There are new enzyme-related techniques emerging to better suit the diverse food

processing environments and meet the development of new food products. Novel
or improved enzymes including proteases, glycoside hydrolases, lipases and
transglutaminases, and so on, with unique properties such as high activity at cold
temperatures or thermal stability have been developed. To discover these specialty
enzymes, metagenomics, recombinant DNA, and protein engineering techniques
have become popular and key tools. Enzymatic processing is developed for new
food applications such as nutrient-dense foods production, texture improved food
products, bioactive compounds extraction, and reduction of safety risks foods.
Innovative enzyme inactivation strategies and enzyme immobilization methods are
emerging. In conclusion, the trend in this field is to make enzymes more specific,
stable and efficient, and easily controlled for foods production.
Introduction Enzymes are biocatalysts that can be applied in the food industry as
an efficient, safe, and environment-friendly alternative to conventional food
processing. The industrial enzymes market was valued US $6.1 billion in 2017 and
is expected to reach US $8.5 billion by 2022 [1]. The food industry holds a large
share of this output. Enzymatic bioprocessing methods are currently used to
produce various foods including dairy, baking, beverages & brewing, fats & oils,
meat, and functional foods. Apart from their usage as processing aids, enzymes
are also naturally present in foods where they elicit both
desirable and undesirable effects. Examples of desirable enzymes and their effects
in foods include, endogenous b-glucosidase, polyphenol oxidase (PPO),
peroxidase (POD), esterase that influence ripening and flavors generation [2,3];
cathepsins in myofibrillar protein hydrolysis in fermented products [4], as well as
proteases and transglutaminases in food texture modification. Examples of the
undesirable effects of enzymes in foods such as dark discolorations in fresh fruits,
vegetables and raw crustacea caused by endogenous PPO or rancidity caused by
endogenous lipase and lipoxygenase (LOX). Enzyme kinetic modeling has been an
important strategy for exploring the behavior of enzymes on substrates under
various reaction conditions to achieve the goal of producing high quality and
affordable food products using classical enzymes. Some recent research on
modeling systems include hydrolases (e.g., pepsin, trypsin, amylase, alcalase,
cellulase, lipase) the hydrolysis/digestion of protein, starch and lipid [5,6];
inactivation of POD, PPO and LOX [7,8]; as well as using specific enzymes to
remove undesirable compounds in foods such as chlorogenic acid lactones in
coffee [9], and biogenic amines in fermented foods [10 ]. There are more than 55
different enzymes products used in food processing [11], and the number is
increasing with the discovery of more novel food enzymes. In this review, some
new trends on enzymatic processing are summarized which include: novel or
improved enzymes that are better suited to particular food processing
environments, as well as recently developed enzyme inactivation and enzyme
immobilization techniques.
Novel food enzymes Novel enzymes are generally associated with unique
properties or functions such as low-temperature activity, thermostability, pH
adaption, and others like tolerance to high salinity, pressure, solvents, metal ions
and inhibitors. These novel enzymes are valuable in food bioprocessing. For
instance, cold-active enzymes enable catalysis to occur at low temperatures. Cold-
active enzymes are generally also heat-liable due to their flexible structures
resulting in their easy inactivation by mild heat treatment. Living organisms
inhabiting extreme environments (a.k. a. extremophiles) have been shown to be
excellent sources of novel enzymes (a.k.a. extremozymes) that can retain their
integrity and function under extreme reaction conditions. Both bioprospecting and
artificial design are used to discover novel or improved food enzymes.
Conventional screening approaches involving enzyme purification and activity
determination are a valuable strategy to obtain new enzymes from animal, plants
and microorganisms. In addition, metagenomic screenings have been used as
effective tools for the discovery of any biotechnological relevant enzymes including
hydrolases and oxidoreductases [12]. For this, environmental samples from
extreme environments are used to establish a metagenomic library from extracted
DNAs to screen the genes encoding new enzymes [1]. Protein engineering has
been used to enzyme design for specific reactions using molecular biological
approaches such as site-specific mutagénesis
In food processing, proteases are widely used for meat tenderization, curdling of
milk, beverage clarification, debittering, texturization, deproteinization, and to
produce flavor. Proteases have been used for decades in the cheese industry for
milk clotting. Nonetheless, there is still increasing demand for novel milk
coagulating enzymes in the dairy industry. In this regard, a new milk-clotting
protease was recently discovered as an alternative to rennet for the development
of cream cheese product [13]. A new milk-clotting enzyme for manufacturing soft
cheese from cow milk and goat milk was reported from sunflower seeds [14].
