter sp., Nocardia sp., Acinetobacter glutaminasificans, Proteus morganii, Aerobacter aerogenes, Proteus vulgaris, Streptomyces netropsis, Erwinia carotovora, Streptomyces olivochromogenes, Escherichia coli, Vibrio cholerae, Flavobacterium flavescens, Vibrio costicola, Micrococcus glutamicus, Xanthomonas juglandis, and Micrococcus lysodeikticus. Yeasts include Cryptococcus albidus, Candida scottii, Cryptococcus nodaensis, Cryptococcus sp. and Debaryomyces sp. and fungi like Aspergillus oryzae, Tilachlidium humicola, and Verticillium malthousei (Nandakumar et al. 2003).
Drugs for cancer treatment show non‐specific toxicity to proliferating normal cells, possess severe side effects, and are not effective against many forms of cancer (Gangadevi and Muthumary 2008). Thus, the treatment of cancer has been enhanced mainly by improvements in diagnosis which allow earlier and more precise therapy (Pasut and Veronesi 2009). The anticancer properties of several secondary metabolites from endophytes have been investigated. Taxol, a diterpenoid, is a well‐known anticancer compound isolated from Taxus brevifolia. These trees are rare, slow growing, and produce small amounts of taxol, and hence expensive when obtained from their natural source (Gangadevi and Muthumary 2008). However, an endophyte called Taxomyces andreanae has provided an alternative approach to obtain cheaper and more available taxol through micro‐organism fermentation. Another important drug for cancer is the alkaloid camptothecin, a potent antineoplastic agent. Camptothecin and 10‐hydroxycamptothecin are two important precursors for the synthesis of the clinically useful anticancer drugs topotecan and irinotecan (Uma et al. 2008). The products were obtained from the endophytic fungi Fusarium solani isolated from Camptotheca acuminata (Kusari et al. 2009b; Shukla et al. 2014).
Phenylpropanoids have attracted much interest for use as anticancer, antioxidant, antimicrobial, anti‐inflammatory, and immunosuppressive agents (Korkina 2007). The endophytic Penicillium brasilianum, found in root bark of Melia azedarach, promotes the biosynthesis of phenylpropanoid amides (Fill et al. 2010). Likewise, two monolignol glucosides, coniferin and syringin, are not only produced by the host plant but were also recognized by the endophytic Xylariaceae species as chemical signals during the establishment of fungus plant interactions. Koshino and his co‐workers in 1988 characterized two phenylpropanoids and lignin from stromata of Epichloe typhina.
Lignans are another kind of anticancer agent originated as secondary metabolites through the shikimic acid pathway and display different biological activities that make them interesting in several lines of research (Gordaliza et al. 2004). Although their molecular backbone consists only of two phenylpropane units (C6‐C3), lignans show enormous structural and biological diversity, especially in cancer chemotherapy (Korkina 2007). Some well‐known alkaloids were first reported to be present in an endophytic fungus, Alternaria sp., isolated from the phloem of Catharanthus roseus that had the ability to produce vinblastine. Later, Zhang et al. (2000) discovered an endophytic Fusarium oxysporum from the phloem of C. roseus that could produce vincristine. Yang et al. (2004) also found an unidentified vincristine‐producing endophytic fungus from the leaves of C. roseus. The aryl tetralin lignans, such as podophyllotoxin, are naturally synthesized by Podophyllum sp., are clinically relevant mainly due to their cytotoxicity and antiviral activities and are also valued as the precursor to useful anticancer drugs like etoposide, teniposide, and etopophos phosphate (Kour et al. 2008; Kusari et al. 2009a).
