fermentation and enzyme technologies in food processing

6 Fermentation and EnzymeTechnologies in FoodProcessingAli Demirci, Gulten Izmirlioglu, and Duygu ErcanDepartment of Agricultural and Biological Engineering, Pennsylvania State University,University Park, Pennsylvania, USA

6.1 Introduction

Fermentation is a microbial biotechnology wherebynatural renewable substrates are converted to value-addedproducts such as enzymes, organic acids, alcohols,polymers, and more. Fermentation end-products suchas ethanol and lactic acid are proton sinks, wherebyNADH is recycled to NAD+, which allows the cell to con-tinue producing energy via glycolysis by substrate-levelphosphorylation. Thus, microorganisms generate manyend- or by-products to maintain energy balance.Today, enhanced production of economically impor-

tant fermentation products has benefited from targetedgenetic engineering techniques to established industrialmicrobial strains (Campbell-Platt, 1994). The formationof end- or by-products is dependent on microbial strainand the environmental conditions employed. For anoptimum fermentation process, a microbial strain shouldbe selected and developed based on the desired product.Strain development technologies include mutation andrecombinant DNA technology. Growth of microorgan-isms and product formation are also affected by temper-ature, pH, dissolved oxygen, and fermentation mediumcomposition. Therefore, the fermentation media andgrowth conditions need to be optimized. Moreover, themetabolic pathways should be determined.Knowledge of biochemical changes in fermented foods

can help producers to manipulate the production bychanging strains and/or conditions. In addition to growthconditions, types of strains, and media, fermentationmodes affect the productivity. Batch, fed-batch, andcontinuous fermentation modes can be selected for

high productivity. Fed-batch and continuous modes canovercome the substrate limitation during fermentationprocesses. Higher productivity can also be achieved by cellimmobilization, which results in increases in the biomassconcentration in the bioreactor, and thus an increasedconcentration of biocatalysts in the reactor. After thefermentation process, recovery methods of the productfrom the fermentation medium need to be evaluatedand optimized, and are often an economic limitationfactor. Indeed, purification of the end-product is often ahigh-cost step of the process. The microbial end-productscan be biomass itself, extracellular products, or intracellu-lar products. Filtration, homogenization, and extraction(liquid and solid) are examples of recovery methods.Enzymes are products of living organisms and have

been used in the industry for many years due to their cat-alytic activities. Enzyme activity depends on temperature,substrate, pH, inhibitors, etc. and should be optimizedfor each process. Since enzyme recovery is difficult, theapplication of enzyme immobilization may reduce proc-ess cost. Enzymes can be isolated from plants and mam-malian tissues, or can be produced by microorganisms.However, microbial enzymes are preferred due toavailability and specificity.Enzymes are used in the production of over 500

commercial products (Johannes & Zhao, 2006). Theyhave a broad range of applications from food to deter-gents. Most enzymes are commercially available and usedto enhance the product quality in food, detergents,leather, paper, cosmetics, and pharmaceuticals. Commer-cial enzymes include lipase, amylases, proteases, pecticenzymes, and milk clotting enzymes (rennet). In this

Food Processing: Principles and Applications, Second Edition. Edited by Stephanie Clark, Stephanie Jung, and Buddhi Lamsal.© 2014 John Wiley & Sons, Ltd. Published 2014 by John Wiley & Sons, Ltd.

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chapter, optimization of media and growth conditionsand criteria for microbial strain selection and straindevelopment are summarized. The strains, productionconditions, and biochemical changes involved in theproduction of fermented foods are explained. The disad-vantages and advantages of different fermentation modesare discussed. Finally, immobilized fermentation princi-ples and basic recovery methods are also summarized.A brief summary of commercial enzymes and theirreactions is provided.

6.2 Fermentation culture requirements

In fermentation processes, environmental conditions,such as temperature, pH, and dissolved oxygen, play acrucial role in microbial growth and product. They needto be carefully evaluated for each fermentation process.Each of these parameters is now briefly discussed.

6.2.1 Culture media

During microbial growth, energy needs are met whilethe metabolites (by-products) are produced. However,the formation of by-products is usually dependent onthe environmental conditions, such as nutrients, pH,and presence of oxygen (Shuler & Kargi, 2008). Optimiz-ing the nutrient requirements of the culture can enhanceboth growth and production of end/by-products.Nutrients used for fermentation can be categorized asmacronutrients and micronutrients. Carbon, nitrogen,oxygen, sulfur, phosphorus, magnesium, and potassiumrepresent major macronutrients. Carbohydrates are themain energy source for the culture. For small-scale fer-mentation, glucose, sucrose, and fructose are commonlyused as a carbon source. However, for industrial-scalefermentation, beet molasses, starch, and corn syrupare the main sources to meet the carbon requirementsof microorganisms (Balat et al., 2008). The secondmajor nutrient in a fermentation medium is nitrogen.Ammonia, ammonium salts, proteins, yeast extract, andpeptone are some of the nitrogen sources that can befound in a typical fermentation medium (Shuler & Kargi,2008). Trace elements are also needed for microbialgrowth and to increase yields and production rates. Iron,zinc, manganese, copper, cobalt, calcium, and sodiumrepresent some examples of trace elements.Each microorganism has specific requirements for

carbon and nitrogen sources, as well as other nutrientssuch as minerals and vitamins. However, a classic media

design approach is used to imitate the elemental compo-sition of the microorganism to be grown. Carbon/nitrogen ratio is used to optimize the amount of carbonand nitrogen in the medium. Magnesium, sulfate,phosphate, and other salts should be equivalent to theirpresence in the cell (Parekh et al., 2000). In general, amicrobial cell is composed of 44–53% carbon for bacteria,yeast, and fungi; 10–14% nitrogen for bacteria, 7–10% foryeasts and fungi; 2–3% phosphorus for bacteria, 0.8–2.6%for yeasts, and 0.4–4.5% for fungi; and 1–4.5% potassiumfor bacteria and yeasts, 0.2–2.5% for fungi (Ertolaet al., 1995).The composition of media is an important issue

when defining the media for a specific product formation.Studies have demonstrated that different end/by-productscan be formed in different culture medium and growthconditions with the same microorganism. For example,various value-added products can be generated byAspergillus niger when different culture media are used(Metwally, 1998). It has been reported that glucoamylaseproduction was suppressed when potassium nitrate wasused as the nitrogen source compared to ammoniumsulfate (Metwally, 1998). On the other hand, Hang andWoodams (1984) reported that the same microorganism,A. niger, produced citric acid when a different carbonsource was used. In their study, apple pomace was usedas the carbon source, and A. niger was able to produce314 g citric acid/kg sugar.In industrial settings, medium design becomes crucial

due to economic feasibility. Moreover, the cost of thedownstream process can be affected by the medium com-position (Kennedy & Krouse, 1999). Demirci et al. (1997)reported that 30% of ethanol cost is due to fermentationmedium costs; in their study, immobilization of cells wasused to decrease the cost while increasing productivity.Another study by Kadam and Newman (1997) on ethanolfermentation reported the use of low-cost fermentationmedium to reduce cost. Their approach was to reducethe cost by replacing yeast extract with urea, and addingcorn steep liquor as a source of vitamins, minerals, andnitrogen. Therefore, medium ingredients play an impor-tant role in achieving an economical product.

6.2.2 Temperature

Each microorganism has a specific temperature foroptimal growth and production rates (g/L/h). Therefore,microorganisms are categorized into three groupsbased on their optimum temperature. Psychrophilesprefer a cold environment, less than 20 �C. Most of the

108 Food Processing: Principles and Applications

psychrophiles are bacteria, Pseudomonas and Vibrio spp.,and Archaea,Methanogenium, andMethanococcus. Mostof the psychrophile bacteria are spoilage microorganismsresponsible for spoilage of refrigerated foods. Mesophilicmicroorganisms require a moderate temperature profileto grow (20–45 �C); Lactobacillus delbrueckii subsp.bulgaricus used in yogurt fermentation and A. niger pro-ducing amylase enzymes are examples of mesophilicmicroorganisms. Thermophiles have an optimal temper-ature greater than 45 �C. Thermomyces lanuginosus andKluyveromyces marxianus are thermophiles that produceamylase and ethanol, respectively. Above the optimaltemperature, growth slows down and thermal deathmay occur. However, optimum temperature for productformationmay differ for microbial growth. Consequently,temperature optimization for a specific product should bedetermined to obtain maximum product yields.Optimization of fermentation temperature for most

value-added products for various microorganisms andmedia has been widely reported. Dutta et al. (2004) stud-ied optimization of temperature for extracellular proteaseproduction from Pseudomonas spp. In their study, threedifferent statistical models were used to optimize cultureparameters. As a result, 38 �C was determined as theoptimum temperature for extracellular protease produc-tion, with 58.5 U/mL enzyme activity. In another study,approximately 34% increase in tartaric acid concentration(2.14 kg/m3 after 96 h fermentation) was reported aftertemperature optimization for tartaric acid productionby Gluconobacter suboxydans (Chandrasker et al.,1999). Furthermore, Kluyveromyces marxianus was cho-sen to investigate the fermentation conditions in glycerolproduction from whey permeate (Rapin et al., 1994).These authors reported that at 37 �C and pH 7, glycerolyield was 9.5% with a 0.15 L/h specific growth rate, whichwas the highest yield among examined conditions.

6.2.3 pH

pH is another parameter requiring optimization for highmicrobial growth and production rates. In general, bacte-ria have an acceptable range of pH 3–8; for yeast, therange narrows down to pH 3–6, and for molds, optimalpH is 3–7 (Shuler & Kargi, 2008). In fermentation pro-cesses, pH can change during microbial growth and prod-uct formation due to production of basic compounds ororganic acids. Furthermore, the source of nitrogen playsa role in pH fluctuation. Ammonia and nitrate can begiven as examples of this; ammonia causes a decrease inpH, while nitrate results in an increase in pH due to

the microbial utilization of hydrogen ions (Shuler &Kargi, 2008).Consequently, pH optimization and control during

fermentation processes is very important for microbialgrowth and value-added product formation. The pHcan be controlled by addition of acid or base during thefermentation (internal buffering and external bufferingshould be distinguished). pH profiles for products havebeen studied. Mohawed et al. (1986) optimized the pHvalue for lipase production by Aspergillus anthecieusgrown in Dox-Olive oil medium by using different buf-fers: citrate-phosphate buffer (ph range from 2.6 to 7.0),borate buffer (pH range from 7.6 to 9.2), and phosphatebuffer (pH range from 5.8 to 8.8). Highest lipase concen-tration (156 μg/mL) was reported at pH 6.6 when phos-phate buffer was used. Ellaiah et al. (2002) optimizedthe initial pH (3.0–7.0) for glucoamylase production bya newly isolated Aspergillus species, and reported thatmaximum glucoamylase production (109.0 U/g) wasobtained at pH 5. It was also reported that glucoamylaseproduction is highly affected by initial pH level for thisparticular species. Malik et al. (2010) reported the effectof initial pH (4.5–6.5) for cellulase production byTrichoderma viride. Interestingly, at pH 4.5, only 0.2 U/mL/min cellulases were produced, whereas cellulase pro-duction reached maximum (1.66 U/mL/min) at an initialpH 5.5. Moreover, further increase in initial pH (>5.5)caused a reduction in cellulase production. Studies haveshown that both initial pH value and pH levels duringfermentation play a very important role in obtainingand maintaining maximum growth and production rates.

6.2.4 Dissolved oxygen

Dissolved oxygen is an essential substrate for aerobicfermentation. Microorganisms can be categorized asaerobic, anaerobic, and facultative anaerobes (Demirci,2002). Aerobic microorganisms require oxygen as an elec-tron acceptor during respiration. Strict anaerobes, on theother hand, are killed by oxygen. Facultative anaerobes,such as yeast, will respire when oxygen is present, and willconvert to fermentation in the absence of oxygen.Whereas many lactic acid bacteria are indifferent to oxy-gen, their growth rate is best anaerobically, but most canalso grow in the presence of oxygen.Because solubility of oxygen in the liquid is very low,

in aerobic settings, oxygen is continuously added to thefermentation medium as a substrate, and improved byagitation. In some cases, oxygen can be the rate-limitingsubstrate, for instance, in continuous stirred tank reactors

6 Fermentation and Enzyme Technologies in Food Processing 109

with high cell population (Shuler & Kargi, 2008). Variousmethods have been reported to overcome this limitation.One is generating oxygen in the reactor by adding hydro-gen peroxide and a catalyst system, or using oxygen-catalyzing microorganisms. Also, co-fermentation (pairof oxygen-consuming and -producing microorganisms)can be applied to increase oxygen availability in the reac-tor. Another approach is utilizingmedia that promote sol-ubility of oxygen by replacing aqueous solutions withorganic solvents (Rols & Goma, 1989). Oxygen vectors,e.g. hydrocarbons and perfluorocarbons, are also intro-duced as alternatives to improve oxygen solubility(Galaction et al., 2004). However, the growth rate is inde-pendent of dissolved oxygen concentration when criticaloxygen concentration is exceeded (Shuler & Kargi, 2008).Therefore, the concentration of dissolved oxygen must besufficient for microbial growth.Oxygen solubility in culture medium is affected by sev-

eral factors. Solubility of oxygen decreases when temper-ature is raised (Demirci, 2002). The presence of otheringredients in the culture medium also has an effect onoxygen solubility. Studies have shown that salts andsugars decrease the solubility of oxygen in the fermenta-tion medium (Demirci, 2002). On the other hand, anincrease in the partial pressure of the oxygen can increasethe solubility of oxygen linearly.Oxygen is supplied to the fermentation broth by spar-

ging air for bench-top fermentors, whereas agitationblades and speed are used to break air bubbles into smallerbubbles for industrial-scale fermentors. It is difficult tokeep the dissolved oxygen at saturation level while it isbeing consumed by growing microorganisms. Further-more, oxygen transfer from gas to the cells is limited bythe oxygen transfer rate (OTR), which must be equal toor higher than oxygen uptake rate (OUR) to ensure thatthe critical oxygen level is achieved. OTR is given as:

OTR = kLa C∗−CLð Þ

where kL is the oxygen transfer coefficient (cm/h), a is thegas–liquid interface area (cm2/cm3), C∗ is saturated dis-solved oxygen concentration (mg/L), and CL is the actualdissolved oxygen concentration in the broth (mg/L). OURcan be given as:

OUR =qO2X

where qO2 is the specific rate of oxygen consumption(mg O2/g dry weight of cells.h), X is the cell concentration(g dry weight of cells/L).