Proteases are also used to produce protein hydrolysates for incorporation in foods
such as infant formula. Such hydrolyzed formula (HF) milks are easier to digest
and have reduced allergenic potential [15]. A novel cold-active serine protease
isolated from Chryseobacterium sp. facilitated tenderization of meat during
refrigerated storage to improve both the texture and taste of the meat [16]. A
thermostable acidic protease with optimal catalytic activity at pH 5.0 and 55 C [17]
was considered to be useful in coagulation of casein in cheese production, and for
the degradation of proteins for juice and wine clarification. Other novel proteases
found recently include a highly thermostable alkaline caseinolytic protease with
maximal activity at pH 7 and 80 C [18], a new alkaline trypsin [19 ] that was a
protease with antithrombotic and anticoagulant properties purified from the edible
medicinal plant Aster koraiensis Nakai [20], an aspartic protease for pork
tenderization and for the preparation of turtle peptides [21], a salt-stable protease
that is useful in low-salt fish sauce fermentation [22], and an inexpensive fungal
protease with high catalytic activity over broad pH and temperature ranges [23].
Glycoside hydrolases hydrolyze glycosidic bonds and are widely used in syrup,
beverage (beer, wine), and dough production. A new low-temperature mashing
system containing diverse amylolytic enzymes was applied to produce wort on a
commercial scale cost-effectively [24]. Amylases hydrolyze starches to produce
simple sugars in varying degrees. For instance, a novel recombinant a-amylase
produced maltose more efficiently from liquefied corn starch than b-amylase [25].
Recombinant amylases, especially from fungi origin or from malted cereals, are
abundantly used presently in baking.
Production of a-amylase can be accomplished inexpensively from agriresidues
such as wheat bran via solid-state fermentation. Amylases have a broad range of
applications in the food and brewing industries, as well as in baking and the
preparation of digestive aids. Amylases also have extensive applications in starch
saccharification, in the textile and detergent industries. Moreover, a novel
thermophilic b-glucosidase showed high selectivity for the synthesis of prebiotic
galactotrisaccharide [26]. A polydatin-b-D-glucosidase that facilitates resveratrol
preparation [27] and an amylase that used in cake formulation [28], were also
reported recently. Lipases act on various kinds of esters to produce acids and
alcohols. They are usually used to enrich flavor and generate meaty or fruity
aromas in meats, cereals, fruit juices, alcoholic beverages, and dairy products. A
new lipase was isolated from the liver of seabass and used to defat seabass skin
more effectively than isopropanol [29]. A new thermostable and solvent-resistant
recombinant lipase was produced from Rhodothermus marinus with potential for
food lipid processing and aroma improvement [30 ]. Transglutaminases (TGases)
are transferases and catalyze acyl transfer reactions between the g-carboxamide
groups of peptide-bound glutamine residues (acyl donors), and primary amines or
water molecules (acyl acceptors). They form inter-molecule or intra-molecule
cross-linkages in proteins via e-(g-Glu)-Lys bonds which is useful for modifying the
physical and functional properties of foods. A cold-active TGase with an optimal
temperature of 0– 10 C was recently purified from Antarctic krill and shown to be
capable of potentiating cold-set gelling of fish gelatin [31 ]. A new bacteria strain
Streptomyces sp. CBMAI 1617, was recently shown to produce high-yield TGase
that enabled the preparation of wheat protein and flour based doughs similar to the
commercial TGase [32]. TGase is very versatile and is used in the food industry to:
improve the texture, appearance, firmness, and shelf life of meat; enhance the
firmness of fish products; improve the quality and texture of dairy products;
improve the mechanical properties of protein-based biodegradable films; and
improve the texture and volume of baked goods. Because of the increasing use of
TGase in the food industry, it is to be expected that some common diets on the
market would contain residual amounts of the enzyme. In addition to the food uses,
TGase is also used extensively as a biocatalyst in the biomedical and
biotechnology fields. Nonetheless, TGase catalyzed reactions in proteins generate
isopeptide bonds that are different from conventional peptide bonds. The
isopeptide bonds may not lend themselves to degradation in the same way as
regular peptide bonds; thus, it is possible that reaction products differ in important
respects in terms of conformation, sizes, degree of cross-linking, digestibility and
possible allergenicity. Studies have established a direct correlation between the
proliferation in celiac disease and the higher consumption of the enzyme ingested
through baked goods such as breads [33], which poses the question as to whether
the use of TGase as a food processing aid has triggered an increase in the
incidence of gluten intolerance in consumers.