Another study reported on a novel fungal endophyte, Trametes hirsuta, that produces podophyllotoxin and other related aryl tetralin lignans with potent anticancer properties (Puri et al. 2006). Novel microbial sources of podophyllotoxin were reported from the endophytic fungi Aspergillus fumigatus. A new compound, ergoflavin, of the ergochrome class, was isolated from endophytic fungi growing on the leaves of an Indian medicinal plant, Mimusops elengi, family Sapotaceae (Deshmukh et al. 2009). Secalonic acid D, a mycotoxin also belonging to the ergochrome class, has shown potent anticancer activities. It was isolated from the mangrove endophytic fungus and demonstrated high cytotoxicity on HL60 and K562 cells by inducing leukemia cell apoptosis (Zhang et al. 2009). Wagenaar and co‐workers reported identification of three novel cytochalasins, bearing antitumor activity, from the endophyte Rhinocladiella sp.
Finally, many compounds with anticancer properties isolated from endophytic microbes have been reported such as cytoskyrins, rubrofusarin B, phomoxanthones A and B, photinides A–F (Ding et al. 2009), and (+)‐ epiepoxydon (Shukla et al. 2014).
Diabetes is a common and sometimes fatal disease that occurs when the supply of insulin is insufficient for the body to break down sugar properly. The majority of insulin used to manage diabetes is produced using biotechnology. Bacterial cells are genetically modified to produce large quantities of human insulin, which is then purified for therapeutic use. Millions of people worldwide now use Humuline, which is a major brand name for “human” insulin produced using genetically modified (GM) bacteria (Bajzer and Seeley 2006).
Researchers have created a strain of non‐pathogenic E. coli bacteria that produce a protein called GLP‐1. This protein triggers cells in the pancreas to make insulin. In one study, scientists fed the engineered bacteria to diabetic mice. After 80 days, the mice went from being diabetic to having normal glucose blood levels. Diabetic mice that were not fed the engineered bacteria still had high blood sugar levels. The promise is that a diabetic could eat yoghurt or drink a smoothie as glucose‐responsive insulin therapy rather than relying on insulin injections. Creating bacteria that produce the protein has a number of advantages over using the protein itself as the treatment. The bacteria can secrete just the right amount of the protein in response to conditions in the host that could ultimately minimize the need for self‐monitoring and allow the patient’s own cells (or the cells of the commensal E. coli) to provide the appropriate amount of insulin when needed (Bronzwaer 2008; Cani et al. 2009).
Gut‐friendly microbes have been engineered to make a specific protein that can help to regulate blood sugar in diabetic mice. Although this research is still in the very early stages, the microbes can be grown in yoghurt and may provide an alternative treatment for people with diabetes (Hossain et al. 2007). Probiotics are cheap, less than a dollar per dose. This is especially helpful in Third World countries where cheaper delivery methods of treatment could be easily distributed.
Interestingly, results from several genomic, metagenomic and metabolomic studies have provided substantial information to target gut microbiota by dietary interventions for the management of type 2 diabetes (T2D). Recent studies have proposed multifactorial interventions including dietary manipulation in the management of T2D. The same interventions have also been recommended by many national and international diabetes associations. These studies are aimed at deciphering the gut microbial influence on health and disease (Turnbaugh et al. 2009).
Probiotics, particularly lactobacilli and bifidobacteria, have recently emerged as prospective biotherapeutics with proven efficacy demonstrated in various in vitro and in vivo animal models adequately supported with their established multifunctional roles and mechanism of action for the prevention and treatment of disease. Dietary intervention in conjunction with probiotics is a novel multifactorial strategy to abrogate progression and development of diabetes that holds considerable promise through improving the altered gut microbial composition and targeting all the possible risk factors (Panwar et al. 2013). However, protein‐based drugs can be very expensive to make and often strike problems as they degrade during digestion (Gupta et al. 2013b, 2014).