6.2.5 Strain selection and straindevelopment

Microbial strain selection is important to achieve thedesired product at optimum productivity levels in the fer-mentation process. Microorganisms can be obtainedfrom culture collections all over the world (Table 6.1)or may be isolated from the environment (Hatti-Kaul,2004). Wild-type strain, that is not mutated, is originallyfound in nature. The characteristics of microbial strainsfor industrial fermentation include effective stable pro-ductivity of large quantities of a single product andgenetic stability. Moreover, the strain should be pureand easy tomaintain and cultivate, and it should be grownin inexpensive culture media. In addition to characteris-tics of strains that are used in industrial production,microorganisms that are used as starter cultures mustbe neither pathogenic nor capable of producing toxins;

Table 6.1 Centers for culture collections

Agencies Abbreviation Web addresses

American TypeCultureCollection

ATCC http://atcc.org

BelgianCo-ordinatedCollections ofMicroorganisms

BCCM http://belspo.be/bccm

Common Access toBiologicalResources andInformation

CABRI http://cabri.org

Czech Collection ofMicroorganisms

CCM http://panizzi.shef.ac.uk/msdn/ccm/ccmi.html

DeutscheSammlung fürMikrorganismenund Zelkulturen

DSMZ http://dsmz.de

Japan Collection ofMicroorganisms

JCM www.jcm.riken.go.jp

NetherlandsCultureCollection ofBacteria

NCCB www.cbs.knaw.nl/NCCB

NationalCollections ofIndustrial andMarine Bacteria

NCIMB www.ncimb.com/culture.html

Northern RegionalResearch Center

NRRL http://nrrl.ncaur.usda.gov/


they should also be able to suppress the growth of specificundesirable microorganisms. The starter cultures shouldbe GRAS (Generally Recognized As Safe) organisms,which are safe for human consumption (Geisen &Holzapfel, 1996).Strain development technologies, such as mutation

or recombinant DNA, are applied to enhance metaboliccapabilities for industrial applications or to make thestrains capable of synthesizing foreign (or heterologous)compounds. Production levels for the wild strains maybe too low for economical production. Thereforeapproaches to increase productivity are typically neces-sary. Productivity can be increased by strain improve-ment. Moreover, strain may be improved to reduceoxygen needs and stabilize foaming. For example, Zhanget al. (2009) reported that proteinase A (PrA), encoded bythe PEP4 gene, promotes cell growth against insufficientoxygen conditions in steady-state cultivation. A stablehead of foam, which is a major consideration in beer qual-ity assessment, was achieved by destruction of the generesponsible for yeast PrA (Wang et al., 2007). The strainmay be developed to metabolize inexpensive substrates.Wild strains may produce a mixture of substances, whichare chemically related. Mutants can be developed to makethe strain able to synthesize one component as the mainproduct. This can simplify product recovery (Parekh et al.,2000; Rowlands, 1984).Classic strain development depends on random muta-

tion and screening. Mutation is achieved by subjectingthe genetic material to physical or chemical agents calledmutagens (Parekh et al., 2000). Nitrosoguanidine (NTG),4-nitroquinolone-1-oxide, methyl methane sulfonate(MMS), ethylmethane sulfonate (EMS), hydroxylamine(HA), γ-ray irradiation, and ultraviolet light (UV) areexamples of mutagen agents (Adrio & Demain, 2006;Clarkson et al., 2001). Moreover, single cells, spores,and mycelia have been used for mutagenesis (Adrio &Demain, 2006). Mutation can be classified based on thelength of the DNA sequence affected by the mutation.While point mutation affects a single nucleotide, segmen-tal mutation affects several adjacent nucleotides (Graur &Li, 2000). After treatment with mutagens, a diversepopulation of cells should be screened to identify thosewith mutations that have desirable traits (Clarksonet al., 2001). Random screening involves the randomselection of survivors from the population and testingfor their ability for enhanced production of the metaboliteof interest by small-scale model fermentation.Assay of fermentation broth for products of interest

and scoring for improved strains is essential for strain

development protocols. This process is applied to confirmthe statistical superiority of microbial strain compared tocontrol strain used prior to mutation (Parekh et al., 2000).For example, classic mutagenesis was used to improvethe yields of α-amylase enzyme production by Bacillussubtilis (Clarkson et al., 2001). Adrio and Demain(2006) reported an increase in the production of penicillinby Penicillium chrysogenum X-1612, which was isolatedafter X-ray mutagenesis.In addition to mutation, alteration in DNA can be

achieved by the application of genetic recombination.Homologous reciprocal recombination involves the evenexchange of homologous sequences between homologouschromosomes. As a result of homologous reciprocalrecombination, new combinations of adjacent sequencesare produced while retaining both variants. Non-reciprocalrecombination involves the uneven replacement of onesequence by another and a process resulting in the lossof one of the variant sequences involved in the recombina-tion (Graur & Li, 2000). Genetic recombination can beachieved by transformation, transduction, or conjugation.A cell takes up isolated DNA molecules from the mediumsurrounding it in transformation, which allows exchangeof genes between non-related organisms. In transduction,the transfer is mediated by bacterial viruses. Anothermethod of genetic recombination is conjugation, whichinvolves the direct transfer of DNA from one cell toanother. Conjugation is a natural process in which, bycontact between cells, transmissible plasmids are trans-ferred among closely related microbial species (Dale &Park, 2010; Geisen & Holzapfel, 1996).While strains obtained by transformation of recombi-

nant DNA are subject to genetic engineering laws andregulations, starter cultures, which were obtained by con-jugation, are not subject to legal regulations (Geisen &Holzapfel, 1996). Genes can also be transferred artificiallyby recombinant DNA technology, in which endonucleaseand ligase enzymes can be employed. The application ofrecombinant DNA technology makes the introduction ofgenes from higher organisms into microbial cells possible.Examples of hosts for such foreign genes includeEscherichia coli, Saccharomyces cerevisiae, Kluyveromyceslactis, and other yeasts as well as filamentous fungi such asAspergillus niger (Stanbury, 2000).The following aspects should be considered when

starter cultures for food production are developed byrecombinant DNA technology: acceptor strains musthave GRAS status; the DNA to be transferred must befully identified and have GRAS status; the vector shouldhave food-grade status; the gene integration site should


be well identified; and integration in this genomic siteshould not cause pleiotropic effects (Geisen & Holzapfel,1996), which means that a single gene can cause multipleeffects (Tandon et al., 2005).Up to now, recombinant DNA technology has been

used as an effective method for strain development toproduce many products, such as human lysozyme,interleukin-1β, hepatitis B surface antigen, human serumalbumin, interferon, insulin, human serum albumin,food-grade β-galactosidase, α-galactosidase, glucoamy-lase, etc. (Merico et al., 2004; Stanbury, 2000; Zhanget al., 2011). In addition to selection and developmentof the strain, culture media, growth conditions, andbioreactor design are important to achieve the maximumyield of desired products.

6.3 Fermentation technologies

6.3.1 Batch fermentation

Batch fermentation is carried out in a continuous stirredtank reactor (CSTR) with an initial amount of medium;during fermentation, no medium addition or removaloccurs (Sanchez & Cardona, 2008). Fermentation is per-formed after sterilization of the medium and adjustmentof pH by either acid or alkali. After inoculation of yeastor bacteria, product formation takes place by controllingtemperature, pH, agitation, and dissolved oxygen, whichdepend on the characteristics of the cultured microorgan-ism. Because no medium addition occurs, the growth ofmicroorganisms follows four main phases of the growthcurve: lag phase, log (exponential) phase, stationaryphase, and death phase (Figure 6.1). In the lag phase, cellgrowth is negligible, because it is an adaptation time forthe cells to adjust to the new environment (i.e. substrates

available in the culture medium, metabolic pathwayinitiation, or synthesizing of specialized enzymes suchas amylases and cellulases) (Shuler & Kargi, 2008). Thelog (exponential) phrase is where microbial growthreaches its maximum rate. The stationary phase is theperiod during which microbial growth rate equals thedeath rate. In this phase, cells are still able to producesecondary metabolites. The last period of batch fermen-tation is the death phase, in which death rate is higherthan growth rate due to lack of nutrients or accumulationof inhibitor primary or secondary metabolites, etc.Malik et al. (2010) reported production of cellulases by

Trichoderma viride using batch fermentation. Maximumproduction of enzyme (CMCase 1.57 U/mL/min, FPase0.921 U/mL/min) was obtained at 30 �C after 72 h of incu-bation. Pseudomonas spp. were investigated for extracel-lular protease production by using statistical optimizationmethods in batch fermentation (Dutta et al., 2004). Theoptimum operating conditions were 38 �C, pH 7.8, andinoculum volume of 1.5% with 58.5 U/mL of proteaseactivity within 24 h of incubation.In the ethanol industry, batch fermentation has a wide

application. Carbohydrate substrates for industrial fer-mentations can be glucose (corn syrup), sucrose (sugarcane molasses), polysaccharide (starch, cellulose or hemi-celluloses) and more. The batch system can be combinedwith pretreatment processes for ethanol fermentationfrom starchy and lignocellulosic materials. Hydrolysisof starch (mostly saccharification) and fermentation canbe performed simultaneously in simultaneous saccharifi-cation fermentation (SSF). Choi et al. (2008) studied eth-anol fermentation from sludge containing cassava mashby applying simultaneous saccharification and batchand repeated batch reactor fermentation. They reportedthat a maximum 85 g/L ethanol concentration wasreached after 42 h of batch fermentation.

0

5

10

15

20

25

30

35

0 10 20 30 40 50 60 70 80

Cell

popula

tion

Time

Lag

phase

Exponential

phase

Stationary

phase

Death

phase

Figure 6.1 A typical batch fermentation curve.


6.3.2 Fed-batch fermentation

In fed-batch processes, one ormore nutrients are added toa reactor continuously or intermittently, while fermenta-tion broth is either removed semi-continuously or notremoved. By addition of medium, two benefits can beobtained: (1) microbial growth will not be affected dueto lack of nutrients, and (2) substrate inhibition will beovercome, if this is a limitation for the process. Ethanolis a metabolic inhibitor for yeast (>10–12% dependingon the strain) and a point of concern in fermentation,which will be eliminated in the fed-batch process byslowly decreasing the concentration of end-productduring the fermentation process. However, to enhanceproductivity and ethanol yield, optimization of feedingshould be done properly (Sanchez & Cardona, 2008).Fed-batch culture is the most common technology inthe ethanol industry in Brazil (Sanchez & Cardona, 2008).Fed-batch fermentation has been studied to increase

the production of cellulase and concentrations ofsubstrate to be consumed by Trichoderma reesei(Hendy et al., 1982). In this study, a maximum of 30.4FPIU/mL was achieved (the highest activity yet reportedfor this strain) with 47.6 g/L soluble protein. Alfenoreet al. (2002) reported a fed-batch fermentation studiedto overcome lack of nutrients during ethanol productionby S. cerevisiae. To overcome nutrient deficiency, vitaminwas added during fermentation. Ethanol productionincreased from 126 g/L to 147 g/L with a maximum pro-ductivity of 9.5 g/L/h. Ozmihci and Kargi (2007) alsostudied fed-batch ethanol fermentation with S. cerevisiae,which produced 63 g/L ethanol and 5.3 g/L/h productivitywith a 125 g/L sugar feed. Moreover, Sanchez et al. (1999)performed production of enzyme from Candida rugosapilot-plant fed-batch fermentations, and characterizedthe crude extracellular enzyme preparations in whichmultiple forms of lipases and esterases were identified.

6.3.3 Continuous fermentation

Continuous fermentation, also known as chemostat orcontinuous stirred tank reactors (CSTR), involves freshsterile media fed into a reactor continuously. In additionto feeding the reactor with fresh nutrients, the effluent isremoved from the reactor and the volume of the reactorremains constant. The rates of feeding and removalstay equal. To avoid wash-out, which means takingaway all the cells from the reactor, growth rate of themicroorganism must equal dilution rates (rate ofremoving cells).

Advantages of continuous fermentation over batch fer-mentation include low construction costs of bioreactors,lower maintenance and operational requirements, higheryield, and better control of the process (Sanchez &Cardona, 2008). Stability of culture, however, is an issuefor continuous fermentation. Even small changes in anyparameter (i.e. temperature, dilution rate, substrate con-centration of feed, etc.) can decrease yield. Differentmethods can be used to overcome drawbacks of continu-ous fermentation. For instance, Sanchez and Cardona(2008) suggest utilization of immobilized cell technologyin which cells are captured in the reactor by biofilms,calcium alginate, chrysolite, etc., to enhance yield.Continuous SSF was studied to produce ethanol from

grains by S. cerevisiae and yielded 2.75 gal/bushel(Madson & Monceaux, 1995). Kobayashi and Nakamura(2004) studied continuous ethanol fermentation froma starch-containing medium by using recombinantS. cerevisiae and reported 7.2 g/L ethanol and 0.23 g/L/hmaximum ethanol productivity at 0.026 h−1 of thedilution rate in the suspended cell culture; however,ethanol productivity increased approximately 1.5-foldin the immobilized cell culture and reached 3.5 g/L/hproductivity at 0.4 h−1 dilution rate.