Enzymes in new foods applications Enzymes can be used to enhance the
production of nutrient-dense foods; that is, foods that are high in nutrients but low
in calories. Enzymes could achieve this by breaking down rigid plant-based foods
to release the nutrients such as vitamins and minerals; hydrolyzing antinutritive
substances such as phytates and enzyme inhibitors; via transformation of
substrates as in the formation of allicin; or by simply concentrating or stabilizing
beneficial components in foods. Peptide ligation mediated by enzymes such as
sortase, butelase or trypsiligase is a valuable strategy to prepare complex peptides
as nutritional food additives [34]. Docosahexaenoic acid (DHA) in fish oils can be
concentrated using commercial Candida antarctica lipase A and TL 100L lipase
due to their selectivity of hydrolysis, which is useful for food fortification and
pharmaceutical applications [35 ]. A new date fruit product enriched in soluble
dietary fiber with antioxidant and prebiotic effects was produced using enzymatic
conversion from insoluble dietary fiber [36]. New food products can be prepared
with improved physicochemical and mechanical properties by enzymatic
treatments. Branching and debranching enzymes affect the ratio of a-1,4 and a-
1,6-glycosidic bonds in starch which decides the structure and performance of
resultant food products. A highly branched dextrin was prepared from high-
amylose maize starch using a waxy rice branching enzyme [37]. TGases were
added in goat’s whey cheese [38] and meat products [39], and so on, and the
corresponding products showed enhanced sensory, texture properties and yield.
Bioactive compounds separation and extraction can be assisted by enzymes to
increase the yield, purity, and efficiency. Bioactive compounds such as phenolics,
flavorings and colorants can be extracted by diverse combination techniques, for
example, enzyme-assisted threephase partitioning, microwave-assisted enzymatic
extraction, ultrasound-assisted enzymatic extraction, or enzyme-assisted
supercritical fluid extraction [40]. Recently, Chinook salmon lipase was immobilized
and used to produce dairy products with distinct flavors by altering free fatty acid
composition [41]. Phenolics extracted from the by-products of winemaking using
enzymes, showed antioxidant and enzyme inhibitory effects (i.e., inhibition of
alpha-glucosidase and lipase activities) [42]. Enzymatic extraction in combination
with pressurized liquids was used to recover phlorotannin from the brown marine
algae. Phlorotannins have important pharmacological activities including
antioxidant, antiHIV, antidiabetic, anticancer, anti-inflammatory and enzyme
inhibitory properties, and are touted as a potential functional ingredient in marine
medicinal foods [43 ]. Chitooligosaccharides (COSs) are bioactive compounds that
have been shown to have antimicrobial and immunostimulating effects with
potential for several applications such as in tumor growth inhibition, mineralization
to increase bone strength, prevention of malaria, wound dressing, and in food
preservation via their antifungal effects [44]. They are water-soluble and are
commonly incorporated into dairy products and beverages. COSs have been
prepared from shrimp chitosan using enzymes such as chitinases and
chitosanases, and also by nonspecific enzymes lysozyme, papain, or cellulase
[45]. Enzyme treatment sometimes aim to relieve the safety risks of food products,
in terms of breaking down the harmful bacteria or undesirable compounds such as
biogenic amines, gluten, lectins, acrylamide, cyanogenic glucosides,
glucosinolates, protease inhibitors, and so on. Some enzymes such as cellulase
combined with surfactants were shown to drastically remove Salmonella biofilm
under meat processing, which could be a new strategy to control Salmonella
outbreak [46]. An immobilized lysozyme exhibited effective lysis of lactic bacteria
Oenococcus oeni in white and red wines [47]. A zearalenone-degrading enzyme
was immobilized as a novel detoxifying agent [48 ]. Furthermore, degradation of
gluten in grain products can be achieved with enzymes such as trypsin,
chymotrypsin, pepsin, and so on [49] which benefits consumers who are gluten
intolerant. High-quality gluten-free products such as sourdough bread, pasta and
beer were produced using new approaches with suitable peptidases [50].