Anemia is more prevalent in developing and Third World countries because of malnutrition, poor availability of iron, and chronic blood loss. The other factors of anemia are deficiency in folic acid, vitamin B12, and iron. Vitamin B12, along with folate, is involved in making the heme molecule, an integral part of hemoglobin. Vitamin B12 is important in living beings and it is used to treat pernicious anemia and peripheral neuritis. Folates perform important roles as co‐factors in 1‐carbon transfer reactions occurring in purine and pyrimidine biosynthesis. They are also required for efficient DNA replication, repair, and methylation. For these key roles in the cellular cycle, tissues with a high cell growth rate or turnover, such as hemopoietic cells and intestinal mucosa, have a high requirement for folate. Deficiency leads to megaloblastic anemia, resulting in inhibition of DNA synthesis in red blood cell production (Bronzwaer 2008).
There are some folic acid‐producing bacteria and yeasts that can be cultured in whey or milk plasma, thereby accumulating high concentrations of folic acid in the medium. The various identified bacteria that produce and enhance the uptake of folic acid are Lactococcus lactis subsp. cremoris, L. lactis subsp. lactis, Bifidobacterium adolescentis, B. pseudocatenulatum, and yeasts like Candida famata, C. guilliermondii, C. glabrata, Yarrowia lipolytica, Saccharomyces cerevisiae, Pichia glucozyma, and Yarrowia lipolytica. The vitamin B12‐producing bacteria are Pseudomonas denitrificans and Propionibacterium shermanii (Kassinen et al. 2007). Application of probiotic bacteria for the prevention of megaloblastic anemia is a novel scientific approach that involves lactic acid‐fermented foods, increases iron absorption by optimization of pH in the digestive tract, activates enzyme phytases, and produces organic acids and other digestive enzymes (Scarpignato 2008).
A strain of probiotic bacteria developed by Swedish firm Probi doubled the absorption of iron from food in women. Lactobacillus plantarum 299v (Lp299v) not only helps our digestive system but also helps our immune system. It is also good for the heart and helpful in reducing gas and bloating conditions. It improves bowel movement by making it more normal and regular. It also reduces the negative effects of antibiotic drugs on colonic fermentation (Liu et al. 2001).
Several medicines are produced using genetically engineered bacteria or fungi that synthesize the medicine in giant bioreactors. Erythropoietin can be man‐made in bioreactors by bacteria and is used to treat anemia but the treatment requires frequent and sometimes daily injections. Researchers have purified specialized cells from human blood that normally repair the lining of blood vessels. These cells were also genetically engineered to express erythropoietin. They were then mixed with mesenchymal stem cells, which are able to form blood vessels. This mixture was then injected underneath the skin of anemic mice. The cell mixture spontaneously formed networks of blood vessels underneath the skin. The vessel lining secreted erythropoietin and cured anemia in both types of mice (Gupta et al. 2014; Kassinen et al. 2007).
Obesity is a major public health concern, caused by a combination of increased consumption of energy‐dense foods and reduced physical activity, with contributions from host genetics, environment, and adipose tissue inflammation. In recent years, the gut microbiome has also been found to be implicated and research in mice and humans has attributed to it both the manifestation and/or exacerbation of this major epidemic and vice versa. At the experimental level, analysis of fecal samples revealed a potential link between obesity and alterations in the gut flora (drop in Bacteroidetes and increase in Firmicutes), the specific gut microbiome being associated with the obese phenotype. Efforts to identify new therapeutic strategies to modulate gut microbiota would be of high priority for public health, and to date, probiotics and/or prebiotics seem to be the most effective tools (Katerina et al. 2014).