6.3.4 Immobilized cell fermentation

Immobilized cell fermentation can be defined as prevent-ing the free migration of cells in a fermentation medium.The advantage of cell immobilization over suspended cellreactors is to increase biomass concentration or biocata-lyst in the reactor, and therefore, enzyme activity as wellas fermentation by-products. Long periods of operation ina fermentation plant due to reuse of cells without recoverycost can also be provided by immobilization. In mostcases, product recovery can be performed easily in immo-bilized cell systems compared to suspended cell bioreac-tors. In continuous fermentation, immobilization caneliminate cell wash-out problems at high dilution rates.Also, better environmental conditions for microbialgrowth can be provided when cells are immobilized.According to the physical mechanism of interest, immo-

bilization can be categorized into four groups: immobiliza-tion on solid carrier surfaces, entrapment within a porousmatrix, aggregation (cell flocculation), and mechanicalcontainment behind a barrier (Kourkoutas et al., 2004;Verbelen et al., 2006).Immobilization on solid carrier surfaces is simply

attachment of cells to a solid support by physicaladsorption because of electrostatic forces or covalent


binding between cells and the surface (Kourkoutas et al.,2004). One of the important aspects here is thickness ofbiofilm. Thick biofilms may cause diffusionally limitedgrowth, whereas thin biofilms may result in low biomassconcentration caused by low rates of conversion (Shuler &Kargi, 2008). Cell attachment and relocation are possiblebecause there is no barrier between cells and the solution.Waste water treatment, water purification, and enhancedproduction of value-added products are some of theapplications of biofilms (Demirci et al., 2007). Althoughwaste water treatment, microbial leaching, and acetic acidproduction (also called the “quick vinegar process”) usingbiofilm reactors have been employed in the industry, mostbiofilm reactors have only been studied at laboratory scale(Bryers, 1994; Cheng et al., 2010). Biofilm reactors havebeen studied to enhance production of ethanol, organicacids, enzymes, cellulose, pullulan, etc.Biofilm formation occurs in three steps (Figure 6.2).

Attachment, the first step, is the rate at which cells aretransported to the surface and is highly dependent onsurface properties. In the second stage, colonization,formation of polymer bridges occurs. Colonization isaffected by growth conditions, nutrient diffusion, andshear force. In the last step, growth, availability of nutrientplays a crucial role (Demirci et al., 2007).Solid supports of biofilm reactors should be favorable

to microorganisms, inexpensive, easily available, andresistant to mechanical force (Cheng et al., 2010). Plasticcomposite supports (PCS) are one of the solid supportsmade from polypropylene, developed at Iowa StateUniversity (Pometto et al., 1997).Entrapment within a porous matrix is one of the

most common cell immobilization techniques. Twomain methods are employed. First, cells are diffusedinto a porous matrix until their mobility is preventedby the other growing cells and the matrix. In thiscase, attachment to the surface is also possible. Someof the materials commonly employed for this method

are sponge, sintered glass, ceramics, silicon carbide,polyurethane foam, and chitosan (Verbelen et al.,2006). Second, entrapment can be obtained in situ. In thistype of entrapment, hydrocolloidal gels are used withnatural gels (Scott, 1987). The gel is formed into dropletsafter adding the suspended cells to a liquid solution of gel-ling material (Scott, 1987), and therefore, the polymericbeads are frequently spherical in a range of 0.3–3 mm(Verbelen et al., 2006). Agar, Ca-alginate, gelatin, polysty-rene, κ-carrageenan, polyurethane, and polyvinylalcoholare some of the gelling agents that have been used forentrapment. However, the chemical and physical insta-bility of gel, non-regenerability of the beads, diffusionlimitations of nutrients, oxygen, and metabolites becauseof the gel matrix, and high densities in the beads are someof the bottlenecks of the entrapment technique (Verbelenet al., 2006).Cell flocculation is the aggregation of cells to form a lar-

ger unit. The ability to aggregate is common in fungi,molds, and plant cells. Aggregation can be induced byartificial flocculating agents or cross-linkers such as poly-electrolytes and inert powders. Cell aggregation is affectedby cell wall composition, pH, dissolved oxygen, agitation,fermentation temperature, microorganism handlingand storage conditions, Ca2+ concentration, and mediumcomposition.

6.4 Downstream processing

The final step of any fermentation process is downstreamprocessing in which the product is separated from otherundesired components of the fermentation broth andfurther purification is carried out. The products can bethe biomass itself, extracellular (present in the fermenta-tion broth) or intracellular (trapped in the cell) products.Since fermentation broth is very complex, recovery of theproduct of interest requires very comprehensive methods

Attachment Colonization

Bulk fluid

Growth

Surface

Figure 6.2 Formation of biofilms (reproducedfrom Cheng et al., 2010, with permission fromSpringer-Verlag).


of recovery and can account for 50–60% of the total pro-duction cost (Cliffe, 1988).For product recovery, the following characteristics

need to be considered during recovery and, in most cases,limit the methods of separation. Commonly, the productsare in low concentration in a very complex liquidmedium, and temperature sensitive. If products are intra-cellular, more complicated steps for separation and puri-fication may be needed. Also, physical or chemicalproperties of the product may be very similar to the otherby-products in the fermentation broth, which makedownstream processing extremely difficult.In general, downstream processing is performed in four

main steps. In most cases, the first step is the removal ofinsolubles, in which solid particles such as biomass areseparated from the liquid medium. Rupturing the cell wallof biomass and extracting the intracellular compounds isthe second step. Removal of solubles from the liquid phasecan be considered as the third step, especially for the pro-ducts present in the medium. The final step is furtherpurification of the product.

6.4.1 Removal of insolubles

In some processes, the product of interest is the biomassitself or the product can be extracellular in the broth;however, in both cases microbial cells must be separated.Typical sizes for cells can be anywhere between 0.5–1 μmfor bacteria, 1–7 μm for yeast, 5–15 μm for molds,20–40 μm for plant cells, and 10–0 μm for suspensionsof animal cell (Dutta, 2008).Removal of insolubles usually involves filtration or cen-

trifugation. Filtration separates the solids from the liquidby forcing the fluid through a filter or solid support onwhich particles are deposited. This method is fairlystraightforward for dilute suspension of large and rigidparticles. However, small particles and deformability ofthe cells can make the process more complicated. Thereare many types of filters available but plate and framepressure filters and rotary vacuum filters are most com-mon. Plate and frame pressure filters are employed forsmall-scale separation of bacteria and fungi from fermen-tation broth. Rotary vacuum filters are preferred for large-scale commercial processes due to lower labor costs(Perry & Chilton, 1973). Even though filtration isstraightforward, for some products pretreatments maybe required. Heating for protein denaturation, additionof electrolytes to promote coagulation and flocculation,and addition of filter aids (earths and perlites) to increase

porosity and decrease the compressibility of cakes aresome pretreatment examples (Belter et al., 1988).On the other hand, centrifugation utilizes the density

difference between the solids and the surrounding fluid,when solid particles settle down and fluid stands as a clearphase. Even though centrifugation requires more expen-sive equipment and produces a wet cake compared tofiltration, it is still an alternative for separation of smallparticles, between 100 and 0.1 μm (Shuler & Kargi,2008). Two basic types of centrifuges are the tubularand the disk centrifuge (Figure 6.3). The tubular centri-fuge consists of a cylindrical rotating element in a station-ary case. Broth is fed through the bottom and clear liquidcollected from top, leaving the solids on the wall of thebowl. The tubular centrifuge can be inexpensive, but isnot suitable for large-scale and continuous processes.The disk centrifuge includes a short, wide bowl that turnson a vertical axis. Closely spaced cone-shaped disksdecrease the distance of particles to be removed andincrease efficiency. This type of centrifuge is utilized forcontinuous and large-scale processes but is more expen-sive than the tubular centrifuge (Belter et al., 1988).

6.4.2 Cell disruption

Although some antibiotics, extracellular enzymes, manypolysaccharides, and amino acids are secreted into thebroth, some products are intracellular, such as lipids,some antibiotics, and most proteins, and trapped in thecell. Cells need to be ruptured to release intracellular pro-ducts. However, rupturing the cells is usually difficultbecause of the high osmotic pressure inside the cell andstrong cell walls (Dutta, 2008). Techniques used have tobe powerful enough to break down the cell wall, but gentleenough not to damage the product.Physical methods include mechanical disruption

by grinding, homogenization, and ultrasonication.Homogenization is effective for mycelia organisms, aswell as animal cell or tissues. However, grinding and ultra-sonication can be employed for most of the suspensioncells. Crushing and orifice type homogenization, on theother hand, are adapted to larger scales (Belter et al.,1988). Chemical methods of cell rupture include treat-ment with detergent, alkalis, organic solvents, or osmoticshock. Alkali treatment is basically converting cell wallcomponents into detergents. Although alkali treatmentis a cheap technique, it is not useful because it is harsh,not specific, and destroys the product (Dutta, 2008).Use of the enzyme lysozyme for digestion of bacterialcell walls is an example of biological methods of cell


disruption. It is an effective method due to selectivenessand gentleness, but the high cost of enzymes makes thismethod impractical for large-scale operations (Shuler &Kargi, 2008).

6.4.3 Removal of solubles

The next step in product recovery is separating intracel-lular (after cell disruption) and extracellular productsfrom the broth and many methods are available for thispurpose. Recovery and purification is a very wide topic,and only a few methods will be explained briefly in thissection (Belter et al., 1988; Shuler & Kargi, 2008).Extraction is the method of separating the solutes of a

liquid solution by contact with another insoluble liquid,the solvent (Dutta, 2008). The desired product can beselectively extracted out of the solution into the solventphase by selecting an appropriate solvent. A good solventshould be easily recovered, have large density differencebetween two phases, non-toxic, and cheap. The solvent-rich phase is called the extract, whereas the residue ofthe liquid is called the raffinate (Dutta, 2008).Adsorption is a technique that relies on the fact that

specific substances in solution can be adsorbed selectively

by certain solids as a result of physical or chemical inter-actions between the molecules of the solid and substanceadsorbed. Adsorption is a very effective method forseparation of dilute substances, because it is very selectivewhile the solute loading on the solid surface is limited(Dutta, 2008). Adsorption can be due to intermolecularforces (van der Waals forces between the molecules andsubstance), chemical interaction between a solute andligand, or ionic forces. Adsorption can occur via stage-wise or continuous-contact methods.Precipitation is a commonly used method for the

recovery of proteins or antibiotics (Belter et al., 1988).It can be induced by addition of salts, organic solvents,or heat. The addition of salts precipitates proteins, sinceprotein solubility is reduced by increase in salt concentra-tion in the solution. This method is effective and cheap,but it causes protein denaturation (Belter et al., 1988).Ammonium sulfate is the most commonly used salt buthas the disadvantage that it is hard to remove fromprecipitated protein. Organic solvents precipitate proteinat a lower temperature by decreasing the dielectric con-stant of the solution. Acetone, ethanol, and methanolare the most commonly employed organic solvents.Heating is another way of promoting precipitation of

InletOultetOultet

Ou

tlet

Feed in

Outle

t

Figure 6.3 Schematics for tubular centrifuge (left) and disk centrifuge (right).


proteins by denaturation. It is used to eliminate unwantedproteins; however, it is difficult to precipitate the unwantedproteins without damaging the desired product.Chromatography employs a mobile phase and a

stationary phase. The mobile phase is the solution con-taining solutes to be separated and eluent that carriesthe solution through the stationary phase. The stationaryphase can be adsorbent, ion exchange resin, porous solidor gel and is mostly packed in a cylindrical column.A solution is injected at one end of the column and eluentcarries the solution through the stationary phase to theother end of the column. Each solute in the originalsolutionmoves at a rate proportional to its relative affinityfor the stationary phase and is separated at the end of thecolumn (Ogez et al., 1989). Chromatography is employedto purify proteins and peptides from a complex solutionat laboratory scale. For large-scale chromatography, astationary phase must be selected which has the requiredstrength and chemical stability (Dutta, 2008).Membrane filtration is used to separate small particles

using polymeric membranes under pressure. Based on thesize of particles, membrane filtration is subcategorizedas microfiltration, ultrafiltration, and reverse osmosis.Microfiltration is employed for colloids, fat globules,and cells with a pore size of 0.01–10 μm (Grandison &Finnigan, 1996). Ultrafiltration employs the sameprinciple but is used for smaller particles with a pore sizeof 10–200 μm. In reverse osmosis, a macromolecule anda solvent are separated by a semi-permeable membraneunder pressure (Belter et al., 1988). The pore size(1–10 μm) of the membrane for reverse osmosis enablesall suspended and dissolved material to be separated(Dutta, 2008). First-generation membranes were madeof cellulose acetate. Nowadays, inorganic and organicmembranes are available (Lewis, 1996). Detailed descrip-tion of membrane filtration can be found in the study ofGrandison and Lewis (1996).

6.4.4 Purification

Especially for pharmaceuticals, finely purified final prod-uct is required. Therefore, crystallization, ultrafiltration,and various chromatographic methods are employedfor final purification. Crystallization operates at lowtemperature and minimizes the thermal degradation ofheat-sensitive materials (Shuler & Kargi, 2008). Dryingis the removal of solvent from purified wet product. Thephysical properties of the product, its heat sensitivity, anddesirable final moisture content have to be consideredto conduct a drying process. Vacuum-tray driers, freeze

driers, rotary drum driers, spray drum driers, andpneumatic conveyor driers are the major types used forpurification (Shuler & Kargi, 2008).