Asparaginase may reduce acrylamide formation in cooked foods by converting
asparagine to aspartic acid, while maintaining sensory quality [51]. Other
innovative enzyme-related effects in food bioprocessing Enzymes need to be
inactivated in food processing to either curtail their undesirable effects (e.g., some
enzymes such as POD, PPO, LOX or lipase), or prevent continued activity of
desirable enzymes in food products after they have been used to achieve the
desired transformation (e.g., proteases, amylases, TGases). Both chemical and
physical procedures are developed for enzyme inactivation. Thermal treatment is
the conventional inactivation method, followed by high pressure, microwave, and
infrared or ultraviolet light treatments. In recent years, some new non-thermal
technologies have emerged. Examples of non-thermal strategies to inactivate
enzymes include the use of high intensity light pulses, pulsed electric fields, and
ultrasound to effectively terminate proteolysis [52]; ohmic heating consisting of
passing an electric field through foods to inactivate PPO
and POD in sugarcane juice [53]; pulsed light has also been used to inactivate
horseradish POD [54]. There are also novel enzyme immobilization approaches
been continuously developed to enable recovery and reuse of enzymes, as well as
their removal from food products to stop continued hydrolysis or pose potential
problems like allergenicity. Different commercial food enzymes such as lipase,
glucose isomerase, lactase, protease, invertase have been immobilized for
producing corresponding food products, namely, interesterification of oils/fats, high-
fructose corn syrup, lactose, amino acids, and invert sugar. The development of
immobilized enzymes is based on three immobilization mechanisms, that is, mild
adsorption using charges or polarity of enzymes, covalent binding/cross-linking of
enzymes to surfaces or to the enzymes themselves, and entrapment/
microencapsulation. Developments in enzyme immobilization include the discovery
of various innovative carriers that are cheaper, inert, stable, reusable, efficient, or
permit catalysis to occur under harsh reaction conditions. Enzymes have been
immobilized onto biotinylaminopropyl celite, thionyl chloride-activated controlled-
pore glass, nylon, and on microelectrodes. Other carriers that have been used are
poly(ethylene glycol) crosslinked to albumin, poly (carbamoyl sulfonate) hydrogels,
and polyethyleneimine-coated magnetite particles [55]. Conclusion Most traditional
enzymes used in food processing are considered as ‘natural’ and biodegradable,
and their use is accompanied by greener outcomes and lower environmental
impacts. Enzymatic reactions are selective, reproducible, fast, efficient, and
practically devoid of undesirable by-products. Thus, enzymes have become
important tools for various markets such as food and animal feed, biofuels,
detergents, textiles, pulp and paper, and so on. Demand for enzymes has grown
steadily over the years, with the global industrial enzymes market projected to
reach $7 billion (US) by Year 2018, and expected to continue to grow for the
foreseeable future. Nonetheless, most of current industrial processes are
performed under extreme conditions of temperature, acidity or alkalinity, and high
salinity; reactions conditions that are detrimental to the integrity of traditional
enzymes which tend to be optimally stable and active within narrow ranges of
physical and chemical conditions. Thus, there is compelling need to discover more
sustainable and environmentally responsible approaches to replace potentially
harmful chemical methods currently in use. In this regard, the discovery of novel
enzymes with attributes that can withstand industrial processing settingsis crucial
to the future of biocatalysis. The increasing use of enzymes as food processing
aids in the food industry is consistent with consumer aversion for foods produced
via chemical modifications. Nonetheless, the use of enzymes as processing aids
should have safety concerns of its own. For example, the potential for release of
enzymes and their carriersinto processing systems have not been well studied to
understand the safety implications. Also, designing new enzymes via recombinant
DNA technologies entail the use of genetically modified microorganisms whose
safety for food use must be verified. Studies on the safety of emerging enzymes as
food processing aids are warranted. Conflict of interest The authors declared no
conflict of interest. References and recommended reading Papers of particular
interest, published within the period of review, have been highlighted as: of special
interest of outstanding interest 1. Ferrer M, Martı´nez-Martı´nez M, Bargiela R,
Streit WR, Golyshina OV, Golyshin PN: Estimating the success of enzyme
bioprospecting through metagenomics: current status and future trends. Microb
Biotechnol 2016, 9:22-34. As environmental genomes have become routine
procedure to screen enzymes, there are challenges for environmental enzymes to
be commercialized. This mini-review article introduced the current status of using
metagenomics for enzyme screening as well as its bottlenecks, followed by an
elaborate analysis of the success for enzyme discovery.