Lactobacillus (L. sporogenes and L. acidophilus NCFB 1748) and Bifidobacterium genus representatives have been reported to play a critical role in weight regulation as an antiobesity effect in experimental models and humans (Mercenier et al. 2002). Lactobacillus sporogenes has the ability to lower cholesterol levels. It produces a significant reduction in low‐density lipoprotein (LDL) levels and a small but significant increase in high‐density lipoprotein (HDL) cholesterol. L. sporogenes can be used as a side effect‐free alternative to drug therapy in the treatment of high cholesterol and heart disease. Clinical studies have revealed that L. sporogenes can be successfully implanted in the intestine. L. sporogenes satisfies the essential requirement of an efficient probiotic. The spores of L. sporogenes are resistant to heat and other adverse environmental conditions. This property of spore formation by L. sporogenes is the main characteristic that makes it the probiotic of choice in clinical applications. Being sporulated, they germinate under favorable conditions and produce sufficient viable cells which proliferate and perform vital healthful functions. In addition, L. sporogenes spores are semi‐resident and are slowly excreted out of the body. L. sporogenes is effective in the form of dietary supplements as well as when added to food products (Gupta et al. 2014; Montrose and Floch 2005).
A literature study was conducted focusing on clinical trials that examined the effect of specific micro‐organisms on body weight control. Analysis of the eligible articles suggested that Lactobacillus gasseri SBT 2055, L. rhamnosus ATCC 53103, and the combination of L. rhamnosus ATCC 53102 and Bifidobacterium lactis Bb12 may reduce adiposity, body weight, and weight gain. This suggests that these microbial strains can be applied in the treatment of obesity. Furthermore, short chain fatty acid production and low‐grade inflammation were found as the underlying mechanisms of action that influence metabolism and affect body weight. These findings might contribute to the development of probiotic treatments for obesity. Further research should be directed to the most effective combination and dosage rate of probiotic micro‐organisms (Mekkes et al. 2014).
Another potential microbiome‐altering strategy is the incorporation of modified bacteria that express therapeutic factors into the gut microbiota. For example, N‐acetylphosphatidylethanolamines (NAPEs) are precursors to the N‐acylethanolamide (NAE) family of lipids, which are synthesized in the small intestine in response to feeding and reduce food intake and obesity. A study demonstrated that administration of engineered NAPE‐expressing E. coli Nissle 1917 bacteria in drinking water for 8 weeks reduced the level of obesity in mice fed a high‐fat diet. Mice that received modified bacteria had dramatically lowered food intake, adiposity, insulin resistance, and hepatosteatosis compared with mice receiving standard water or control bacteria (Zhongyi et al. 2014).
Plants also represent the most studied natural source of antiobesity bioactives. Camellia sinensis is the most representative species exerting several antiobesity effects. Well‐known probiotics (bacteria which bestow health benefit), such as strains of Bifidobacterium and Lactobacillus families, and certain prebiotics (non‐viable food components that confer a health benefit on the host associated with modulation of microbiota effects), such as insulin‐type fructans, have also shown capability to combat obesity. Finally, compounds from animal sources, in particular bioactive peptides from milk‐derived whey and casein protein digestion, high dietary calcium and ω‐3s polyunsaturated fatty acids (ω‐3 PUFA) present in fish oils, have also shown potential antiobesity effects (Cristina et al. 2013).
Another study reported that there is a virus that causes obesity. Adenovirus‐36 (Adv 36), commonly known as a human “common cold virus,” is easily caught from an infected person who is coughing or sneezing. It is believed that at least one‐third of people affected by obesity have been infected. Researchers at the University of Wisconsin began experimenting with Adv36 around 1995 and found that when they experimentally infected chickens and mice, the animals increased their body fat by 50–150%. Compared to uninfected animals, about 60–70% of infected animals became obese. The mode of action of this virus is that the DNA (genetic material) of the virus gets into the fat cells of the person or animal and causes them to bring in more fat and glucose from the blood and to make fat out of the glucose. The viral DNA also causes adult stem cells in the fat tissue to turn into fat cells, so the total fat cell number increases. Thus, an infected person will have bigger fat cells and more of them. Scientists have figured out the sequence of the DNA in Adv36, which gene in the virus causes the effect and how this gene changes the chemistry inside fat cells to cause obesity. Research is ongoing to identify antiviral agents that appear to work against Adv36 infection. A vaccine has been developed that is still in very early studies, to prevent infection with Adv36. More research is needed, but it appears that eventually we will be able to prevent Adv36‐induced obesity (Cristina et al. 2013)).