6.5 Fermented foods

Fermentation of foods dates back many thousands ofyears, with grape and barley fermentation for alcoholicbeverage production (Campbell-Platt, 1994). Fermentationcan improve the sensory characteristics, increase the shelflife, and enhance the nutritional value of food. This sectionfocuses on someof the results of actions ofmicroorganisms,which cause biochemical and physical changes during theproduction of fermented foods.

6.5.1 Fermented dairy products

Lactic acid bacteria play an important role in the productionof fermented dairy products. They ferment lactose and pro-duce lactic acid, which decreases the pH of milk. As a result,pH reaches the isoelectric point of casein, which is themajorprotein in milk. At the isoelectric point, the total numbersof positive and negative charges on the amino acids are inequilibrium. At that point, casein precipitates and coagulumis formed (Üçüncü, 2005). This causes both physical andbiochemical changes in the product. In addition to lacticacid, starter cultures may produce organic molecules, suchas acetaldehyde, diacetyl, acetic acid, ethanol, exopolysac-charides, etc., which have effects on textural and flavor pro-files of the product (Hutkins, 2006). Therefore, differenttypes of cultures and production conditions create differentcharacteristics for specific products.

6.5.1.1 Yogurt

Yogurt is a dairy product produced by fermentationof lactose in pasteurized milk. The starter cultures of yogurtare Streptococcus thermophilus and Lactobacillus delbrueckiisubsp. bulgaricus (Üçüncü, 2005). At the beginning of fer-mentation, S. thermophilus grows faster than L. delbrueckiisubsp. bulgaricus and decreases pH and Eh (oxidation/reduction potential) to levels suitable for the growth ofL. delbrueckii subsp. bulgaricus. Moreover, S. thermophilusproduces formic acid and CO2, which are the primegrowth factors for L. delbrueckii subsp. bulgaricus(Gürakan & Altay, 2010). S. thermophilus has lowerproteolytic activity than L. delbrueckii subsp. bulgaricus.L. delbrueckii subsp. bulgaricus produces a proteinasethat makes peptides and amino acids available for allorganisms, including S. thermophilus (Hutkins, 2006).


During manufacturing, S. thermophilus and L.delbrueckii subsp. bulgaricus are prepared separately,because when the conditions are suitable for the growthof L. delbrueckii subsp. bulgaricus, they can produce moreacid than can be tolerated by S. thermophilus and suppressthe growth of S. thermophilus. The growth of streptococciis inhibited at pH of 4.2–4.4, but lactobacilli toleratepH values in the range of 3.5–3.8 (Lourens-Hattingh &Viljoen, 2001). The fermentation for yogurt productionoccurs at 40–45 �C for approximately 6 h, until the pHis about 4.4–4.6 or a titratable acidity (as lactic acid) of0.8–0.9% is reached (Gürakan & Altay, 2010; Hutkins,2006). Since the growth of L. delbrueckii subsp. bulgaricusis favored at higher temperature than the growth ofS. thermophilus, changes in incubation temperature mayinfluence the growth rates of the two organisms, as wellas the metabolic products they produce (Hutkins, 2006).For example, the acidity of the product may increase ifthe growth of L. delbrueckii subsp. bulgaricus is favored.Streptococcus thermophilus and L. delbrueckii subsp. bul-

garicus are thermophilic and use a secondary transport sys-tem (called LacS) for lactose uptake. Transport of lactoseoccurs via a symport mechanism, in which the proton gra-dient serves as the driving force (Hutkins, 2006). Then, S.thermophilus and L. delbrueckii subsp. bulgaricus hydro-lyze the intracellular lactose by β-galactosidase and releaseglucose and galactose. Glucose is subsequently phosphory-lated (using ATP as the phosphoryl group donor) by glu-cokinase (or hexokinase) to form glucose-6-phosphate.Glucose-6-phosphate is used to produce lactic acid viathe Embden Meyerhof glycolytic pathway. Most strainsof S. thermophilus and L. delbrueckii subsp. bulgaricus donot have an enzyme to metabolize galactose. Therefore,galactose is secreted back into the milk (Gürakan & Altay,2010; Hutkins, 2006). In addition to lactic acid, both S.thermophilus and L. delbrueckii subsp. bulgaricus can pro-duce acetaldehyde. In one pathway, an aldolase enzymehydrolyzes the amino acid threonine, and acetaldehydeand glycine are formed directly. In the second pathway,the pyruvate can either be decarboxylated, forming acetal-dehyde directly, or converted first to acetyl-CoA and thenoxidized to acetaldehyde. Moreover, diacetyl, acetoin, andpolysaccharides may also be produced by the yogurtculture (Hutkins, 2006).

6.5.1.2 Cultured buttermilk

Traditionally, cultured buttermilk was made from thefermentation of fluid remaining after cream is churnedinto butter. Now, low-fat milk is cultured to produce

cultured buttermilk. The starter culture for buttermilkusually contains a combination of acid-producingbacteria including Lactococcus lactis subsp. lactis and/orL. lactis subsp. cremoris and flavor-producing bacteria,such as L. lactis subsp. lactis (diacetyl-producing strains),Leuconostoc mesenteroides subsp. cremoris or Leuconostoclactis, and Streptococcus lactis subsp. diacetylactis in aratio of about 5:1 (Hutkins, 2006; Oberman, 1985).The acid-producing bacteria are homolactic, producing

two lactic acid molecules per glucose molecule. The flavor-producing bacteria are heterofermentative and producelactic acid as well as small amounts of acetic acid, ethanol,and carbon dioxide, which contribute to the flavor. More-over, these bacteria may ferment citric acid from the Krebscycle to diacetyl (Hutkins, 2006; Oberman, 1985). Diacetylis the major flavor compound of cultured buttermilk(Hutkins, 2006). Citrate is transported by a citrate per-mease (CitP) with an optimum activity between pH 5.0and 6.0, then acetate and oxaloacetate are produced byhydrolyzation of the intracellular citrate by citrate lyase.The oxaloacetate is decarboxylated by oxaloacetate decar-boxylase and produces pyruvate and carbon dioxide, andthe acetate is released into the medium. In citrate fermen-tation, acetaldehyde-TPP is formed from the oxidativedecarboxylation of excess pyruvate by thiamine pyrophos-phate (TPP)-dependent pyruvate decarboxylase and then,acetaldehyde-TPP condenses with another pyruvatemolecule to form α-acetolactate. α -Acetolactate is decar-boxylated in the presence of oxygen and forms diacetylnon-enzymatically.Acetoin and 2,3-butanediol may be formed by the fur-

ther reduction of diacetyl and these products have no effecton the flavor. Therefore, it is critical to adjust the optimumconditions to obtain desired amount of diacetyl in culturedbuttermilk. Oxygen slows down the rate at which diacetyl isreduced to acetoin or 2,3-butanediol. Therefore, it may beuseful to introduce air into the final product by agitation.Moreover, after diacetyl is produced, the low pH inhibitsthe reduction reactions that convert diacetyl to acetoinand 2,3-butanediol. If the temperature during incubationis greater than 24 �C, the citrate fermentors may be inhib-ited by the growth of the homolactic lactococci, which maycause too much acid production. In contrast, if the incuba-tion temperature is lower than 20 �C, diacetyl formationmay not occur due to lack of acid (Hutkins, 2006).

6.5.1.3 Kefir

Kefir is a self-carbonated fermented pasteurized milkdrink. Kefir grains consist of bacterial and yeast


microorganisms, which grow in a symbyotic relationship.The flavor compounds of plain kefir are lactic andacetic acids, diacetyl, and acetaldehyde, produced byhomofermentative and heterofermentative lactic acidbacteria (Lactobacillus brevis, Lactabacillus fermentum,Lactobacillus kefiri, Lactobacillus plantarum, Leuconostocmesenteroides subsp. cremoris, Streptococcus thermophilus,Lactococcus lactis subsp. cremoris), and ethanol, which isproduced when the yeasts (Candida kefir, Candida maris,Kluyveromyces marxianus, Saccharomyces cerevisiae, etc.)ferment lactose (Guzel-Seydim et al., 2010; Hutkins,2006). Acetic acid-producing bacteria, such as Acetobacteraceti, can involve kefir grains (Hutkins, 2006). The fermen-tation takes place at 25�C for 24 h with agitation. Whenthe pH reaches 4.6, fermentation ends (Guzel-Seydimet al., 2010).

6.5.1.4 Cheese

Cheese making is a very old method for the preservationof unpasteurized milk. When fresh milk becomes sour,the caseins aggregate to form a gel. Whey separation gen-erally occurs when the gelled or clotted milk is kept forsome time. These steps are the origin of cheese making.However, for centuries, milk has also been clotted bythe addition of specific agents, especially chymosin (alsocalled rennet), an enzyme extracted from the stomach ofa calf, and starter cultures (Walstra et al., 2006). Thereare numerous cheese varieties that follow different pro-duction processes. Variations at one or more steps duringproduction of cheese cause different textures and flavors(Gunasekaran & Ak, 2003). Microorganisms have alsobeen used to produce milk-clotting enzymes (rennet).Microbial rennets can be of bacterial or fungal origin.Bacillus subtilis, Bacillus polymyxa, Bacillus cereus, andEscherichia coli can be used by bacterial rennetproducers (Kandarakis et al., 1999; Üçüncü, 2005).Kandarakis et al. (1999) reported that the fermentationproduced by chymosin from E. coli can be considered asuccessful alternative to calf rennet used for the manufac-ture of feta cheese, based on firmness, syncretic andsensory properties. Mucor pusillus, Mucor miehei, andEndothia parasitica are used to produce fungal originrennets (Üçüncü, 2005). In acid-coagulated cheesemaking, acid may be formed from lactose by lactic acidbacteria (Fox et al., 2000; Walstra et al., 2006).The composition of the starter culture used to make

cheese depends on the type of end-product desired. Forexample, for curds that will be exposed to high tempera-tures, thermophilic lactic acid bacterial culture should be

used to withstand high temperatures during production.Mesophilic cultures, such as L. lactis subsp. lactis andL. lactis subsp. cremoris, grow within a temperature rangeof about 10–40 �C. Thermophilic cultures, mainlyLactobacillus helveticus, L. delbrueckii ssp. bulgaricusand S. thermophilus, grow at a temperature around42–45 �C. Culture inoculum is about 1% (w/w), whichgives an initial cell concentration of about 5 × 106 cellsper mL of milk in Cheddar type cheese production.Mozzarella is normally made with a 2% starter and Swisscheese is made with 0.5% culture (Hutkins, 2006).Moreover, microorganisms, including adventitious

bacteria, yeast, and molds, are present in cheese through-out the curd ripening process and contribute eitherdirectly through their metabolic activity or indirectlythrough the release of enzymes into the cheese matrixthrough autolysis (Beuvier & Buchin, 2004). Scott et al.(1998) suggested that the primary factors in ripeningare (i) storage temperature and humidity, (ii) chemicalcomposition of the curd (fat content, level of amino acids,fatty acids, and other by-products of enzymatic action),and (iii) residual microflora of the curd.The microflora of cheese and milk have effects on

cheese quality. The microflora associated with cheeseripening are extremely diverse. Cheese ripening can bedefined as the storage of the cheese at a controlled temper-ature and humidity until the desired texture and flavorhave developed. The microflora are divided into twogroups, which are the starter lactic acid bacteria and thesecondary microflora (commonly known as non-starterlactic acid bacteria). The secondary microflora, includingnon-starter lactobacilli, Pediococcus, Enterococcus, andLeuconostoc, propionic acid bacteria, molds, yeasts, etc.,are involved in the ripening process (Beresford &Williams, 2004). Crow et al. (2001) suggested thatmesophilic lactobacilli are important in the maturationof cheeses, because they can catabolize citrate and couldbe involved in proteolysis and other enzymatic pro-cesses during cheese ripening (Vidojevic et al., 2007).Leuconostoc spp. also have the ability to co-metabolizesugars and citrate. In addition, Enterococci spp.metabolize citrate and can form acetaldehyde, acetoin,and diacetyl. Ray (1995) also reported that pediococcican produce diacetyl from glucose. Moreover, non-starterlactic acid bacteria contribute to protein breakdown.Lactic acid bacteria could be involved in amino acid

breakdown by using the α-ketoglutarate dependent trans-aminase pathway. The resultant α-keto acids are subjectedto further enzymatic or chemical reactions to formhydroxy acids, aldehydes, alcohols and carboxylic acids,


which are important for cheese flavor (Beresford &Williams, 2004). Lipase and esterase activities are foundin non-starter lactobacilli and effect cheese characteristics.Tsakalidou et al. (1993) reported the beneficial effect ofenterococci in cheese making as a contribution to thehydrolysis of milk fat by esterases. Methyl ketones andthioesters, which have effects on cheese flavor, can beformed by the conversion of released fatty acids(Beresford & Williams, 2004).Lactic acid bacteria can protect the cheese against

spoilage bacteria by producing lactic acid, H2O2, andantibiotics such as nisin. Giraffa (2003) reported thatEnterococcus faecium is capable of producing a varietyof bacteriocins, called enterocins, with activity againstListeria monocytogenes, which has an important impacton the safety of cheeses made from raw milk (Abdiet al., 2006). Strains of L lactis subsp. lactis andLeuconostoc spp. metabolize citrate to diacetyl in the pres-ence of a fermentable sugar during production and earlyripening. Therefore, diacetyl is a very significant aromacompound for unripened cheeses (Fox et al., 2000).Acetic acid may be the result of the catabolism of lactose,citrate, amino acids, or propionic acid fermentation.Moreover, propionic acid, butyric acid, hexanoic acid,and branched chain volatile fatty acids are importantfor cheese flavor.Propionic acid is the end-product of lactic acid fermen-

tation by propionibacteria. Butyric acid can originatefrom the catabolism of triglycerides and from lactatefermentation by clostridia (Beuvier & Buchin, 2004).Metabolism of fatty acids in cheese by Penicillium spp.involves release of fatty acids by the lipolytic systems,oxidation of β-ketoacids, decarboxylation to methylketone with one less carbon atom and reduction of methylketones to the corresponding secondary alcohol underaerobic conditions (Fox et al., 2000). Therefore, theirlevels depend on the balance between production anddegradation, which depends on the degree of maturityof the cheese (Beuvier & Buchin, 2004). Ketones,especially methyl ketones, have typical odors and areimportant for the aroma of surface-mold ripened andblue veined cheeses (Curionia & Bossetb, 2002). Theconcentration of methyl ketones is related to lipolysis.In surface-ripened cheese, these compounds originatefrom the enzymatic activity of Penicillium roqueforti,Penicillium camemberti or Geotrichum candidum onketoacids (Curionia & Bossetb, 2002; Fox et al., 2000).Indole and skatole are nitrogen-containing compoundsthat contribute to the aroma profile of some cheeses(Curionia & Bossetb, 2002).

6.5.2 Meat fermentation

Fermented ground meat (typically beef or pork) productsare made from comminutedmeat and fat, mixed with salt,curing agents such as NaNO2, KNO3, and spices, andfilled into casings. The fermentation is carried out bylactic acid bacteria and enhances the microbiologicalstability and organoleptic properties of meat. The fermen-tation temperature is usually 15–26 �C and the relativehumidity is around 90% (Lücke, 1985). The primaryorganisms which are responsible for the fermentationinclude species of Lactobacillus. Pediococcus acidilactici,P. pentosaceus, and P. cerevisiae are the bacteria usedfor fast, consistent, large-scale production of fermentedmeat products. Lactobacillus sake and L. curvatus are con-sidered psychrotrophic, so they are suitable for fermenta-tions conducted at cool ambient temperatures. Most ofthe bacteria used as starter cultures for fermented sausage,such as P. acidilactici, L. plantarum, and L. sakei, are ableto produce bacteriocins (Hutkins, 2006). The main prod-uct of carbohydrate fermentation in meat fermentation islactic acid. The rate and extent of lactic acid productionfrom glucose, sucrose, or maltose are higher than fromlactose, starch, or dextrin. In addition to lactic acid, aceticacid, butyric acid, propionic acid, acetoin, 2,3-butanediol,and/or diacetyl may be produced in minor amountsduring fermentation (Lücke, 1985).The genus of Micrococcaceae is also used in starter

culture mixture. They are used in meat fermentation toconvert nitrate to nitrite via expression of the enzymenitrate reductase. This also causes formation of flavorand enhancement of color (Hutkins, 2006). They arethe main agents of lipid degradation in the fermentationprocess. Lipids are the precursor of many odoriferouscompounds, such as aldehydes, ketones, and short chainfatty acids. Molds and yeasts, which show lipolytic activ-ity, contribute to the flavor of fermented meat products(Lücke, 1985).

6.5.3 Vegetable fermentation

The production principle of vegetable fermentation is theconversion of sugars to acids by natural lactic microflora,such as Leuconostoc mesenteroides, Weisella kimchii,Lactobacillus plantarum, Lactobacillus brevis, Pediococcuspentosaceus, etc. (Hutkins, 2006; Vaughn, 1985).Sauerkraut is made by the fermentation of salted

cabbage and includes three stage of microbial succession,which are initiated by heterofermentative Leuconostocmesenteroides, followed by heterofermentative rods such


as Lactobacillus brevis, and homofermentative rods(Lactobacillus plantarum) and cocci (Pediococcuscerevisiae). The optimum temperature of sauerkrautfermentation is around 21 �C. In the production ofsauerkraut, 2.0–2.5% of salt is added. L. mesenteroides issalt tolerant and can grow fast and metabolize sugarsvia the heterofermentative pathway, yielding lactic andacetic acids, carbon dioxide, and ethanol. The environ-ment created by Leuconostoc mesenteroides inhibits thegrowth of microorganisms except lactic acid bacteria(Battcock & Azam-Ali, 1998; Hutkins, 2006; Vaughn,1985). Then Lactobacillus brevis, L. plantarum, andPediococcus cerevisiae begin to grow and produce lacticacid, acetic acid, carbon dioxide, and ethanol (Vaughn,1985). Moreover, mannitol, diacetyl, acetaldehyde, andother volatile flavor compounds may be produced duringfermentation of sauerkraut. Mannitol is produced fromfructose via the NADH-dependent enzyme mannitoldehydrogenase by heterofermentative leuconostocs andlactobacilli. The fermentation process completes whenthe acidity is at about 1.7% (w/v), with a pH of 3.4–3.6(Hutkins, 2006).Pickling technology, such as cucumber fermentation,

also includes lactic acid fermentation. Unlike sauerkrautfermentation, growth of Leuconostoc spp. is inhibited withthe addition of salt up to 8% (Vaughn, 1985). The fermen-tation is initiated by Pediococcus cerevisiae, L. brevis, andL. plantarum (Battcock & Azam-Ali, 1998; Hutkins, 2006;Vaughn, 1985).In olive fermentation, in addition to lactic acid bacteria,

non-lactic organisms (e.g. Pseudomonas and Enterobac-teriaceae) may affect the fermentation process and thefinal product consists of lactic acid, but also acetic acid,citric acid, malic acid, carbon dioxide, and ethanol atpH 4.5 and acidity less than 0.6% (Hutkins, 2006).

6.5.4 Vinegar

Vinegar is an aerobic fermentation of various ethanolsources to produce a dilute solution of acetic acid(Adams, 1985). The minimum acetic acid concentrationin vinegar should be 4% (w/v). The name of the vinegarindicates its juice source, such as red wine vinegar, applecider vinegar, malt vinegar, etc. (Hutkins, 2006).The manufacture of vinegar includes two stages of

fermentation. First, ethanol is produced by yeast, such asS. cerevisiae, via the fermentation of sugars. Then, theethanol is oxidized to acetic acid by Acetobacter, Glucono-bacter, and Gluconoacetobacter (Adams, 1985; Hutkins,2006). In the first stage, high sugar concentration and

low pH (~4.5) favor ethanol production and as a resultof alcoholic fermentation, anaerobic conditions are created,and acid and ethanol concentrations increase. Then, aero-bic conditions are re-established at the surface for theoxidation of ethanol to acetic acid (Adams, 1985). Thepathway of acetic acid production includes two main steps.In the first step, acetaldehyde is produced by oxidation ofethanol. Then, the acetaldehyde is oxidized to acetic acid.The acetic acid fermentation is performed by obligate aero-bes in the periplasmic space and cytoplasmic membrane(Hutkins, 2006). The conversion of ethanol substrate toacetic acid can be performed by the open vat method,the trickling generator process (quick vinegar process),and aerobic submerged fermentation process (Adams,1985; Hutkins, 2006).

6.5.5 Alcoholic beverages

6.5.5.1 Wine

Grape wine fermentation is initiated by crushing thegrape, producing the juice, removing the pomace andthen inoculating with S. cerevisiae. The acid and sugarlevels of fruit juice and low pH (~4.5) enhance the growthof yeasts and production of ethanol. The ethanol pro-duced restricts the growth of bacteria and fungi. At thebeginning of fermentation, several species of yeast,including Kloeckera, Hanseniaspora, Candida andMetschnikowia, may grow and produce small amountsof acids, esters, glycerol, and other potential flavor com-ponents (Battcock & Azam-Ali, 1998). The optimumtemperature is between 10 �C and 18 �C for white wineand between 20 �C and 30 �C for red wine fermentation(Battcock & Azam-Ali, 1998; Hutkins, 2006).Flavor components, such as 4-methyl-4-mercaptopen-

tan-2-one and 3-mercapto-1-hexanol, can be producedduring fermentation and the production of these com-pounds depends on the strain. Moreover, some commonyeast by-products from ethanol fermentations (e.g.glycerol, and acetic acid) affect the flavor of wine.Acetaldehyde is also formed from pyruvate duringvinification and is a precursor metabolite for acetate,acetoin, and ethanol synthesis and binds to proteins orindividual amino acids to produce aroma compounds.During yeast fermentation, many medium- and long-chain fatty acids, which contribute to the wine aroma,are also formed via the fatty acid synthesis pathway fromacetyl-CoA (Styger et al., 2011).Malolactic fermentation by lactic acid bacteria is

desired for high-acid wine. It involves the deacidification


of wine via conversion of dicarboxylic L-malic acid tomonocarboxylic L-lactic acid and carbon dioxide.Oenococcus oeni, Lactobacillus spp., Leuconostoc spp.,and Pediococcus spp. carry out the malolactic fermenta-tion (Hutkins, 2006; Styger et al., 2011).

6.5.5.2 Beer

Beer is created by the fermentation of malted barley that isheated with hops to produce the wort for yeast fermenta-tion. The main process stages of beer making are malting,milling, mashing, brewing, cooling, fermentation, matu-ration, filtering, and packaging. In the fermentationprocess, yeast ferments the sugars and produces ethanoland CO2. The strains of S. cerevisiae are used for “ale”or “top-fermenting yeast” beers. Saccharomyces pastoria-nus is used for lager beers or “bottom-fermenting yeast.”Top-fermenting yeasts form low-density clumps orflocs, which cause the entrapment of CO2 and rise tothe surface. In lager beer fermentations, S. pastorianusflocculate, and the flocs settle to the bottom of the fermen-tation tank. The optimum fermentation temperatures are18–27 �C for top-fermenting yeast and below 15 �C forbottom-fermenting yeast. The inoculum of yeast popula-tion is around 106 cells per mL of wort. The fermentationvessel should be sparged with sterile air before inoculationto decrease the lag phase. Moreover, biosynthesis ofcell membrane lipids, which are essential for growthof yeast in wort, requires oxygen. Sugar metabolism byyeast occurs via the Embden-Meyerhoff-Parnas (EMP)glycolytic pathway. The ethanol percentage on a weightbasis can vary based on the process and is about 3.4%in a regular US beer (Hutkins, 2006).

6.5.6 Bread

Saccharomyces cerevisiae is used for bread production.It should have special characteristics, such as ability to pro-duce good bread flavor, stability and viability during stor-age. The seed growth medium includes molasses, variousammonium salts such as ammonium phosphate, magne-sium sulfate, calcium sulfate, and lesser amounts of thetrace minerals (e.g. zinc and iron). The optimum growthconditions are aerobic, 30 �C, pH of 4.0–5.0. Then, yeastis added to flour as yeast cream that contains about 20%total yeast solids. The fermentation process includes theuse of glucose via aerobic and/or anaerobic pathways(Hutkins, 2006). Yeast produces CO2, which causes an elas-tic, airy texture. Yeast also produces acid, which can affectthe flavor of the bread (Hutkins, 2006). In addition to yeast,

Lactobacillus brevis var. lindneri, L. fructivorans, and L.farciminis are used as a starter culture in sour dough ryebread. These cultures also produce acetic acid and someflavor compounds (Hutkins, 2006; Sugihara, 1985).

6.5.7 Tempeh

Tempeh is a popular fermented soybean product that iscommon in Far East countries such as Indonesia. Tempehproduction includes four main steps: soaking, boiling,inoculating of soybean with the fungal organism Rhizopusmicrosporus subsp. oligosporus, and incubating at roomtemperature (Astuti et al., 2000; Hutkins, 2006). The pro-duction of tempeh takes about 48 h. During the steepingperiod, endogenous lactic acid bacteria grow andreduce the pH. This causes inhibition of the growth ofundesirable spoilage bacteria and pathogens. Rhizopusoligosporus is inoculated at levels from 107 to 108 sporesper kg of beans. The recommended strains of Rhizopusoligosporus are NRRL 2710 and DSM 1964, which wereisolated from Indonesian tempeh (Hutkins, 2006). Inaddition to R. oligosporus, R. oryzae, R. arhizus, and R. sto-lonifer are also used in Indonesia (Astuti et al., 2000).Alternatively, “usar,” which is produced by inoculatingwild Rhizopus spores onto the surface of leaves obtainedfrom the indigenous Indonesian Hibiscus plant, can beused to inoculate soybeans.The fermentation process is a solid-state fermentation,

which includes soybeans held together by the mold myce-lium that grows throughout and in between the individualbeans. The fermentation conditions include little excesswater and are microaerophilic. The fermentation isconsidered to be completed when the beans are coveredwith white mycelium before the mold begins to sporulate.During fermentation, R. oligosporus excretes lipases andproteinases. As a result of lipid hydrolysis of oil insoybeans, mono- and diglycerides, free fatty acids, anda small amount of free glycerol are produced. Whileoxidation of most of the free fatty acids occurs due toR. oligosporus acitivity, only about 10% of the releasedamino acids and peptides are oxidized by R. oligosporus(Hutkins, 2006). Astuti et al. (2000) reported that3.6–27.9% of the total amino acids decrease occurs duringfermentation due to consumption of amino acids as asource of nitrogen for growth by strains of Rhizopus. R.oligosporus also produces polysaccharide-hydrolyzingenzymes. These enzymes hydrolyze pectin, cellulose,and other fiber constituents, and various pentoses (xylose,arabinose) and hexoses (glucose, galactose) are released(Hutkins, 2006).