An allergy is a hypersensitivity disorder of the immune system. Symptoms include redness, itchiness, and runny nose, eczema, hives, or an asthma attack. Allergies can play a major role in conditions such as asthma. In some people, severe allergies to environmental or dietary allergens or to medication may result in life‐threatening reactions called anaphylaxis (Kay 2000).
The rapid increase in immune‐mediated disorders such as allergic disease is strongly linked to reduced early microbial exposure (Bach 2002). The gut microbiota represents the greatest microbial exposure by far and is central to the development of immune regulation. The specific composition of the gut microbiota may affect the risk of developing allergic disease (Penders et al. 2007). This finding provided the foundation for intervention studies designed to modify gut microbial composition for the treatment of allergic disease. The effects of beneficial bacteria (probiotics) or resistant starches or fiber (prebiotics) that selectively stimulate a limited number of beneficial bacteria have been evaluated in allergy treatment studies.
Studies have shown that probiotics and prebiotics can be used for the treatment of allergic diseases (Johannsen and Prescott 2009; Ozdemir 2010; Tang et al. 2010; Thomas and Greer 2010; Yao et al. 2010). In an experimental mouse model, researchers discovered a highly interesting effect that mice who were very susceptible to neurodermatitis developed the disease less frequently when a lyzate of certain pathogens was applied to the skin. The susceptibility of the lyzate‐treated mice to developing neurodermatitis was considerably lower than that of control mice. More detailed analyses showed that the animals had a higher concentration of immune‐modulating interleukin‐10 and a lower concentration of the proinflammatory mediator interferon‐γ. Interferon‐γ is secreted when the immune system recognizes foreign invaders and fine‐tunes the immune system to effectively get rid of foreign intruders. The secretion of interferon‐γ causes inflammatory reactions that create a hostile environment for the intruders, thereby initiating their elimination (Thomsen et al. 2007). Non‐pathogenic and probiotic bacteria do not cause inflammation, but are nevertheless recognized by the human immune system. The elevated production of interleukin‐10 in certain immune system cells leads to anti‐inflammatory reactions, which in turn create an active immunological tolerance. This means that the human body is able to learn and tolerate self‐peptides and harmless allergens when it is exposed to specific non‐pathogenic micro‐organisms (Thomsen et al. 2007; Yan et al. 2007).
It has also been observed that use of probiotic bacteria or lyzates of non‐pathogenic micro‐organisms leads to a significant and permanent improvement in neurodermatitis. The application of non‐pathogenic or probiotic bacteria to the skin is more effective in reducing the recurrence of diseases following the successful primary treatment of diseases such as neurodermatitis. The bacterium leads to permanent stabilization of the immune system once glucocorticoid treatment has alleviated the acute reaction (Sicherer and Sampson 1999).
Probiotics are perceived to exert beneficial effects in the prevention and treatment of allergic diseases by modifying the gut ecosystem. The effect of ingestion of fermented milk containing Lactobacillus paracasei‐33 (LP‐33) was observed in patients with perennial allergic rhinitis and it was found that ingestion of LP‐33‐fortified fermented milk can effectively and safely improve the quality of life of patients with allergic rhinitis, and may serve as an alternative treatment (Wang et al. 2004).
Microbes have great significance as food ingredients. They can be used as a source of proteins, vitamins, enzymes, preservatives, antiallergens, anticancer, antidiabetes, anemia, obesity, and cholesterol agents. Thus it can be safely concluded that microbes have tremendous potential as nutraceuticals and in designing various disease‐targeting preventive functional foods and dietary supplements.
Thhe authors are grateful to Dr Ashok K. Chauhan, Founder President, Amity Group of Institutions, and Mr. Atul Chauhan, Chancellor, Amity University UP, Noida, for encouragement, research facilities, and financial support.