6.6 Enzyme applications

Enzymes are proteins (MW of 15,000 to several millliondaltons) and catalyze the specific biochemical reactionsin biological systems (plants, animal, and microbial)(Aurand et al., 1987; Berk, 1976; Shuler & Kargi, 2008).Many enzymes are conjugated proteins containing spe-cific prosthetic groups. The complete enzyme is called ahaloenzyme; the protein part is called the apoenzyme,and the non-protein moiety the prosthetic group (Berk,1976; deMan, 1996). The prosthetic group is often onlyloosely attached to the protein and can be separated bydialysis. In this case, the protein moiety is termed theapoenzyme and the prosthetic group the co-enzyme orco-factor (Aurand et al., 1987; Berk, 1976). Nicotinamideadenine dinucleotide (NAD) is an example of a co-enzyme, which binds to enzyme less strongly than pros-thetic groups; thus co-enzymes dissociate from theenzyme during or at the termination of a catalytic reaction(Aurand et al., 1987).At the first stage of enzyme mechanism, the substrate

combines with the catalyst into a substrate-enzyme com-plex. The complex is unstable under reaction conditionsand is quickly decomposed. The enzyme is regenerated.The substrate, which emerges from the reaction, can bein the final, changed form or in a highly activated state.When an enzyme reacts with its substrate, only the “activesite,” which is certain regions of the protein molecule,participates in the process. Special groups of amino acidresidues produced by the sequence and particular foldingof the enzyme protein form active sites. The portion of theprotein molecule outside the active regions provides thebackbone and support for the active sites.The rate of enzyme-catalyzed reaction is proportional

to the concentration of the enzyme. The rate also dependson the concentration of the substrate. At low substrateconcentration, reaction rate is related to substrate concen-tration linearly and complies with first-order kinetics(Aurand et al., 1987). Substrate binding is a prerequisitefor catalysis. Therefore, enzymes have specificity and sus-ceptibility to inhibition by certain substances. Substratespecificity means that the enzyme is selective towardsthe substrate. For example, the hydrolytic enzyme argi-nase catalyzes cleavage of the amino acid L-arginine, intourea and L-ornithine, but will not do so with D-arginine.Enzyme inhibitors slow down the rate of enzymatic reac-tions. Inhibitors, termed antimetabolites, may blockimportant metabolic pathways. For example, Bowman-Birk trypsin isoinhibitors present in soybean may inhibit

the proteolytic action of trypsin (Tan-Wilson et al., 1987).Moreover, the number of active sites available for bindingthe substrate may decrease due to the effect of a substancewhich is sufficiently similar in structure to the substrate.For example, if malonic acid is added to succinic acid,it will compete with succinic acid for the active site,which makes the succinic acid dehydrogenase unavailablefor converting the succinic acid into fumaric acid(Berk, 1976).Temperature is a factor of the rate of enzyme-catalyzed

reactions. An increase in temperature increases the veloc-ity of the reaction within definite limits (Aurand et al.,1987). Most enzymes are thermolabile and applicationof 70–80 �C for 2–5 min can destroy their activity(Berk, 1976). Initially the reaction rate increases withincreasing temperature to an optimum, but then as thetemperature increases further, the reaction rate decreaseseventually to zero. Above about 45 �C, thermal denatura-tion of proteins starts, and the activity of the enzymesgradually decreases (Aurand et al., 1987). The enzymeactivity is also greatly affected by changes in pH of itsmedium, which cause changes in the ionization of theenzyme, substrate, or enzyme-substrate complex. Moreo-ver, low or high pH can cause inactivation due to denatur-ation of the enzyme protein (Aurand et al., 1987).Instead of quantification of the enzyme in terms of

direct concentration, the quantity is expressed as “activ-ity” or the rate at which the specific reaction proceedsin the presence of specific concentrations of enzyme prep-aration, under standard conditions (Berk, 1976). Thereaction velocity can be measured by static or dynamicmethods. In the static method, the substrate and enzymeare mixed and the reaction is stopped at a certain time andthe concentration of remaining substrate or of productformed is determined. In the dynamic method, the sub-strate concentration is continuously monitored for aspecific time period and the change in the substrate orproduct concentration from zero time to time t representsthe enzyme activity (Aurand et al., 1987).The names of enzymes are formed by adding the suffix

-ase to the name of the substrate, e.g. lipases, proteinases,amylase, maltase, etc. (Berk, 1976; Eskin, 1990). TheEnzyme Commission number (EC number) of theInternational Union of Biochemists allows systematicclassification, which divides the enzymes into six maingroups according to the type of reactions involved, asnow briefly described.

Oxidoreductase enzymes catalyze the oxidation-reduction reactions. This group includes the enzymes


commonly known as dehydrogenases, reductases, oxi-dases, oxgenases, hydroxylases, and catalases (Berk,1976). For example, glucose oxidase is used to minimizethe Maillard reaction, catalase is used to remove H2O2 inthe milk industry, and lipoxygenase is used in the bakingindustry (Eskin, 1990). An example for this reaction is:

L-iditol +NAD! L-sorbose +NADH2Boyce & Tipton, 2001ð Þ

Transferases are enzymes that catalyze the transfer ofvarious chemical groups (methyl, acetyl, aldehyde,ketone, amine, phosphate residues, etc.) from one sub-strate to another (Berk, 1976). Transminases and kinasesare typical examples of this group (Aurand et al., 1987).An example for this reaction is:

S-adenosyl-L-methionine+ 3-hexaprenyl-4,5-dihydroxybenzoate!

S-adenosyl-L-homocysteine + 3-hexaprenyl-4-hydroxy-5-methoxybenzoate Boyce & Tipton, 2001ð Þ

Hydrolases are responsible for the hydrolytic cleavage ofbonds by the addition of a water molecule. For example,amylases are used for the production of glucose syrupsfrom cornstarch, pectinases are used to clarify juices,and proteases are used as meat tenderizers. An examplefor this reaction is:

4-hydroxybenzoyl-CoA+H2O!4−hydroxybenzoate +CoA Boyce & Tipton, 2001ð Þ

Lyases catalyze the non-hydrolytic cleavage of C-C, C-O,and C-N bonds, and other bonds by elimination, leavingdouble bonds or rings or conversely, adding groups todouble bonds. They remove groups from their substrates,leaving double bonds. Examples are carboxylases, aldolases,hydratases, and phenylalanine ammonia lyase (Aurandet al., 1987; Berk, 1976). Pectate lyases have potential appli-cations in the medicine, textile, and food industries toimprove the surface properties of natural fibers (Hanet al., 2011). An example for this reaction is:

2-hydroxyisobutyronitrile! cyanide + acetoneBoyce & Tipton, 2001ð Þ

Isomerases transform their substrates from one isomericform to another. Racemases and epimerases are membersof this group (Berk, 1976). For example, glucose

isomerase is used for the production of high-fructose cornsyrups. An example of this reaction is:

2Sð Þ-2-methylacyl-CoA! 2Rð Þ-2-methylacyl-CoABoyce & Tipton, 2001ð Þ

Ligases catalyze reactions that join two moleculestogether (C-O, C-S, C-N, or C-C bonds), which representsa synthesis (Berk, 1976). For example, aminoacyl-tRNAsynthetases catalyze the linking together of two substratemolecules with the breaking of a pyrophosphate bond(Aurand et al., 1987). An example for this reaction is:

4-chlorobenzoate +CoA+ATP! 4-chlorobenzoyl-CoA+AMP+diphosphate Boyce & Tipton, 2001ð Þ

Although there are six main divisions of enzyme, eachenzyme group has many subgroups. Enzymes are givena code number, such as EC 3.1.1.2 (carboxylic ester hydro-lases) and EC 4.2.1.20 (tryptophan synthase). The ECnumber contains four elements separated by dots. Thefirst number symbolizes the division of enzyme out ofsix main enzyme groups. The second and third numbersshow the subclass and sub-subclass, respectively. Thefourth number indicates the serial number of the enzymein its sub-subclass (Moss, 1992).Some of the traditional enzymes are prepared from ani-

mal and plant sources. For example, papain from papaya(Carcia papaya), bromelain from pineapple (Ananassativa), and ricin from the castor oil plant (Ricinuscommunis) have a high proteolytic effect (Üçüncü,2005). Rennet, an animal derivative, is extracted fromthe fourth stomach of a calf or young goat (Carroll,1994). For isolation of enzyme, the tissue is usually homo-genized in the presence of an extraction buffer that oftencontains an ingredient to protect the enzyme from oxida-tion and from traces of heavy metal ions. Then, proteinimpurities are removed by fractional precipitation, ionexchange chromatography, etc. (Belitz et al., 2004).Nowadays, most industrial enzymes are of microbial

origin. Microbial enzymes can be heat stable and have abroader pH optimum. Most of these enzymes are preparedby batch cultivation of highly developed strains ofmicroorganisms. Moreover, gene transfer to carriermicroorganisms makes microbial enzyme productionpossible (deMan, 1996). For example, the gene forprochymosin (rennet) has been cloned in Escherichia coli,Saccharomyces cerevisiae, Kluyveromyces marxianus,Aspergillus nidulans, Aspergillus niger, and Trichodermareesei. As a result of recombination, enzymatic properties


of the recombinant enzymes are indiscernible from thoseof calf chymosin (rennet). The cheese-making propertiesof recombinant chymosins have been assessed in manycheese varieties, mostly with very satisfactory results (Foxet al., 2000). Moreover, Mucor pusillus and Aspergillusoryzae are used to produce proteinase (Belitz et al., 2004).Enzyme preparations are expensive. Although the

enzyme is regenerated in the process and could be usedover and over again, its separation from the reactionmixture is not usually economically feasible. Thus immo-bilized enzymes are employed for enzyme reuse. Immobi-lization is the attachment of enzymes to insolublematrices. Therefore, the enzyme can be easily separatedfrom the reaction mixture (Berk, 1976). Immobilized orbound enzymes have been prepared by physically orchemically binding the enzyme to an insoluble support.Immobilized enzymes can be used in batch processesand then recovered by filtration or centrifugation forfurther use. Applications of this technology facilitaterepeated use of the same enzyme preparation in a batchprocess. Immobilized enzymes can also be used in contin-uous processes in specially designed reactors, such as con-tinuous feed stirred tank, packed-bed reactor, fluidizedbed reactor, and more. Immobilized enzymes are gener-ally more stable to heat than soluble enzymes. Immobi-lized enzymes have been used in the food industry(Eskin, 1990). For example, an immobilized lactase sys-tem has been developed by adsorption of enzyme onporous glass beads and stainless steel, covalent linkageto organic polymers, entrapment within gels or fibers,and microencapsulation within nylon or cellulose micro-capsules to produce low-lactose milk (Eskin, 1990).Detailed examples of the application of immobilizedenzymes in foods can be found in Biochemistry of Foodsby Eskin (1990).

6.6.1 Industrially important enzymes

Microbial enzymes have been used in the food industryfor centuries. They also had applications in the leatherindustry, such as using dung for preparation of hides(Underkofler et al., 1958). In the 1930s, enzyme technol-ogy was used for the first time in the food industry, toclarify fruit juice (Adler-Nissen, 1987). Since then,enzymes have found uses in the food, detergent, textile,leather, cosmetic, and pharmaceutical industries, dueto their benefits, such as specificity, mild conditions,environmentally friendly, and less waste generation(Hasan et al., 2006). In this section, some major industrialenzymes will be presented.

6.6.1.1 Lipases

Lipases (triacylglycerol acylhydrolase, EC 3.1.1.3) acton carboxylic ester bonds, and require no co-factor.Long-chain fatty acids are the natural substrates for lipase(Ghosh et al., 1996). Lipases are of interest for industrybecause of their natural function of hydrolyzing triglycer-ides into diglycerides, monoglycerides, fatty acids, andglycerol (Ghosh et al., 1996). Furthermore, substratespecificity, regioselectivity, and enantioselectivity areproperties of lipases that attract industrial interest(Hasan et al., 2006). Protein engineering allows theimprovement of properties of lipase, including active site,specific activity, stability, specificity, calcium binding, andsurfactant compatibility (Svendsen, 2000). Lipases aremonomeric proteins with a molecular weight of 19–60kDa and the characteristics of the enzyme are highlydependent on position of the fatty acids in the glycerolbackbone, chain length, degree of unsaturation, and otherfactors (Aravindan et al., 2007).Lipase activity is pH dependent but studies have shown

that lipases are stable in a pH range of 4–8. On theother hand, the temperature profile of lipases varies basedon the source of the enzyme. For example, lipases derivedfrom mesophilic microorganisms are active at 30–35 �C,whereas this value reaches 40–60 �C when the microbialsource is a thermophile (Aravindan et al., 2007).Lipases have applications in several industries and

most lipases are commercially available (Houde et al.,2004). The advantages of microbial lipases over plant-or animal-derived enzymes include availability of largeamounts of purified lipase, higher stability, activity atelevated temperatures, reduced by-products, cheaperdownstream processing, and activity in organic solvents(Hasan et al., 2006).Infant formulas are a good alternative to breast milk

asfar as they have the same properties as human milk.However, the fat in infant formulas is derived from plantsand differs from human milk, and binds the calciumand causes constipation. Lipase is used to modify the tri-glyceride to increase the palmitic acid proportion of themilk and the absorption capability (Houde et al., 2004).Cocoa butter fat is a high-value product with desired

properties such as melting point (37 �C), snap, and gloss.It also provides a cooling sensation and smoothness forchocolate (Hasan et al., 2006; Houde et al., 2004). How-ever, a cheaper substitute for cocoa butter, palm oil, hasa melting point of 23 �C, is liquid at room temperature,and is a low-value product. Lipases are used to convertpalm oil into cocoa butter substitute by transesterification


reactions (Aravindan et al., 2007). Such modification ofless expensive fats, such as shea butter, salt fat, and palmoil, can be obtained by transesterification and providescheap substitutes for cocoa butter (Houde et al., 2004).In the baking industry, lipases are widely used to

improve flavor, texture and softness, as well as to increaseloaf volume (van Oort, 2010). Lipase esterifies triglycer-ides to mono- and diglycerides to enhance the flavorcontent of bakery products (Aravindan et al., 2007).Studies have shown that a lipid film surrounding thegas cell increases gas cell stability (van Oort, 2010),which can be provided by esterification of triglycerides(Hamer, 1995).Lipases are commonly used in the dairy industry to

hydrolyze milk fat, and current applications of lipasesin the dairy industry include cheese ripening, flavorenhancement, manufacturing cheese-like products, andlipolysis of cream and butterfat (Aravindan et al., 2007).Cheese texture is dependent on fat content so lipases thatrelease short-chain fatty acids (C4 and C6) develop thesharp and tangy flavor, whereas release of medium-chainfatty acids (C12 and C14) causes a soapy taste in theproduct (Hasan et al., 2006). Lipases are also used forenzyme-modified cheeses (EMC) to liberate fatty acidsat sn-1 and sn-3 positions on the glycerol backbone(Houde et al., 2004). EMC find applications in the foodindustry to add cheese flavor to salad dressings, dips,soups, sauces, and snacks (Aravindan et al., 2007).The detergent industry is another example of lipase

application for lipid degradation (Gandhi, 1997). In fact,lipases find their most significant application in the deter-gent industry, approximately 30% of the total enzymemarket (Houde et al., 2004). Common commercial usesof lipases for detergents are dish washing, bleaching com-position, dry-cleaning solvents, liquid leather cleaner,contact lens cleaning solutions, cleaning of lipid-cloggeddrains, and so on (Gandhi, 1997). Lipases follow a two-step mechanism: the first step is binding of the enzymesurface and the second step is hydrolysis of carboxyl esterbonds (Galante & Formantici, 2003). Lipases thereforehydrolyze the water-insoluble triglycerides into water-soluble products (mono- and diglycerides, and glycerol)(Galante & Formantici, 2003). Enzymes are preferredin the detergent industry because they can reduce theenvironmental load of detergent products, enable lowerwash temperatures, reduce undesired chemicals in thedetergent formulation, leave no harmful residues, andare safe for aquatic life (Hasan et al., 2006).In the leather industry, fat residues have to be removed

during hide and skin processing. Conventional methods

involve use of organic solvents and surfactants whichare of environmental concern (Hasan et al., 2006). Lipasesreplace chemical substances to remove the fats andgrease. Enzymes are used in the bating step in whichnon-collagenous, globular proteins are degraded to softenhides before tanning (Galante & Formantici, 2003).Lipases endow a more uniform color and cleaner appear-ance. They also improve the production of waterproofleather (Hasan et al., 2006). Muthukumaran and Dhar(1982) reported that lipase derived from Rhizopusnodosus was used for the production of suede clothingleathers from sheepskins.Biodiesel is a renewable, environmentally safe, and

energy-efficient bioenergy. In the production of biodiesel,plant and animal lipids can be treated with lipase to pro-duce free fatty acids, which are esterified to methanol toproduce methyl ester fatty acids and glycerin. Althoughany fatty acid can be a raw material for the productionof biodiesel, waste vegetable oil and animal fats are pre-ferred due to the fact that they are not food-value pro-ducts (Refaat, 2010). Production of biodiesel from wasteoil starts with pretreatment of raw material, whichincludes filtration to remove dirt, food residues, andnon-oil materials. After determining the concentrationof fatty acid, transesterification is performed to obtainbiodiesel. Korus et al. (1993) reported that some catalystscould be used to improve transesterification such aspotassium hydroxide, sodium hydroxide, sodium meth-oxide, or sodium ethoxide.Applications of lipases are not limited to the examples

given above. They have found applications in cosmetics,for instance in hair waving preparations, antiobesitycreams, wax esters in personal care products, retinol,and so on (Hasan et al., 2006). The meat, beverage, paper,and healthy food industries also employ lipases for fatremoval and flavor development, aroma improvement,hydrolysis, and transesterification, respectively (Lanttoet al., 2010).

6.6.1.2 Amylases

Enzymes which are capable of hydrolyzing theα-1,4-glucosidic linkages of starch are called amylases(Vihinen &Mantsiila, 1989). Although amylases are foundin plants and animals, microbial amylases are most com-mon in industry. α-Amylase (EC 3.2.1.1) and glucoamylase(EC 3.2.1.3) are two major amylases (Pandey et al., 2000).Industry has taken advantage of the starch-degradingproperties of these enzymes (Bigelis, 1993). Starch isproduced mainly in higher plants, and is water insoluble


(Aiyer, 2005). It is composed of two components:amylose and amylopectin. Amylose is a mainly linearpolysaccharide which is formed by α-1,4 linked D-glucoseresidues and some α-1,6-branching points. Amylopectinhas a highly branched tree-like structure. The proportionof branches is an important property of the substratebecause enzymes hydrolyze different substrates with differ-ing specificities.Several amylolytic enzymes hydrolyze starch or its deg-

radation products. The actions of these enzymes can bedivided into two categories. Endoamylases break downlinkages randomly in the interior of the starch molecule.Exoamylases hydrolyze the polysaccharide from thenon-reducing end, which produces short end-products.α-Amylase (endo-α-1,4-D-glucan glucohydrolase)hydrolyzes the α-1,4-D-glucosidic linkages in the linearamylase chain, randomly. However, glucoamylase(exo-α-1,4-α-D-glucan glucohydrolase) cleaves theα-1,6-linkages at the branching points of amylopectinas well as α-1,4-linkages (Pandey et al., 2000). β-Amylase(α-1,4-glucan maltohydrolase, EC 3.2.1.2) is originallyderived from plants although there are a few microbialstrains which can produce it. β-Amylase can cleavenon-reducing ends of amylase and amylopectin, whichresults in incomplete degradation (Pandey et al., 2000).Amylases are extensively employed for starch processing

in which gelatinization, liquefaction, and saccharificationoccur. Gelatinization is the dissolution of starch granulesat above 60 �C in the presence of thermostable α-amylasederived from Bacillus subtilis or B. licheniformis (Bigelis,1993; Maarel, 2010). In this step, pH adjustment is impor-tant to provide the optimum environment for amylase tofunction. In addition to pH adjustment, addition ofcalcium is also necessary to stabilize the enzyme (calciumprevents unfolding of the enzyme) (Maarel, 2010). Thenext step in starch hydrolysis is liquefaction at which slurryis held at 95–100 �C for 1–2 h. During liquefaction, ther-mostable amylase breaks down the α-1,4-linkages in bothamylose and amylopection and generates dextrins (Maarel,2010). The liquefaction process is continued until thedesired dextrose equivalent (DE) value is reached(Bigelis, 1993). The DE value is determined as the numberof reducing ends relative to a pure glucose of the sameconcentration. The DE value is 100 for glucose and 0 forstarch. In liquefaction, usually, DE of 10–15 is obtained(Bigelis, 1993).The final step of starch hydrolysis is saccharification.

The liquefied slurry is processed at about 60 �C with theaddition of glucoamylase. In saccharification, pullunase,glucoamylase, β-amylase, or α-amylase can be employed.

Glucoamylases hydrolyze α-1,4-linkages with additionalactivity on α-1,6-linkages. Moreover, addition of pullu-nase in glucoamylase increases the glucose yield about2%, while a cocktail of pullunase and β-amylase producesmaltose (Maarel, 2010). Pullunase addition has morebenefits such as shorter saccharification time, reductionof glucoamylase amount used, and increase in dry mattercontent (Bigelis, 1993). Maltose, glucose, or mixed syrupsare end-products of saccharification according to theenzyme used.Amylases are mainly used in the food processing

industry such as for baking, brewing, high-fructosecorn syrup, alcoholic beverages, cakes, and fruit juices(Couto & Sanroman, 2006). Amylases produced fromAspergillus oryzae, A. niger, A. awamori or species ofRhizopus are used in dough making to degrade starchin the flour into dextrins and provide fermentable sugarsfor yeast (Souza & Magalhaes, 2010). The addition ofamylase therefore reduces the viscosity of dough,increases fermentation rate, and improves taste and crustcolor by generating sugar (Bigelis, 1993). Volume andanti-firming are the other benefits of using amylases inbaking (Hamer, 1995).High-fructose corn syrup (HFCS) is used as a sweet-

ener in beverages and foods. It has fewer calories and ischeaper than sucrose (Bigelis, 1993). In the United States,corn is the main raw material used to produce glucosesyrup by hydrolysis of starch in the presence of amylases(Pomerantz, 1991). After glucose syrups are produced,glucose isomerases (EC 5.3.1.18) can convert glucosesyrup into glucose-fructose mixture. Immobilization ofglucose isomerase reduces the cost of HFCS and increasesthe production of glucose-fructose syrup. However,glucose isomerase only converts 50% of the glucose intofructose (Maarel, 2010).Most alcoholic beverages, such as whiskey, vodka, and

brandy, are produced from sugar-containing raw materi-als. Malted barley, corn, milo, and rye are common rawmaterials for alcohol fermentation in the United States(Bigelis, 1993). The raw material is cooked to gelatinizethe starch for enzymatic degradation, and cooled to roomtemperature before saccharification by amylase is per-formed. Fungal amylase, from Aspergillus or Rhizopusspp., is used for saccharification, because it increasesthe reaction rate while a complete saccharification is per-formed. It also produces fewer by-products, for example,maltose, isomaltose, and oligosaccharides, that are notfermentable by yeast (Bigelis, 1993).Amylases are used in the detergent industry for laundry

and dish washing to degrade the starch-containing


residues to smaller oligosaccharides and dextrins(Olsen & Faholt, 1998). It is reported that 90% of liquidlaundry detergents contain α-amylase (Galante & For-mantici, 2003; Souza & Magalhaes, 2010). Amylases areactive at lower temperature and alkaline pH (Souza &Magalhaes, 2010). Oxidative-stable amylases are alsodeveloped by protein engineering of proteases in Bacilluslicheniformis. Therefore, among all known microbialamylases, B. licheniformis enzyme is the most appropriatebecause of its thermostability and resistance to proteolyticdigestion (Galante & Formantici, 2003).In the textile industry, amylase is used for desizing.

Sizing is defined as coating the yarn before fabric weavingwith a layer of removable material to enhance a fast andsecure weaving process (Souza &Magalhaes, 2010). How-ever, sizing agents have to be removed before bleaching,dying, or finishing. Sizing agents can be starch, cellulose,gelatin, polyester, etc.; starch is extensively employedbecause it is cheap, easily available, and can be removedeasily (Galante & Formantici, 2003). Amylases are used toremove starch from yarn. Low-temperature (20–40 �C)and high-temperature (90–105 �C) amylases are used.Amylases require calcium to maintain stability at hightemperatures but in the textile industry the use of calciumis not practical because free calcium availability is an issuein the presence of cellulose (Galante & Formantici, 2003).However, engineered amylases from B. licheniformis haveovercome this problem with a wide range of pH and ther-mal stability (van der Laan, 1995).Ethanol can be produced synthetically and naturally by

microorganisms. Various feedstock and chemicallydefined media can be used for ethanol fermentation.The most commonly used types of feedstock for ethanolproduction are corn, sugar cane, and wheat (Balat et al.,2008). Corn is the main raw material for ethanol pro-duction in the US, accounting for around 97% of the totalethanol production, whereas in Brazil sugar cane is themain raw material. However, corn is a starchy materialand requires starch hydrolysis before fermentation sinceS. cerevisiae cannot ferment polysaccharides.Amylases have been applied in the fuel industry to

convert starch-containing raw material into glucosemonomers before fermentation. In the pulp and paperindustry, amylases are used to modify the starch forpaper coating. Coating provides a smooth and strongpaper surface, which improves writing quality. Starchused for paper coating should have low viscosity and highmolecular weight (Gupta et al., 2003). α-Amylase is usedto partially degrade starch polymers to decrease the vis-cosity (Souza & Magalhaes, 2010).

6.6.1.3 Pectic enzymes

These enzymes can hydrolyze the long and complicatedmolecules named pectins that are structural polysacchar-ides in the plant cell and maintain integrity of the cellwall (Bigelis, 1993). Pectic enzymes are common com-mercially used enzymes and account for 25% of foodenzymes sales worldwide (Jayani et al., 2005). Aspergillusspecies are the most common microbial source forthese enzymes but Coniothyrium diplodiella, Sclerotinialibertiana, Penicillum spp., and Rhizopus spp. arealternative producers (Bigelis, 1993). Pectic substancesare high molecular weight (30,000–300,000 Da),negatively charged complex polysaccharides, with abackbone of galacturonic acid residues linked by α-1,4-linkages (Kashyap et al., 2001). The monomers ofgalacturonic acid are esterified by methyl groups orgalactans, arabinogalactans, arabinans, and rhamnoga-lacturonans (Bigelis, 1993).The American Chemical Society has categorized pectic

substances into four main groups. The first group, calledprotopectins, are water-soluble pectic substances com-posed of pectin or pectic acid. The second group is pecticacid and polymers of galacturonans that contain negligi-ble amounts of methoxyl groups. Pectinic acid, the thirdgroup, is the polygalacturonan chain with variousamounts of methoxyl groups (0–75%). Pectin is the lastgroup and defines the mixture of differing compositionsof galacturonate units esterified with methanol (Jayaniet al., 2005; Kashyap et al., 2001; Lea, 1995). As pecticsubstances are categorized, pectinolytic enzymes are alsoclassified and divided into three groups in general. Proto-pectinases are enzymes that catalyze the degradation ofinsoluble propectin to highly polymerized soluble pectin(protopectin + H2O! pectin). Pectinesterases are thesecond major group of pectic enymzes that catalyze thede-esterification of pectin by removal of methoxy esters.Pectin methyl esterase (EC 3.1.1.11) is an example of thisgroup of enzymes and hydrolyzes pectin into pectic acidand methanol. The last group are depolymerases whichcatalyze the hydrolytic cleavage of the α-1,4-glycosidicbonds in the galacturonic acid (Grassin & Coutel, 2010;Jayani et al., 2005). Lyases are subcategorized underdepolymerases but differ from hydrolases in terms ofcleaving α-1,4-glycosidic bonds by transelimination andproduce galacturonide with an unsaturated bond betweenC4 and C5 at the non-reducing end of the galacturonicacid formed (Kashyap et al., 2001).Pectic enzymes have been used since the 1930s in

manufacturing of wines and fruit juices (Kashyap


et al., 2001). Recently, there have been new applicationsin several industrial processes beside juice and wine, suchas textiles, tea, coffee, oil extraction, waste water treat-ment, etc. In juice and wine making, Aspergillus niger-derived pectic enzymes are commonly used. Sparklingclear juices, cloudy juices, and unicellular products arecreated by pectic enzyme applications in the juice indus-try. Enzymes for sparkling juices are employed toincrease the yield and clarification of juice (Grassin &Coutel, 2010; Kashyap et al., 2001). Filtration time isreduced up to 50% when fruit juices are processed withpectic enzymes (Jayani et al., 2005). Clarification isaffected by pH, temperature, enzyme concentration,and enzyme contact time. Lower pH will induce clarifi-cation rather than high pH, while elevated temperaturewill also increase the clarification rate as long as theyenzyme is not denatured (Kilara, 1982). Apple, pear,strawberry, raspberry, blackberry, and grape juice aresome examples of sparkling clear juices (Grassin &Coutel, 2010).Another use of pectic enzymes in juice industry is

stabilizing the cloud of citrus juices, purees, and nectars.Orange, lemon, mango, apricot, guava, papaya, pineapple,and banana are processed with enzymes to maintain acloudy texture (Kashyap et al., 2001). Enzyme applicationdiffers for each fruit due to its specific requirements andproperties. For example, oranges naturally contain pectinesterase and in the presence of calcium, an undesirablesedimentation of cloud particles occurs. Enzymatic treat-ment increases the stability of cloudiness by degrading thepectin without catalyzing the insoluble pectin that main-tains cloud stability (Kashyap et al., 2001). Production ofunicellular products is another application of pecticenzymes. In this process, a pulpy product is producedwith the addition of enzymes, also called maceration, touse as material for baby foods, puddings, and yogurts.Maceration is transformation of tissue into a suspendedcell. A mechanical treatment is performed before enzymehydrolysis of pectin to enhance product properties(Kashyap et al., 2001).Application of pectic enzymes is not limited to the fruit

juice industry. Pectinases are commercialized to removesizing agents from cotton with a combination of amylases,hemicellulases, and lipases (Hoondal et al., 2000). Pecti-nases are also an eco-friendly and economic alternativefor degumming (removing gums from fibers) comparedto chemical treatments in which pollution and toxicityare of concern (Kapoor et al., 2001). Waste water fromvegetable processing plants contains pectin, and pecti-nases facilitate elimination of those by-products

(Hoondal et al., 2000). Pectinases are also used for pro-duction of animal feed (Hoondal et al., 2000), paper mak-ing (Reid & Richard, 2004), and extraction of citrus oil(Scott, 1978).

6.6.1.4 Proteases

Proteases are protein-degrading enzymes and catalyzethe cleavage of peptide bonds in the proteins. Proteinsare linear polymers of amino acids with a general formulaof R-CHNH2-COOH in which peptide bonds are formedby the covalent binding of nitrogen atoms of the aminogroup to the carbon atoms of the preceding carboxylgroup with the release of water (Adler-Nissen, 1993).Proteases are classified according to their catalytic action

into endopeptidases and exopeptidases. The EC systemsubcategorizes the enzymes as 3.4.11–19 for exopeptidases,and 3.4.21–24 for endopeptidases. Endopeptidases act onthe polypeptide chain at specifically susceptible peptidebonds, while exopeptides hydrolyze the release of a singleamino acid at a time from either the N terminus orC terminus (Adler-Nissen, 1993). Endopeptidases are ser-ine proteases (EC 3.4.21), cysteine proteases (EC 3.4.22),aspartic proteases (EC 3.4.23), and metalloproteases (EC3.4.24). Serine, cysteine, and aspartic proteases containserine, cysteine, and aspartic acid, respectively, whereasmetalloproteases contain an essential metal atom, usuallyzinc (Zn), at the active sites (Adler-Nissen, 1993).Exopeptidases, on the other hand, are divided into threemajor groups: aminopeptidases, carboxypeptidases, andomegapeptidases. Aminopeptidases generate a singleamino acid residue, di- or tripeptide, by attacking theN terminus of the polypeptide chain. Carboxypeptides,in contrary, act at the C terminus of the polypeptide andliberate a single amino acid or dipeptide (Rao et al., 1998).Proteases are found in plants, animals, and microor-

ganisms. Papain, extracted from Carica papaya, has beenused as a meat tenderizer for a long time. This enzyme isactive at pH 5–9 and stable up to 90 �C, but the perfor-mance of the enzyme is highly dependent on plant source,climate conditions for growth, and extraction and purifi-cation methods. Papain is used for preparation of solubleand flavored protein hydrolyzates in industry (Rao et al.,1998). Bromelain, extracted from pineapples, is a cysteineprotease active up to 70 �C in a range of pH 5–9. The othercommon plant protease is keratinase produced by abotanical plant. Keratinases degrade hair and wool, andfind applications in prevention of clogging of waste watersystems (Rao et al., 1998).


Well-known animal proteases are trypsin, chymotrypsin,pepsin, and rennin (Boyer, 1971). Trypsin is a digestiveenzyme that hydrolyzes food proteins. However, use oftrypsin in the food industry is not common because it gen-erates a very bitter taste so it is used for bacterial mediaformulation and some medical applications (Rao et al.,1998). Chymotrypsin is found in animal pancreases andis expensive, and thus used mainly for analytical applica-tions. Milk protein hydrolyzates are commonly dealler-genized by chymotrypsin. Pepsin is an acidic proteaseand catalyzes the hydrolysis of peptide bonds betweentwo hydrophobic amino acids (Rao et al., 1998).Rennet (rennin, chymosin) cleaves a single peptide bondin κ-casein and produces insoluble para-κ-casein andC-terminal glycopeptides (Law, 2010).Bacteria, fungi, and viruses are excellent protease

sources. Microbial proteases represent about 40% ofenzyme sales globally (Godfrey & West, 1996). Bacterialproteases are active at alkaline pH and stable up to60 �C which allows the detergent industry to use them.Fungal proteases also have a wide pH range (4–11) buttheir reaction rate and heat tolerance are lower comparedto bacterial enzymes (Rao et al., 1998).Proteases have a broad application in the food industry.

In the dairy industry, milk-coagulating enzymes (animalrennin, microbial coagulants, engineered chymosin) areextensively used for cheese making. Chymosin hasadvantages over animal rennin due to its specific activityand availability. The protease-producing GRAS micro-organisms areMucormichei, Bacillus subtilis, and Endothiaparasitica. Proteases are used in the baking industry tomodify insoluble wheat protein gluten, which determinesthe characteristics of dough. Enzymatic treatment of doughimproves the handling and machining, as well as timereduction and increase in loaf volume. Aspergillus oryzaeenzymes have been used in the baking industry (Raoet al., 1998). However, proteases can generate a bitter taste,which limits their use in the food industry. Bitterness isrelative to the number of hydrophobic amino acids inthe hydrolyzate. A combination of endoprotease andamonipeptidase can reduce bitterness (Rao et al., 1998).Proteases have several applications in the pharmaceu-

tical industry as a digestive aid, in the health food industryfor synthesis of aspartame, and in infant formulas fordebittering of protein hydrolyzate.Use of proteases in the detergent industry has grown

and the largest application is in household laundry deter-gent formulation. Proteases remove proteinaceous stains,such as food, blood, and other body secretions (Guptaet al., 2002). Proteases used in detergent industry should

be active at a wide range of pH and temperature profiles,and compatible with chelating and oxidizing agents (Raoet al., 1998). Bacillus strains are employed for productionof proteases used in the detergent industry. In the leatherindustry, a major problem is removal of skin and hair,which are proteinaceous substances. Therefore, use ofproteases having elastolytic and keratinolytic activity isan alternative to chemical treatment in the leatherindustry in which environmental impacts are of concern.Proteases are employed in the soaking, dehairing, and bat-ing stages of leather processing to destroy undesirable pig-ments and maintain hair-free leather (Gupta et al., 2002).

6.6.1.5 Oxidoreductases

Oxidoreductases are another group of enzymes that cat-alyze the oxidation/reduction reaction. Oxidoreductasesplay a crucial role in foods in terms of taste, texture,shelf life, appearance, and nutritional value. Polyphenoloxidase is a copper-containing enzyme widely found inplants, animals, and humans, and can catalyze phenoliccompounds involved in reactions (Hammer, 1993). Oneof the subclasses of oxidoreductases is peroxidase. Hydro-gen peroxide is the main substrate for peroxidases thatusually generates colored end-products. Horseradish per-oxidase is the well-known enzyme of this group because ofits use as an indicator in spectrophotometric and immu-noassay techniques (Hammer, 1993). Lactoperoxidase is ahemoprotein that catalyzes the oxidation of thiocyanateand iodine ions in the presence of hydrogen peroxideto produce highly reactive oxidizing agents. Lactoperoxi-dase is antimicrobial and kills gram-negative and-positive bacteria, and fungi. Catalase, a hemoprotein,catalyzes the decomposition of hydrogen peroxide intooxygen and water (Inamine & Baker, 1989). Oxidationof thiols to their subsequent disulfides is catalyzed by sulf-hydryl oxidase using molecular oxygen as the electronacceptor. This enzyme is isolated from Myrotheciumwerrucaria, Aspergillus sojae, and Aspergillus niger (de laMotte & Wagner, 1987; Katkochin et al., 1986; Mandel,1956). Lipoxygenase is another subgroup of oxidoreduc-tases which catalyzes the oxidation of polyunsaturatedfatty acids by using oxygen; hydroperoxides are producedwhich are converted into acids, aldehydes or ketones, andother compounds (Matheis & Whitaker, 1987).Some other applications of oxidoreductases include

alcohol oxidase, which catalyzes the oxidation of short-chain linear aliphatic alcohols to aldehydes and H2O2.However, alcohol oxidase may not have applications inthe alcoholic beverages industry because the optimum


pH for this enzyme is 7.5–8. Also, ethanol and methanolhave inhibitory effects on the enzyme (Hammer, 1993).Oxidoreductases are also employed in the pasteurizationof eggs and cheese by H2O2, desugaring of eggs prior tospray drying, preservation of raw milk, and eliminationof cooked flavor of UHT (ultra high temperature) pas-teurized milk (Szalkucki, 1993).Because of the limitations of space, only the most impor-

tant enzymes that have applications in the food industryhave been mentioned. However, there are many otherenzymes that have been extensively used, including lactases,invertases, cellulases, hemicellulases, dextranases, andlysozyme. More details on enzymes used in food and otherindustries can be found in Reed (1966), Nagodawithanaand Reed (1993), Tucker and Woods (1995), Whitehurstand van Oort (2010), Chen et al. (2003), and Bhat (2000).

6.7 Sustainability

The definition of sustainability varies depending on whodefines it. Sustainability can be defined as “… a process ofchange in which the exploitation of resources, the direc-tion of investments, the orientation of technologicaldevelopment, and institutional change are all in harmonyand enhance both current and future potential to meethuman needs and aspirations…” according to theBrundtland Report (1987). Therefore, sustainable pro-cesses utilize renewable resources and decrease green-house gases and other pollutants.Microbial fermentation has been used for such pur-

poses for many commercial products and enables noveltechniques to support sustainable manufacturing. Utiliza-tion of non-food value feedstocks, genetically engineeredmicroorganisms, and cost- and energy-efficient fermenta-tion technologies promotes sustainability. In the vitaminproduction industry, for example, complex vitamin B2production has come down to a simple fermentationprocess that has also lowered environmental impact(Gebhard, 2009). Furthermore, production increases300,000 fold when genetically engineered bacteria(Bacillus subtilis) are used (Gavrilescu & Chisti, 2005).Enzymes are commonly used in the chemical industryas catalysts. The specificity of enzymes increases demandcompared to conventional chemical catalysts. Enzymesalso work under non-toxic and non-corrosive conditions(Gavrilescu & Chisti, 2005).Therefore, more study isneeded to develop cost-competitive fermentation andenzyme technologies to reduce material, water and energyconsumption and pollutant disposal.

6.8 Concluding remarks and future trends

Fermentation is extensively employed by the food indus-try to produce value-added products. The success of thefermentation is dependent on the microorganism/strainselection and optimization of growth parameters for theselected microorganism. Fermentation has also been usedto enhance the quality and flavor of food. Optimization offermentation, for a consumer-acceptable fermented food,also should be well considered. On the other hand,enzymes have their own application in the industry dueto their catalytic activity.There is still the need for isolation of new microbial

strains and strain development for high yields in thefermentation process. Moreover, genetic and metabolicengineering is an important tool to develop strains,which can produce heterologous products. As a result,products, which are not currently industrially availablemay become affordable for industrial use. Because somemicroorganisms do not produce exopolysaccharide,which creates difficulties with attachment to supports,immobilization reactors should be tested for each productto test the effect of immobilization on yield. In addition toyield, the strains and the production parameters of tradi-tional fermented food should be studied. The natural floraimprove the flavor and texture of the fermented food.Therefore, microorganisms in traditional foods shouldbe isolated and the functions of these microorganismsshould be studied. As a result, the diversity of traditionalfermented foods can be protected.There is always a need to study microbial production

and applications of enzymes. The characteristics ofenzymes are being improved by protein engineering toincrease their potential with respect to their temperatureand pH profiles, stability, specificity, etc. Furthermore,novel immobilization techniques can be studied in orderto use enzymes more efficiently.

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